Synthesis of [14C]-labelled repinotan hydrochloride and its major metabolite M-6

Article abstract of DOI:10.1002/jlcr.629

For studies of pharmacokinetics and drug metabolism of the new 5-HT_1A agonist repinotan, the ¹⁴C-labelled version was synthesized. Starting from [U-¹⁴C]phenol, a 10-step synthesis led to 457mg (1.58 GBq) of [U-¹⁴

Full text of DOI:10.1002/jlcr.629

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 1115–1132.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.629
Research Article
Synthesis of [14C]-labelled repinotan
hydrochloride and its major metabolite M-6
D. Seidel*, M. Conrad, Y. Schoof and R. Schohe-Loop
Bayer AG, Pharmaceutical Business Group, Wuppertal, Germany
Summary
For studies of pharmacokinetics and drug metabolism of the new 5-HT1A
agonist repinotan, the 14C-labelled version was synthesized. Starting
from [U-14C]phenol, a 10-step synthesis led to 457 mg (1.58 GBq) of
[U-14C]repinotan hydrochloride, labelled uniformly in the aromatic ring of
the chromane moiety. For a study in man, a mono-carbon-14 labelled
substance was required. Therefore a 7-step synthesis was performed starting
from [carbonyl-14C]2-hydroxy-acetophenone. The yield was 106 mg (0.396
GBq) of [4-chromane-14C]repinotan hydrochloride. The carbon-14 labelled
major metabolite, hydroxylated in the 6-position of the chromane moiety,
was synthesised as reference compound. Copyright # 2002 John Wiley &
Sons, Ltd.
Key Words: chromane; uniform labelling; mono 14carbon-labelling; optical
resolution; semi-preparative chromatography
Introduction
Repinotan is a new, enantiomerically pure aminomethyl chromane
derivative with a saccharinylbutyl substituent. It demonstates neuro-
protective properties, especially for the treatment and prevention of
ischemic diseases, such as stroke.1
*Correspondence to: D. Seidel, Bayer AG, PH-PD-P Drug Metabolism Isotope Chemistry,
D-42096 Wuppertal, Germany.
Copyright # 2002 John Wiley & Sons, Ltd.
Received 24 April 2002
Revised 18 June 2002
Accepted 21 June 2002

1116
D. SEIDEL ET AL.
For studies of pharmacokinetics and drug metabolism of repinotan,
the version with a metabolically stable 14carbon-label in the chromane
moiety was required. A first radiosynthesis was planned giving uniform
labelling in the aromatic ring of the chromanyl part of the molecule.
Repinotan is administered in a very low dose. In order to facilitate
mass spectral analysis of metabolites in the course of an ADME study in
healthy volunteers, mono-labelling with 14carbon was needed.2 So a
second radiosynthesis of repinotan hydrochloride (A) was performed,
locating the label in the 4-position of the chromane moiety.
For evaluation and interpretation of the study the 14carbon labelled
metabolite M-6 of repinotan, hydroxylated in the 6-position of the
chromane moiety, was synthesised as the hydrochloride. The synthesis
was started with an appropriate intermediate of the second radio-
synthesis.
Results and discussion
Uniformly labelled [14C]-phenol is a readily available starting com-
pound. In the first labelling synthesis it was used for the known
preparation of chromane rings via Michael adduct formation with
dimethyl acetylene-dicarboxylate, hydrogenation of the dimethyl
phenoxyfumarate/maleate, hydrolysis of the ester and cyclization to
the chromanone system. The Michael addition is described in the
literature using non-labelled compounds under different conditions.3 In
some cases the resulting 1-phenoxy-ethylene-1,2-dicarboxylates were
saponified and cyclized under dehydrating conditions.4 Cox and co-
workers4 pointed out that only the fumaric acid derivative underwent
cyclization. To avoid this selectivity problem, the double bond was
hydrogenated to obtain the corresponding dimethyl phenoxy-succinate
(III) (Reaction scheme 1) which was hydrolysed and subsequently
cyclized. The 14carbon-labelled 4-oxo-4H-chromane-2-carboxylic acid (V)
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1115–1132

SYNTHESIS OF [14C]-LABELLED
1117
Reaction scheme 1.
was reduced with triethylsilane/trifluoroacetic acid as described for
other systems by Swenton and Raynolds.5 For optical resolution the
amide VII was formed with the optically active (1R)-1-phenylethyla-
mine, the amide was reduced to the corresponding amine (VIII) and
the diastereomers were separated by chromatography on silica gel. In
the penultimate step alkylation with 4-bromobutylsaccharine led to
the tertiary amine X. The reaction was performed solvent free, using
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1115–1132

