Radiosynthesis of [11C]ximelagatran via palladium catalyzed [11C]cyanation

Article abstract of DOI:10.1002/jlcr.1461

N-hydroxyamidines (amidoximes) may be used in prodrug technology in improving oral bioavailability of drugs containing amidino functional groups. In the body, amidoximes are reduced quickly to amidines by enzymes that are present in several organs. Ximela

Full text of DOI:10.1002/jlcr.1461

Research Article
Received 19 June 2007,
Revised 13 August 2007,
Accepted 21 August 2007
Published online 3 January 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1461
Radiosynthesis of [11C]ximelagatran via
palladium catalyzed [11C]cyanation
Ã
Anu J. Airaksinen,a Jan Andersson,a Phong Truong,a Olle Karlsson,b and
Christer Halldina
N-hydroxyamidines (amidoximes) may be used in prodrug technology in improving oral bioavailability of drugs containing
amidino functional groups. In the body, amidoximes are reduced quickly to amidines by enzymes that are present in
several organs. Ximelagatran is a benzamidoxime and ethyl ester prodrug of melagatran, which is a thrombin inhibitor. Our
aim was to develop a fast and efficient labeling route for the synthesis of [11C]ximelagatran ([11C]3) with a label in a
metabolically stable position. [11C]3 was synthesized via a two-step synthesis sequence, starting from palladium catalyzed
[
11C]cyanation of its corresponding bromide precursor (2-[2-(4-bromo-benzylcarbamoyl)-azetidin-1-yl]-1-cyclohexyl-2-oxo-
ethyl amino-acetic acid ethyl ester) (1), followed by a reaction with hydroxylamine. [11C]3 was synthesized with 27 17%
total overall decay corrected yield (specific radioactivity of 2360 165 Ci/mmol at EOS), with a total synthesis time of
45 min. A fast and efficient labeling route for the synthesis of [11C]3 was developed.
Keywords: N-hydroxyamidine; amidoxime; serine protease inhibitor; radiosynthesis; PET; ximelagatran
body, after absorption from the gastrointestinal tract, ximelagatran
is metabolized quickly in two steps, via hydrolysis of the ethyl ester
Introduction
N-hydroxyamidine functionalities (amidoximes) may be used in
prodrug technology in improving oral bioavailability of drugs
containing amidino functional groups.1–4 Amidino groups are
very strong bases and they are protonated under physiological
conditions, which causes poor absorption of amidino-based
drugs from the gastrointestinal tract. In N-hydroxyamidines, an
oxygen atom is introduced on one of the nitrogen atoms of the
amidino group, which leads to lower pKa and thus higher
lipophilicity, increasing permeability across epithelial cells. In the
body, the hydroxyamidine group is reduced quickly to its active
amidino moiety by enzymes that are present in several organs.1
Amidino groups are a common feature of many serine
protease inhibitors.5 The functional group binds to the
enzymatic pocket of the protease, inhibiting binding of the
endogenous substrate.6,7 Many serine proteases, such as
thrombin and factors VIIa, IXa, Xa, XIa and XIIa, are involved in
the blood coagulation cascade.8 Serine protease inhibitors
targeting especially the inhibition of the function of thrombin
have been popular targets in the recent development of novel
drugs in the treatment of thromboembolic diseases.9
group and reduction of the hydroxyamidine moiety, to the active
drug melagatran. Melagatran itself is eliminated through urine and
faeces without further metabolism.13
Positron emission tomography is a useful, but still rarely used
tool in evaluating the bioavailability of prodrugs in vivo.14 The
technique enables simultaneous observation of the biodistribu-
tion of the investigated prodrug, metabolized intermediates and
the active drug, which provides important information about
possible accumulation of the prodrug or the intermediates in
non-target organs. The technique also enables analysis of
kinetics of the metabolic processes liberating the drug from its
prodrug moiety. All this may provide crucial information on in
vivo behavior of a novel prodrug in early clinical stages.
11
11
´
[ C]Amidination of [ C]nitriles was reported by Simeon et al.
in the radiosynthesis of [11C](Z,Z)-BABCH.15 According to our
knowledge, [11C](Z,Z)-BABCH is the only 11C-labeled benzami-
dine reported so far. However, synthesis of [11C]benzamidox-
imes has not been reported before and our aim was to develop
a fast and efficient labeling route for the synthesis of 11C-labeled
benzamidoximes with a label in a metabolically stable position.
We used ximelagatran as a model compound.
