Synthesis of 3H- and 14C-labeled AR-A000002, a new and selective 5-HT1B/1D ligand

Article abstract of DOI:10.1002/jlcr.810

AR-A000002 is a novel and selective high-affinity 5-HT_1B/1D receptor antagonist. The compound has been shown to enhance 5-HT turnover in the guinea pig brain in vivo and to increase the extracellular concentration of 5-HT and the metabolite 5-h

Full text of DOI:10.1002/jlcr.810

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 175–180.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.810
Research Article
3
14
Synthesis of H- and C-labeled AR-A000002, a new
and selective 5-HT1B/1D ligand
Tom Werner, Stefan Berg and Rolf Johansson*
. .
Local Discovery, Research Area CNS & Pain Control, AstraZeneca R&D Sodertalje,
. .
SE-15185 Sodertalje, Sweden
Summary
AR-A000002 is a novel and selective high-affinity 5-HT1B/1D receptor antagonist.
The compound has been shown to enhance 5-HT turnover in the guinea pig
brain in vivo and to increase the extracellular concentration of 5-HT and the
metabolite 5-hydroxyindoleacetic acid (5-HIAA) in guinea pig frontal cortex. The
observed effects suggest that the compound could be used for the treatment of
affective disorders.
The syntheses of labeled AR-A000002 analogues as needed for the further
pharmacological evaluation of this selective 5-HT1B/1D antagonist, are described.
Copyright # 2004 John Wiley & Sons, Ltd.
3
Key Words: 5-HT1B; 5-HT1D
;
14C; H; Rosenmund–von Braun
Introduction
A disturbed serotonergic signaling has been implicated in the pathogenesis of
psychiatric diseases such as anxiety and depression. The release of 5-HT from
the raphe region in the brain is mainly regulated by the 5-HT1A receptor
located on the soma and dendrites of the serotonergic neurons, and by the
pre-synaptic 5-HT1B receptor located at the nerve terminal.1 Inhibition of
the pre-synaptic 5-HT1B receptor gives an increased availability of serotonin in
the synaptic cleft, and potentially an alleviation of, e.g. symptoms associated
with anxiety and depression. The 5-HT1B/1D antagonist AR-A000002 (1) is
%
currently being developed for testing of this hypothesis in man. In order to
delineate the biological properties of this compound, a labeled version of the
material was needed.
*Correspondence to: R. Johansson, Local Discovery, Research Area CNS & Pain Control, AstraZeneca
.
R&D Sodertalje, SE-15185 Sodertalje, Sweden. E-mail: rolf.johansson@astrazeneca.com
.
.
.
Copyright # 2004 John Wiley & Sons, Ltd.
Received 29 September 2003
Revised 14 November 2003
Accepted 18 November 2003

176
T. WERNER ET AL.
Results and discussion
[3H]AR-A000002 (5) with a specific activity of 833 GBq/mmol, was obtained
%
in three steps from (R)-2-amino-5-methyl-8-(4-methylpiperazin-1-yl)-1,2,3,
4-tetrahydronaphthalene.2 (R)-2-Amino-5-methyl-8-(4-methylpiperazin-1-yl)-
1,2,3,4-tetrahydronaphthalene was selectively brominated in the 7-position.
The primary amine 3 was acylated with 4-morpholinobenzoic acid and 1,10-
%
carbonyldiimidazole3 (CDI) in DMF affording the brominated precursor 4.
Catalytic debromination in tritium gas gave the desired product 5 (Scheme 1).
%
3H NMR at 426 MHz, giving a doublet at d 7.0 ppm (J=8 Hz), confirmed the
incorporation of the tritium atom.
3
Scheme 1. (a) Br2 (b) 4-morpholinobenzoic acid/CDI (c) H2/PdO
14C-Labeled 4-cyanophenylmorpholine was synthesized via the Rosen-
mund–von Braun4 reaction of 4-bromophenylmorpholine5 with freshly
prepared Cul4CN in N-methylpyrrolidone at elevated temperature (Scheme 2).
Acidic hydrolysis of the nitrile afforded labeled 4-morpholinobenzoic acid.
l4
The benzoic acid-derivative was then condensed with 2 as above, to give C-
%
labeled AR-A000002 with a specific activity of 2057 MBq/mmol.
Experimental
Tritium gas was purchased from RC Tritec AG, Switzerland, and potassium
[l4C]cyanide was purchased from Amersham, England. Radiochemical purity
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 175–180

