Labelling of the guanylate cyclase activator cinaciguat (BAY 58-2667) with carbon-14, tritium and stable isotopes

Article abstract of DOI:10.1002/jlcr.1738

For studies of pharmacokinetics and drug metabolism of the new soluble guanylate cyclase activator cinaciguat (BAY 58-2667) the ¹⁴C-labelled compound was synthesized. The tritiated compound was required to elucidate the mode of action and the s

Full text of DOI:10.1002/jlcr.1738

Research Article
Received 24 September 2009,
Revised 25 November 2009,
Accepted 30 November 2009
Published online 12 January 2010 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1738
Labelling of the guanylate cyclase activator
cinaciguat (BAY 58-2667) with carbon-14,
tritium and stable isotopes
Ã
D. Seidel and U. PleiX
For studies of pharmacokinetics and drug metabolism of the new soluble guanylate cyclase activator cinaciguat (BAY
58-2667) the 14C-labelled compound was synthesized. The tritiated compound was required to elucidate the mode of action
and the stable labelled compound was required for bio-analytical studies by quantitative mass spectrometry as well. Two
radiosyntheses are described with different formation of the labelled intermediate 1-(chloro[14C]methyl)-4-(2-pheny-
lethyl)benzene. The first one started with 14C-carboxylation of 1-bromo-4-(2-phenylethyl)benzene yielding the desired
product in 5 steps. In the second synthesis intermediate 1-(chloro[14C]methyl)-4-(2-phenylethyl)benzene was formed by
chloromethylation of bibenzyl with [14C]paraformaldehyde/hydrochloric acid subsequently resulting in the final product in
three steps. Tritium labelling was performed by tritium exchange of the diester intermediate using an organo-iridium
catalyst and subsequent saponification. The stable labelled compound was synthesized via a convergent synthesis
starting with 13C,15N-cyanation of 1-(chloromethyl)-2-{[4-(2-phenylethyl)benzyl]oxy}benzene and 13C-cyanation of methyl
4-bromobenzoate, respectively. The labelled product was obtained after 7 chemical steps.
Keywords: guanylate cyclase activator; carbon-14; carbon-11; tritium; carbon-13; nitrogen-15; synthesis
for studies of the mode of action tritium-labelled substance
was synthesized. The labelling with tritium was realized
Introduction
Soluble guanylate cyclase (sGC) is a heterodimeric enzyme with
a heme moiety as the prosthetic group. Activated by nitric oxide
(NO), it generates the second messenger cGMP. Impaired
bioavailability and/or responsiveness to endogenous NO have
been implicated in the pathogenesis of especially cardiovascular
diseases. Therapies with organic nitrites and other NO donors
are limited by non-specific interactions of NO with various
biomolecules, lack of response and development of tolerance.
The new guanylate cyclase activator cinaciguat (BAY 58-2667)
acts in a NO-independent manner.1 This provides considerable
therapeutic advantages. Cinaciguat activates sGC with EC50 and
Kd values in the low nanomolar range. This renders the
compound the most potent NO-independent sGC activator
reported to date2 (Figure 1).
by an organo-iridium exchange using the ester form of
cinaciguat. As an internal standard for quantitative mass spectral
N
O
O
OH
O
OH
In contrast to hem-dependent sGC stimulators, cinaciguat
produces an additive, non-synergistic effect when combined
with NO donors. It relaxes blood vessels with potency that is
several orders of magnitude greater than the NO donors sodium
nitroprusside and 3-morpholinosydnonimine.3 The compound
reduces coronary perfusion pressure in the rat Langendorff heart
preparation and remains active in the tissues made tolerant to
gylceryl trinitrate.4
For studies of pharmacokinetics and drug metabolism of
cinaciguat, the version with a metabolically stable carbon-14
label was required. Two carbon-14 labelling syntheses were
performed using different synthetic routes to the carbon-14
labelled key intermediate 5. The objective of the second
synthesis was to reduce the number of reaction steps and
to avoid a critical by-product of the reduction step. Additionally
Figure 1. Structure of BAY 58-2667.
Isotope Chemistry, Bayer Schering Pharma, Global Drug Discovery, Global Early
Development, Drug Metabolism and Pharmacokinetics, Development Pharma-
cokinetics, Bayer Schering Pharma AG, Aprather Weg 18a, D-42096 Wuppertal,
Germany
*Correspondence to: D. Seidel, Isotope Chemistry, Bayer Schering Pharma,
Global Drug Discovery, Global Early Development, Drug Metabolism and
Pharmacokinetics, Development Pharmacokinetics, Bayer Schering Pharma AG,
Aprather Weg 18a, D-42096 Wuppertal, Germany.
E-mail: dietrich.seidel@bayerhealthcare.com
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Copyright r 2010 John Wiley & Sons, Ltd.

