Synthesis of N, N-diethyl-4-[phenyl-(piperidin-4-ylidene)methyl]- [carboxy-14C]benzamide, a potent δ opioid receptor agonist

Article abstract of DOI:10.1002/jlcr.651

The synthesis of carbon-14 labelled N,N-diethyl-4-[phenyl-(piperidin-4-ylidene)methyl]-benzamide is described. The radioisotope is introduced via an aryllithium reaction with ¹⁴CO₂ to form the labelled acid, which is subsequently tra

Full text of DOI:10.1002/jlcr.651

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 131–138.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.651
Research Article
Synthesis of N, N-diethyl-4-[phenyl-
(piperidin-4-ylidene)methyl]-
[carboxy-14C]benzamide, a potent d opioid
receptor agonist
Lars Gawell*
! ! !
Department of Chemistry, AstraZeneca R&D Montreal 7171, Frederick-
Banting, Ville Saint-Laurent, Quebec, H4S 1Z9, Canada
Summary
The synthesis of carbon-14 labelled N,N-diethyl-4-[phenyl-(piperidin-4-ylide-
ne)methyl]-benzamide is described. The radioisotope is introduced via an
aryllithium reaction with 14CO2 to form the labelled acid, which is
subsequently transformed into the amide. Copyright # 2002 John Wiley &
Sons, Ltd.
Key Words: d agonist; benzamide; carbon-14; aryllithium
Introduction
N; N-Diethyl-4-[phenyl-(piperidine-4-ylidene)methyl]-benzamide [1, AR-
M 390, Figure 1] is a potent, orally active opioid agonist, currently
under investigation at AstraZeneca as one of a series of 4-[aryl-
(piperidine-4-ylidene)methyl]-benzamides. It binds to the d opioid
receptor with high affinity, is a potent full agonist and has exceptional
!
*Correspondence to: L. Gawell, Department of Chemistry, AstraZeneca R&D, Montreal, 7171,
Quebec, H4S 1Z9, Canada.
E-mail: lars.gawell@astrazeneca.com
Copyright # 2002 John Wiley & Sons, Ltd.
Received 11 March 2002
Revised 29 May 2002
Accepted 11 June 2002

132
L. GAWELL
Figure 1.
selectivity with respect to m and k opioid receptors1. During the
pharmacological evaluation of AR-M 390, the compound was required
labelled with carbon-14 to be used in autoradiographic studies. The
synthesis of [14C]AR-M 390 described here constitutes the general
method used for the preparation of 14C-labelled benzamides from this
series.
Results and discussion
Results from metabolic studies of this class of compounds led us to
choose to label AR-M 390 (1) with carbon-14 in its amide carbonyl
group (Figure 1). This functionality remained attached to the main
molecular structure in all the metabolites that were identified, suggesting
that a label in this position would be ideal for elucidating the
distribution of the compound.
The radioactive carbon atom at this position could be in-
corporated by the reaction between an organolithium precursor
and 14CO2, forming the corresponding labelled carboxylic acid, which
would serve as the precursor for the preparation of the amide. The
starting material was prepared from the t-Boc-protected amino ketone 2
(Scheme 1).
The Grignard reaction between 4-bromo-phenylmagnesium bromide
and ketone 2 produced the corresponding alcohol, which was
dehydrated in refluxing benzene in the presence of p-TsOH. Under
these reaction conditions, the N-protecting group was also cleaved off
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LABELLING OF A POTENT DELTA OPIOID RECEPTOR AGONIST
133
Scheme 1.
to furnish amine 3, which was re-protected with di-tert-butyl
dicarbonate to give compound 4.
The remaining steps of the synthesis, which constitute the radio-
synthesis, were first validated with non-radioactive material. The
protected amino bromide 4 was subjected to halogen–lithium exchange
by treatment with n-butyllithium at ꢀ788C to give the corresponding
aryl–lithium reagent. This was reacted with carbon dioxide, generated
by the action of concentrated sulfuric acid on barium carbonate, to
provide the acid 5. The reaction was performed on a vacuum manifold.
Treatment of the acid 5 with thionyl chloride/DMAP at ꢀ208C, formed
the acid chloride which was coupled, in situ, with diethylamine to give
amide 6.
