Synthesis and activity of 2-[4-(4-[3H]-2-cyanophenyl) piperazinyl]-N-(2,4,6-[3H]3-3-methylphenyl)acetamide: A selective dopamine D4 receptor agonist and radioligand

Article abstract of DOI:10.1016/j.bmcl.2004.07.068

The first selective dopamine D₄ agonist radioligand is described. The synthesis of these piperazine radioligands relied on the transformation of brominated precursors 4a and 4b with tritium gas in the presence of a sensitive cyano functional gr

Full text of DOI:10.1016/j.bmcl.2004.07.068

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5095–5098
Synthesis and activity of 2-[4-(4-[3H]-2-cyanophenyl)piperazinyl]-
N-(2,4,6-[3H]3-3-methylphenyl)acetamide: a selective dopamine D4
receptor agonist and radioligand
Mark A. Matulenko,a,* Bruce Surber,b Leimin Fan,b Teodozyi Kolasa,a Masaki Nakane,a
Marc A. Terranova,a Marie E. Uchic,a Loan N. Miller,a Renjie Chang,a
Diana L. Donnelly-Roberts,a Marian T. Namovic,a Robert B. Moreland,a
Jorge D. Brionia and Andrew O. Stewarta
aNeuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, AP9A/L16, 100 Abbott Park Road,
Abbott Park, IL 60064-6115, USA
bDrug Metabolism, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA
Received 23 April 2004; revised 27 July 2004; accepted 29 July 2004
Available online 20 August 2004
Abstract—The first selective dopamine D4 agonist radioligand is described. The synthesis of these piperazine radioligands relied on
the transformation of brominated precursors 4a and 4b with tritium gas in the presence of a sensitive cyano functional group. The
specific activity of these two radioligands was measured and [3H]6b found to be suitable for use in D4 saturation and competition
binding studies. The synthesis, biological, and radioactivity of this new agonist radioligand as well as preliminary SAR will be
discussed.
Ó 2004 Elsevier Ltd. All rights reserved.
Dopamine, the predominant catechol neurotransmitter
in the brain, exerts its actions via two classes of G-pro-
tein coupled receptors: the Gs-coupled D1-like family
(D1 and D5) and the Gi/o-coupled D2-like family (D2,
D3, D4).1,2 These receptors have been characterized by
cloned gene expression in stable cell lines, by immunoh-
istochemistry and by use of D4 selective ligands.1–3
While the localization of the dopamine D4 receptor in
the cortex suggests an important function in psychiatric
disorders like schizophrenia, this hypothesis has not
been validated at the clinical level.4,5 In addition, recent
publications have demonstrated that selective D4 agon-
ists play a role in facilitating penile erection.6,7 In view
of these important pharmacological effects associated
with this receptor, the availability of selective D4 radio-
ligands would be useful in understanding the localiza-
tion of dopamine D4 receptors in specific areas of the
brain. These selective radioligands could be used not
only to determine relevant affinity (competition binding
assays) dependent on the functional profile of the tar-
geted ligand but also to measure receptor abundance
and localization in brain (saturation binding assays).
Ultimately, the specific localization of D4 receptors in
brain could be determined either in vitro (autoradiogra-
phy) or in vivo with positron emission tomography
(PET) techniques using the proper radiolabels such as
carbon (11C) or fluorine (18F).
Recently, published reports8 have appeared utilizing
both selective and nonselective antagonist radioligands
in an effort to characterize the D4 receptor. As a result
of nonspecific ligand binding and low abundance of
D4 receptor in brain, characterization in vitro and in
vivo has been difficult. Several groups report the need
for higher affinity, higher specific activity, and more
selective D4 radioligands for these studies.9–11
In our previous efforts to identify new dopamine D4
agonists,12 we identified compound 5a (Scheme 1) as a
potent, selective13 agonist ligand. Acetamide 5a showed
selectivity of >400-fold over D2L and >700-fold overall
other dopamine receptor subtypes. A general screen of
Keywords: Dopamine; Agonist; Radioligand.
