Synthesis and biological evaluation of vinylogous combretastatin A-4 derivatives

Article abstract of DOI:10.1039/b505955k

Stereospecific syntheses of the Z-E and E-Z vinylogues of combretastatin A-4, and two B-ring related analogues, were achieved through a Suzuki-Miyaura coupling. As compared to CA4, the derivative with a phenyl moiety has shown increased potency in its abi

Full text of DOI:10.1039/b505955k

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A R T I C L E
Synthesis and biological evaluation of vinylogous combretastatin
A-4 derivatives
Julia Kaffy, Rene´e Pontikis,* Jean-Claude Florent and Claude Monneret
UMR 176 CNRS, Institut Curie-Section de Recherche, 26 rue d’Ulm, 75248, Paris cedex 05,
France. E-mail: rene´e.pontikis@curie.fr
Received 28th April 2005, Accepted 26th May 2005
First published as an Advance Article on the web 21st June 2005
Stereospecific syntheses of the Z–E and E–Z vinylogues of combretastatin A-4, and two B-ring related analogues,
were achieved through a Suzuki–Miyaura coupling. As compared to CA4, the derivative with a phenyl moiety has
shown increased potency in its ability to inhibit tubulin polymerisation.
Introduction
Combretastatin A-4 (CA4), a natural product isolated1 by Pettit
et al. in 1989 from the South African willow tree, Combretum
caffrum, strongly inhibits the polymerisation of tubulin by
binding to the colchicine site.2 It has been shown that tumour
vessels are more susceptible to the disruptive effect of CA4 than
normal vasculature. The result, for the tumour’s blood vessels,
is a morphological change in their endothelial cells leading
to increased permeability and rapid vascular shutdown.3–5 In
experimental tumours, anti-vascular effects of CA4, which are
observed at 10% of its maximum tolerated dose, rapidly lead
to extensive ischemic necrosis in areas that are often resistant
to conventional anti-cancer treatments. A disodium phosphate
prodrug form (CA4P) has already entered clinical trials for the
treatment of solid tumours.6 AVE8062, a water-soluble analogue
of CA4,7,8 is currently under clinical evaluation as tumour
vascular targeting agent.9
Given the encouraging antivascular/anticancer activity of
Scheme 1 Retrosynthetic analysis of ZE and EZ vinylogues of CA4
CA4P, the syntheses of numerous analogues have been reported
in order to have a better understanding of the structure–activity
relationships.10 A key structural factor for tubulin affinity is the
presence of the double bond (or of a suitable linker) forcing
the two aromatic rings to be within an appropriate distance.2,11
In order to evaluate the influence of the length of the bridge
between both aromatic rings of CA4, the syntheses of analogues
with a E,Z-dienic moiety were undertaken. Herein are described
the preparation and biological activities of a series of vinylogues
of CA4 (Fig. 1).
(derivatives a: Ar = 3,4,5-trimethoxyphenyl, b: Ar = 3-hydroxy-
4-methoxyphenyl).
3a–b were elaborated from two common intermediates, the 1,1-
dibromoalkenes 4a–b which have been previously synthesised13,14
from the commercially available aldehydes 6a–b.
(E)-3,4,5-Trimethoxystyryl boronic acid 3a, required for
the synthesis of diene 1EZ (Scheme 2), was prepared by
hydroboration15 of the appropriate acetylenic precursor. The
known arylacetylene 5a was synthesized, via the dibromide 4a,
by Corey–Fuchs conversion16 of 3,4,5-trimethoxybenzaldehyde
6a. Hydroboration of 5a with catecholborane (1 equiv.) in
refluxing THF, followed by in situ hydrolysis of the intermediate
adduct, stereoselectively afforded the (E)-vinylboronic acid 3a
in 60% yield. The other partner, (Z)-b-styrylbromide 2b, was
prepared from isovanillin 6b (60% yield) according to Gaukroger
et al.,14 by stereoselective hydrogenolysis of the dibromide 4b
with tributyltin hydride in the presence of a catalytic amount of
Pd(PPh3)4.17 The cross-coupling of vinylbromide 2b and boronic
acid 3a, in the presence18 of Pd(PPh3)4 and EtONa as a base,
afforded the desired diene 1EZ in 80% yield with retention of
configuration.
