Synthesis of a 11C-labelled taxane derivative by [1- 11C]acetylation

Article abstract of DOI:10.1002/jlcr.1059

The ¹¹C-labelling of the taxane derivative BAY 59-8862 (1), a potent anticancer drug, was carried out as a module-assisted automated multi-step synthesis procedure. The radiotracer [¹¹C]1 was synthesized by reacting [1-¹¹C

Full text of DOI:10.1002/jlcr.1059

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 357–365.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1059
Research Article
11
Synthesis of a C-labelled taxane derivative by
[1-11C]acetylation
P. Mading1,*, J. Zessin1, U. Pleiꢀ2, F. Fuchtner1 and F. Wust1
1 Institut fur Radiopharmazie, Forschungszentrum Rossendorf, Postfach 510119,
¨
01314 Dresden, Germany
2 Bayer HealthCare AG, Drug Metabolism & Isotope Chemistry, Aprather Weg 18a,
42096 Wuppertal, Germany
Summary
The 11C-labelling of the taxane derivative BAY 59-8862 (1), a potent anticancer drug,
was carried out as a module-assisted automated multi-step synthesis procedure. The
radiotracer [11C]1 was synthesized by reacting [1-11C]acetyl chloride (6) with the lit-
hium salt of the secondary hydroxy group of precursor 3 followed by deprotection.
After HPLC purification of the final product [11C]1, its solid-phase extraction,
formulation and sterile filtration, the decay-corrected radiochemical yield of [11C]1
was in the range between 12 and 23% (related to [11C]CO2; n=10). The total synthesis
time was about 54 min after EOB. The radiochemical purity of [11C]1 was greater than
96% and the chemical purity exceeded 80%. The specific radioactivity was
16.8 ꢀ 4.7 GBq/mmol (n=10) at EOS starting from 80 GBq of [11C]CO2. Copyright
# 2006 John Wiley & Sons, Ltd.
Key Words: taxane; anticancer drug; [1-11C]acetyl chloride; [11C]BAY 59-8862;
positron emission tomography
Introduction
Taxanes are an important class of antitumor agents. These compounds bind to
the microtubuli and inhibit their depolymerization into tubulin. Subsequently,
taxanes arrest cell-cycle progression in mitosis by interacting directly with
tubulin and interfering with microtubule spindle function.1
The non-invasive investigation of such antitumor agents, labelled with a
positron emitting radionuclide, by means of positron emission tomography
(PET) has proved as valuable tool to assess general pharmacokinetics of the
*Correspondence to: P. Mading, Institut fur Radiopharmazie, Forschungszentrum Rossendorf, Postfach
510119, 01314 Dresden, Germany. E-mail: p.maeding@fz-rossendorf.de
Copyright # 2006 John Wiley & Sons, Ltd.
Received 22 October 2005
Revised 11 November 2005
Accepted 20 January 2006

358
P. MADING ET AL.
Scheme 1. Synthesis of the precursor 3
drug in vivo. Moreover, PET also allows monitoring the body’s response after
treatment with such drugs.
The taxane derivative paclitaxel (Taxol1) is an effective anticancer drug
against solid tumors, which was labelled with carbon-11 by reacting
[a-11C]benzoyl chloride with the corresponding primary amine precursor.2
Recently, another 11C-labelling procedure involving a free amino group
of a taxane derivative has been reported by van Tilburg et al.3 Docetaxel
(Taxotere1) as an accepted chemotherapeutic agent for the treatment of
breast cancer and non-small cell lung cancer was labelled with 11C in
the carbamate moiety using [11C]tert-butanol via [11C]tert-butyl-1,2,2,2-
tetrachloroethyl carbonate.3
This report describes the 11C-labelling of the taxane derivative BAY 59-8862
(1) using [1-11C]acetyl chloride. Compound 1, 5b, 20-epoxy-1,2a,4,7b,13a,
14b-heptahydroxytax-11-en-9-one-1,14-carbonate-4,10-diacetate-2-benzoate
13-[(2R,3S)-3-(N-tert-butoxycarbonyl)-amino-2-hydroxy-5-methylhexanoate]
(see Scheme 1) was shown to be a new potent anticancer drug4 which is
intended for the therapy of brain metastases of breast cancer.