D. Seidel and U. PleiX
analysis a stable labelled compound was prepared as well. conditions was described earlier by Speer and Hill.7 Common
A convergent synthesis was performed to introduce carbon-13 reduction with triethylsilane in trifluoroacetic acid performed in
and nitrogen-15.
this synthesis led to 1-bromo-4-(2-phenylethyl)benzene in a
better yield under mild conditions. In spite of the mild
conditions, the dehydration of the hydroxyl intermediate could
not be fully avoided giving 1-bromo-4-[(E)-2-phenylvinyl]ben-
Results and discussion
The first radiosynthesis of cinaciguat was performed as given in zene in a small amount as by-product which was very difficult to
Scheme 1.5 Intermediate 5 was prepared by a new synthetic remove. The carbon-14 was introduced by halogen–metal
route, different from the technical synthesis.6
exchange with n-butyllithium followed by carboxylation with
Commercially available 1-(4-bromophenyl)-2-phenylethanone 14CO2. The obtained 4-(2-phenylethyl)[carboxy-14C]benzoic acid
was reduced to the corresponding bibenzyl derivative. The (3) was then reduced to the alcohol derivative 4 with lithium
conversion with red phosphorus in hydroiodic acid under drastic aluminium hydride as described by M. Carrara et al. for the
O
14C
Br
Br
Et3SiH,
TFA
1) n-BuLi
2) 14CO2
OH
O
1
3
2
LiAlH4
H2
14C
H2
14C
Cl
OH
SOCl2
4
5
N
O
ONa
O
O
O
6
N
N
OH
O
O
O
H124
C
H124
C
O
O
NaOH
O
OH
O
O
8
[14]C]cinaciguat
7
Scheme 1. First radiosynthesis of [14C]cinaciguat.
J. Label Compd. Radiopharm 2010, 53 130–139
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D. Seidel and U. PleiX
non-labelled methyl ester.8 The direct reduction of the acid formaldehyde in reasonable limits incomplete conversion was
proceeded as good as in the case of the ester. By common accepted. Nevertheless the reaction needed a long time (49 h at
chlorination with thionyl chloride intermediate 4 was converted 601C). The obtained labelled 1-[chloro[14C]methyl]-4-(2-pheny-
to 1-[chloro[14C]methyl]-4-(2-phenylethyl)benzene (5).
lethyl)benzene had to be purified intensively by repeated
The last but one step of the radiosynthesis was the chromatography.
O–alkylation of intermediate 6 with the chloro[14C]methyl
derivative 5 using sodium methoxide as base. Intermediate 6 ester saponification were performed in a one-pot procedure.
In difference to the first synthesis alkylation and subsequent
was prepared according to the technical synthesis.6 Subsequent
This second synthesis proceeded much quicker than the first
ester saponification yielded the carbon-14 labelled cinaciguat. one and delivered the product in a radiochemical yield (9.6%)
Final analytics revealed a small contamination with the stilbene better than that of the first one (5.7%). Nevertheless due to new
derivative of the product, which made the chromatographic by-products the final purification was also problematic.
purification difficult.
With the aim to shorten the radiosynthesis an alternative was necessary. Therefore, the compound was labelled with
synthetic route to intermediate 5 was elaborated (Scheme 2). tritium. Labelling was performed by an organo-iridium catalyzed
For research purposes a receptor binding study of cinaciguat
In 1960 F. Vismara described the formation of intermediate 5 hydrogen exchange.10,11 The advantage of this method is the
by chloromethylation of bibenzyl (9) with 37% formaldehyde possibility of direct introduction of tritium into the final
solution (formalin).9 Experiments with paraformaldehyde compound. Under several iridium catalysts such as (1,5–cyclooc-
showed superiority over the solution. To keep the excess of tadiene) bis-(triphenylphosphine)-iridium(I) tetrafluoroborate
H2
14C
Cl
(14CH2O)n
HCl/H2SO4
9
5
N
O
OH
1) NaOCH3
O
2) NaOH
O
O
10
N
OH
O
H124C
O
O
OH
8
[14C]cinaciguat
Scheme 2. Second radiosynthesis of [14C]cinaciguat.
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Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 130–139

D. Seidel and U. PleiX
and (1,5–cyclooctadien) bis-(methyldiphenylphosphine)-iridium(I) chlorine against [13C, 15N]cyanide which was reduced with
hexafluorophosphate (1,5-cyclooctadiene)(pyridine)(tricyclohexyl- hydrogen and raney nickel as catalyst under a pressure of 400 kPa.
phosphine)-iridium(I) hexafluorophosphate was the most suitable
The synthesis of the second part of the labelled molecule
to get an acceptable labelling degree. The diester (11) of started from 4-bromobenzoic acid (19). Under Rosenmund–von
cinaciguat was used in the reaction due to the insufficient Braun conditions [13C]cyanide was introduced. Boiling DMF and
solubility of cinaciguat in dichloromethane. After the labelling a reaction time of about 11 h were necessary to complete the
process the tritiated diester 11 was saponified yielding [3H]cina- reaction. The following reduction with Raney-nickel alloy in
ciguat. Due to the small radioactive yield a 3H NMR spectrum was formic acid (75%) led to methyl 4-[13C]formylbenzoate (21) in a
not performed. The 3H NMR spectrum of a very similar compound yield of 42%.