1118
D. SEIDEL ET AL.
sodium bicarbonate as HBr trapping agent. Critical in this step was the
liability of the saccharine moiety towards hydrolysis. The crude
substance was purified chromatographically on silica gel. The final
product was obtained by cleavage of the phenethyl–N bond and acidic
work up of the labelled secondary amine; purification was achieved via
recrystallization from ethanol.
The second labelling route was started from commercially available
[carbonyl-14C]-2-hydroxy-acetophenone (XI) (Reaction scheme 2).
Condensation with dimethyl oxalate and subsequent ring closure gave
compound XII. The synthesis of the non-labelled compounds have been
reported.6 So also has the hydrogenation to the chromane-2-carbox-
ylate.7 In this second synthesis optical resolution was achieved at the
Reaction scheme 2.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1115–1132

SYNTHESIS OF [14C]-LABELLED
1119
stage of the chromane-2-carboxylate. This procedure had the advantage
that the undesired enantiomer can be racemized and again used in the
enantiomeric separation. The separation of the enantiomers by semi-
preparative HPLC on a chiral stationary phase was changed from
reversed phase to normal phase conditions during the synthesis, because
of steam-volatility of the methyl ester. The amine XV was obtained in
excellent yield by aminolysis of the methyl ester without any loss of
enantiomeric excess. Lithium iodide was used as catalyst. The
corresponding amine was obtained by reduction with a complex
aluminium hydride as in the first synthesis. The same amine in racemic
and non-labelled form was prepared by Meshaw and co-workers by
alkylation of chroman-2-ylmethylamine with benzylchloride,8 but the
yield was poor. Van Lommen and co-workers used similar route9
alkylating benzylamine with 3,4-dihydro-2H-chromen-2-ylmethyl tosy-
late. The optically active, but non-labelled N-benzyl-N-(3,4-dihydro-
2H-chromen-2-ylmethyl)amine was prepared by the same group by
reductive amination of the (2R)-3,4-dihydro-2H-chromene-2-carbalde-
hyde.9 Both synthetic routes were not attractive because of poor
yields. Some experiments were performed for optimisation of the
alkylation of chroman-2-ylmethylamine with bromobutylsaccharin
to obtain the final product more easily. However the preparation of
chroman-2-ylmethylamine gave a lower yield than the synthesis of
the benzyl protected amine, and furthermore double alkylation and
side reactions of the saccharin moiety with the primary amine were
observed.
In the present synthesis the alkylation of XVI with bromobutylsac-
charin proceeded at a lower temperature and more rapidly than in the
case of the phenethyl substitution, probably due to steric reasons. The
final product was formed by hydrogenolytical debenzylation and
subsequent addition of hydrochloric acid. The presence of several by-
products required a two-fold chromatographic purification, indicating
that purification of the precursor XVII is advantageous prior to the
hydrogenolysis.
To aid in the interpretation of the results of the study with [4-
chromane-14C]repinotan hydrochloride in humans the 14carbon-labelled
6-hydroxy metabolite [14C]M-6 was required as reference compound.
The synthesis started from the labelled intermediate XVII (Reaction
scheme 3). After chromatographic purification XVII was subjected
to a Friedel–Crafts acylation with acetyl chloride. The acetylation took
place very selectively in position 6 of the chromane ring. Subsequent
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1115–1132

1120
D. SEIDEL ET AL.
Reaction scheme 3.
Baeyer–Villiger oxidation with 3-chloroperbenzoic acid/trifluoroacetic
gave intermediate XIX, the O-acetyl and N-benzyl protected metabolite.
The deacetylation and the debenzylation steps were carried out using
HCl, Pd/C in the first and H2, Pd/C in the second step. The desired
product was obtained after purification by semi-preparative HPLC in
good quality.
Experimental
General methods
1
The H NMR spectra were recorded on a Bruker AM 300 nuclear
magnetic resonance spectrometer. Radiochemical GC purities were
determined with the GC system HP 5890 equipped with radioactivity
detector FHT 7000. HPLC analyses of the final products were
performed on a HP 1050 Series II instrument. For non-chiral analysis
Nucleosil1 100 C-18 was used as stationary phase and acetonitrile/
phosphate buffer (pH 2.5) as mobile phase. For chiral analysis
Chiralpak AD1 was used as stationary phase and n-heptane/ethanol
(0.2% NEt3) as mobile phase. Radioactivity and UV signals (radio-
activity detector Ramona1 92) were recorded by the work station of the
chromatograph. The radioactivity signal was digitised by the A/D-
converter HP 35900 and evaluated by the work station. Radiochemical
counting was performed on a Liquid Scintillation Analyzer TRI-
CARB1 2500 TR using Ultima GoldTM cocktail. Radio thin layer
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1115–1132

