Carbon-14 radiosynthesis of combretastatin A-1 (CA1) and its corresponding phosphate prodrug (CA1P)

Article abstract of DOI:10.1002/JLCR.1676

The natural product combretastatin A-1 (CA1) is isolated from the African bush willow tree, a member of the Combretaceae family. CA1 has important medicinal value, due in part to its ability to inhibit tubulin assembly. The prodrug combretastatin A-1 diph

Full text of DOI:10.1002/JLCR.1676

Research Article
Received 1 June 2009,
Revised 8 August 2009,
Accepted 11 August 2009
Published online 21 October 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/JLCR.1676
Carbon-14 radiosynthesis of combretastatin
A-1 (CA1) and its corresponding phosphate
prodrug (CA1P)
Ã
Rodney T. Brown,a Victor L. Murrell,a Austin McMordie,a Madhavi
Sriram,b Kevin G. Pinney,b Suman Sharma,c and David J. Chaplinc
The natural product combretastatin A-1 (CA1) is isolated from the African bush willow tree, a member of the Combretaceae
family. CA1 has important medicinal value, due in part to its ability to inhibit tubulin assembly. The prodrug
combretastatin A-1 diphosphate (CA1P; OXi4503) is currently in human Phase I clinical trials as a vascular disrupting agent.
This paper describes the carbon-14 radiosynthesis of [40-14C]CA1 and the corresponding phosphate prodrug salt
[40-14C]CA1P in high specific activity (55 mCi/mmol). The carbon-14 label was introduced by methylation of the C-40
protected phenolic moiety of the CA1 precursor following removal of the tert-butyldimethylsilyl protecting group in the
presence of [14C]methyl iodide. This was accomplished in excellent yield without significant Z to E isomerization. The [14C]-
precursor ((Z)-1-[30,[40-14C],50-trimethoxyphenyl]-2-[200,300-di-[(isopropyl)oxy]-400-methoxyphenyl] ethene) was subjected to a
de-isopropylation reaction with TiCl4. The tetrabenzyl phosphate derivative of the resulting diol was prepared using fresh
dibenzyl phosphite. Debenzylation with trimethylsilylbromide, followed by hydrolysis of the trimethylsilyl ester and
adjustment of the pH with dilute aqueous hydrochloric acid yielded [40-14C]CA1P with an overall radiochemical yield of
8.4%.
Keywords: tumour; combretastatin; OXi4503; CA1P; CA1; CA4
are not present and the VDA causes the endothelial cell to
become spherical. As these cells build up, they block the
Introduction
The hydroxyl stilbenoid natural products combretastatin A-1
(CA1) and combretastatin A-4 (CA4) (Figure 1) are microtubule
depolymerization agents. They are members of a family of 17
compounds, first isolated from the bark of the African bush
willow tree Combretum caffrum by George R. Pettit and co-
workers in the early 1980s.1
OXiGENE Inc. (Waltham, MA) is currently developing the
phosphate ester prodrugs combretastatin A-1 diphosphate
(CA1P; OXi4503) and combretastatin A-4 phosphate (CA4P;
ZybrestatTM, fosbretabulin) for the treatment of advanced solid
tumors.2
flow of blood, leading to the tumor and kill it by starving it of
oxygen.4
In contrast to CA4P, OXi4503 also possesses direct cytotoxic
activity. Preclinical studies have demonstrated that the addi-
tional phenolic moiety in CA1 as compared to A-4 markedly
changes the redox properties of the molecule and introduces a
completely different chemical functionality.5 The oxidative
enzymes (tyrosinases and peroxidases), which are present at
elevated levels in various solid and liquid tumors, cause OXi4503
to undergo metabolic in vivo activation. This oxidative
metabolism process results in the creation of reactive ‘radical’
oxygen species and an orthoquinone metabolite, which
covalently binds to proteins and the nucleic acids in DNA,
producing direct cytotoxic effects, leading eventually to tumor
necrosis.2
CA1P and CA4P are classed as vascular disrupting agents
(VDAs)3 and have associated cancer therapy properties. These
compounds show selective activity against the vasculature of
cancerous tumors.
