Synthesis of 3H-, 14C-, and stable-isotope-labelled galantamine

Article abstract of DOI:10.1002/jlcr.589

Reminyl is a newly approved drug, used in the treatment of mild to moderate Alzheimer disease. The active compound, galantamine, was initially isolated from the bulbs of certain Narcissus species, but is at the moment also produced synthetically. In the process leading to the final approval, the synthesis of tritium-, carbon-14- and stable-isotope-labelled galantamine for pharmacokinetic studies was required. Racemic (±)-1-bromonarwedine, a compound available as intermediate from the commercial synthesis, was transformed to racemic 1-bromo-galantamine. Catalytic bromo-tritium exchange, followed by HPLC purification and resolution afforded tritium-labelled galantamine. The [¹⁴C]-label was introduced on the nitrogen as well as on the oxygen-methyl position. This was achieved by N- and O-demethylation of galantamine and reaction of the thoroughly purified intermediate with [¹⁴C]-methyl iodide. Stable-isotope-labelled galantamine was obtained likewise by ¹³CD₃OD-methylation of O-demethylated galantamine under Mitsunobu conditions. Copyright

Full text of DOI:10.1002/jlcr.589

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 841–855.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.589
Research Article
3
14
Synthesis of H-, C-, and stable-isotope-
labelled galantamine
Cor. G. M. Janssen*, Jos B. A. Thijssen and Willy L. M. Verluyten
Department of Non-clinical Pharmacokinetics, Johnson & Johnson
Pharmaceutical Research & Development, B-2340 Beerse, Belgium
Summary
Reminyl1 is a newly approved drug, used in the treatment of mild to moderate
Alzheimer disease. The active compound, galantamine, was initially isolated
from the bulbs of certain Narcissus species, but is at the moment also produced
synthetically. In the process leading to the final approval, the synthesis of
tritium-, carbon-14- and stable-isotope-labelled galantamine for pharmacoki-
netic studies was required.
Racemic ( Æ )-1-bromonarwedine, a compound available as intermediate
from the commercial synthesis, was transformed to racemic 1-bromo-
galantamine. Catalytic bromo-tritium exchange, followed by HPLC
purification and resolution afforded tritium-labelled galantamine. The [14C]-
label was introduced on the nitrogen as well as on the oxygen-methyl position.
This was achieved by N- and O-demethylation of galantamine and reaction of
the thoroughly purified intermediate with [14C]-methyl iodide. Stable-isotope-
labelled galantamine was obtained likewise by 13CD3OD-methylation of
O-demethylated galantamine under Mitsunobu conditions. Copyright # 2002
John Wiley & Sons, Ltd.
Key Words: carbon-14; tritium; stable-isotope; synthesis; galantamine
*Correspondence to: Cor. G. M. Janssen Department of Non-clinical Pharmacokinetics, Johnson
& Johnson Pharmaceutical Research, B-2340 Beerse, Belgium. E-mail: cjansse1@prdbe.jnj.com
Copyright # 2002 John Wiley & Sons, Ltd.
Received 19 February 2002
Accepted 7 March 2002

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C. G. M. JANSSEN ET AL.
Introduction
Galantamine (I) is a tertiary alkaloid that has been isolated from the
bulbs of the Caucasian snowdrop (Galanthus woronowi),1 from the
common snowdrop (Galanthus nivalis),2 and also from a number of
other sources e.g. from various species of Narcissus.3,4
Galantamine shows reversible, competitive acetylcholinesterase inhibit-
ing and nicotinic acetylcholinergic receptor modulatory properties5–7
and readily crosses the blood–brain barrier. It is generally accepted that
cognitive and functional improvement can be achieved with compounds
that increase cholinergic neurotransmission in the central nervous
system. Based on documented improvement of cognitive function in
several animal species, galantamine has been developed for the
symptomatic treatment of mild to moderate Alzheimer’s disease, and
was subsequently shown to have a beneficial therapeutic effect in a
number of clinical trials.8–11 In early 2001, the US Food and Drug
Administration approved the compound’s hydrobromic acid salt as
treatment for mild to moderate Alzheimer’s disease under the name
Reminyl1. Pharmacokinetic research in support of the approval
extensively used 3H-, 14C-, and stable-isotope-labelled galantamine,
the synthesis of which is described here.
