[14C] and [3H]-labelling of Ragaglitazar: A dual acting PPARα and PPARγ agonist with hypolipidemic and anti-diabetic activity

Article abstract of DOI:10.1002/jlcr.690

Currently, Ragaglitazar is being developed as a drug for the treatment of hyperglycaemia and hyperlipidemia in patients with type 2 diabetes. Here, we report the labelling of Ragaglitazar with carbon-14 and tritium for in vivo and in vitro investigations.

Full text of DOI:10.1002/jlcr.690

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 475–488.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.690
Research Article
[14C] and [3H]-labelling of Ragaglitazar:
A dual acting PPARa and PPARg agonist
with hypolipidemic and anti-diabetic activity
Jesper B. Kristensen*, Steen K. Johansen, Jacob S. Valsborg,
Lars Martiny, and Christian Foged
(
Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Maløv, Denmark
Summary
Currently, Ragaglitazar is being developed as a drug for the treatment of
hyperglycaemia and hyperlipidemia in patients with type 2 diabetes. Here, we
report the labelling of Ragaglitazar with carbon-14 and tritium for in vivo and
in vitro investigations. Two different carbon-14 labelled as well as two different
tritium labelled tracers of Ragaglitazar were synthesised. The carbon-14 label
was introduced from either ethyl bromo[2-14C]acetate (5 steps/33% overall
yield) or [U-14C]phenoxazine (4 steps/48% overall yield). Tritium was
incorporated either by catalytic tritiation of an alkene precursor followed by
chiral HPLC separation (2 steps/17% overall yield) or by catalytic tritium–
halogen exchange of an aryl bromide precursor (2 steps/68% overall
yield). Copyright # 2003 John Wiley & Sons, Ltd.
Key Words: carbon-14; tritium; type 2 diabetes; PPARa and PPARg agonist;
Ragaglitazar
Introduction
Ragaglitazar is being developed as a new anti-diabetic drug for the
treatment of type 2 diabetes. Ragaglitazar acts by increasing the
sensitivity towards insulin, thus normalizing blood glucose levels.
(
*Correspondance to: J. B. Kristensen, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Malꢀv,
Denmark. E-mail: jbkr@novonordisk.com
Copyright # 2003 John Wiley & Sons, Ltd.
Received 22 October 2002
Revised 11 November 2002
Accepted 18 November 2002

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J. B. KRISTENSEN ET AL.
On the molecular level, the target is the peroxisome proliferator-
activator receptors (PPAR), for which Ragaglitazar is an agonist
targeting both the PPARg and the PPARa receptor subtypes. These are
linked to the plasma glucose and plasma triglycerides lowering effect,
respectively.1 Ragaglitazar (Figure 1) is a phenoxazine analogue of
phenyl propionic acid and belongs to a new class of compounds, which
possess this unique dual acting ability.2
Drug candidates in development must be submitted for a series of
metabolism studies, for which a tracer labelled with carbon-14 is the
preferred choice. The labelling position is of paramount importance,
since the amount of information, that can be obtained, will depend on
this decision. In order to achieve as detailed information as possible, it
was decided to synthesize a tracer labelled with carbon-14 in the ethoxy
spacer as well as a tracer labelled in the phenoxazine moiety.
Tritium has less metabolic stability than carbon-14 in vivo, but it
offers the advantage of high specific radioactivity and is very useful for
exploratory in vitro investigations. In most cases, tritium also offers the
added advantage of shorter and faster synthetic routes compared to
carbon-14. Two tritium labelled tracers were prepared: one labelled in
the phenyl propionic acid part and one labelled in the phenoxazine
moiety.
Results and discussion
[Ethoxy-2-14C]Ragaglitazar
For the carbon-14 labelling of Ragaglitazar in the ethoxy spacer, we
decided to introduce the label starting from ethyl bromo[2-14C]acetate,
which is a commercially available starting material. Reaction of
Figure 1. Structure of Ragaglitazar
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[
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Scheme 1. Synthesis of [ethoxy-2-14C]Ragaglitazar
phenoxazine with ethyl bromo[2-14C]acetate proceeded to give the ester
1 in high yield (84%) (Scheme 1). It was necessary to run the reaction
with an excess of phenoxazine (5 eq.) in order to achieve full conversion
of ethyl bromo[2-14C]acetate. However, this necessitated a purification
step by HPLC.