When this procedure was applied to the preparation of the carbon-14
labelled material, the reaction with 14CO2 produced acid 7 in 44% yield.
The acid 7 was transformed into the amide 8 (90% yield), which was
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L. GAWELL
subsequently treated with TFA=CH2Cl2 to provide [14C]AR-M 390.
The compound was purified by column chromatography, from which
one fraction was treated with HCl/EtOH to produce the hydrochloride
9 with a specific activity of 48:7 mCi=mmol.
Experimental
Melting points were determined on a Thomas–Hoover melting point
apparatus and are uncorrected. IR spectra were recorded on a Perkin-
Elmer Pragon 100 FT-IR spectrometer. NMR spectra were recorded on
a Varian Unity Plus 400 MHz spectrometer with ðCH3Þ4Si (TMS) as the
internal reference. Elemental analyses were performed by Canadian
Microanalytical Service Ltd., Delta, British Columbia and the results
were within ꢁ0:4% of the theoretical values unless otherwise indicated.
Radiochemical purity was determined by TLC using a Raytest Rita 92
detector or with HPLC connected to a Packard Radiomatic 500TR
detector. The HPLC-system was a Luna C18-column with CH3CN
containing 0.04% HCOOH in H2O as eluent (18:82% at t ¼ 0 and
25:75% at t ¼ 6 min, postrun 3 min). Flow rate: 0:6 mL=min. Radio-
activity was measured in a Packard Tri-Carb 2300TR liquid scintillation
spectrometer using Packard Ultima Gold as counting medium. n-BuLi
was purchased from Aldrich and the concentration determined by
titration with diphenylacetic acid2. Drying of organic extracts was
performed with Na2SO4.
4-Bromo-[phenyl-(piperidin-4-ylidene)methyl]-benzene,(3)
To a solution of N-tert-butoxycarbonyl-4-benzoylpiperidine (2)1,
ð6:65 g; 23 mmolÞ in THF ð30 mLÞ cooled by an ice-water bath, was
added 4-bromo-phenylmagnesium bromide (1 M in THF, 25 mL).
When the addition was completed, the cooling bath was removed and
the reaction mixture refluxed for 2 h. The mixture was cooled to room
temperature and saturated aqueous NH4Cl was added followed by
diethyl ether. The organic layer was separated and washed with brine,
dried and evaporated to dryness. The residual oil was mixed
with benzene ð150 mLÞ and p-TsOH ꢂ H2O ð8:6 gÞ and heated to reflux
with water removal by means of a Dean–Stark trap. After 2 h the water
had ceased to form and the reaction mixture was cooled to room
temperature. 2 M HCl was added followed by diethyl ether. The
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aqueous layer was separated, washed with diethyl ether and made basic
ðpH ꢃ 10Þ with solid NaOH. Extraction with diethyl ether gave, after
drying and evaporation of the solvent, a solid residue of crude
compound 3 (6:10 g, 81% yield). An analytical sample was converted
to the HCl-salt and recrystallized from EtOH–diethyl ether. M.p.
1
187–1898C. H NMR (CDCl3, TMS): d 2.69 (4 H, m), 3.21 (4 H, m),
6.88–6.98 (2 H, m), 6.99–7.09 (2 H, m), 7.20–7.34 (3 H, m), 7.36–7.45
(2 H, m), 9.77 (2 H, br s, ). 13C NMR (CDCl3, TMS): d 28.11, 45.09,
121.32, 127.40, 128.50, 129.17, 129.42, 130.90, 131.59, 138.97, 140.44.
Anal. ðC18H19BrClNÞ: C, H, N.
