The synthesis of tritium labelled neurokinin-1 receptor ligands

Article abstract of DOI:10.1002/jlcr.802

Radiolabelled neurokinin-1 (NK₁) receptor antagonists 1b and 2b were required for in vitro/in vivo characterization to support the development of 1a and 2a as fluorine-18 labelled PET ligands. These tritium labelled compounds were synthesized f

Full text of DOI:10.1002/jlcr.802

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 99–106.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.802
Research Article
The synthesis of tritium labelled neurokinin-1 receptor
ligands
Terence G. Hamill* and H. Donald Burns
Imaging Research Department, Merck Research Laboratories, West Point,
PA 19486, USA
Summary
Radiolabelled neurokinin-1 (NK1) receptor antagonists 1b and 2b were required for
in vitro/in vivo characterization to support the development of 1a and 2a as fluorine-18
labelled PET ligands. These tritium labelled compounds were synthesized from aryl
iodide precursors giving the final tritiated tracers with specific activities of 28 (1b) and
14 (2b) Ci/mmol. Copyright # 2004 John Wiley & Sons, Ltd.
Key Words: neurokinin-1; substance P; tritium
Introduction
The agonist substance P, acting through the neurokinin-1 (NK1) receptor, is
believed to play an important role in pain, emesis, inflammation and
psychiatric disorders such as depression.1 Work is ongoing to identify NK1
antagonists as therapeutic agents to treat conditions such as chemotherapy-
induced emesis (CIE) and depression.2 As part of an NK1 clinical program for
Figure 1. Chemical structures of neurokinin-1 compounds
*Correspondence to: T. G. Hamill, Merck Research Laboratories, WP44C-2, West Point, PA 19486, USA.
E-mail: terence hamill@merck.com
Copyright # 2004 John Wiley & Sons, Ltd.
Received 11 June 2003
Revised 29 September 2003
Accepted 3 November 2003

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T. G. HAMILL, H. D. BURNS
CIE and depression, we were interested in developing tracers for positron
emission tomography (PET) studies of the NK1 receptor to determine receptor
occupancy of clinical candidates. To support the development of the fluorine-
18 labelled PET ligands,3 the tritiated compounds were required for in vivo/
in vitro characterization. This paper describes the synthesis of the tritium
labelled compounds 1b and 2b.
Results and discussion
At the time this work was undertaken, data on [18F]1 as a potential NK1 PET
ligand was very promising,3 so in addition to specifically targeting 1b, a route
giving an intermediate that could be used to make other fluorine-containing
alkyl ether analogs of 1b was also desired. For this reason, we also targeted the
synthesis of tritium labelled phenol 6 (Sch 1), which could be a common
precursor for a variety of tritium labelled, fluorine-containing alkyl aryl ether
analogs.
Scheme 1 summarizes the chemistry that was followed to synthesize 1b, 2b
and their precursors. Phenol 3 (Compound 3 was synthesized at Merck
Research Laboratories) was chosen as the starting point. Halogenation of 3
would ultimately allow incorporation of tritium by catalytic reductive
dehalogenation. Iodination of 3 was attempted using various conditions
(Other conditions used were ICl, NaI with either chloramine-T or NaOCl and
iodine/morpholine) with the best results coming when IPy2BF44 in methylene
chloride was used. The intermediate iodophenol 4, itself a precursor to 2b, was
Scheme 1. Synthetic routes for tritium labelled neurokinin-1 compounds
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TRITIUM LABELLED NEUROKININ-1 RECEPTOR LIGANDS
101
alkylated using fluoromethyliodide5 and deprotected to give the desired
iodoaryl ether 5.
When a model hydrogenation reaction of 5 was carried out using 10% Pd/C
in DMF with triethylamine at room temperature, the desired product 1a
formed, as determined by HPLC coelution with the authentic standard.
Repeating this reaction using tritium gas gave crude 1b with a radiochemical
purity of ꢀ95%. HPLC purification of this material gave 1b with a
radiochemical purity of >99% and a specific activity of 28 Ci/mmol.
In an analogous fashion, gas tritiation of 4 gave crude 6. This material was
first deprotected using trifluoroacetic acid in methylene chloride to give 7, and
was then alkylated using 1-bromo-2-fluoroethane to give 2b. This sequence
was carried out in a one-pot procedure without purifying 7. However, the
specific activity of 7 was determined by purifying an aliquot and its specific
activity was determined to be 14.2 Ci/mmol. Purification of crude 2b by HPLC
gave 2b with a radiochemical purity of >98% and an estimated specific
activity of 14 Ci/mmol based on the specific activity of 7. If this deprotection/
alkylation sequence was reversed so that the alkylation was carried out first,
followed by the deprotection step, a product resulted which did not coelute
with the authentic standard. Treatment of unlabelled 2a with trifluoroacetic
acid showed that this compound is stable to trifluoroacetic acid and that the
unknown product did not arise from decomposition of the desired product.