The ester 1 was reduced to the alcohol 2 with lithium aluminium
hydride. It was necessary to add excess lithium aluminium hydride
(4 eq.) in order to achieve complete radiochemical conversion. Reaction
of 2 with methanesulfonyl chloride gave the activated sulfonate 3. The
reaction proceeded smoothly and quickly when using an excess (5 eq.) of
methanesulfonyl chloride and triethylamine and provided 3 in 84%
yield (two steps).
The sulfonate 3 was coupled to isopropyl (S)-3-(4-hydroxyphenyl)-2-
ethoxypropionate (4) in the presence of K2CO3 (toluene, 1008C, 16 h) to
give the ester 5 in 93% yield. Finally, the ester 5 was hydrolysed in
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J. B. KRISTENSEN ET AL.
aqueous NaOH. Purification by semi-preparative HPLC gave [ethoxy-
2-14C]Ragaglitazar (6) (51%) in an overall yield of 33% and with a
radiochemical purity of >98%. Analysis of the enantiomeric purity (ee
>97%) showed that no racemization had taken place during the
synthesis, neither in the coupling nor in the hydrolysis step. The specific
activity was determined by mass spectroscopy to be 55 mCi/mmol.
[Phenoxazine-U-14C]Ragaglitazar
The labelling of Ragaglitazar with carbon-14 in the phenoxazine moiety
started from [U-14C]phenoxazine (7), which was acquired from
Amersham Biosciences. Although it would be possible to use the same
synthetic strategy as for labelling in the ethoxy spacer, this would not be
practical, as the first step requires the use of five equivalents of
phenoxazine. Thus, a new synthetic strategy was needed for this
synthesis.
The reaction between [U-14C]phenoxazine (7) and ethylene oxide
offered the opportunity of a simple one step route to 2-([phenoxazine-
U-14C]-10-yl)ethanol (8) (Scheme 2). However, it was difficult to control
the addition of the ethylene oxide gas and excess ethylene oxide was
found to produce a large amount of impurities. We found that by
employing a large excess of n-butyllithium (25 eq.) that this sensitivity
towards ethylene oxide was negated, and 8 was easily produced in high
yield (100% crude yield). Excess n-butyllithium most likely acts by
quenching excess ethylene oxide, thus preventing it from reacting with 8.
Conversion of the alcohol 8 to the sulfonate 9 was carried out in
quantitative yield similar to the procedure for the preparation of 3.
Although the coupling reaction using K2CO3 proceeded without
major problems, we felt that room for improvement still existed.
Especially, we wanted to shorten the reaction time, if this could be
performed without compromising yield and purity. For this reason, we
decided to investigate the use of a different base in the coupling step.
Winters et al. have compared the alkylation of potassium and cesium
phenolates with different alkylating agents.3 They found the use of
Cs2CO3 as the base superior to K2CO3, as it gave accelerated reaction
rates, higher yields, and also provided for the introduction of a wider
range of alkyl groups. This is attributed to the greater polarizability and
reduced solvation of the cesium cation.3
This proved to be true in our case as well. The sulfonate 9 was
coupled with 4 using Cs2CO3 in toluene at 1008C. These conditions
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[
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Scheme 2. Synthesis of [phenoxazine-U-14C]Ragaglitazar
proceeded to give 10 in quantitative yield within a relatively short
reaction time (2–4 h). Whilst the crude yield proved higher (100% vs.
93%), the purity was slightly lower (76% vs 87%) making the two
reactions comparable, when the two factors were combined.
The final hydrolytic step toward [phenoxazine-U-14C]Ragaglitazar
(11) was carried out as described above to give 11 in an overall yield of
48% and with a radiochemical purity of >98%. The enantiomeric
purity was found to be >97%, which again showed that no
racemization had taken place during synthesis. The specific activity
was determined by mass spectroscopy to be 81 mCi/mmol.
[Propionic-2,3-3H2]Ragaglitazar
Whereas labelling with carbon-14 often requires lengthy syntheses in
order to incorporate the label in a metabolically stable position, tritium
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J. B. KRISTENSEN ET AL.
