Synthesis of [14C]-labelled vardenafil hydrochloride and metabolites

Article abstract of DOI:10.1002/jlcr.736

For studies of pharmacokinetics and drug metabolism of the new orally active, selective phosphodiesterase type V (PDE V) inhibitor vardenafil (Levitra), the ¹⁴C-labelled version was synthesised. Starting from the cyanation of 2-iodophenol with

Full text of DOI:10.1002/jlcr.736

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 1019–1032.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.736
Research Article
Synthesis of [14C]-labelled vardenafil hydrochloride
and metabolites
y
.
D. Seidel*, P. Brehmer, Y. Schoof, U. Weinberg, U. Niewohner and
M. Nowakowski
Bayer AG, HealthCare Division, Wuppertal D-42096, Germany
Summary
For studies of pharmacokinetics and drug metabolism of the new orally active,
selective phosphodiesterase type V (PDE V) inhibitor vardenafil (Levitra1),
the 14C-labelled version was synthesised. Starting from the cyanation of 2-
iodophenol with K14CN, an 8-step synthesis led to two batches with 0.727 g
(2.857 GBq) and 2.199 g (5.497 GBq) of [triazinone-14C]vardenafil hydrochlo-
ride with different specific radioactivities. The label was located in position 2 of
the imidazotriazinone moiety. Several carbon-14 labelled metabolites were
synthesised as reference compounds for metabolism studies. Copyright
# 2003 John Wiley & Sons, Ltd.
Key Words: Levitra1; PDE V inhibitor; carbon-14; cyanation; nucleophilic
substitution; regioselective chlorosulfonation; semi-preparative chromato-
graphy
Introduction
Vardenafil (Levitra1) is a new orally active, selective phosphodiesterase
type V (PDE V) inhibitor marketed for the treatment of male erectile
dysfunction. It demonstrates excellent in vivo activity at the subnano-
molar level.1
*Corresponding author: D. Seidel, Bayer AG, HealthCare Division, Wuppertal D-42096, Germany
y Deceased September 2001.
Copyright # 2003 John Wiley & Sons, Ltd.
Received 9 April 2003
Revised 23 May 2003
Accepted 26 May 2003

1020
D. SEIDEL ET AL.
O
O
S
HN
N
N
N
O
N
O
HCl
N
Vardenafil hydrochloride
For studies of pharmacokinetics and drug metabolism of vardenafil,
the version with a metabolically stable carbon-14 label was required. In
contrast to other piperazine containing molecules effective and quickly
performed labelling in the piperazine moiety did not give a suitable
label, because the piperazine moiety turned out to be the major site of
metabolic degradation.2 An 8-step radiosynthesis was necessary to
insert the carbon-14 label in a stable position in the imidazotriazinone
system of the molecule.3
For evaluation and interpretation of the metabolic studies several
carbon-14 labelled metabolites were synthesized. The syntheses initiated
from an appropriate intermediate of the main radiosynthesis.
Results and discussion
2-Hydroxybenzonitrile is the starting compound for the common
vardenafil synthesis.4 So we investigated the formation of the labelled
version for introducing the carbon-14 label. The corresponding
synthesis of the non-labelled compound has been described.5 The
authors used cuprous cyanide in the high boiling solvent DMF. Starting
from readily available potassium [14C]cyanide and 2-iodophenol we
performed the reaction successfully in N-methylpyrrolidinone (NMP)
at a temperature of 1808C with the less reactive reagent system
K14CN/CuI (reaction scheme 1). The subsequent phenol ether forma-
tion was carried out by conventional reaction with ethyl iodide and
potassium carbonate. The 2-hydroxybenzo[14C]nitrile was introduced
into the etherification reaction without isolation and purification. The
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1019–1032

SYNTHESIS OF [14C]-LABELLED
1021
Reaction Scheme 1.
resulting 2-ethoxybenzo[14C]nitrile (III) was then purified chromato-
graphically on silica gel. The desired amidine derivative IV was obtained
by reaction with trimethylaluminium and ammonium chloride. It was
isolated in good yield as the hydrochloride as described in the
literature.4 The above steps were carried out in three separate batches
due to the high level of radioactivity employed (starting activity 67GBq).
Hydrazinolysis to the intermediate V was performed with hydrazine
hydrate at a maximum temperature of 08C within some minutes. The
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1019–1032