*
Corresponding author. Tel.: +1 847 938 2460; fax: +1 847 937 9195;
e-mail: mark.a.matulenko@abbott.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.068

5096
M. A. Matulenko et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5095–5098
R2
were further shown to be potent antagonists. We were
R2
H
N
interested in developing an agonist radioligand in order
to more accurately characterize competitive binding for
agonists at the D4 receptor. It was decided to incorpo-
rate tritium into our lead agonist in the acetamide series,
5a. Tritium offers predictable biochemical activity,
greater analog stability as well as rapid and facile syn-
thesis. In addition, we would be able to easily modulate
the required specific activity needed for binding studies
by the substitution of multiple tritium atoms. This re-
port describes the identification of the first selective dop-
amine D4 agonist radioligand.
Me
R1
Me
R1
NHR3
R2
N
CN
b
O
N
R2
R3
c
3a R1=Br; R2=R3=H (77%)
4a R1=R3=Br; R2=H (61%)
4b R1=R2=R3=Br (57%)
1a R1=Br; R2=R3=H
1b R1=R2=Br; R3=H
a
2a (71%)
2b (49%)
d
5a R1=R2=R3=H (78%)
6a R1=R3=T; R2=H
6b R1=R2=R6=T
R3=C(O)CH2Br
Scheme 1. Reagents and conditions: (a) 2a: ClC(O)CH2Br, 2N NaOH,
CH2Cl2, rt; 2b: ClC(O)CH2Br, PhMe, 100°C; (b) piperazine, i-Pr2EtN,
PhMe, 60°C; (c) 10% Pd/C, H2 (50psi), MeOH, rt, 1h; (d) T2, Pd/C,
Et3N, EtOAc, 2.5h.
Our strategy was to incorporate the radioactive labels by
dehalogenation of aryl bromides in the presence of tri-
tium gas. Compound 5a (Scheme 1) was chosen as the
representative agonist for tritiation. A concern was the
reductive conditions required in the presence of a sensi-
tive cyano functional group. As a result, it was decided
to attempt hydrogenation on a model system, bromide
3a. If the reduction was successful, then the tritiation
could be carried out on dibromide 4a or perhaps tetra-
bromide 4b. The synthesis of 3a, 4a, and 4b is shown
in Scheme 1. Commercially available anilines, 1a and
1b, were acylated with chloroacetyl bromide using either
basic, biphasic conditions or refluxing toluene15 to pro-
vide bromides 2a and 2b in moderate yields. Model com-
pound 3a was completed by reaction of 2a with
commercially available 1-(2-cyanophenyl)piperazine in
5a, in approximately 70 receptors,14 showed little activ-
ity at other nondopaminergic receptors. There was sig-
nificant activity at the 5-HT1A receptor but even in
that case, the dopamine D4 selectivity was >350-fold.
