Preparation of carbon-14 labeled (3R)-7-hydroxy-N-(1S)-1-{[(3R,4R)-4-(3- hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl-1,2,3, 4-tetrahydroisoquinolinecarboxamide (JDTic)

Article abstract of DOI:10.1002/jlcr.1560

Starting with [¹⁴C]-D-tyrosine, carbon-14 labeled JDTic dihydrochloride with specific activity 15 mCi/mol was prepared in 5% radiochemical yield. Separation of the (3R)- and (3S)-diastereomers was carried out via the 3-phenyl-2,3,10,10a-tetrahy

Full text of DOI:10.1002/jlcr.1560

Research Article
Received 30 April 2008,
Revised 18 September 2008,
Accepted 30 September 2008
Published online 7 November 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1560
Preparation of carbon-14 labeled (3R)-7-
hydroxy-N-(1S)-1-{[(3R,4R)-4-(3-
hydroxyphenyl)-3,4-dimethyl-1-
piperidinyl]methyl}-2-methylpropyl-1,2,3,4-
tetrahydroisoquinolinecarboxamide (JDTic)
Ã
Bertold D. Berrang, Anita H. Lewin, and F. Ivy Carroll
Starting with [14C]-D-tyrosine, carbon-14 labeled JDTic dihydrochloride with specific activity 15 mCi/mol was prepared in 5%
radiochemical yield. Separation of the (3R)- and (3S)-diastereomers was carried out via the 3-phenyl-2,3,10,10a-tetrahydro-
5H-imidazo[1,5-b]isoquinolin-1-ones formed by reaction with benzaldehyde.
Keywords: JDTic; k antagonist; carbon-14
Spengler reaction was carried out in the presence of strong
acid(s) under anhydrous conditions. Thus, cyclization of 2b using
Introduction
The potent and selective
k opioid receptor antagonist
a
small excess of paraformaldehyde in the presence of
(3R)-7-hydroxy-N-(1S)-1-{[(3R;4R)-4-(3-hydroxyphenyl)-3;4-dimethyl-
1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-iso-
quinolinecarboxamide (JDTic) has been found to have long
lasting antinociceptive effects; mandating metabolism and
distribution studies.1 To this end the preparation of carbon-14
labeled JDTic was undertaken.
Considerations of metabolic stability precluded the labeling of
pendant groups (e.g. i-propyl or methyl). Based on the synthesis
of JDTic2 it was decided to incorporate carbon-14 at the
isoquinoline moiety, i.e. to use [14C]-D-tyrosine as the starting
point for the synthesis (Scheme 1).
hydrobromic acid in trifluoroacetic acid afforded 3b in 89%
when the reaction was carried out for 15 h at 501C. Similar
results were obtained with the diiodo analog 2a although the
purity of 3a was somewhat lower than that of 3b. Improved
yields (490%) were achieved in the dehalogenation reaction by
replacing the triethylamine3 (used to scavenge the acid formed
in the dehalogenation) by cesium carbonate.
Based on these encouraging results the carbon-14 synthesis
was carried out using commercially procured carboxyl carbon-
14 labeled D-tyrosine ([14C]-1). Bromination gave the hydro-
bromide salt of [14C]-2b in 96% yield and reaction with
paraformaldehyde in the presence of trifluoroacetic acid and
hydrobromic acid in acetic acid for 12 h at 801C gave [14C]-3b
hydrobromide in 88% yield. Protection followed by reductive
dehalogenation at atmospheric pressure afforded the required
[14C]-5 in 50% overall yield (from [14C]-1). Coupling with 3-[1-(2S-
amino-3-methylpropyl)-3R,4R-dimethyl-4-piperidinyl]phenol (6)
using O-(7-azabenzotriazol-1-yl)-N,N,N0,N0,-tetramethyluronium
hexafluorophosphate (HATU reagent) proceeded in 56% yield.