Scheme 3. Synthesis of [propionic-2,3-3H2]Ragaglitazar
labelling more often relies on the availability of a suitable precursor,
which in a few steps can lead to the desired molecule.
Our first strategy for the synthesis of tritium labelled Ragaglitazar
was based on the catalytic tritiation of acrylic acid 12, which in one step
would lead to tritium labelled Ragaglitazar, albeit in the racemic form.
Reaction of 12 with tritium gas and Pd/C in DMF proceeded smoothly
to give crude racemic 13 (66% } based on 12) (Scheme 3). Separation
by chiral HPLC followed by purification by reverse phase HPLC
provided the desired enantiomer of [propionic-2,3-3H2]Ragaglitazar
(13) in 17% overall yield with a radiochemical purity >98% and an
enantiomeric purity >97%. The specific radioactivity was determined
to be 27 Ci/mmol (MS).
[Phenoxazine-6,14-3H2]Ragaglitazar
The relatively low specific activity obtained in the synthesis of 13
combined with the need for chiral separation prompted us to develop
a second method based on catalytic debromination. In our experience,
this routinely gives high specific activities (25–30 Ci/mmol per
atom incorporated), whereas tritiation of double bonds often
gives lower incorporation of tritium (10–15 Ci/mmol per atom
incorporated).
The synthetic strategy was to obtain a brominated precursor of the
Ragaglitazar isopropyl ester with the correct chiral configuration using
much of the chemistry described above. Tritium would be introduced by
catalytic dehalogenation followed by hydrolysis of the ester to complete
the synthesis.
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[
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Scheme 4. Synthesis of [phenoxazine-6,14-3H2]Ragaglitazar
Experimentation showed that direct bromination of the phenoxazine
ring system in 14 was a feasible approach and with a little optimization
it was possible to isolate the dibrominated compound 15 in one simple
step in high yield (88%) (Scheme 4). Assignment of structure was
performed by 2D NMR (1H-13C HSQC and HMBC), which clearly
demonstrated the incorporation of bromine at positions 6 and 14. The
ester 17 was synthesized using the same methodology employed in the
syntheses of 5 and 10 and provided 17 in 77% yield (two steps).
Catalytic dehalogenation using tritium gas and Pd/C in THF proceeded
smoothly to give crude 18 (75%), which was hydrolysed directly to
provide [phenoxazine-6,14-3H2]Ragaglitazar (19) (90%) in 68% overall
yield for the steps employing radioactivity. The radiochemical purity
was >98% with the specific radioactivity determined to be 53 Ci/mmol
(MS).
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Conclusion
In summary, we have developed synthetic methods for the labelling of
the dual acting PPARa and PPARg agonist Ragaglitazar with carbon-
14 and tritium. Ragaglitazar was labelled with carbon-14 in the ethoxy
spacer part of the molecule as well in the phenoxazine moiety, whereas
the tritium label was introduced in the phenyl propionic acid part as
well as in the phenoxazine ring. The same methodology could be
employed in most of the synthetic sequences, thus ensuring a high
degree of synergy. Some improvements in the individual synthetic steps
were also developed in the process. In all cases, Ragaglitazar was
obtained in high radiochemical and enantiomeric purity as well as with
high specific activity.
Experimental
General
Ethyl bromo[2-14C]acetate (specific activity: 55 mCi/mmol) and
[U-14C]phenoxazine (specific activity: 84 mCi/mmol) were supplied by
Amersham Biosciences, UK. Reactions using tritium gas were
performed on a custom built tritium handling unit from RC Tritec
AG, Switzerland, who also supplied the tritium gas. Tritium gas was
stored in the form of U3H3 and released in situ by heating the uranium
bed. Phenoxazine, 2-(phenoxazine-10-yl)ethanol, and isopropyl (S)-3-
(4-hydroxyphenyl)-2-ethoxypropionate were all supplied by Novo
Nordisk A/S. 3-[4-[2-(Phenoxazine-10-yl)ethoxy]phenyl]-2-ethoxypro-
peonic acid was supplied by Dr Reddy’s Research Foundation, India.
All reagents and solvents were of analytical grade and used without
further purification.