During the course of our studies, it was discovered that
substitution on the aryl piperazine portion of the mole-
cule had a significant effect on the functional activity. If
functional groups such as the cyano group in 5a were
moved to the para position (e.g., R5 in Table 1), a loss
in agonist activity was observed. Additional characteri-
zation revealed that these para-substituted analogs dis-
played good binding affinities using [3H]spiperone and
Table 1. Pharmacologic characterization of analogs 3–6a,b and 10–17 in the human D4.4 receptor
R2
H
Me
R1
N
N
R3
O
N
R4
R5
R2
R1
R2
R3
R4
R5
EC50
%Eb
[3H]6b Kic
[3H]spiperone Kid
a
Compound
3a
4a
Br
Br
Br
H
T
H
H
Br
H
H
T
CN
CN
CN
CN
CN
CN
OMe
H
H
H
Br
Br
H
T
96.0 22.8
>10,000
>10,000
7.5 0.8
NDe
69
––
––
80
ND
ND
60
––
––
––
––
––
––
––
ND
ND
H
14.0 3.5
113.5 14.4
14.5 2.1
ND
4.0 0.7f
1.8 0.3
39.1 4.2
88.9 9.1
>10,000
54.2 5.9
936 118
2.7 1.0
ND
4b
5a
H
H
6a
6b
H
T
H
T
ND
ND
10
11
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
26.8 6.7
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
0.7 0.1g
46 2.2
101 12
ND
OMe
H
12
13
H
OMe
Ph
Bn
H
H
14
15
H
H
>10,000
ND
ND
H
H
CHPh2
F
––
174.5 37.8
90.7 6.7
>10,000
16
17h
H
––
H
––
61.9 1.6
>10,000
28.4 0.4
1.4 0.04
Clozapine
Haloperidol
30.4 4.7
1.9 0.3
>10,000
––
a Mean values for agonists (EC50 in nM) calculated from at least three determinations standard error of the mean (SEM) in the calcium flux assay
(agonist mode) using HEK-293 cells co-transfected with human D4.4 receptor and Gaqo5
.
b Efficacy relative to 10lM dopamine (100%).
c Mean values for binding affinity (Ki in nM) calculated from at least three determinations SEM versus [3H]6b.
d Mean values for binding affinity (Ki in nM) calculated from at least three determinations SEM versus [3H]spiperone.
e ND = not determined.
f Kd.
g In the human dopamine D4.2 allele.
h 4-Pyridyl piperazine.

M. A. Matulenko et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5095–5098
5097
with [3H]6b on a number of analogs shown in Table 1.
The ortho methoxy substituted agonist, 10, showed good
binding affinity (Ki = 1.8nM) as did the regioisomers 11
and 12. Previous work showed that 11 and 12 were
antagonists.12 Comparison of [3H]6b and [3H]spiperone
with agonists 5a and 10, however, did not show signifi-
cant differences in calculated Ki values for either D4
agonist. As a result, we are currently expanding our
investigation of this series as well as other structurally
diverse D4 agonists within our program in order to
uncover potential differences in measured Ki values
when using [3H]6b.
CN
BocN
CN
HN
CN
F
N
N
a
b
62%
89%
Br
Br
Br
7
8
9
Scheme 2. Reagents and conditions: (a) Boc-piperazine, DMSO, Et3N,
90°C, 24h; (b) concentrated HCl, rt, 15min.
the presence of a tertiary amine base and toluene at ele-
vated temperatures in 77% yield. In order to complete 4a
and 4b, bromo piperazine 9 was required (Scheme 2).
This compound was obtained from commercially avail-
able fluoride, 7, by a two-step procedure. First, nucleo-
philic addition of t-butyl 1-piperazinecarboxylate in
DMSO provided compound 8 in 62% yield. Deprotec-
tion of the piperazine nitrogen under acidic conditions
gave the desired intermediate, 9. Reaction of 9 with 2a
or 2b at elevated temperatures in toluene in the presence
of a tertiary amine base furnished acetamides 4a and 4b
in 61% and 57% yield, respectively.
Another aspect of our SAR study was to explore size
limitations in the aryl piperazine portion of the D4
receptor. We examined relatively encumbered ligands
13, 14, and 15 and less sterically hindered analogs 16
and 17. The fluoro analog, 16, showed moderate binding
affinity for D4 and was functionally active as an antag-
onist.12 The para-substituted phenyl compound, 13,
showed poor binding affinity as did the benzyl analog,
14 thus suggesting a disfavored ligand–receptor interac-
tion in this region of the molecule. The larger benzhyd-
ryl compound (15), however, displayed modest affinity
(Ki = 174nM) while no binding was observed for the
much smaller pyridyl analog, 17.
Biochemical evaluation of bromides 3a, 4a, and 4b in the
calcium flux assay duplicated (Table 1) the trend re-
ported earlier.12 Substitution in the para position of
the benzonitrile ring caused a loss in D4 agonist activity.