Fig. 1
The synthesis of the isomeric diene 1ZE by the same approach
(Scheme 3) required the (E)-alkenylboron derivative 3b and
the (Z)-3,4,5-trimethoxystyrylbromide 2a which was prepared
from dibromide 4a.19 Reaction of dibromostyrene 4c with n-
butyllithium, followed by hydrolysis, afforded arylacetylene 5c
(93% yield). Difficulties were encountered for hydroboration
of compound 5c; three equivalents of catecholborane were
necessary for completion of the reaction. Moreover, after in
situ hydrolysis of the boronate, the corresponding boronic
acid proved to be very difficult to separate from the catechol
Results and discussion
The route adopted to prepare these dienes (Scheme 1) was
based on the Suzuki–Miyaura cross-coupling12 of stereodefined
alkenyl boron derivatives with stereodefined haloalkenes. Diene
1ZE was constructed from the (Z)-vinylbromide 2a and the
(E)-vinylboronic acid 3b, diene 1EZ from derivatives 2b and
3a. These four requisite olefin partners were elaborated from
two common intermediates, 1,1-dibromoalkenes 4a–b. The
required (Z)-vinylbromides 2a–b and (E)-vinylboronic acids
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 5 7 – 2 6 6 0
2 6 5 7
©

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isomerically pure diene 1ZE in an unoptimized 37% yield after
chromatography.
A molecular modelling study of vinylogue 1ZE and CA4,
using HyperChem software,20 suggested close geometric similar-
ities (Fig. 2). Nevertheless, for the dienic derivative, substituents
of the B-ring were out of the conformational space of CA4.
These results prompted us to synthesize dienic derivatives
(Scheme 4) with no substituent at the 4-position of the B-ring.
So, using the afore-mentioned procedure, vinylbromide 2a was
coupled to commercially available (E)-vinylboronic acids 7 and
8, giving dienes 9 and 10 in 75 and 54% yields, respectively.
Scheme 2 Synthesis of vinylogue 1EZ. Reagents and conditions:
(i) ref. 13: n-BuLi (2 equiv.), THF, 0 ◦C to rt; (ii) catecholborane, THF,
reflux then H2O; (iii) ref 14: Bu3SnH, Pd(PPh3)4, benzene, 0 ◦C to rt;
(iv) Pd(PPh3)4, EtONa, THF, reflux.
Fig. 2 Superimposition of diene 1ZE (gray) with CA4 (black).
Scheme 4 Synthesis of dienes 9 and 10. Reagents and conditions:
(i) Pd(PPh3)4, EtONa, THF, reflux.
The synthesized diarylbutadienes were evaluated (Table 1)
for their antitubulin polymerisation activities and for their in
vitro cytotoxicity against the human colon carcinoma cell line
(HCT116).
Vinylogues 1ZE and 1EZ inhibit tubulin polymerisation with
IC50 values of 2.3 and 2.0 lM, respectively, values comparable
to that of CA4, but both these compounds, in acidic media, are
prone to isomerise into the more stable (EE)-butadiene.21 This
isomerisation resulted in a marked loss in antitubulin activity
(>10-fold) that confirms, in this series, the necessity of a cis-
double bond for bioactivity. Derivative 9, with no substituent
Scheme 3 Synthesis of vinylogue 1ZE. Reagents and conditions:
(i) n-BuLi (2 equiv.), THF,
0
◦C to rt; (ii) TBAF, THF, rt;
(iii) catecholborane, THF, reflux then chromatography; (iv) Pd(PPh3)4,
EtONa, THF, reflux.