Biological mode of action
aIsotope Chemistry Group, Almac Sciences, Almac House, 20 Seagoe Industrial
Estate, Craigavon BT63 5QD, Northern Ireland
In the case of CA1P and CA4P, the mode of action has been
shown to be initial dephosphorylation, followed by binding of
the free combretastatin to the tubulin heterodimer. This results
in an irreversible process of microtubule depolymerization,
and a cascade of cell signaling events. In normal blood vessels,
this tubulin binding has little effect because these vessels
also have a well-organized ‘scaffolding’ system to keep them
intact. However, in the microvessels feeding abnormal cells
(tumors), or on the retina in some eye diseases, such supports
bDepartment of Chemistry and Biochemistry, Baylor University, Waco, TX 76798,
USA
cOXiGENE Inc., 300 Bear Hill Road, Waltham, MA 02451, USA
*Correspondence to: Rodney T. Brown, Isotope Chemistry Group, Almac Sciences,
Almac House, 20 Seagoe Industrial Estate, Craigavon BT63 5QD, Northern
Ireland.
E-mail: rodney.brown@almacgroup.com
J. Label Compd. Radiopharm 2009, 52 567–570
Copyright r 2009 John Wiley & Sons, Ltd.

R. T. Brown et al.
MeO
MeO
MeO
MeO
O
O
MeO
MeO
OH
P
OH
OH
O
.2K+
OMe
OMe
OH
OMe
O
OMe
OMe
OMe P
O
O
CA1
CA4
OH
site of carbon-14 label
Figure 1. Combretastatins CA1 and CA4.
Figure 2. Position of carbon-14 label in CA1P.
In July 2007, the biotechnology company OXiGENE initiated a
180-patient phase III clinical trial of Zybrestat in combination
with carboplatin for the treatment of anaplastic thyroid cancer
(Study of Combretastatin and Paclitaxel/Carboplatin in the
Treatment of Anaplastic Thyroid Cancer). There is currently no
fully FDA approved treatment for this form of cancer.2 OXi4503
(CA1P) is currently being evaluated as a monotherapy in a Phase 1
dose-escalation clinical trial in patients with advanced solid
tumors.6a
remove potential titanium impurities. The removal of these
impurities avoids possible isomerization during the salt
formation step. After chromatography, the resultant yellow oil
was recrystallized giving tan colored crystals of [14C]CA1 (3), of
good radiochemical purity by radio-TLC and 1H NMR.
The treatment of deprotected combretastatin (3) with dibenzyl
chlorophosphate, generated in situ from dibenzyl phosphite
and carbon tetrachloride, gave the tetrabenzyl protected
phosphate product (4). The commercially available dibenzyl
phosphite did not perform well in this step, thus the
reagent was prepared fresh for use in the radiosynthesis.10
This method of phosphorylation is a higher yielding alter-
native to the published method using N-chlorosuccinimide,8
although it has the drawback of utilizing toxic carbon
tetrachloride.
Finally, debenzylation of stilbene (4) using a solution of
trimethylsilyl bromide in acetonitrile resulted in the tetra-
trimethylsilyl phosphate ester, which was quenched into a
solution of excess potassium methoxide in methanol to afford
the tetra potassium salt along with other mono-, di-, and tri-
potassium substituted salts. The mixture of salts was then
dissolved in water, and the resultant aqueous solution was
carefully adjusted to a pH between 4.75 and 4.85 using dilute
hydrochloric acid, followed by precipitation of the di-potassium
salt [14C]CA1P with ethanol. The final product was characterized
by 1H NMR, analytical HPLC, and specific activity. [14C]CA1P was
obtained in an overall yield of 8.4%, with 97.5% chemical purity,
96.7% radiochemical purity by analytical HPLC area %, and with
a specific activity of 55 mCi/mmol.