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D. SEIDEL ET AL.
2-ethoxybenzene[14C]carboximidohydrazide (V) was subsequently re-
acted with the enolester VI to form the triazinone derivative VII. VI was
synthesised via the Dakin-West reaction according to Hartley et al.6
who described also some similar condensations. Optimisation experi-
ments showed that the highest yields were achieved when both V and VI
were used without isolation and purification. The obtained VII was
purified by low pressure chromatography on silica gel due to the high
amounts of by-products formed and to obtain a better yield in the
subsequent cyclisation step.
Cyclisation of VII with phosphorus oxychloride gave the imidazo-
triazinone system VIII in good quality and yield. Due to its stability an
aliquot of the compound was separated as starting compound for the
synthesis of the carbon-14 labelled metabolites. Most of it was
Reaction Scheme 2.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1019–1032

SYNTHESIS OF [14C]-LABELLED
1023
converted to the sulfochloride derivative IX with chlorosulfonic acid.
The reaction proceeded under very mild conditions with complete
regioselectivity at the 5-position which seemed to be typical for this
substitution pattern.7 Final coupling with N-ethylpiperazine afforded
the free base of X which was converted to [14C]vardenafil hydrochloride
by addition of a defined amount of hydrochloric acid. Purification was
performed by recrystallization. Most of the product was diluted with
non-labelled vardenafil in order to reduce the specific radioactivity for
use in the ADME studies and to ensure better stability against
radiolysis.
To aid in the interpretation of metabolic studies with
[triazinone-14C]vardenafil hydrochloride some important metabolites
were required as carbon-14 labelled reference compounds. The
syntheses of the derivatives with different functionalisation of the
sulfonyl group started from the labelled intermediate VIII (reaction
scheme 2). Sulfochloride IX was obtained from freshly purified VIII by
a known procedure and used as a key intermediate for coupling with the
different amines and water, respectively (reaction scheme 2).
The N-oxide metabolite [14C]M-2 was synthesized directly by
oxidation of the free base of X with 3-chloroperbenzoic acid (reaction
scheme 1).
Most of the labelled metabolites had to be purified chromatographi-
cally.
Experimental
General methods
1
The H NMR spectra were recorded on a Bruker DRX 400 nuclear
magnetic resonance spectrometer. Radiochemical purities were deter-
mined with the GC system HP 5890 equipped with a radioactivity
detector FHT 7000. HPLC analyses of the final products were
performed on a HP 1050 Series II instrument. Nucleosil1 120 C-18
was used as stationary phase and acetonitrile/phosphate buffer (pH 6.5)
as mobile phase. Radioactivity and UV signals (radioactivity detector
Ramona1 92) were recorded. Radiochemical counting was performed
on a liquid scintillation analyzer TRI-CARB1 2500 TR using Ultima
GoldTM cocktail. GC/MS detection was performed on a HP 5890
equipped with MS HP 5970. LC mass spectrometric analysis was
performed on a PE/Sciex API III mass spectrometer with MacIntosh
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1019–1032