by-product. Consequently, the reaction was repeated using
arylacetylene 5b bearing a free phenol (obtained in 75% yield
after TBAF treatment). Flash chromatography of the crude
boronate was performed, but concomitant hydrolysis of the
product occurred, so boronic acid 3b was isolated in only modest
yield (35% yield). Finally, subsequent Suzuki coupling of the
free phenol derivative 3b with the (Z)-vinylbromide 2a produced
on the B-ring, is 4-fold more potent than vinylogue 1ZE (IC50
0.5 lM), whereas compound 10 (IC50 = 3.5 lM), possessing a
meta-methoxy group, is slightly less potent.
=
Table 1 Inhibition of tubulin polymerisation and cellular proliferation for dienes 1, 9 and 10
IC50
Compound
R1
R2
Antitubulin activity/lMa
HCT116/lMb
1ZE
1EZ
1EE
9
OH
OH
OH
H
OCH3
—
OCH3
OCH3
OCH3
H
H
—
2.3
2.0
24.9
0.5
3.5
1.2
0.163
0.062
ND
0.503
6.32
10
CA4
0.016c
a Drug concentration that inhibits tubulin polymerisation by 50%. b Drug concentration that inhibits cancer cells growth by 50%. c Reference 10h.
2 6 5 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 5 7 – 2 6 6 0

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From the cellular tests, it appears that cytotoxicities are
not well correlated with the inhibitory ability of tubulin
polymerisation. Against HCT116, vinylogue 1EZ showed good
antiproliferative effect (IC50 = 62 nM), while the unsubstituted
derivative 9 showed a 8-fold reduction in potency. This reduced
cytotoxicity was presumed to be due to its poor cellular
permeability. Adding a methoxy group at the C-3 position of
the phenyl ring decreased the observed activity.
5.1. C9H8O2 requires C, 73.0; H, 5.4%);dH (300 MHz, CDCl3)
7.05 (2H, m, H-2,6), 6.78 (1H, d, J 8.1, H-5), 5.60 (1H, s, OH),
3.90 (3H, s, OCH3), 2.97 (1H, s, CH); MS (CI/NH3) m/z 166
(M + NH4)+.
(E)-2-(3,4,5-Trimethoxyphenyl)vinylboronic acid (3a). To a
solution of trimethoxyphenylacetylene 5a (250 mg, 1.3 mmol)
in dry THF (1 mL) under argon at rt was added dropwise
catecholborane (140 lL, 1.3 mmol). The mixture was heated
under reflux for 3 h, then partially concentrated in vacuo. Cold
water (2 mL) was added and the white suspension was stirred for
2 h at 0 ◦C to hydrolyze the ester. The resulting solid was filtered,
and rinsed with cold water. Recrystallization from diethylether
afforded acid 3a as a white solid (185 mg, 60% yield); mp 136–
138 ◦C; dH (300 MHz, CDCl3) 7.23 (1H, d, J 16.6, ArCH), 6.70
(2H, s, H-2,6), 6.04 (1H, d, J 16.6, CHB), 3.83 (6H, s, OCH3 ×
2), 3.81 (3H, s, OCH3); dC (75 MHz, CDCl3) 153.0 (C × 2),
146.9 (C), 138.3 (C), 133.5 (C), 120.7 (CH), 103.8 (CH × 2),
60.7 (CH3), 55.9 (CH3 × 2).
Conclusion
We have demonstrated that replacement of the cis double bond
between the two aromatic rings of CA4, by an (E,Z)butadiene
moiety, gave compounds with equal or greater depolymerising
microtubule activity than CA4. Surprisingly however, this does
not correlate with the cytotoxicities observed. Nevertheless,
separating the cytotoxic activity from the ability to inhibit the
polymerisation of tubulin represents an interesting goal in the
search for potent antivascular agents.
(E)-2-(3-Hydroxy-4-methoxyphenyl)vinylboronic acid (3b).
