Synthesis of deramciclane* labeled with tritium in various positions

Article abstract of DOI:10.1002/jlcr.964

[(1R)-endo]-(+)-3-bromocamphor was dehalogenated with tritium gas to [3-³H]camphor and via [3-³H]phenylborneol converted to [3-³H]deramciclane isolated as the fumarate salt (specific activity 51.8 GBq/mmol). This three ste

Full text of DOI:10.1002/jlcr.964

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 693–700.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.964
Research Article
Synthesis of deramciclane* labeled with tritium in
various positions
Ja
and Imre Klebovich2,3
nos Szammer1, Edit Simon-Trompler1,*, Zoltan Banka1, Jo nyog2
´ ´ ´
zsef Szu´
1 Institute of Chemistry, Chemical Research Center, Hungarian Academy of Sciences,
H-1525 Budapest, P.O. Box 17, Hungary
2 EGIS Pharmaceuticals Ltd., H-1475 Budapest, P.O. Box 100, Hungary
3 Department of Pharmaceutics, Semmelweis University, H-1092 Budapest, Ho+gyes E.
Str. 7., Hungary
Summary
[(1R)-endo]-(+)-3-bromocamphor was dehalogenated with tritium gas to [3-3H]cam-
phor and via [3-3H]phenylborneol converted to [3-3H]deramciclane isolated as the
fumarate salt (specific activity 51.8 GBq/mmol). This three step synthesis from
[3-3H]camphor gave an overall yield of 22%.
Benzyloxy-acetic acid methyl ester was reduced with sodium-borotritide to 2-
benzyloxy-ethanol-[1-3H], and through a four step procedure was converted to 2-
dimethylaminoethyl-[2-3H] chloride. The latter was condensed with the sodium
derivative of 2-phenylborneol giving rise to [2-dimethylamino-[2-3H]ethoxy]deramci-
clane isolated as the fumarate (specific activity 8.177 GBq/mmol). This six step
synthesis from [3H]NaBH4 gave an overall yield of 6%. Copyright # 2005 John
Wiley & Sons, Ltd.
Key Words: tritium; anxiolytic agents* 1R,2S,4R-(–)-2-(dimethylaminoethoxy)-2-
phenyl-1,7,7-trimethylbicyclo[2.2.1]heptane; [3-3H]deramciclane; [2-dimethylamino-
[2-3H]ethoxy]deramciclane
Introduction
Deramciclane fumarate (EGIS-3886) (Figure 1) is a new putative non-
benzodiazepin-type anxiolytic compound, synthesized by EGIS Pharmaceu-
tical Ltd.1
Deramciclane possesses high affinity to 5-HT2 receptors, where it exerts
antagonistic effects.2–4 Previously published pharmacokinetic studies in several
*Correspondence to: Institute of Chemistry, Chemical Research Center, Hungarian Academy of Sciences,
H-1525 Budapest, P.O. Box 17, Hungary. E-mail: simone@chemres.hu
Copyright # 2005 John Wiley & Sons, Ltd.
Received 18 January 2005
Revised 5 May 2005
Accepted 9 May 2005

694
J. SZAMMER ET AL.
O
CH3
HOOC
.
N
CH3
COOH
Deramciclane fumarate
Figure 1. Deramciclane fumarate
different species, including man, have shown extensive metabolism, with
cleavage of the side chain the major metabolic pathway.5–10 The resulting
metabolite and derivatives show increased distribution and significant plasma
levels for a longer biological half-life than the parent compound.11,12 Two
tritium labelled forms of deramciclane were required with the label in the
camphor skeleton, ([3-3H]deramciclane (3)) and the side chain ([2-dimethyla-
mino-[2-3H]ethoxy]deramciclane (9)) for in vitro and in vivo studies to further
investigate various aspects of the metabolism and pharmacokinetics of
3
deramciclane.11–19 Previous studies with H and 14C labelled deramciclane
had showed that the metabolism of the camphor skeleton and side chain could
be differentiated by virtue of their significantly different biological half-life
values.11
Discussion
Preliminary ‘in vitro’ studies of the biotransformation of deramciclane
required the synthesis of two separate forms of tritium labelled deramciclane,
in order to fully trace the fate of the camphor skeleton and the side chain.