Results and discussion
The total synthesis of galantamine (1a), epigalantamine (1b) and
narwedine (1c) is described in numerous papers.12–21 Most approaches
are based on a biomimetic scheme,12 applying a phenolic oxidative
cyclization process leading to the formation of the tetracyclic frame-
work. All of the mentioned multistep sequences resulted in a very low
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SYNTHESIS OF 3H-, 14C- AND STABLE
843
overall yield of at best 5%.13 Only in 1988, a procedure was described,
in which a preparatively interesting 11% overall yield of racemic
galantamine was attained.19 Later on, the same researchers reported a
stereoselective reduction with l-selectride which eliminated the forma-
tion of epigalantamine, thus improving the overall yield of racemic
galantamine to 24%.21 Synthetic procedures for the synthesis of
optically pure (À)-galantamine on kilogram scale were disclosed only
recently.22,23
A total synthesis of galantamine would offer the possibility of
incorporating a carbon-14 label on a favourable skeleton-position.
However, considering the tedious and time-consuming synthesis, such
approach was refrained from. Moreover, since the proposed reaction
sequence does not allow the introduction of a 14C-unit in the elaborated
pathway via an easily attainable labelled fragment, the label should be
incorporated into one of its precursors. This would increase the number
of reaction steps and inevitably lead to substantially diminished yields.
In order to meet the pharmacokinetic demands, instead of introdu-
cing a 14C-label in the galantamine-skeleton, the introduction of a 14C-
label in the exterior methyl groups and also the introduction of a tritium
label on the phenyl ring system was opted for. This approach would
offer the opportunity to start from optically pure material which gives a
direct, one-step access to optically pure galantamine. If a (partial)
racemization of one of the chiral centres would occur (the most likely
position would be the allylic alcohol position, leading to epigalanta-
mine), the formed diastereomeric impurity could easily be detected and
removed via HPLC.
N-[14C]-methyl-labelled galantamine
The first step in the synthesis of N-[14C]-methyl galantamine was the
cleavage of the aminomethyl group (see Scheme 1). To effect these
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C. G. M. JANSSEN ET AL.
Scheme 1. Synthesis of N-[14C]-methyl-galantamine. Reagents and conditions:
(i) m-chloroperbenzoic acid, ferrous sulphate, dichloromethane and (ii) diisopro-
pylamine, [14C]methyl iodide, methanol
N-demethylations, the use of chloroformate reagents is reported to give
high yields under mild reaction conditions.24 Vinyl chloroformate was
reported to demethylate 3,14-diacetyloxymorphone in essentially
quantitative yield.25 a-Chloroethyl chloroformate was used by the same
authors in the demethylation of oxycodone with near quantitative
yield.26 When applied to galantamine, only a poor 3% of nor-
galantamine (2a) could be isolated. A selective N-demethylation of
galantamine via a non-classical Polonovski reaction27 was reported to
afford nor-galantamine in a 76% preparative yield. This method of
choice gave in our hands a 51% isolated yield as the HCl Á H2O salt; the
isolation of the desired nor-galantamine proved to be very laborious. In
order to obtain N-[14C]-methyl galantamine it seemed a matter of course
to use the commercially available [14C]-methyl iodide. Cold test
reactions under various conditions revealed a preference for the
quarternization of the secondary amine. This problem could only be
addressed by using a four-fold excess of nor-galantamine, to provide
after purification a 36.8% yield of labelled compound (3a), based on
[14C]-methyl iodide.
O-[14C]-methyl-and O-stable-isotope-methyl-labelled galantamine
O-[14C]-methyl galantamine was obtained in an analogous approach
(see Scheme 2). Methods for O-demethylation are numerous,28 but most
suffer from lack of selectivity. In order to preserve stereochemistry and
taking into account the labile nature of the oxygen bridge and the allylic
alcohol function, a mild procedure was required. O-demethylation of
codeine, a structure highly resembling galantamine, was reported by
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SYNTHESIS OF 3H-, 14C- AND STABLE
845
Scheme 2. Synthesis of O-[14C]- and O-[13CD3]-methyl-galantamine. Reagents
and conditions: (I) n-Butyllithium, butanethiol, hexamethylphosphoramide;
(ii) potassium hydroxide, [14C]-methyl iodide, 1,3-dimethyl-2-imidazolidinone
for 3b or triphenylphosphine, 13CD3OD, diisopropyl azodicarboxylate, tetra-
hydrofuran for 3c
Rice29 and Han30 to be a high yield reaction on gram scale using boron
tribromide in dichloromethane. When applied to galantamine, change
of variables like stoichiometry, solvent and concentration always gave
unsavoury reaction mixtures with at best a 13% isolated yield of the
desired phenol (2b). Reaction of thiolates in N,N-dimethylformamide
with codeine, as reported by Lawson,31 gave better results in so far that
the final reaction mixture only existed of galantamine and the
demethylated product with the highest isolated yield of 21%. The low
yields were ascribed to a poor solubility of the compounds in DMF.