To a solution of alkyne 5b (665 mg, 4.49 mmol) in dry THF
(10 mL) under argon at rt was added dropwise catecholborane
(1.43 mL, 13.45 mmol). The mixture was heated under reflux
for 5 h then evaporated in vacuo. Column chromatography on
silica gel (cyclohexane–EtOAc 7 : 3) of the crude ester afforded
boronic acid 3b as a foam (305 mg, 35%);dH (300 MHz, CDCl3)
7.21 (1H, d, J 18.0, ArCH), 7.02 (1H, d, J 1.8, H-2), 6.94 (1H,
dd, J 8.1, 1.8, H-6), 6.87 (1H, d, J 8.1, H-5), 6.14 (1H, d, J
18.0, CHB), 3.86 (3H, s, OCH3); dC (75 MHz, CDCl3) 149.9
(C), 149.5 (C), 147.7 (CH), 132.7 (CH), 120.9 (CH), 113.9 (CH),
112.4 (CH), 56.3 (CH3).
Experimental
General
NMR spectra were recorded on a Bruker AM-300 spectrometer
at 300 MHz (1H) and at 75 MHz (13C) using CDCl3 as the
solvent. The residual proton-solvent peak is used as the internal
standard [(d 7.25 (1H) and 77.0 ppm (13C)] and J values are
given in Hz. Multiplicities are reported using the following
abbreviations: s = singlet, d = doublet, dd = doublet of doublets,
br = broad, m = multiplet. Chemical ionization (CI) mass
spectra were recorded on a Nermag R10-10-C spectrometer.
High-resolution mass spectra (HRMS) were obtained on a Jeol-
700 spectrometer. Melting points were determined using an
Electrothermal 9200 apparatus and are uncorrected. Elemental
analyses were performed by the “Service de Microanalyses du
CNRS” (Vernaison-Lyon, France). The thin-layer chromato-
graphic analyses were performed using pre-coated silica gel
(Merck, 60F254) plates and the spots were examined with
UV light and phosphomolybdic acid spray. Preparative column
chromatographies were carried out on Merck silica gel (230–240
mesh). THF was distilled from sodium benzophenone ketyl prior
to use. Derivatives 2b,14 4a19 and 5a13 were prepared according
to published procedures.
(1E,3Z )-1-(3ꢀ,4ꢀ,5ꢀ -Trimethoxyphenyl)-4-(3ꢀꢀ -hydroxy-4ꢀꢀ -
methoxyphenyl)-1,3-butadiene (1EZ). To a stirred solution
of boronic acid 3a (61 mg, 0.25 mmol) and vinylbromide 2b
(59 mg, 0.25 mmol) in degassed THF (1 mL) under argon were
successively added Pd(PPh3)4 (12 mg, 0.01 mmol) then 21%
sodium ethoxide in ethanol (190 lL). The reaction mixture
was stirred at room temperature for 2 h then heated under
reflux for 1 h. Dilution with EtOAc was followed by filtration
through Celite. The filtrate was washed with a saturated NaCl
solution and the combined organic layers were dried (MgSO4),
filtered and concentrated in vacuo. Purification of the crude
product by silica gel chromatography (cyclohexane–EtOAc 7 :
3) provided diene 1EZ as a foam (71 mg, 80%); dH (300 MHz,
CDCl3) 7.22 (1H, dd, J 15.4, 10.7, H-2), 7.00 (1H, d, J 1.6,
H-2ꢀꢀ), 6.87 (2H, m, H-5ꢀꢀ,6ꢀꢀ), 6.62 (2H, s, H-2ꢀ,6ꢀ), 6.59 (1H,
d, J 15.4, H-1), 6.35 (2H, m, H-3,4), 5.63 (1H, s, OH), 3.92
(3H, s, OCH3), 3.87 (6H, s, OCH3 × 2), 3.85 (3H, s, OCH3); dC
(75 MHz, CDCl3) 153.3 (C × 2), 145.8 (C), 145.4 (C), 138.0
(C), 134.3 (CH), 133.2 (C), 131.2 (C), 129.8 (CH), 129.1 (CH),
125.0 (CH), 121.2 (CH), 115.1 (CH), 110.4 (CH), 103.7 (CH ×
2), 60.9 (CH3), 56.1(CH3 × 2), 56.0 (CH3); HRMS (CI/NH3)
calcd. for C20H23O5 (MH+): 343.1546. Found: 343.1544.