SYNTHESIS OF 3H- AND 14C-LABELED
177
Scheme 2. (a) Cu14CN (b) HCl/HOAc (c) 2/CDI
%
was determined by thin layer chromatography (TLC) using a Bioscan System
200 Imaging Scanner or with a Packard 500 TR Flow Scintillation Analyzer
connected to a HPLC instrument. TLC was performed on TLC precoated
plates, silica gel60 F254 (Merck) and column chromatography were performed
on silica gel 60 (230–400 mesh ASTM, Merck). Gas chromatographic analysis
was performed on a Hewlett Packard 5890 apparatus equipped with a
10 m ꢀ 0.15 mm fused silica capillary column coated with 0.12 mm CP-Sil 5.
Radioactivity was measured in a Packard 1000 liquid scintillation spectro-
3
1
meter using Packard Ultima Gold as a counting medium. H NMR, H and
13C NMR spectra were obtained on either a Varian 300 or a 400 MHz NMR
3
spectrometer. The samples were dissolved in CDCl3 and the 14C- and H-
labeled samples were all run in Teflon1-tubes (Wilmad Glass Co., Inc. USA).
Mass spectra were recorded on a Finnigan Mat SSQ 710 instrument operated
at an electron energy (EI) of 70 eV and connected to a GC HP5890 inlet or a
Finnigan Mat SSQ 7000 TSP instrument. Specific rotations were determined
on an AA-10 from Optical Activity Ltd. The organic extracts of crude
products were dried over anhydrous sodium sulfate.
(R)-2-Amino-7-bromo-5-methyl-8-(4-methylpiperazin-1-yl)-1,2,3,4-tetrahydro-
naphthalene, 3
%
To a solution of (R)-2-amino-5-methyl-8-(4-methylpiperazin-l-yl)-1,2,3,4-
tetrahydronaphtalene2 (2) (280 mg, l.l mmol) in ethanol (12 ml) was added
%
HCl (2 M, 14 ml) followed by the dropwise addition of bromine (58 ml,
1.1 mmol). The reaction mixture was stirred for 1 h, cooled on an ice-bath,
then made alkaline with NaOH (2 M) and extracted with diethyl ether. The
organic phase was dried, filtered, evaporated in vacuo, and purified by
chromatography (SiO2, CHCl3/MeOH/NH4OH 95:5:0.5) affording the title
compound (170 mg, 47%) as colorless oil. [a]2D1=+28(c 1, CHCl3), H NMR
1
(300 MHz), d 7.19 (s, 1 H), 3.65–3.58 (m, 1 H), 3.53–3.43 (m, 1 H), 3.22 (dd,
J=17, 5 Hz, 1 H), 3.14–3.02 (m, 1 H), 2.91–2.80 (m, 2 H), 2.78–2.31 (m, 9 H),
Copyright # 2004 John Wiley & Sons, Ltd.
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T. WERNER ET AL.
2.35 (s, 3 H), 2.14 (s, 3 H), 2.07–1.95 (m, 1 H), 1.63 (br s, 2 H), 1.68–1.45 (m,
1 H), 13C NMR (75 MHz) d 145.2, 137.5, 135.4, 134.8, 132.1, 120.4, 56.0, 55.9,
48.6, 46.9, 46.5, 36.9, 31.9, 26.0, 18.9, MS 337/339 (M+).