Further evaluation of compounds 4a and 4b in a compe-
tition binding assay using [3H]spiperone revealed that
both analogs bound to the D4 receptor with good
affinity.
These preliminary results show that a careful examina-
tion of this region of the molecule is required to under-
stand the critical factors associated with both functional
activity and binding affinity. More subtle factors of lig-
and–receptor interaction (both steric and electronic), the
possibility of different binding orientations within the
same series of analogs and the possibility for high- and
low-affinity binding conformations of each analog are
clearly relevant.17 As this acetamide series is expanded
to address these questions, the use of both functional as-
says and competition binding studies using [3H]6b will
play a critical role. In addition to in vitro SAR develop-
ment using [3H]6b, efforts in our laboratories are cur-
rently underway to expand the scope of this
radioligand in both brain tissue saturation binding stud-
ies and autoradiography to further characterize the
localization and function of the D4 receptor.13
With the desired bromides in hand, hydrogenation stud-
ies of compound 3a; aimed at nitrile stability and the
potential of undesired side products; were initiated.
Upon hydrogenation of 3a, compound 5a was isolated
and found to match that of previously reported mate-
rial.12 We were confident that the tritiation of dibromide
4a would proceed without disturbing the nitrile func-
tionality or produce any undesired side products. Upon
tritiation of 4a, compound [3H]6a was isolated and
determined to have a specific activity of 23.7Ci/mmol.
This specific activity was inadequate for accurate cell tis-
sue saturation and competition binding assays due to
the low expression levels of the D4 receptor in rat brain.
Therefore, the tritiation of 4b was carried out to provide
tetra tritiated compound, [3H]6b. This material had
88.1Ci/mmol specific activity and performed well in a
saturation binding assay using human D4 receptor
transfected HEK-293 cells (Kd = 4.0nM). In addition,
compound [3H]6b showed equipotency for the three ma-
jor human D4 variants as well as rat D4.13
In conclusion, we have synthesized and evaluated the
first selective D4 agonist radioligand. Brominated ana-
logs 4a and 4b were successfully transformed into
[3H]6a and [3H]6b. Subsequent biochemical evaluation
of these radioligands showed [3H]6b to possess sufficient
selectivity and specific activity to be used in competition
binding studies. Initial evaluation of acetamide D4 agon-
ists with both [3H]6b and the [3H]spiperone, did not
show the same shift in binding affinities as reported
for the D2L receptor. Additional examples in the aceta-
mide series are required to further evaluate use of [3H]6b
to determine agonist affinities for the putative high-affin-
ity receptor conformations. Structure–activity relation-
ships with [3H]6b have shown that the aryl piperazine
region of the D4 receptor tolerates larger groups (like
15) but the lack of binding affinity in 17 suggesting a
subtle electronic element or larger changes in ligand
binding orientation.
Previous work by Van Vliet et al.16 reported a dramatic
difference in binding affinities with D2L selective agonists
using the antagonist radioligand, [3H]spiperone and the
D2L agonist radioligand, N-0437. The conclusion was
that because the D2L receptor can exist in a high- and
low-affinity binding state, an agonist radioligand more
accurately measured relevant agonist binding affinities
for the high-affinity receptor conformation. We specu-
lated that the same observation might be made for the
D4 receptor. Competition binding studies were initiated

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M. A. Matulenko et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5095–5098
Mikusa, J. D.; Mcvey, J. M.; Marsh, K. C.; Sullivan, J. P.;
Acknowledgements
Brioni, J. D. J. Pharmacol. Exp. Ther. 2004, 308, 330.
8. See the supplementary data section of this article for
structures and references of these selective and nonselec-
tive radioligands as well as experimental procedures for all
compounds reported in this paper.
The author would like to thank Mr. Adam Huffman and
Dr. Tom Pagano for their helpful NMR characteriza-
tions and Dr. Steven Hannick and Mr. Bryan Macri
for high-pressure hydrogenation.