Results and discussion
The acetyl moiety of compound 1 at position 10 (Scheme 1) was selected to
introduce carbon-11 by reaction of a suitable 10-deacetyl precursor compound
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SYNTHESIS OF A 11C-LABELLED TAXANE
359
with [1-11C]acetyl chloride. With respect to drug metabolism, experiments with
human hepatocytes revealed sufficient metabolic stability of the selected acetyl
group.
For the synthesis of the required precursor 3 taxane 1 (BAY 59-8862) was
selectively deacetylated in position 10 by treatment with sodium hydrogen-
carbonate and hydrogen peroxide to give compound 2 in 46% yield (Scheme 1).
To avoid the known epimerization of the hydroxy group in position 7 and to
stabilize the ester group in position 13 the 20 and 7 hydroxy groups of 2 were
selectively protected as triethylsilyl ethers.5 This protection procedure was
carried out using triethylsilyl chloride and imidazole in DMF to afford com-
pound 3 in 59% yield. Precursor 3 contains the sterically hindered hydroxy
group in position 10.
[11C]BAY 59-8862 ([11C]1) was synthesized in a multi-step procedure
according to Scheme 2 using an automated synthesis module. The schematic
diagram of this module is demonstrated in Figure 1. The capability of
this module includes the radiosynthesis of [1-11C]acetyl chloride (6) and its
conversion with the lithium salt 4 of precursor 3 into intermediate 5, acidic
hydrolysis of 5 to crude [11C]1, HPLC purification and solid-phase extraction
of the final product [11C]1 as well as its formulation.
Scheme 2. Synthesis of [11C]1
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P. MADING ET AL.
Figure 1. Schematic diagram of the module for synthesizing [11C]1
The first step of this procedure was the radiosynthesis of [1-11C]acetyl chlo-
ride (6) by conversion of [11C]CO2 with MeMgBr in diethylether followed by
quenching the resulting [11C]acetate solution with phthaloyl dichloride and
2,6-di-t-butylpyridine6 according to Scheme 2. The synthesis of 6 is described
in literature using different Grignard reagents before addition of phthaloyl
dichloride: MeMgBr in diethylether,6 MeMgBr in THF,7 MeMgCl or MeLi in
diethylether8 and MeMgCl in THF.9 We have tested the several Grignard/
solvent combinations and found out, that only the use of MeMgBr in
diethylether6 gave a radiochemical pure 6. The other combinations caused a
non-identified radioactive by-product of up to 50%. The reason for undesir-
able labelled products can be a side reaction of carbonated methylmagnesium
halide with excess methylmagnesium halide as discussed by Pike et al.10.
The release of 6 out of the reaction mixture was carried out by means of a
fractionated distillation.11 The first fraction up to 858C (reactor temperature)
contained the diethylether with interfering phthaloyl dichloride and was evap-
orated in the exhaust. The main radioactivity of 6 was released at 85–1008C,
although the boiling point of acetyl chloride is 518C. This issue was probably
caused by boiling point elevation due to the presence of phthaloyl dichloride
and 2,6-di-t-butylpyridine.
It was shown that traces of phthaloyl dichloride substantially prevent the
[11C]acetylation reaction. Therefore phthaloyl dichloride contaminations
should be avoided, and distillation of pure 6 was achived by carefully
flushing a helium gas stream over the surface of the quenched Grignard
reaction mixture. Mixing by direct bubbling the He stream through
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SYNTHESIS OF A 11C-LABELLED TAXANE
361
the quenched Grignard reaction mixture was only performed within very short
time intervals. This bubbling was necessary for satisfactory radiochemical
yields of 6.
After transfer of the purified gaseous 6 into a cooled THF solution of
precursor 3 and lithium hexamethyldisilazide (LiHMDS) in a second reaction
vessel, the [11C]acetylation reaction to form intermediate 5 was carried out at
room temperature. After evaporation of the solvent in vacuo, the triethylsilyl
protecting groups of 5 were removed by means of hydrochloric acid in MeOH/
dioxane at room temperature. After neutralization of the reaction mixture,
crude [11C]1 was purified by semi-preparative RP HPLC and solid-phase
extraction12 using an RP–18-cartridge. The resulting ethanolic solution of
[11C]1 was transferred through a sterile filter followed by a isotonic saline
solution to obtain the final formulation of [11C]1 in pharmaceutical quality.