When changing to HMPT we succeeded in obtaining 69% isolated yield
of O-demethylated galantamine (98% pure) on a multigram scale. As
for the N-[14C] methylations, [14C]-methyl iodide was also used for the
O-methylations. Unlabelled test reactions revealed even more problems
than in the case of the N-methylations. Change of base (sodium
hexamethyldisilazane, potassium carbonate, diisopropyl amine or
potassium hydroxide), solvent (N,N-dimethylformamide, N,N-dimethyl-
acetamide, toluene or 1,3-dimethyl-2-imidazolidinone) or even the use
of phase transfer methods gave reaction mixtures which mainly
consisted of quarternary amino compound and starting material, with
small amounts of the desired product. The combination of potassium
hydroxide in 1,3-dimethyl-2-imidazolidinone proved to be optimal and
when applying the [14C]-methyl iodide in substoichiometric amounts the
O-[14C]-methyl galantamine (3b) was obtained in a 18% yield, based on
[14C]-methyl iodide.
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A certain vulnerability of the O- and the N-methyl label positions in
metabolic studies might be expected. This was indeed proven for both
labelled compounds (observed N-demethylation in humans between 4
and 7%, O-demethylation in humans between 5 and 33%), limiting their
use to non-metabolic in vitro research.32
Modern high-throughput analysis is shifted substantially towards the
use of LC–MS. In the development of a suitable high-throughout
analytical method the synthesis of stable-isotope-labelled galantamine
as an internal standard was desired. The compound was required to
have a molecular mass raised with 4 units (M+4), a purity of >98%
and with less than 0.1% of unlabelled galantamine (M+0). Galanta-
mine was O-demethylated as described and the product was meticu-
lously purified from traces of remaining galantamine. The observations
made in the synthesis of the O-[14C]-methyl label refrained us from using
labelled methyl iodide. Instead, the material was reacted with 13CD3OD
under Mitsunobu reaction conditions33 thus circumventing the pro-
blems of N-quarternization. In this way, the stable-isotope-labelled
galantamine (3c) was obtained in a 36.1% yield, keeping the amount of
unlabelled galantamine below detection limit.
Tritium-labelled galantamine
In order to obtain radiolabelled galantamine which could be used in in
vivo studies, efforts were redirected towards the introduction of a tritium
label on a metabolically stable position (see Scheme 3). A literature
search revealed the investigation of two alternative routes.34 Aromatic
bromine–tritium exchange with LiAlT4 was shown to be theoretically
Scheme 3. Synthesis of 1-tritio-galantamine. Reagents and conditions: (i)
l-Selectride, tetrahydrofuran and (ii) triethylamine, palladium 10% on carbon,
butanethiol catalyst poison, tetrahydrofuran, tritium gas
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SYNTHESIS OF 3H-, 14C- AND STABLE
847
feasible, but this approach was left because of the expected very lengthy
reaction times (several days) and low tritium incorporation. The second
alternative was the introduction of a tritium label on the allylic 6-
position, effected by the reduction of (À)-narwedine with tritiated
l-Selectride1. Since investigations of the metabolism showed extensive
in vivo oxidation to (À)-narwedine, which, in turn, is reduced to both
(À)-galantamine and (À)-epigalantamine,32 this label position is by its
nature unstable and synthesis of this compound to be used for in vivo
studies was not taken into consideration. Instead, a simple catalytic
bromine–tritium exchange was tried. For this reason, racemic ( Æ )-1-
bromonarwedine (4), a compound available as intermediate from
the commercial synthesis, was reduced with l-selectride to racemic
1-bromogalantamine (5). The bromo-tritium exchange took place in
tetrahydrofuran using a palladium catalyst poisoned with butanethiol in
order to avoid a possible reduction of the allylic double bond. HPLC
purification and resolution produced tritium-labelled galantamine (6).
