Isotope labelling by reduction of nitriles: Application to the synthesis of isotopologues of tolmetin and celecoxib

Article abstract of DOI:10.1002/jlcr.3492

The aryl methyl group is found in many drug-like compounds, but there are limited ways of preparing compounds with an isotope label in this methyl position. The process of cyanation of an aryl halide followed by complete reduction of the nitrile to a methyl group was investigated as a route for preparing stable and radiolabelled isotopologues of drug-like compounds. Using this methodology, carbon-13, deuterium, carbon-14, and tritium labelled isotopologues of the nonsteroidal anti-inflammatory drug tolmetin were produced, as well as carbon-13, deuterium, and carbon-14 labelled isotopologues of another nonsteroidal anti-inflammatory drug, celecoxib. The radiolabelled compounds were produced at high specific activity and the stable isotope labelled compounds with high incorporation making them suitable for use as internal standards in mass spectrometry assays. This approach provides a common synthetic route to multiple isotopologues of compounds using inexpensive and readily available labelled starting materials.

Full text of DOI:10.1002/jlcr.3492

Isotope Labelling by Reduction of Nitriles: Application to the
Synthesis of Isotopologues of Tolmetin and Celecoxib
Short title: Isotope Labelling by Reduction of Nitriles
Authors: Kate Ellis-Sawyer, Ryan A. Bragg, Nick Bushby*, Charles S. Elmore and Michael
J. Hickey
Corresponding author:
Nick Bushby
AstraZeneca UK Ltd.
Mereside
Alderley Park
Macclesfield
Cheshire SK10 4TG
United Kingdom
Email: nick.bushby@astrazeneca.com
Abstract
The aryl methyl group is found in many drug-like compounds, but there are limited ways of
preparing compounds with an isotope label in this methyl position. The process of cyanation
of an aryl halide followed by complete reduction of the nitrile to a methyl group was
investigated as a route for preparing stable and radiolabelled isotopologues of drug-like
compounds. Using this methodology, carbon-13, deuterium, carbon-14 and tritium labelled
isotopologues of the non-steroidal anti-inflammatory drug (NSAID) tolmetin were produced,
as well as carbon-13, deuterium and carbon-14 labelled isotopologues of another NSAID,
celecoxib. The radiolabelled compounds were produced at high specific activity and the
stable isotope labelled compounds with high incorporation making them suitable for use as
internal standards in mass spectrometry assays. This approach provides a common synthetic
route to multiple isotopologues of compounds using inexpensive and readily available
labelled starting materials.
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doi: 10.1002/jlcr.3492
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Introduction
The complete reduction of aromatic nitrile groups under palladium-catalysed hydrogenation
conditions to give a methyl group is a known but little-used process.1 The aromatic nitrile
compounds required for this process are readily formed by palladium-catalysed cross
coupling reaction of the corresponding aromatic halides with cyanide.2 Combining these two
reactions leads to a synthetic route in which the carbon of the methyl group can be
isotopically labelled by the use of labelled cyanides and the hydrogen positions by carrying
out the reduction with hydrogen isotopes (Scheme 1). A common route can therefore be used
to make carbon-14, stable isotope and tritium labelled isotopologues of organic compounds.
The tolyl group is a common motif found in many drug-like compounds (Figure 1), including
the non-steroidal anti-inflammatory drugs (NSAIDs) tolmetin 1a and celecoxib 2a, the
tyrosine kinase inhibitor imatinib (Gleevec) 3, and the local anaesthetic lidocaine 4.
Labelling in the methyl position of a tolyl group should provide labelled compounds suitable
for a variety of applications. Synthesis of a stable isotope labelled compound with both
carbon-13 and three deuterium atoms would give a mass increase of +4 over the unlabelled
compound. In most cases this would make it suitable for use as an internal standard in mass
spectrometry bioanalysis assays, as long as sufficient levels of deuterium incorporation were
achieved. The methyl position is known to be susceptible to metabolism involving oxidation
to the alcohol and the carboxylic acid by the cytochrome P450 enzymes CYP3A4 and
CYP2C9.3,4 This means that in metabolism studies hydrogen isotopes could be lost from the
methyl position, and for this reason compounds with tritium labels in the methyl position
would often need to find applications in studies that do not involve metabolism. It is rare for
an aryl methyl to be removed by metabolism, so a carbon-14 labelled methyl would in
general be suitable for ADME studies.