SYNTHESIS OF [14C]-LABELLED
1121
chromatography was performed using silica gel plates Si 60 F254 and a
Raytest Rita1-3200 analyser. Mass spectrometric analysis was per-
formed on a PE/Sciex API III mass spectrometer with MacIntosh
Quadra1 900. Semipreparative chromatography was performed with
Latek-P 402 in the first synthesis and WellChrom Maxi-Star1 K 1000 in
the second synthesis and in the labelling synthesis of the metabolite,
Knauer Variable Wavelength monitor and Merck/Hitachi D-2500A
Chromato-Integrator.
First labelling procedure
Dimethyl (2E,Z)-2-[U-14C]phenoxy-2-butenedioate, (II). [U-14C]-
Phenol (I) was obtained from Forschungszentrum Rossendorf e.V.
with a specific radioactivity of 2.49 GBq/mmol and a total quantity of
6.07 mmol. Both the chemical and radiochemical purities were 98%, as
established by GC and radio-GC, respectively.
Non-labelled phenol (3.92 mmol) was added to the labelled
compound. The resulting specific activity was calculated to be
1.51 GBq/mmol.
A solution of the labelled phenol, 2.29 g (16 mmol) of dimethyl
acetylenedicarboxylate and 6 drops of Triton B1 (40% solution in
methanol) in 8 ml of dioxane was refluxed for 40 minutes. The brown
solution was cooled in an ice bath, diluted with 35 ml of water, acidified
with 25% hydrochloric acid and further diluted with 20 ml of water. The
product was extracted with diethyl ether (4 ꢀ 25 ml). The combined
organic layers were washed with water (5 ꢀ 10 ml), dried over sodium
sulphate and evaporated to dryness to yield 3.5 g of an oily residue with
a chemical purity (GC) of ꢁ 91% and a radiochemical purity (radio-
GC) of ꢁ 99%.
For purification the crude II was dissolved in 23 ml of acetonitrile and
purified in 7 runs by low pressure chromatography on Lobar1 column,
LiChroprep1 RP-18, size B with the eluent acetonitrile/water 70:30 and
a flow of 5 ml/min. The product containing fractions were evaporated
under reduced pressure. The residue was extracted with diethyl ether.
The organic layer was dried over sodium sulphate and evaporated to
dryness.
Yield: 2.71 g of II, chemical purity (GC): ꢁ 99%.
Dimethyl 2-[U-14C]phenoxysuccinate, (III). To 400 mg of 10% palla-
dium on charcoal and 5 g of ammonium formate in 8 ml of methanol,
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1115–1132

1122
D. SEIDEL ET AL.
a solution of II (2.71 g) in 4 ml of methanol was added dropwise and the
reaction mixture was stirred for 1 h at room temperature. For work up
the mixture was filtered over kieselguhr and the filter cake was washed
several times with methanol. The filtrate was evaporated under reduced
pressure. To the concentrated solution 40 ml of water was added and the
mixture was extracted several times with 15 ml portions of diethyl ether.
The ether layers were washed with water, dried over sodium sulphate
and evaporated to dryness under reduced pressure.
Yield: 2.57 g (8.85 mmol with respect to the purity) of compound III,
chemical purity (GC): ꢁ 82%, radiochemical purity (radio-GC):
ꢁ 83%. This amount corresponds to 88% of the theoretical related
to the starting phenol.
The crude product was used without further purification in the next
step.
2-[U-14C]Phenoxysuccinic acid, (IV). The ester III (2.57 g) was
dissolved in 30 ml of 25% hydrochloric acid and stirred for 3 h at
1308C bath temperature. After cooling to room temperature 50 ml of
water was added and the product was extracted with 20 ml of diethyl
ether. The aqueous layer was saturated with 1 g of sodium chloride. The
organic layer was removed and the extraction was repeated twice, each
with 10 ml of diethyl ether. For purification 5 ml of water was added to
the combined organic layers and basified with sodium carbonate. The
organic layer was discarded and the aqueous layer was washed with
diethyl ether. For liberation of the desired acid IV the aqueous solution
was acidified with concentrated hydrochloric acid and the product was
extracted with diethyl ether (4 ꢀ 15 ml). The organic layers were dried
over sodium sulphate and evaporated to dryness under reduced
pressure.
Yield: 2.84 g of compound IV, chemical purity (GC): 96%, radio-
chemical purity (radio-GC): 98%.
4-Oxo-[U-14C]chromane-2-carboxylic acid, (V). Polyphosphoric acid
(30 g) was mixed with 2 ml of xylene (isomeric mixture) and heated to
708C. Compound IV (2.84 g) was dissolved in 4 ml of hot xylene and
added to the mixture. The reaction mixture was stirred intensively for
5 h at 708C. Then water was added until two layers were obtained. After
cooling to room temperature the product was extracted with diethyl
ether (4 ꢀ 20 ml). Purification and isolation were performed by acidic–
alkaline treatment as described above.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1115–1132

