A short synthesis of [14C]-labelled levamisole and its major metabolite

Article abstract of DOI:10.1002/jlcr.573

Levamisole (I) is the levo isomer of tetramisole. It is a broad spectrum anthelmintic which is used extensively as a veterinary drug for food producing animals. Metabolism and environmental studies necessitated the synthesis of ¹⁴C-labelled levamisole (6) and of its major metabolite (12). ¹⁴C-Tetramisole was obtained in two steps from ¹⁴C-thiourea. Resolution via salt formation and crystallization afforded ¹⁴C-levamisole and ¹⁴C-dexamisole. Racemisation followed again by resolution made it possible to improve the over-all radiochemical yield of ¹⁴C-levamisole to 51.0%. The compound had a specific activity of 73.6 MBq/mmol, a HPLC purity of 99.8% and an enantiomeric excess of 99.4%. The ¹⁴C-labelled metabolite of levamisole (II) was obtained from ¹⁴C-potassium cyanate in 4 consecutive steps. Resolution via chiral HPLC afforded the desired compound in a 16.2% overall radiochemical yield. It had a specific activity of 895 MBq/mmol, a HPLC purity of 98.7% and an enantiomeric excess of 100%. Copyright

Full text of DOI:10.1002/jlcr.573

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 591–600.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.573
Research Article
A short synthesis of [14C]-labelled levamisole
and its major metabolite
Cor G.M. Janssen*, Jos B.A. Thijssen and Willy L.M. Verluyten
Department of Non-clinical Pharmacokinetics, Johnson & Johnson
Pharmaceutical Research and Development, B-2340 Beerse, Belgium
Summary
Levamisole (I) is the levo isomer of tetramisole. It is a broad spectrum
anthelmintic which is used extensively as a veterinary drug for
food producing animals. Metabolism and environmental studies
necessitated the synthesis of 14C-labelled levamisole (6) and of its major
metabolite (12).
14C-Tetramisole was obtained in two steps from 14C-thiourea.
Resolution via salt formation and crystallization afforded 14C-levami-
sole and 14C-dexamisole. Racemisation followed again by resolution
made it possible to improve the over-all radiochemical yield of 14C-
levamisole to 51.0%. The compound had a specific activity of
73.6 MBq/mmol, a HPLC purity of 99.8% and an enantiomeric excess
of 99.4%.
The 14C-labelled metabolite of levamisole (II) was obtained from 14C-
potassium cyanate in 4 consecutive steps. Resolution via chiral HPLC
afforded the desired compound in a 16.2% overall radiochemical yield.
It had a specific activity of 895 MBq/mmol, a HPLC purity of 98.7%
and an enantiomeric excess of 100%. Copyright # 2002 John Wiley &
Sons, Ltd.
Key Words: carbon-14; synthesis; levamisole; levamisole metabolite; resolution
*Correspondence to: Cor. G. M. Janssen, Department of non-clinical Pharmacokinetics, Johnson
& Johnson Pharmaceutical Research and Development, B-2340 Beerse, Belgium.
Copyright # 2002 John Wiley & Sons, Ltd.
Received 25 January 2002
Accepted 31 January 2002

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Cor. G. M. JANSSEN ET AL.
Introduction
Levamisole (I) is the levo isomer and the most active form of the broad
spectrum anthelmintic d,l-tetramisole.1–4 It is used extensively as a
veterinary drug for food producing animals.5 For research
reasons, specifically tritium-labelled levamisole at high specific activity
has been synthesized according to a procedure already described
in this Journal.6 Later, for additional metabolism studies, the need
arose to obtain levamisole with a carbon-14 label located in the
imidazo[2,1-b]thiazolidine ring system. Because of extensive use of
levamisole-containing drug, environmental safety studies on its major
metabolite (II) in ruminants were indicated. Of this compound one
desired a synthetic approach which was preferably aimed towards the
introduction of the carbon-14 label at the same position as had the
metabolite formed from the parent drug; the synthesis of which is
described herewith.
