Syntheses of [14C]BAY 59-7939 and its radiolabeled metabolite M-4

Article abstract of DOI:10.1002/jlcr.1103

BAY 59-7939 is a novel, oral, direct Factor Xa inhibitor in clinical development for the prevention and treatment of thromboembolic diseases. Radiolabeled BAY 59-7939 was required for drug absorption, distribution, metabolism and excretion (ADME studies). The BAY 59-7939 was labeled with carbon-14 in the carboxamide group in one step in an overall radiochemical yield of 85% starting from 4-{4-[(5S)-5-(aminomethyl)-2-oxo-1,3-oxazolidin-3-yl] phenyl}mor-pholin-3-one and 5-chlorothiophene-2-[¹⁴C]carboxylic acid. The radiolabeled metabolite M-4 was prepared in 77% yield starting from [1-¹⁴C]glycine and 5-chlorothiophene-5-carboxylic acid. Copyright

Full text of DOI:10.1002/jlcr.1103

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd. Radiopharm 2006; 49: 929–934.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1103
Research Article
1
4
Syntheses of [ C]BAY 59-7939 and its radiolabeled
metabolite M-4
1
U. Pleiss *, D. Seidel and R. Grosser
,
1
2
1
Drug Metabolism and Isotope Chemistry, Global Drug Discovery, Bayer HealthCare
AG, 42096 Wuppertal, Germany
2
Medicinal chemistry, Global Drug Discovery, Bayer HealthCare AG, 42096
Wuppertal, Germany
Summary
BAY 59-7939 is a novel, oral, direct Factor Xa inhibitor in clinical development for
the prevention and treatment of thromboembolic diseases. Radiolabeled BAY 59-
7
939 was required for drug absorption, distribution, metabolism and excretion
ADME studies). The BAY 59-7939 was labeled with carbon-14 in the carboxamide
group in one step in an overall radiochemical yield of 85% starting from 4-{4-[(5S)-5-
aminomethyl)-2-oxo-1,3-oxazolidin-3-yl]phenyl}mor-pholin-3-one and 5-chlorothio-
(
(
1
4
phene-2-[ C]carboxylic acid. The radiolabeled metabolite M-4 was prepared in 77%
1
4
yield starting from [1- C]glycine and 5-chlorothiophene-5-carboxylic acid. Copyright
2006 John Wiley & Sons, Ltd.
#
Received 1 June 2006; Revised 6 June 2006; Accepted 6 June 2006
Key Words: Factor Xa inhibitor; carbon-14 synthesis; amide formation
Introduction
Factor Xa has been identified as a potential target for intervention in the
coagulation cascade. It is integral to both the intrinsic and extrinsic pathways
of the coagulation cascade, and is the point at which the two pathways
converge. Approximately one molecule of Factor Xa results in the generation
1
of up to 1000 thrombin molecules. BAY 59-7939 (see Figure 1) has been
shown to selectively and directly inhibit Factor Xa, block thrombin generation
2
and prolong clotting times. Furthermore, results from in vivo studies have
shown reduced thrombus formation with BAY 59-7939 administered
*Correspondence to: U. Pleiss, Bayer HealthCare AG, Aprather Weg 18a, 42096 Wuppertal, Germany.
E-mail: ulrich.pleiss@bayerhealthcare.com
Copyright # 2006 John Wiley & Sons, Ltd.

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U. PLEISS ET AL.
O
N
O
O
N
O
H
N
Cl
S
O
Figure. 1. BAY 59-7939
2
prophylactically in rat and rabbit thrombosis models. In support of a
program to develop this new Factor Xa inhibitor synthesis of carbon-14
labeled BAY 59-7939 was required. The radiolabeled metabolite M-4 was
synthesized for use as reference standard. This paper describes the syntheses of
the two compounds.
Results and discussion
1
4
[
C]BAY 59–7939
The approach taken for the synthesis of carbon-14 labeled BAY 59-7939 relies
3
on the previously reported synthesis of BAY 59-7939. The carboxamide
1
4
formation in the final compound 3 with 5-chloro-2-thiophene[ C]carboxylic
acid (1) and 4-{4-[(5S)-5-(aminomethyl)-2-oxo-1,3-oxazolidin-3-yl]phenyl}-
morpholin-3-one (2) was realized using N’-(3-dimethylaminopropyl)-N-ethyl-
carbodiimide hydrochloride (EDC.HCl) and 1-hydroxy-1-benzotriazole
(
HOBT) in a yield of 85% as shown in Scheme 1. The final compound was
1
4
purified using a suitable chiral HPLC system to give 7.3 mCi of [ C]BAY
9-7939 (3) with high chemical and radiochemical purity and an enantiomeric
excess of >99%.
5
1
4
C-labeled Metabolite M-4 of BAY 59-7939
For the carboxamide formation in compound 7 the acid chloride (5), prepared
4
from 5-chlorothiophenecarboxylic acid (4) using thionyl chloride, was reacted
1
4
with [1- C]glycine (6) yielding the metabolite M-4 (7) in 77% as shown in
Scheme 2. Due to the hydrolytic stability of the acid chloride (5) the reaction
was carried out in water in the presence of sodium bicarbonate.
Experimental
Materials
1
4
5
-Chloro-2-thiophene[ C]carboxylic acid was purchased from Amersham
1
Pharmacia Biotech, Cardiff, UK and [1- C]glycine was purchased from
4
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 929–934
DOI: 10.1002/jlcr