Carbon-14 radiosynthesis
To elucidate the mechanism of metabolism of CA1P in ADME-
toxicology studies it was important to incorporate a radio-
isotope into the structure. Carbon-14 was chosen as the
radiotracer due to its long half-life period (ꢀ5730 years) and
the fact that it could be introduced without changing the
chemical structure. The ease of replacement of the methoxy
group at the 40-position in the substrate 30,40,50-trimethoxy-
phenyl (Figure 2) made it a feasible location to incorporate a
carbon-14 labelled methoxy moiety, although similar trimethox-
yphenyl VDA compounds have been shown to undergo
metabolic demethylation.6b [14C]Methyl iodide was used as the
methylating agent.
This paper describes the synthesis of [14C]CA1P, using the
route shown in Scheme 1. The synthetic methodology was
based on the unlabelled route established by Pinney and
co-workers at Baylor University.7–9
The orthogonally protected stilbene starting material (Z)-1-
[30,50-dimethoxy-40-[(tert-butyldimethylsilyl)oxy]phenyl]-2-[200,300-
di-[(isopropyl)oxy]-400-methoxyphenyl] ethene (1) was reacted
with the fluorinating reagent TBAF and one equivalent of
[14C]methyl iodide, selectively introducing the carbon-14 label at
the C40 phenolic position. The original unlabelled synthesis used
four equivalents of methyl iodide, but in this case, to avoid
excess radiochemical waste and a low radiochemical yield, the
number of equivalents was reduced to one. As there was no
excess of [14C]methyl iodide, it was important that the reaction
solution was completely anhydrous to avoid protonation at C40.
Conclusion
The carbon-14 labelled compound CA1P was prepared in four
steps following the route developed by the Pinney group at
Baylor University. The material was prepared in a radiochemical
purity of greater than 96% by HPLC area % and an overall
radiochemical yield of 8.4%.
˚
This was achieved by the addition of activated 3 A molecular
sieves. Subsequently, both isopropyl groups of (Z)-1-[30,
[40-14C],50-trimethoxyphenyl]-2-[200,300-di-[(isopropyl)oxy]-400-metho-
Experimental
xyphenyl] ethene (2) were removed using the Lewis acid The reaction steps were performed under inert atmosphere
titanium tetrachloride in dichloromethane, under carefully using nitrogen gas unless specified differently. Chemical
controlled temperature conditions. This deprotection procedure reagents used in the synthetic procedures were obtained from
gave variable yields, particularly on a small scale. Utilizing a various chemical suppliers. [14C]Methyl iodide was purchased
freshly opened, highly pure (99.999%) bottle of titanium from Perkin Elmer (497%, 40–60 mCi/mmol). Anhydrous acet-
tetrachloride, the reaction was completed within 40 min, onitrile (99.8%) and titanium tetrachloride (99.999%) were
although [14C]CA1 (3) was obtained in lower yield than purchased from Aldrich and used without further purification.
during the cold synthesis (27% cf. 45%).9 The [14C]CA1 A fresh bottle of titanium tetrachloride was used for each de-
obtained after the deprotection of the isopropyl groups was protection step. Anhydrous dichloromethane was freshly
purified using silica gel capped with a plug of Florisils, to distilled from P2O5 followed by distillation from calcium hydride
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Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 567–570

R. T. Brown et al.
MeO
MeO
H
MeO
14CH3I (1 eq.)
TBAF(1.1 eq.)
H 14
H
C
O
O
TiCl4 (4.4 eq.)
CH2Cl2
H
O
O
O
TBSO
OMe
H
14C
OH
OH
O
H
OMe
Anhydr. CH3CN
OMe
OMe
OMe
OMe
(1)
(2)
(3)
OBn
OBn
O
O
MeO
H
MeO
O
OH
P
(BnO)2P(O)H
P
H
H 14
H
C
O
CCl4, CH3CN,
1. TMS-Br, CH3CN
O
H
14C
O
O
H
.2K+
2. KOMe, MeOH,
3. HCl, H2O, EtOH
DIPEA, DMAP
OMe
OMe
O
O
OMe P OBn
OMe P
O
O
OH
OBn
O
(4)
[
14C]-CA1P
Scheme 1. Synthesis of [14C]-CA1P.