HPLC was performed using a Merck Hitachi Intelligent pump L6200
equipped with a Merck Hitachi Column Thermostat T6300 with a
Rheodyne Injector and Merck Hitachi UV detector L4000 (detection at
245 nm). Detection of [14C] and [3H] was performed on a Canbarra
Packard Flow Detector 500 TR. Reactions were monitored using the
following system: RP C18 column (4.6 ꢀ 250 mm, 5 mm, OdDMeSi
(
120 A, Novo Nordisk) with a gradient running from 60:40 to 20:80 A/B
over 30 min followed by 0:100 A/B for 10 min (A: 10% acetonitrile in
0.1% aqueous TFA, B: 90% acetonitrile in 0.1% aqueous TFA).
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Enantiomeric purity was analysed using the following system: Chiralcel
OJ column (4.6 ꢀ 250 mm, Diacel Chemical Industries) with isocratic
elution (hexane:ethanol:AcOH 90:10:0.3; 1.2 ml/min). Radioactivity
measurements were performed on a Packard Tri-Carb 1000 liquid
scintillation analyzer using Ultima FloTM M (Packard Bioscience) as
liquid scintillation cocktail. Specific activities were determined on a Sciex
1
API 300 mass spectrometer equipped with an ionspray interface. H
NMR spectra were recorded on a Bruker DRX 300 spectrometer. Flash
(
chromatography was performed with silica gel 60 A (Merck, 230–400
mesh), and TLC was performed on Merck F254 precoated silica plates.
Ethyl (phenoxazine-10-yl)[2-14C]acetate ð1Þ
Phenoxazine (332 mg, 1.8 mmol) was dissolved in NMP (0.6 ml) in a
1 ml reaction vial. Ethyl bromo[2-14C]acetate in NMP (175 ml, 20 mCi,
0.36 mmol) was added, and the vial was filled with NMP and sealed
tightly to prevent evaporation. The reaction mixture was stirred
overnight at 808C. The crude product was purified by semi-preparative
(
HPLC using a RP C18 column (10 ꢀ 250 mm, 10 mm, OdDMeSi 120 A,
Novo Nordisk) and a gradient running from 100:0 to 20:80 A/B over
30 min (A: 10% acetonitrile in 0.1% aqueous TFA, B: 90% acetonitrile
in 0.1% aqueous TFA) to provide 1 (12.6 mCi). A total of 12 repetitions
of this procedure provided 1 in a total yield of 219 mCi (84%) with a
radiochemical purity of >98%.
2-(Phenoxazine-10-yl)[2-14C]ethanol ð2Þ
Ethyl (phenoxazine-10-yl)[2-14C]acetate (1) (219 mCi, 4.0 mmol) in THF
(50 ml) was added to a slurry of lithium aluminium hydride (0.20 g,
5.3 mmol) in THF (10 ml). The reaction mixture was stirred at room
temperature, and additional lithium aluminium hydride (0.20 g,
5.3 mmol) was added after 1 and 2 h. The reaction was quenched after
2.5 h by addition of water (200 ml) and aqueous HCl (2N, 15 ml), and
the mixture was extracted with EtOAc (2 ꢀ 100 ml). The combined
organic layers provided 2 (185 mCi, 84%) with a radiochemical purity
of >98%.
2-(Phenoxazine-10-yl)[2-14C]ethanol methanesulfonate ð3Þ
Triethylamine (0.6 ml, 4.3 mmol) and methanesulfonyl chloride (0.3 ml,
3.8 mmol) were added to a solution of 2-(phenoxazine-10-yl)[2-14C]
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J. B. KRISTENSEN ET AL.
ethanol (2) (42 mCi, 0.77 mmol) in dichloromethane (25 ml). The
reaction mixture was stirred for 3 h at room temperature and quenched
by addition of water (25 ml). The mixture was extracted with
dichloromethane (2 ꢀ 25 ml), the combined organic layers were con-
centrated to dryness in vacuo and the residue dissolved in toluene
(25 ml). This provided 3 (42 mCi, 100%) with a radiochemical purity
of 95%.