2-(3-t-Butyldimethylsilyloxy-4-methoxyphenyl)ethyne
(5c).
Following the method of Corey and Fuchs,16 a solution of the
dibromoalkene 4c (2.50 g, 5.9 mmol) in anhydrous THF (35
mL) at −78 ◦C under argon was treated with n-butyllithium
(7.8 mL, 1.6 M in hexane, 12.5 mmol). The reaction mixture
was stirred at −78 ◦C for 1 h then warmed slowly to rt and
stirred for a further 1 h. Saturated aqueous NH4Cl solution
was added and the mixture extracted with ether. The combined
organic fractions were dried (MgSO4), filtered and evaporated
in vacuo to give a brown oil. After column chromatography on
silica gel (cyclohexane–EtOAc 99 : 1), ethyne 5c was isolated as
a clear oil (1.44 g, 93%); dH (300 MHz, CDCl3) 7.08 (1H, dd, J
8.3, 2.0, H-6), 6.98 (1H, d, J 2.0, H-2), 6.77 (1H, d, J 8.3, H-5),
3.81 (3H, s, OCH3), 2.97 (1H, s, CH), 0.99 [9H, s, C(CH3)3],
0.15 [6H, s, Si(CH3)2].
The following compounds were prepared in a similar manner.
(1Z,3E)-1-(3ꢀ,4ꢀ,5ꢀ -Trimethoxyphenyl)-4-(3ꢀꢀ -hydroxy-4ꢀꢀ -
methoxyphenyl)-1,3-butadiene (1ZE). From vinylbromide 2a
(410 mg, 1.50 mmol) and boronic acid 3b (189 mg, 0.97 mmol)
was obtained, after chromatography (cyclohexane–EtOAc 8 :
2), compound 1ZE as a foam (122 mg, 37%); dH (300 MHz,
CDCl3) 7.22 (1H, dd, J 15.4, 9.5, H-3), 7.00 (1H, d, J 1.9, H-2ꢀꢀ),
6.88 (1H, dd, J 8.3, 1.9, H-6ꢀꢀ), 6.79 (1H, d, J 8.3, H-5ꢀꢀ), 6.61
(1H, d, J 15.4, H-4), 6.59 (2H, s, H-2ꢀ,6ꢀ), 6.37 (2H, m, H-1,2),
5.59 (1H, s, OH), 3.88 (9H, s, OCH3 × 3), 3.87 (3H, s, OCH3); dC
(75 MHz, CDCl3) 153.0 (C × 2), 146.5 (C), 145.8 (C), 137.3 (C),
134.4 (CH), 133.4 (C), 131.1 (C), 130.2 (CH), 129.4 (CH), 123.7
(CH), 119.2 (CH), 111.8 (CH), 110.7 (CH), 106.1 (CH × 2),
2-(3-Hydroxy-4-methoxyphenyl)ethyne (5b). To a solution of
5c (2.90 g, 11 mmol) in THF (25 mL) was added Bu4NF (11 mL,
1.0 M in THF). After stirring at rt for 1 h, water was added and
the mixture was extracted with ether. The combined organic
fractions were dried (MgSO4), filtered and evaporated under
reduced pressure. The crude product was purified by column
chromatography on silica gel (cyclohexane–EtOAc 8 : 2), to
yield phenol 5b as a white solid whic◦h was recrystallized in
ether–hexane (1.23 g, 75%); mp 62–64 C (Found: C, 73.3; H,
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 5 7 – 2 6 6 0
2 6 5 9

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61.0 (CH3), 56.1 (CH3 × 2), 56.0 (CH3); HRMS (CI/NH3)
Acknowledgements
calcd. for C20H23O5 (MH+): 343.1546. Found: 343.1542.
