Fast and efficient tritium labelling of the nonsteroidal anti-inflammatory drugs naproxen, tolmetin, and zomepirac

Article abstract of DOI:10.1002/jlcr.950

Fast and efficient tritium labelling of the nonsteroidal anti-inflammatory drugs naproxen, tolmetin and zomepirac is reported. Naproxen along with its (R)-enantiomer were labelled by catalytic tritium-halogen exchange of the corresponding 5-bromo derivatives providing [³H]naproxen with a specific activity of 25.4 Ci/mmol. Tolmetin and zomepirac were labelled by the hydrogen isotope exchange reaction using Crabtree's catalyst. This provided [³H]tolmetin and [3H]zomepirac with specific activities of 80.8 and 64.3 Ci/mmol, respectively. All compounds were obtained in high radiochemical purity (>98%). Copyright

Full text of DOI:10.1002/jlcr.950

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 569–576.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.950
Research Article
Fast and efficient tritium labelling of the nonsteroidal
anti-inflammatory drugs naproxen, tolmetin, and
zomepirac
Steen K. Johansen*, Lone Sꢀrensen and Lars Martiny
Department of Isotope Chemistry, Novo Nordisk A/S, Novo Nordisk Park, DK-2760
˚
Maløv, Denmark
Summary
Fast and efficient tritium labelling of the nonsteroidal anti-inflammatory
drugs naproxen, tolmetin and zomepirac is reported. Naproxen along with its
(R)-enantiomer were labelled by catalytic tritium–halogen exchange of the corre-
sponding 5-bromo derivatives providing [3H]naproxen with a specific activity of
25.4 Ci/mmol. Tolmetin and zomepirac were labelled by the hydrogen isotope
exchange reaction using Crabtree’s catalyst. This provided [3H]tolmetin and
[3H]zomepirac with specific activities of 80.8 and 64.3 Ci/mmol, respectively. All
compounds were obtained in high radiochemical purity (>98%). Copyright # 2005
John Wiley & Sons, Ltd.
Key Words: tritium; tolmetin; zomepirac; naproxen; Crabtree’s catalyst; NSAID
Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) are an important class of
drugs with widespread applications. Members of this class include ibuprofen,
naproxen, and tolmetin. They are used to reduce the pain and inflammation
caused by many conditions such as osteoarthritis and rheumatoid arthritis as
well as general pain relief (ibuprofen). Zomepirac, another member of the
class, was voluntarily withdrawn from use due to adverse allergic reactions in
1983.1
Information on the metabolism of these compounds in man and
animals and identification of the metabolic pathways can give important
information on the cause of the adverse reactions this class of drugs gives
rise to.2
*Correspondence to: Steen K. Johansen, Department of Isotope Chemistry, Novo Nordisk A/S, Novo
Nordisk Park, DK-2760 Malꢀv, Denmark. E-mail: skjo@novonordisk.com
Copyright # 2005 John Wiley & Sons, Ltd.
Received 10 February 2005
Revised 14 March 2005
Accepted 15 March 2005

570
S. K. JOHANSEN ET AL.
Figure 1. Structure of naproxen, tolmetin, and zomepirac
Labelling with radioactive isotopes such as tritium or carbon-14 is one of
the most efficient and convenient tools for the labelling of biological
compounds. In general, tritium labelling offers the advantage of short and
fast synthetic routes compared to carbon-14. This, combined with the high
specific radioactivity obtainable with tritium, makes tritium labelling very
useful for exploratory in vitro and in vivo investigations.
Here, we report on the tritium labelling of naproxen, tolmentin, and
zomepirac as well as the (R)-enantiomer of naproxen for use in in vitro and
in vivo investigations (Figure 1).
Results and discussion
Carbon-14-labelled naproxen is commercially available with the labelling
positioned in the O-methyl group. However, the O-methyl group is readily
metabolized making this particular tracer unsuited for the intended use.
Furthermore, we also wanted to label the (R)-enantiomer of naproxen, and
therefore, we decided to look at an alternative labelling route.