Isopropyl
(S)-3-[4-[2-(phenoxazine-10-yl)[2-14C]ethoxy]phenyl]-2-
ethoxypropionate ð5Þ
Isopropyl (S)-3-(4-hydroxyphenyl)-2-ethoxypropionate (4) (544 mg,
2.2 mmol) and K2CO3 (372 mg, 2.7 mmol) were added to a solution of
2-(phenoxazine-10-yl)[2-14C]ethanol methanesulfonate (3) (15 mCi,
0.27 mmol) in toluene (10 ml). The reaction mixture was stirred
overnight at 1008C. The reaction was quenched by addition of water
(50 ml) and EtOAc (50 ml) followed by careful addition of aqueous HCl
(2N), until pH 2–3 was reached. The mixture was extracted with EtOAc
(2 ꢀ 50 ml), the combined organic layers were concentrated in vacuo,
and the residue was dissolved in 2-propanol (15 ml). This gave 5
(14 mCi, 93%) with a radiochemical purity of 87%.
(S)-3-[4-[2-(Phenoxazine-10-yl)[2-14C]ethoxy]phenyl]-2-ethoxypro-
pionic acid ð6Þ
Aqueous NaOH (5 N, 20 ml) was added to a solution of isopropyl (S)-3-
[4-[2-(phenoxazine-10-yl)[2-14C]ethoxy]phenyl]-2-ethoxypropionate (5)
(14 mCi, 0.26 mmol) in 2-propanol (15 ml), and the reaction mixture
stirred for 5 h at 308C. The reaction was quenched by addition of water
(50 ml) and EtOAc (50 ml) followed by addition of aqueous HCl (2N)
until pH 2. The mixture was extracted with EtOAc (3 ꢀ 50 ml), and the
combined organic layers were concentrated to dryness in vacuo. The
crude product was purified by semi-preparative HPLC using a RP C18
(
column (10 ꢀ 250 mm, 10 mm, OdDMeSi 120 A, Novo Nordisk) and
isocratic elution (acetonitrile:water:AcOH 58:42:0.1, 5 ml/min). This
provided [ethoxy-2-14C]Ragaglitazar (6) (7.1 mCi, 51%) in 33% overall
yield with a radiochemical purity >98%. The enantiomeric purity was
determined to be >97% (HPLC), and the specific radioactivity was
determined to be 55 mCi/mmol (MS).
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2-([U-14C]Phenoxazine-10-yl)ethanol ð8Þ
[U-14C]Phenoxazine (7) (15 mCi, 0.18 mmol) was dissolved in TBME
(8 ml) under a N2 atmosphere and cooled to ꢁ108C to -20C8. n-
Butyllithium (1.6 M in hexane, 2.8 ml, 4.5 mmol) was slowly added and
the mixture stirred for 7 minutes. Then, ethylene oxide gas was bubbled
directly into the reaction mixture for 30 seconds. The reaction mixture
was stirred for 1 hour at room temperature and quenched by addition of
aqueous HCl (1N, 3 ml) and water (45 ml). The mixture was extracted
with toluene (30 ml), and the organic layer was concentrated in vacuo
and the residue dissolved in dichloromethane (15 ml). This gave 8 (15
mCi, 100%) with a radiochemical purity of 80%.
2-([U-14C] Phenoxazine-10-yl)ethanol methanesulfonate ð9Þ
2-([U-14C]Phenoxazine-10-yl)ethanol (8) (15 mCi, 0.18 mmol) was
reacted according to the procedure for 3. This provided 9 (15 mCi,
100%) with a radiochemical purity of 81%.
Isopropyl (S)-3-[4-[2-([U-14C]phenoxazine)-10-yl)ethoxy]phenyl]-2-
ethoxypropionate ð10Þ
2-([U-14C]Phenoxazine-10-yl)ethanol methanesulfonate (9) (15 mCi,
0.18 mmol) was reacted for 4 h at 1008C according to the procedure
for 5 using Cs2CO3 instead of K2CO3. This gave 10 (15 mCi, 100%)
with a radiochemical purity of 76%.