The stepwise transfer of these solutions prevented the loss of [11C]1 by
absorption on the membrane of the sterile filter.
The decay-corrected radiochemical yield of [11C]1 ranged from 12 to 23%
(related to [11C]CO2; n=10) in its final formulation. The total synthesis time of
the process was 54 min. The radiochemical purity of [11C]1 was higher than
96%. The chemical purity ranged between 80 and 90% based on UV absorp-
tion at 235 nm. The specific radioactivity of the final product was
16.8 ꢀ 4.7 GBq/mmol (n=10) at EOS starting from 80 GBq of [11C]CO2.
Experimental
Materials and methods
1
The H-NMR spectrum of the precursor 3 was recorded on a Bruker DRX
NMR spectrometer at 400 MHz. Chemical shifts are reported in ppm relative
to TMS as internal standard.
The LC-MS analyses for the compounds 2 and 3 were performed using a
PE/API III with MacIntosh Quadra1 900 from Perkin-Elmer Sciex Instru-
ments.
To determine the radiochemical and chemical purity of the reaction prod-
uct, and the specific radioactivity of [11C]1, an Agilent 1100 HPLC system with
an UV detector (235 nm) coupled in series with a radioactivity detector GABI
(raytest, Straubenhardt, Germany) was used. The HPLC analyses were per-
formed on a Purospher RP-18 column (5 mm, 125 mm ꢁ 3 mm, Merck) eluted
isocratically with acetonitrile/water (60/40) containing 0.1 M ammonium
formate at a flow rate of 0.5 ml/min.
The specific radioactivity of [11C]1 was calculated by means of a calibration
curve using different concentrations of the nonradioactive 1 in relation to their
UV absorbance response and the final radioactivity of [11C]1.
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P. MADING ET AL.
The used chemicals were reagent grade obtained from commercial suppliers.
BAY 59-8862 (1) and the reference compound of 2 (10-deacetyl BAY 59-8862)
was delivered by Indena (I-20090 Milan, Italy).
The THF (Fluka, waterfree) for the radiochemical conversions was freshly
distilled over Na/benzophenone in a nitrogen atmosphere. The 0.15 M solu-
tion of methylmagnesium bromide in diethyl ether was made from a 3 M
ethereal MeMgBr solution (Aldrich) by dilution with dry diethyl ether (Fluka,
99.8%, puriss., over molecular sieve) under nitrogen. The grade and source of
the other reagents for the synthesis of the final product [11C]1 were as follows:
phthaloyl dichloride, for synthesis, Merck; 2,6-di-t-butylpyridine, 597%,
Aldrich; lithium hexamethyldisilazide, 1 M in THF, Aldrich; hydrochloric
acid, 4 M in dioxane, Fluka; methanol, 599.5%, extra pure, Merck; NaOH,
1 M, Ph Eur, Merck; acetonitrile, p.a., Merck; water, for injection use,
Serumwerk Bernburg AG, Germany; ammonium formate, 99%, Acros
Organics; ethanol, 96%, extra pure, Merck; isotonic saline solution, for
intravenous infusion, DeltaSelect GmbH, Pfullingen, Germany.
The sterile filtration were done by means of a sterile filter ‘Millex1-GP’
(Millipore, 0.22 mm, Ø=25 mm).
The used module, outlined in Figure 1, was a commercially available
module for 11C methylation from Nuclear Interface (Munster, Germany). It
was modified in terms of program and hardware.
Before starting the two-pot synthesis, a manual cleaning procedure and two
cleaning programs for the module were carried out:
The two contaminated reaction vessels were removed and manually purified
by means of acetone, ethanol (96%) and water. The purified reaction vessels
were filled with acetone (extra pure, Merck) and installed at the module. All
contaminated tubing and valves were rinsed with acetone from storage vessel 1
via valve 1–reaction vessel 1–valve 18–valve 20a–exhaust and via valve
1–reaction vessel 1–valve 9–valve 10–reaction vessel 2–valve 19–valve
20a–exhaust.
The first cleaning program rinsed the synthesis part of the module with
acetone (storage vessels 1 and 2) and water (storage vessel 3). The second
cleaning program rinsed the synthesis part of the module with acetone (storage
vessels 1–3) and the solid-phase extraction part of the module with ethanol
(storage vessels 4–6). The module was dried by means of a helium gas stream
and vacuum.