One obvious way of introducing the tritium label would be by reduction of
2-(6-methoxy-2-naphthyl)propenoic acid. However, a number of issues arise
with this synthetic strategy. First, although impressive enantioselectivities have
been obtained in the asymmetric reduction of this compound using chiral
ruthenium catalysts,3,4 the reaction is highly dependant on the hydrogen
pressure. In general, high pressures (>100 atm) are needed in order to achieve
high ee’s,4 which is incompatible with the use of tritium manifold equipment.
Secondly, 2-(6-methoxy-2-naphthyl)propenoic acid is not commercially
available, and while a number of procedures for the preparation of this
compound have been published,5 this makes a racemic approach (nonselective
reduction with tritium gas followed by chiral separation) less appealing.
Previously, we have achieved good results with labelling of activated
aromatics by introduction of bromine followed by tritium–halogen ex-
change.6,7 If this could be applied to the present task, then, naproxen could
be tritium-labelled in two simple steps, namely bromination and catalytic
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FAST AND EFFICIENT TRITIUM
571
tritium–halogen exchange. A further advantage of this approach would be the
elimination of a chiral purification step, as the chiral center in naproxen is not
affected in this synthetic sequence. This approach would also be easily
applicable to the labelling of the (R)-enantiomer, as both enantiomers are
commercially available.
Treatment of naproxen ((S)-1) with an excess of bromine in dichloro-
methane gave the crystalline bromo compound (S)-2 in quantitative yield
(Scheme 1). Bromination was expected to take place in the 5-position
analogous to the bromination of 2-naphthol8 and this was confirmed by 2D
1H-NMR (HMBC). Subsequent catalytic dehalogenation using tritium gas
and Pd/C in THF proceeded smoothly to give crude (S)-3, which was purified
by RP HPLC to give [3H]naproxen ((S)-3) in 70% overall yield with a
radiochemical purity >98%. The specific activity was determined to be
25.4 Ci/mmol as determined by mass spectroscopy.
Scheme 1. Tritium labelling of naproxen and the (R)-enantiomer of naproxen
Then, the (R)-enantiomer of naproxen ((R)-1) was tritium-labelled in exactly
the same manner. Conversion into the bromo compound (R)-2 was performed
in quantitative yield followed by catalytic dehalogenation giving (R)-3 in 60%
overall yield with a radiochemical purity >98% and a specific activity of
25.8 Ci/mmol.
Tolmetin9 and zomepirac10 have been labelled with carbon-14 as part of the
development of these drugs. Both compounds were labelled in the ketone
carbonyl group, however, no information on the labelling routes are available.
Likewise, no information on the labelling position or route is available for the
reported tritium labelling of tolmetin.11 Deuterium12- and tritium13-labelled
zomepirac have also been reported. Deuterium-labelled zomepirac was
synthesized in five steps with the label being introduced from D2O. Tritium
labelling using this method would be feasible; however, it did not seem
appealing due to the multi-step sequence and the practical issues concerning
use of tritium-labelled water. Tritium-labelled zomepirac was synthesized
by alkylation of N-desmethyl zomepirac with tritium-labelled methyl iodide.
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S. K. JOHANSEN ET AL.
This is a short and practical route; however it still requires the synthesis of the
N-desmethyl precursor.
A popular alternative to tritium labelling via multi-step synthetic routes is
the use of the iridium-catalysed hydrogen isotope exchange reaction. A
popular choice of catalyst for this reaction is Crabtree’s catalyst ([Ir(cod)
(Cy3P)(Py)]PF6) due to its stability and commercial availability. This reaction
has the benefit of working directly on the target compound, thus negating the
need for preparation of a precursor. Furthermore, the position of labelling is
generally very predictable with the labelling taking place predominantly in the
ortho-position(s) of aromatic systems possessing suitable directing groups such
as a carbonyl.14
We considered both tolmetin and zomepirac to be ideal candidates for the
hydrogen isotope exchange reaction, as the keto and acid carbonyl moieties
should be able to direct the labelling. Thus, treatment of tolmetin (4) with
tritium gas and one equivalent of Crabtree’s catalyst in dichloromethane
proceeded smoothly to give crude 5, which was purified by RP HPLC to give
[3H]tolmetin (5) in 28% yield with a radiochemical purity >98% (Scheme 2).