Pilot deprotection of a small portion of the product [14C]-7
afforded material that was ꢀ85% radiochemically pure. How-
ever, HPLC analysis indicated that the product consisted of a
4:1 mixture of diastereomers, which were identified as D- and
Results and discussion
Conversion of tyrosine (1) to N-Boc-7-hydroxy-1,2,3,4-tetrahy-
droisoquinoline-3-carboxylic acid (5) has been carried out via
3,5-dihalogenated tyrosine derivatives using the Pictet Spengler
reaction (Scheme 1).3 Thus, 3,5-diiodotyrosine (2a) is reported to
afford,
6,8-diiodo-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid (3a) hydrochloride in ꢀ55% yield3–5 while the
dibromo analog 2b gave 3b in 30% yield.3 Preparation of the
required Boc-protected intermediate 5 has been reported to
proceed in 54% yield by Boc protection of 3a followed by
dehalogenation. Experience in the synthesis of JDTic in our
laboratories essentially replicated these results. Based on this
information the radiochemical yield of 5 would have been
ꢀ29%, at best. In light of the expense of the starting carbon-14
labeled D-tyrosine, it was sought to improve the overall yield of
5. After some experimentation it was found that the yield of
cyclized product was significantly improved when the Pictet
Center for Organic and Medicinal Chemistry, Research Triangle Institute, P.O.
Box 12194, Research Triangle Park, NC 27709-2194, USA
*Correspondence to: Anita H. Lewin, Research Triangle Institute, P. O. Box 12194,
Research Triangle Park, NC 27709-2194, USA.
E-mail: ahl@rti.org
J. Label Compd. Radiopharm 2008, 51 440–443
Copyright r 2008 John Wiley & Sons, Ltd.

B. D. Berrang et al.
Scheme 1.
L-isomers of the tetrahydroisoquinoline carboxylic acid moiety. further even with addition of a large excess of the carbonyl
This was surprising since it had been reported that virtually no containing compound. Condensation with benzaldehyde gave
racemization occurred when reaction of 2a with formaldehyde the most favorable results in that it furnished a high yield of
was carried out at 721C for longer time (18.5 h).3 In subsequent adducts. Fractional crystallization separated an adduct that,
studies it was found that 7% racemization occurred when the when hydrolyzed, yielded JDTic (8), virtually free of JLTic (9)
cyclization was performed at 551C for 72 h.6 The reason(s) for accompanied by several minor lipophilic impurities. These were
this discrepancy are unknown.
readily removed by normal phase chromatography. X-ray
Attempts to separate the diastereomers by use of chiral acids crystallography identified the benzaldehyde adduct 10 as
failed, as did attempted reversed phase chromatography. resulting from condensation with both the isoquinoline nitro-
During some of these investigations it was noted that treatment gen and the amide nitrogen. In a tracer run carbon-14 labeled
of the mixture of diastereomers with acetone led to the JDTic-adduct [14C]-10 was obtained in 75% yield and 495%
formation of two distinctly different adducts and that adduct purity. Encouraged by these results the main fraction of [14C]-7
formation was reversible. Other carbonyl containing com- was deprotected and the resulting carbon-14 labeled product, a
pounds, e.g. acetophenone, cyclohexanone and benzaldehyde mixture of 80% [14C]-8 and 20% [14C]-9 was converted to the
also reacted to give adducts. In most cases significant amounts benzaldehyde adduct. Fractional crystallization gave the pure
of starting material remained unreacted and failed to react D-isomer [14C]-10 with specific activity 57.5 mCi/mmol (53 mg,
J. Label Compd. Radiopharm 2008, 51 440–443
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B. D. Berrang et al.
5.5 mCi) in good agreement with the specific activity of the 10 h. The solvent was evaporated and, the residue was vacuum
starting material [14C]-1. Careful hydrolysis furnished [14C]-8 in dried to give crude [14C]-3b as a light gray solid (360 mg, 40 mCi,
85% radiochemical purity and 65% chemical purity. The 83%) that was radiochemically ꢀ88% pure based on TLC-
lipophilic benzaldehyde-related byproducts were removed by radioscan using SiO2, EtOAc–7% MeOH (Rf 0.16). The crude
repeated chromatographies, to afford pure carbon-14 labeled material was used in the next step without purification. 1H NMR
JDTic ([14C]-8) in 50% yield. The product (2.4 mCi) was diluted to (300 MHz, MeOH-d4) d (ppm): 3.2–3.4 (m, 2H, H4); 4.2–4.5 (m, 3H,
specific activity 15 mCi/mmol with unlabeled 8 and then H1, H3); 7.5 (s, 1H, H5).
converted to the dihydrochloride salt. The diluted
.
product 8 2HCl was formulated in 50% aqueous ethanol at 6,8-Dibromo-N-t-butoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-3-
0.5 mCi/mL.