(the O-substituent at the phenol was 4-azidophenyl-benzyl
Reductive amination starting from 18 to 21 was performed in
instead of phenyl-ethyl-benzyl in compound (13), which was boiling toluene giving the Schiff base derivative 22 in excellent
labelled with tritium under exactly the same conditions, showed yield. Platinum catalyzed hydrogenation lead to the secondary
the tritium in the ortho-position of the carboxyl group in the amine 23 in moderate yield. Alkylation of 23 with methyl
benzoic acid substituent. Because of the same aromatic 5-bromovalerate turned out to be the most critical step. The
methoxycarbonyl directing group in both of the molecules it best conditions were found by using potassium carbonate as
was assumed that the labelling in compound (13) was in the base in boiling acetonitrile. Nevertheless the yield was only 27%.
ortho-position as well. The labelling degree was determined to be Subsequent ester saponification and final purification by semi-
18.7% by mass spectrometry (Scheme 3).
preparative liquid chromatography yielded stable labelled
For quantitative mass spectrometry cinaciguat labelled with cinaciguat in the desired quality (Scheme 4).
stable isotopes was required as internal standard (ISTD) with
three mass units difference in comparison to the non-labelled
Experimental
compound.
For stable labelling a new synthetic way was developed. The
convergent synthesis (Scheme 4) started from the substituted
Materials
benzylchloride (14), which was converted to the 13C and 15N Barium [14C]carbonate was delivered by Izotop VO, Moscow,
labelled amine (18) in 4 reaction steps.6 The labelling positions Russia. [14C]Paraformaldehyde was obtained from GE Healthcare
were introduced by crown ether supported substitution of UK Ltd., Buckinghamshire, UK. Tritium was delivered by RC Tritec
+
P
Ir
N
N
-
PF6
N
O
O
O
O
3H
O
O
tritium gas
O
O
O
O
11
12
NaOH
N
OH
O
3H
O
O
OH
13
[3H]cinaciguat
Scheme 3. Tritium labelling of cinaciguat.
J. Label Compd. Radiopharm 2010, 53 130–139
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D. Seidel and U. PleiX
Scheme 4. Stable labelling of cinaciguat.
Ltd., Teufen, Switzerland (in uranium storage). Potassium USA. All remaining solvents and reagents were obtained from
[13C,15N]cyanide and potassium [13C]cyanide were both commercial sources and used without further treatment unless
obtained from Cambridge Isotope Laboratories, Inc., Andover, indicated otherwise.
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J. Label Compd. Radiopharm 2010, 53 130–139

D. Seidel and U. PleiX
Liquid scintillation counting
spectrometer (Bruker, Rheinstetten, Germany). The NMR spec-
trum of [14C]cinaciguat of the second radiosynthesis was
recorded on a Bruker Avance 500 magnetic resonance spectro-
meter (Bruker, Rheinstetten, Germany).
Quantification of radioactivity was performed using a Perkin-
Elmer TRI-CARBs 2500 TR liquid scintillation analyzer, with
Ultima GoldTM cocktail used throughout.
High-performance liquid chromatography
First radiosynthesis of cinaciguat
[14C]Cinaciguat (first radiosynthesis) and [13C2,15N]cinaciguat
were analyzed by HPLC using a HP 1050 system Series II
(Hewlett-Packard, Waldbronn, Germany) with a Ramonas Cell 5
(Raytest, Straubenhardt, Germany) for radioactivity detection.
The following HPLC system was used for the purity check:
Prodigys ODS (3) 100 A, 250 Â 3.2 mm, 5 mm (Phenomenex;
Hoesbach, Germany), column temperature: 451C, flow rate:
0.6 mL/min, eluent; A: phosphate buffer (0.61 g KH2PO410.78 g
Na2HPO4/L H2O), B: CH3CN, gradient: 0 min 30% B, 40 min 70% B,
50 min 80% B, UV 210 nm.
[14C]Cinaciguat of the second radiosynthesis was analyzed
by HPLC using a HP 1100 (Hewlett-Packard; Waldbronn,
Germany) with a detector Ramonas Star (Raytest, Strauben-
hardt, Germany) for radioactivity detection. The following HPLC
system was used for the purity check: Synergys MAX-RP
250 Â 4.6 mm, 4 mm (Phenomenex; Hoesbach, Germany), column
temperature: 451C, flow rate: 1.5 mL/min, eluent; A: ammonium
acetate buffer pH 4.7, B: CH3CN, gradient: 0 min 40% B, 15 min
90% B, 35 min 90% B, 50min 90% B, UV 230 nm.
[3H]Cinaciguat was analyzed by HPLC using a HP 1050
system Series II (Hewlett-Packard, Waldbronn, Germany) with
a Ramonas Cell 5 (Raytest, Straubenhardt, Germany) for
radioactivity detection. The following HPLC system was used
for the purity check: Phenomenexs Aqua C18 250 Â 4.6 mm,
5 mm (Phenomenex; Hoesbach, Germany), flow rate: 1.0 mL/min,
eluent; A: 0.05% HClO4, B: CH3CN, gradient: 0 min 30% B, 5 min
60% B, 20 min 60% B, 35 min 90% B, UV 210 nm.
1-Bromo-4-(2-phenylethyl)benzene (2)
Benzyl 4-bromophenyl ketone (1.88 g, 32.2 mmol) was dissolved
in 28 mL of trifluoroacetic acid. Triethylsilane (28 mL) was added
and the mixture was stirred for 42 h at ambient temperature.