Hence using synthetic methodology supplied by EGIS Pharmaceuticals Ltd.
[3-3H]deramciclane (3) and [2-dimethylamino-[2-3H]ethoxy]deramciclane (9)
were prepared.
The synthesis of [3-3H]deramciclane was realized as shown in Figure 2.
[3-3H]camphor was prepared from [(1R)-endo]-(+)-3-bromocamphor by
tritiation with 3H2 in the presence of Pd/C catalyst in dioxane. The remainder
of the synthesis was carried out using the procedure2 described earlier for
unlabelled deramciclane. The [3-3H]camphor (1) was condensed with phenyl
magnesium bromide in diethylether giving rise to [3-3H]phenylborneol (2). The
latter, after conversion into its sodium salt, was reacted with dimethylami-
noethyl chloride to give [3-3H]deramciclane (3), which was isolated as the
fumarate salt.
For the synthesis of [2-dimethylamino-[2-3H]ethoxy]deramciclane (see
Figure 3), the benzyloxy-acetic acid methyl ester was reduced with
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 693–700

SYNTHESIS OF DERAMCICLANE* LABELED WITH TRITIUM
695
3H2
C6H5MgBr
T
T
Br
OH
Pd / C
O
O
1
2
1. NaH
2. (CH3)2NCH2CH2Cl
3. Fumaric acid
T
O
CH3
CH3
HOOC
.
N
COOH
3
[3-3H]-Deramciclane fumarate
Figure 2. [3-3H]-Deramciclane fumarate
sodium-borotritide to 2-benzyloxy-ethanol-[1-3H] (4) and then converted to 2-
benzyloxy-ethylchloride-[1-3H] (5) with thionyl chloride. The 5 was aminated
with dimethylamine to 2-benzyloxy-1-dimethylamino-ethane-[1-3H] (6) and
then hydrolyzed to 2-dimethylaminoethanol-[2-3H] (7), which was chlorinated
with thionyl chloride to 2-dimethylaminoethyl-[2-3H] chloride (8). It was
condensed with the sodium derivative of 2-phenylborneol giving rise to
[2-dimethylamino-[2-3H]ethoxy]deramciclane (9) isolated as the fumarate.
Experimental
Materials
All reagents were analytical grade and used without further purification.
Tritium gas and sodium-borotritide were purchased from the Institute of
Isotopes Ltd. (Budapest, Hungary). Column chromatography was performed
on silica gel 60 (0.063–0.200 mm, Merck).
Copyright # 2005 John Wiley & Sons, Ltd.
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696
J. SZAMMER ET AL.
CH2
O
COOCH3
C6H5
CH2 CHT OH
O
CH2 CHT Cl
O
[ H]-NaBH
SOCl
CH2
C6H5
C6H5
CH2
CH2
4
5
(CH ) NH
CH2 CHT N(CH3)2
O
SOCl
HCl / MeOH
CHT N(CH3)2
CH2
Cl
CH2 CHT N(CH3)2
OH
C6H5
CH2
7
6
8
1.
ONa
2. Fumaric acid
O
CHT
HOOC
CH3
CH3
.
N
COOH
9
[2-dimethylamino-[2-3H]ethoxy]deramciclane fumarate
Figure 3. [2-dimethylamino-[2-3H]ethoxy]deramciclane
Synthesis
[3-3H]phenylborneol (2). In the presence of 10% Pd/C (5 mg) catalyst the
[(1R)-endo]-(+)-3-bromocamphor (10 mg) was hydrogenated with tritium gas
in 1 ml of dioxane for 3 h using a tritium manifold system. To the dioxane
solution of [3-3H]camphor (1) formed in the reaction, 5 ml of diethylether
containing 40 mg of unlabelled camphor was added. This solution was added
under an argon atmosphere to the Grignard solution prepared from 80 mg of
bromobenzene and 12 mg of Mg in 5 ml of ether. The reaction mixture was
stirred at room temperature for 1 h then refluxed for 30 min. The cooled
reaction mixture was quenched with water and acidified with conc.