The characterization of fluorine-18 labelled 1 has been reported3 and the
in vitro and in vivo characterization of 2 labelled with tritium and fluorine-18
and its suitability as an NK1 receptor ligand and PET radiotracer will be
reported elsewhere.
Conclusion
The synthesis of two tritiated neurokinin-1 receptor ligands has been carried
out. Each ligand was synthesized from an iodoaryl precursor giving the final
products with specific activities of 14 or 28 Ci/mmol. These fluorine-containing
compounds also lend themselves to fluorine-18 labelling chemistry and use as
neurokinin-1 receptor PET ligands.
Experimental
Materials and methods
1H NMR spectra were recorded using a Varian Infinity-300 spectrometer
operating at 300 MHz or a Varian Unityplus operating at 500 MHz. Mass
spectral analyses were carried out using a VG 7070E mass spectrometer.
Analytical and preparative HPLC was carried out using a Waters 600E
Powerline Multi Solvent Delivery System with 100 ml heads with a Rheodyne
7125 injector and a Waters 990 Photodiode Array Detector with a Gilson
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T. G. HAMILL, H. D. BURNS
FC203 Microfraction collector. The acetonitrile used for the HPLC analyses
was Fisher Optima grade. The HPLC radiodetector used was a Beckman 171
Radioisotope detector with a Beckman 110B solvent delivery system and
Beckman Ready Flow III scintillation cocktail. A Vydac C-18 column,
4.6 Â 250 mm (The Nest Group) was used for analytical and preparative
HPLC. Solutions of radioactivity were concentrated using a Jouan vacuum
centrifuge. Calibration curves and chemical concentrations used to determine
specific activities were determined using a Hewlett Packard Model 8452A UV/
Vis Diode Array Spectrophotometer. Sample radioactivities were determined
in an LKB Wallac 1410 liquid scintillation counter. The identity of labelled
compounds was determined by HPLC coelution with authentic compounds.
Reagents were purchased from Aldrich Chemical Co. The IPy2BF4 was
prepared according to the literature procedure.4 Compound 3 was synthesized
at Merck Research Laboratories.
Nonradioactive syntheses
[2-Fluoromethoxy-5-(5-trifluoromethyl-tetrazol-1-yl)-benzyl]-([2S,3S]-2-phe-
nyl-piperidin-3-yl)-amine (1a): A room temperature solution of 3 (100 mg,
0.19 mmol) in DMF (6.6 ml) was treated with Cs2CO3 (313 mg, 0.96 mmol)
giving a yellow mixture. After stirring for several minutes at room temperature
a solution of fluoromethyl iodide (0.09 ml) in DMF (1.2 ml) was added
discharging the yellow color. After stirring at room temperature for 30 min,
the reaction was diluted with H2O/brine/aq. saturated NH4Cl. The aqueous
layer was extracted with ethyl acetate and the organic layers were combined,
dried (MgSO4), filtered and concentrated in vacuo to give 113 mg of the crude
1
alkylated compound: H NMR (d, CDC13): 7.54 (3H, m), 7.38–7.22 (6H, m),
5.72 (1H, m), 5.71 (1H, m), 5.48 (1H, m), 3.95 (3H, m), 3.02 (2H, m), 1.87–1.26
(3H, m), 1.39 (9H, s); MS m/z (relative intensity) for C26H30F4N6O3: 551
(M+1, 100%).
A room temperature solution of the crude alkylated compound (113 mg,
0.205 mmol) in methylene chloride (5 ml) was treated with trifluoroacetic acid
(2.5 ml) and stirred at room temperature for 50 min. The reaction was
concentrated in vacuo to give a yellow oil and diluted using ethyl acetate/aq.