Chemistry
Synthesis of compound 2. A solution of compound 1 (500 mg, 573 mmol) in
tetrahydrofurane (48 ml), hydrogenperoxide (30%, 24 ml) and sodium hydro-
gencarbonate (48 mg, 56.9 mmol) were stirred at room temperature for 30 min.
Ethyl acetate (50 ml) and a sodium thiosulfate solution (5%, 50 ml) were added
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SYNTHESIS OF A 11C-LABELLED TAXANE
363
and the phases were separated. The organic phase was washed twice with a
sodium thiosulfate solution (5%, 50 ml), following with a sodium chloride
solution (50 ml) and subsequently with water (20 ml). After drying with
sodium sulfate the solution was evaporated to dryness and the crude product
was purified by HPLC in 14 portions under the following conditions: column:
Nucleosil1 C18, 120 ꢁ 16 mm, (Knauer GmbH, Berlin, Germany), eluent:
acetonitrile/water 60/40, flow rate: 6 ml/min, UV detection: 228 nm, retention
time for 2: about 8 min. The separated fractions of 2 were combined
and evaporated to dryness. Yield: 220 mg (265 mmol, 46%); LC-MS: m/z
[M+H]+=831, calculated for C42H55O16N: M=830.
The HPLC retention time of 2 was identical to the reference compound IDN
5236 (10-deacetyl BAY 59-8862) from Indena.
Synthesis of precursor 3. A mixture of compound 2 (155 mg, 186 mmol),
imidazole (508 mg, 75 mmol) and triethylsilyl chloride (1.25 ml, 75 mmol) in
dimethyl formamide (14 ml) was stirred under an argon atmosphere at room
temperature for 1 h. After addition of water (15 ml) the mixture was extracted
three times with ethyl acetate (each 5 ml). The organic phases were combined,
washed with sodium chloride solution (5 ml) and water (5 ml), dried over
sodium sulfate and evaporated to dryness. The raw material was purified by
HPLC in 12 portions using the following conditions: column: LiChrosorb1
RP18, 250 ꢁ 25 mm, (Merck, Darmstadt, Germany), eluent: acetonitrile/water
95/5, flow rate: 15 ml/min, UV detection: 230 nm, retention time for 3: about
26 min.The separated fractions of 3 were combined and evaporated to dryness.
Yield: 116 mg (110 mmol, 59%); LC-MS: m/z [M+H]+=1059, calculated for
C48H71O16NSi2: M=1058; 1H-NMR (400 MHz, CDCl3)d8.04 (d, 2H,
J=8.4 Hz, benzoylortho), 7.63 (t, 1H, J=7.4 Hz, benzoylpara), 7.50 (q, 2H,
J=8.4 Hz, benzoylmeta), 6.40 (d, 1H, J=7.2 Hz, H-13), 6.11 (d, 1H, J=7.4 Hz,
H-2), 5.10 (s, 1H, H-10), 4.95 (dd, 1H, J = 9.7 Hz, 2.4 Hz, H-5), 4.86 (d, 1H,
J = 7.2 Hz, H-14), 4.61 (d, 1H, J=10.0 Hz, NH-30), 4.41 (d, 1H, J=3.9 Hz,
H-20), 4.40 (dd, 1H, J=10.9, 6.7 Hz, H-7), 4.33 (d, 1H, J=8.7 Hz, H-20a), 4.26
(d, 1H, J=8.7 Hz, H-20b), 4.17 (tt, 1H, J=10.0, 4.2 Hz, H-30), 3.80 (d, 1H,
J=7.4 Hz, H-3), 2.54 (s, 3H, COCH3), 2.49 (m, 2H, H-6), 1.96 (s, 3H, H-18),
1.80 (s, 3H, H-19), 1,70 (m, 1H, H-50), 1.54 (m, 2H, H-40), 1.43 (s, 9H,
OC(CH3)3), 1.35 (s, 3H, H-16), 1.25 (s, 3H, H-17), 1.01 (d, 6H, J=6.7 Hz,
H-60, 70), 0.98 (m, 18H, SiCH2CH3), 0.74–0.64 (m, 12H, SiCH2CH3).
