A practical radiosynthesis of a tritium-labelled fluorocombretastatin

Article abstract of DOI:10.1002/jlcr.2939

Zybrestat (combretastatin A-4 disodium diphosphate) is currently in clinical trials as an antivascular anticancer agent. A similar fluorinated agent has shown promise as an antivascular agent, and a radiolabelled version would enable further understanding of its biological activities. Herein is described the synthesis of a tritiated fluorinated analogue of Zybrestat. Copyright

Full text of DOI:10.1002/jlcr.2939

Research Article
Received 30 March 2012,
Revised 24 May 2012,
Accepted 24 May 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.2939
A practical radiosynthesis of a tritium-labelled
fluorocombretastatin
a
a
b
*
John A. Hadfield, Nicholas Hirst, Keira Gaukroger,
Nicholas J. Lawrence,c and Alan T. McGowna
Zybrestat (combretastatin A-4 disodium diphosphate) is currently in clinical trials as an antivascular anticancer agent. A similar
fluorinated agent has shown promise as an antivascular agent, and a radiolabelled version would enable further understanding
of its biological activities. Herein is described the synthesis of a tritiated fluorinated analogue of Zybrestat.
Keywords: Zybrestat; combretastatin; tritiation
bond of the stilbene. In one resonance form, the anion is localised
at the benzylic position. This allows rotation around the single
bond in this resonance form leading to a more favoured trans
configuration as illustrated in Figure 3.
Introduction
Combretastatin A-4 phosphate
2 (CA4P, Zybrestat) and
combretastatin A-1 phosphate 4 (CA1P, OXi4503) (Figure 1) are
presently in Phase III and Phase I clinical trials for cancer, respec-
tively.1 CA4P is also in Phase II clinical trial for age-related macular
degeneration1 (a major cause of blindness for those aged over
50 years). These two agents, along with AVE8062 (Ombrabulin)2
and BNC105,3 are the leading cancer vascular damaging drugs
presently in clinical trials. The availability of a biologically active
radiolabelled combretastatin would greatly enhance the under-
standing of the pharmacokinetics and pharmacodynamics of these
types of molecules. Methods for the syntheses of radiolabelled
analogues of CA4P4 and CA1P5 have been developed, and the
synthesis of 14C-labelled combretastatin A-1 (CA1) and CA1P has
been accomplished.6
A fluorinated derivative (5) of combretastatin A-4 1 is also a
potent tubulin inhibitor,7,8 which shows antitumour effects in vivo
(Figure 2). Magnetic resonance imaging studies show that this
fluorocombretastatin also damages tumour vasculature (Hadfield
JA and McGown AT, unpublished). A radiolabelled version of this
fluorocombretastatin will provide a useful tool to understand the
pharmacokinetics and pharmacodynamics of this type of agent.
Also, a radiolabelled fluorocombretastatin could replace tritiated
colchicine in a tubulin colchicine displacement assay9 and would
enable a direct comparison between the binding affinities for
tubulin of combretastatin A-4 and the fluorocombretastatin. This
paper describes the synthesis of a tritiated fluorocombretastatin.
Before attempting the radiosynthesis of the desired
fluorinated combretastatin, model reactions were performed
using ‘cold’ methylating agents. The Wittig reaction of 3-fluoro-
4-methoxybenzaldehyde 6 with phosphonium bromide 7 (ref.10)
afforded the two silyl ethers, 8 and 9, in 41% and 29% yield, respec-
tively, following separation by column chromatography (Figure 4).
Deprotection of the silyl ethers 8 and 9 using tetrabutylammonium
fluoride (TBAF) gave the Z and E phenols 10 and 11 in high yields,
respectively. The compounds were obtained isomerically pure as
indicated by their olefinic coupling constants in their 1H NMR
spectra. For example, stilbene 11 is configured E as indicated by
the signals at d 6.84 and 6.90 ppm, which have a coupling constant
of 16.2 Hz. The corresponding Z isomer 10 olefinic protons are
observed as a 2 H singlet at higher field (d 6.45 ppm). For 8 and
9, the olefinic signals are observed as 2 H singlets at 6.86 (E) and
6.44 ppm (Z) consistent with previous findings8,11,12 that the
olefinic signals of stilbenes of the Z isomer are observed at
higher field.
