Synthesis of tritium labelled L783483 - A PPARδ ligand

Article abstract of DOI:10.1002/jlcr.687

The potent peroxisome proliferator-activated receptor (PPAR) δ ligand L783483 (3-chloro-4-(3-(7-propyl-3-trifluoromethyl-benzisoxazol-6-oxy) pro-pylsulfanyl)phenylacetic acid) has been labelled with tritium via selective tritium/bromine exchange of 5-brom

Full text of DOI:10.1002/jlcr.687

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 459–464.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.687
Research Article
Synthesis of tritium labelled L783483 - a
PPARd ligand
Gunnar Grue-Sꢀrensen* and Nicolaj Hꢀj
LEO Pharma A/S, Medicinal Chemistry Research, Industriparken 55, DK-2750
Ballerup, Denmark
Summary
The potent peroxisome proliferator-activated receptor (PPAR) d ligand
L783483 (3-chloro-4-(3-(7-propyl-3-trifluoromethyl-benzisoxazol-6-oxy)pro-
pylsulfanyl)phenylacetic acid) has been labelled with tritium via selective
tritium/bromine exchange of 5-bromo-6-(3-bromopropyloxy)-7-propyl-3-tri-
3
fluoromethyl-benzisoxazole. [ H]-L783483 had a specific activity of 529 GBq/
mmol (14.3 Ci/mmol) and a radiochemical purity of 98%. Copyright # 2003
John Wiley & Sons, Ltd.
Key Words: L783483; PPARd; tritium–bromine exchange
Introduction
The peroxisome proliferator-activated receptors (PPARs) have been
1
studied intensely for more than a decade. There is a need for
radiolabelled ligands with high specific activities to elucidate and
characterize the role of the various subtypes (a, d, g) of these receptors in
*Correspondence to: G. Grue-Sꢀrensen, LEO Pharma A/S, Medicinal Chemistry Research,
Industriparken 55, DK-2750 Ballerup, Denmark. E-mail: gunnar.grue-soerensen@leo-pharma.com
Copyright # 2003 John Wiley & Sons, Ltd.
Received 17 September 2002
Revised 24 October 2002
Accepted 29 October 2002

4
60
G. GRUE-SØRENSEN AND N. HØJ
metabolic processes and diseases. These radiolabelled ligands are also
useful in screening of new ligands with optimal pharmacological
properties for the treatment of PPAR related diseases. L783483 (5a) is
2
3
such a potent PPARd-ligand synthesised by Merck & Co. [ H] -
2
L783483 with undisclosed method and sites of labelling has also been
3
synthesised by Merck & Co (17 Ci/mmol) (cf. reference ). Here we
3
report our synthesis of [ H]-L783483 (5b).
Results and discussion
Our goal was to synthesize the PPAR d ligand L783483 (5a) labelled
with tritium and with a specific activity >10 Ci/mmol (>370 GBq/
mmol). Halogen–tritium exchange is a common way of introducing
tritium in aromatic compounds, provided no other functionality
susceptible to catalytic reduction is present. Compound 5a contains
an aromatic chlorine substituent and selective halogen–tritium exchange
of a halogen derivative of 5a to give 5b would most likely be difficult to
control. Thus, it was decided to introduce halogen in the benzisoxazole
moiety before combination with the chlorine containing part of the
compound.
Bromination of 6-hydroxy-7-propyl-3-trifluoromethyl-benzisoxazole
(1) with NBS in CCl in the presence of AIBN gave the expected
4
bromo-derivative (2) in 67% yield (Scheme 1). Alkylation with an
2
excess of 1,3-dibromopropane (cf. reference ) gave the dibromo-
derivative 3 in 84% yield. Selective exchange of the aromatic
bromine with tritium was achieved by reaction with tritium gas
in ethanol using 10% Pd/C at atmospheric pressure for 30 min.
Prolonged reaction times (>3 h) resulted in a mixture where 55%
of the activity belonged to the desired compound 4b. 3-Chloro-4-
4
(dimethylaminocarbonylsulphanyl)-phenylacetic acid methyl ester
was deprotected with 0.5 M MeONa/MeOH at 638C for 70 min
to give the sodium salt of 3-chloro-4-sulphanyl-phenylacetic acid
methyl ester. This mixture was alkylated with 4b in methanol
at 638C for 60 min. Finally, the reaction mixture was treated with
1 M LiOH at 638C for 15 min to give 5b (radiochemical purity: 98%,
specific activity: 14.3 Ci/mmol) in 76% isolated radiochemical yield in
step d).
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 459–464