SYNTHESIS OF [14C]-LABELLED
1123
Yield: 1.49 g (6.8 mmol with respect to the purity) of compound V,
chemical purity (GC): ꢁ 88%, radiochemical purity (radio-GC):
ꢁ 87%. This corresponds to 77% of the theory over 2 steps.
The crude product was used without further purification in the next
step.
(2R,S)-[U-14C]Chromane-2-carboxylic acid, (VI). Compound
V
(1.49 g) was dissolved in 6 ml of trifluoroacetic acid. Triethylsilane
(2 ml) was added and the solution was stirred overnight at room
temperature. Addition of 2 ml of triethylsilane was repeated twice every
25 h. For work up trifluoroacetic acid and excessive triethylsilane were
removed by evaporation at aspirator pressure. To the residue 10 ml of
water and 20 ml of diethyl ether were added. The aqueous layers were
extracted with diethyl ether and the desired compound VI was purified
and isolated by acidic-alkaline treatment as described above.
Yield: 1.61 g of compound VI, chemical purity (GC): 92%, radio-
chemical purity (radio-GC): 86%.
The crude product was used without further purification in the next
step.
N-[(1R)-1-Phenylethyl]-(2R,S)-[U-14C]chromane-2-carboxamide, (VII).
Compound VI (1.61 g) was dissolved in 6 ml of THF and
N,N0-carbonyldiimidazole (2.27 g) was added in portions. After stirring
for one hour at reflux the mixture was cooled in an ice bath and 2 ml of
(1R)-1-phenylethylamine was added. After stirring overnight at room
temperature 20 ml of water and 10 ml of diethyl ether were added. The
aqueous layer was extracted with diethyl ether and the organic layer was
dried over sodium sulphate and evaporated to dryness under reduced
pressure.
Yield: 2.8 g of compound VII, chemical purity (GC): 77%, radio-
chemical purity (radio-GC): 98%.
The crude product was used without further purification in the next
step.
(2R,S)-2-{N-[(1R)-1-Phenylethyl]}aminomethyl-[U-14C]chromane, (VIII).
The labelled VII (2.8 g) was dissolved in 6 ml of toluene and 10 ml of sodium
bis(methoxyethoxy)aluminumhydride (70%in toluene, Red-Al1) were added
under ice bath cooling. The reaction mixture was stirred for one hour at
reflux. After cooling to room temperature the reaction mixture was added
carefully to 20 ml of a 16% sodium hydroxide solution. The mixture was
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1115–1132

1124
D. SEIDEL ET AL.
diluted with water and the desired amine was extracted with diethyl ether.
The organic layer was washed with water, dried over sodium sulphate and
evaporated to dryness under reduced pressure.
Yield: 2.88 g of compound VIII, chemical purity (GC): 51%
radiochemical purity (radio-GC): 97%.
The crude product was used without further purification in the optical
resolution.
(2R)-2-{N-[(1R)-1-Phenylethyl]}aminomethyl-[U-14C]chromane, (IX).
The mixture of the diastereomers VIII (2.88g) was dissolved in 18ml of
cyclohexane/isopropanol 14:1. The chromatographic separation of the
diastereomers was performed in 11 runs on a column Lobar1 Si 60 size B
using n-heptane/isopropanol 40:1 as eluent, a flow of 6 ml/min and UV
detection at 230 nm. The desired R,R-diastereomer was eluted first. The
product containing fractions were combined and evaporated to dryness
under reduced pressure.
Yield: 0.745 g (2.79 mmol) of compound IX. The diastereomeric
excess was determined by GC (Chiraldex1) as 98.4%.
2-(4-{[(2R)-3,4-dihydro-2H-[U-14C]chromen-2-ylmethyl][(1R)-1-
phenylethyl]amino}butyl)-1,1-dioxo-1,2-benzisothiazol-3(2H)-one, (X).
Compound IX (0.745 g, 2.79 mmol) was mixed with 1.77 g (5.59 mmol)
of 4-bromobutylsaccharin, 0.704 g (8.39 mmol) of sodium bicarbonate
and 0.24 g (1.42 mmol) of powdered potassium iodide. The mixture was
stirred for 6.5 h at a bath temperature of 1458C. Then the mixture was
cooled to 1008C and 10 ml of pinacolone were added. After cooling to
room temperature the salts were removed by filtration over quartz wool.
The filter was washed colourless with pinacolone. The filtrate was
evaporated under reduced pressure and 3 ml of ethyl acetate and 15 ml
of 25% hydrochloric acid were added to the brownish residue. After
extraction with diethyl ether (4 ꢀ 15 ml) the organic layers were
discarded and the acidic layer was neutralised with 45% sodium
hydroxide solution to pH 6.5. The product was extracted with ethyl
acetate (4 ꢀ 15 ml). The organic layer was dried over sodium sulphate
and evaporated to dryness under reduced pressure. Crude X was
obtained as a residue of 1.43 g.
For purification the crude product was dissolved in 6 ml of
THF/isopopanol 1:1. Chromatographic purification was performed in
4 runs on a column Lobar1 Si 60 size B using n-heptane/isopropanol
9:1 as eluent, a flow of 6 ml/min. and UV detection at 230 mm. The
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1115–1132