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D. SEIDEL ET AL.
Quadra1 900. Semi-preparative chromatography was performed with
WellChrom Maxi-Star1 K 1000, Knauer variable wavelength monitor
and Merck/Hitachi D-2500A Chromato-Integrator.
Labelling procedure
2-Hydroxy-benzo[14C]nitrile, (II). Potassium [14C]cyanide was obtained
from Amersham International plc with a specific radioactivity of 30
MBq/mg. The chemical purity was 99.3%.
An aliquot of 28.9 GBq (13.7 mmol) of K14CN was suspended in
17 ml of 1-methyl-2-pyrrolidinone (NMP) and 2.64 g (12 mmol) of 2-
iodophenol together with 2.53 g (13.3 mmol) of cuprous iodide were
added. The reaction mixture was stirred for 5 h at 1808C, then the
conversion was checked as complete by GC. Two additional syntheses
were performed in the same manner starting with 18.42 and 19.6 GBq of
K14CN, respectively. The product containing mixtures were used
without isolation or purification in the subsequent etherification.
2-Ethoxy-benzo[14C]nitrile, (III). Potassium carbonate (7.81 g, 56.5 mmol)
and ethyl iodide (1.37 ml, 17.1 mmol) were added to the mixture of
crude II of the first batch. Then 35 ml of acetone was added and the
reaction mixture was heated at 808C for 70 min. The acetone was
evaporated off and 150 ml of water and 250 ml of dichloromethane were
added to the residue. A precipitate was removed by filtration and the
filter cake was washed four times with 20 ml of dichloromethane. The
organic layer of the mother liquor was separated and evaporated to
dryness. The oily residue contained III and NMP. Two additional
syntheses were performed in the same manner with the second and third
batch of II. The residues of the three 2-ethoxy-benzo[14C]nitrile batches
were combined (total of 38.4 g), dissolved in 35 ml of dichloromethane
and purified chromatographically in 17 runs on a column Lobar1 Si 60
size C using CH2Cl2/CH3OH 9:1 as eluent, a flow of 15 ml/min. and UV
detection at 230 nm.The product containing fractions were combined
and evaporated.
Yield: 6.14 g of compound III, radiochemical purity (radio-GC):
95%, chemical purity (GC): 65.5%. Residual NMP was detected as
30%. With this value the yield of III was calculated as 27 mmol which
corresponds to 85% of theoretical related to K14CN which was used in
slight excess. The structure was confirmed by GC/MS with m/z = 149
[14C-M]+.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1019–1032