Other potential methods for labelling these methyl positions include the use of labelled
methyl iodide following metal-halogen exchange on the aryl ring,5 Stille coupling of the
organotin compound with labelled methyl iodide,6 or Suzuki-type coupling with labelled
methyl iodide7 or a labelled methyl borinate.8
Due to the relative ease of preparing the halogenated starting materials, the drugs tolmetin 1a
and celecoxib 2a were chosen as examples to test this synthetic route. Tolmetin 1a has
previously been labelled with carbon-14 in the ketone carbon9 and with tritium in multiple
positions on the toluene and pyrazole rings.10,11 Celecoxib has been labelled with carbon-14
in the toluene12 and pyrazole rings13 and with deuterium in the toluene14 and
phenylsulfonamide rings.15
Results and Discussion
Tolmetin
The iodinated tolmetin analogue 7 was prepared by the condensation of 4-iodobenzoyl
chloride 5 and the substituted pyrrole 6 in refluxing xylene (Scheme 2).16 The reaction
proceeded in moderate yield (43%) but attempts to improve on this by the addition of DBN17
or LiI18 led to lower recoveries. For the cyanation step, initially potassium cyanide was
investigated as the cyanide source as this is the cheapest source of isotopically labelled
cyanide. Although this worked in acceptable yield (typical conditions: KCN, Pd(PPh3)4, CuI,
70°C for 18 h) it was found that the subsequent reduction step did not work with material
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prepared in this way despite purification of the nitrile by both normal and reverse phase
chromatography. For this reason, alternative conditions using zinc cyanide as the cyanide
source along with palladium catalyst and Xantphos ligand under microwave heating were
used to give the nitrile derivatives 8a-c in good yield (72-76%).19 In the case of the carbon-13
labelled nitrile 8b, zinc [13C,15N]cyanide was used despite the fact that the nitrogen-15 label
would be lost in the reduction step.
The conditions for the chemoselective reduction of the nitrile were optimised with different solvents
and reduction times. In protic solvents the reduction of the nitrile 8a proceeded smoothly with
palladium on carbon under an atmosphere of hydrogen; however, extended reaction times led to
reduction of the ketone. When the reduction of the carbon-13 labelled nitrile 8b was carried out with
deuterium gas, isotope incorporation of only 0.5 d/mol was achieved. Changing the solvent to
d4-methanol gave 9b with incorporation of 5 deuterium atoms, the extra two being alpha to the ester.
These exchanged out rapidly during purification and in the subsequent hydrolysis step to give the
[13C, 2H3]tolmetin 1b with no unlabelled tolmetin detected by mass spectrometry. The reduction of the
unlabelled nitrile compound 8a with tritium gas was carried out at sub-atmospheric pressures and
after hydrolysis gave [3H]tolmetin 1d with a specific activity of 20.5 Ci/mmol, with tritium
incorporated exclusively in the methyl position. Synthesis of [14C]tolmetin 1c by cyanation with zinc
[14C]cyanide to give the nitrile 8c, followed by reduction with hydrogen gas and hydrolysis gave
material at high specific activity (55.6 mCi/mmol) and radiochemical purity.
Celecoxib
The iodinated celecoxib analogue 14 was prepared in a two-step process (Scheme 3).20,21
Condensation of the iodoacetophenone 10 with ethyl trifluoroacetate 11 gave the 1,3-dicarbonyl 12
which cyclised regioselectively with the hydrazine 13 to give the iodinated intermediate 14. The rest
of the reaction sequence was initially trialled with unlabelled reagents. The same cyanation protocol
as used for tolmetin worked well to give the nitrile 15a, although longer reaction times were required
for complete reaction. The subsequent hydrogenation gave mainly partial reduction to the amine 16
after 4.5 h. Resubmitting the mixture to the reaction conditions for an additional 22 h resulted in
complete reduction to the methyl 2a.
Using this methodology, [13C,2H3]celecoxib 2b was produced with high deuterium
incorporation (2.9 d/mol) when d4-methanol was used as the solvent in the reduction step.
[14C]Celecoxib 2c was prepared in high specific activity (55.8 mCi/mmol) and radiochemical
purity (>99%). [3H]Celecoxib was not prepared, but this methodology should provide a route
to this compound if required.