Results and discussion
Levamisole is a potent anthelmintic which is already marketed since
1968. Although the use of levamisole-racemate (tetramisole)
carrying the 14C-label on the 7a-position7 or on the 6-position8 has
been mentioned, only one preparative procedure with a 14C-label
uniformly distributed in the phenyl group was revealed in the
literature.9 Our choice to introduce a carbon-14 label situated on the
7a-position proved to be most fruitful in providing a short two-
step synthetic approach towards tetramisole. Resolution with (+)-(L)-
2-[[(4-methoxyphenyl)sulfonyl]amino]-pentane-dioic acid gave an al-
most quantitative isolation of the desired levamisole at an 99.4%
enantiomeric excess. The undesired dexamisole was easily racemized,
offering the possibility to improve the over-all radiochemical yield to
51.0% by repeating the racemization–resolution cycle up to four
times (Scheme 1).
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A SHORT SYNTHESIS OF [14C]-LABELLED
593
Scheme 1. Synthesis of [14C]-labelled levamisole. Reagents and conditions: (i)
HCI 0.1 N, reflux, 12 j; (ii) conc. H2SO4, 08C, 5 min; HPLC-purification; (iii)
Sodium salt formation of optically pure acid, then 3, unlabelled 6, careful
acidification with HCl; (iv) Base liberation with NaOH 50%, then toluene, DMF,
KO-t-Bu, 708C, 11/2 h. Isolation of 3-base in toluene, then HCl; (v) NaOH 10 N,
isolation of base, then toluene and HCl
In vivo, the metabolite in question is formed via hydroxylation of the
thiazolidine ring system, followed by oxidation and hydrolysis of
the tautomeric thiolactone form of the thiohydantoic acid metabolite.7
The synthetic approach comprised four radiochemical steps, which
were followed by HPLC purification and resolution to offer the
optically pure compound in an overall radiochemical yield of 16.2%.
Identification of the correct isomer was performed by co-chromato-
graphy of the isolated enantiomers with a radioactive sample of the
desired metabolite, stemming from in-vivo samples of a 14C-levamisole
metabolism study (Scheme 2).
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Cor. G. M. JANSSEN ET AL.
Scheme 2. Synthesis of [14C]-labelled levamisole. Reagents and conditions: (i)
Base liberation: Na2CO3, 4-methyl-2-pentanone, 18 h, RT, then+7, 4 h, RT,
isolation, then HCl; (ii) KO*CN, MeOH-H2O (70-30, v/v), 1 h RT, 1 h 1008C,
18 h RT, 1 h 08C; (iii) H2, Pd-C, 10%, MeOH, 18 h, 508C; (iv) Lawesson’s
reagent, toluene; (v) NaOH 1 N, H2O, resolution
Experimental
Analytical HPLC was performed on an apparatus consisting of a Gilson
model 305 (master) pump and a Gilson model 306 (slave) pump, each
equipped with a 10 SC pumphead. The samples were injected by a
Rheodyne 7125 injector. Chemical and radiochemical purity were
determined by on-line UV detection on a Gilson 218 detector and
radioactivity detection on a Berthold Radioactivity monitor LB 506 C
system with a flow-through cell of 0.500 ml. The eluate was mixed with
Pico-Fluor TM30 (Packard, used as a scintillation cocktail) in an FMI
ultrograd mixing unit. The normalized areas of the radioactivity peaks
were computed and visualized by a Compaq Deskpro computer unit.
Preparative HPLC and resolution were performed with only on-line UV
detection. Conditions of analytical and preparative HPLC are described
in the text. Radioactivity of known aliquots was measured by liquid
scintillation counting (Packard Tri-Carb 4530), using Ultima Gold
(Packard) as scintillation cocktail. The specific activity was determined
by measuring the UV absorbance (on HPLC) relative to the absorbance
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A SHORT SYNTHESIS OF [14C]-LABELLED
595
of known amounts of injected unlabelled standards and the radio-
activity contents in the HPLC-eluate by means of liquid scintillation
1
counting. H-NMR spectra were obtained on a 400 MHz spectrometer
.
(Bruker, AMX 400). Chemical shifts are reported in ppm with
tetramethylsilane as internal reference.
2-Imino-a-phenyl-3-[2-14C]thiazolidineethanol hydrochloride (2)
A mixture of a-[2-chloroethyl)aminomethyl]benzenemethanol hydro-
chloride (1, 1.65 g, 7.00 mmol), [14C]-thiourea (0.532 g, 7.00 mmol,
14.1 GBq)10 and hydrochloric acid 0.1 N (10.5 ml) was stirred at
reflux for 12 h. The mixture was then cooled to 808C and sodium
chloride (700 mg) was introduced. The mixture was brought to
reflux and was then allowed to slowly cool to room temperature.