The authors are pleased to thank Dr Alain Commerc¸on and Dr
Patrick Mailliet for helpful suggestions and discussions (Aventis
Pharma, Medicinal Chemistry Department, 94400 Vitry-sur-
Seine, France). Laboratories Aventis Pharma are gratefully
acknowledged for a doctoral grant to Julia Kaffy and for
biological evaluation. This work was financially supported
by the Centre National de la Recherche Scientifique and the
Institut Curie (Programme Incitatif et Coope´ratif: angiogene`se
tumorale).
(1Z,3E)-1-(3ꢀ,4ꢀ,5ꢀ-Trimethoxyphenyl)-4-phenyl-1,3-butadiene
(9). From vinylbromide 2a (250 mg, 1.68 mmol) and (E)-vinyl-
boronic acid 7 (230 mg, 0.84 mmol) was obtained, after chro-
matography (cyclohexane–EtOAc 9 : 1) and recrystallisation
from EtOAc–pentane, compound 9 as a white solid (187 mg,
75%); mp 85–87 ◦C; dH (300 MHz, CDCl3) 7.40 (2H, m,
H-2ꢀꢀ,6ꢀꢀ), 7.32 (1H, m, H-4ꢀꢀ), 7.29 (2H, m, H-3ꢀꢀ,5ꢀꢀ), 7.26 (1H,
m, H-3), 6.71 (1H, d, J 15.6, H-4), 6.60 (2H, s, H-2ꢀ,6ꢀ), 6.42
(2H, m, H-1,2), 3.89 (3H, s, OCH3), 3.88 (6H, s, OCH3 × 2);
dC (75 MHz, CDCl3) 153.0 (C × 2), 137.2 (C × 2), 134.7 (CH),
133.2 (C), 130.3 (CH), 130.1 (CH), 128.7 (C × 2), 127.7 (CH),
126.4 (CH × 2), 125.1 (CH), 106.1 (CH × 2), 61.0 (CH3),
56.1 (CH3 × 2); HRMS (CI/NH3) calcd. for C19H21O3 (MH+):
297.1491. Found: 297.1483.
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(1Z,3E)-1-(3ꢀ,4ꢀ,5ꢀ-Trimethoxyphenyl)-4-(3ꢀꢀ-methoxyphenyl)-
1,3-butadiene (10). From vinylbromide 2a (410 mg, 1.50
mmol) and boronic acid 8 (189 mg, 0.97 mmol) was obtained,
after chromatography (cyclohexane–EtOAc 8 : 2), compound
10 as a foam (122 mg, 37%); dH (300 MHz, CDCl3) 7.36 (1H,
dd, J 15.5, 10.6, H-3), 7.22 (1H, m, H-5ꢀꢀ), 6.99 (1H, d, J 7.8,
H-6ꢀꢀ), 6.92 (1H, m, H-2ꢀꢀ), 6.79 (1H, m, H-4ꢀꢀ), 6.68 (1H, d,
J 15.6, H-4), 6.60 (2H, s, H-2ꢀ,6ꢀ), 6.42 (2H, m, H-1,2), 3.89
(3H, s, OCH3), 3.88 (6H, s, OCH3 × 2), 3,80 (3H, s, OCH3); dC
(75 MHz, CDCl3) 159.8 (C), 153.1 (C × 2), 138.7 (C), 137.3 (C),
134.6 (CH), 133.2 (C), 130.5 (CH), 129.9 (CH), 129.8 (CH),
125.4 (CH), 119.1 (CH), 113.3 (CH), 111.8 (CH), 106.1 (CH ×
2), 61.0 (CH3), 56.1 (CH3 × 2), 56.0 (CH3); HRMS (CI/NH3)
calcd. for C20H23O4 (MH+): 327.1596. Found: 327.1588.