˚
and storage over 3 A molecular sieves. Trimethylsilyl bromide separated and dried over anhydrous Na2SO4. The solvent
(98%, Aldrich) was freshly distilled from calcium hydride. Silica was concentrated until a solid precipitated. Ethyl acetate
˚
gel (200–400 mesh, 60 A) was used for column chromatography. (50 mL) was added. The solid was removed by vacuum filtration
TLC plates (precoated glass plates with silica gel 60 F254, and washed with ethyl acetate (2 ꢁ 50 mL). The filtrates
0.25 mm thickness) were used to monitor reactions. Intermedi- from both batches were combined and concentrated in vacuo
1
ates and products synthesized were characterized based on H to give a yellow oil. The residue was purified by flash
NMR (Bruker Avance operating at 500 MHz). All the chemical chromatography eluting with 96:4 hexane:ethyl acetate. The
shifts are expressed in ppm (d), coupling constants (J, Hz), and combined batches gave 367 mCi of the title compound (75%
peak patterns are reported as broad (br), singlet (s), doublet (d), yield). 1H NMR (CDCl3) d6.93 (1 H, d, J = 8.6 Hz, H-600), 6.63 (1H, d,
triplet (t), quartet (q), septet (sep), and multiplet (m). Purity of J = 12.3 Hz, H-2), 6.49 (3H, m, H-20, H-60, H-500), 6.43 (1H, d,
the final compound was further analyzed using an Agilent J = 12.2 Hz, H-1), 4.70 (1H, sep, J = 6.2 Hz, C-200 OCH(CH3)2), 4.43
1100 HPLC system with UV- and radiochemical detection; a (1H, sep, J = 6.2 Hz, C-300 OCH(CH3)2), 3.82 (3H, s, C-400 OCH3), 3.79
Supelco Discovery C18 HPLC column 12.5 cm ꢁ 4.6 mm, 5 mm; (3H, s, C-40 OCH3), 3.65 (6H, s, C-30, C-50 OCH3), 1.29 (12H, d,
T = 251C; eluants, solvent A, 25 mM tetrabutylammonium J = 6.2 Hz, C-200, C-300 OCH (CH3)2).
bromide (TBAB) with 0.1% trifluoroacetic acid (TFA) in water,
solvent B, 25 mM TBAB with 0.08% TFA in water/acetonitrile
(2/8 v/v); gradient, 80% A/20% B to 5% A/95% B over 0–45 min;
flow rate, 0.7 mL/min; injection volume 25 mL; monitored at
264 nm wavelength.
Synthesis of (Z)-1-[30,[40-14C],50-trimethoxyphenyl]-2-[200,300-
dihydroxy-400-methoxyphenyl]ethene; Z-[40-14C]CA1, (3)
(Z)-1-[30,[40-14C],50-Trimethoxyphenyl]-2-[200,300-di-[(isopropyl)oxy]-400
-methoxyphenyl] ethene (2) (1.35 g; 3.23 mmol; 183.7 mCi; 1.0 eq.)
Synthesis of (Z)-1-[30,[40-14C], 50-trimethoxyphenyl]-2-[200,300-
was dissolved in anhydrous dichloromethane (27 mL) and cooled
to o01C in an ice/acetone bath. TiCl4 (1.57 mL; 14.19 mmol; 4.4 eq.)