SYNTHESIS OF [14C]-LABELLED
1125
product-containing fractions were combined and evaporated to dryness
under reduced pressure.
Yield: 1.09 g (2.16 mmol) of compound X. This amount corresponds
to a yield of 77%. The radiochemical purity was determined by radio
TLC as 97%.
(-)-R-2-{4-[([U-14C]Chroman-2-ylmethyl)amino]butyl}-1,1-doxo-1,2-
benzisothiazole-3(2H)-one monohydrochloride, [U-14C]repinotan hydro-
chloride. Compound X (1.09 g) was dissolved in 16 ml of methanol
and 8 ml of acetic acid and 1.1 g of Pearlman’s catalyst (Pd(OH)2)
was added. Hydrogen was bubbled through the solution for 4 h
under intensive stirring. Then the mixture was filtered over kieselguhr
and the filtrate was evaporated under reduced pressure. The residue
was dissolved in 15 ml of concentrated hydrochloric acid and
the product was extracted with dichloromethane. The organic
layer was evaporated to dryness leading to 0.843 g of crude product
with a purity of 90%, it was recrystallised from 3 ml of ethanol. The
product was filtered off, washed with ethanol and dried at 708C under
vacuum.
Yield: 0.457 g of [U-14C]repinotan hydrochloride (1.05 mmol), radio-
chemical purity: ꢁ 99% by radio-HPLC and >98% by radio-TLC
(ROI), chemical purity (HPLC): ꢁ 98%. The optical purity was
determined as 99% e.e.
Determination of the specific radioactivity:
For determining the specific radioactivity a weighed amount
of the labelled compound was dissolved in acetonitrile/water
and the total radioactivity was determined by LS counting. By this
method the specific activity was determined as 1.51 GBq/mmol
(3.46 MBq/mg).
The total radioactivity of 1.581 GBq corresponds to a yield of 10% of
the theory, based on the starting [U-14C]phenol.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1115–1132

1126
D. SEIDEL ET AL.
NMR (methanol-d4) d (ppm); 1.93 (m, 6H, a), 2.86 (m, 4H, b), 3.15
(m, 2H, c), 3.89 (t, 2H, d), 4.30 (m, 1H, e), 6.87+7.08 (multiplets, 4H, f),
8.01 (m, 4H, g).
Second labelling procedure
Methyl 4-oxo-4H-[4-14C]chromene-2-carboxylate, (XII). The starting
[carbonyl-14C]2-hydroxyacetophenone (XI) was obtained from Isotop-
chim, France, (batch no. 97113) with a total radioactivity of 11.1 GBq
and a specific radioactivity of 1.903 GBq/mmol, corresponding to
5.8 mmol (0.789 g). The chemical and radiochemical purities were 97%,
as established by HPLC and radio-HPLC, respectively.
The labelled 2-hydroxyacetophenone (5.8 mmol) was added to a
solution of 0.85 g (7.2 mmol) dimethyl oxalate in 2.2 ml of methanol.
This solution was added to solution of 1.177 g (21.8 mmol) sodium
methoxide in 4 ml of methanol within 1.5 h at a temperature of 608C.
The thick yellow suspension was stirred for 3 h at reflux. After cooling
to room temperature 4.8 ml of concentrated hydrochloric acid was
added without external cooling. A new slightly coloured solid was
obtained. After stirring for 30 min at room temperature 16 ml of water
was added and stirring was continued for 45 min in an ice bath. The
product was filtered off, washed intensively with water and dried in an
evacuated desiccator over blue gel. A first product batch of 1.03 g was
obtained.
A second precipitate was obtained from the mother liquor overnight
which was filtered over the frit containing the first product batch. The
combined solids were washed with water and dried at reduced pressure
over blue gel, leading to 1.2 g of product. By GC analysis 83% of the
desired methyl ester and 16.2% of the corresponding carboxylic acid
was detected. Complete conversion to the desired ester was performed
with diazomethane.
Yield: 1.0 g (4.9 mmol) of XII. The chemical purity was determined as
99% by GC. The amount corresponds to 84% of the theory related to
the starting labelled 2-hydroxyacetophenone.
Methyl [4-14C]chroman-2-carboxylate, (XIII). Compound XII (1.0 g)
was dissolved in 75 ml of acetic acid. Palladium catalyst (2 g of 10%
palladium on charcoal) and hydrogen were added. The mixture was
filtered over kieselguhr and the filter was washed with methanol
(4 ꢀ 15 ml). The filtrate was evaporated to dryness.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1115–1132