saturated NaHCO3. The layers were separated and the aqueous layer was
extracted with ethyl acetate. The organic layers were combined, dried
(MgSO4), filtered and concentrated in vacuo to give 136 mg of crude 1a as a
yellow oil. TLC (5:93:2 methanol:ethyl acetate:triethylamine) shows 1a at
0.35Rf. This material was dissolved in chloroform and purified by radial
chromatography (5:93:2 methanol:ethyl acetate:triethylamine) to give 41.2 mg
of pure 1a along with 50 mg of 1a containing small amounts of the
1
corresponding phenol. H NMR (d, CDCl3): 7.29 (2H, dm, Jꢀ7.7 Hz), 7.21
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TRITIUM LABELLED NEUROKININ-1 RECEPTOR LIGANDS
103
(1H, dd, J=8.6, 2.7 Hz), 7.16 (2H, t, Jꢀ7.6 Hz), 7.11 (1H, d, J=8.6 Hz), 7.05
(1H, tt, Jꢀ7.3, 1.2 Hz), 6.97 (1H, d, J=2.7 Hz), 5.59 (1H, dd, J=53.5, 3.2 Hz),
5.57 (1H, dd, J=54.0, 3.2 Hz), 3.94 (1H, d, J=2.6 Hz), 3.76 (1H, d,
J=16.1 Hz), 3.61 (1H, d, J=16.1 Hz), 3.29 (1H, dm, J=11.8 Hz), 2.83 (1H,
ddd, J ꢀ 12.4, 12.4, 3.1Hz), 2.78 (1H, q, Jꢀ2.7 Hz), 2.10 (1H, dm,
Jꢀ13.6 Hz), 1.94 (1H, qt, Jꢀ13.1, 3.8 Hz), 1.62 (1H, dddd, Jꢀ13.6, 13.6, 4.3,
3.1 Hz), 1.53 (1H, dm, J=13.7 Hz). A standard NOESY sequence with a 500-
ms mixing period showed a strong NOE between the fluoromethylene protons
and the ortho aromatic proton at d 7.11. MS m/z (relative intensity) for
C21H22F4N6O1: 451 (M+1, 100%).
[2-Hydroxy-3-iodo-5-(5-trifluoromethyl-tetrazol-1-yl)-benzyl]-([2S,3S]-1-t-
butoxycarbonyl-2-phenyl-piperidin-3-yl)-amine (4): A room temperature solu-
tion of 3 (30 mg, 0.06 mmol) in methylene chloride (3 ml) was treated with
IPy2BF4 (26 mg, 0.066 mmol). The reaction flask was shielded from light and
stirred for 1 h at room temperature, giving a slightly white opaque mixture.
The reaction mixture was concentrated in vacuo to remove the methylene
chloride and the residue was rinsed with ethyl acetate and placed in a
separately funnel containing 10% aqueous Na2S2O3. The layers were
separated and the aqueous layer was extracted with ethyl acetate. The organic
layers were combined, dried (MgSO4), filtered and concentrated in vacuo to
1
give 41 mg of crude 4 as a yellow oil: H NMR (d, CDCl3): 7.75 (1H, d,
J=2.44 Hz), 7.5–7.33 (5H, m), 7.08 (1H, d, J=2.44 Hz), 5.42 (1H, d,
J=6.1 Hz), 4.22–3.99 (3H, m), 3.21–3.07 (2H, m), 2.0–1.25 (4H, m), 1.37
(9H, s); MS m/z (relative intensity) for C25H28F3IN6O3: 645 (M+1, 100%).
[2-Fluoromethoxy-3-iodo-5-(5-trifluoromethyl-tetrazol-1-yl)-benzyl]-([2S,3S]-
2-phenyl-piperidin-3-yl)-amine (5): A room temperature solution of 4 (41 mg,
0.064 mmol) in DMF (2 ml) was treated with Cs2CO3 (98 mg, 0.3 mmol) giving a
yellow mixture. After stirring for several minutes a solution of fluoromethyl
iodide (0.03 ml) in DMF (0.5 ml) was added giving an off-white opaque mixture.
After stirring for 2.5 h at room temperature, the reaction was diluted with brine/
saturated aqueous NH4Cl/H2O/ethyl acetate and placed in a separatory funnel.
The layers were separated and the aqueous layer was extracted with ethyl acetate.