Conclusion
Cyanation followed by complete reduction has proven to be a useful approach to the
synthesis of drug-like compounds containing aryl methyl groups. It allows for carbon-14,
carbon-13, deuterium and tritium labelled isotopologues suitable for use in ADME and mass
spectrometry assays to be prepared using a common synthetic route and using readily
available and inexpensive labelled starting materials.
Acknowledgments
The authors would like to thank Dr Eva M. Lenz for NMR spectrometry analysis.
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Experimental
Microwave reactions were carried out using a CEM Explore (CEM Corporation, NC, USA).
Column chromatography was carried out using pre-packed silica gel cartridges (SiliCycle,
Quebec, Canada) on an Isco Companion (Teledyne Isco, NE, USA). 1H NMR spectra were
recorded on a Bruker (600 MHz) using the stated solvent. Chemical shifts (δ) in ppm are
quoted relative to CDCl3 (δ 7.26 ppm) and DMSO-d6 (δ 2.50 ppm). Liquid chromatography-
mass spectrometry (LC-MS) data was collected using a Waters Alliance LC (Waters
Corporation, MA, USA) with Waters ZQ mass detector. Analytical HPLC data was recorded
using Agilent 1200 HPLC system with a β-Ram Flow Scintillation Analyser, using the
following conditions: Waters Sunfire C18, 3.5 µm, 4.6 x 100 mm column at 40°C, eluting
with 5% acetonitrile/water + 0.1% TFA to 95% acetonitrile/water + 0.1% TFA over a 32
minute gradient. Specific activities were determined gravimetrically with a Packard TriCarb
2100CA Liquid Scintillation Analyser (Packard Instrument Company Inc., IL, USA) using
Ultima GoldTM cocktail. Reactions with tritium gas were carried out on a steel manifold
obtained from RC Tritec AG (Teufen, Switzerland). LCMS and specific activity
measurements were obtained by electrospray ionization using a single quadrupole mass
spectrometer with Masslynx v3.5 acquisition and data manipulation software. Specific
activity was calculated by comparison of the ratio of tritium/hydrogen or carbon-14/carbon-
12 for the tracer against the unlabelled reference.
Zinc [14C]cyanide (gravimetric specific activity: 796 µCi/mg equivalent to 95.9 mCi/mmol)
was obtained from Quotient Bioresearch (Radiochemicals) (Cardiff, UK) and zinc
[13C2,15N2]cyanide was obtained from Sigma-Aldrich. Tritium gas was supplied absorbed
onto a depleted uranium bed by RC Tritec AG (Teufen, Switzerland). All other reagents and
solvents were obtained from Sigma-Aldrich and Fisher and were used without further
purification.
Synthesis of Tolmetin Isotopologues
Methyl 2-(5-(4-iodobenzoyl)-1-methyl-1H-pyrrol-2-yl)acetate (7)
4-Iodobenzoyl chloride 5 (5.33 g, 20.0 mmol) was added to a solution of methyl 2-(1-methyl-
1H-pyrrol-2-yl)acetate 6 (1.53 g, 10.0 mmol) in xylenes (15 ml). The reaction mixture was
heated to reflux and stirred for 24 h then cooled to room temperature, concentrated and
diluted with dichloromethane (15 ml). N,N-Dimethyl-1,3-propanediamine (3 ml) was added
and the solution stirred at room temperature for 30 min. The solution was washed with water
(15 ml), the organic layer was diluted with DCM (25 ml) and washed sequentially with 1M
HCl (15 ml), 10% potassium carbonate solution (15 ml), and saturated brine (15 ml), dried
over MgSO4, filtered and concentrated. The precipitate was collected by filtration and
product remaining in the filtrate was purified by flash silica chromatography, elution gradient
0 to 40% EtOAc in heptane. Fractions containing product were pooled and evaporated and
combined with the precipitate collected earlier. The combined batches were crystallised from
EtOAc and heptane to yield 7 (1.66 g, 4.32 mmol, 43%) as a tan solid. LCMS (ESI) m/z 384
[M+H]+. 1H NMR (DMSO-d6): δ 3.67 (s, 3H), 3.82 (s, 3H), 3.92 (s, 2H), 6.14 (d, J = 4.1 Hz,
1H), 6.60 (d, J = 4.1 Hz, 1H), 7.49 (d, J = 8.5 Hz, 2H), 7.88 (d, J = 8.5 Hz, 2H).