(R)-N-[(7-Bromo-1,2,3,4-tetrahydro-5-methyl-8-(4-methylpiperazin-1-yl)-
2-naphthyl]-4-morpholinobenzamide, 4
%
A solution of 4-morpholinobenzoic acid (115 mg, 0.55 mmol) and 1,10-
carbonyldiimidazole (98 mg, 0.603 mmol) in DMF (20 ml) was heated at
808C for 30 min. The mixture was cooled, and (R)-2-amino-7-bromo-5-
methyl-8-(4-methylpiperazin-1-yl)-1,2,3,4-tetrahydronaphthalene (3) (170 mg,
%
0.50 mmol) in DMF (5 ml) was added and stirring was continued at room
temperature for 24 h. The solvent was then evaporated in vacuo and the residue
was extracted with CH2Cl2. The organic phase was dried, filtered, evaporated
and the crude solid was purified on a column (SiO2, CHCl3/EtOH/NH4OH
100:3:0.1) affording the title compound (172 mg, 65%) as white crystals, mp
263–2648C, [a]2D1=ꢁ708(c 0.25, CHCl3) 1H NMR (300 MHz) d 7.71 (d,
J=9 Hz, 2 H), 7.23 (s, 1 H), 6.89 (d, J=9 Hz, 2 H), 6.09 (d, J=8 Hz, 1 H),
4.42–4.37 (m, 1 H), 3.86 (t, J=5 Hz, 4 H), 3.63–3.52 (m, 2 H), 3.34–3.2 (m,
1 H), 3.24 (t, J=5 Hz, 4 H), 2.87–2.81 (m, 2 H), 2.75–2.60 (m, 4 H), 2.4–2.3 (m,
1 H), 2.35 (s, 4 H), 2.17 (s, 4 H), 1.84–1.71 (m, 1 H), 13C NMR (75 MHz) d
166.6, 153.4, 145.4, 136.6, 135.7, 134.7, 132.6, 128.4, 125.0, 120.6, 114.1, 66.6,
55.9, 55.8, 48.5, 48.4, 48.0, 46.4, 45.2, 33.4, 28.4, 25.5, 19.0.
(R)-N-[(1,2,3,4-Tetrahydro-7-[3H]-5-methyl-8-(4-methylpiperazin-1-yl)-2-
naphthyl]-4-morpholinobenzamide, 5
%
A solution of (R)-N-[(7-bromo-1,2,3,4-tetrahydro-5-methyl-8-(4-methylpiper-
azin-1-yl)-2-naphthyl]-4-morpholinobenzamide (4) (2.46 mg, 4.66 mmol) and
%
palladium oxide (4.07 mg) in DMF (0.3 ml) was stirred in an atmosphere of
carrier-free 3H2 in a tritium manifold system (RC TRITEC AG, Switzerland).
After 18 h the reaction mixture was freeze-degassed, filtered and the solvent
distilled in vacuo. Labile tritium was removed by repeated lyophilization from
ethanol, leaving a residue containing 6.4 GBq of the desired product. The
radiochemical purity of the product, determined by TLC (SiO2, CHCl3/EtOH/
NH4OH 95:5:0.1) was 70%. A portion of crude material above (ca 2.2 GBq)
was purified by preparative HPLC (Phenomex C18 column 300 ꢀ 3.9 mm,
phosphate buffer (pH=3)/acetonitrile 70:30) with UV detection at 290 nm.
The product obtained (992 MBq) had a specific radioactivity of 833 GBq/
mmol determined by MS and a radiochemical purity of 98% in the HPLC
3
system above. H NMR (426 MHz) d 7.0 (d, J=8 Hz).
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 175–180