Recently, a procedure was described to obtain 1-bromogalantamine
directly from galantamine,35 which would avoid the final resolution
step. The tritium label on the phenyl ring proved to be metabolically
stable after oral dosing in rats (no formation of tritiated water was
observed).32
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 118 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
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848
C. G. M. JANSSEN ET AL.
by measuring the UV absorbance (on HPLC) relative to the absorbance
of known amounts of injected unlabelled standards and the radio-
activity contents in the HPLC-eluate by means of liquid scintillation
counting. LC/MS/MS data were obtained on a Perkin-Elmer Sciex API
3000 triple quadrupole LC/MS/MS mass spectrometer.
(À)-[4aS-(4aa,6b,8aR*)]-4a,5,9,10,11,12-hexahydro-3-methoxy-6H-
benzofuro [3a,3,2-e,f] [2]-benzazepin-6-ol hydrochloride (N-desmethyl
galantamine, 2a)
The free base of galantamine (1a) was liberated from its HBr salt (4.9 g,
13.4 mmol) in a mixture of water–chloroform (55 ml, 10:1; v/v) which
was made alkaline with conc. aqueous ammonium hydroxide solution
(2 ml). The phases were separated and the water layer was extracted
with chloroform (12 ml). The combined organic layers were washed with
brine (6 ml), dried on magnesium sulphate, filtered and concentrated.
m-Chloroperbenzoic acid (70% aqueous, 3.4 g, 13.7 mmol) was
dissolved in dichloromethane (100 ml), the water layer was removed
and this solution was added to the stirred galantamine base in
dichloromethane (125 ml). Stirring was continued for 75 min, methanol
(125 ml) was added and the clear reaction mixture was cooled on ice. At
28C, ferrous sulphate Á 2 aq (7.5 g, 27 mmol) was added in one portion,
causing an immediate brown colouration and the temperature rise to
108C. The mixture was stirred for 90 min on ice and then for 1 h at room
temperature. It was filtered over dicalite, concentrated and dissolved in
a mixture of dichloromethane–chloroform (160 ml, 4:1; v/v). Concen-
trated aqueous ammonium hydroxide solution (5 ml) was added, which
caused the formation of a brown gelatinous precipitate. Water (200 ml)
and an extra amount of dichloromethane (60 ml) were added. After
5 min, the mixture was transferred to a separation funnel and the layers
were allowed to separate for approx. 1 h. The lower, organic, layer was
taken and filtered over dicalite. The middle layer with incomplete
separation was taken and centrifuged for 15 min at 2500 rpm. The
completely separated organic layer was pipetted off and combined with
the original lower layer. It was dried on magnesium sulphate, filtered
and concentrated to leave a product containing residue (4.3 g). This was
purified via gravity chromatography over silica (column dimensions:
ID: 52 mm, height: 240 mm) using a dichloromethane–methanol eluate
with increasing polarity (1200 ml 95:5, v/v; 800 ml 80:20, v/v; 400 ml
70:30, v/v; 1000 ml 50:50, v/v); TLC retention time: 0.27; eluate:
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SYNTHESIS OF 3H-, 14C- AND STABLE
849
dichloromethane–ammonia saturated methanol (94:6, v/v) to yield
2.25 g of N-desmethyl galantamine base. This material was suspended in
hot acetonitrile (20 ml) and HCl-saturated 2-propanol was added until
the solution turned acidic. The material shortly solubilized and quickly
thereafter a precipitate formed. The mixture was stirred for 5 min and
was then, while stirring, cooled on ice. The precipitate was filtered,
washed with acetonitrile and dried under vacuum at 908C to afford N-
desmethyl galantamine Á HCl Á H2O, 2.2 g (2a, 51% overall yield).