1
4
SYNTHESIS OF [ C]BAY 59-7939 AND ITS RADIOLABELED METABOLITE
931
O
N
O
HO14
O
N
O
+
NH2
C
Cl
S
O
2
1
88 %
EDC.HCl, HOBT,
THF, DMF
O
O
O
N
N
O
H
N
1
4
C
Cl
S
1
4
O
3
([ C]BAY 59-7939)
14
Scheme 1. Synthesis of [ C]BAY 59-7939
OH
SOCl2
Cl
Cl
O
Cl
S
S
1
4
97 %
O
H N
C
4
O
2
5
OH
6
7
7 % water, NaHCO3
O
1
4
H
C
N
OH
Cl
S
O
7
Scheme 2. Synthesis of the metabolite M-4 of BAY 59-7939
American Radiolabeled Compounds, Inc., St. Louis, USA. Compounds 2 and
were obtained from the Chemical Development or Chemical Research
4
Departments, Bayer HealthCare AG. All remaining reagents and solvents
were purchased from commercial suppliers and were used as received.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 929–934
DOI: 10.1002/jlcr

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U. PLEISS ET AL.
Liquid scintillation counting
Quantification of radioactivity was performed using a PerkinElmer TRI-
1
TM
CARB 2500 TR liquid scintillation analyzer, with Ultima Gold
cocktail
used throughout.
High-performance liquid chromatography
All final compounds were analyzed by HPLC using a HP 1050 system Series II
1
(Hewlett-Packard, Waldbronn, Germany) with a Ramona
Cell 2270
(
Raytest, Straubenhard, Germany) for radioactivity detection. The LC-MS
1
analytics were obtained at a PE Sciex/API III with MacIntosh Quadra 900
PerkinElmer Sciex Instruments, Thornhill, Canada). The following HPLC
(
system was used for the purity check:
1
(1) Cosmosil
C18-5 AR, 250 ꢀ 4.6 mm (Waters GmbH, Eschborn
Germany), column temperature: 458C, flow rate: 1 ml/min, gradient: A:
0
.2% phosphoric acid, B: acetonitrile, 0 min: 30% B, 0–20 min: 40% B,
2
0–35 min 90% B, 35–40 min 90% B, detection: UV 250 nm.
1
(
2) Chiracel OD-H AD, 250 ꢀ 4.6 mm (J.T. Baker B.V., Deventer, Nether-
lands), column temperature: 50 8C, flow rate: 1 ml/min, eluent:
n-heptane/ethanol 70+30 (v+v), detection UV 250 nm.
NMR spectra
The NMR spectra were recorded on a Bruker DRX 400 spectrometer (Bruker,
Rheinstetten, Germany).
Synthesis of 5-chloro-N-({(5S)-2-oxo-3-[4-(3-oxo-4-morpholinyl)phenyl]-1,3-
oxa-zolidin-5-yl}methyl)-2-thiophene-[ C]carboxamide (3)
14
14
A solution of 5-chloro-2-thiophene[ C]carboxylic acid (1) (544 mg, 3.3 mmol,
total radioactivity: 200 mCi, specific activity: 59 mCi/mmol, radiochemical
purity: 99.1%), 1-hydroxy-1-benzotriazole (498 mg, 3.7 mmol) and N’-(3-
dimethylaminopropyl)-N-ethyl
.7 mmol), dissolved in 10 ml tetrahydrofuran and 6.7 ml dimethyl formamide
was stirred at room temperature for 1 h. A solution of 4-{4-[(5S)-5-
aminomethyl)-2-oxo-1,3-oxazolidin-3-yl]-phenyl}morpholin-3-one (2) (975mg,
.3 mmol) in 8.6 ml dimethyl formamide and 4.5 ml tetrahydrofuran, was
carbodiimide
hydrochloride
(706 mg,
3
(
3
slowly added. The reaction mixture was stirred for 20 h at room temperature
and subsequently for 1 h at 458C. After addition of water (60 ml) the reaction
mixture was stirred for 1 h. The precipitate was filtered off and washed several
times with about 100 ml of water overall. The white crystals were dried under
high vacuum yielding 1260 mg of compound 3 (88%) with a total radioactivity
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 929–934
DOI: 10.1002/jlcr