di-[(isopropyl)oxy]-400-methoxyphenyl] ethene (2)
was added drop-wise to the reaction mixture, while maintaining
temperature at o01C, and stirred vigorously. The reaction mixture
was stirred for 40 min at 01C, and then quenched by slowly adding
water, keeping the internal temperature o101C. The aqueous
layer was separated and extracted with dichloromethane. The
combined organic layers were washed with brine (50 mL). The
organic layer was dried over anhydrous Na2SO4. This layer was
concentrated on to silica and the compound was purified by flash
chromatography through a silica column with a 0.5 cm plug of
Florosils, eluting with 75:25 hexane:ethyl acetate. The yellow oil
was recrystallized from 50:50 hexane:ethyl acetate giving tan
colored crystals. This reaction was performed in two batches of
183.7 mCi each and gave 100 mCi of the title compound (27%
This reaction was performed in two batches (total activity of
490 mCi of [14C]methyl iodide with a specific activity range of
40–60 mCi/mmol). For each batch, (Z)-1-[30,50-dimethoxy-40-
[(tert-butyldimethylsilyl)oxy]phenyl]-2-[200,300-di-[(isopropyl)oxy]-
400-methoxyphenyl] ethene (1) (2.22 g; 4.30 mmol; 1.0 eq.) was
dissolved in anhydrous acetonitrile (32 mL) and dried over
14
˚
activated 3 A molecular sieves (9.5 g). [ C]Methyl iodide (0.62 g;
4.30 mmol; 245 mCi; 1.0 eq.) was transferred to the flask using
a manifold line and stirred for 20 min. TBAF, 1.0 M in THF
(4.73 mL; 4.73 mmol; 1.1 eq.), was added and the reaction
stirred at 01C for 30 min. The reaction was quenched by the
addition of water (15 mL). Volatile carbon-14 radioactive
components were flushed from the system with nitrogen gas
and collected in a flask containing DMF, which was then
frozen in a Dewar of liquid nitrogen and stoppered. The
reaction mixture was filtered to remove molecular sieves and
these were washed with ethyl acetate. The organic layer was
1
yield). H NMR (CDCl3) d6.76 (1 H, d, J=8.7Hz, H-600), 6.59 (1H, d,
J=12.2Hz, H-2), 6.54 (3H, m, H-1, H-20, H-60), 6.38 (1H, d, J=8.7Hz,
H-500), 5.40 (2H, s, C-200, C-300 OH), 3.86 (3H, s, C-40 OCH3), 3.83 (3H, s,
C-40 OCH3), 3.67 (6H, s, C-30, C-50 OCH3).
J. Label Compd. Radiopharm 2009, 52 567–570
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org

R. T. Brown et al.
C-30, C-50 OCH3); HPLC retention time is 16.82 min (consistent
with an authentic sample); 97.5% chemical purity by HPLC area
% (UV), 96.7% radiochemical purity by HPLC area %, specific
activity 55 mCi/mmol (determined by gravimetry).
Synthesis of (Z)-1-[30,[40-14C],50-trimethoxyphenyl]-2-[200,300-
di-[([bis-[(benzyl)oxy]]phosphoryl)oxy]-400-methoxyphenyl]
ethene (4)
[40-14C]-CA1 (3) (0.58 g; 1.75mmol; 100 mCi; 1.0 eq.) was dissolved
in anhydrous acetonitrile (13 mL). The solution was cooled to
–201C and carbon tetrachloride (3.4mL; 35mmol; 20eq.) was
added. The reaction mixture was stirred for a few minutes,
then di-isopropylethyl amine (1.2mL; 7.0 mmol; 4.0 eq.) and
4-dimethylaminopyridine (42 mg; 0.34mmol; 0.2 eq.) were added.
After stirring for further 2 min, freshly prepared dibenzyl
phosphite (1.16 mL; 5.22mmol; 3.0 eq.) was added drop-wise
and the reaction mixture stirred for 1 h. The reaction was
quenched by the addition of 0.5 M KH2PO4 (4.2mL) and ethyl
acetate (16.9mL). The organic phase was separated and washed
with brine, then dried over Na2SO4. The organic layer was
concentrated on the rotary evaporator to leave 1.8 g of yellow oil.