SYNTHESIS OF [14C]-LABELLED
1025
2-Ethoxy-benz[14C]amidine hydrochloride, (IV). Ammonium chloride
(2.5 g, 46.7 mmol) was suspended in 32 ml of toluene under an argon
atmosphere. The mixture was cooled in an ice bath and 24 ml (48 mmol)
of trimethylaluminium (2 M in toluene) was added dropwise. The
mixture was stirred without further cooling until gas formation had
ceased. Then an aliquot of III (2 g, 8.8 mmol) was added and the
reaction mixture was stirred for 9 hs under reflux. After cooling to room
temperature 1.6 g of silica gel was added and excess trimethylaluminium
quenched by careful addition of 16 ml of methanol with ice bath
cooling. The resulting suspension was diluted with 16 ml of dichlor-
omethane and stirred without further cooling until hydrogen formation
had ceased. The solid was removed by filtration and washed four times
with 8 ml of methanol. After the addition of 5 drops of 37%
hydrochloric acid the mother liquor was evaporated, the residue was
dissolved in 24 ml of CH2Cl2/CH3OH (9:1) and filtered off from a small
amount of insoluble solid. The solution was evaporated to dryness and
the semi-solid residue was dissolved in 3 ml of CH2Cl2/CH3OH 9:1.
Then 1 ml of 37% hydrochloric acid was added and the mixture was
evaporated again. The residue was suspended in 25 ml of diethyl ether
and stirred for 30 min at room temperature. The clear supernatant was
decanted and the residue washed twice with 3 ml of diethyl ether and
dried under vacuum.
Yield: 2.43 g of compound IV.
The rest of compound III was converted in two separate syntheses in
the same manner.
N-{1-[3-(2-Ethoxy-phenyl)-5-oxo-4,5-dihydro-1H-[3-14C][1,2,4]triazin-6-
ylidene]-ethyl}-butyramide, (VII). An aliquot of compound IV (3.15 g)
was dissolved in 16 ml of anhydrous ethanol. The mixture was cooled in
an ice bath and 0.79 ml (15.3 mmol) of hydrazine hydrate was added
over the course of 15 min. Ammonium chloride was precipitated. Then
19 g of oxalic acid 2–butyrylamino-1-ethoxycarbonyl-propenyl ester
ethyl ester (VI), diluted with 10 ml of ethanol, was added. The reaction
mixture was stirred for 30 min at room temperature and subsequently
for 5 hs under reflux. The solvent was removed by evaporation and the
oily residue was partitioned between 50 ml of dichloromethane and
20 ml of water. The organic layer was evaporated to dryness and the
residue dried azeotropically twice by addition and evaporation of 25 ml
of ethanol. The crude VII (16.55 g) was dissolved in 33 ml of
dichloromethane and purified chromatographically in 21 runs on a
Copyright # 2003 John Wiley & Sons, Ltd.
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1026
D. SEIDEL ET AL.
column Lobar1 Si 60 size C using CH2Cl2/CH3OH (95:5) as eluent, a
flow of 25 ml/min. and UV detection at 230 nm.The product containing
fractions were combined and evaporated.
Yield: 2.35 g (6.48 mmol) of compound VII as a thicky yellow oil,
chemical purity (GC): 91%.
A second batch started from 3.15 g of IV and gave 1.99 g (5.55 mmol)
of compound VII, chemical purity (GC): 92%.
2-(2-Ethoxy-phenyl)-5-methyl-7-propyl-3H-[2-14C]imidazo[5,1-f]triazin-
4-one, (VIII). The first batch of compound VII (2.35 g) was dissolved
in 27 ml of dichloroethane. Phosphoryl chloride (1.35 ml, 14.37 mmol)
was added and the reaction mixture was stirred for 2 hours under reflux.
Then it was evaporated, the residue was dissolved in 25 ml of
dichloromethane, washed twice with 10 ml of saturated sodium
bicarbonate solution and subsequently with water, dried over sodium
sulphate and evaporated carefully due to vigorous foaming of the
product.
Yield: 1.717 g (5.23 mmol) of compound VIII, chemical purity (GC):
92%, radiochemical purity (radio-GC): 86%.
A second batch starting from 1.99 g of VII, gave 1.69 g (4.93 mmol) of
compound VIII, chemical purity (GC): 91%.
Both batches were combined, dissolved in dichloromethane and
diluted to exactly 25 ml.The total radioactivity was determined by LS
counting to be 17.953 GBq. This amount corresponds to 26.8% of the
starting radioactivity. With a specific radioactivity of 2.035 GBq/mmol
a product content of 8.82 mmol (2.75 g) was calculated.
4-Ethoxy-3-(5-methyl-4-oxo-7-propyl-3,4-dihydro-[2-14C]imidazo[5,1-
f][1,2,4]-triazin-2-yl)-benzene-sulfonyl chloride, (IX). An aliquot of
compound VIII (14.31 GBq, 2.19 g, dissolved in 20 ml of dichloro-
methane) was cooled in an ice bath and 3.5 ml (52.5 mmol) of
chlorosulfonic acid was added dropwise whilst cooling. The mixture
was stirred for one hour in an ice bath and subsequently for 16 hs at
room temperature. Then 12 ml of ice cold water was added carefully
whilst cooling. The organic layer was washed four times with 8 ml of
cold water, dried over sodium sulphate and evaporated to dryness under
reduced pressure.
Yield: 2.95 g of compound IX.
The crude product was used without further purification in the next
step.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1019–1032