SYNTHESIS OF TRITIUM LABELLED L783483
461
CF3
CF3
Br
(
b)
(
a)
N
N
O
O
HO
HO
1
2
CF3
N
CF
3
Br
X
(
c)
N
O
O
Br
O
Br
O
1
3
4a: X = H
4
3
b: X = H
CF
3
X
HOOC
(
d)
N
O
S
O
Cl
1
5
5
a: X = H
3
b: X = H
3
Scheme 1. Synthesis of [ H]-L783483. Reagents and conditions: (a) NBS, AIBN,
CCl , 738C, 2 days; (b) 1,3-dibromopropane, K CO , 2-butanone, reflux, 2 h; c)
4
2
3
1
either [ H ], 10% Pd/C, ethanol, room temperature, 1 atm, 6 h to give 4a or
2
3
[
H ], 10% Pd/C, ethanol, room temperature, 1 atm, 30 min to give 4b; (d) 3-
2
chloro-4-(dimethylaminocarbonylsulphanyl)-phenylacetic acid methyl ester was
treated with 0.5 M MeONa/MeOH, 638C, 70 min, this mixture was added to 4a
or 4b, 638C, 60 min, then addition of 1 M LiOH, 638C, 15 min, HPLC-
purification
Experimental
General
A Packard Tri-Carb 2900TR liquid scintillation analyzer was used to
determine the radioactivity in liquid samples using Pico-Fluor 40
(Packard) as scintillation cocktail. Chemical and radiochemical purity
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 459–464

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62
G. GRUE-SØRENSEN AND N. HØJ
was determined by HPLC on a Merck Hitachi apparatus (L-6200
pump) on a Spherisorb ODS1 column (150 mm ꢀ 4.6 mm) with MeCN/
0
.05 M phosphoric acid 73:27 as eluent (0.8 ml/min) and UV-detection
(254 nm) (L-4250 UV-detector) or radioactivity detection (Packard
Flow System Analyzer Model D525F1 with Ultima-Flo M (Packard) as
scintillation liquid). Concentrations and specific activities were deter-
mined by HPLC by comparison of peak areas of radio-inactive
1
13
reference compounds. H and C NMR spectra were obtained on a
Bruker ARX300 spectrometer. Chemical shifts are reported in ppm with
tetramethylsilane as internal reference. ES Mass spectra were obtained
on a VG Quattro II mass spectrometer in negative ion electrospray
mode and EI spectra were obtained on a VG AutoSpec mass
spectrometer.
5
-Bromo-6-hydroxy-7-propyl-3-trifluoromethyl-benzisoxazole (2). 6-Hy-
2
droxy-7-propyl-3-trifluoromethyl-benzisoxazole (1) (4.05 g, 16.5 mmol),
N-bromosuccinimide (3.30 g, 18.5 mmol) and 2,2’-azobisisobutyronitrile
(62 mg) were stirred in carbon tetrachloride (60 ml) under argon at 738C
for 2 days. The reaction mixture was cooled to room temperature and
filtered. Solvent was removed from the filtrate in vacuo. The residue was
suspended in petroleum ether (bp 35–508C) (100 ml) and the suspension
was applied to a column of silica gel (43–60 m) (8 g). The column was
eluted with petroleum ether (400 ml) and the solvent was removed from
the eluate to produce 5-bromo-6-hydroxy-7-propyl-3-trifluoromethyl-
1
benzisoxazole (2) (3.59 g, 11.1 mmol, 67%). H NMR (DMSO-d ) d
6
1
0.49 (bs, OH), 7.98 (m, 1 H), 2.94 (m, 2 H), 1.65 (m, 2 H), 0.95 (t, 3 H).
1
3
C NMR (DMSO-d ) d 163.9, 154.4, 148.3 (q, J ¼ 38:0 Hz), 120.9,
6
1
19.9 (q, J ¼ 271:6 Hz), 112.3, 112.2, 109.7, 25.9, 21.6, 13.7. MS:
M-H] = 322.
ꢁ
[
5
-Bromo-6-(3-bromopropyloxy)-7-propyl-3-trifluoromethyl-
benzisoxazole (3). A mixture of 2 (3.32 g, 10.2 mmol), 1,3-dibromopro-
pane (6.56 g, 32.5 mmol), potassium carbonate (4.48 g, 32.4 mmol) and
2
-butanone (50 ml) was stirred at 808C for 4 h. After cooling to room
temperature ethyl acetate was added and the mixture was washed with
potassium phosphate buffer (pH 4.0) (400 ml) and water (400 ml).
Drying over sodium sulphate, filtration and evaporation of solvent in
vacuo followed by removal of residual 1,3-dibromopropane at 1008C
1
in vacuo gave 3 (3.82 g, 8.6 mmol, 84%). H NMR (DMSO-d ) d 8.15 (s,
6
1
H), 4.11 (t, 2 H), 3.80 (t, 2 H), 2.97 (m, 2 H), 2.39 (m, 2 H),
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 459–464