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SYNTHESIS OF [ C]BAY 59-7939 AND ITS RADIOLABELED METABOLITE
933
of 170.2 mCi. The UV purity was 98.3%, the radiochemical purity was 98.2%
and the enantiomeric excess was >87.1%.
þ
14
þ
LC-MS : m=z ¼ 436 ½M þ Hꢁ ð3:1%Þ and 438 ½ C-M þ Hꢁ ð96:9%Þ
A portion of compound 3 (1080 mg), dissolved in 29 ml tetrahydrofuran and
2
5 ml dimethyl formamide, was diluted with non-radioactive BAY 59–7939
(900 mg). After addition of 108 ml water the precipitate was filtered off,
washed with 200 ml water and dried under high vacuum yielding 1898 mg of
compound 3 showing a specific activity of 32.4 mCi/mmol. From this batch
1
0 mg aliquots were purified using the following chiral HPLC conditions:
column: filled with poly(N-methacryloyl-L-leucine-tert-butylamide)silica com-
5
5
posite, 10 mm, 250 ꢀ 20 mm (Bayer AG, Wuppertal, Germany), eluent: ethyl
acetate; flow rate: 13 ml/min; detection: UV 270 nm; retention time: 13.7 min.
Using this method approximately 130 mg of crude product was purified to give
compound 3 (98 mg, 7.3 mCi) with chemical purity, radiochemical purity and
enantiomeric excess of >99%.
1
H-NMR (400 MHz, D -DMSO) d 8.95 (t, 1H), 7.69 (d, 1H), 7.56 (d, 2H),
6
7
.40 (d, 2H), 7.19 (d, 1H), 4.83 (m, 1H) 4.23–4.14 (m, 3H), 3.95 (t, 2H), 3.84
(m, 1H), 3.70 (t, 2H), 3.59 (t, 2H) ppm.
1
Synthesis of N-[(5-chloro-2-thienyl)carbonyl][1- C]glycine (7)
4
A mixture of 5-chlorothiophene-2-carboxylic acid (4) (500 mg, 3.1 mmol) and
2
.5 ml thionyl chloride was heated to 60 8C for 2 h under stirring. The reaction
mixture was evaporated to dryness under vacuum. The residue was dissolved
in 5 ml toluene and evaporated to dryness. This process was repeated yielding
5
41 mg of compound 5 (97%). The GC analysis showed a chemical purity of
>
99%.
Non-radioactive glycine (48.5 mg, 0.65 mmol) and sodium bicarbonate
(
279 mg, 3.32 mmol) and subsequently compound 5 (147 mg, 0.81 mmol) and
14
1
ml water were added to a solution of [1- C]glycine (6) (1.36 mg, 18.1 mmol,
total radioactivity: 1 mCi, specific activity: 55 mCi/mmol, radiochemical purity
99%) in 1 ml 0.01% hydrochloric acid. The reaction mixture was stirred for
3 h at room temperature. The pH value was adjusted to pH = 1 with half
>
2
concentrated hydrochloric acid. The precipitate was filtered off, washed four
times with each 0.5 ml water and dried under high vacuum giving 113 mg of
compound 7 with a total radioactivity 0.77 mCi and a specific activity of
1
.5 mCi/mmol corresponding to 77% yield.
12
þ
GC ꢂ MS : m=z ¼ 219 ½ C ꢂ Mꢁ
1
H-NMR (400 MHz, D -DMSO) d 12.69 (s, 1H), 8.96 (t, 1H), 7.66 (d, 1H),
6
7
.20 (d, 1H), 3.89 (d, 2H) ppm.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 929–934
DOI: 10.1002/jlcr

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U. PLEISS ET AL.
Acknowledgements
The authors would like to thank Ute Weinberg, Stefan Noesel and Udo Balzer
for skillfully performing all syntheses. Thanks are also due to Harald
Schnorbus for performing HPLC and MS analyses and to Dr Peter Schmitt
for the NMR spectra.
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31(12): 405–411.
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1
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 929–934
DOI: 10.1002/jlcr