Synthesis of carbon-14 labeled 4-[4-[2-[2-[bis(4-chlorophenyl)methoxy]ethyl sulfonyl][1-14C]ethoxy]phenyl]-1,1,1-trifluoro-2-butanone

Article abstract of DOI:10.1002/jlcr.655

Carbon-14 labeled 4-[4-[2-[2-[bis(4-chlorophenyl)methoxyethylsulfonyl] [1-¹⁴C]-ethoxy]phenyl]-1,1,1-trifluoro-2-butanone was prepared in a six step radioactive synthesis from 2-bromo[1-¹⁴C]acetic acid. The overall radiochemical yield was 2.2%. The specific activity of the final product was found to be 42 μCi/mg with a radiochemical purity of > 98%. Copyright

Full text of DOI:10.1002/jlcr.655

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 167–174.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.655
Research Article
Synthesis of carbon-14 labeled 4-[4-[2-[2-
[bis(4-chlorophenyl)methoxy]ethyl
sulfonyl][1-14C]ethoxy]phenyl]-1,1,1-
trifluoro-2-butanone
Douglas D. Dischino1,*, Jacques Banville2 and Roger Remillard2
1 Department of Chemical Synthesis, Bristol-Myers Squibb, 5, Research
Parkway, Wallingford, CT 06492, USA
2 Bristol-Myers Squibb Pharmaceutical Research Institute, Candiac, Quebec,
Canada
Summary
Carbon-14 labeled 4-[4-[2-[2-[bis(4-chlorophenyl)methoxyethylsulfonyl] [1-14C]-
ethoxy]phenyl]-1,1,1-trifluoro-2-butanone was prepared in a six step radioactive
synthesis from 2-bromo[1-14C]acetic acid. The overall radiochemical yield was
2.2%. The specific activity of the final product was found to be 42 mCi/mg with
a radiochemical purity of >98%. Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: carbon-14; cPLA2; anti-inflammatory
Introduction
Leukotrienes and prostaglandins are derived from arachidonic acid and
are potent lipid mediators of inflammation and pain. Cytosolic
phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic
acid from the sn-2 position of phospholipids and is believed to play a
key cellular role in the generation of arachidonic acid.1 4-[4-[2-[2-[bis
(4-chlorophenyl)methoxy]ethylsulfonyl]ethoxy]phenyl]-1,1,1-trifluoro-
*Correspondence to: D. D. Dischino, Department of Chemical Synthesis, Bristol-Myers Squibb,
5 Research Parkway, Wallingford, CT 06492-7600, USA. E-mail: douglas.dischino@bms.com
Copyright # 2002 John Wiley & Sons, Ltd.
Received 24 July 2002
Revised 6 August 2002
Accepted 16 August 2002

168
D. D. DISCHINO ET AL.
Cl
Cl
O
O
S
O
O
F
C
3
O
1a
Figure 1. 4-[4-[2-[2-[bis(4-chlorophenyl)methoxy]ethylsulfonyl]ethoxy]phenyl]-1,1,
1-trifluoro-2-butanone
2-butanone 1a, was found to be a selective inhibitor of cPLA2
(IC50=2.8 mM) in that it did not inhibit secreted phospholipase A2
in vitro, nor phospholipase C and phospholipase D in cells (Figure 1).1
This compound was also active in inhibiting arachidonate and
eicosanoid production in U937 cells, neutrophils, platelets, monocytes,
and mast cells.1
Compound 1a represents a novel inhibitor of cPLA2, which
partitions into the phospholipid bilayer and competes with phospho-
lipid substrate for the active site.1 This potent inhibition of the enzyme
translated into anti-inflammatory activity when applied topically (5%,
w/v) to a phorbol ester- induced chronic inflammation model in mouse
ears, inhibiting edema and neutrophil infiltration, as well as, prosta-
glandin and leukotriene levels in the skin.1
This paper reports the synthesis of 4-[4-[[2-bis(4-chlorophenyl)-
methoxy]ethyl]sulfonyl][1-14C]ethoxyphenyl]-1,1,1-trifluoro-2-butanone,
1b, for use in subsequent metabolism and pharmacokinetic studies, as
well as, experimental skin flux studies (Scheme 1).