Radiochemistry
The storage vessels 1–6, the mixing vessel and the two reaction vessels of the
module were supplied with the needed reagent solutions or solvents according
to Figure 1.
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P. MADING ET AL.
[11C]CO2 was produced by the 14N(p,a)11C nuclear reaction on the PET
cyclotron ‘CYCLONE 18/9’ (IBA, Belgium). The nitrogen target was irradi-
ated with a beam current of 25 mA for 40 min giving, on average, 80 GBq of
[11C]carbon dioxide at the end of the bombardment.
After trapping at ꢂ1658C the [11C]carbon dioxide was released from the
cooling trap at –208C using a helium gas flow of 8 ml/min into reaction vessel 1
containing a cooled solution (–208C) of methylmagnesium bromide in diethyl
ether (500 ml; 0.15 M). When the radioactivity in the vessel reached a plateau,
phthaloyl dichloride (100 ml; 692 mmol) and 2,6-di-t-butylpyridine (100 ml;
445 mmol) were added. By means of a helium gas stream (50 ml/min) the di-
ethyl ether was evaporated up to 858C in the exhaust (via V18 and V20a).
After reaching 858C, the helium gas stream was switched from the exhaust to
reaction vessel 2 (via V9 and V10). Then in the range of 85–1008C gaseous 6
was transferred from reaction vessel 1 into reaction vessel 2 containing a
cooled (ꢂ508C) THF solution (250 ml) of 3 (1.8 mg; 1.7 mmol) and lithium
hexamethyldisilazide (4 ml; 1 M in THF). The helium gas stream (50 ml/min) in
reaction vessel 1 was driven over the quenched Grignard reaction mixture for
the most time. It was driven through the quenched Grignard reaction mixture
only in nine intervals of each with 4 s over a time of 6 min. The [11C]acetylation
took place at room temperature for 6 min. After evaporation of the THF in
vacuo at room temperature a solution of hydrochloric acid in dioxane (60 ml;
4 M) and MeOH (400 ml) were added for cleavage of the triethylsilyl protecting
groups at room temperature. After 6 min, the reaction mixture was neutralized
with NaOH (200 ml; 1 M), diluted with HPLC eluent (1.5 ml) and injected to a
semipreparative HPLC column (Kromasil-100, RP-18, 7 mm, 300 ꢁ 8 mm,
Knauer, including a precolumn 30 ꢁ 8 mm). The final product [11C]1 was
eluted with acetonitrile/water (65/35) containing 0.1 M ammonium formate
(pH 7) at a flow rate of 6 ml/min with a retention time of about 8 min. The
detection was carried out by means of a UV detector (235 nm) and a radi-
oactivity detector.
The separated fraction of [11C]1 was diluted with water (20 ml) and collected
by solid-phase extraction at an RP–18 cartridge. After washing the cartridge
with water (10 ml), [11C]1 was eluted with ethanol (1.4 ml). Then the ethanolic
solution of [11C]1 and a isotonic saline solution (12.6 ml) were transferred
successively through a sterile filter ‘Millex1-GP’ to obtain the formulated final
solution of [11C]1. In this way a clear, colorless, sterile, pyrogen-free, isotonic
NaCl solution of [11C]1 within a volume of 14 ml and containing 10% ethanol
was obtained.
Conclusion
[11C]BAY 59-8862 ([11C]1) was synthesized in a four-step procedure using
a commercially available module for 11C methylation. The base of the
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SYNTHESIS OF A 11C-LABELLED TAXANE
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11C-labelling was the synthesis of [1-11C]acetyl chloride (6) in high radio-
chemical and chemical purity. The [1-11C]acetylation using 6 was carried
out with the lithium salt of the suitable taxane precursor 3 at the sterically
hindered, secondary hydroxy group in position 10. After a deprotection
step, crude [11C]1 was purified by semi-preparative RP HPLC and solid-phase
extraction. The yield, the radiochemical and chemical purity and the specific
radioactivity of the formulated [11C]1 was suitable for human PET studies.
Acknowledgements
The authors would like to thank Mrs A. Hentges and Mr T. Krauꢀ for
technical assistance. Thanks are also due to the crew of the PET cyclotron,
Mr S. Preusche and Mr H. Roꢀ, for radionuclide production. General support
from Indena is gratefully acknowledged as well.
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