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Methyl 2-(5-(4-cyanobenzoyl)-1-methyl-1H-pyrrol-2-yl)acetate (8a)
N,N,N′,N′-Tetramethylethylenediamine (TMEDA) (16.0 µL, 0.10 mmol), Zn(CN)2 (36.8 mg,
0.31 mmol), Pd2(dba)3 (1.20 mg, 1.30 µmol) and 4,5-bis(diphenylphosphino)-9,9-
dimethylxanthene (Xantphos) (3.02 mg, 5.22 µmol) were added to 7 (200 mg, 0.52 mmol) in
DMF (1.2 ml) and sealed into a microwave tube. The reaction mixture was heated to 160°C
for 200 s in the microwave reactor. The reaction mixture was cooled to room temperature,
filtered and washed through with DCM. The solution was concentrated and the residue was
azeotroped with toluene. The crude product was purified by flash silica chromatography,
elution gradient 0 to 40% EtOAc in heptane to yield 8a (112 mg, 0.40 mmol, 76% based on
7, 65% based on Zn(CN)2) as a white solid. LCMS (ESI) m/z 283 [M+H]+. 1H NMR (DMSO-
d6): δ 3.67 (s, 3H), 3.86 (s, 3H), 3.94 (s, 2H), 6.17 (d, J = 4.1 Hz, 1H), 6.59 (d, J = 4.1 Hz,
1H), 7.83 (d, J = 8.1 Hz, 2H), 7.97 (d, J = 8.1 Hz, 2H).
Methyl 2-(5-(4-cyanobenzoyl)-1-methyl-1H-pyrrol-2-yl)acetate (9a)
8a (30.0 mg, 0.11 mmol) and 10 % palladium on carbon (10% Pd/C) (18.1 mg, 0.02 mmol)
were suspended in methanol (5 ml). The reaction mixture was stirred under hydrogen gas for
3 h. The reaction mixture was filtered to give a crude reaction mixture containing 9a (69%
conversion by analytical HPLC) which was used directly in the next stage. LCMS (ESI) m/z
272 [M+H]+.
2-(1-Methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl)acetic acid (tolmetin 1a)
9a (37.3 mg, 0.13 mmol) was dissolved in water (350 µL), methanol (500 µL) and THF (500
µl) and 1M NaOH (157 µL, 0.16 mmol) were added. The reaction was stirred at room
temperature for 16 h. The reaction was concentrated and the aqueous mixture was acidified
with 1M HCl to pH 6 then extracted with DCM (2 x 10 ml), the organic layer was dried over
MgSO4, filtered and evaporated to afford 1a (23.6 mg, 0.092 mmol, 70%) as a white solid.
LCMS (ESI) m/z 258 [M+H]+. 1H NMR (DMSO-d6): δ 2.42 (s, 3H), 3.78 (s, 2H), 3.95 – 3.97
(m, 3H), 6.14 (d, J = 4.0 Hz, 1H), 6.69 (d, J = 4.0 Hz, 1H), 7.25 (d, J = 7.9 Hz, 2H), 7.71 (d,
J = 7.9 Hz, 2H).
Methyl 2-(5-(4-[13C,15N]cyanobenzoyl)-1-methyl-1H-pyrrol-2-yl)acetate (8b)
TMEDA (16.0 µL, 0.11 mmol), Zn([13C,15N]CN)2 (38.0 mg, 0.31 mmol), Pd2(dba)3 (1.20 mg,
1.30 µmol) and Xantphos (3.02 mg, 5.22 µmol) were added to 7 (200 mg, 0.52 mmol) in
DMF (1.2 ml) and sealed into a microwave tube. The reaction mixture was heated to 160°C
for 200 s in the microwave reactor. The reaction mixture was cooled to room temperature,
filtered and washed through with DCM. The solution was evaporated and the crude product
was purified by flash silica chromatography, elution gradient 0 to 40% EtOAc in heptane to
yield 8b (108 mg, 0.38 mmol, 73% based on 7, 61% based on Zn([13C,15N]CN)2) as a cream
solid. LCMS (ESI) m/z 285 [M+H]+.