SYNTHESIS OF [14C]-LABELLED
1027
[14C]Vardenafil hydrochloride, 2-[2-ethoxy-5-(4-ethyl-piperazine-1-sul-
fonyl)-phenyl]-5-methyl-7-propyl-3H-[2–14C]imidazo[5,1-f][1,2,4]tria-
zin-4-one hydrochloride, (X). The labelled IX (2.95 g) was dissolved in
14 ml of dichloromethane and 1.06 g (8.25 mmol) of N-ethyl-diisopro-
pylamine and subsequently a solution of 0.82 g (7.18 mmol) of N-ethyl-
piperazine in 4 ml of dichloromethane was added. The reaction mixture
was stirred for 16 hs at room temperature. The solution was washed
three times with 12 ml of water, twice with 12 ml of 5% sodium
bicarbonate solution and finally with 12 ml of water. The organic layer
was dried over sodium sulphate and evaporated to dryness giving 2.99 g
of crude X.
An aliquot of 2.8 mg was dissolved in acetonitrile and diluted exactly
to 10 ml. The content of pure X was determined as 2.25 mg by UV
quantification using defined non-labelled reference compound. From
this result the total amount of pure X was calculated as 2.4 g
(4.91 mmol). The total radioactivity was 10.782 GBq which corresponds
to a total yield of 20.1%, based on the starting K14CN.
For formation of the desired hydrochloride the crude product
was dissolved in 17 ml of boiling acetone and 0.64 ml (4.93 mmol) of
25% hydrochloric acid was added. The product crystallised during
cooling to room temperature. An additional 2 ml of acetone was added
and the suspension stored for 65 hs in a refrigerator. The product was
filtered off, washed three times with 6 ml of acetone (a remarkable
solubility was observed) and dried in an evacuated desiccator over
Blaugel.
Yield: 0.73 g of [triazinone-14C]vardenafil hydrochloride (1.38 mmol),
radiochemical purity: ꢀ 97% by radio-HPLC, chemical purity
(HPLC): ꢀ 99%.
From the above mother liquor and washing liquors three batches of
free base [triazinone-14C]vardenafil were isolated by concentration,
dissolution in water and addition of saturated sodium bicarbonate
solution. The combined amounts (767 mg+392 mg+418 mg) were
purified by recrystallisation from 17.5 ml of acetone/water (1:1) giving
1.28 g (2.61 mmol) of [triazinone-14C]vardenafil (free base), radio-
chemical purity: ꢀ 98% by radio-HPLC, chemical purity (HPLC):
ꢀ 96%.
For formation of the desired hydrochloride and reduction of
the specific radioactivity the total amount was dissolved in 80 ml
of methanol and 0.815 g (1.55 mmol) of non-labelled vardenafil
hydrochloride and 2.55 ml of 1 M hydrochloric acid (2.55 mmol)
Copyright # 2003 John Wiley & Sons, Ltd.
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1028
D. SEIDEL ET AL.
were added. Very small amounts of insoluble components were
removed by filtration and the solution was used for characterisation
and storage.
The total radioactivity was 5.5 GBq. The weight was calculated as
2.199 g (4.17 mmol) of [triazinone-14C]vardenafil hydrochloride with a
specific radioactivity of 2.50 MBq/mg. The radiochemical purity was
determined as ꢀ 97%; the chemical purity (HPLC) was ꢀ 97%.
Determination of the specific radioactivity
The specific radioactivity of both product batches was determined by
two different methods. A weighed amount of the labelled compound
was dissolved in water and the total radioactivity was determined by LS
counting. Secondly the concentration of [triazinone-14C]vardenafil
hydrochloride in solution was determined by HPLC/UV and quantified
using non-labelled reference compound. The specific radioactivity of
batch 1 (non-diluted) was 2.063 GBq/mmol (3.93 MBq/mg) and of
batch 2 (diluted) as 1.313 GBq/mmol (2.50 MBq/mg).
The total radioactivity of 8.354 GBq (2.87 GBq of batch 1 and
5.5 GBq of batch 2) corresponds to a yield of 16% of the theory, based
on the starting K14CN.
e
O
N
j
c
O
S
HN
14C
N
N
k
f
m
l
d
O
N
a
HCl
O
b
N
g
h
i
NMR (DMSO-d6) d (ppm); 0.93 (m, 3H, a), 1.20 (t, 3H, b), 1.33 (t,
3H, c), 1.74 (m, 2H, d), 2.50 (s, 3H, e), 2.80 (multiplets, 4H, f+g),
3.09+3.42 (multiplets, 6H, i+h), 3.81 (d, 2H, g’), 4.22 (q, 2H, j), 7.41
(d, 1H, k), 7.93 (s+d, 2H, l+m), 10.68 (broad, 1H, HCl), 11.84 (s, 1H,
NH).
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1019–1032