4-Bromo-[(phenyl-(N-tert-butoxycarbonyl)piperidin-4-ylidene)
methyl]-benzene, (4)
To a mixture of amine 3 ð6:10 g, 18:6 mmol) and KHCO3 (4:0 g,
40 mmol) in H2O=THF ð20 mL=15 mLÞ was added di-tert-butyl
dicarbonate ð4:9 g; 22:5 mmolÞ. The mixture was refluxed for 1 h and
after cooling to room temperature, EtOAc ð100 mlÞ was added. The
organic layer was separated and washed with H2O ð2 ꢂ 75 mLÞ
and dried. Evaporation to dryness left a solid residue, which was
crystallized from EtOAc–hexane to give 5:50 g (69% yield) of
1
compound 4. M.p, 120–1238C. H NMR (CDCl3, TMS): d 1.46 (9 H,
s), 2.30–2.33 (4 H, m), 3.44–3.46 (4 H, m), 6.98 (2 H, d, J ¼ 8:0 Hz),
7.05–7.13 (2 H, m), 7.18–7.32 (3 H, m), 7.40 (2 H, d, J ¼ 8:0 Hz). 13C
NMR (CDCl3, TMS): d 28.44, 31.56, 45.12, 79.57, 120.57, 126.75,
128.18, 129.64, 131.52, 135.17, 136.19, 141.05, 141.63, 154.80. Anal.
ðC23H26BrNO2Þ: C, H, N.
4-[(Phenyl-(N-tert-butoxycarbonyl)piperidin-4-ylidene)methyl]-
benzoic acid, (5)
On a vacuum manifold, n-BuLi (1:4 M in hexane, 0:7 mL, 0:98 mmol)
was added at ꢀ788C to a solution of aryl bromide 4 ð402 mg; 0:94
mmolÞ in THF ð5 mLÞ. The mixture was stirred at ꢀ788C for 1 h after
which CO2 (from BaCO3, 200 mg, 1 mmol and conc. H2SO4) was
introduced during 15 min. The temperature was maintained for another
15 min and then the stirring was continued at room temperature. After
4 h, H2O was added to the reaction mixture followed by 1 M citric acid
to pH ꢃ 3. The organic layer was separated, washed with H2O and
dried. After evaporation of the solvent to dryness, the residue was
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recrystallized from EtOAc–heptane to give acid 5 (123 mg, 33% yield).
M.p. 216–2178C. IR (KBr): n 2974, 1719, 1692 cmꢀ1. 1H NMR (CDCl3,
TMS): d 1.43 (9 H, s), 2.23–2.36 (4 H, m), 3.38–3.50 (4 H, m), 7.04–7.09
(2 H, m), 7.18 (2 H, d, J ¼ 8:6 Hz), 7.20–7.30 (3 H, m), 7.99 (2 H, d,
J ¼ 8:6 Hz).). 13C NMR (CDCl3, TMS): d 28.44, 31.60, 45.03, 79.69,
126.90, 127.43, 128.27, 129.69, 129.86, 130.07, 135.94, 136.50, 141.33,
147.85, 154.83, 171.11. Anal. ðC24H27NO4Þ: C, H, N.
N,N-Diethyl-4-[(phenyl-(N-tert-butoxycarbonyl)piperidin-4-ylidene)
methyl]-benzamide, (6)
To a solution of DMAP (83 mg, 0:68 mmol) in CH2Cl2 ð2:5 mLÞ at
ꢀ208C, SOCl2 (40 mL, 0:54 mmol) was added dropwise. A precipitate
was formed while the mixture was stirred for 15 min. To the slurry, the
acid 5 ð120 mg, 0:31 mmolÞ in CH2Cl2 ð1 mLÞ was added and after 15
min at ꢀ208C the mixture had become a clear solution. Diethylamine
ð150 mL; 1:48 mmolÞ was added and the cooling bath removed. After
stirring overnight, the reaction mixture was washed with 1 M citric acid,
H2O and dried. Evaporation to dryness provided the amide 6 (104 mg,
1
75% yield). M.p. 121–1238C, (EtOAC–hexane). IR (KBr): n cmꢀ1. H
NMR (CDCl3, TMS): d 1.13 (3 H, br s), 1.23 (3 H br s), 1.46 (9 H, s),
2.25–2.40 (4 H, m), 3.29 (2 H, br s), 3.40–3.52 (4 H, m), 3.54 (2 H br s),
7.08–7.12 (2 H, m), 7.14 (2 H, d, J ¼ 8:0 Hz), 7.20–7.26 (1 H, m),
7.26–7.30 (2 H, m), 7.31 (2 H, d, J ¼ 8:0 Hz). 13C NMR (CDCl3, TMS):