Then the solution was evaporated and the residue was dissolved
in 50 mL of cyclohexane and washed with 50 mL of water. The
organic layer was dried over sodium sulfate and evaporated to
dryness. Flash chromatography on silica gel with cyclohexane as
the mobile phase afforded 6.9 g (26.4 mmol) of 2 with a purity of
97.3% (GC). The yield was 82%.
4-(2-Phenylethyl)[carboxy-14C]benzoic acid (3)
The non-labelled 2 (4.1 g, 15.7 mmol) was dissolved in 20 mL of
diethyl ether (dried over sodium/lead alloy) and cooled in an ice
bath. Butyl lithium solution (12 mL, 15% in n-hexane) was added.
The solution was stirred for 30 min at ice bath temperature and
1.5 h at ambient temperature. Then the reaction flask was
connected over a vacuum line with an equipment of a reaction
flask with the carbon-14 labelled barium carbonate (3.1 g,
15.7 mmol, SA: 2080.6 MBq/mmol, corresponding to 32.67 GBq)
and a dropping funnel with concentrated sulfuric acid (25 mL).
The solution of the bromo derivative was frozen with liquid
nitrogen and the equipment was evacuated. Then the equip-
ment was closed and the carbon-14 labelled carbon dioxide was
liberated by drop wise addition of the sulfuric acid and
condensed to the frozen bromo derivative solution. For
complete carbon dioxide liberation the carbonate mixture was
heated after addition of the total amount of the sulfuric acid
until a clear liquid was obtained. Then the vacuum was broken
and the reaction mixture was stirred for 1 h at ice bath
temperature and for 16 h at ambient temperature. The labelled
acid 3 was liberated by addition of 20 mL of 2 M hydrochloric
acid. The resulting suspension was diluted with 25 mL of water
and the product was extracted with 125 mL of dichloromethane.
The organic layer was washed with 25 mL of water. For
formation of the sodium salt 2.5 mL of 45% sodium hydroxide
solution were added and the mixture was stirred for 18 h at
ambient temperature. The solid was filtered off and washed
three times with each 10 mL of water. The solubility of the salt in
water is very low. For liberation of the free acid the solid was
stirred with 20 mL of 2 M hydrochloric acid. The obtained acid
was filtered off, washed three times with each 10 mL of water
and dried for 16 h in an evacuated desiccator over blue gel
yielding 2.88 g of 3. This was dissolved in 25 mL of THF and
filtered off from insoluble solid. By LS counting the total
radioactivity of the solution was determined to 16.284 GBq.
From the specific radioactivity of the starting Ba14CO3
(2080.6 MBq/mmol) the content of 3 was calculated to 1.776 g
(7.8 mmol). The radiochemical yield was 49.7%. The purity
was 99.2% (GC, after derivatization to the methyl ester).
Radio-TLC
[14C]Cinaciguat (first radiosynthesis) was analyzed on silica gel
plate 60 F254, 10 Â 20 cm, Merck; Darmstadt, Germany) with the
eluent: dichloromethane/methanol/acetic acid = 85:10:5 (v/v)
and radiodetection by a Raytest Ritas-3200 analyzer (Raytest,
Straubenhardt, Germany). [14C]Cinaciguat of the second radio-
synthesis was analyzed under the same conditions with radio-
detection by a Phosphorimager BAS 5000 (Fuji Photofilm Ltd.,
Tokyo, Japan).
Mass spectral analysis
[14C]Cinaciguat (first radiosynthesis), [3H]cinaciguat and
[13C2,15N]cinaciguat were analyzed by PE/API III with MacIntosh
Quadras 900 (Perkin-Elmer Sciex Instruments; Thornhill,
Canada). [14C]Cinaciguat of the second radiosynthesis) was
analyzed by HP 1100 MSD (Agilant; Waldbronn, Germany).
Gas chromatography and GC-MS analysis
GC and GC-MS analyses were recorded on a HP 5890 equipped
with MS HP 5970 (Agilent; Waldbronn, Germany).
NMR spectra
The NMR spectrum of [14C]cinaciguat (first radiosynthesis) Product formation and labelling were confirmed by GC-MS with
was recorded on a Bruker DRX 400 magnetic resonance m/z = 242 [M]1 (methyl ester).
J. Label Compd. Radiopharm 2010, 53 130–139
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D. Seidel and U. PleiX
[4-(2-Phenylethyl)phenyl][14C]methanol (4)
Purification by re-crystallization from methanol or ethanol
failed.
The above solution of the labelled 3 was diluted with 10 mL of
THF and heated to 501C. Lithium aluminium hydride solution
(16 mL, 1 M in THF) was added drop wise. The reaction mixture
was stirred for 2 h at 501C. After cooling in an ice bath 2 mL of
water and 0.6 mL 10% potassium hydroxide solution were
added drop wise followed by evaporation. The residue was
suspended in 80 mL of boiling diisopropyl ether and filtrated
over silica guhr. The filter cake was washed with 45 mL of
diisopropyl ether. The mother liquor was evaporated to dryness
under reduced pressure yielding 1.91 g of 4 (17% more than the
theoretical yield). Product formation and labelling were con-
firmed by GC-MS with m/z = 214 [M]1.