Ximelagatran is a prodrug of an oral direct thrombin inhibitor
melagatran.10,11 Melagatran belongs to the class of serine
protease inhibitors with
a benzamidine functional group
for binding to the arginine binding S1 pocket of thrombin
(Figure 1). Melagatran is a peptidomimetic, mimicking the
D-Phe-Pro-Arg sequence. The pKa’s of the benzamidine
(pKa 5 11.5) and the carboxylic acid (pKa 5 2.0) groups of
melagatran cause its zwitterionic character at physiological pH,
leading to poor oral bioavailability of the drug.12 The prodrug
ximelagatran (Exanta, AstraZeneca) was designed to overcome this
problem by using ester and amidoxime (pKa 5.2) functionalities in
order to avoid ionization of the drug at physiological pH. In the
aKarolinska Institutet, Department of Clinical Neuroscience, Psychiatry Section,
Karolinska Hospital, S-17176 Stockholm, Sweden
bAstrazeneca R&D, Department of Medicinal Chemistry, S-431 83 Mo¨lndal,
Sweden
*Correspondence to: Anu J. Airaksinen, Karolinska Institutet, Department of
Clinical Neuroscience, Psychiatry Section, Karolinska Hospital, S-17176 Stock-
holm, Sweden.
E-mail: anu.airaksinen@ki.se
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A. J. Airaksinen et al.
Figure 1. Structures of melagatran and its prodrug ximelagatran.
Results and discussion
Production of [11C]HCN
Radiosynthesis of [11C]ximelagatran ([11C]3)
For [11C]cyanation of the bromide-precursor (1), we considered
two synthetic strategies, cyanation via traditional copper
catalyzed Rosenmund reaction or via palladium catalyzed
reaction.19,20 We decided to utilize the palladium-catalyzed
[11C]Hydrocyanic acid was produced according to previously
published methods with minor modifications.16 In short,
[11C]CH4 was converted online in a platinum oven (T 5 9901C)
to [11C]ammonium cyanide ([11C]NH4CN), which was immedi-
ately bubbled through H2SO4 to generate [11C]HCN.17 The
[11C]HCN gas was passed through a P2O5 column to a solution
containing KOH and Kryptofix 2.2.2 in dimethyl sulfoxide
(DMSO) to generate K11CN (Figure 2).
NH3 concentration is very important for the [11C]HCN
formation, and in our system 10–15 vol% was found to be
sufficient. A furnace temperature of 9901C gave significantly
better conversion (42717%, n 5 3) than a temperature of 9501C
(1172%, n 5 2), which is not in accordance with previously
published data.18 The over-all conversion yields were lower than
what has been previously reported. Explanations for the low
yields could be the contact time for the gas with platinum or
possibly the platinum amount. It has been reported that
platinum amount below 3 g reduces the conversion yield
linearly.18 The amount, 1.3 g, which was used in our system,
may not be sufficient. Another possible reason is that oxygen or
H2O is interfering with the catalytic reaction of [11C]CH4 with
NH3 on Pt. Iwata et al. reported the effect of absorbers to
improve the yield, achieving close to quantitative conversion
when metallic sodium coated on quartz wool was used.
approach in the radiosynthesis of [11C]3 (Scheme 1), because in
11
the synthesis of [ C](Z,Z)-BABCH Simeon et al. reported a
´
comparison of copper-catalyzed and palladium-catalyzed
[11C]cyanations and in their labeling conditions, they found
the palladium-catalyzed reaction more efficient.
The K11CN solution, which was produced as described
above, was transferred into a vial containing the bromide
precursor (1, 2-[2-(4-bromo-benzylcarbamoyl)-azetidin-1-yl]-1-
cyclohexyl-2-oxo-ethyl amino-acetic acid ethyl ester) and
Pd(PPh3)4 dissolved in DMSO and heated for 4 min at 1351C.
The reaction provided the 11CN-intermediate ([11C]2) with good
incorporation yield, with only small variations between synthesis
repetitions (between 70 and 80%) (Figure 3). The high-
performance liquid chromatography (HPLC) analysis of the
reaction mixture revealed one unidentified radiolabeled
side product eluting before the intermediate ([11C]2). The
intermediate ([11C]2) was identified by comparing with the
retention time of the corresponding non-labeled analogue (2).
In classical organic chemistry, hydroxyamidines are synthe-
sized from nitriles by treatment with hydroxylamine at room
temperature. To complete the reaction, reaction times from
several hours to days are often needed.3,21 However, in tracer
Scheme 1
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A. J. Airaksinen et al.
Figure 2. Schematic diagram of the synthesis apparatus for H11CN production and for the synthesis of [11C]3.
conditions, the reaction between the 11CN-intermediate (2) and ethyl amino-acetic acid ethyl ester (1) and the references of 2-[2-
hydroxylamine was complete in 3 min at 1351C in a mixture of (4-cyano-benzylcarbamoyl)-azetidin-1-yl]-1-cyclohexyl-2-oxo-
EtOH and EtOAc in the presence of a small amount of EDTA. Two ethyl amino-acetic acid ethyl ester (2) and ximelagatran (3) were
¨
unidentified radiolabeled side products were observed in the provided by Astrazeneca R&D, Molndal, Sweden. Radiosynthesis
reaction mixture corresponding to 12 and 10% of the total was performed in a homemade semi-automated synthesis
radioactivity (Figure 3). These side products were most probably module. A carbolite furnace, MTF 10/15, and platinum wire
originating from the previous reaction step, followed by further (1.3 g, 0.127 mm, 99.99%) from Sigma-Aldrich were used for
reaction. No 11CN-intermediate (2) was left in the reaction [11C]cyanide production.
mixture.