SYNTHESIS OF [14C]-LABELLED
1029
Metabolite syntheses
2-[2-Ethoxy-5-(4-ethyl-4-oxo-piperazine-1-sulfonyl)-phenyl]-5-methyl-
7-propyl-3H-[2–14C]imidazo-[5,1–f][1,2,4]triazin-4-one,[14C]M-2,(XI).
The free base [triazinone-14C]vardenafil was liberated from 25.7 mg
(0.049 mmol) of X with sodium bicarbonate solution, extracted with
dichloromethane and isolated by evaporation. The solid was dissolved
in 1.5 ml of dichloromethane and
a
solution of 20 mg of
3-chloroperbenzoic acid (50–60%) in 0.5 ml of dichloromethane
was added. The reaction mixture was stirred for 5 hs at room
temperature, then the solvent was removed by passing argon over the
surface of a warm solution. The remaining solid was dissolved in 2 ml of
acetonitrile and purified in 4 runs by semipreparative HPLC on
Nucleosil1 100 C 18 (125 ꢁ 16 mm; 7 mm) with the eluent acetonitrile/
0.02% NEt3-solution 50:50 (v/v), a flow of 5 ml/min. and UV detection
at 230 nm. The combined product containing fractions were evaporated
to dryness.
Yield: 18 mg (0.035 mmol) of XI, radiochemical purity = 98% by
radio-HPLC, chemical purity (HPLC) = 99%.
The specific radioactivity was calculated from the ratio of the
molecular ion peaks [14C–M+H]+ = 394 and [12C-M+H]+ = 392 as
2.034 GBq/mmol (4.03 MBq/mg).
2-[2-Ethoxy-5-(piperazine-1-sulfonyl)-phenyl]-5-methyl-7-propyl-
3H-[2–14C]imidazo-[5,1–f][1,2,4]triazin-4-one, [14C]M-1,(XII). The la-
belled intermediate IX was freshly prepared from the above
aliquot of VIII. Piperazine (69 mg, 0.8 mmol) and N-ethyl-diisopropy-
lamine (70 ml, 0.4 mmol) were dissolved in 1.4 ml of dichloromethane.
A solution of 129 mg of the sulfochloride IX in 1.5 ml of dichlor-
omethane was added over a course of 5 min. at room temperature.
The mixture was stirred for 16 hours at room temperature. Then 2 ml
of water was added, the organic layer separated, washed with 2 ml
of water, dried over sodium sulphate and evaporated. The crude
XII (148 mg) was dissolved in 7 ml of acetonitrile and purified in
14 runs by semipreparative HPLC on Nucleosil1 100 C 18
(125 ꢁ 16 mm; 7 mm) with the eluent acetonitrile/0.1% ammonia
30:70 (v/v), a flow of 6 ml/min. and UV detection at 230 nm.
The combined product containing fractions were concentrated to
approximately 15 ml. The product was extracted three times with
10 ml of dichloromethane. The combined extracts were evaporated to
dryness.
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1030
D. SEIDEL ET AL.
Yield: 91 mg (0.198 mmol) of XII, radiochemical purity = 98% by
radio-HPLC, chemical purity (HPLC) = 97%.
The specific radioactivity was determined by the above mentioned
gravimetric method as 2.023 GBq/mmol (4.39 MBq/mg). The structure
was confirmed by electrospray-MS with m/z = 467 [14C-M+H]+ and
1
by H NMR.
d
O
N
f
b
O
S
HN
14C
N
N
g
e
i
h
c
O
a
N
O
HN
e
e
NMR (DMSO-d6) d (ppm); 0.92 (m, 3H, a), 1.31 (t, 3H, b), 1.72 (m,
2H, c), 2.49 (s, 3H, d), 2.71+2.80 (m+m, 10H, e), 4.20 (q, 2H, f), 7.38
(d, 1H, g), 7.80 (s, 1H, h), 7.83 (d, 1H, i).
N-(2-Aminoethyl)-4-ethoxy-3-(5-methyl-4-oxo-7-propyl-3,4-dihydro[2–14C]
imidazo-[5,1–f][1,2,4]triazin-2-yl)benzenesulfonamide, [14C]M-5, (XIII).
XIII was prepared according to the synthesis of XII using 16.7 ml
(0.25 mmol) of 1,2-diaminoethane and 22.6 ml (0.13 mmol) of N-ethyl-
diisopropylamine in 0.5 ml of dichloromethane and a solution of 41 mg
of freshly prepared sulfochloride IX in 1 ml of dichloromethane. The
eluent of the chromatography was changed to acetonitrile/0.1%
ammonia 22:78. The isolated product (40 mg) was digested in 7.5 ml
of methanol and some insoluble components were removed by filtration.
The filtrate was evaporated.
Yield: 27.6 mg (0.064 mmol) of XIII, radiochemical purity = 97% by
radio-HPLC, chemical purity (HPLC) = 97%.
The specific radioactivity was determined by the above mentioned
gravimetric method as 2.017 GBq/mmol (4.64 MBq/mg). The structure
was confirmed by electrospray-MS with m/z = 437 [14C-M+H]+.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1019–1032