Methyl 2-[1-methyl-5-[4-[2H3,13C]methylbenzoyl]pyrrol-2-yl][2-2H]acetate (9b)
8b (108 mg, 0.38 mmol) and 10% Pd/C (64.7 mg, 0.06 mmol) were suspended in
d4-methanol (16 ml) and stirred under deuterium gas for 5 h. The reaction mixture was
filtered and evaporated then purified by preparative HPLC using decreasingly polar mixtures
of water (containing 0.1% NH3) and acetonitrile as eluents to yield 9b (42.8 mg, 0.15 mmol,
41%) as a white solid. LCMS (ESI) m/z 276 [M+H]+.
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2-[1-Methyl-5-[4-[2H3,13C]methylbenzoyl]pyrrol-2-yl]acetic acid ([2H3,13C]tolmetin, 1b)
9b (42.8 mg, 0.15 mmol) was dissolved in water (575 µL) and THF (825 µL) and 1M NaOH
(185 µL, 0.19 mmol) was added. The reaction mixture was stirred at room temperature for 2
h then concentrated and acidified with 1M HCl to pH 6. The mixture was extracted with
DCM (2 x 10 ml), the organic layer was dried with MgSO4, filtered and evaporated to afford
1b (39.3 mg, 0.15 mmol, 97%). HPLC purity >99% (UV detection at 254 nm). LCMS (ESI)
m/z 262 [M+H]+. 1H NMR (CDCl3) δ 3.78 (s, 2H), 3.96 (s, 3H), 6.14 (d, J = 4.0 Hz, 1H), 6.69
(d, J = 4.0 Hz, 1H), 7.24 (dd, J = 4.5, 8.1 Hz, 2H), 7.71 (d, J = 8.0 Hz, 2H). Isotope
incorporaton 3.9 d/mol, atom% abundance 98.7%.
Methyl 2-(5-(4-[14C]cyanobenzoyl)-1-methyl-1H-pyrrol-2-yl)acetate (8c)
TMEDA (16.0 µL, 0.10 mmol), Zn([14C]CN)2 (30.1 mCi, 38.0 mg, 0.31 mmol), Pd2(dba)3
(1.30 mg, 1.30 µmol) and Xantphos (3.02 mg, 5.22 µmol) were added to 7 (200 mg, 0.52
mmol) in DMF (1.2 ml) and sealed into a microwave tube. The reaction mixture was heated
to 160°C for 200 s in the microwave reactor then cooled to room temperature, filtered and
washed through with DCM. The crude product was purified by flash silica chromatography,
elution gradient 0 to 40% EtOAc in heptane to yield 8c (106 mg, 21.1 mCi, 0.38 mmol, 70%
radiochemical yield) as a white solid. LCMS (ESI) m/z 285 [M+H]+.
Methyl 2-(1-methyl-5-(4-[14C]methylbenzoyl)pyrrol-2-yl)acetate (9c)
8c (106 mg, 21.1 mCi, 0.37 mmol) and 10% Pd/C (63.5 mg, 0.06 mmol) were suspended in
methanol (18 ml) and stirred under hydrogen gas for 5 h. The reaction mixture was filtered
and evaporated to afford crude product which was purified by preparative HPLC using
decreasingly polar mixtures of water (containing 0.1% NH3) and acetonitrile as eluents to
yield 9c (49.1 mg, 10 mCi, 0.18 mmol, 48% radiochemical yield) as a white solid. LCMS
(ESI) m/z 274 [M+H]+.
2-[1-Methyl-5-[4-[14C]methylbenzoyl]-1H-pyrrol-2-yl]acetic acid ([14C]tolmetin, 1c)
9c (49.0 mg, 10 mCi, 0.18 mmol) was dissolved in water (650 µL) and THF (1.45 ml) and
1M NaOH (0.22 ml, 0.22 mmol) added. The reaction mixture was stirred at room temperature
for 3 h. The reaction mixture was concentrated and acidified with 1M HCl to pH 6 then
extracted with DCM (2 x 10 ml), dried with MgSO4, filtered and evaporated to yield 1c (7.37
mCi, 34.9 mg, 0.14 mmol, 74% radiochemical yield). Radiochemical purity >98% by HPLC.