9. Tarazi, F. I.; Kula, N. S.; Baldessarini, R. J. Neuroreport
1997, 8, 3423.
10. Kula, N. S.; Baldessarini, R. J.; Kebabian, J. W.;
Bakthavachalam, V.; Xu, L. Eur. J. Pharmacol. 1997,
331, 333.
Supplementary data
11. Kula, N. S.; Tarazi, F. I.; Baldessarini, R. J.; Xu, L.;
Bakthavachalam, V.; Pounds, S.; True, C. D. Eur. J.
Pharmacol. 1999, 367, 139.
12. Matulenko, M. A.; Hakeem, A. A.; Kolasa, T.; Nakane,
M.; Terranova, M. A.; Uchic, M. E.; Miller, L. N.; Chang,
R.; Donnelly-Roberts, D. L.; Namovic, M. T.; Moreland,
R. B.; Brioni, J. D.; Stewart, A. O. Bioorg. Med. Chem.
2004, 12, 3471.
Supplementary data associated with this article can
be found, in the online version, at 10.1016/
j.bmcl.2004.07.068.
References and notes
13. Moreland, R. B.; Terranova, M. A.; Chang, R.; Uchic, M.
E.; Matulenko, M. A.; Surber, B. W.; Stewart, A. O.;
Brioni, J. D. Eur. J. Pharmacol., in press.
1. Missale, C.; Nash, S. R.; Robinson, S. W.; Jaber, M.;
Caron, M. G. Physiol. Rev. 1998, 78, 189.
2. Vallone, D.; Picetti, R.; Borrelli, E. Neurosci. Behav. Rev.
2000, 24, 125.
14. Screened at Cerep, Le Bois lÕEveque-B.P. 1-86600 Celle
lÕEvescault, France.
3. Oak, J. N.; Oldenhof, J.; Van Tol, H. H. M. Eur. J.
Pharmacol. 2000, 405, 303.
4. Hrib, N. J. Drugs Fut. 2000, 25, 587.
15. Thyes, M.; Lehmann, H. D.; Gries, J.; Konig, H.;
Kretzschmar, R.; Kunze, J.; Lebkucher, R.; Lenke, D. J.
Med. Chem. 1983, 26, 800.
5. Corrigan, M. H.; Gallen, C. C.; Bonura, M. L.; Merchant,
K. M. Biol. Psychiat. 2004, 55, 445.
16. Van Vliet, L. A.; Tepper, P. G.; Dijkstra, D.; Damsma, G.;
Wikstrom, H.; Pugsley, T. A.; Akunne, H. C.; Heffner, T.
G.; Glase, S. A.; Wise, L. D. J. Med. Chem. 1996, 39,
4233.
17. Stewart, A. O.; Cowart, M. D.; Moreland, R. B.; Latshaw,
S. P.; Matulenko, M. A.; Bhatia, P. A.; Wang, X.;
Daanen, J. F.; Nelson, S. F.; Terranova, M. A.; Namovic,
M. T.; Donnelly-Roberts, D. L.; Miller, L. N.; Nakane,
M.; Sullivan, J. P.; Brioni, J. D. J. Med. Chem. 2004, 47,
2348.
6. Brioni, J. D.; Moreland, R. B.; Cowart, M.; Hsieh, G. C.;
Stewart, A. O.; Hedlund, P.; Donnelly-Roberts, D. L.;
Nakane, M.; Lynch, J. J.; Kolasa, T.; Polakowski, J. S.;
Osinski, M. A.; Marsh, K.; Andersson, K. E.; Sullivan, J.
P. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 6758.
7. Hsieh, G. C.; Hollingsworth, P. R.; Martino, B.; Chang,
R.; Terranova, M. A.; OÕ Neill, A. B.; Lynch, J. J.;
Moreland, R. B.; Donnelly-Roberts, D. L.; Kolasa, T.;