(À)-[4aS-(4aa,6b,8aR*)]-4a,5,9,10,11,12-hexahydro-11-methyl-6H-
benzofuro [3a,3,2-e,f] [2]-benzazepin-3,6-diol hydrochloride
(O-desmethyl galantamine, 2b Á HCl)
A solution of butanethiol (9.7 g, 11.5 ml, 108 mmol) in hexamethylphos-
phoramide (50 ml) was stirred under argon at À108C. A solution of
n-butyllithium 2.5 M in hexanes (39 ml, 97.5 mmol) was added dropwise,
maintaining the temperature below 158C. After the addition, the
temperature was allowed to rise to room temperature and galantamine Á
HBr (1a, 12.0 g, 32.5 mmol) was added as a solid in one portion. The
reaction flask was immersed into an oil bath, warmed to 1008C and
solvents were distilled off until the reaction mixture reached a
temperature of 928C. The remaining volume was cooled to 158C and
a mixture of dichloromethane (150 ml) and methanol (40 ml) was
introduced. The material was divided into two equal portions and each
portion was filtered over silica (diameter: 90 mm, height: 70 mm) using
dichloromethane–methanol (80:20, v/v) as eluate. At first the elution
was hampered by the formation of a gelatinous mass. The product
containing fractions, as verified by means of TLC (Rf=0.25; eluate:
dichloromethane–methanol 80:20, v/v) were combined and the solvent
was expelled at aspirator pressure. The residue (8.3 g) was chromato-
graphed carefully over silica (column dimensions: ID: 50 mm, height:
130 mm) with dichloromethane–methanol (80:20, v/v) as elution solvent.
The fractions containing both product and trace amounts of galanta-
mine were combined and concentrated to give 3.35 g of product which
contained 1.2% of starting material, according to HPLC (Kromasil
RP18 100-10, 4.6 mm ID Â 300 mm; flow rate: 1.0 ml/min; UV.
detection at 288 nm; programmed run consisting of a linear gradient
run for 25 min starting with aqueous 0.1 M ammonium acetate solution
(pH 7.0) methanol–acetonitrile (93–3.5–3.5, v/v/v) to solvent composi-
tion (64–18–18, v/v/v), followed by a 5 min linear gradient run to solvent
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C. G. M. JANSSEN ET AL.
composition (10–45–45, v/v/v) and a 10 min isocratic run at the latter
solvent composition; method A). The fractions which were pure
according to TLC were combined and gave after concentration 2.85 g
of material. Of this residue, HPLC indicated 0.15% of starting material
to be present. For use as internal standard the amount of unlabelled
galantamine was required to be less than 0.1% of the amount of labelled
material. For this reason, the latter residue (2.85 g) was chromato-
graphed again over silica (column dimensions: ID: 30 mm, height:
135 mm) with dichloromethane–methanol–ammonia-saturated metha-
nol (90:5:5, v/v/v). The collected fractions were analysed by means of
LC/MS/MS (Chromatographic conditions: Column: Water Symmetry-
ShieldTM RP18 3.5 mm, 4.6 mm ID Â 50 mm; flow rate: 1.5 ml/min;
isocratic run for 2 min with eluate: aqueous 0.01 M ammonium acetate
solution (pH 7.0)–acetonitrile (85–15, v/v)). The fractions still contain-
ing galantamine were combined and concentrated to yield 2b-base with
0.1% of galantamine (1.7 g). The galantamine-free fractions were
combined and concentrated to give 765 mg of 2b-base. This residue
was dissolved in boiling 2-propanol (9.0 ml) and HCl-saturated
2-propanol (0.52 ml) was added. The formed crystals were filtered and
washed twice with small amounts of 2-propanol to yield 2b Á HCl
(777 mg).
(À)-[4aS-(4aa,6b,8aR*)]-4a,5,9,10,11,12-hexahydro-3-methoxy-11-
[14C]-methyl-6H-benzofuro-[3a,3,2-e,f[2]-benzazepin-6-ol
(N-[14C]-methyl-galantamine, 3a)
To a suspension of N-desmethyl galantamine 2a (0.900 mmol, 319 mg)
in methanol (2.0 ml) diisopropylamine (0.9 mmol, 126 ml) was added.
The reaction vial was immediately stoppered, the reaction mixture was
stirred to obtain a clear solution and [14C]methyl iodide (0.225 mmol,
14 ml, containing 441 MBq of radioactivity and with a 92% radio-
chemical purity) was introduced. The reaction vessel was stoppered and
the contents were stirred for 18 h at room temperature. Then the solvent
was taken off at aspirator pressure, water (100 ml) was added and
purification was performed by means of 50 ml injections on preparative
HPLC (Hypersil ODS (5 mm), 7.1 mm ID Â 300 mm; flow rate: 4.0 ml/
min; UV detection at 275 nm; isocratic run with eluate: acetonitrile–
water–diisopropylamine (23–77–0.2, v/v/v)) to yield after concentration
and dilution with ethanol 162 MBq of compound 3a (radiochemical
yield: 40.0%) at a purity of 99.3% (HPLC conditions as described in the
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SYNTHESIS OF 3H-, 14C- AND STABLE
851
synthesis of O-desmethyl galantamine, 2b; method A), with an
enantiomeric excess of 100% (Chiralcel OD (10 mm), 4.6 mm
ID Â 300 mm; flow rate: 0.9 ml/min; column temperature 08C; isocratic
run with eluate: hexane–ethanol (80–20, v/v); method B). The
compound had a specific activity of 1.91 MBq/mmol.
