Synthesis and radioiodination of selective ligands for the dopamine D3 receptor subtype

Article abstract of DOI:10.1016/j.bmcl.2004.05.052

Starting from FAUC 365, a series of iodine substituted heteroaryl carboxamides has been synthesized revealing high affinity and selectivity for the dopamine D3 receptor. Binding data showed a 15-560-fold selectivity for the dopamine D3 over D2. A 2,3-dich

Full text of DOI:10.1016/j.bmcl.2004.05.052

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3963–3966
Synthesis and radioiodination of selective ligands for the dopamine
D3 receptor subtype
a
b
b
b
Carsten Hocke,a, Olaf Prante, Stefan Lober, Harald Hubner, Peter Gmeiner
*
€
€
and Torsten Kuwerta
aDepartment of Nuclear Medicine, Krankenhausstrasse 12, D-91054 Erlangen, Germany
bDepartment of Medicinal Chemistry, Emil Fischer Center, Schuhstrasse 19, D-91052 Erlangen,
Friedrich-Alexander University, Germany
Received 6 April 2004; revised 19 May 2004; accepted 21 May 2004
Available online 17 June 2004
Abstract—Starting from FAUC 365, a series of iodine substituted heteroaryl carboxamides has been synthesized revealing high
affinity and selectivity for the dopamine D3 receptor. Binding data showed a 15–560-fold selectivity for the dopamine D3 over D2. A
2,3-dichloro substitution pattern on the phenylpiperazine moiety led to the highest subtype selectivity, whereas the 2-methoxy
substituted compounds showed superior D3 affinity. Suitable precursors were radioiodinated with high radiochemical yields (53–
85%) leading to potential imaging agents for the D3 receptor by SPET.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Since the dopamine D3 receptor was identified by So-
koloff et al.1 great progress in the validation of this
putative drug target has been made. As a result of these
investigations, a couple of disorders are believed to be
associated with the D3 receptor signaling pathway,
including schizophrenia, Parkinson’s disease and
cocaine craving. Very recently, BP 897 (1), a preferential
D3 receptor partial agonist (Fig. 1), was found to inhibit
cocaine-seeking behavior without revealing any intrin-
sic, primary rewarding effect.2 Based on these findings,
we adopted an interactive drug discovery process lead-
ing to the super-potent benzothiophene and benzofuran
derivatives FAUC 346 (3), FAUC 365 (4) and 5, 6,
respectively, and to the pyrazolo[1,5-a]pyridine analog
N
O
N
O
N
2 (FAUC 329)
N
OMe
N
OMe
N
H
N
H
N
O
N
1 (BP 897)
N
O
R'
N
O
R'
R
N
R
N
H
N
H
S
5: R= OMe, R'= H
6: R, R'= Cl
3 (FAUC 346): R= OMe, R'= H
4 (FAUC 365): R, R'= Cl
Figure 1. Potent dopamine D3 receptor ligands.
Keywords: Dopamine; D3 receptor; Radioligand; SPET.
* Corresponding author. Tel.: +49-9131-8533699; fax: +49-9131-8539262; e-mail: carsten.hocke@nuklear.imed.uni-erlangen.de
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.052

