Synthesis of [3H] vardenafil, Levitra, using a new labeling technique

Article abstract of DOI:10.1002/jlcr.782

Tritium was introduced into the new orally active, selective phosphodiesterase type V (PDE V) inhibitor vardenafil (Levitra), by reduction of a suitable amide precursor with freshly prepared lithium aluminum tritide. A specific activity of 52.7 Ci/mmol (1

Full text of DOI:10.1002/jlcr.782

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 1241–1247.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.782
Research Article
Synthesis of [3H] vardenafil, Levitra1, using a new
labeling technique
U. Pleiss*
Drug Metabolism and Isotope Chemistry, Bayer Health Care AG,
42096 Wuppertal, Germany
Summary
Tritium was introduced into the new orally active, selective phosphodiesterase
type V (PDE V) inhibitor vardenafil (Levitra1), by reduction of a suitable
amide precursor with freshly prepared lithium aluminum tritide. A specific
activity of 52.7 Ci/mmol (1.95 TBq/mmol) was achieved. In order to overcome
the usual technical difficulties during the preparation of complex tritides a new
and easy labeling technique which has considerable potential for various tritia-
tion procedures, was developed. Copyright # 2003 John Wiley & Sons, Ltd.
Key Words: Levitra1; PDE V inhibitor; complex tritide; tritium labeling
technique
Introduction
Receptor binding studies of vardenafil,1 Levitra1, a phosphodiesterase
type V inhibitor against erectile dysfunction, required the synthesis of
the tritium labeled compound 4. The complete synthesis is depicted in
Scheme 1. For that very reason a special amide precursor 3 was
synthesized in order to reduce the amide to the tertiary amine with
lithium aluminum tritide, which was freshly prepared starting from
butyl lithium.2 Hydrogenation of butyl lithium with tritium in the
presence tetramethylethylenediamine (TMEDA) yielded lithium tritide,
*Correspondence to: U. Pleiss, Drug Metabolism and Isotope Chemistry, Bayer Health Care AG,
Aprather Weg 18a, D-42096 Wuppertal, Germany. E-mail: Ulrich.Pleiss.UP@bayer-ag.de
Copyright # 2003 John Wiley & Sons, Ltd.
Received 14 July 2003
Revised 18 August 2003
Accepted 19 August 2003

1242
U. PLEISS
O
O
N
O
HN
O
HN
N
N
SOCl2
N
N
N
Cl
S
O
1
HO
S
O
1a
O
O
H
N
1.) TMEDA
2.) AlBr3
BuLi + 3H2
LiAl3H4
N
2
O
N
O
O
O
HN
N
O
HN
N
N
N
N
3H
x HCl
N
N
S
O
3
3H
O
N
N
S
O
4
O
O
Scheme 1. Synthesis of [3H]vardenafil
which was subsequently reacted with aluminum tribromide, yielding
lithium aluminum tritide.
During this process a gas mixture of tritiated butane and excess tritium
gas and solvents (mainly an ether/cyclohexane mixture) had to be
removed. For disposal of this radioactive mixture, the reaction vessel is
usually frozen in liquid nitrogen and the remaining tritium gas absorbed
on a tritium uranium getter and the solvent vacuum transferred to a
cooled trap.3 This procedure has the disadvantage that the remaining
tritium gas contains traces of [3H]butane and solvents which inhibit
reabsorption of the tritium gas on the getter. Furthermore, the disposal of
highly radioactive solvents is difficult. Furthermore, additional tritium
was formed due to the decomposition of the excess lithium aluminum
tritide. This tritium gas contained hydrogen and also traces of solvents. A
safe disposal of this radioactive waste is also difficult.
To overcome these problems we developed a special trap made from
stainless steel (Figure 1) which is filled with a mixture of platinum oxide
and charcoal. The trap is evacuated before the start of the labeling
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1241–1247

SYNTHESIS OF [3H]VARDENAFIL
1243
3
5
3
4
1
2
Figure 1. Tritium labeling apparatus: 1, steel trap; 2, two-neck reaction vessel; 3,
connections to manometers, 4 reservoir, 5 connection to tritium storage
process and is cooled with liquid nitrogen during the synthesis. The
capacity is big enough to absorb all volatile components including
tritium gas, solvents and even the air to evacuate the labeling apparatus.
Pumps for the vacuum were not required during the whole synthesis.
After the labeling all radioactive volatiles, including the solvents from
the exchange of the labile tritium, were trapped in the steel container.
The trap is connected via 1/16 in steel capillary and Swagelok1
connectors to the labeling apparatus. At the end of the labeling the
trap was disconnected and closed tightly by cutting the capillary with
diagonal pliers. The tightness of the trap after cutting was tested by
many experiments conducted before. But for safety reasons, the head of
the trap was additionally sealed with epoxide resin. We tried to use
a 1/8 in capillary because the diameter would have been better for the
transfer into the trap but this capillary could not be closed tightly during
the cutting process.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1241–1247