LCMS (ESI) m/z 260 [M+H]+. 1H NMR (CDCl3) δ 2.42 (s, 3H), 3.77 (s, 2H), 3.96 (s, 3H),
6.14 (d, J = 4.0 Hz, 1H), 6.68 (d, J = 4.0 Hz, 1H), 7.24 (d, J = 7.8 Hz, 2H), 7.71 (d, J = 8.0
Hz, 2H). Specific activity, by mass spectrometry: 55.6 mCi/mmol, gravimetric: 211 μCi/mg.
Methyl 2-(5-(4-[3H]methylbenzoyl)-1-methyl-1H-pyrrol-2-yl)acetate (9d)
8a (1.4 mg, 4.96 µmol) and 10% Pd/C (1.2 mg) were mixed in methanol (0.2 ml) in a 1.3 ml
flask, which was fitted to the tritium manifold. The mixture was freeze-pump-thaw degassed
and then stirred under tritium gas (1800 mCi, 167 mbar) for 3 hours. Palladium was removed
by filtration through a PTFE filter, washing through with ethanol (5 ml). Labile tritium was
remove by lyophilisation and the residue was dissolved in ethanol (6 ml) to give crude 9d
(59.1 mCi) which was used without further purification. LCMS (ESI) m/z 274 [M+H]+.
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2-(1-Methyl-5-(4-[3H]methylbenzoyl)-1H-pyrrol-2-yl)acetic acid ([3H]tolmetin, 1d)
A portion (4 ml, 39 mCi) of the crude 9d solution was purified by preparative HPLC (Waters
XBridge Prep C18 OBD column, 5µ silica, 19 mm diameter, 100 mm length), using
decreasingly polar mixtures of water (containing 0.1% NH3) and acetonitrile as eluents.
Fractions containing the desired compound were combined and 1 M NaOH (100 µl, 0.10
mmol) was added and the mixture was kept at -20°C in the dark for 18 h. The solution was
freeze-dried then purified by reverse-phase preparative HPLC (Waters XBridge Prep C18
OBD column, 5µ silica, 19 mm diameter, 100 mm length), using decreasingly polar mixtures
of water (containing 0.1% TFA) and acetonitrile as eluents. Fractions containing product
were combined and freeze-dried then re-dissolved in acetonitrile / water (1:1, 3 ml) to give a
solution of 1d (6.3 mCi, 16% radiochemical yield). Radiochemical purity >98% by HPLC.
LCMS (ESI) m/z 260 [M+H]+. 1H NMR (600 MHz, CDCl3) δ 2.42 (s, 2H), 3.78 (s, 2H), 3.96
(s, 3H), 6.15 (d, J = 3.6 Hz, 1H), 6.69 (d, J = 4.2 Hz, 1H), 7.24 (d, J = 7.4 Hz, 2H), 7.71 (d, J
= 7.7 Hz, 2H). 3H NMR (640 MHz, CDCl3) δ 2.32 - 2.43 (m). Specific activity by mass
spectrometry: 20.5 Ci/mmol.
Synthesis of Celecoxib Isotopologues
4,4,4-Trifluoro-1-(4-iodophenyl)butane-1,3-dione (12)
Sodium hydride (0.358 g, 8.94 mmol) 60% in mineral oil was added portionwise to a solution
of 1-(4-iodophenyl)ethanone 10 (1.85 g, 7.53 mmol) in THF (20 ml) at 0°C, under nitrogen.
The resulting solution was stirred at 0°C for 0.5 h then ethyl 2,2,2-trifluoroacetate 11 (1.27 g,
8.94 mmol) was added and the solution stirred at room temperature for 3 h. The reaction
mixture was cooled to 0°C and quenched with 1M HCl (10 ml) then extracted with EtOAc (2
x 50 ml) and the combined organic layers were washed with water (50 ml), dried with
MgSO4, filtered and evaporated to afford 12 (2.56 g, 7.48 mmol, 99%) as a brown oil. 1H
NMR (DMSO-d6) δ 6.97 (s, 1H), 7.85 (d, J = 8.4 Hz, 2H), 7.95 (d, J = 8.4 Hz, 2H).