The final purification was performed by semi-preparative
HPLC. The total amount of crude 8 (1.68 g) was dissolved in
66 mL of acetonitrile and 7 mL of concentrated hydrochloric
acid. Liquid chromatography was performed on a Nucleosils
C
18 phase (7 mm, 250 Â 25 mm) with acetonitrile/0.1 M hydro-
chloric acid 55:45 (v/v) as mobile phase, a flow of 12 mL/min and
UV-detection at 230 nm. The chromatographic separation was
difficult due to near running by-products. The product was
isolated and characterized as the hydrochloride. The yield was
547 mg of 8 as hydrochloride (0.9 mmol). The radiochemical
purity was 98.9%, the chemical purity (210 nm) was 99.0%.
Radio-TLC revealed a purity of 99.0% (total). The specific
radioactivity was 3.41 MBq/mg (2062 MBq/mmol) determined
by LC counting/UV quantification. The total radiochemical yield
1-[Chloro[14C]methyl]-4-(2-phenylethyl)benzene (5)
1
was 5.7% based on the starting barium [14C]carbonate. H NMR
Intermediate 4 (1.91 g, maximal 7.86 mmol) was dissolved in
18 mL of chloroform. A solution of thionyl chloride (1.31 mL,
18.05 mmol) in 3 mL of chloroform was added. After stirring for
2 h at ambient temperature the solution was washed three
times with each 10 mL of water and evaporated yielding 2.13 g
of 5 (about 20% more than the theoretical yield).
(400MHz, DMSO-d6) d = 7.94 (d, 2 H), 7.63 (d, 2 H), 7.33 (t, 4 H),
7.29–7.16 (m, 7 H), 7.04 (d, 1 H), 6.89 (t, 1 H), 5.03 (s, 2 H), 4.12 (s,
2 H), 2.95 (m, 10 H), 2.14 (m, 2 H), 1.60 (m, 2 H), 1.43 (m, 2 H).
Second radiosynthesis of [14C]cinaciguat
Sodium 2-(2-{(5-ethoxy-5-oxopentyl)[4-(methoxycarbonyl)benzyl]- 1-[Chloro[14C]methyl]-4-(2-phenylethyl)benzene (5)
amino}ethyl)-phenolate (6)
Bibenzyl (9, 2.08 g, 11.43 mmol) was submitted in a two-necked
Methyl 4-({(5-ethoxy-5-oxopentyl)[2-(2-hydroxyphenyl)ethyl]amino}-
methyl)-benzoate6 was purified by flash chromatography on
silica gel using n-heptane/ethyl acetate = 1:1 (v/v) as the mobile
phase before starting the formation of the sodium salt. Purified
6 (7.95 g, 19.2 mmol) was dissolved in 25 mL of methanol.
Sodium methoxide solution (3.8 mL, 30% in methanol,
19.9 mmol) was added. The solution was stirred for 3 h at
ambient temperature, evaporated and the residue was evapo-
rated again from 25 mL of toluene. Then the oily residue was
evaporated from 25 mL of acetonitrile and dried under oil pump
vacuum yielding 8.12 g of 6. The substance was used without
further characterization in the next chemical step.
100 mL-reaction tube. The tube was transferred into a single-use
glove box. The total amount of [14C]paraformaldehyde
(29.6 GBq, 0.444 g, 14.29 mmol, at a specific radioactivity of
2.07 GBq/mmol) was added. One neck of the tube was closed
tightly with a cold finger combined with an orifice for gas
emission. The second neck was closed with a septum. Then the
single-use glove box was evacuated to remove the volatile
radioactivity. Acetic anhydride (5.23 mL, 91.45 mmol) was added
with a syringe through the septum to the reactants in the
reaction tube leading to
a suspension. Then 4.26 mL
(51.44 mmol) of 37% hydrochloric acid were added with a
syringe through the septum giving a very bulky suspension. On
heating the reaction mixture to 651C, an emulsion was obtained.
Methyl 4-{[(5-ethoxy-5-oxopentyl){2-[2-({[4-(2-phenylethyl)phenyl]- Concentrated sulfuric acid (3.51 mL, 62.87 mmol) was added
with a syringe through the septum at 651C within 1 h. The
[14C]methyl}oxy)-phenyl]ethyl}amino]methyl}benzoate (7)
reaction mixture was stirred for 20 h at 601C. HPLC analysis
Non-labelled 6 (4 g) was added to a solution of 5 (2.12 g,
revealed 26.4% of product and 65.1% of bibenzyl. Additional
maximal 7.86 mmol) in 25 mL of DMF. The reaction mixture was
concentrated hydrochloric acid (2.15 mL, 25.96 mmol) was
stirred for 3 h at 551C. LC-MS revealed the presence of the
added followed by the addition of 1.72 mL (30.81 mmol) of
expected ethylmethyl ester (7) and a greater part of dimethyl
concentrated sulfuric acid within 10 min. HPLC analysis after a
ester. Due to the subsequent ester hydrolysis this conversion
total time of 49 h at 601C revealed 36.8% of product and 48.7%
was not critical. The reaction mixture was evaporated. Product
of bibenzyl. After cooling to ambient temperature a slight
formation and labelling were confirmed by LC-MS with m/z
stream of air was passed for 1 h through the reaction mixture to
596 [M1H]1 (dimethylester) and 610 [M1H]1 (7). The crude
remove volatile radioactivity. Then the reaction mixture was
product mixture was used without purification in the next
cooled in an ice sodium chloride cooling bath and 15 mL of ice-
chemical step.
cold water and subsequently 22 mL of dichloromethane were
added. After intensive stirring the layers were separated. The
4-{[(4-Carboxybutyl)(2-{2-4-phenethylphenyl[14C]methoxy]phenyl}-ethyl)-
organic layer was washed with half-saturated sodium bicarbo-
amino]-methyl}benzoic acid, [14C]cinaciguat (8)
nate solution (18 mL) and subsequently with 10 mL of water and
evaporated to dryness yielding 2.95 g of crude product 5. HPLC
analysis revealed 41.6% of product and 41.2% of bibenzyl.