After 5 h, the precipitate was filtered, washed with acetone (18 ml) and
1
dried to the air to leave 2 (1.40 g, 10.9 GBq, yield 77.4%). H-NMR
(DMSO): 3.78 (3H, s), 4.06 (2H, s), 4.83 (2H, s), 7.59–8.03 (5H, m),
9.88 (1H, br).
( ꢀ )-2,3,5,6-Tetrahydro-6-phenyl[7a-14C]imidazo[2,1-b]thiazole
hydrochloride (3, tetramisole hydrochloride)
To nitrogen-covered, ice cooled and stirred concentrated sulphuric acid
(5.0 ml), 2 (1.40 g, 5.42 mmol) was slowly added. After addition, the
solution was stirred for 5 min more at ice bath temperature and was
then allowed to slowly come to room temperature. Crushed ice
was added and the mixture was made strongly alkaline with ammonium
hydroxide (conc.), while maintaining the temperature below 258C.
The mixture was extracted with chloroform (50 ml) and the
organic layers were washed with brine (10 ml), dried on magnesium
sulphate and filtered. HCl-saturated 2-propanol was added
to the filtrate, which was evaporated to yield impure tetramisole
hydrochloride. The solid was dissolved in water (4.0 ml)-
methanol (0.3 ml) and purified via preparative HPLC (Hypersil
ODS (5 mm), column 7.1 mm ID ꢁ 300 mm; flow rate: 3.6 ml/min;
eluate: acetonitrile–water–diisopropylamine (32–68–0.2, v/v/v)). The
purified fractions were combined, evaporated, dissolved in methanol
and acidified with HCl-saturated 2-propanol to pH 1, evaporated and
dried for 18 h at 608C under vacuum to yield HPLC-pure 3 (1.16 g,
9.73 GBq, yield 89.2%).
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Resolution of 3 into levamisole hydrochloride (6) and dexamisole
hydrochloride (5) and racemization of dexamisole to tetramisole
A mixture of (+)-(l)-2-[[(4-methoxyphenyl)sulfonyl]amino]-pentane-
dioic acid (3.19 g, 10.1 mmol) and sodium hydroxide 2 N (10.3 ml,
20.6 mmol) was stirred for 10 min at 608C. A mixture of 3 (1.16 g,
4.82 mmol, 9.73 GBq), unlabelled tetramisole hydrochloride (1.16 g,
4.82 mmol) and unlabelled levamisole hydrochloride (1.25 g, 5.18 mmol)
was introduced. After 5 more minutes, sodium citrate 2H2O (60 mg,
0.26 mmol) was added. The solution was removed from its heat source
and slowly acidified at 408C with concentrated hydrochloric acid to pH
4.5 and then with hydrochloric acid 1 N to pH 4.2. Stirring was
continued for 2 h more after which the precipitate was filtered, washed
with water (3 ꢁ 5.0 ml) and dried to the air to give 4 (4.90 g, 94%).
The filtrate was, under nitrogen atmosphere, strongly alkalinized
with 50% sodium hydroxide, stirred for 5 min and extracted with
chloroform (12 ml). The chloroform layers were dried on magne-
sium sulphate and evaporated under nitrogen to give a residue of
dexamisole (5-base, 0.93 g). The residue was dissolved in toluene
(10.0 ml) and N,N-dimethyl-formamide (0.23 ml). Potassium-tert-but-
oxide (0.16 g) was added under nitrogen at 708C. The mixture was
stirred for 90 min at 708C after which it was cooled to 508C. The
reaction mixture was washed twice with water and was separated after
10 min. The toluene layer was dried, filtered, to the filtrate was slowly
added a solution of HCl-saturated 2-propanol (2 ml) and the whole was
evaporated under aspirator pressure at 408C to give tetramisole
hydrochloride (3, 1.07 g, 4.40 mmol). With this material the above
described resolution cycle was repeated five times. Combination of all
the diastereomeric salts (4, 18.2 g) and crystallization from nearly
boiling water (180 ml) gave pure 4 (16.9 g, 32.4 mmol) with a radio-
activity of approx. 7.2 GBq.