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C. Hocke et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3963–3966
FAUC 329 (2),3 which proved to be protective against
MPTP-induced dopamine depletion.4 Aspiring to utilize
these type of potent dopamine D3 receptor ligands for
noninvasive drug target imaging procedures, we aimed
for the development of radiolabeled piperazinylbutyl
substituted heteroaryl carboxamides. Although, several
radioligands are at present available to evaluate the D2
receptor mediated neurotransmitter system by single-
photon emission tomography (SPET), there is still need
for the development of suitable radiopharmaceuticals to
investigate the CNS located dopamine D3 receptors
in vivo.
Stannyl precursors were prepared from 9a–f by the use
of Pd(0) as the catalyst and hexabutyldistannane via tin-
for-bromo exchange reaction.8 The resulting tributyl-
stannyl derivatives 10a–f were readily reacted with 1 M
iodine solution in tetrahydrofuran at room temperature
to give the iodo derivatives 11a–f in yields of 35–71%.9;10
These compounds were subjected to receptor binding
studies as described below.
Starting from the aryl stannanes 10a–f, radioiodinations
were achieved by iododestannylation reactions using
hydrogen peroxide as the oxidant giving access to the
desired radioiodinated ligands [131I]11a–f11 (Scheme 2) in
high radiochemical yields (RCY) (53–87%) and with
greater than 95% radiochemical purities.12
In view of the high selectivity of the benzothiophene and
benzofuran derivatives 3–6, these ligands served as lead
compounds for the development of radioligands. In
analogy to radiolabeling procedures previously devel-
oped in our laboratory for the synthesis of potential D4
receptor tracers using iodine-131,5 we aimed for the
synthesis and biological in vitro evaluation of new 131I-
substituted derivatives of 3–6 to evaluate their suitability
for application as SPET ligands.
In Table 1, the resulting radiochemical yields were
shown for the various compounds. Alternative solvent
systems (methanol/acetic acid or phosphate buffer sys-
tems) and oxidants (chloramine-T) were examined, but
best results were obtained employing the reaction con-
For the synthesis of SPET tracers, aryl stannanes are
known as very powerful precursors, allowing regio-
specific radioiodination and, thus, giving access to the
corresponding iodo standards. For the synthesis of these
organo tin derivatives, we started from the commercially
available phenylpiperazines 7a,b. Alkylation with
4-bromobutyronitrile and subsequent reduction with
LiAlH4 in THF afforded the primary amines 8a,b in
more than 60% over-all yield (Scheme 1). Amide bond
formation with either 5-bromothiophene carboxylic
acid, 5-bromobenzothiophene-2-carboxylic acid6 or
5-bromobenzofuran-2-carboxylic acid7 by thionyl chlo-
ride induced activation in CH2Cl2 and addition of the
primary amine in the presence of triethylamine afforded
the bromo substituted amides 9a–f in 74–87% yield.
Ar'
O
N
N
Bu3Sn
N
H
Ar
10a-f
[
131I]iodide, H2O2,
EtOH, 1N HCl (v/v 99/1)
Ar'
N
O
131I
N
N
H
Ar
[
131I]11a-f
Scheme 2. Radiosynthesis of [131I]11a–f starting from the stannyl
precursors 10a–f.
a, b
O
N
R'
c
N
R'
N
R'
N
R
Br
N
R
Br
Br
N
H
HN
R
H2N
Ar
COOH
O
S
7a: R= OMe, R'= H
7b: R, R'= Cl
8a: R= OMe, R'= H
8b: R, R'= Cl
or
9a: R= OMe, R'= H, Ar= benzofuran
9b: R, R'= Cl, Ar= benzofuran
COOH
9c: R= OMe, R'= H, Ar= benzothiophene
9d: R, R'= Cl, Ar= benzothiophene
9e: R= OMe, R'= H, Ar= thiophene
9f: R, R'= Cl, Ar= thiophene
or
COOH
Br
S
O
N
R'
N
R
Bu3Sn
d
N
H
Ar
10a: R= OMe, R'= H, Ar= benzofuran
10b: R, R'= Cl, Ar= benzofuran
10d: R, R'= Cl, Ar= benzothiophene
10e: R= OMe, R'= H, Ar= thiophene
10f: R, R'= Cl, Ar= thiophene
10c: R= OMe, R'= H, Ar= benzothiophene
Scheme 1. Reagents and conditions: (a) 4-bromobutylnitrile, DMF, 100 ꢁC, 5 h, (85–90%); (b) LiAlH4, THF, 0 ꢁC–reflux, 5 h (80%); (c) SOCl2,
CH2Cl2, NEt3, rt (74–87%); (d) hexabutyldistannane, (Ph3P4)Pd, toluene, rt–reflux, 16 h (12–41%).