SYNTHESIS OF TRITIUM LABELLED L783483
463
1
3
1
.73 (m, 2 H), 0.98 (t, 3 H). C NMR (DMSO-d ) d 163.5, 155.5, 148.5
6
(
q, J¼ 38:3 Hz), 121.8, 120.5, 119.6 (q, J¼ 271:8 Hz), 116.4, 113.7, 71.9,
+
2.6, 30.9, 26.4, 22.1, 13.9. MS: M =443.
3
3
5- H]-6-(3-Bromopropyloxy)-7-propyl-3-trifluoromethyl-benzisoxazole
[
(4b). Compound 3 (3.8 mg, 8.5 mmol) was dissolved in ethanol (0.5 ml),
1
0% Pd on carbon (2.0 mg) was added and the mixture was treated with
tritium gas for 30 min.Volatile tritium compounds were removed by
lyophilisation with methanol (3 ꢀ 1 ml). Catalyst was removed by
filtration and the filtrate was concentrated in vacuo. The residue was
applied to a 20 ꢀ 20 ꢀ 0.05 (cm) silica gel TLC plate and eluted with n-
hexane/ethyl acetate 4:1 (v/v). The strongest UV-band (254 nm) (Rf
0
.43) containing compound 4b was eluted with ethyl acetate, followed
by removal of solvent in vacuo. The residue was chromatographed on a
Merck Lichrosper RP-18 250-10 column with acetonitrile/0.05 M
phosphoric acid 73:27 (v/v) (4 ml/min, UV-detection at 254 nm) as
eluent. The fraction from 28.3–32.3 min was collected, neutralized with
2
M sodium hydroxide (0.10 ml) and concentrated to a volume of
approximately 4 ml.The remaining aqueous phase was extracted with
ethyl acetate (3 ꢀ 2 ml). Drying over sodium sulphate, filtration and
evaporation of solvent in vacuo gave [5- H]-6-(3-bromopropyloxy)-7-
3
propyl-3-trifluoromethyl-benzisoxazole (4b), which was dissolved in
ethanol (5.00 ml). The analysis of this solution showed a total
radioactivity of 2990 MBq and a radiochemical purity of 99%. For
use in the following reaction ethanol was removed in vacuo, and
compound 4b was transferred to a 1 ml vial with ethyl acetate followed
by removal of solvent in a gentle stream of argon. Unlabelled 6-(3-
2
bromopropyloxy)-7-propyl-3-trifluoromethyl-benzisoxazole (4a) - used
1
as a reference compound - had the following spectroscopic data: H
NMR (DMSO-d ) d 7.76 (d, 1 H), 7.39 (d, 1 H), 4.29 (t, 2 H), 3.72
6
1
3
(t, 2 H), 2.87 (m, 2 H), 2.34 (m, 2 H), 1.65 (m, 2 H), 0.91 (t, 3 H).
C
NMR (DMSO-d ) d 164.0, 158.9, 149.0 (q, J¼ 37:8 Hz), 120.1 (q,
6
J¼ 271:3 Hz), 119.0, 112.9, 112.1, 109.9, 67.0, 31.8, 31.0, 25.0, 21.7,
+
3.8. MS: M =465.
1
3
-Chloro-4-(3-(7-propyl-3-trifluoromethyl-[5- H]-benzisoxazol-6-oxy)-
3
2
propylsulphanyl)phenylacetic acid (5b) (cf. reference ). 3-Chloro-4-
4
(
dimethylaminocarbonylsulphanyl)-phenylacetic acid methyl ester
6.4 mg, 0.022 mmol) was stirred under argon with a solution of 0.5 M
(
sodium methoxide in methanol (60 ml, 0.030 mmol) at 638C for
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 459–464

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G. GRUE-SØRENSEN AND N. HØJ
7
0 min.After cooling to room temperature this solution was transferred
3
to the vial containing [5- H]-6-(3-bromopropyloxy)-7-propyl-3-trifluoro-
methyl-benzisoxazole (4b), (2990 MBq) in an argon atmosphere (see
above). The mixture was stirred at 638C for 1 h. An aqueous solution of
1
M lithium hydroxide (30 ml, 0.030 mmol) was added and stirring was
continued at 638C for 15 min. After cooling to room temperature
acetonitrile/0.05 M phosphoric acid 73:27 (v/v) (1 ml) was added and the
product was isolated by preparative HPLC on a Merck Licrospher RP-
1
7
8 column (250 mm x 10 mm) with acetonitrile/0.05 M phosphoric acid
3:27 (v/v) (4 ml/min, 254 nm). The eluate containing the desired
product (20.0–22.5 min, 10 ml) was concentrated to a volume of approx.
ml and extracted with ethyl acetate (4 ꢀ 1 ml). The combined organic
2
phases were dried over sodium sulphate and solvent was removed in
vacuo. The residue was dissolved in ethanol (5 ml) and this solution of 5b
was characterized by liquid scintillation counting and HPLC (by
2
comparison with authentic unlabelled compound ). Radiochemical
yield: 2270 MBq (61 mCi); specific activity: 529 GBq/mmol, (14.3 Ci/
mmol); radiochemical purity: 98%.
Acknowledgements
We are indebted to Grethe Aagaard (NMR), Malene Mohr (MS),
Dr. Karen Margrethe Engell, Dr. Lene Merete Hansen and Peter Anker
Jꢀrgensen (HPLC) (Leo Pharma A/S) and to Dr. Ewald Bannwart
(catalytic tritiation) (RC TRITEC AG, Teufen, Switzerland) for
invaluable assistance.
References
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&
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. Belanger PC, Fortin R, Guidon Y, Rokach J, Yoakim C. (1984) Merck
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Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 459–464