The obtained 4 was suspended in water (25 ml) and, under nitrogen
atmosphere, strongly alkalinized with sodium hydroxide 10 N. The
mixture was extracted with chloroform (50 ml), the chloroform layers
were washed once with brine, dried on magnesium sulphate, filtered and
evaporated to give levamisole 6-base (6.51 g, 3.19 mmol). Unlabelled
levamisole hydrochloride (16.1 g, 67.0 mmol) was dissolved in water
(25 ml), alkalinized and isolated as above to give unlabelled levamisole
(13.5 g, 66.1 mmol). Both the labelled and the unlabelled levamisole
were dissolved in toluene and combined (200 ml). The solution was
acidified with HCl-saturated 2-propanol and the mixture was stirred
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A SHORT SYNTHESIS OF [14C]-LABELLED
597
for 20 h. The precipitate was filtered and dried for 8 h at 758C under
vacuum to supply levamisole hydrochloride 6 (23.5 g) with a total
radioactivity of 7.20 GBq (radiochemical yield: 51.0%). The material
had a specific activity of 73.6 MBq/mmol. The radiochemical purity, as
determined via HPLC (Hypersil ODS (5 mm), 4.6 mm ID ꢁ 300 mm;
flow rate: 1.5 ml/min; eluate: acetonitrile-water-diisopropylamine
(35-65-0.2, v/v/v)) was 99.8% with an enantiomeric excess (Chiralcel
OJ (10 mm), 4.6 mm ID ꢁ 300 mm; flow rate: 0.7 ml/min; eluate: hexane–
ethanol (80–20, v/v)) of 99.4%. The material was HPLC identical to
unlabelled material.
Glycine, N-(2-oxo-2-phenylethyl) methyl ester hydrochloride (8)
Glycine methyl ester hydrochloride (1.24 g, 10.0 mmol) was dissolved
in 4-methyl-2-pentanone (20.0 ml) and liberated from its salt by
stirring it for 18 h at room temperature with sodium carbonate
(3.52 g, 30 mmol). To the stirred mixture was dropped a solution
of a-bromo acetophenone (7, 1.79 g, 9.0 mmol) in 4-methyl-2-pentanone
(10.0 ml). After 1 h, the mixture was filtered and the solids were washed
with hot 4-methyl-2-pentanone (14.0 ml). To the filtrate was added
a 6 N solution of hydrochloric acid in 2-propanol (1.6 ml), causing
the product to precipitate as the HCl salt. It was filtered, washed
with 4-methyl-2-pentanone and dried to the air. Crystallisation from
acetonitrile (60 ml) afforded 8 (812 mg, 37.2%).
Methyl[12-14C]-2-oxo-4-phenyl-4,5-didehydroimidazolidineacetate (9)
To a solution of 14C-labelled potassium cyanate (161 mg, 2.0 mmol,
containing 1.68 GBq of radioactivity)y in a mixture of methanol–
water (70–30, v/v; 1.75 ml) was dropped a solution of 8 (487 mg,
2.0 mmol) in the same solvent mixture (2.25 ml). The solution
was stirred for 1 h at room temperature, causing a precipitate to
form. The mixture was brought to reflux temperature and was
stirred for 1 h. It was then stirred for 18 h at room temperature and
then for 1 h at 08C. The precipitate was filtered and washed with ice-
cooled water to yield 9 (330 mg, 71%). The material was used as such in
the next reaction step.
y The material was purchased from NEN, Brussels, Belgium.
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Methyl (ꢀ)-[12-14C]-2-oxo-4-phenyl-1-imidazolidineacetate (10)
A mixture of 9 (330 mg, 1.42 mmol), palladium on carbon (10%, 50 mg)
and methanol (15 ml) was hydrogenated at 508C for 18 h. It was filtered
hot over celite, and the celite was rinsed several times with small
portions of hot methanol. The filtrate was concentrated at aspirator
pressure and the residue was recrystallized from 2-propanol (5.0 ml) to
yield 10 (323 mg, 98%, containing 1.05 GBq of radioactivity). It was
92.7% HPLC pure (Kromasil KR100-10 (5 mm), 4.6 mm ID ꢁ 300 mm;
flow rate: 1.0 ml/min; programmed run consisting of an isocratic run for
20 min with aqueous 0.1 M ammonium acetate solution brought to pH
9.0–methanol–acetonitrile (64–18–18, v/v/v), followed by a 5 min linear
gradient run to solvent composition (10–45–45, v/v/v) and again a
10 min isocratic run at the latter composition).