The next stage required an efficient procedure for the methylation
of the Z phenol 10 with methyl iodide. Following the method of
Songca13 methyl iodide was added to a solution of the phenol
10 and potassium carbonate in dimethylformamide (DMF). After
aKidscan Laboratories, Biomedical Research Centre, School of Environment and
Life Sciences, University of Salford, Manchester, M5 4WT, UK
Results and discussion
bDepartment of Drug Development and Imaging, Paterson Institute for Cancer
Research, Wilmslow Road, Manchester, M20 4BX, UK
The radiolabelling strategy adopted involves the methylation of a
free phenolic group on a cis-stilbene using tritiated methyl iodide.
Methylation was carried out on the A-ring 3-position as the initial
cDepartment of Drug Discovery, Moffitt Cancer Center, 12902 Magnolia Drive,
formation of a phenoxide on either the 4-position or the 40-position Tampa, FL 33612, USA
would be expected to lead to Z to E isomerisation of the alkene.5
For related examples, this is thought to be due to delocalisation
of the negative charge of the intermediate phenoxide anion, which
*Correspondence to: John A. Hadfield, Kidscan Laboratories, Biomedical Research
Centre, School of Environment and Life Sciences, University of Salford, Manchester,
M5 4WT, UK.
can be delocalised over the aromatic system and onto the olefin E-mail: j.a.hadfield@salford.ac.uk
J. Label Compd. Radiopharm 2012
Copyright © 2012 John Wiley & Sons, Ltd.

J. A. Hadfield et al.
MeO
MeO
MeO
MeO
R
R
OMe
OMe
R
OMe
1 Combretastatin A-4 R = OH
2 Combretastatin A-4P R = OP(=O)(ONa)2
OMe
3 Combretastatin A-1 R = OH
4 Combretastatin A-1P R = OP(=O)(ONa)2
Figure 1. Structures of Combretastatins.
1
signals in the H NMR spectrum confirmed the presence of the
extra methoxy group (3.72 ppm), and observation of the olefinic
coupling constants (J 12.4 Hz) confirmed that no isomerisation to
the E stilbene had occurred.
The reaction was then carried out using tritiated methyl iodide
(3.52 mCi) under Ando’s reaction conditions.15 (Figure 6) Cold
methyl iodide was added at the end of the experiment to ensure
that the reaction went to completion. The fluorostilbene 12 was
isolated in 98% yield (by mol). This was single spot by TLC (UV
MeO
MeO
OMe
F
OMe
5
Figure 2. Structure of Fluorocombretastatin 5.
1 h, the reaction was complete (determined by TLC), and after work and radio detection) and co-ran with unlabelled compound 5.
up, the desired Z-1-(30,40,50-trimethoxyphenyl)-2-(300-fluoro-400- Determination of the number of decays per minute indicated
methoxyphenyl)ethene 5 was obtained in 95% yield (Figure 5).
However, DMF is a difficult solvent to remove owing to its high
boiling point, so the methylation was attempted using more
volatile solvents. Nyemba14 reported using acetone under reflux
with potassium carbonate and methyl iodide for the methylation
of the phenols. When this reaction was performed with a large
excess (10 mol equivalents) of potassium carbonate at room
temperature, the methylation was complete after 4 h. The longer
time for the reaction and the need for a large excess of base were
largely attributed to the poor solubility of the potassium carbo-
nate in acetone. This reaction time was shortened by changing
75% tritium incorporation into the molecule.
Experimental
General
All reagents and chromatography grade solvents were obtained from
commercial sources and used without further purification unless indi-
cated. Flash column chromatography was performed on silica gel [Fluka,
silica gel 60 220–440 mesh (35–70 mm)], and TLC was carried out using
silica (0.2 mm, 60 F254) pre-coated, aluminium-backed plates. Mass spectra
the solvent to acetonitrile: the reaction was then complete after were recorded on VG70-70 EQ (FAB, CI+, EI+) and MS50 (FAB) spectro-
meters, with only major peaks being reported. Melting points were deter-
mined on a Gallenkamp melting point apparatus and are uncorrected.
Elemental analyses were carried out by the Microanalytical Department
at UMIST and Manchester University and are within 0.3% of theoretical
values. 1H NMR spectra were recorded at 300 MHz on a Bruker AC-300
instrument or at 400 MHz on a Bruker AC-400 MHz instrument. 13C NMR
spectra were recorded at either 75.5 MHz on a Bruker AC-300 instrument
or at 100 MHz on a Bruker AC-400 instrument. Chemical shifts (d) are
quoted in ppm, relative to tetramethylsilane and are referenced to the
CDCl3 unless otherwise stated. Coupling constants (J) are reported to 1
2 h. Even so, the use of a boiling solvent needs to be avoided if
possible when handling radioactive materials, so another method
was sought.