SYNTHESIS OF [14C]-LABELLED
1127
Yield: 0.73 g (3.8 mmol) of compound XIII. The chemical purity was
determined as 97.4% by GC. The amount corresponds to 77.6% of the
theory related to XII.
Methyl (-)-(2R)-[4-14C]chroman-2-carboxylate, (XIV). The first op-
tical resolution by semi-preparative HPLC on Chiralcel1 OD
(250 ꢀ 20 mm) with the eluent acetonitrile/water 50:50 (v/v) failed due
to isolation problems. Unexpectedly the chromane ester was found to be
very steam-volatile.
Interestingly the chiral stationary phase could also be used under
normal phase conditions. The racemic ester was dissolved in n-heptane/
ethanol 1:1 (v/v) and chromatographed in 10 runs on Chiralcel1 OD,
250 ꢀ 20 mm with the eluent n-heptane/ethanol 35:65 (v/v), a flow of
8 ml/min. and UV detection at 230 nm. The desired R-enantiomer was
eluted at approximately 21 min and the S-enantiomer at approximately
10 min. The fractions of both enantiomers were combined separately
and evaporated under reduced pressure.
Yield: 0.403 g of the R-enantiomer XIV (2.1 mmol). In addition,
0.288 g of the S-enantiomer was isolated.
The S-enantiomer (0.288 g) was dissolved in methanol and racemized
with 30% sodium methoxide solution at 508C. The obtained racemate
was resolved into the enantiomers (2 runs) as described above. This
recycling was repeated once with the new S-enantiomer.
Total yield: 0.538 g (2.8 mmol) of compound XIV. The chemical
purity was determined as 99% by HPLC. The optical purity was
determined as 99% e.e. by HPLC. The amount corresponds to 73% of
the amount of the racemate XIII.
(-)-(2R)-[4-14C]Chromane-2-carboxylic acid benzylamide,(XV). Com-
pound XIV (0.538 g), benzylamine (1.53 ml, 14 mmol) and lithium
iodide (54 mg) were stirred for 2 h at 1008C. For work up the mixture
was cooled to room temperature and 6 ml of 0.1 m hydrochloric acid
added. After dilution of the suspension with 3 ml of 0.1 m hydrochloric
acid the desired amide was filtered off, washed with 0.1 m hydrochloric
acid (2 ꢀ 2 ml) and dried in an evacuated desiccator over blue gel.
Yield: 0.702 g (2.63 mmol) of XV, chemical purity (GC): 93%,
radiochemical purity (radio-GC): 99%. The optical purity was
determined as >99% e.e. by HPLC. The structure was confirmed by
GC/MS with m/z=269 [14C-M]+ and 267 [12C-M]+. The amount
corresponds to 94% of theory.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1115–1132

1128
D. SEIDEL ET AL.
(-)-(2R)-2-(Benzylaminomethyl)-[4-14C]chromane, (XVI). Compound XV
(0.702 g) was suspended in 6 ml of toluene. Red-Al1 (1.44 ml, 4.8 mmol)
was added and the reaction mixture was stirred for 30 min at room
temperature, 30 min at 508C and subsequently 1 h at 608C. Then an
additional 0.7 ml and subsequently 0.4 ml of Red-Al1 were added and
stirring was continued at 608C. The conversion was still incomplete, but
due to the formation of by-products the reaction was stopped by
cooling to room temperature and careful addition of 7 ml of 2 m sodium
hydroxide solution. The organic layer was washed with water (2 ꢀ 4 ml)
and evaporated to dryness under reduced pressure.
Yield: 0.52 g of compound XVI (1.64 mmol), chemical purity (GC):
79%, radiochemical purity (radio-GC): 72%. This corresponds to 62%
of the theory.
The crude product was used without further purification in the next
step.
2-(4-{Benzyl[(-)-(2R)-3,4-dihydro-2H-[4-14C]chromen-2-ylmethyl]amino}
butyl)-1,1-dioxo-1,2-benzisothiazole-3(2H)-one, (XVII). A solution of
compound XVI (0.52 g, 1.64 mmol), 4-bromobutylsaccharin (0.79 g,
2.47 mmol) and N-ethyl-diisopropylamine (0.538 ml, 3.09 mmol) in
2.5 ml of N-methylpyrrolidinone was stirred for 3 h at 1208C. After
cooling to room temperature 3 ml of water was added. The clear
supernatant was removed and the remaining oily residue was dissolved
in 3 ml of dichloromethane. The solution was washed with water
(2 ꢀ 3 ml) and evaporated to dryness under reduced pressure. For
aceotropic drying 10 ml of ethanol was added and also removed by
evaporation.
Yield: 1.5 of compound XVII. The chemical purity was determined
by HPLC as 67%.
An aliquot of the crude substance was used without further
purification in the next chemical step.
(-)-R-2-{4-[([4-14C]Chroman-2-ylmethyl)amino]butyl}-1,1-doxo-1,2-ben-
zisothiazole-3(2H)-one monohydrochloride, [4-chromane-14C]repinotan
hydrochloride. The major part of XVII (1 g) was dissolved in 14 ml of
methanol and 7 ml of acetic acid. Pearlman’s catalyst (Pd(OH)2; 1 g) was
added. Hydrogen was bubbled through the solution under intensive
stirring. After 4 hours an additional 0.2 g of Pearlman’s catalyst was
added and hydrogenolysis was continued for 3 h. Then the mixture was
filtered over kieselguhr and the filter was washed with methanol
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1115–1132