(S)-3-[4-[2-([U-14C]Phenoxazine-10-yl)ethoxy]phenyl]-2-ethoxypro-
pionic acid ð11Þ
Isopropyl (S)-3-[4-[2-([U-14C]phenoxazine-10-yl)ethoxy]phenyl]-2-eth-
oxypropionate (10) (15 mCi, 0.18 mmol) was hydrolysed for 1 h at
608C according to the procedure for 6. The crude product was purified
by semi-preparative HPLC using a RP C18 column (10 ꢀ 250 mm,
(
10 mm, OdDMeSi 120 A, Novo Nordisk) and isocratic elution (acetoni-
trile:water:AcOH 44:56:0.1; 5 ml/min). This provided [phenoxazine-
U-14C]Ragaglitazar (11) (7.2 mCi, 48%) in 48% overall yield with a
radiochemical purity >98%. The enantiomeric purity was determined
to be >97% (HPLC), and the specific radioactivity was determined to
be 81 mCi/mmol (MS).
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J. B. KRISTENSEN ET AL.
ðSÞ-3-[4-[2-(Phenoxazine-10-yl)ethoxy]phenyl]-2-[2,3-di-3H]-ethoxy-
propionic acid ð13Þ
Pd/C (10%, 1.2 mg) was added to a solution of 3-[4-[2-(phenoxazine-10-
yl)ethoxy]phenyl]-2-ethoxypropeonic acid (12) (2.3 mg, 5.5 mmol) in
DMF (0.55 ml). The reaction mixture was degassed by three freeze/thaw
cycles and then stirred overnight with tritium gas (7.9 Ci) at room
temperature. Excess tritium gas was re-absorbed on to an uranium
waste bed followed by filtration of the reaction mixture and lyophilisa-
tion with ethanol (3 ꢀ 1 ml) in order to remove labile tritium. Finally,
the residue was dissolved in ethanol (5 ml) to give crude 13 (97 mCi,
66%) with a radiochemical purity of 81%.
Purification by chiral HPLC using a Chiralcel OJ column
(4.6 ꢀ 250 mm, Diacel Chemical Industries) and isocratic elution
(hexane:ethanol:AcOH 90:10:0.3; 1.2 ml/min) gave the (S)-enantiomer
of 13 (27 mCi) in 96% radiochemical purity. 3.4 mCi of this was further
purified by HPLC using a RP C18 column (4.6 ꢀ 250 mm, 5 mm,
(
OdDMeSi 120 A, Novo Nordisk) and isocratic elution (ethanol:water
57:43; 0.5 ml/min). This provided [propionic-2,3-3H2]Ragaglitazar (13)
(3.2 mCi, 17%) with a radiochemical purity >98%. The compound was
formulated in the HPLC pool, as it is unstable towards concentration.
The enantiomeric purity was determined to be >97% (HPLC), and the
specific radioactivity was determined to be 27 Ci/mmol (MS).
2-(6,14-Di-bromo-phenoxazine-10-yl)ethanol ð15Þ
A solution of Br2 in dichloromethane (0.18 g/ml, 1.0 ml, 1.1 mmol) was
added to a solution of 2-(phenoxazine-10-yl)ethanol (14) (0.12 g,
0.53 mmol) in dichloromethane (10 ml). The reaction mixture was
stirred for 1 hour at room temperature and concentrated in vacuo.
Purification by flash chromatography (EtAOc/heptane 4:6) gave 15 as
1
colourless crystals (0.18 g, 88%). H NMR (300 MHz, CDCl3) d 3.67
(2 H, t, J 6 Hz), 3.88 (2 H, t, J 6 Hz), 5.29 (1 H, s), 6.45 (2 H, t, J 8.5 Hz),
6.74 (2 H, d, J 2 Hz), 6.89 (2 H, dd, J 2.0, 8.5 Hz).
2-(6,14-Di-bromo-phenoxazine-10-yl)ethanol methanesulfonate ð16Þ
2-(6,14-Di-bromo-phenoxazine-10-yl)ethanol (15) (97 mg, 0.25 mmol)
was reacted according to the procedure for 3. This provided 16 as off-
white crystals (0.13 g, 100%), which were used directly in the following
step.