Likewise, zomepirac (6) was labelled using the same procedure to give
[3H]zomepirac (7) in 22% yield with a radiochemical purity >98%. The
specific activities were determined to be 80.8 and 64.3 Ci/mmol for 5 and 7,
respectively.
Scheme 2. Tritium labelling of tolmetin and zomepirac
The labelling was determined by tritium NMR to have taken place in the
two ortho-positions in the benzene moiety as well as the available positions in
the pyrrole moiety (two for 5 and one for 7) as shown in Scheme 2. Thus, the
labelling had been influenced by both directing groups, and is consistent with
the proposed 5- and 6-membered transition states.15ꢀ17
Conclusion
In summary, we have developed fast and efficient synthetic routes for tritium
labelling of the nonsteroidal anti-inflammatory drugs naproxen, tolmetin, and
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FAST AND EFFICIENT TRITIUM
573
zomepirac with high specific activities. Naproxen and its (R)-enantiomer were
labelled with tritium in two steps by catalytic tritium–halogen exchange of the
corresponding 5-bromo derivatives. This provided [3H]naproxen (25.4 Ci/
mmol) as well as the (R)-enantiomer (25.8 Ci/mmol) in 70 and 60% overall
yield, respectively. Tolmetin and zomepirac were labelled in one step by the
hydrogen isotope exchange reaction using Crabtree’s catalyst. This provided
[3H]tolmetin (80.8 Ci/mmol) and [3H]zomepirac (64.3 Ci/mmol) in 28 and 22%
overall yield, respectively. In all cases the labelled compounds were obtained in
high radiochemical purities. The cases presented highlight two of the fastest
and most efficient methods for tritium labelling, namely halogenation of
activated aromatic systems followed by catalytic tritium–halogen exchange
and the iridium-catalysed hydrogen isotope exchange reaction.
Experimental
General
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 was prepared in situ by
heating the uranium bed. Tolmetin, sodium salt dihydrate and zomepirac,
sodium salt were supplied by Sigma, naproxen by Riedel-de Haen, the
¨
(R)-enantiomer of naproxen by The Pharmaceutical University of Denmark
and Crabtree’s catalyst ([Ir(cod)(Cy3P)(Py)]PF6) by Aldrich. The free acids of
tolmetin and zomepirac were prepared by extraction with ethyl acetate from
an aqueous HCl solution of the corresponding salts. All other reagents and
solvents were of analytical grade and used without further purification. HPLC
was performed using a Merck Hitachi Intelligent Pump L6200A equipped with
a Supertherm Column Thermostat (Mikrolab Aarhus) (set at 408C), a Merck
LC Organizer with a Rheodyne injector, and a Merck Hitachi UV Detector
L4000 (detection at 248 nm for tolmetin/zomepirac and 276 nm for naproxen).
Detection of tritium was performed on a Canbarra Packard flow detector
500TR. Analytical and preparative HPLC was performed on a RP C18
column (4.6 ꢁ 250 mm, 5 mm, OdDMeSi 120 A, Novo Nordisk) with a flow of
1.0 ml/min. using one of the two following systems. System 1: 75% A 0–30 min
followed by 0% A for 10 min; and system 2: 65% A 0–30 min followed by 0%
A for 10 min (A: 10% acetonitrile in 0.1% aq. TFA; B: 90% acetonitrile in
0.1% aq. TFA). Quantitative radioactivity measurements were performed on a
Packard Tri-Carb 1000 liquid scintillation analyzer using Ultima FloTM
M
(PerkinElmer) as liquid scintillation cocktail. Specific activities were deter-
mined on a Sciex API 300 mass spectrometer equipped with an ionspray
1
3
interface. H- and H-NMR spectra were recorded on a Bruker DRX400
spectrometer.
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(2S)-2-(5-Bromo-6-methoxy-2-naphthyl)propanoic acid ((S)-2)
Bromine (263 mg, 1.64 mmol) in dichloromethane (1.2 ml) was added to a
solution of (2S)-2-(6-methoxy-2-naphthyl)propanoic acid ((S)-1) (76.7 mg,
0.333 mmol) in dichloromethane (15 ml). The reaction mixture was stirred for
1 h at room temperature and then concentrated to provide (S)-2 as colourless
crystals (104 mg, quantitative).