1244
U. PLEISS
Here we describe in considerable detail the development of this new
technology which is likely to have wide application for various tritiation
procedures. The example chosen is that of the tritiation of vardenafil.
Results and discussion
In order to introduce tritium into vardenafil a suitable starting
compound had to be synthesized. The sulfonic acid 1 was reacted with
thionyl chloride yielding the sulfochloride 1a as intermediate which was
directly converted with N-acetylpiperazine 2 to give the starting material
3 for the radiosynthesis. The acetyl moiety was reduced with freshly
prepared lithium aluminum tritide [1] to the ethyl functionality.
The total radioactivity was determined by LS counting to be 15.5 mCi
(573 MBq). The distribution of tritium was determined by LC–MS
(Table 1).
According to this distribution the specific activity was calculated as
52.7 Ci/mmol (1.95 TBq/mmol). The MS fragmentation pattern corre-
sponds to that for the non-labeled reference compound. The chemical
and radiochemical purities were both >99.0%. The low yield was most
likely caused by traces of moisture in one of the solvents because in pre-
experiments with deuterium significantly better yields were achieved.
However, this yield was sufficient for the further studies. The most
important result of this experiment was the safe handling of tritium and
its volatile compounds during this multistep synthesis. The whole
volatile radioactivity during the labeling was completely captured in a
tightly closed stainless steel trap which is accepted by official deposits
for radioactive waste in Germany.
Experimental
General
All reagents were purchased from commercial suppliers and were used
without further purification. The cylindrical trap made from stainless
Table 1.
m/z
489
491
493
Ion
Percentage
[M+H]+
1.8%
[3HM+H]+
15.2%
[3H2M+H]+
83.0%
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1241–1247

SYNTHESIS OF [3H]VARDENAFIL
1245
steel with a wall thickness of 1.6 mm, and prepared at Bayer AG was
12 cm long and had an inner diameter of 2.4 cm which gives a total
volume of about 54 ml. This trap was filled with a mixture of charcoal
(5 g) (Merck AG, Darmstadt, Germany) and platinum oxide (250 mg)
(Sigma-Aldrich, Taufkirchen, Germany). A metal frit U10C-413, 10 mm
(ass-CHEM GmbH, Bad Homburg, Germany) in the Swagelok1
connector ensured that no charcoal or catalyst powder could escape
from the trap during the evacuation. The trap is connected via its 1/4 in
tube (3 cm long), the Swagelok1 connector and the 1/16 in steel
capillary with an inner diameter of 1 mm to the labeling apparatus. For
cutting the capillary an ordinary diagonal pliers was used. The epoxide
was UHU plus1 (UHU Vertrieb GmbH, Buehl, Germany) consisting of
two components which were mixed short before. The epoxide is solid
after 5 min.
Analytical methods
A Supelcosil ABZ Plus column, 5 mm, 250 Â 4.6 mm, (Sigma-Aldrich,
GB Supelco, Deisenhofen, Germany), flow rate: 1.0 ml/min, and UV
detection (Hewlett Packard 1050 diode array spectrophotometer) at
254 nm and an on-line radioactivity flow detector Ramona1 (Raytest,
Straubenhardt, Germany) were employed with the following gradient:
Eluent A: 0.2% trifluoroacetic acid; eluent B: acetonitrile; 0 min 10%
B; 0–30 min 20% B; 30–40 min 40% B; 40–45 min 90% B; 45–50 min
10% B; retention time for vardenafil: 36.3 min.
LC–MS was performed using a PE/Sciex/API III with MacIntosh
Quadra1 900 (Perkin-Elmer Sciex Instruments; Thornhill, Ont.,
Canada); Infusion: acetonitrile/0.2% trifluoroacetic acid 90+10
(v+v); LC–MS conditions: the interface was maintained at 4.8 kV
and the nebulizing nitrogen pressure at 265 kPa. The atmospheric
pressure ion source was used in the positive ion mode. The scan rate was
2.5 s per scan with data collected in 0.2 amu steps from 480 to 500.
Radiochemical counting was performed on a Liquid Scintillation
Analyzer TRI-CARB1 2500 TR using Ultima GoldTM cocktail.
2-[2-Ethoxy-5-(4-acetylpiperazine-1-sulfonyl)-phenyl]-5-methyl-7-pro
pyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one, (3)
A mixture of sulfonic acid 1 (2.0 g, 5.1 mmol) dried under vacuum at
608C for 1 h, thionyl chloride (4 ml) and dimethylformamide (20 ml) was
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1241–1247