(À)-[4aS-(4aa,6b,8aR*)]-4a,5,9,10,11,12-hexahydro-3-[14C]-meth-
oxy-11-methyl-6H-benzofuro-[3a,3,2-e,f][2]-benzazepin-6-ol
(O-[14C]-methyl-galantamine, 3b)
To a solution of galantamine-free O-desmethylgalantamine 2b Á HCl
(0.100 mmol, 30 mg) in 1,3-dimethyl-2-imidazolidinone (1.0 ml) was
added freshly ground potassium hydroxide powder (0.3 mmol, 17.7 mg)
and the reaction mixture was stirred for 4 h under nitrogen atmosphere.
The yellow solution was then added to the [14C]-methyl iodide
(0.006 mmol, containing 12 MBq of radioactivity), the reaction vessel
was stoppered and the reaction mixture was stirred for 18 h at room
temperature. The purification of the reaction mixture was performed by
means of 50 ml injections on preparative HPLC using the same
conditions as for the N-[14C]-methyl galantamine. The product fractions
were combined, evaporated, diluted with methanol–water (20:80, v/v)
and again purified in two portions via the same preparative HPLC
procedure. After concentration, the product which contained 2.16 MBq
of radioactivity (radiochemical yield 19.6%) was diluted with ethanol.
The batch had a purity of 98.7% (HPLC conditions as described in the
synthesis of O-desmethyl galantamine, 2b; method A), with an
enantiomeric excess of 100% (HPLC conditions as described in the
synthesis of N-[14C]-methyl-galantamine, 3a; method B). The compound
had a specific activity of 1.91 MBq/mmol.
(À)-[4aS-(4aa,6b,8aR*)]-4a,5,9,10,11,12-hexahydro-3-[13CD3]-
methoxy-11-methyl-6H-benzofuro-[3a,3,2-e,f][2]-benzazepin-6-ol
hydrobromide (O-[13CD3]-methyl-galantamine hydrobromide, 3c)
To a solution of galantamine-free 2b Á HCl (410 mg, 1.5 mmol) in freshly
distilled dry tetrahydrofuran (30 ml) was added polymer bound
triphenylphosphine (1.1 g, containing 3.3 mmol of triphenylphosphine)
and 13CD3OD-labelled methanol (0.55 ml, 13.3 mmol) from a freshly
opened ampoule. The reaction vessel was flushed with argon and
sealed with a rubber septum. Diisopropyl azodicarboxylate (0.62 ml,
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852
C. G. M. JANSSEN ET AL.
3.15 mmol) was slowly dropped to the reaction mixture. After stirring
for 20 h at room temperature, the mixture was filtered over a fritted P4
glass filter and the remaining material was thoroughly rinsed with,
respectively, tetrahydrofuran (5 ml), methanol (4 Â 5 ml), and dichloro-
methane–methanol (1:1, v/v, 5 ml). The combined filtrates were
concentrated at aspirator pressure to leave 1.14 g of residue. This was
taken up in a small volume of dichloromethane–methanol (90:10, v/v)
and purified over silica (column dimensions: ID: 30 mm, height:
180 mm) with dichloromethane–methanol (90:10, v/v) as eluate. The
product-containing fractions were combined and the solvents were
expelled at aspirator pressure. The residue (184 mg) was dissolved in
warmed ethanol and to the stirred solution was dropped slowly a
solution of 48% aqueous hydrobromic acid (80 ml, 0.70 mmol, 1.1 Eq)
dissolved in ethanol (100 ml), causing the product to precipitate as the
HBr salt. The mixture was stirred for 1.5 h, the crystals were filtered off
and were washed twice with ethanol (2 Â 125 ml). The material was dried
in air to yield 98.5% pure [13CD3]-galantamine Á HBr salt (3c Á HBr,
204 mg, 36.1%) with unlabelled galantamine below the LC–MS
detection limit of 0.02% (LC/MS/MS conditions as described for
compound 2b).