C. Hocke et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3963–3966
3965
Table 1. Radiochemical yields [%] of radioiodinated compound
131I]11a–f (100 lL EtOH/1 N HCl (99/1), 10 lL H2O2 (30%), nca [131I]
iodide ca. 2 MBq, t ¼ 2 min)
ing porcine striatal membranes and the D1 selective
radioligand [3H]SCH 23390.16 Affinities to the serotonin
receptors 5HT1A, 5HT2, and the adrenergic a1 receptor
were evaluated utilizing [3H]8-OH-DPAT, [3H]ketan-
serin and [3H]prazosin and porcine cortical membranes,
respectively.
[
H
N
R2
N
N
R1
O
Compound
R1
R2
RCY [%]
The resulting Ki values are listed in Table 2 compared to
the D3 selective lead compound FAUC 365. Most
compounds (11a,c,e, and 11f) showed high affinities in
the low nanomolar range to the different dopamine
receptor subtypes preferentially recognizing the D3
receptor with Ki values of 1.4, 0.86, 2.4, and 1.4 nM,
respectively. The 2,3-dichlorophenylpiperazinyl substi-
tuted derivatives 11b and 11d exhibited substantial D3
selectivity when binding only in high nanomolar or
micromolar concentrations to D2long, D2short and D4 but
showing D3 affinities of 5.7 and 4.5 nM, respectively.
The dichlorophenylpiperazine moiety induced clear
selectivity for the D3 receptor especially emphasized
when combined to a spatially demanding heteroaryl
moiety as shown for 11b and 11d, which are character-
ized by a D3 selectivity of 560- and 100-fold over D2
and 390- and 120-fold over D4.
I
I
[
[
[
[
[
131I]11a
131I]11b
131I]11c
131I]11d
131I]11e
2-MeO
56
58
53
55
83
4
3
5
7
3
O
O
S
S
2,3-Dichloro
2-MeO
I
I
2,3-Dichloro
2-MeO
S
S
I
I
[
131I]11f
2,3-Dichloro
87
3
With respect to the serotonin receptor binding, the Ki
values of 10–130 nM indicate a preferred binding to the
5-HT1A subtype compared to the Ki values of 210–
790 nM for 5-HT2.
ditions described above. The RCYs of radioiodinated
benzofuranes and benzothiophenes ([131I]11a–d) were
about 55% when significant differences in the radio-
chemical yields between both bicyclic systems could not
be observed. Radioiodination of the thiophene deriva-
tives [131I]11e–f resulted in RCYs of over 80%. The
RCYs were not influenced by the 2-methoxy or 2,3-di-
chloro substitution of the phenylpiperazine. All radio-
labeled compounds ([131I]11a–f) could be obtained in
sufficient high yields to investigate the tracer behavior
in vivo and in vitro for further studies.
Whereas serotonergic binding is less affected by the
different substitution pattern of the phenylpiperazine
moiety, the affinity to the adrenergic a1 receptor is
clearly influenced by these residues resulting in Ki values
of 5.9, 5.8, and 1.7 nM for the 2-methoxyphenylpiper-
azinyl substituted compounds 11a,c, and 11e and 300,
49, and 42 nM for the appropriate 2,3-dichloro substi-
tuted derivatives, respectively.
Radioligand binding assays were employed to investi-
gate the affinity and selectivity of the target compounds
11a–f to the different subtypes of dopamine receptors.
Binding data were based on the displacement of the
Comparing the binding profile of the test compounds
11a–f to that of the lead FAUC 365, incorporation of a
bulky iodine substituent in position 5 of the heteroaryl
moiety decreases affinity and selectivity to the dopamine
D3 receptor. The benzofuran derivative 11b displayed
promising binding data justifying an application as a
radioligand for further SPET imaging studies.
radioligand [3H] spiperone from the cloned human
13
dopamine receptors D2long, D2short
,
D3,14 and D4.415
all stably expressed in Chinese hamster ovary cells
(CHO).16 D1 receptor affinities were determined utiliz-
Table 2. Receptor binding data (Ki values [nM] based on the for the human D2long, D2short, D3, and D4 and the bovine D1 receptors in correlation to
the reference compound 4 (FAUC 365) (2–4 experiments each performed in triplicate)
Compound
Ki values (nM)
[3H]SCH
23990
[3H]spiperone
[3H]8-OH-
DPAT
[3H]ketan-
serin
[3H]prazosin
D2long
D2short
D3
D4.4
D2l/D3–D2s/
D3–D4/D3
D1
5-HT1A
5-HT2
a1
11a
11b
11c
11d
11e
11f
4
59
3200
48
27
430
15
1.4
5.7
46
2200
42
42–19–33
560–75–390
56–17–49
420
3300
520
33
130
27
230
790
250
290
690
210
3000
5.9
300
5.8
0.86
4.5
450
75
140
35
550
31
100–31–120
31–15–13
64–23–93
1400
600
46
10
49
2.4
1.4
1.7
89
3600
32
2600
130
340
820
8800
24
360
42
370
0.50
7200–5200–680