Results and discussion
The synthesis of carbon-14 labeled 4-[4-[2-[2-[bis(4-chlorophenyl)
methoxy]ethylsulfonyl][1-14C]ethoxy]phenyl]-1,1,1-trifluoro-2-butanone,
1b, was achieved starting from 2-bromo[1-14C]acetic acid in six steps. In
this procedure, 2-[bis(4-chlorophenyl)methoxy]ethane thioacetate 2 was
deprotected to yield 2-[bis(4-chlorophenyl)methoxy]ethanethiol 3 which
was then reacted immediately with 2-bromo[1-14C]acetic acid to yield 2-
[bis(4-chlorophenyl)methoxy]ethanethio[1-14C]acetic acid 4. Reduction
of the radiolabeled acid 4 to the corresponding alcohol 5 with LiAlH4
yielded 2-[2-[bis(4-chlorophenyl)methoxy]ethylthio][1-14C] ethanol 5.
Coupling of the alcohol with methyl 3-(4-hydroxyphenyl)propionate
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SYNTHESIS OF CARBON-14 LABELLED
169
Scheme 1. Synthesis of Carbon-14 labeled 4-[4-[2-[2-bis(4-chlorophenyl)methoxy]
ethylsulfonyl][1-14C]ethoxy]phenyl]-1,1,1-trifluoro-2-butanone
yielded methyl 3-[4-[2-[2-[bis(4-chlorophenyl)methoxy]ethylthio][1-14-
C]ethoxy]phenyl]propionate (6). Saponification of 6 with potassium
hydroxide yielded 3-[4-[2-[2-[bis(4-chlorophenyl)methoxy]ethylthio]
[1-14C] ethoxy]phenyl]propionic acid 7. Conversion of carboxylic acid
7 to the trifluoromethyl ketone 8 via trifluoroacetic anhydride and
pyridine yielded 4-[4-[2-[2-[bis(4-chlorophenyl) methoxy]ethylthio][1-14-
C]ethoxy]- phenyl]-1,1,1-trifluoro-2-butanone 8.2 Subsequent oxidation
of the sulfide 8 via 3-chloroperoxybenzoic acid, yielded the crude final
product 1b which was purified via HPLC.
The radiochemical yield of this synthesis was 2.2% which resulted
from numerous reactions proceeding in only modest yield and
difficulties encountered during purification procedures. Flash chroma-
tography was used to purify several of the radiolabeled intermediates,
however subsequent radio-HPLC analysis indicated that some closely
eluting radioactive impurities could not be removed via flash
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D. D. DISCHINO ET AL.
chromatography and were thus carried on to the next reaction. LC-MS
and NMR studies have shown that in the presence of water the
trifluoromethyl ketone of compounds 8 and 1b exists in equilibrium
with the corresponding gem-diol moiety.3 This equilibrium causes
serious tailing of the peak for both 8 and 1b during reverse phase HPLC
using conventional aqueous buffer/acetonitrile conditions.3 Purification
of the final product, 1b, from close eluting radiolabeled impurities of
unknown structure was only possible using reverse-phase HPLC, which
resulted in significant tailing of the desired compound and incomplete
recovery of product.
Materials and methods
Previously prepared non-radioactive proprietary precursors and final
product were synthesized according to published procedures and used as
standards throughout this study.4 2-Bromo[1-14C]acetic acid (52 mCi/
mmol) was purchased from ViTrax Co. All other reagents were
obtained from Aldrich and were either ACS grade or the highest
quality material commercially available. HPLC purification and
analysis was performed on a Rainin Dynamax HPLC system consisting
of two SD-200 pumps, a Rainin UV-I detector and an INUS b-RAM
radioactive flowthrough detector. Zorbax Rx C-18 columns were
obtained from VWR Scientific Products. YMC basic 5 mm ODS (2)
columns were obtained from Waters.
High-performance liquid chromatography
Method A. In this method samples are loaded onto a Zorbax RxC-18
column (4.6 Â 250 mm2) with a mobile phase of 60% CH3CN and 40%
H2O containing 0.1% TFA at a flow rate of 1.0 ml/min. The UV-1
detector was set at 265 nm.
Method B. In this method samples are loaded onto a Zorbax RxC-18
column (4.6 Â 250 mm2) with a mobile phase of 80% CH3CN and 20%
H2O containing 0.1% TFA at a flow rate of 1.0 ml/min. The UV-1
detector was set at 265 nm.
Method C. In this method samples are loaded onto a Zorbax RxC-18
column (4.6 Â 250 mm2) with a mobile phase of 70% CH3CN and 30%
H2O containing 0.1% TFA at a flow rate of 1.0 ml/min. The UV-1
detector was set at 240 nm.
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SYNTHESIS OF CARBON-14 LABELLED
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Method D. In this method samples are loaded onto a YMC basic 5 mm
ODS (2) column (100 Â 250 mm2) with a mobile phase of 60% CH3CN
and 40% H2O at a flow rate of 4.0 ml/min. The UV-1 detector was set at
230 nm.