Separation from bibenzyl was performed by low liquid pressure
chromatography on a pre-packed column Lobars LiChropreps
Si 60 (440 Â 37 mm) with n-heptane as mobile phase, a flow
of 20 mL/min and UV-detection at 254 nm yielding 1.20 g.
The purity was 84.8% (GC). The second chromatographic
The amount of 7 was added with 50 mL of 10% sodium
hydroxide solution. The reaction mixture was stirred for 4 h at
1001C. Then the solution was cooled in an ice bath and 30 mL of
37% hydrochloric acid were added adjusting the pH to 4. The
solid precipitate was filtered off, washed with 20 mL of water
and dried in an evacuated desiccator over blue gel yielding
4.98 g of crude 8. The radiochemical purity was 87.2%.
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D. Seidel and U. PleiX
purification was performed on Nucleosils
C 18 (7 mm, the solvent was adsorbed in a trap filled with platinum oxide and
250 Â 21 mm) with acetonitrile/water 45:55 (v/v) as mobile charcoal at the temperature of liquid nitrogen. The residue was
phase, a flow of 20 mL/min and UV-detection at 210 nm yielding dissolved in 1.0 mL of dichloromethane/methanol 4:1 and evapo-
0.698 g. As the purity was not sufficient, a third chromatography rated to dryness. This procedure was repeated three times with
was performed on Synergy Polar-RP 8 (4 mm, 150 Â 21.2 mm) 1.0mL of the same mixture of dichloromethane/methanol in order
with methanol/water 65:35 (v/v) as mobile phase, a flow of to remove the labile tritium. The dry crude product was taken up in
20 mL/min and UV-detection at 210 nm yielding 4349.9 MBq of 1 mL of acetonitrile. Due to the huge amount of labile radioactivity
5 (2.10 mmol calculated with the starting specific radioactivity the crude material was dissolved with a mixture of acetonitrile/
of 2.07 GBq/mmol) The radiochemical purity (radio-HPLC) was water and subsequently lyophilized twice resulting in 166 mCi
96.8%. Product formation and carbon-14 labelling were (6 GBq) of crude product 12. Chromatographic purification was
confirmed by GC/MS with m/z = 232 [M(14C)]1 and the chlorine performed on Phenomenexs Aqua C18 (5 mm, 250 Â 10 mm) with
isotope pattern. The radiochemical yield was 14.6% based on acetonitrile/0.05 M hydrochloric acid 65:35 (v/v) as mobile phase, a
the amount of [14C]paraformaldehyde. The excess of 25% for flow of 4 mL/min and UV-detection at 206 nm yielding inter-
the labelled paraformaldehyde in the chloromethylation was mediate 12 as hydrochloride. The amount was not determined.
disregarded in the yield calculation. The chemical yield was
18.4% based on bibenzyl.
4-({(4-Carboxybutyl)[2-(2-{[4-(2-phenylethyl)benzyl]oxy}phenyl)-
ethyl]amino}methyl)-[2-3H]benzoic acid (13)
4-{[(4-Carboxybutyl)(2-{2-4-phenethylphenyl[14C]methoxy]phenyl}-
ethyl)amino]-methyl}benzoic acid, [14C]cinaciguat (8)
To the hydrochloride of 12 a mixture of 1 mL of dioxane, 0.15 mL
of water and 0.15 mL of 1 M sodium hydroxide solution were
added and refluxed for 2 h. The pH of the mixture was adjusted
to pH 3 with 25% hydrochloric acid. The solution was
evaporated to dryness and the residue was dissolved with a
mixture of 1 mL acetonitrile/0.05 M hydrochloric acid 1:1.
Chromatographic purification was performed on Phenomenexs
Aqua C18 (5 mm, 250 Â 10 mm) with acetonitrile/0.05 M hydro-
chloric acid 50:50 (v/v) as mobile phase, a flow of 4 mL/min and
UV-detection at 206 nm yielding the 510 MBq of [3H]cinaciguat
as hydrochloride. The radiochemical purity was 499%. The
specific radioactivity was 200 GBq/mmol determined by mass
spectrometry under the following conditions; mass spectro-
meter: PE/Sciex/API III with MacIntosh Quadras 900, infusion
with acetonitrile/0.2% trifluoroacetic acid 9:1 (v/v), interface:
4.8 kV, nebulizing nitrogen pressure: 265 kPa, ion source in
positive ion mode, scan rate: 1.0 s per scan, data collection:
0.2 amu steps from 560 to 580 amu.
Intermediate 5 (2.10 mmol), intermediate 10 (1.042 g, 2.52 mmol)
and 2.5 mL of dry N-methylpyrrolidinone were heated to 55 1C.