1H-NMR (400 MHz, CDCl3): d 1.61 (3 H, d, J ¼ 7:1), 3.92 (1 H, q, J ¼ 7:1),
4.03 (3 H, s), 7.28 (1 H, d, J ¼ 9:6, partly hidden by the CDCl3 peak), 7.54
(1 H, dd, J ¼ 2:0, 9.1), 7.71 (1 H, d, J ¼ 1:5), 7.79 (1 H, d, J ¼ 9:0), 8.19 (1 H,
d, J ¼ 9:1).
(2S)-2-(5[3H]-6-Methoxy-2-naphthyl)propanoic acid ([3H]naproxen) ((S)-3)
Pd/C (10%, 3.98 mg) and Et3N (10 ml, 0.072 mmol) were added to a solution
of (2S)-2-(5-bromo-6-methoxy-2-naphthyl)propanoic acid ((S)-2) (3.17 mg,
0.010 mmol) in THF (0.4 ml). The reaction mixture was degassed by three
freeze/thaw cycles and stirred overnight with tritium gas (8.7 Ci). Excess
tritium gas was reabsorbed on to a 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 ethanol (5 ml) to
give crude (S)-3 (238 mCi). Approximately 16.6 mCi of this solution was
purified by HPLC (System 2). This provided (S)-3 (12.34 mCi, 70% based on
(S)-2 with a radiochemical purity >98% (System 2). The specific radioactivity
was determined to be 25.4 Ci/mmol by MS.
(2R)-2-(5-Bromo-6-methoxy-2-naphthyl)propanoic acid ((R)-2)
(2R)-2-(6-Methoxy-2-naphthyl)propanoic acid ((R)-1) (43.7 mg, 0.190 mmol)
was reacted according to the procedure for (S)-2 to give (R)-2 as colourless
1
crystals (60.0 mg, quantitative). H-NMR as for compound (S)-2.
(2R)-2-(5[3H]-6-Methoxy-2-naphthyl)propanoic acid ((R)-3)
(2R)-2-(5-Bromo-6-methoxy-2-naphthyl)propanoic acid ((R)-2) (3.64 mg,
0.012 mmol) was tritiated according to the procedure for (S)-2 to give crude
(R)-3 (317 mCi). Approximately 22.2 mCi of the crude product was purified by
HPLC (System 2). This provided (R)-3 (12.94 mCi, 60% based on (R)-2 with a
radiochemical purity >98% (System 2). The specific radioactivity was
determined to be 25.8 Ci/mmol by MS.
[3H]1-Methyl-5-(p-toluoyl)pyrrole-2-acetic acid ([3H]tolmetin) (5)
Crabtree’s catalyst ([Ir(cod)(Cy3P)(Py)]PF6) (11.0 mg, 0.014 mmol) and 1-
methyl-5-(p-toluoyl)pyrrole-2-acetic acid (tolmetin) (4) (3.6 mg, 0.014 mmol)
were dissolved in dichloromethane (0.4 ml). The reaction mixture was degassed
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by three freeze/thaw cycles and stirred overnight with tritium gas (8.5 Ci).
Excess tritium gas was reabsorbed on to a 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 ethanol
(5 ml) to give crude 5 (1836 mCi). Approximately 46 mCi of this solution was
purified by HPLC (System 1). This provided 5 (7.95 mCi, 28% based on 4)
with a radiochemical purity >98% (System 1). The specific radioactivity was
determined to be 80.8 Ci/mmol by MS.
[3H]5-(p-Chlorobenzoyl)-1,4-dimethylpyrrole-2-acetic acid ([3H]zomepirac)
(7)
5-(p-Chlorobenzoyl)-1,4-dimethylpyrrole-2-acetic acid (zomepirac) (6) (2.1 mg,
0.007 mmol) was tritiated according to the procedure for 5 to give crude 7
(1609 mCi). Approximately 96.5 mCi of the crude product was purified by
HPLC (System 2) to provide 7 (6.02 mCi, 22% based on 6) with a
radiochemical purity >98% (System 2). The specific radioactivity was
determined to be 64.3 Ci/mmol by MS.
Acknowledgements
We are grateful to Mr Ole Wassmann for performing the mass spectra
analyses and Dr Henrik Olsen for performing the NMR analyses.
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