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF DERAMCICLANE* LABELED WITH TRITIUM
697
hydrochloric acid. The layers were separated and the aqueous phase was
extracted with ether. The organic phase was dried, evaporated and the crude
[3-3H]phenylborneol (2) was purified by column chromatography (eluent:
ether–hexane=1:1).
[3-3H ]-1R, 2S, 4R-(–)-2-(dimethylaminoethoxy)-2-phenyl-1, 7, 7-trimethylbi-
cyclo [2.2.1] heptane (deramciclane) (3). The pure [3-3H]phenylborneol
(35 mg, 7.6 GBq) was converted into its sodium salt by addition of 20 mg of
NaH (55% in mineral oil suspension) and refluxing in toluene (3 ml) for 2 h
under an argon atmosphere. To this reaction mixture 60 mg of dimethylami-
noethyl chloride was added in 2 ml of toluene and the reaction refluxed for 3 h.
After cooling it was evaporated to dryness and the product was purified
by column chromatography (eluent: chloroform–methanol–ammonia-sol.=
95:5:0.5) to give pure [3-3H]deramciclane (3), which was isolated as the
fumarate salt. This three step synthesis from [3-3H]camphor gave an overall
yield of 22%. The specific radioactivity was 51.8 GBq/mmol and the
radiochemical purity was better than 98%.20
2-Dimethylaminoethyl-[2-3H] chloride hydrochloride (8). To the stirred
reaction mixture containing benzyloxy-acetic acid methyl ester (20 mg) and
sodium-borotritide (3.7 GBq, 6 mg) in tert-butanol (2 ml), 0.2 ml of methanol
was added at reflux temperature and was further refluxed for 1 h. The solvent
was distilled off and the residue (2-benzyloxy-ethanol-[1-3H] (4)) was dissolved
in chloroform, and purified by column chromatography (eluent: benzene–
methanol=8:2). The isolated pure 4 (2.8 GBq) was then refluxed with 0.5 ml of
thionyl chloride for 30 min and then evaporated to dryness. The formed crude
2-benzyloxy-ethylchloride-[1-3H] (5) was dissolved in 1 ml of methanol,
transferred to a glass tube and 0.1 mmol of dimethylamine was added. The
tube was sealed and warmed at 808C for 1 h resulting in the formation of 2-
benzyloxy-1-dimethylamino-ethane-[1-3H] (6). After allowing to cool, and
opening the tube, 10 ml of 2 N hydrochloric acid was added to the methanolic
solution of 6 and the reaction mixture was refluxed for 1 h. The solvent was
then distilled off and the residue was rendered alkaline with 40% sodium
hydroxide and steam-distilled. About 50 ml of distillate was collected, acidified
with hydrochloric acid and evaporated. The obtained 2-dimethylaminoetha-
nol-[2-3H] hydrochloride (7) was refluxed with thionyl chloride (1 ml) for
30 min then evaporated, resulting in 2-dimethylaminoethyl-[2-3H] chloride
hydrochloride (8) (405 MBq), which was used directly in the next step.
1R, 2S, 4R-(–)-2-(2-dimethylamino-2-[3H]ethoxy)-2-phenyl-1, 7, 7-trimethyl-
bicyclo [2.2.1] heptane (deramciclane) (9). The water solution of 8 was made
alkaline with 50% sodium hydroxide solution and extracted with 3 Â 4 ml of
toluene. The toluene phase was dried with potassium carbonate, filtered and
Copyright # 2005 John Wiley & Sons, Ltd.
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698
J. SZAMMER ET AL.
concentrated to about 3 ml and condensed with the sodium derivative of 2-
phenylborneol as previously described giving rise to [2-dimethylamino-
[2-3H]ethoxy]deramciclane (9) isolated as the fumarate (222 MBq). This six
step synthesis from [3H]NaBH4 gave an overall yield of 6%. The specific
radioactivity was 8.177 GBq/mmol and the chemical and radiochemical purity
were greater than 98%.16
Analysis
Radioactivity measurements and determination of chemical and radiochemical
purity of [3-3H]deramciclane and [2-dimethylamino-[2-3H]ethoxy]deramci-
clane were performed by the following methods.