Then 0.48 mL of sodium methoxide solution (30% in methanol,
2.52 mmol) was added. The reaction mixture was stirred for 1 h
at 551C. Additional compound 10 (0.26 g, 0.63 mmol) was added
and stirring at 551C was continued for 1 h. Additional sodium
methoxide solution (0.072 mL, 0.38 mmol) was added and
stirring at 551C was continued for 3 h. Then 3 mL of 10%
sodium hydroxide solution were added and stirring at 551C was
continued for 1 h. The milky suspension gave a clear yellow
solution after 30 min. Work-up was performed on a pre-packed
column Lobars LiChropreps RP–18 (440 Â 37 mm) with acet-
onitrile/2.5% ammonium chloride solution 40:60 (v/v) as mobile
phase, a flow of 20 mL/min and UV-detection at 230 nm yielding
0.792 g of 8. Due to a very near eluting by-product a second
chromatography was necessary. This was performed on
Synergys MAX-RP (4 mm, 250 Â 21.1 mm) with acetonitrile/
acetate buffer (8.2 g NaOAc1acetic acid/L H2O-pH 4.75)
30:70 (v/v) as mobile phase, a flow of 18 mL/min and UV-
detection at 240 nm yielding 0.792 g of 8. The radiochemical
purity and the chemical purity (230 nm) both were determined
to 99.8%. Radio-TLC revealed a purity of 98.7%. The specific
radioactivity of 3.60 MBq/mg (2039 MBq/mmol) was determined
by LC counting/UV quantification. The total radiochemical yield
was 9.6% based on the starting [14C]paraformaldehyde. LC-MS:
m/z 568 (M1H)1. 1H NMR (500 MHz, DMSO-d6) d = 7.84 (d,
J = 8.2 Hz, 2 H), 7.34 (d, J = 8.2 Hz, 2 H), 7.27 (t, J = 7.3 Hz, 4 H),
7.25–7.20 (m, 4 H), 7.20–7.10 (m, 3 H), 7.00 (d, J = 8.2 Hz, 1 H),
6.85 (t, J = 7.4 Hz, 1 H), 4.99 (s, 2 H), 3.62 (s, 2 H), 2.86 (s, 4 H),
2.77–2.70 (m, 2 H), 2.62–2.55 (m, 2 H), 2.43 (t, J = 6.8 Hz, 2 H), 2.12
(t, J = 7.1 Hz, 2 H), 1.55–1.33 (m, 4 H).
Stable labelling of cinaciguat
(2-{[4-(2-phenylethyl)benzyl]oxy}phenyl)methanol (15)
2-Hydroxybenzyl alcohol (1.76 g, 14.2 mmol), 1-[chloromethyl]-4-
(2-phenylethyl)benzene (14, 3.26 g, 14.2 mmol) and potassium
carbonate (1.95 g, 14.2 mmol) were dissolved/suspended in
240 mL of acetonitrile and refluxed for 16 h. Water (100 mL)
was added and exhaustive extraction was performed with
dichloromethane. The extracts were dried over sodium sulfate
and evaporated to dryness. The crude material was purified by
flash chromatography on silica gel with cyclohexane/ethanol 9:1
(v/v) as mobile phase resulting in 3.68 g (11.6 mmol) of 15 with a
purity of 499% (GC) and a yield of 82%.
This step was repeated under the same conditions.
Tritiation of cinaciguat
2-(Chloromethyl)phenyl 4-(2-phenylethyl)benzyl ether (16)
Methyl 4-({(5-methoxy-5-oxopentyl)[2-(2-{[4-(2-phenylethyl)benzyl]-
oxy}phenyl)ethyl]-amino}methyl)[2-3H]benzoate (12)
Thionyl chloride (1.38 mL, 19.0 mmol) was added to a solution of
15 (4.3 g, 13.5 mmol) in 70 mL of dichloromethane at 01C. The
To a solution of diester 11 (3.8 mg, 6.4 mmol) in 1.0 mL of mixture was stirred for 1 h at this temperature. Ice water (50 mL)
dichloromethane 3.5 mg of [(cod)Ir(pyridine)(tricyclohexylpho- was added and the organic phase was washed repeatedly with
sphine)] PF6 were added. This mixture was stirred in a tritium water and subsequently with sodium hydrogencarbonate
labelling apparatus12 for 1 h with 9.1 Ci (337 GBq) of tritium gas at a solution. The organic layer was dried over sodium sulfate and
partial pressure of about 118 kPa (1188 mbar). After freezing of the evaporated to dryness yielding 4.3 g (12.8 mmol) of 16. The
reaction mixture with liquid nitrogen the non-reacted tritium and purity was 496% (GC) and the yield was 95%.
J. Label Compd. Radiopharm 2010, 53 130–139
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

D. Seidel and U. PleiX
(2-{[4-(2-Phenylethyl)benzyl]oxy}phenyl)[1-13C,15N]acetonitrile (17)
for 22 h. The catalyst was removed by filtration over silica guhr.
The filtrate was evaporated yielding 1.88 g of 23 with a purity of
87% (GC).