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C. Hocke et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3963–3966
9. To a solution of 10a–f (0.19 mmol) in THF (5 mL) was
added a solution of iodine in THF (0.5 mL, 1 M) at room
temperature. The mixture was stirred at room temperature
over night. After addition of aqueous NaHSO3 solution,
the mixture was stirred for 5 min and the organic layer was
separated. The organic phase was dried (Na2SO4), filtered,
and concentrated to give 11a–f (12–45% yield).
10. 1H NMR (CDCl3, 360 MHz) 11a: d (ppm): 1.7 (4H), 2.5
(2H), 2.7 (4H), 3.15 (4H), 3.55 (2H), 3.85 (3H), 6.8–7.0
(4H), 7.1 (1H), 7.25 (2H), 7.7 (1H), 8.05 (1H). Compound
11b: d (ppm): 1.7 (4H), 2.5 (2H), 2.65 (4H), 3.1 (4H), 3.55
(2H), 6.95 (1H), 7.0 (1H), 7.15 (2H), 7.25 (1H), 7.5 (1H),
7.65 (1H), 8.0 (1H). Compound 11c: d (ppm): 1.65 (4H),
2.45 (2H), 2.6 (4H), 3.05 (4H), 3.5 (2H), 3.65 (3H), 6.75–
6.95 (4H), 7.0 (1H), 7.55 (1H), 7.65 (2H) 8.15 (1H).
Compound 11d: d (ppm): 1.65 (4H), 2.45 (2H), 2.6 (4H),
3.0 (4H), 3.5 (2H), 6.65 (2H), 7.1 (1H), 7.15 (1H), 7.5 (1H),
7.65 (2H), 8.15 (1H). Compound 11e: d (ppm): 1.5 (4H),
2.3 (2H), 2.9 (4H), 3.2 (2H), 3.45 (4H), 3.75 (3H), 6.8–7.0
(4H), 7.4 (2H), 8.5 (1H). Compound 11f d (ppm): 1.5 (4H),
2.35 (2H), 2.95 (4H), 3.2 (2H), 3.45 (4H), 7.1 (1H), 7.25
(2H), 7.35 (1H), 7.45 (1H), 8.55 (1H).
In conclusion, highly selective dopamine D3 receptor
radioligands were synthesized using FAUC 365 as lead
compound. Using radiodestannylation reactions the
obtained radiochemical yields of [131I]11a–f were suffi-
ciently high. These compounds are potential radio-
ligands for imaging of the D3 receptor by SPET.
Especially, the selectivity and affinity of [131I]11b for the
D3 receptor stimulate further studies to determine its
biodistribution and metabolic stability in vivo.
Acknowledgements
We thank Dr. J.-C. Schwartz and Dr. P. Sokoloff (IN-
SERM, Paris), Dr. H. H. M. Van Tol (Clarke Institute
of Psychiatry, Toronto), and Dr. J. Shine (The Garvan
Institute of Medical Research, Sydney) for providing
D3, D4.4, and D2 receptor expressing cell lines. Special
thanks are due to Mrs. S. Pachaly, Mrs. H. Szczepanek,
11. Na131I, code no. IBSSO, was obtained from Amersham
Buchler (Braunschweig, Germany). Typical radiolabeling
procedures were as follows: To the stannyl precursor
(150 nmol) dissolved in 100 lL ethanol/HCl (v/v; 99/1), no
carrier added (nca) [131I]iodide (typically 2 MBq) and
10 lL hydrogen peroxide solution (30%) as oxidant at
room temperature were added. After 2 min maximal RCY
were observed to be 53–89% (radio-HPLC and radio-
TLC) of substituted product, exclusively.
€
Mrs. P. Schmitt, and Mrs. P. Hubner for skillful tech-
nical assistance.
This work was supported by a grant from the ELAN-
Fonds of the Friedrich-Alexander University Erlangen
(02.08.06.1) and by the Deutsche Forschungsgemein-
schaft (DFG, PR 677/2).
12. The radioiodinated compounds [131I]11a–f were identified
by reversed-phase chromatography (RP 18 Select B5
column (250 · 4 mm) eluted with methanol/aqueous buffer
(70/30 v/v, 0.1 N ammonium formiate buffer, pH 6.8) and a
flow rate of 1 mL/min). Analytical HPLC was performed
on the following system: HPLC Hewlett Packard (HP
1100) with a quarternary pump and variable wavelength
detector (HP 1100) connected to a radio-HPLC detector
D505TR (Canberra Packard). Computer analysis of the
HPLC data was performed using FLO-One software
(Canberra Packard). Electronic autoradiography (Instant-
Imagere, Canberra Packard) was used to analyse radio-
TLC data.
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