[14C]carboxylic acid [14C]-4b
A solution of crude [14C]-3b (360 mg, 40 mCi, 0.83 mmol) in
anhydrous DMF (2 mL) was diluted with H2O (0.5 mL) and Et3N
Conclusions
Pure (99%) JDTic ([14C]-8) dihydrochloride was prepared in eight
steps in overall 5% yield from carboxyl carbon-14 labeled
(400 mg, 4 mmol) and di-t-butyl carbonate (380 mg, 1.7 mmol)
were added. The resulting brown solution was stirred at ambient
temperature for 3–4 h. The solvent was evaporated under a N2
stream at ꢀ401C. The residual brown solid was partitioned
between EtOAc and 1 N HCl. The aqueous layer was re-extracted
tyrosine ([14C]-1). Although the reason(s) that partially racemized
product was obtained in the preparation is unknown, it has
been determined that the diastereomers are separable via a
cyclic benzaldehyde adduct (10).
with EtOAc, and the combined extract was evaporated. The
residue was chromatographed on a silica gel column (10 g)
using a gradient of EtOAc with 1 to 4% MeOH. This afforded
[14C]-4b as a glassy solid (370 mg, ꢀ25 mCi, 63% yield), which
Experimental
Proton NMR spectra were recorded on a Bruker Avance 300 MHz
NMR spectrometer. TLC analyses were carried out on commer-
cial pre-coated silica gel 60F254 or C18F254 glass plates (E.
Merck: 5 ꢁ 10 cm and 5 ꢁ 20 cm). The plates were scanned using
a Bioscan System 200 Imaging Scanner Automatic Linear
Analyzer equipped with a Gateway P5-133 computer for data
reduction. Chromatographies were carried out using 230–400
mesh silica gel 60 (E. Merck).
HPLC analyses were performed on a dual pump (Rainin HPX)
system equipped with Rheodyne injector, variable wavelength
monitor (Knauer) followed by a b-RAM detector (IN/US) using a
liquid scintillate cell. Samples were counted on a Tri-Carb
Scintillation Analyzer (Packard Bioscience Company model
2100TR) using Optima Gold counting cocktail.
was 97% radiochemically pure by TLC-radioscan (SiO2,
EtOAc–10% MeOH–1% HOAc) (Rf 0.70). 1H NMR (300 MHz,
MeOH-d4) d (ppm): 1.5 (s, 9H, C(CH3)3); 3.1 (m, 2H, H4); 4.4, 4.6 (d,
dd, J = 16, 7.5 Hz, 2H, H1); 5.0 (m, 1H, H3); 7.4 (s, 1H, ArH).
N-t-butoxycarbony-1,2,3,4-tetrahydroisoquinoline-3-[14C]carboxylic
acid [14C]-5
A solution of [14C]-4b (360 mg, 0.52 mmol) in DMF (7 mL) was
treated with cesium carbonate (1.3 g), which had been vacuum
dried at 1501C for 15 min. The suspension was stirred at ambient
temperature under vacuum until gas evolution slowed notice-
ably (ꢀ20 min). Hydrogenation over 10% Pd/C (50 mg) was
performed at atmospheric pressure for 8 h. The suspension was
filtered through celite and, the filter cake was washed with
EtOAc. The filtrate was concentrated under N2 to ꢀ0.5 mL and,
the residue was partitioned between EtOAc and 1 N HCl. The
organic layer was evaporated to a light brown glassy solid that
was chromatographed on silica gel (3.5 g) using CH2Cl2 with a
gradient of 1–10% of MeOH containing 10% NH4OH. This
furnished [14C]-5 as a white amorphous solid, (124 mg, 22.8 mCi,
91% yield). The radiochemical purity, determined by TLC-
radioscan (C18, CH3CN-H2O 1:1 with 0.1% HOAc and 0.05%
Et3N), was 94%. 1H NMR (300 MHz, MeOH-d4) d (ppm): 1.48, 1.52
(2s, 9H, C(CH3)3); 3.05 (m, 2H, H4); 4.5 (m, 3H, H1, H3); 6.5 (m, 2H,
H6, H8); 6.9 (m, 1H, H5).
30,50-Dibromo-[14C-CO2]-D-tyrosine hydrobromide [14C]-2b
To
a
suspension of [14C-CO2]tyrosine [14C]-1 (Amersham
Biosciences/GE Healthcare; 50 mCi, 161 mg, 0.89 mmol; specific
activity 57 mCi/mmol) in HOAc (2 mL) and H2O (0.5 mL) at 01C
was added Br2 (275 mg, 1.72 mmol) dropwise, with stirring, at a
rate such that Br2 was absorbed before further addition. A light
yellow solution resulted after 25 min. Further Br2 (25 mg,
0.15 mmol) in HOAc (300 mL) was added over a 10 min period.