Intermediate 16 (2.0 g, 5.9 mmol), 18-crown-6 (95 mg) and
potassium [13C,15N]cyanide (597 mg, 8.9 mmol) were submitted
in 25 mL of acetonitrile and refluxed for 3 h. The suspension was
filtered of and the filtrate was evaporated. The chromatographic
purification was performed on a pre-packed column Lobars
LiChropreps Si 60 (310 Â 25 mm) with cyclohexane/ethanol
98.5:1.5 (v/v) as mobile phase, a flow of 8 mL/min and
UV-detection at 254 nm yielding 1.57 g of 17 with a purity of
498% (GC) and a yield of 80%.
4-{[(4-Carboxybutyl)[2-(2-{[4-(2-phenylethyl)benzyl]oxy}phenyl)}-
[1-13C]ethyl]-[15N]amino][13C]methyl}benzoic acid (24)
Intermediate 23 (1.88 g), methyl 5-bromovalerate (694 mg,
3.5 mmol) and potassium carbonate (2.2 g, 16.0 mol) were
refluxed in 15 mL of acetonitrile for 17 h. Remaining solid was
filtered off and the filtrate was evaporated to dryness giving an
oily residue (1.67 g). Chromatographic purification was per-
formed on a Hibars RP18 (7 mm, 250 Â 25 mm) with acetonitrile/
0.1% triethylamine 90:10 (v/v) as mobile phase, a flow of
15 mL/min. and UV-detection at 230 nm yielding 529 mg of the
dimethyl ester of the labelled product. For saponification 2N
sodium hydroxide solution (14 mL) was added and the reaction
mixture was stirred for 14 h at a temperature of 1001C. Later
190 mg of sodium hydroxide and 1 mL of dioxane were added.
Stirring was continued for 2 h at 1301C. The almost clear
solution was filtrated and the filtrate was acidified with
concentrated hydrochloric acid to pH 2–3. The precipitate
was finely dispersed with the aid of ultrasonic, filtered off,
washed and dried. Purification was performed by conversion to
the hydrochloride. Therefore the crude product was suspen-
ded in 40 mL of a mixture of acetonitrile/0.1% hydrochloric
acid 55:45 (v/v). The solution was filtrated to remove small
amounts of insoluble solids. The filtrate was evaporated
2-(2-{[4-(2-Phenylethyl)benzyl]oxy}phenyl)[1-13C]ethan[15N]amine (18)
Raney-nickel (4 g) was washed several times with THF to remove
water, then added to a solution of 17 (830 mg, 2.5 mmol) in
45 mL of THF. Hydrogenation was performed in an autoclave at
a hydrogen pressure of 4.0 bar for 4 h. The mixture was filtered
off over silica guhr. The obtained clear product solution was
evaporated giving 830 mg (2.4 mmol) of 18. The purity was
494% (GC) and the yield was 96%.
This reaction was repeated under the same conditions.
Methyl 4-[13C]cyanobenzoate (20)
Methyl 4-bromobenzoate (2.15 g, 10.0mmol), potassium [13C]-
cyanide (726mg, 11.0mmol) and cuprous iodide (2.09g, 11mmol)
were submitted in 14mL of DMF and refluxed for 11h. The clear
solution was evaporated. Dichloromethane (150 mL) was added
to the residue yielding a suspension which was filtered off. The
filtrate was washed with 150 mL of water, dried over sodium
sulfate and evaporated to dryness resulting in 1.48g (9.1mmol) of
20. The purity was 497% (GC) and the yield was 73%.
until
a complete precipitation was observed. The solid
was filtered off, washed with water and dried in a desiccator
yielding 428 mg (0.71 mmol) of 24 as hydrochloride. Product
formation and labelling were confirmed by LC-MS with m/
z = 569 [M1H]1. The molecular ion of the non-labelled molecule
was not found in detectable amounts. The total yield was 18.6%
referred to the reaction of the compounds 18 and 21 to the
Schiff base.
Methyl 4-[13C]formylbenzoate (21)
Intermediate 20 (1.48 g, 9.1 mmol) and nickel-aluminium-alloy
(1.68 g) were refluxed in 29 mL of 75% formic acid for 3 h. The
alloy was filtered off, washed with ethanol and the filtrates were
evaporated. The residue was refluxed for 1.5h in 14.8mL of water
and 7 mL of methanol. The solution was evaporated to remove
the methanol, then 50mL water were added and extracted with
dichloromethane several times. The combined organic layers were
dried over sodium sulfate and evaporated. Chromatographic
purification was performed on a pre-packed column Lobars
LiChropreps Si 60 (440 Â 37 mm) with dichloromethane as mobile
phase, a flow of 17 mL/min and UV-detection at 254 nm yielding
0.63 g of 21. The purity was 499% and the yield was 42%.
Acknowledgements
The authors would like to thank Yvonne Schoof, Stefan Noesel,
Udo Balzer and Gregor Bredlich for skilfully performing the
syntheses. Special thanks go to Harald Schnorbus and
Mark Breyer for performing GC, HPLC and MS analyses and to
Dr. Peter Schmitt and Detlef Bauer for the NMR spectra.
References
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[15N]imino}-[13C]methyl]benzoate (22)
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J. Label Compd. Radiopharm 2010, 53 130–139

D. Seidel and U. PleiX
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J. Label Compd. Radiopharm 2010, 53 130–139
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org