(1E,3E)-1-(3ꢀ,4ꢀ,5ꢀ -Trimethoxyphenyl)-4-(3ꢀꢀ -hydroxy-4ꢀꢀ -
methoxyphenyl)-1,3-butadiene (1EE). A solution of dienic
derivative 1ZE (28 mg, 0.081 mmol) in CDCl3 was stirred,
under argon, for 4 d. Compound 1EE was obtained as a foam
(19.5 mg, 70%) after chromatography (CH2Cl2); dH (300 MHz,
CDCl3) 7.07 (1H, d, J 1.9, H-2ꢀꢀ), 6.90 (1H, dd, J 8.4,1.9, H-6ꢀꢀ),
6.80 (3H, m, H-2, H-3, H-5ꢀꢀ), 6.59 (2H, s, H-2ꢀ,6ꢀ), 6.58 (d,
1, J 14.8, H-1 or H-4), 6.55 (d, 1, J 14.8 H-1 or H-4), 5.60
(1H, s, OH), 3.90 (9H, s, OCH3 × 3), 3.85 (3H, s, OCH3); dC
(75 MHz, CDCl3) 153.3 (C × 2), 146.4 (C), 145.7 (C), 137.7
(C), 133.3 (C), 132.4 (CH), 131.8 (CH), 131.1 (C), 129.0 (CH),
127.5 (CH), 119.2 (CH), 111.6 (CH), 110.6 (CH), 103.2 (CH ×
2), 61.0 (CH3), 56.1 (CH3 × 2), 56.0 (CH3); HRMS (CI/NH3)
calcd. for C20H23O5 (MH+): 343.1546. Found: 343.1543.
Biological test methods 22
12 N. Miyaura and A. Suzuki, Chem. Rev., 1995, 95, 2457–2483.
13 N. J. Lawrence, F. A. Ghani, L. A. Hepworth, J. A. Hadfield and
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14 K. Gaukroger, J. A. Hadfield, L. A. Hepworth, N. J. Lawrence and
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15 H. C. Brown and S. K. Gupta, J. Am. Chem. Soc., 1972, 94, 4370–
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16 E. Corey and P. L. Fuchs, Tetrahedron Lett., 1972, 36, 3769–3772.
17 J. Uenishi, R. Kawahama, Y. Shiga and O. Yonemistu, Tetrahedron
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Tubulin polymerisation. Porcin brain tubulin was prepared
as previously reported. Tubulin polymerisation was monitored
by turbidimetry at 350 nM with a Uvikon 931 spectropho-
tometer (Kontron) equipped with a thermostatically-regulated
cuvette holder. The products were dissolved at 10 mM in DMSO
and added at variable concentrations (0.5 to 10 lM) to the
tubulin solution before polymerisation. The IC50 value is defined
as the concentration of product which inhibits the rate of
polymerisation by 50%.
18 N. Miyaura, H. Suginome and A. Suzuki, Tetrahedron, 1983, 39,
3271–3277.
19 H.-G. Herz, M. J. Queiroz and G. Maas, Synthesis, 1999, 6, 1013–
1016.
Cytotoxicity assays. Human colon carcinoma HCT116 cell
line was obtained from the American Tissue Culture Collec-
tion. The cell proliferation was evaluated by measuring the
incorporation of 14C-thymidine. The IC50 value is defined as
the concentration of product which reduces the radioactivity by
50% compared with an untreated control.
20 HyperChem version 7.0, (HyperCube Inc.).
21 This isomerisation was observed with compounds 1ZE and 1EZ in
solution in CDCl3.
22 C. Combeau, J. Provost, F. Lancelin, Y. Tournoux, F. Prod’homme, F.
Herman, F. Lavelle, J. Leboul and M. Vuilhorgne, Mol. Pharmacol.,
2000, 57, 553–563.
2 6 6 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 5 7 – 2 6 6 0