The resulting light brown solution was evaporated under a
nitrogen jet at ambient temperature. After vacuum drying, [14C]-
2b was obtained as a light brown solid (48 mCi, 360 mg, 96%)
1
which was used in the next step without purification. H-NMR
(300 MHz, MeOH-d4) d (ppm): 3.1–3.2 (m, 2H, CH2), 4.2 (m, 1H,
CH); 7.4 (s, 2H, ArH).
(3R)-7-Hydroxy-N-(1S)-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-
1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-t-butoxy-
carbonylamido-3-isoquinoline-[14C]carboxamide [14C]-7
To a solution of [14C]-5 (124 mg, 0.42 mmol) in CH2Cl2 (3 mL) was
added 6 (132 mg, 0.45 mmol) followed by O-(7-azabenzotriazole-
6,8-Dibromo-1,2,3,4-tetrahydroisoquinoline-3-[14C]carboxylic acid
hydrobromide [14C]-3b
1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophosphate
To a solution of [14C]-2b (360 mg, 0.86 mmol) in TFA (5 mL) was
added paraformaldehyde (33 mg, 1.1 mmol) and 45% HBr in
HOAc (160 mg, 0.89 mmol). The mixture was heated to 801C with
stirring in a teflon sealed tube. The course of the reaction was
monitored by taking aliquots for H NMR analysis after 3, 8, 10,
12 h. After 8 h more paraformaldehylde (5 mg, 0.01 mmol) was
added to the reaction mixture. No change was observed after
(182 mg, 0.48 mmol). The suspension was stirred at 01C for
1.2 h under N2 atmosphere, then DIEA (127 mg, 1.07 mmol) was
added and stirring was continued at 01C until a clear solution
resulted (ꢀ10 min). Analysis by TLC-radioscan after stirring at
ambient temperature for 1.5 h showed a mixture of products
(C18, CH3CN–H2O–HOAc–Et3N 60:40:0.1:0.05) containing ꢀ50%
of [14C]-7 (Rf 0.44). The crude product was partitioned between
1
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J. Label Compd. Radiopharm 2008, 51 440–443

B. D. Berrang et al.
H2O and CH2Cl2. The aqueous phase was re-extracted with was added at ambient temperature and the mixture was stirred
CH2Cl2 followed by extraction with EtOAc. The combined extract for 17 h. Reaction progress was monitored by TLC (SiO2, CH2Cl2–
was evaporated to a light brownish yellow glass (385 mg, MeOH–NH3 9:1:0.1, Rf 0.70). Over a 28 h period HOTs (10 mg,
15.8 mCi), which contained solvents as well as radioactive and 0.05 mmol) was added twice while the temperature was
non-radioactive materials. For purification this product was gradually raised to 501C for the first 4 h of the reaction. The
chromatographed on silica gel (7 g) conditioned with resulting product consisted of ꢀ65% [14C]-8, 2.5% [14C]-9 and
CH2Cl2–NH4OH 8:2:0.2, then washed with CH2Cl2. Crude [14C]-7 several lipophilic by-products. The yellow green fluorescent
was applied to the column in CH2Cl2 solution and eluted with solution was evaporated to a yellow glass, which was treated
CH2Cl2 (20 mL) followed by
a gradient of CH2Cl2 with with a mixture of 5% NaHCO3 solution and EtOAc–10% MeOH.
(MeOH–10% NH4OH) up to 9% of the latter solvent. Combina- The organic phase was separated and the aqueous phase was
tion of fractions with highest purity on evaporation gave an re-extracted with EtOAc–10% MeOH. The combined extract was
amorphous solid, which consisted of ꢀ85% of [14C]-7 (TLC- evaporated to a gum that was dissolved in CH2Cl2–10% MeOH
radioscan, as above Rf 0.43). The recovered [14C]-7 (14 mCi) was and chromatographed on silica gel (3 g) using a gradient of
used in the next step without further purification.
CH2Cl2ꢂ2 to 15% MeOH. Combination of the purest fractions
furnished [14C]-8 (2.4 mCi). HPLC analysis (C18 column,
250 ꢁ 4.6 mm, 20 min gradient of 80% Aꢂ20% B, 40% Aꢂ60%
B [A = H2O, 0.2% dichloroacetic acid, triethylamine]; B = metha-
nol 1 mL/min, 275 mm, b-RAM) indicated 98% chemical purity
and 99% radiochemical purity. The identity of [14C]-8 was
confirmed by its coelution with authentic unlabeled material (Rt
15.5 min). Impure fractions containing mostly non-polar by
products were combined and re-chromatographed on silica gel.