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U. PLEISS
warmed up to a temperature of 608C and stirred for 1.5 h at that
temperature. After addition of p-xylene (10 ml) the mixture was stirred
at 858C for 15 min and subsequently evaporated to dryness. To remove
traces of thionyl chloride p-xylene (10 ml) was added and repeatedly
evaporated to dryness. To the sulfochloride 1a dissolved in p-xylene
(1.5 ml), N-acetylpiperazine 2 (1.5 g, 11.7 mmol) in p-xylene (3.2 ml) was
added over the course of 30 min. This reaction mixture was stirred at
408C for 2 h and over a weekend at room temperature. After addition of
water (12 ml) the pH was adjusted to 1.0 with concentrated hydrochloric
acid (1.5 ml). The aqueous phase was separated and acetone (4 ml) was
added. The pH was adjusted to 7.5 with sodium hydroxide solution and
the mixture cooled in an ice bath for 1.5 h. The precipitate was filtered
off, washed with a mixture of acetone: water 3:1 and dried in a
desiccator. Yield: 1.33 g 3 corresponding to 53% of the theory, the
chemical purity was >99% as determined by HPLC.
LC–MS: [M+H]+ m/z=503.
2-[2-Ethoxy-5-(4-[1-3H2]ethyl-piperazine-1-sulfonyl)-phenyl]-5-methyl-
7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one hydrochlorid, [3H]var-
denafil, (4)
The labeling apparatus (Figure 1) was evacuated by cooling the pre-
evacuated steel trap in liquid N2. A vacuum of 10À1 mbar was achieved
within 10–15 min. Subsequently tritium gas from the storage was
transferred to the frozen mixture of butyl lithium/hexane solution
(125 ml, 1.6 mol) in hexane (0.9 ml) and tetramethylethylenediamine
(TMEDA) (400 ml, 250 mmol) in a 5 ml two-neck vessel attached to the
apparatus. The mixture was warmed up to room temperature and
stirred for 60 min at a tritium partial pressure of about 38 kPa
(380 mbar). Then the non-reacted tritium and the solvent was adsorbed
in the trap filled with platinum oxide and charcoal at the temperature of
liquid nitrogen. To the solid residue in the evacuated two-neck vessel
aluminum bromide (14.2 mg, 50 mmol) dissolved in ether (500 ml) was
added by means of a syringe and via the septum of the second neck of
the vessel. After stirring for 5 min compound 3 (5.3 mg, 10 mmol),
dissolved in tetrahydrofuran (1125 ml) was added in the same way. The
reaction mixture was stirred for 15 min. To decompose the surplus
lithium aluminum tritide 200 mls of methanol were added. The released
tritium and the solvent were adsorbed in the same trap used for the
preparation of the lithium tritide. To remove the labile tritium the dry
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1241–1247

SYNTHESIS OF [3H]VARDENAFIL
1247
residue was dissolved/suspended in ethanol (1 ml) and the solvent was
adsorbed in the trap as well. This process was repeated twice. At the end
all the solvents and the tritium gas used were in the trap. The trap was
disconnected by diagonal pliers and sealed with epoxide resin.
The dry residue was dissolved with 1.0 ml of a mixture of acetonitrile/
0.2% trifluoroacetic acid 20:80 (v+v) and purified in 18 equal portions
under the following conditions: column: SupelcosilTM, SPLC-ABZ,
250 Â 10 mm, (Sigma-Aldrich, GB Supelco, Deisenhofen, Germany);
eluent: A=acetonitrile, B=0.2% trifluoroacetic acid, gradient: 0 min
18% A, 0–40 min 20% A, 40–41 min 50% A, 41–45 min 50% A, 45–
46 min 15% A, 46–50 min 15% A; flow rate: 5 ml/min; UV detection:
254 nm; retention time for [3H]vardenafil: 22.5–23.5 min. The eluates
containing [3H]vardenafil were fractionated on the basis of the UV
signal. The eluates were evaporated under vacuum and hydrochloric
acid (10 ml 0.5 mol) was added to the residue in order to prepare the
hydrochloride. The solution was lyophilized and the remaining solid
was dissolved in 10 ml ethanol to form the stock solution.
Yield: 144 mg (0.29 mmol) 3 corresponding to 2.9% of theory, the
chemical purity was >99% as determined by HPLC; specific activity:
52.7 Ci/mmol (1.95 TBq/mmol); total radioactivity: 15.5 mCi (573MBq).
Acknowledgements
The author would like to thank H. Heinrichs for fruitful discussions
regarding the development of the steel trap, S. Noesel for the skilful
realization of the synthesis and H. Schnorbus for performing the LC–
MS measurements.
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3. Pleiss U. In Synthesis and Applications of Isotopically Labelled Compounds,
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Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1241–1247