This was purified by column chromatography on silica gel, eluting
with 70:30 hexane:ethyl acetate. This gave 72.3mCi of the title
compound (72% yield). 1H NMR (CDCl3) d7.24 (20H, m, C-200, C-300
OP(O)(OCH2C6H5)2), 7.00 (1H, d, J = 8.7 Hz, H-600), 6.66 (1H, d,
J = 8.5 Hz, H-500), 6.65 (1H, d, J = 12.0Hz, H-2), 6.51 (1H, d,
J = 12.0Hz, H-1), 6.45 (2H, s, H-20, H-60), 5.16 (4H, m, C-200,
C-300OP(O)(OCH2C6H5)2), 5.04–5.11 (4H, m, C-200 C-300 OP(O)-
(OCH2C6H5)2), 3.79 (3H, s, C-400 OCH3), 3.76 (3H, s, C-40 OCH3),
3.62 (6H, s, C-30, C-50 OCH3).
Acknowledgement
Almac Sciences gratefully acknowledge sponsorship of this work
by OXiGENE and also thank Professor Kevin Pinney and Dr
Madhavi Sriram of Baylor University for helpful discussions
throughout the course of this project and supply of (Z)-1-[30,50-
dimethoxy-40-[(tert-butyldimethylsilyl)oxy]phenyl]-2-[200,300-di-[(iso-
propyl)oxy]-400-methoxyphenyl] ethene (1). K. G. P. is appreciative
of the support from OXiGENE Inc. and The Welch Foundation
(Grant No. AA-1278) that funded, in part, the original synthetic
methodology development (with carbon-12).
References
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Synthesis of (Z)-1-[30,[40-14C],50-trimethoxyphenyl]-2-[200,300-
di-[(monopotassium)phosphate]-400-methoxyphenyl] ethene;
Z-[40-14C] -CA1P
The tetrabenzyl phosphate ester of [40-14C]CA1 (4) (0.52g;
0.61 mmol; 35mCi; 1.0eq.) was dissolved in anhydrous acetonitrile
(16.1 mL). The solution was cooled to –101C and trimethylsilyl
bromide (freshly distilled from CaH2) (0.40 mL; 3.05mmol; 5.0eq.)
was added drop-wise. The reaction mixture was stirred for
1.5 h at –101C.
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In a separate flask, potassium methoxide (0.43 g; 6.10 mmol;
10.0 eq.) was dissolved in anhydrous methanol (7.3 mL). The
solution was cooled to –101C and the phosphate solution was
added slowly, drop-wise, over 2.5 h using a syringe pump. The
reaction mixture was then allowed to warm to room tempera-
ture and stirred for 15 min. The solution was concentrated to
dryness at 301C on a rotary evaporator. The brown solid was
dissolved in de-ionized water (3.2 mL) to give a cloudy, brown
colored solution. The pH of the solution was measured at 13.4.
The pH was carefully titrated to ꢀ5.5 by drop-wise addition of
1.0 M aqueous HCl to the vigorously stirred solution. The
titration was continued until pH 4.84 using 0.1 M HCl. The
solution was then filtered to remove a brown scum and the flask
and funnel were rinsed with de-ionized water (2.3 mL). Absolute
ethanol (18 mL) was added to the filtrate causing a white solid to
precipitate. The suspension was cooled at –201C for 30 min, and
then the solid was collected by vacuum filtration and washed
with ethanol (6.5 mL). After 30 min air-drying in the funnel, a
white solid, [40-14C]CA1P was obtained. This gave 20.5 mCi of the
title compound (58% yield). 1H NMR (D2O) d6.84 (1H, d,
J = 8.7 Hz, H-600), 6.68 (1H, d, J = 11.9 Hz, H-2), 6.64 (1H, d,
J = 8.8 Hz, H-500) 6.61 (2H, s, H-20, H-60), 6.58 (1H, d, J = 12.0 Hz, H-
1), 3.76 (3H, s, C-400 OCH3), 3.68 (3H, s, C-40 OCH3), 3.62 (6H, s,
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J. Label Compd. Radiopharm 2009, 52 567–570