SYNTHESIS OF [14C]-LABELLED
1129
(3 ꢀ 6 ml). The filtrate was evaporated under reduced pressure. The oily
residue was dissolved in 20 ml of concentrated hydrochloric acid and
extracted with dichloromethane (3 ꢀ 10 ml). The HCl layer was
discarded. The organic layers were combined and evaporated to dryness
giving 0.39 g of crude product.
A first purification was performed by low pressure liquid chromato-
graphy on Lobar1 RP-18 size B with the eluent acetonitrile/0.1 m
hydrochloric acid 40:60 (v/v).
Final purification
Final purification was performed by low pressure liquid chromato-
graphy on Lobar1 Si 60 size B with the eluent dichloromethane/
methanol 96:4 (v/v), a flow of 10 ml/min. and UV detection at 230 nm.
The substance (0.222) g was dissolved in 10 ml of dichloromethane and
purified in 24 runs. To each 0.7 ml of the product solution was added 1
drop of 2 m sodium hydroxide solution to liberate the free base. The
organic layer was injected. The basic compound was eluted at
approximately 21 min. To the combined product an excess of
hydrochloric acid (4 m dioxane) was added and the solution evaporated
to dryness.
Yield: 0.106 g of [4-chromane-14C]repinotan hydrochloride with a
radiochemical purity of 99.4% and a chemical purity of 99.4%.
The optical purity was determined by HPLC on Chiralpak AD1 as
>99% e.e.
Determination of the specific radioactivity:
The specific radioactivity was determined by two different
methods. A weighed amount of the labelled compound was dissolved
in acetonitrile/water and the total radioactivity was determined
by LS counting. On the other hand the concentration of
[4-chromane-14C]repinotan hydrochloride in a solution was deter-
mined by HPLC/UV quantification using non-labelled reference
compound and the total radioactivity was also measured by LSC.
The specific radioactivity was determined as 1.63 GBq/mmol
(3.74 MBq/mg).
With respect to the use of only 2/3 of the yield of the precursor XVII
and disregarding the resolution of the ester XIII the obtained total
radioactivity of 396 MBq corresponds to a yield of 6.3% of theory,
related to carbon-14 labelled 2-hydroxyacetophenone.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1115–1132

1130
D. SEIDEL ET AL.
Synthesis of the labelled metabolite M-6
2-(4-{Benzyl[(-)-(2R)-6-acetyl-3,4-dihydro-2H-[4-14C]chromen-2-
ylmethyl]amino}butyl)-1,1-dioxo-1,2-benzisothiazole-3(2H)-one, (XVIII).
The crude XVII (450 mg) was dissolved in 60 ml of acetonitrile/water 75:25
and purified in 16 runs on Nucleosil1 100 C-18 (120 ꢀ 16 mm, 7 mm) with
the eluent acetonitrile/water 75:25 (v/v), a flow of 6 ml/min. and UV
detection at 254 nm. The desired product was eluted at approximately
15 minutes. The product containing fractions were evaporated to dryness
giving 138 mg of VXII.
A solution of XVII (138 mg) in 1.1 ml of dichloromethane was cooled
in an ice bath and 171 mg (1.28 mmol) of anhydrous aluminium chloride
was added. The mixture was stirred for 20 minutes at room temperature,
then it was cooled again and 55 ml (0.77 mmol) of acetyl chloride added.
The mixture was refluxed for 2 h and worked up with careful addition of
1.4 g of ice and 1.4 ml of water under ice bath cooling. The organic layer
was washed twice with water (2 ꢀ 1.6 ml), subsequently with 1.1 ml of
2.2% sodium bicarbonate solution and finally with water (2 ꢀ 1.1 ml).
Then the organic layer was evaporated to dryness yielding 164 mg of
XVIII. The purity was not determined.
(2R)-2-({Benzyl[4-(1,1-dioxido-3-oxo-1,2-benzisothiazole-2(3H)-yl)-
butyl]amino}methyl)-3,4-dihydro-2H-[4-14C]chromen-6-yl acetate, (XIX).
Compound XVIII (164 mg) was dissolved in 1.7 ml dichloromethane and
170 mg of 3-chloroperbenzoic acid (57–86%) was added. The mixture was
cooled in an ice bath and 22.3 ml of trifluoroacetic acid added. The reaction
mixture was stirred for 18 h at room temperature in the dark. For work up
1 ml of dichloromethane was added and the mixture washed with 1.7 ml of
saturated sodium bicarbonate solution. The dichloromethane layer was
washed with of 0.1 m sodium hydroxide solution (2 ꢀ 6.1 ml) and
subsequently with water (2 ꢀ 6.1 ml). The organic layer was evaporated
to dryness giving 124 mg of XIX. The purity was not determined.
(-)-R-2-{4-[(6-Hydroxy-[4-14C]chroman-2-ylmethyl)amino]butyl}-1,1-
dioxo-1,2-benzisothiazole-3(2H)-one monohydrochloride, [14C]M-6 HCl.
Compound XIX (124mg) was dissolved in 3 ml THF. Concentrated
hydrochloric acid (1ml) and 10% palladium (30 mg) on charcoal were
added. The mixture was stirred for 2.5 h at room temperature and filtered
over kieselguhr. The filter was washed with THF and the filtrate was
evaporated to dryness. The oily residue was dissolved in 2 ml of methanol
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1115–1132