d 12.85, 14.24, 28.40, 31.50, 39.15, 43.26, 45.41, 79.50, 126.21, 126.64,
128.08, 129.65, 129.69, 135.13, 135.26, 136.58, 141.71, 143.03, 154.75,
171.08. Anal. ðC28H36N2O3Þ; C, H, N.
4-[(Phenyl-(N-tert-butoxycarbonyl)piperidin-4-ylidene)methyl]-
[carboxy-14C]benzoic acid, (7)
To a stirred solution of bromide 4 ð431 mg; 1 mmolÞ in THF ð5 mLÞ, in
a flask attached to a vacuum manifold, was added dropwise at ꢀ788C, a
solution of n-BuLi (1 mL, 1:6 M in hexane). After 75 min at this
temperature, 14CO2 (from Ba14CO3, 50 mCi, 55 mCi=mmol) was
introduced, the cooling bath removed and the reaction mixture left
overnight to reach room temperature. To the yellow solution, 1 M
aqueous citric acid was added ðpH ꢃ 3Þ followed by diethyl ether
ð5 mLÞ. The organic layer was separated, washed with H2O, dried and
evaporated to dryness. The residue was purified by flash chromato-
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graphy on a SiO2 column using EtOAc: n-hexane (1:1) with HOAc (2%)
as eluent. The main fraction (210 mg, 19:7 mCi) of an oil, crystallized
by the addition of EtOAc providing the acid 7 ð158 mgÞ with
a radiochemical purity of 96%, as determined by TLC in the same
system.
N,N-Diethyl-4-[(phenyl-(N-tert-butoxycarbonyl)piperidin-4-ylidene)
methyl]-[carboxy-14C]benzamide, (8)
To a solution of DMAP (85 mg, 0:7 mmol) in CH2Cl2 ð2:5 mLÞ
was added SOCl2 ð50 ml; 0:67 mmolÞ at ꢀ208C. A white precipitate
was formed and after 15 min of stirring, the acid 7 (158 mg, 0.40 mmol)
in CH2Cl2 ð3 mLÞ was added, dropwise, keeping the temperature
between ꢀ25 and ꢀ208C. The stirring was continued at the
same temperature while the precipitate dissolved. After 15 min,
diethylamine ð150 mg; 2 mmolÞ was added and the stirring continued
for 1 h. 2 M HCl ð2 mLÞ was added and the organic layer separated,
washed with 1 M aqueous citric acid, H2O and dried. Evaporation to
dryness left benzamide 8 as a glass [162 mg, 14:9 mCi, radiochemical
purity: 95.7%, as determined by TLC: SiO2/hexanes–EtOAc, (1:1)]
which was used in the next step.
N,N-Diethyl-4-[(phenyl-(piperidin-4-ylidene)methyl]-[carboxy-14C]
benzamide hydrochloride, (9)
The compound 8 was dissolved in CH2Cl2 ð1 mLÞ and trifluoroacetic
acid (TFA) ð100 mLÞ was added. The mixture was stirred at
room temperature and after 2 h, TLC indicated 29% conversion
and more TFA ð50 mLÞ was added. After another hour, TLC indicated
53% conversion and 100 mL of TFA was added and the stirring
continued. After 1 h; CH2Cl2 ð3 mLÞ was added and the pH adjusted
to ꢃ 10 with 2 M NaOH. The organic layer was separated, washed
with H2O and dried. Evaporation to dryness left a residue ð109 mgÞ
which was purified on a SiO2 column using CH2Cl2 (w. drops of
conc. NH3) with a gradient of MeOH as eluent. Three fractions
containing the base of compound 9 were collected (tot. 88 mg). The
first fraction ð55 mgÞ was treated with HCl in EtOH to furnish
the compound 9 ð6:6 mCi, spec. act: 48:7 mCi=mmol, radiochemical
purity: 99.4% by HPLC and 98.1% by TLC, ½SiO2=CH2Cl2–MeOH–
NH4OH (9:1:drps)].
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Acknowledgements
.
The author is grateful to Mr Peter Strom, AstraZeneca R&D Lund,
Sweden, for providing laboratory space and equipment.
References
1. Wei Y-Z, Brown W, Takasaki B, et al. J Med Chem 2000; 43:3895–3905.
2. Kofron WG, Baclawski LM. J Org Chem 1976; 41:1879–1880.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 131–138