4-(5-(4-Iodophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide (14)
A suspension of 4-hydrazinylbenzenesulfonamide hydrochloride 13 (1.84 g, 8.23 mmol) and
12 (2.56 g, 7.48 mmol) in ethanol (17 ml) was heated to reflux for 23 h. The reaction mixture
was evaporated and the solid was then redissolved in EtOAc (50 ml) and washed sequentially
with water (50 ml) and brine (50 ml). The organic layer was dried with MgSO4, filtered and
evaporated and the crude product purified by flash silica chromatography, elution gradient 20
to 60% EtOAc in heptane to afford 14 (3.06 g, 6.19 mmol, 83%) as a cream foam. LCMS
(ESI) m/z 494 [M+H]+. 1H NMR (DMSO-d6) δ 7.12 (d, J = 8.4 Hz, 2H), 7.27 (s, 1H), 7.51 (s,
2H), 7.56 (d, J = 8.6 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.6 Hz, 2H).
4-(5-(4-Cyanophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide (15a)
TMEDA (16.0 µL, 0.11 mmol), Zn(CN)2 (25.7 mg, 0.22 mmol), Pd2(dba)3 (0.835 mg, 0.91
µmol) and Xantphos (2.11 mg, 3.65 µmol) were added to 14 (180 mg, 0.36 mmol) in DMF
(0.84 ml) and sealed into a microwave tube. The reaction was heated to 160°C for 200 s in
the microwave reactor and cooled to room temperature. The reaction was incomplete so
additional reagents were added: TMEDA (16.0 µL, 0.11 mmol), Zn(CN)2 (25.7 mg, 0.22
mmol), Xantphos (4.22 mg, 7.30 µmol) and Pd2(dba)3 (1.67 mg, 1.82 µmol) and the mixture
was heated at 160°C for an additional 200 s in the microwave reactor. The reaction mixture
was cooled, filtered and the microwave tube and compound were washed through with DCM.
The resulting mixture was evaporated to afford crude product which was purified by flash
silica chromatography, elution gradient 0 to 60% EtOAc in heptane to afford 15a (74.3 mg,
0.189 mmol, 52% based on 14, 43% based on Zn(CN)2) as a cream solid. LCMS (ESI) m/z
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391 [M+H]+. 1H NMR (DMSO-d6) δ 7.41 (s, 1H), 7.51 - 7.55 (m, 4H), 7.57 (d, J = 8.7 Hz,
2H), 7.89 (d, J = 8.7 Hz, 2H), 7.92 (d, J = 8.5 Hz, 2H).
4-(5-(p-Tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide (celecoxib, 2a)
15a (30.0 mg, 0.08 mmol) and 10% Pd/C (13.0 mg, 0.01 mmol) were mixed in methanol (5
ml) and stirred under hydrogen gas for 16 h. The reaction mixture was filtered and evaporated
to dryness to afford 2a (14.9 mg, 0.039 mmol, 51%) as a white solid. HPLC purity >98%
(UV detection at 254 nm). LCMS (ESI) m/z 382 [M+H]+. 1H NMR (DMSO-d6) δ 2.32 (s,
3H), 7.18 (s, 1H), 7.19 - 7.23 (m, 4H), 7.36 (br s, 2H), 7.52 (d, J = 8.6 Hz, 2H), 7.86 (d, J =
8.6 Hz, 2H).
4-(5-(4-[13C,15N]Cyanophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide
(15b)
TMEDA (16.0 µL, 0.11 mmol), Zn([13C,15N]CN)2 (26.6 mg, 0.22 mmol), Pd2(dba)3 (1.67 mg,
1.82 µmol) and Xantphos (4.22 mg, 7.30 µmol) were added to 14 (180 mg, 0.36 mmol) in
DMF (0.84 ml) and sealed into a microwave tube. The reaction was heated to 160°C for 8
min in the microwave reactor and cooled to room temperature. The reaction mixture was
filtered and the microwave tube and compound were washed through with DCM. The
resulting mixture was evaporated to afford crude product which was purified by flash silica
chromatography, elution gradient 10 to 60% EtOAc in heptane to afford 15b (101 mg, 0.256
mmol, 70% based on 14, 58% based on Zn([13C,15N]CN)2) as a cream solid. LCMS (ES-) m/z
393 [M-H]-. 1H NMR (DMSO-d6) δ 7.41 (s, 1H), 7.51 - 7.55 (m, 4H), 7.57 (d, J = 8.5 Hz,
2H), 7.89 (d, J = 8.4 Hz, 2H), 7.92 (dd, J = 5.2, 8.4 Hz, 2H).