Semi-preparative HPLC system consisted of Merck-Hitachi L-
The synthesized [11C]3 was purified with semi-preparative 7100 Pump, L-7400 UV-detector and GM-tube for radioactivity
HPLC and evaporated to dryness under reduced pressure detection. Waters mBondapak C18 column (300 Â 7.8 mm,
(Figure 4). The evaporation residue, containing the product, 10 mm) was used, eluted with 30% acetonitrile in HCO2NH4
was dissolved in isotonic phosphate buffer solution (pH 7.4). The (0.1 M) with a flow of 6 mL/min. A wavelength of 235 nm was
total overall yield starting from [11C]CH4 was 27716% (decay used. The analytical HPLC system included Merck-Hitachi L-7100
corrected yield (DCY) at EOS) with specific radioactivity Pump, L-7400 UV-detector, D-7000 interface and Beckman
8776 GBq/mmol (23607165 Ci/mmol) in a total synthesis time radiodetector (Model 170). The system was controlled by
of 45 min. The relatively high specific radioactivity was achieved Merck-Hitachi Chromatography Data Station Software D-7000
due to the in-target produced [11C]CH4, which was used as a (version 4.1). Waters mBondapak C18 column (300 Â 3.9 mm,
starting material.22 The difficulties in the H11CN production 10 mm) was used, eluted with 35% acetonitrile in HCO2NH4
caused the large variation in the overall yields. Radiochemical (0.1 M) with a flow of 2 mL/min and the chromatogram was
purity of the final product was 9870.7% (Figure 4). The product monitored with a wavelength of 235 nm.
was identified by co-injection with the corresponding non-
labeled ximelagatran.
Production of [11C]HCN
[11C]CH4 was produced in-target with a GEMS PETtrace cyclotron
using 16.4 MeV protons in the 14N(p,a)11C reaction on N2(g) and
10% H2(g). The target gas was irradiated for 20 min with a beam
intensity of 35 mA. NH3 gas (flow of 20–30 mL/min) was added to
Experimental
General
All the chemicals were purchased from commercial suppliers the produced [11C]CH4 (flow of 200 mL/min) and the mixture
and used without further purification. The starting material 2-[2- was passed through a carbolite furnace containing a quartz tube
(4-bromo-benzylcarbamoyl)-azetidin-1-yl]-1-cyclohexyl-2-oxo-
containing a heated platinum wire (9901C). The produced
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A. J. Airaksinen et al.
Figure 4. HPLC chromatograms of (A) the semi-preparative purification,
Rt([11C]3) 5 9.5 min (semi-prep HPLC conditions) and (B) the purified [11C]3, co-
injected with ximelagatran reference (3) , Rt([11C]3) 5 4.7 min (analytical HPLC
conditions).
Figure 3. HPLC chromatograms of (A) the reaction mixture after cyanation co-
injected with unlabeled reference 2, Rt([11C]2) 5 7.2 min (analytical mBondapak
C18, 20% acetonitrile, 80% H3PO4 (0.01 M), flow of 3 mL/min) and (B) reaction
mixture after reaction with hydroxylamine co-injected with ximelagatran reference
(3), Rt([11C]3) 5 2.2 min (analytical mBondapak C18, 5% acetonitrile, 95% H3PO4
(0.01 M), flow of 3 mL/min). SP 5 radiolabeled un-identified side product.
(5.0 mg, 13.3 mmol) in DMSO (400 mL). The K11CN/Kryptofix
solution was transferred into a vial containing the bromide
precursor (1, 2-[2-(4-bromo-benzylcarbamoyl)-azetidin-1-yl]-1-
cyclohexyl-2-oxo-ethyl amino-acetic acid ethyl ester, 4.0 mg,
8.1 mmol) and Pd(PPh3)4 (5.5 mg, 4.8 mmol) dissolved in DMSO
(400 mL). The reaction mixture was heated for 4 min at 1351C.
The reaction vial was cooled to room temperature and a mixture
of EDTA (1.0 mg, 3.4 mmol), 50% wt hydroxylamine in H2O
(100 mL, 1.63 mmol), EtOH (180 mL) and ethylacetate (100 mL)
was added. The reaction mixture was heated at 1351C for 3 min,
cooled to 401C after which 800 mL of the mobile phase was
added and the diluted reaction mixture was injected into the
semi-preparative HPLC. The fraction, which contained the
product (Rt 5 9.5 min), was evaporated to dryness under
[11C]NH4CN was bubbled through H2SO4 (50%, 2 mL) at 651C to
generate [11C]HCN.
Synthesis of [11C]3
[11C]HCN gas was directed through a P2O5 column to a solution
of powdered KOH (1.0 mg, 17.8 mmol) and Kryptofix 2.2.2
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A. J. Airaksinen et al.
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A fast and efficient method for synthesizing [11C]3 was
developed. [11C]3 was synthesized with good yield (27717%,
total overall DCY), giving 1.7571.0 GBq (47727 mCi) of radio-
chemically pure compound (497%) with a total synthesis time
of 45 min and relatively high specific radioactivity. The devel-
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