SYNTHESIS OF [14C]-LABELLED
1031
4-Ethoxy-N-(2-ethylamino-ethyl)-3-(5-methyl-4-oxo-7-propyl-3,4-dihy-
dro[2–14C]imidazo-[5,1–f][1,2,4]triazin-2-yl)benzenesulfonamide,
[14C]M-4, (XIV). XIV was prepared according to the synthesis of XII
using 26.3 ml (0.25 mmol) of 2-ethylamino-ethylamine, 22.6 ml
(0.13 mmol) of N-ethyl-diisopropylamine and 41 mg of freshly prepared
sulfochloride IX. The eluent of the chromatography was varied to
acetonitrile/0.1% ammonia 20:80. The isolated product was digested in
7.5 ml of methanol and some insoluble components were removed by
filtration. The filtrate was evaporated.
Yield: 14.7 mg (0.032 mmol) of XIV, radiochemical purity = 97% by
radio-HPLC, chemical purity (HPLC) = 97%.
The specific radioactivity was determined by the above mentioned
gravimetric method as 1.964 GBq/mmol (4.24 MBq/mg). The structure
was confirmed by electrospray-MS with m/z = 465 [14C-M+H]+.
4 - Ethoxy - 3-(5-methyl-4-oxo-7-propyl-3,4-dihydro-[2-14C]imidazo[5,1-
f][1,2,4]triazin-2-yl)-benzenesulfonamide, [14C]M-7, (XV). Due to the
fact that the very pure intermediate VIII provides pure sulfochloride IX,
which gives pure compounds XV and XVI, the crude starting
intermediate VIII (378 mg) was purified chromatographically on
Lobar1 Si 60 (310 ꢁ 25 mm) with the eluent dichloromethane/methanol
95:5 (v/v), a flow of 6 ml/min. and UV detection at 254 nm (14 runs)
giving 303 mg of pure VIII, radiochemical purity ꢀ97% by radio-
HPLC, chemical purity (HPLC) ꢀ97%.
For formation of the desired amide 0.5 ml of 25% ammonia solution
was added to 33 mg (0.08 mmol) of fresh sulfochloride IX, prepared
from purified VIII. The mixture was stirred for 2 h at room temperature,
then water and ammonia were removed by passing argon over the
surface of a warm solution. The solid residue was suspended in 1 ml of
ice-cold water, filtered off, washed twice with 1 ml of ice-cold water and
dried for 18 hours in an evacuated desiccator over Blaugel.
Yield: 26.6 mg (0.068 mmol) of XV, radiochemical purity = 96% by
radio-HPLC, chemical purity (HPLC) = 95%.
The specific radioactivity was calculated from the ratio of the
molecular ion peaks [14C-M+H]+ = 394 and [12C-M+H]+ = 392 as
2.100 GBq/mmol (5.37 MBq/mg).
4-Ethoxy-3-(5-methyl-4-oxo-7-propyl-3,4-dihydro-[2-14C]imidazo[5,1-
f][1,2,4]triazin-2-yl)-benzenesulfonic acid, [14C]M-6, (XVI). Sulfochlo-
ride IX from the above batch (33 mg, 0.08mmol) was dissolved in 0.5 ml
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1019–1032

1032
D. SEIDEL ET AL.
of acetonitrile and 1 ml of water was added. The mixture was stirred for
18hs at room temperature, then the solvents were removed by passing
argon over the surface of a warm solution.
Yield: 28 mg (0.071 mmol) of XVI, radiochemical purity = 97% by
radio-HPLC, chemical purity (HPLC) = 97%.
The specific radioactivity was calculated from the ratio of the
molecular ion peaks [14C–M+H]+ = 395 and [12C-M+H]+ = 393 as
2.100 GBq/mmol (5.35 MBq/mg).
Acknowledgements
We thank H. Schnorbus for performing the GC, HPLC analyses, and
MS measurements and Dr. P. Schmitt for performing the NMR
analyses.
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Copyright # 2003 John Wiley & Sons, Ltd.
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