4-[5-([13C,2H3]Methyl)phenyl]-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide
([13C,2H3]celecoxib, 2b)
15b (30 mg, 0.08 mmol) and 10% Pd/C (13.0 mg, 0.01 mmol) were mixed in d4-methanol (5
ml) and stirred under deuterium gas for 16 h. The reaction mixture was filtered and
evaporated to dryness to afford 2b (27.2 mg, 0.071 mmol, 93%) as a white solid. LCMS
(ESI) m/z 286 [M+H]+. HPLC purity >98% (UV detection at 254 nm). 1H NMR (DMSO-d6)
δ 7.18 - 7.25 (m, 5H), 7.51 (s, 2H), 7.54 (d, J = 8.7 Hz, 2H), 7.87 (d, J = 8.7 Hz, 2H). Isotope
incorporaton 3.9 d/mol, atom% abundance 96.1%.
4-(5-(4-[14C]Cyanophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide
(15c)
TMEDA (10.6 µl, 0.07 mmol), Zn([14C]CN)2 (20.1 mCi, 25.4 mg, 0.21 mmol), Pd2(dba)3
(1.60 mg, 1.74 µmol) and Xantphos (4.04 mg, 6.97 µmol) were added to 14 (172 mg, 0.35
mmol) in DMF (0.81 ml) and sealed into a microwave tube. The reaction was heated to
160°C for 8 min in the microwave reactor and cooled to room temperature. The reaction
mixture was filtered and the microwave tube and compound were washed through with
DCM. The resulting mixture was evaporated to afford crude product which was purified by
flash silica chromatography, elution gradient 10 to 60% EtOAc in heptane to afford 15c (100
mg, 14.2 mCi, 0.254 mmol, 71% radiochemical yield) as a cream solid. LCMS (ESI) m/z 395
[M+H]+.
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4-[5-([14C]Methyl)phenyl]-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide
([14C]celecoxib, 2c)
15c (100 mg, 0.25 mmol, 14.2 mCi) and 10% Pd/C (43.2 mg, 0.04 mmol) in methanol (17
ml) were mixed under hydrogen gas for 16 h. The reaction mixture was filtered and then
evaporated to dryness to afford crude product which was purified by preparative HPLC using
decreasingly polar mixtures of water (containing 0.1% NH3) and acetonitrile as eluents to
afford 2c (48.6 mg, 7.08 mCi, 0.127 mmol, 50% radiochemical yield) as a white foam.
Radiochemical purity >99% by HPLC. LCMS (ESI) m/z 384 [M+H]+. 1H NMR (DMSO-d6) δ
2.31 (s, 3H), 7.21 (dt, J = 6.3, 12.6 Hz, 5H), 7.49 (s, 2H), 7.54 (d, J = 8.5 Hz, 2H), 7.87 (d, J
= 8.5 Hz, 2H). Specific activity, by mass spectrometry: 55.8 mCi/mmol, gravimetric: 146
μCi/mg.
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`
D2
H2
T2
Scheme 1 Proposed labelling of methyl group with different isotopes by cyanation then reduction
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`
reflux
xylene
5
6
7
Zn(CN)2
Pd2(dba)3
or Zn(13C15N)2
or Zn(14CN)2
Xantphos
DMF
8a X =
8b X =
8c X =
H2, methanol
or D2, d4-methanol
or T2, methanol
Pd/C
9a X = CH3
Y= H
9b X = 13CD3 Y= D
9c X = 14CH3 Y= H
9d X = C[T]H3 Y= H
NaOH
THF / water
1a X = CH3
1b X = 13CD3
1c X = 14CH3
1d X = C[T]H3
Scheme 2 Common synthetic route to tolmetin 1a and three of its isotopologues
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`
13
NaH
THF
ethanol
10
11
12
14
Pd2(dba)3
Zn(CN)2
or Zn(13C15N)2
or Zn(14CN)2
Xantphos
DMF
Pd/C
H2, methanol
or D2, d4-methanol
16
2a X = CH3
15a X =
15b X =
15c X =
2b X = 13CD3
2c X = 14CH3
Scheme 3 Common synthetic route to celecoxib 2a and two of its isotopologues
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tolmetin 1a
celecoxib 2a
imatinib 3
lidocaine 4
Figure 1 Important marketed drugs containing aryl methyl groups
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