It has been reported15 that fluoride impregnated on alumina is
an effective reagent for promoting the alkylation of phenols and
alcohols. Indeed, when the reaction of methyl iodide and the
phenol 10 was performed using acetonitrile as the solvent and
potassium fluoride on alumina as the base, the reaction was com-
plete in 30 min. Filtering the product through a short silica column
afforded the fluorostilbene 5 in high yield (96%). Key diagnostic decimal place.
H
O
O
OH
OH
Figure 3. Z to E Isomerisation Mechanism of 4-Hydroxystilbenes.
www.jlcr.org
Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012

J. A. Hadfield et al.
PPh3Br
TBDMSO
MeO
O
n-BuLi
+
OMe
F
MeO
OTBDMS
F
OMe
OMe
OMe
8
+
6
7
OMe
F
TBDMSO
MeO
OMe
9
HO
MeO
OMe
F
TBAF
TBAF
HO
8
9
OMe
F
MeO
OMe
OMe
10
11
Figure 4. Synthesis of Phenols 10 and 11.
(1 H, d, J 2.0 Hz, H-20), 6.84 (1 H, t, J 8.7Hz, H-500), 6.99 (1 H, dd, J 1.8 and
8.7 Hz, H-600), 7.04 (1 H, dd, J 1.8 and 12.3 Hz, H-200).
HO
MeO
MeO
MeI
MeO
E-1-(30,40-Dimethoxy-50-t-butyldimethylsilyloxyphenyl)-2-
OMe
F
OMe
(300-fluoro-400-methoxyphenyl)ethene 9
F
OMe
OMe
Further elution afforded 9 as an off-white solid (0.57 g, 29%). Found: C,
65.9; H, 7.4. M+, 418.1974. C23H31FO4Si requires C, 66.0; H, 7.4%;
418.1975. m.p. 117–119 ꢁC. Rf 0.75 (SiO2, hexane : ethyl acetate 4:1 v/v);
dH (CDCl3; 300 MHz) 0.226 [6 H, s, Si-(CH3)2], 1.04 [9 H, s, C(CH3)3], 3.92
(3 H, s, OCH3), 3.92 (3 H, s, OCH3), 3.93 (3 H, s, OCH3), 6.64 (1 H, d, J
2.1 Hz, H-60), 6.70 (1 H, d, J 2.1 Hz, H-20), 6.86 (2 H, s, CH=CH), 6.94
(1 H, t, J 8.4 Hz, H-200), 7.18 (1 H, dd, J 1.7 and 8.4 Hz, H-600), 7.30 (1 H,
dd, J 1.7 and 12.9 Hz, H-200).
10
11
Figure 5. Methylation of 10.
3H3CO
MeO
HO
Z-1-(30,40-Dimethoxy-50-hydroxyphenyl)-2-(300-fluoro-400-
methoxyphenyl)ethene 10
1. KF-Alumina
MeO
2. C3H3I
3. MeI
OMe
OMe
F
F
To a stirred solution of the Z silyl ether 8 (0.949 g, 2.27 mmol) in dry THF
(25 cm3) was added (TBAF) (6 cm3 of 1.0 M solution in THF, 6 mmol). The
resulting yellow solution was stirred for 20 min and then treated with
water (50 cm3). The mixture was extracted with chloroform (3 ꢂ 25 cm3)
and the combined organic extracts washed with water (2 ꢂ 25 cm3),
dried (MgSO4) and concentrated in vacuo. The resulting colourless oil
was purified by flash column chromatography (SiO2, ethyl acetate :
hexane, 1:4 v/v) to yield the stilbene 10 as a yellow oil (609 mg, 100%).
Found: M+, 304.1113. C17H17FO4 requires 304.1111. Rf 0.57 (SiO2, petrol :
EtOAc 1:1 v/v); dH (CDCl3; 400 MHz) 3.70 (3 H, s, OCH3), 3.89 (3 H, s,
OCH3), 3.92 (3 H, s, OCH3), 5.72 (1 H, s, OH), 6.41 (1 H, d, J 1.4 Hz, H-60),
6.45 (2 H, s, CH=CH), 6.54 (1 H, s, J 1.4 Hz, H-20), 6.85 (1 H, d, J 8.4 Hz,
H-500), 7.01 (1 H, dd, J 2.0 and 8.4 Hz, H-600), 7.07 (1 H, dd, J 2.0 and
12.4 Hz, H-200).
OMe
OMe
10
12
Figure 6. Synthesis of tritiated fluorocombretastatin 12.