SYNTHESIS OF 3H- AND 14C-LABELED
179
4-[14C]Cyanophenylmorpholine, 7
%
To a stirred aqueous solution of copper(II)sulfate (1.33 M, 1.0 ml) sodium
bisulfite solution (1.33 M, 0.75 ml) was added dropwise at 478C . After 2 min,
potassium [14C]cyanide (1.85 GBq, 0.91 mmol) in water (1 ml) was added to
form a white precipitate. The mixture was stirred at 47–49 8C for 10 min,
cooled and centrifuged (10 min, 4000 r/m). The precipitate was washed with
water (2 ꢀ 1 ml), ethanol (2 ꢀ 1 ml), dried in vacuo to yield copper(I)[14C]cya-
nide as a white powder (79 mg, 86%). To the powder 4-bromophenylmorpho-
line (5) (243 mg, 1.0 mmol) in N-methylpyrrolidone (1.4 ml) was added and the
%
reaction mixture was heated at 170–175 8C for 135 min. After cooling, NaOH
(2 M, 3 ml) was added and the mixture filtered. The remainder was extracted in
ethyl acetate/diethyl ether (4 ꢀ 1.5 ml). The combined organic phase was then
washed with water (2 ꢀ 1.5 ml), brine (2 ꢀ 1.5 ml), dried, filtered and
evaporated in vacuo, to give the title compound (230 mg) as a yellow oil
(GC purity ca 64%) which was used without further purification in the next
step.
4-Morpholino-1-[14C]-benzoic Acid, 8
%
The crude oil from above (230 mg) was dissolved in HCl (20%, 3 ml) and acetic
acid (2 ml) and then the mixture was heated at reflux overnight. The solution
was taken to dryness and the residue dissolved in NH4OH (2 M, 6 ml) and
washed with diethyl ether (3 ꢀ 1.5 ml). The alkaline solution was acidified with
HCl (5 M) to pH 2 and the precipitate centrifuged and collected. The
precipitate was crystallized from water (0.5 ml) and ethanol (3 ml) to give after
drying in vacuo, the title compound (66 mg, 35% from 7) as white crystals. The
%
radiochemical purity determined by TLC (SiO2, CH2Cl2/MeOH/acetic acid
90:6:3) was 99%. MS/TSP m/z: 208 (M+).
(R)-N-[1,2,3,4-tetrahydro-5-methyl-8-(4-methylpiperazin-1-yl)-2-naphthyl]-
4-morpholino-1-[14C]-benzamide, 9
%
A solution of the above acid (66 mg, 0.32 mmol) and 1,10-carbonyldiimidazole
(56 mg, 0.33 mmol) in DMF (0.65 ml) was heated at 708C for 30 min. The
mixture was cooled, and (R)-2-amino-5-methyl-8-(4-methylpiperazin-1-yl)-
1,2,3,4-tetrahydronaphthalene (2) (79 mg, 0.30 mmol) in DMF (0.3 ml) added,
%
and stirring was continued overnight at room temperature. The precipitated
product was centrifuged as above, and the residue washed with diethyl ether
and dried in vacuo, affording 47 mg of crude material. The supernatant was
taken to dryness, and NaOH (2 M, 3 ml) added and the mixture extracted with
CH2Cl2 (4 ꢀ 1 ml).
The combined organic phase was washed with water (2 ꢀ 1 ml), brine
(1.5 ml), dried, filtered and evaporated to give another 79 mg of crude 9. The
%
Copyright # 2004 John Wiley & Sons, Ltd.
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180
T. WERNER ET AL.
solid products were crystallized from methanol (0.9, then 1.2 ml) to give first
35 and then 48 mg of white crystals. The material was combined and re-
crystallized from methanol to give the title compound (58.9 mg, 42%) as white
crystals. The radiochemical purity determined by TLC (SiO2, CHCl3/EtOH/
NH4OH 90:10:0.1) was 98% and by HPLC 97%. The chemical purity (HPLC)
was 99%. The specific radioactivity was 2057 MBq/mmol measured by liquid
scintillation counting. 1H NMR (300 MHz): d 7.77 (d, J=8.7 Hz, 2 H), 7.01 (d,
J=8.l Hz, 1 H), 6.89 (d, J=8.1 Hz, 1 H), 6.86 (d, J=8.7 Hz, 2 H), 5.98 (d,
J=7.3 Hz, 1 H), 4.43 (m, 1 H), 3.84 (dd, J= 4.5, 4.5 Hz, 4 H), 3.46 (s, 3 H),
3.21 (dd, J=4.5, 4.5 Hz, 5 H), 2.87 (m, 3 H), 2.77 (dd, J=6.3, 6.3 Hz, 2 H), 2.54
(br s, 3 H), 2.32 (s, 3 H), 2.18 (s, 4 H), 1.88 (m, 1 H), 13C NMR (75 MHz): d
166.5, 153.3, 149.8, 135.1, 131.8, 129.6, 128.3, 127.9, 125.2, 117.1, 114.1, 66.6,
55.6, 52.1, 48.2, 46.1, 45.1, 31.8, 28.7, 25.4, 19.3, MS 451 (M+).
References
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Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 175–180