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Isopropyl (S)-3-[4-[2-(6,14-dibromo-phenoxazine-10-yl)ethoxy]
phenyl]-2-ethoxypropionate ð17Þ
Isopropyl (S)-3-(4-hydroxyphenyl)-2-ethoxypropionate (4) (129 mg,
0.50 mmol) and Cs2CO3 (230 mg, 0.71 mmol) were added to a solution
of 2-(6,14-di-bromo-phenoxazine-10-yl)ethanol methanesulfonate (16)
(112 mg, 0.25 mmol) in toluene (10 ml). The reaction mixture was stirred
for 2 h at 1008C. Then, the reaction was quenched by addition of water
(10 ml) and toluene (15 ml), and the mixture extracted with toluene
(2 ꢀ 25 ml). The combined organic layers were washed with brine
(10 ml), dried (MgSO4), and concentrated in vacuo. Purification by flash
chromatography (EtOAc/heptane 1:4) gave 17 as a clear oil (0.12 g,
1
77%). H NMR (300 MHz, CDCl3) d 1.15 (3 H, t, J 7.0 Hz), 1.18 (3 H,
d, J 6.5 Hz), 1.24 (3 H, d, J 6.5 Hz), 2.93 (2 H, m), 3.33 (1 H, m), 3.58
(1 H, m), 3.86 (2 H, t, J 6.5 Hz), 3.92 (1 H, dd, J 6.0, 7.0 Hz), 4.12 (2 H, t,
J 6.5 Hz), 5.04 (1 H, m), 6.47 (2 H, d, J 8.5 Hz), 6.73 (2 H, d, J 2.0 Hz),
6.77 (2 H, d, J 8.5 Hz), 6.89 (2 H, dd, J 2.0, 8.5 Hz), 7.16 (2 H, d, J
8.5 Hz).
Isopropyl (S)-3-[4-[2-([6,14-di-3H-phenoxazine]-10-yl)ethoxy]phenyl]-
2-ethoxypropionate ð18Þ
Pd/C (10%, 7.0 mg) and Et3N (10 ml, 72 mmol) were added to a solution
of isopropyl (S)-3-[4-[2-(6,14-dibromo-phenoxazine-10-yl)ethoxy]phe-
nyl]-2-ethoxypropionate (17) (7.7 mg, 12 mmol) in THF (0.4 ml). The
reaction mixture was degassed by three freeze/thaw cycles and stirred
overnight with tritium gas (9.5 Ci). Excess tritium gas was re-absorbed
on to an uranium waste bed followed by filtration of the reaction
mixture and lyophilization with ethanol (3 ꢀ 1 ml) in order to remove
labile tritium. Finally, the residue was dissolved in acetonitrile (5 ml) to
give 18 (470 mCi, 75%) with a radiochemical purity of 88%.
(S)-3-[4-[2-([6,14-Di-3H]-phenoxazine-10-yl)ethoxy]phenyl]-2-ethoxy-
propionic acid ð19Þ
Aqueous NaOH (1 N, 0.3 ml) was added to a solution of isopropyl (S)-
3-[4-[2-([6,14-di-3H]-phenoxazine-10-yl)ethoxy]phenyl]-2-ethoxypropio-
nate (18) (70.5 mCi, 1.3 mmol) in acetonitrile (1.5 ml), and the reaction
mixture was stirred for 5 hours at 608C. The reaction was quenched by
addition of aqueous HCl (2N, 0.3 ml). Approximately 20 mCi of this
solution was purified by HPLC using a RP C18 column (4.6 ꢀ 250 mm,
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 475–488

488
J. B. KRISTENSEN ET AL.
(
5 mm, OdDMeSi 120 A, Novo Nordisk) and isocratic elution (acetoni-
trile:water:AcOH 42:58:0.1; 1 ml/min). This provided [phenoxazine-
6,14-3H2]Ragaglitazar (19) (18 mCi, 90%) in 46% overall yield with a
radiochemical purity >98%. The specific radioactivity was determined
to be 53 Ci/mmol (MS).
Acknowledgements
We are grateful to Mr Henrik Olsen for the structural elucidation of
compound 15, Mr Ole Wassmann for mass spectra analyses, and Dr
Reddy’s Research Foundation, India for supplying compound 12. We
would also like to thank Mrs Lone Sꢀrensen and Mrs Anne-Mette Dall
for invaluable technical assistance.
References
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2. Sauerberg, P, Pettersson I, Jeppesen L, et al. J Med Chem 2002; 45: 789–804.
3. Winters RT, Sercel AD, Showalter HDH. Synthesis 1988; 712–714.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 475–488