Methyl (ꢀ)-[12-14C]-2-thiooxo-4-phenyl-1-imidazolidineacetate (11)
A suspension of 10 (323 mg, 1.38 mmol) and Lawesson’s reagent
(279 mg, 0.69 mmol) in toluene (7.0 ml) was stirred for 10 h at 908C.
The obtained light yellow solution was allowed to cool to room
temperature, causing the product to crystallize. The crystals were
filtered, washed with toluene (1.0 ml) and diisopropyl ether (4.0 ml) and
dried to the air to give 11 (235 mg). The HPLC radiochemical purity
(Kromasil KR100-10 (5 mm), 4.6 mm ID ꢁ 300 mm; flow rate: 1.0 ml/
min; programmed isocratic run for 10 min with aqueous 0.1 M
ammonium acetate solution (pH 9.0)–methanol–acetonitrile (48–26–
26, v/v/v), followed by a 5 min linear gradient run to solvent
composition (20–40–40, v/v/v) and again a 10 min isocratic run at the
latter solvent composition) was 92.6%.
(S)-[2-14C]-2-thiooxo-4-phenyl-1-imidazolidineacetic acid (12)
The obtained 11 (293 mg) was stirred with sodium hydroxide 1 N
(1.25 ml) for 1.5 h at 808C. The solution was then acidified with conc.
HCl (0.12 ml), causing the product to precipitate. The mixture
was stirred for 30 min and the precipitate was then filtered,
washed with water and diisopropyl ether and dried to the air to
give the racemate of 12 (183 mg). Part of this material (130 mg) was
dissolved in methanol (1.2 ml) and purified via preparative HPLC
by injection of 50 ml portions with an extra added 200 ml of
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599
water (Kromasil RP18 100-10, 7.1 mm ID ꢁ 300 mm; flow rate: 4.0 ml/
min; UV detection at 235 nm, eluate: water-acetonitrile-formic acid (72-
28-0.2; v/v/v)). The combined product peaks were concentrated at 408C
(aspirator pressure), dissolved in acetonitrile (20.0 ml) and again
thoroughly concentrated at 308C (aspirator pressure). The residue was
dissolved in ethanol (10.0 ml) and resolved by means of chiral HPLC by
multiple 30 ml injections (Chiralpak AD (10 mm), 4.6 mm ID ꢁ 50 min;
flow rate 3.0 ml/min; UV detection at 250 nm, eluate: hexane–ethanol–
trifluoroacetic acid (76–24–0.03; v/v/v)). The desired material was the
first eluting peak, as verified with material obtained from in vivo
samples. Those fractions were combined and concentrated at 308C
(aspirator pressure), dissolved in acetonitrile (20.0 ml) and again
thoroughly concentrated at 308C (aspirator pressure). The residue was
dissolved in acetonitrile (25.0 ml) and was stored as such. It contained
the desired metabolite at a purity of 98.7% (Kromasil RP18 100-5,
4.6 mm ID ꢁ 300 mm; flow rate: 1.0 ml/min; programmed run consisting
of an isocratic run for 20 min with aqueous 0.1 M ammonium acetate
solution (pH 9.0)–methanol-acetonitrile (80–10–10, v/v/v), followed by
a 5 min linear gradient run to solvent composition (10–45–45, v/v/v) and
again a 15 min isocratic run at the latter solvent composition) and with
an enantiomeric excess of 100% (Chiralpak AD (10 mm), 4.6 mm
ID ꢁ 50 mm; flow rate: 0.7 ml/min; eluate: hexane–ethanol–trifluoroa-
cetic acid (80–20–0.03, v/v/v)). It contained 194 MBq of radioactivity,
overall radiochemical yield: 16.2%. The specific activity was 895 MBq/
mmol. The material was HPLC and NMR identical to unlabelled
material.
Acknowledgements
The authors wish to thank B. Van Hoof for carrying out NMR analysis
and S. Van Hoegaerden for performing HPLC analyses.
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