Chemical and radiochemical purity of the products were checked by thin
layer/overpressured layer – (TLC/OPLC), high-performance liquid – (HPLC)
and gas chromatography, on the basis of comparison with deramciclane
standard.
All chemicals and solvents were of analytical or HPLC-solvent grade.
Radioactivity of samples was measured by liquid scintillation with
RackBeta Liquid Scintillation Counter Model 1217-001 (LKB-Wallac, Turku,
Finland) in a cocktail of benzyl alcohol (1000 ml)–ethanol (200 ml) and PPO
(6 g).
Thin layer chromatography. TLC-sheet: DC Alufolien Kieselgel 60 F254 or
HPTLC layer (Merck, Darmstadt).
Eluent: chloroform:methanol:ammonia-sol.=95:5:0.5.
Detection: UV light and Digital Autoradiograph (DAR) [(EG&G Berthold
LB 287, WinDAR Software – Vildbad, Germany), run time: 30 min, gas flow:
5 ml/min, counter voltage: 2040 V].13,15,20,21
Overpressured layer chromatography. OPLC separation: Aluminium-backed
HPTLC Kiselgel 60 F254 layer (Merck, Darmstadt) (20 Â 20 cm; 0.20 mm;
sealed on all four edges).
OPLC system: OPLC 50 Instrument (optimum-performance laminar
chromatography) Bionisis SA (Le Plessis-Robinson, France).
Mobile phase:
1-butanol–acetic acid–water (6:1:1 v/v/v);
external pressure 50 Mpa;
flow rate 250 ml/ml.
Detection: Digital Autoradiography (DAR) [(EG&G Berthold LB 287,
WinDAR Software – Vildbad, Germany), run time: 30 min, gas flow: 5 ml/
min, counter voltage: 2040 V].13,15,20,21
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF DERAMCICLANE* LABELED WITH TRITIUM
699
High-performance liquid chromatography. ISCO HPLC-system (Model 2360
Gradient Programmer, Model 2350 HPLC-Pump, V4 Variable Wavelength
Absorbance Detector; Lincoln, Nebraska, USA) was used under the following
conditions:
Column: Hypersil ODS (5 mm, 250 Â 4 mm ID).
Mobile phase: 60% acetonitrile – 40% water adjusted to pH3 with [0.1%
(70% perchloric acid) and triethylamine].
Detection: At 220 nm.
Radioactivity was measured by a HPLC-Radioactivity Monitor (Hewlett-
Packard 1090M-EG&G Berthold LB506-C-1), parameters: Scintillator cock-
tail Quickscint Flow 301 (Zinsser Analytic), detector cell type: Z 1000 4 (1 ml).
Gas chromatography. Hewlett-Packard 5890 Series II gas chromatograph
(HP, Palo Alto, USA) connected to Raytest RAGA-93 Radioactivity Gas
Analyzer (Raytest Isotopenmessgerate GmbH, Straubenhardt, Germany). The
reactor of the RAYTEST RAGA-93 radioactive gas analyser (detector; RD)
was connected to the capillary column in the oven of the gas chromatograph.
The packing of the reactor consisted of approximately 6 g of platinum wire
shavings. The packing was placed into the 2 mm ID, 165 mm long fused silica
reactor tube so that it was located in the middle third of the catalyst. Platinum
shavings were fixed with asbestos wool. Reduction was carried out at 7508C in
8 ml/min hydrogen stream. High purity (4.5) methane at a 15 ml/min flow rate
was used as counter gas. The proportional counter was operated at 3400 V and
the splitter was set to 100%.
GC column: SUPELCO SPB-5 (30 m  0.25 mm, film thickness: 0.25 mm).
Temperature: 1008C (0.4 min), 138C/min: 1858C (20 min),
508C/min: 2508C (4 min), 708C/min: 1008C.
Carrier (He): 340 kPa (0.05 min), 500 kPa/min: 190 kPa (7 min),
400 kPa/min: 100 kPa (21 min), 100 kPa/min: 340 kPa.
Injector:
Split (10 ml/min), 2408C.
Injected vol.: 2 ml.
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J Label Compd Radiopharm 2005; 48: 693–700