Z-1-(30,40-Dimethoxy-50-t-butyldimethylsilyloxyphenyl)-2-
(300-fluoro-400-methoxyphenyl)ethene 8
To a slurry of the phosphonium bromide 7 (3.94 g, 6.331 mmol) in THF
(30 cm3) was added nBuLi (5 cm3 of 1.6 M solution, 8.0 mmol) at ꢀ15 ꢁC.
The bright red anion was stirred for 20 min before 3-fluoro-4-
methoxybenzaldehyde 6 (1.0g, 6.487 mmol) was added. The mixture was
stirred for 1 h. Water was added to the mixture, and the resulting solution
was extracted with diethyl ether (3ꢂ 60 cm3). The combined organic
extracts were dried (MgSO4) and concentrated in vacuo to furnish a brown
oil. This was purified by flash column chromatography (SiO2, ethyl acetate :
E-1-(30,40-Dimethoxy-50-hydroxyphenyl)-2-(300-fluoro-400-
methoxyphenyl)ethene 11
In a similar manner, the E silyl ether 9 (0.662g, 1.58mmol) was deprotected
hexane, 1:20v/v) to furnish 8 as a yellow oil (1.12 g, 41%). Found: C, 65.8; H, using TBAF (3cm3 of 1.0 M solution in THF, 3mmol) to yield 11 as a white
7.5. M+, 418.1979. C23H31FO4Si requires C, 66.0; H, 7.5%; 418.1975. Rf 0.82
solid (398 mg, 83%) after flash column chromatography (SiO2, ethyl acetate :
(SiO2, petrol:EtOAc 4:1v/v); dH (CDCl3; 300 MHz) 0.10 [6 H, s, Si-(CH3)2],
0.96 [9 H, s, C(CH3)3], 3.72 (3 H, s, OCH3), 3.79 (3 H, s, OCH3), 3.87
(3 H, s, OCH3), 6.40 (1 H, d, J 2.0 Hz, H-60), 6.44 (2 H, s, CH=CH), 6.47
hexane, 1:4v/v). Found: C, 67.0; H, 5.7. M+, 304.1116. C17H17FO4 requires C,
67.1; H, 5.6%; 304.1111. m.p. 132–134 ꢁC. Rf 0.56 (SiO2, petrol:EtOAc 1:1v/v);
dH (CDCl3; 300 MHz) 3.92 (3 H, s, OCH3), 3.93 (3 H, s, OCH3), 3.94
J. Label Compd. Radiopharm 2012
Copyright © 2012 John Wiley & Sons, Ltd.
www.jlcr.org

J. A. Hadfield et al.
(3 H, s, OCH3), 5.79 (1 H, s, OH), 6.61 (1 H, d, J 2.3Hz, H-60), 6.78 (1 H, d,
J 2.3 Hz, H-60), 6.84 (1 H, d, J 16.2Hz, C=CH), 6.90 (1 H, d, J 16.2Hz, C=CH),
6.95 (1 H, t, J 8.7Hz, H-500), 7.18 (1 H, dd, J 2.7 and 8.7 Hz, H-600), 7.27
(1 H, dd, J 2.7 and 12.3 Hz, H-200).
Acknowledgements
The authors would like to thank Cancer Research UK and the
International Association for Cancer Research for financial
support for this work.
Z-1-(30,40,50-Trimethoxyphenyl)-2-(300-fluoro-400-methoxyphenyl)
ethene 5
Following a similar method with that of Ando and co-workers,15 the
Conflict of Interest
phenol 10 (10mg, 0.032mmol) was stirred with KF-alumina (25 mg) in The authors did not report any conflict of interest.
acetonitrile (1 mL) for 20min before iodomethane (3mL, 0.042mmol) was
added. This was stirred for 30 min before being run down a short silica
column, eluting with ethyl acetate. Evaporation of the solvent afforded
References
the fluorocombretastatin 5 as a white solid (10 mg, 96%). m.p. 76–78 ꢁC
(ref.8 75–78 ꢁC); Rf 0.62 (SiO2, hexane : ethyl acetate 1:1v/v); dH
(CDCl3; 300MHz) 3.72 (6 H, s, 2ꢂ OCH3), 3.72 (3 H, s, OCH3), 3.86 (3 H,
s, OCH3), 6.42 (1 H, d, J 12.4 Hz, C=CH), 6.49 (1 H, d, J 12.4Hz, C=CH), 6.51
(2 H, s, H-20 and H-60), 6.85 (1 H, t, J 8.3 Hz, H-500), 7.01 (1 H, dd, J 2.4 and
8.3 Hz, H-600), 7.07 (1 H, dd, J 2.4 and 12.6 Hz, H-200).
[1] http://www.oxigene.com/pipeline/clinical_trials [22 March 2012].
