M. J. Hickey et al.
Waters Sunfire C18, 3.5 μm, 4.6 × 100 mm, column temperature: 40ºC,
flow rate: 1.2 mL/min, eluent; A: 0.1% aq TFA, B: 0.1% TFA in acetonitrile,
gradient: 0min 5% B, 2 min 5% B, 18 min 95% B, 20min 95% B, UV 254nm.
Quantification of radioactivity was performed using a Perkin–Elmer TRI-
CARB 2500 liquid scintillation analyzer, with Ultima GoldTM cocktail.
Figure 1. Ticlopidine structure.
2-Chloro-[carboxyl-14C] benzoic acid (2)
To a solution of 1-bromo-2-chlorobenzene (396 mg, 2.07 mmol) in dry
tetrahydrofuran (4 mL) at 0°C, was added dropwise, iPrMgCl·LiCl complex
(1.3 M in tetrahydrofuran) (1.59 mL, 2.07 mmol). The solution was stirred
at 0°C for 1 h, submerged in liquid nitrogen and degassed twice by a
freeze-thaw cycle. The reaction flask was evacuated and [14C]carbon
dioxide (118 mCi, 2.07 mmol) gas was transferred in vacuo to the reaction
mixture. The mixture was warmed to 0°C and after stirring for 1 h, a
mixture of aq 2M HCl (30 mL) and methyl t-butyl ether (30 mL) was
added. The aqueous was extracted with methyl t-butyl ether (30 mL)
and combined organic layers were washed with water (30 mL), sat. NaCl
(30 mL) and dried over MgSO4. After filtering, the solvent was removed
under reduced pressure to give the product as a white solid (0.24 g,
81 mCi, 69% radiochemical yield) that was used directly in the next
reaction. LCMS m/z: 159 ([M + H]+); the material cochromatographed with
an authentic sample by HPLC (Rt = 9.0 min).
Figure 2. Ticlopidine metabolites identified from glutathione trapping experiments.
the material was found to be unstable as the free base with the
formation of a 3% radiochemical impurity after storing for 1 h at
room temperature; the MS of the impurity showed a mass
decrease of two when compared with the parent compound, but
was not further characterised. Consequently, [methylene-14C]
ticlopidine was converted to the hydrochloride salt directly after
silica gel purification to provide 55 mCi of [methylene-14C]ticlopidine
HCl 1 (47% overall radiochemical yield) with a specific activity of
[methylene-14C]Ticlopidine hydrochloride (1)
2-Chloro-[carboxyl-14C]benzoic acid
2
(0.24 g, 1.51 mmol) in
tetrahydrofuran (4 mL) was treated with N,N-carbonyldiimidazole
(0.26 g, 1.59 mmol) and stirred at room temperature for 2 h. 4,5,6,7-
Tetrahydro thieno[3,2-c]pyridine hydrochloride (0.29 g, 1.67 mmol) was
added followed by triethylamine (0.63 mL, 4.53 mmol). The mixture was
heated at 55°C for 16 h, cooled to 0°C and borane–tetrahydrofuran
complex (1M) (12 mL, 12.0 mmol) was added dropwise. The mixture
was heated at 55°C under nitrogen for 6 h and cooled to room
temperature. 2M aq HCl (30 mL) was added and the solution stirred at
55°C for 18 h. The volatiles were evaporated in vacuo, and the residue
diluted with water (30 mL) and washed with methyl t-butyl ether
(30 mL). The aqueous layer was basified to pH 12 with aq 2M NaOH,
extracted twice with methyl t-butyl ether (30 mL each), and the
combined organic layers were washed with water (30 mL), sat. NaCl
(30 mL) and dried over MgSO4. The slurry was then filtered and the
filtrate concentrated under reduced pressure. The crude product
(292 mg) was dissolved in dichloromethane (2 mL) and purified by flash
chromatography on silica (70 g) eluting with 5% ethyl acetate in
53.4 mCi/mmol and
a radiochemical purity of 98.5%. The
hydrochloride salt was stored as an ethanol solution (1 mCi/ml)
at À20ºC. Under these conditions, the rate of radiochemical
degradation was measured to be 1.4% over 6 months.
Experimental
Materials and methods
[
14C]Carbon dioxide (specific activity 57 mCi/mmol) was generated
from a [14C]carbon dioxide manifold system purchased from RC TRITEC
AG, Teufen, Switzerland. All other reagents and anhydrous solvents
were obtained from Sigma Aldrich and were used without further
purification. 1H and 13C nuclear magnetic resonance (NMR) spectra
were recorded on a Bruker (500 MHz). Chemical shifts (δ) in parts per
million are quoted relative to CDCl3 (δ = 7.26). Flash column isohexane to give a clear oil that was immediately dissolved in ethanol
chromatography was performed using pre-packed SiliSepTM silica gel
cartridges (SiliCycle, Quebec, Canada). Analytical thin layer
chromatography was carried out on Merck 5785 Kieselgel 60F254
fluorescent plates. LCMS data were obtained on a Waters Acquity ultra
performance liquid chromatography with a Waters Micromass ZQ ESCi
probe mass detector. Radiochemical purity checks were determined on
(20 mL). 4M HCl in dioxane (3 mL) was added, and the solution was
evaporated in vacuo to afford a white solid (312 mg, 55 mCi, 68%
radiochemical yield, 98.5% RCP by HPLC). The solid was dissolved in
ethanol (55 mL) and stored under nitrogen at À20°C.
1H NMR (CDCl3) δ 13.25 (br s, 1H) 8.40 (dd, J= 7.57, 1.63 Hz, 1H), 7.47
(m, 2H), 7.43 (dd, J= 7.64, 1.74 Hz, 1H), 7.25 (d, J= 5.22 Hz, 1H), 6.75
an Agilent series 1100 HPLC system coupled to a β-Ram Flow (d, J= 5.22 Hz, 1H), 4.58 (m, 2H), 4.41 (d, J= 15.25 Hz, 1H), 4.04 (dd, J= 15.14,
Scintillation Analyser (Lab Logic, UK) using the following system: 6.31 Hz, 1H), 3.78 (m, 1H), 3.62 (m, 1H), 3.35 (m, 1H), 3.13 (m, 1H)
Scheme 1. a) iPrMgCl·LiCl, THF, 0°C then [14C]carbon dioxide; b) N,N-carbonyldiimidazole (CDI), THF, then 4,5,6,7-tetrahydro thieno[3,2-c]pyridine hydrochloride, Et3N,
55 °C; c) BH3-THF, 55°C then 2M aq HCl, purification, 4M HCl in dioxane.
J. Label Compd. Radiopharm 2014, 57 172–174
Copyright © 2014 John Wiley & Sons, Ltd.
Hickey, Michael J.