Experimental
2-[Bis(4-chlorophenyl)methoxy]ethanethiol 3
%
Into a 100 ml RB flask containing 2-[bis(4-chlorophenyl)methoxy]
ethane thioacetate 2 (0.679 g, 1.91 mmol) was added anhydrous THF
(9 ml), absolute EtOH (9 ml) and the solution allowed to stir at 228C
under argon. To this solution was added a freshly prepared solution of
NaOH (4.38 ml, 1 M, 4.38 mmol) in deoxygenated water and stirred for
1 h after which time the reaction mixture was then acidified with HCl
(5.25 ml, 1 N), and extracted with toluene (90 ml). The organic layer was
rinsed with H2O (10 ml, 3x), brine (10 ml), dried (MgSO4), filtered and
concentrated in vacuo to yield 3 (0.6 g, 1.91 mmol) as an oil which was
used immediately in the next step.
2-[Bis(4-chlorophenyl)methoxy]ethanethio[1-14C]acetic acid 4
%
Into a 100 ml RB flask containing 3 (0.6 g, 1.91 mmol) was added
anhydrous THF (6.7 ml), absolute EtOH (6.7 ml) and the solution
allowed to stir at 228C under argon. To this solution was added a freshly
prepared solution of NaOH (4.5 ml, 1 M, 4.5 mmol) in deoxygenated
water and a mixture of 2-bromo[1-14C]acetic acid (50 mCi, 52 mCi/
mmol, 0.133 g, 0.96 mmol) and non-radioactive 2-bromoacetic acid
(0.133 g, 0.96 mmol) in one portion. The reaction mixture was stirred at
228C for 1 h and then diluted with H2O (45 ml), acidified with HCl
(5.35 ml, 1 N) and extracted with EtOAc (90 ml). The organic phase was
rinsed with H2O (25 ml, 2 Â ), brine (25 ml), dried (MgSO4), filtered and
concentrated in vacuo to yield 4 (38 mCi, 0.568 g, 1.53 mmol, 76%).
HPLC analysis of 4 (Method A) showed a radiochemical purity of 97%.
In this system 4 has a retention time of approximately 10.5 min.
2-[2-[Bis(4-chlorophenyl)methoxy]ethylthio][1-14C]ethanol (5).
Into a 250 ml RB flask containing 4 (38 mCi, 0.568 g, 1.53 mmol)
dissolved in anhydrous THF (60 ml) was added LiAlH4 (0.20 g,
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D. D. DISCHINO ET AL.
5.3 mmol) in small portions. After the evolution of hydrogen had
ceased, the reaction mixture was heated to reflux for 1 h. The reaction
mixture was then cooled to 08C and treated successively with H2O
(0.2 ml added slowly), NaOH (0.2 ml, 15% solution) and H2O (0.6 ml).
After 15 min, the resulting solid was filtered and the filtrate diluted with
EtOAc (200 ml), and the organic layer washed with brine (25 ml), dried
(MgSO4) and filtered. The filtrate was concentrated in vacuo and
purified via flash chromatography (SiO2, 20% EtOAc/hexane to 30%
EtOAc/hexane) to yield 5 (20.2 mCi, 0.293 g, 0.816 mmol, 53%). HPLC
analysis of 5 (Method A) showed a radiochemical purity of 90%. In this
system 5 has a retention time of approximately 11.7 min.
Methyl 3-[4-[2-[2-[bis(4-chlorophenyl)methoxy]ethylthio]
[1-14C]ethoxy]phenyl] propionate, 6
%
Into a 25 ml RB flask containing 5 (20.2 mCi, 0.293 g, 0.816 mmol)
dissolved in toluene (4.5 ml) was added methyl 3-(4-hydroxyphenyl)-
propionate (0.200 g, 1.110 mmol), and triphenylphosphine (0.285 g,
1.110 mmol) and stirred at room temperature. To this solution was
added diethyl azodicarboxylate (175 ml, 1.110 mmol) over 5 min and the
reaction stirred at room temperature for 18 h after which time, the
reaction mixture was diluted with EtOAc (40 ml), washed with saturated
NaHCO3 (20 ml), dried (MgSO4), and filtered. The filtrate was
concentrated in vacuo and tritiated with hexane:toluene (6:4, 10 ml)
and the resulting triphenylphosphine oxide filtered and the filtrate
concentrated in vacuo. The residue was dissolved in a minimum amount
of 10% EtOAc/hexane and sonicated to assist in solublization. The
crude product was then purified via flash chromatography (SiO2, 5–10%
EtOAc/hexane) to yield 6 (12.4 mCi, 0.260 g, 0.500 mmol, 61%). The
balance of activity was isolated as 5. HPLC analysis of 6 (Method B)
showed a radiochemical purity of 91%. In this system 6 has a retention
time of approximately 12.3 min.