[2] A. Delmonte, C. Sessa. Expert Opin. Invest. Drugs 2009, 18, 1541–1548.
[3] G. Kremmidiotis, A. F. Leske, T. C. Lavranos, D. Beaumont, J. Gasic, A.
Hall, M. O’Callaghan, C. A. Matthews, B. Flynn. Mol. Cancer Ther. 2010,
9, 1562–1573.
[4] G. R. Pettit, M. D. Minardi, F. Hogan, P. M. Price. J. Nat. Prod. 2010, 73,
399–403.
[5] A. Shirali, M. Sriram, J. J. Hall, B. L. Nguyen, R. Guddneppanavar, M. B.
Hadimani, J. F. Ackley, R. Siles, C. J. Jelinek, P. Arthasery, R. C. Brown,
V. L. Murrell, A. MeMordie, S. Sharma, D. J. Chaplin, K. G. Pinney. J.
Nat. Prod. 2009, 72, 414–421.
[6] R. T. Brown, V. L. Murrell, A. McMordie, M. Sriram, K. G. Pinney, S. Sharma,
D. J. Chaplin. J. Labelled Compd. Radiopharm 2009, 52, 567–570.
[7] J. A. Hadfield, A. T. McGown, S. P. Mayalarp, E. J. Land, I. Hamblett, K.
Gaukroger, N. J. Lawrence, L. A. Hepworth, J. Butler. Substituted
stilbenes and their reactions. US Patent, 2007, 7, 220-784.
[8] N. J. Lawrence, L. A. Hepworth, D. Rennison, A. T. McGown, J. A.
Hadfield. J. Fluorine Chem. 2003, 123, 101–108.
[3H]Z-1-(30[3H],40,50-Trimethoxyphenyl)-2-(300-fluoro-400-
methoxyphenyl)ethene 12
The phenol 10 (7mg, 0.023 mmol) was stirred with KF-alumina (18mg) in
acetonitrile (1 mL) for 20 min before [3H]iodomethane (800 mL, 3.52 mCi,
5.5 ꢂ 10ꢀ8 M in toluene, specific activity: 80Ci/mmol) was added. This was
stirred for 4 h before iodomethane (3mL, 0.042 mmol) was added. The
mixture was allowed to stir for a further 30min before being evaporated
to dryness and eluting down a short silica column with ethyl acetate to
furnish 12 as a white solid (7 mg, 2.64mCi, 75% incorporation). Rf 0.61
(SiO2, hexane:ethyl acetate 1:1v/v).
[9] J. A. Woods, J. A. Hadfield, G. R. Pettit, B. W. Fox, A. T. McGown. Br. J.
Cancer 1995, 71, 705–711.
[10] G. R. Pettit, S. B. Singh. Can. J. Chem. 1987, 65, 2390–2396.
[11] K. Gaukroger, J. A. Hadfield, L. A. Hepworth, N. J. Lawrence, A. T.
McGown. J. Org. Chem. 2001, 66, 8135–8138.
[12] K. Gaukroger, J. A. Hadfield, N. J. Lawrence, S. Nolan, A. T. McGown.
Org. Biomol. Chem. 2003, 3033–3037.
[13] S. P. Songca, R. Bonnett, C. M. Maes. South African J. Chem. 1997, 50, 40–47.
[14] A. M. Nyemba, T. N. Mpondo, S. F. Kimbu, J. D. Connolly. Phytochemistry
1995, 39, 895–898.
[15] T. Ando, J. Yamawaki, T. Kawate, S. Sumi, T. Hanafusa. Bull. Chem. Soc.
Japan 1982, 55, 2504–2507.
Conclusions
In conclusion, we have developed three methods for the potential
incorporation of a radiolabel into the fluorocombretastatin 5. One
of these methods was used to successfully incorporate a tritium
label into the molecule. Also, with a quick (0.5h) reaction time, it
should be feasible to incorporate an 11C label for use in positron
emission tomography studies using this methodology.
www.jlcr.org
Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012