The organic layers were combined, dried (MgSO4), filtered and concentrated to
give 41 mg of a yellow oil. A room temperature solution of this crude material
(40 mg, 0.059 mmol) in methylene chloride (2.2 ml) was treated with trifluor-
oacetic acid (1.1 ml) and stirred at room temperature. After 20 min at room
temperature, the reaction was concentrated in vacuo, treated with methylene
chloride/aqueous saturated NaHCO3 and placed in a separatory funnel. The
layers were separated and the aqueous layer was extracted with methylene
chloride. The organic layers were combined, dried (MgSO4), filtered and
concentrated to give 30 mg of 5 as a yellow oil. The crude 5 was dissolved in
chloroform and purified by radial chromatography (3:96:1 methanol:ethyl
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T. G. HAMILL, H. D. BURNS
acetate:triethylamine) to give 11.4 mg (31%) of 5 as an oil: 1H NMR (d, CDC13):
7.65 (1H, d, J=2.45 Hz), 7.28 (2H, m), 7.09 (1H, t, J=7.57 Hz), 6.92 (2H, m),
5.59 (1H, dd, J=53.96, 2.44 Hz), 5.55 (1H, dd, J=54.3, 2.4 Hz), 3.87 (1H, s), 3.80
(2H, s), 3.25 (1H, m), 2.79 (1H, m), 2.67 (1H, m), 2.1–1.5 (6H, m); MS m/z
(relative intensity) for C21H21F4IN6O: 577 (M+1, 97%).
[2-Fluoroethoxy-5-(5-trifluoromethyl-tetrazol-1-yl)-benzyl-([2S,3S]-2-phenyl-
piperidin-3-yl)-amine (2a): A room temperature solution of 2-hydroxy-5-(5-
trifluoromethyl-tetrazol-l-yl)-benzyl]-([2S,3S]-2-phenyl-piperidin-3-yl)-amine, bis
HC1 salt (50 mg, 0.102 mmol) in DMF (2 ml) was treated with cesium carbonate
(230 mg, 0.714 mmol). After stirring for several minutes, bromofluoroethane
(9 ml, 0.122mmol) was added and the reaction was stirred at room temperature
overnight giving a yellow slurry. The reaction was diluted with aqueous
saturated NH4Cl/brine/H2O and ethyl acetate. The layers were separated and
the aqueous layer was extracted with ethyl acetate. The organic layers were
combined, washed with H2O, dried over MgSO4, filtered and concentrated in
vacuo to give 44 mg of a yellow oil. Purification by rotary chromatography (5%
MeOH/EtOAc to 10% MeOH/EtOAc) gave 33.2 mg (70%) of 2a: 1H NMR (d,
CDC13): 7.25 (2H, m), 7.17 (1H, d  d, J=8.7 Hz, 2.8Hz), 7.14 (2H, m), 7.02
(1H, m), 6.87 (1H, d, J=2.8 Hz), 6.82 (1H, d, J=8.7 Hz), 4.64 (1H, m), 4.63
(1H, m), 4.2–4.0 (2H, m), 3.87 (1H, d, J=2.4 Hz), 3.75 (1H, d, J=16 Hz), 3.58
(1H, d, J=16 Hz), 3.25 (1H, m), 2.80 (1H, m), 2.74 (1H, m), 2.10 (1H, m), 1.89
(1H, m), 1.59 (1H, m), 1.48 (1H, m). A standard NOESY sequence with an 800-
ms mixing time showed a correlation between the d 4.11 methylene on the
fluoroethyl sidechain and the phenyl proton at d 6.82 (ortho to the fluoroethoxy
chain). There is also a correlation between the d 6.87 phenyl proton (ortho to the
tetrazole and aminomethyl moieties) and the d 7.25 proton of the phenyl group.
MS m/z (relative intensity) for C22H24F4N6O: 465 (M+l, 100%).
Radioactive syntheses
[2-Fluoromethoxy-3-tritio-5-(5-trifluoromethyl-tetrazol-1-yl)-benzyl]-([2S,3S]-
2-phenyl-piperidin-3-yl)-amine (1b): A room temperature solution of 5 (8.5 mg,
0.015 mmol) in DMF (2ml) was treated with triethylamine (0.08 ml) and 10%
Pd/C (6.5 mg). The reaction vessel and its contents were degassed by a single
evacuation cycle and then exposed to 5 Ci of tritium gas. After 2.8 h at room
temperature and atmospheric pressure, the reaction was passed through a short
column of celite and rinsed with 3 Â 1 ml of DMF and 3 Â 1 ml of methanol.
The solvents were removed in vacuo and labile tritium was washed out by
evaporation with methanol (3Â 2 ml). The residue was dissolved in ethanol
(20 ml) to give 200 mCi of activity. An aliquot (15 mCi) was concentrated in
vacuo, dissolved in ethanol (75 ml) for purification by HPLC (C18 Vydac
protein and peptide column, 4.6 Â 250 mm, 1 ml/min, linear gradient of 10%
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TRITIUM LABELLED NEUROKININ-1 RECEPTOR LIGANDS
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MeCN:H2O (0.1% TFA) to 95% MeCN over 15min, hold at 95% MeCN for
10min, 254 nm). Fractions (0.2ml) were collected beginning at 9 min and the
product eluted at 14min. The center cut fractions were pooled, concentrated in
vacuo and diluted with 91% ethanol/H2O (6.6 ml) to give 5 mCi of 1b with a
specific activity of 27.8Ci/mmol and a radiochemical purify >98%.