Applying the conditions described above an additional 350 mCi
of [14C]-8 was recovered.
(3R)-7-Hydroxy-N-(1S)-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-
1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoqui-
noline-[14C]-carboxamide [14C]-8 dihydrochloride
The amorphous solid [14C]-7 (140 mg, 0.25 mmol) was dissolved
in THF-40% 6 N HCl (7 mL) and stored at ambient temperature
for 5–6 h. The solvents were evaporated under a N2 jet leaving a
light yellow solid (11 mCi, 78% crude return), which based on
HPLC analysis, [C18, H2O–0.1% Cl2CHCO2H–0.1% Et3N (solvent A)
and MeOH (Solvent B) using a gradient 0–20 min 20% B–60% B,
20–30 min 60% B, with a flow rate of 1 mL/min, and detection by
UV at 275 nm] (Rt 17.8 min) consisted of a 4:1 mixture of [14C]-8
(Rt 16.8 min) and its L-isomer [14C]-9 (Rt 17.7 min). The mixture
was dissolved in CH2Cl2ꢂ20% MeOH and chromatographed on
silica gel (7 g) as described above. Collected fractions consisted
of [14C]-8 and [14C]-9 in a ratio of 1:1 increasing to 35:1. Fractions
containing p20% [14C]-9 were combined, evaporated to a pale
yellow-brown solid (8.7 mCi), which contained ꢀ90% of [14C]-8.
The earlier eluting chromatography fractions containing 420%
of [14C]-9 were combined and rechromatographed on silica gel.
Recovery of [14C]-8 containing ꢀ5% [14C]-9 was only 250 mCi.
(3R)-7-Hydroxy-N-(1S)-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-
1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoqui-
noline-[14C]carboxamide dihydrochloride ([14C]-8 ꢃ 2HCl)
For final formulation, 2.4 mCi of [14C]-8 (20 mg) was diluted with
a solution of unlabeled 8 (55 mg) in MeOH (2 mL). The resulting
solution was treated with 1 N HCl (1 mL). Evaporation of the
solvent gave [14C]-8 2HCl as a white solid. The specific activity,
.
determined gravimetically, was 15 mCi/mmol. The material was
dissolved in 50% ethanol and stored at ꢂ201C.
Benzaldehyde adduct [14C]-10
Acknowledgement
The chromatographed mixture of [14C]-8 and [14C]-9 (80–90% at
a ratio of 7:1) was analyzed after storage in the freezer for ꢀ3
months. Radiochemical purity had decreased by ꢀ5% and
chemical purity was o50% due to formation of lipophilic matter
of low radioactivity. A solution of this mixture (8.7 mCi,
0.15 mmol) in MeOH (2 mL) was stirred with benzaldehyde
(50 mg, 0.5 mmol) and HOTs (3 mg, 0.015 mmol) at 401C for 2
days. The solvent was evaporated to dryness, and the residue
was re-dissolved in MeOH (0.8 mL). Upon seeding, a solid
crystallized. The product [14C]-10 was isolated after cooling at
ꢂ201C overnight, (53 mg 5.5 mCi, S.A 57.5 mCi/mmol, 63% yield)
of mp: 215–2171C, radiochemical purity of 495%. 1H NMR
(MeOH-d4) d (ppm): 0.75–0.88 (m, 9H, CH(CH3)2; 3- CHCH3); 1.26
(s, 3H, CCH3); 1.5(m, 1H, CH CH3); 2.0, 2.3, 2.8, 3.0, 3.2, 3.3 (6 m,
10H, (CH2)3, 3H, 4H, CH); 5.0 (s, 1H, C6H5–CH); 6.3, 6.6, 6.8, 7.0, 7.1
(5 m, 7H, C6H3, C6H4); 7.4, 7.7 (2m, 5H, C6H5).
The authors are grateful to NIDA for support (Contract No. N01-
DA-3-7736 and grant No. DA09045) and to Dr J. Deschamps
(Naval Research Laboratory) for determining the X-ray crystal
structure.
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(3R)-7-hydroxy-N-(1S)-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-
1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoqui-
noline-[14C]carboxamide [14C]-8 from hydrolysis of [14C]-10
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dissolved by heating in MeOH (4 mL), HOTs (25 mg, 0.13 mmol)
J. Label Compd. Radiopharm 2008, 51 440–443
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org