( Æ )-(4aa,6b,8aR*)]-4a,5,9,10,11,12-hexahydro-1-bromo-3-
methoxy-11-methyl-6H-benzofuro-[3a,3,2-e,f] [2]-benzazepin-6-ol
(racemic 1-bromogalantamine, 5)
To a stirred, argon-covered solution of ( Æ )-(4aa)-4a,5,9,10,11,12-
hexahydro-1-bromo-3-methoxy-11-methyl-6H-benzofuro-[3a,3,2-e,f]
[2]-benzazepin-6-one (racemic 1-bromonarwedine, 4, 4.18 g, 12.0 mmol)
in dry tetrahydrofuran (40 ml) was added, in one portion at À108C,
L-selectride 1 M solution in tetrahydrofuran (13.2 ml, 13.2 mmol). An
exothermic effect causing a temperature rise to À28C was observed. The
reaction mixture was stirred for 30 min at 08C, was then allowed to
come to room temperature and was stirred for 2 h at ambient
temperature. Methanol (1.0 ml) was added and the solvent was
evaporated at aspirator pressure. The residue was dissolved in a
mixture of 2 N aqueous hydrochloric acid (40 ml) and ethyl acetate
(40 ml). The layers were separated and chloroform (40 ml) was added to
the water layer, which was then basified (pH>10) with 10 N aqueous
sodium hydroxide solution. The organic layer was separated and the
water layer was extracted with chloroform (40 ml). The combined
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 841–855

SYNTHESIS OF 3H-, 14C- AND STABLE
853
organic layers were dried on magnesium sulphate and evaporated at
aspirator pressure. Gravity chromatography over silica (column
dimensions: ID: 40 mm, height: 160 mm) with chloroform–methanol
(97:3, v/v) as eluate afforded after thorough evaporation compound 5
(2.07 g). This material was used as such in the next reaction step.
(À)-[4aS-(4aa,6b,8aR*)]-1-tritio-4a,5,9,10,11,12-hexahydro-3-
methoxy-11-methyl-6H-benzofuro-[3a,3,2-e,f] [2]-benzazepin-6-ol
(1-tritio-galantamine, 6)
A mixture of racemic 1-bromogalantamine (5, 25 mg, 0.068 mmol),
triethylamine (0.040 ml), palladium 10% on carbon (40.0 mg) and a 4%
n-butanethiol–diisopropylether solution (0.040 ml) in tetrahydrofuran
(4.0 ml) was stirred and cooled on ice. It was then brought under tritium
atmosphere (740 GBq of tritium gas) and bromine–tritium exchange
was allowed to occur for 46 h at room temperature. Catalyst and labile
tritium were removed and the residue was dissolved in methanol
(100 ml). The solution contained 12.2 GBq of radioactivity with 27.5%
of racemic galantamine. Part of the crude material (30 ml) was purified
and resolved via HPLC (purification: Kromasil RP18 100-10, 4.6 mm
ID Â 300 mm; flow rate: 2.0 ml/min; UV detection at 288 nm; isocratic
run with eluate: aqueous 0.1 M ammonium acetate solution (pH 7.0)–
methanol–acetonitrile (80–10–10, v/v/v); resolution: HPLC conditions
as described in the synthesis of N-[14C]-methyl-galantamine, 3a; method B.
The first eluting compound is the desired enantiomer) to afford 1-tritio-
galantamine 6 (238 MBq) with a radiochemical purity of 99.2%
(Hypersil ODS (5 mm), 4.6 mm ID Â 300 mm; flow rate: 1.0 ml/min;
UV detection at 288 nm; eluate conditions: a linear gradient run for
30 min starting from water–diisopropylamine (100–0.2, v/v) to acetoni-
trile–diisopropylamine (100–0.2, v/v) followed by an isocratic run for
10 min at the latter solvent composition) and an enantiomeric excess of
99.6% (HPLC conditions as described in this synthesis; method B). The
material had a specific activity of 94.4 GBq/mmol.
Acknowledgements
The authors wish to thank S. Van Hoegaerden for performing HPLC
analyses and L. Diels for carrying out the LC/MS/MS analysis.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 841–855

854
C. G. M. JANSSEN ET AL.
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