[2-Hydroxy-3-tritio-5-(5-trifluoromethyl-tetrazol-1-yl)-benzyl]-([2S,3S]-1-t-
butoxycarbonyl-2-phenyl-piperidin-3-yl)-amine (6): A room temperature solu-
tion of 4 (9 mg, 0.015mmol) in DMF (1.8 ml) was treated with triethylamine
(0.035 ml) and transferred to a 2 ml reaction containing 10% Pd/C (8 mg). The
reaction vessel and its contents were degassed by a single evacuation cycle and
then exposed to 5 Ci of tritium gas. The reaction was terminated after a total of
1 h at room temperature and atmospheric pressure. The catalyst was removed
through a short column of celite and the celite was rinsed with DMF (3 Â 1 ml)
and methanol (3 Â 1 ml). The solvents were removed in vacuo and any labile
tritium was washed out by evaporation with methanol (3 Â 2 ml). The residue
was dissolved in ethanol to give 334 mCi (ꢀ13 mCi/ml) of 6.
[2-Hydroxy-3-tritio-5-(5-trifluoromethyl-tetrazol-1-yl)-benzyl]-([2S,3S]-2-
phenyl-piperidin-3-yl)-amine (7): An aliquot containing of crude 6 (ꢀ6.5 mCi)
was concentrated to dryness and treated with 1:1 CH2Cl2:TFA (0.1 ml) and
stirred at room temperature for 30 min. The reaction was partially
concentrated in vacuo, diluted with ethanol (0.08 ml) and purified by HPLC
[C18 Vydac, 4.6 Â 250 nm, 15 min linear gradient @ 1 ml/min, 10%
acetonitrile:H2O (0.1% TFA) to 95% acetonitrile, retention time
ꢀ12.5 min]. The center cut fractions were pooled, concentrated in vacuo and
diluted with ethanol (3 ml) to give 2.8 mCi of 7 with a specific activity of
14.2 Ci/mmol and radiochemical purity >98%.
[2-Fluoroethoxy-3-tririo-5-(5-trifluoromethyl-tetrazal-1-yl)-benzyl]-([2S,3S]-
2-phenyl-piperidin-3-yl)-amine (2b): An aliquot of crude 6 (ꢀ13mCi) was
concentrated to dryness and treated with methylene chloride (0.1ml) and TFA
(0.1 ml) and stirred at room temperature for 15min. The reaction mixture was
concentrated to dryness and treated with DMF (0.05ml) and several milligrams
of Cs2CO3. A 10 ml aliquot of a solution of 1-bromo-2-fluoroethane (3 ml) in
DMF (0.5 ml) was added to the reaction solution. After stirring overnight at
room temperature an additional 10 ml of the 1-bromo-2-fluoroethane/DMF
solution was added and the reaction was stirred for two additional hours. The
reaction was quenched with H2O (20ml) and purified by HPLC [C18 Vydac, 4.6
 250 mm, 15min linear gradient @ 1 ml/min, 10% acetonitrile:H2O (0.1%
TFA) to 95% acetonitrile, retention time ꢀ13.5 min]. The center cut fraction
(earlier fractions were contaminated with the unalkylated phenol which elutes at
ꢀ12.5min under these conditions) was concentrated and diluted with ethanol
(1 ml) to give 0.97mCi of 2b. The specific activity was estimated to be 14 Ci/
mmol based on the specific activity of 7. The radiochemical purity was >98%.
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T. G. HAMILL, H. D. BURNS
Acknowledgements
The authors wish to thank Chemsyn Science Laboratories for carrying out the
gas tritiation reactions described here, Marc Berridge (3D Imaging) for
providing the fluoromethyl iodide, Gerard Kieczykowski (Merck Research
Laboratories) for providing 3 and 2-hydroxy-5-(5-trifluoromethyl-tetrazol-1-
yl)-benzyl]-([2S,3S]-2-phenyl-piperidin-3-yl)-amine, bis HC1 salt (compound 7,
unlabelled) used to synthesize 2a and to determine the specific activity of 7 and
S. Pitzenberger and J. Murphy (Merck Research Laboratories) for carrying
1
out the NOESY H NMR analysis of 1a and 2a.
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