SYNTHESIS OF [14C]-LABELLED
1131
and 1 ml acetic acid, and 124 mg of Pearlman’s catalyst (20% Pd(OH)2
on charcoal) were added. Hydrogen was bubbled through the solution
for 4 hours at room temperature. The mixture was filtered over
Kieselguhr, the filter washed with methanol and the filtrate evaporated
to dryness. The oily residue was dissolved in 5 ml of concentrated
hydrochloric acid and extracted with dichloromethane (2ꢀ 4 ml and
2 ꢀ 2.5 ml). The aqueous layer was neutralised with saturated sodium
bicarbonate solution and extracted with dichloromethane (3ꢀ 8 ml). The
dichloromethane layer was washed with water (2 ꢀ 5 ml) and evaporated
to dryness. To remove water the evaporation was repeated twice with
each 5 ml of ethanol giving 86mg of crude [14C]M-6 HCl with a
radiochemical purity (radio-HPLC) of 87.6%.
Purification
The product was dissolved in acetonitrile/water and chromatographed
in 6 runs on Nucleosil1 7 C-18 (250 ꢀ 10 mm, 5 mm) with the eluent
acetonitrile/0.05 M hydrochloric acid 30:70 (v/v), a flow of 5 ml/min and
UV detection at 230 nm. The desired product eluted at approximately
9 min. The product containing fractions were evaporated to dryness.
Yield: 28 mg of [14C]M-6 HCl, radiochemical purity (radio-HPLC):
97.8%, chemical purity (HPLC): 98.5%.
Determination of the specific radioactivity
The specific radioactivity was determined by two different methods.
First the content of [14C]M-6 HCl in a solution was determined by
HPLC/UV quantification using non-labelled reference compound and
the total radioactivity was also measured by LSC. The specific
radioactivity was found to be 1.614 GBq/mmol (3.568 MBq/mg). This
value was confirmed by electrospray mass spectrometry. The specific
radioactivity calculated from the ratio of the 14C-molecular ion
m/z=419 [M+H]+ and the 12C-molecular ion m/z=417 [M+H]+
was found to be 1.665 GBq/mmol (3.680 MBq/mg).
Acknowledgements
We thank H. Schnorbus for performing the GC and HPLC analyses and
the MS measurements and Dr. P. Schmitt for performing the NMR
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1115–1132

1132
D. SEIDEL ET AL.
analysis. Thanks to Dr. G. Franckowiak for providing the non-labelled
intermediate bromobutylsaccharin.
References
1. De Vry J, Dietrich H, Glaser T, et al. Drugs Future 1997; 22: 341.
2. Seidel D. J. Labelled Cpmd Radiopharm 1999; 42: 1002.
3. Stoermer MJ, Fairly DP. Aust J Chem 1995; 48: 677; Kodomari M,
Sakamoto T, Yoshitomi S. J Chem Soc Chem Commun 1990; 701.
4. Ruhemann S, Beddow F. J Chem Soc 1900; 77: 1119; Cairns H, Fitzmaurice
C, Hunter D, et al. J Med Chem 1972; 15: 583; Davis SG, Mobbs BE. J
Chem Soc Perkin Trans 1987; 2597.
5. Swenton JS, Raynolds PW. J Chem Soc 1978; 61.
6. Ellis GP, Thomas IL. J Chem Soc Perkin Trans 1974; 1: 2570.
7. Baddeley G, Cooke JR. J Chem Soc 1958; 2797; Augstein J, Monro AM,
Potter GWH, Sholfield P. J Med Chem. 1968; 11: 844.
8. Mewshaw RE, Kavanagh J, Stack G, et al. J Med Chem 1997; 40: 4235.
9. Van Lommen GRE, De Bruyn MFL, Janssens WJJ. WO 93/17017; PCT/
EP 93/00435.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1115–1132