3-[4-[2-[2-[Bis(4-chlorophenyl)methoxy]ethylthio][1-14C]ethoxy]phe-
nyl]propionic acid, 7
%
Into a 25 ml RB flask containing 6 (12.4 mCi, 0.260 g, 0.500 mmol) and
aqueous EtOH (80%, 11 ml) was added KOH (150 mg, 2.673 mmol)
dissolved in H2O (1 ml) and the resulting mixture heated at 608C for 1 h.
The solvent was then removed in vacuo and the residue diluted with H2O
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SYNTHESIS OF CARBON-14 LABELLED
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(10 ml), acidified to pH 2 with HCl (2 N) and extracted with CH2Cl2
(20 ml, 2 Â ). The organic layers were combined, washed with brine and
dried (MgSO4), filtered and the filtrate concentrated in vacuo to yield 7
(10.7 mCi, 218 mg, 0.432 mmol, 86%) as a white solid. HPLC analysis of
7 (Method B) showed a radiochemical purity of 78%. In this system 7
has a retention time of approximately 6.3 min. The balance of activity
was found as a single peak eluting before the desired product. The
identify of this impurity was not determined.
4-[4-[2-[2-[Bis(4-chlorophenyl)methoxy]ethylthio][1-14C]ethoxy]
phenyl]-1,1,1-trifluoro-2-butanone, 8
%
Into a 25 ml RB flask containing crude 7 (10.7 mCi, 218 mg,
0.432 mmol) dissolved in anhydrous CH2Cl2 (5.5 ml) was added oxalyl
chloride (0.34 g 2.67 mmol), and DMF (30 ml) and the reaction mixture
stirred at room temperature for 1 h. The solvent was then removed
in vacuo and the residue dissolved in toluene (17 ml) and cooled (0–58C,
ice-bath). To this solution was added trifluoroacetic anhydride (0.70 g,
4.77 mmol), and pyridine (0.22 g, 2.78 mmol). The reaction mixture was
then allowed to warm to room temperature and stirred for 2 h. The
reaction mixture was diluted with H2O (1 ml), stirred for 15 min, and
then extracted with EtOAc (60 ml). The organic layer was washed with
H2O (15 ml), saturated NaHCO3 (15 ml), brine (15 ml), dried (MgSO4),
and filtered. The filtrate was concentrated in vacuo and purified via flash
chromatography (SiO2, 15–35% EtOAc/hexane) to yield 8 (3.1 mCi).
HPLC analysis of 8 (Method C) showed a radiochemical purity of 91%.
In this system 8 has a retention time of approximately 16.5 min, while 7
has a retention time of approximately 11.5 min. In this chromatographic
system there is significant tailing of the peak for 8. Radiochemical purity
was determined by including all of the area under the peak until the
peak returned to baseline (ꢀ3 min).
4-[4-[2-[2-[Bis(4-chlorophenyl)methoxy]ethylsulfonyl][1-14C]
ethoxy]phenyl]-1,1,1-trifluoro-2-butanone, 1b
Into a 50 ml RB flask containing 8 dissolved in CH2Cl2 (17 ml) was
added 3-chloroperoxybenzoic acid (0.32 g, 0.57 mmol) and the mixture
stirred at room temperature for 2 h, diluted with EtOAc (80 ml), the
organic layer washed with saturated NaHCO3 (15 ml), brine (15 ml),
dried (MgSO4) and filtered. The filtrate was concentrated in vacuo and
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D. D. DISCHINO ET AL.
the crude product 1b was purified via HPLC (Method D) to yield 1b
(1.1 mCi, 26.2 mg, specific activity 42 mCi/mg). During this purification,
there is significant tailing of the peak as it elutes from the HPLC column
and the product was collected from 21 to 29 min post-injection. HPLC
analysis (Method C) of 1b indicated a radiochemical purity of >98%).
In this system 1b has a retention time of approximately 5.8 min while 8
has a retention time of approximately 16.5 min.
References
1. Burke JR, Davern LB, Stanley PL, et al. J Pharm Exp Therap 2001; 298:
376–385.
2. Boivin J, Kaim LE, Zard SZ. Tetrahedron Lett 1992; 33: 1285–1288.
3. Rourick R. Internal Communication, Bristol-Myers Squibb, 27 April, 1998.
4. Banville J, Gai Y, Johnson G, Zusi FC, Burke JR. International Patent
Application, WO 99/15129. April 1 1999.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 167–174