Click chemistry based synthesis of dopamine D4 selective receptor ligands for the selection of potential PET tracers

Article abstract of DOI:10.1016/j.bmcl.2013.09.026

Taking advantage of click chemistry, a library of N-arylpiperazinylmethyl triazoles bearing fluoro substituted appendages was synthesized and the target compounds were investigated for dopamine and serotonin receptor binding. With the aim to bias their hydrophilicity and to optimize their D4 receptor affinity and selectivity, a concise series of triazoles containing fluoroalkyl, fluoroalkoxy, fluoroalkoxyphenyl, and deoxyfluoroglucosyl substituents was studied. The D4 receptor affinity and selectivity could be tuned by altering the chemical moiety attached to the triazole unit. Among the test compounds, the fluoroethoxyphenyl derivative 15b showed weak partial agonism at D4 and a K _i value of 14 nM, while its fluoropropoxyphenyl homologue 16a turned out to act as a neutral D4 antagonist (K_i = 5.1 nM). Both, 15b and 16a revealed an excellent balance between D4 receptor affinity and subtype selectivity, providing lead candidates for the development of ¹⁸F-labeled radioligands for D4 receptor imaging studies by positron emission tomography (PET).

Full text of DOI:10.1016/j.bmcl.2013.09.026

Bioorganic & Medicinal Chemistry Letters xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Click chemistry based synthesis of dopamine D4 selective receptor
ligands for the selection of potential PET tracers
Ashutosh Banerjee a,b, Simone Maschauer a, Harald Hübner b, Peter Gmeiner b, Olaf Prante a,
⇑
a Molecular Imaging and Radiochemistry, Department of Nuclear Medicine, Friedrich-Alexander University, Schwabachanlage 6, D-91054 Erlangen, Germany
b Department of Medicinal Chemistry, Emil Fischer Center, Friedrich-Alexander University, Schuhstrabe 19, D-91052 Erlangen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Taking advantage of click chemistry, a library of N-arylpiperazinylmethyl triazoles bearing fluoro substi-
tuted appendages was synthesized and the target compounds were investigated for dopamine and sero-
tonin receptor binding. With the aim to bias their hydrophilicity and to optimize their D4 receptor
affinity and selectivity, a concise series of triazoles containing fluoroalkyl, fluoroalkoxy, fluoroalkoxyphe-
nyl, and deoxyfluoroglucosyl substituents was studied. The D4 receptor affinity and selectivity could be
tuned by altering the chemical moiety attached to the triazole unit. Among the test compounds, the flu-
oroethoxyphenyl derivative 15b showed weak partial agonism at D4 and a Ki value of 14 nM, while its
fluoropropoxyphenyl homologue 16a turned out to act as a neutral D4 antagonist (Ki = 5.1 nM). Both,
15b and 16a revealed an excellent balance between D4 receptor affinity and subtype selectivity, provid-
ing lead candidates for the development of 18F-labeled radioligands for D4 receptor imaging studies by
positron emission tomography (PET).
Received 1 August 2013
Revised 4 September 2013
Accepted 9 September 2013
Available online xxxx
Keywords:
Click chemistry
Triazole
Fluorine
PET
Dopamine
D4 receptor
Ó 2013 Elsevier Ltd. All rights reserved.
The dopamine D4 receptor (D4R) belongs to the subfamily of
D2-like dopamine receptors which in turn is a member of the super
family of G-protein-coupled receptors (GPCRs). The D4R is believed
to play a role in various neurobehavioral and psychiatric disorders,
such as attention-deficit hyperactivity disorder (ADHD) and
schizophrenia.1–3 However, the exact regional distribution of the
D4R in the brain is still a matter of debate. Keeping this in mind,
there is an urgent requirement of D4 subtype selective radioli-
gands for the successful imaging of the D4 receptor distribution
in vivo.4 Positron emission tomography (PET) is one of the most ad-
vanced and highly sensitive techniques available for noninvasive
in vivo imaging studies of cerebral receptor expression for studying
normal and pathological brain function and diseases and for drug
development research.5–7 One of the main hindrances in this goal
is the availability of highly selective D4R ligands that fulfill various
other requirements such as high D4 receptor affinity, high stability
in vivo, good penetration of the blood–brain-barrier, suitable lipo-
philicity with low unspecific binding in brain combined with a
suitable binding potential in vivo. In this context, the positron
emitter F-18 has been frequently used for radiolabeling and repre-
sents the isotope of choice due to its favorable properties (e.g. lim-
ited patient dose by low positron energy and sufficient time span
for radiosynthesis due to the half life of 120 min).
From a library of D4R selective ligands that had been previously
reported by our group,8 the N-arylpiperazine triazole derivative 1
was expected to be a good starting point since it had a high affinity
for the D4R (Ki = 2.7 nM) besides being moderately selective over
the D2 and D3 receptor subtypes (Fig. 1).8 The Cu(I)-catalyzed
azide-alkyne cycloaddition (CuAAC) to give triazoles, reported by
Sharpless and Fokin, is one of the most popular reactions within
the click chemistry concept.9 The triazole moiety should allow an
easy and straightforward synthesis by the use of click chemistry
between an alkyne and an appropriate azide (Fig. 1). Therefore,
we decided to synthesize a new series of N-arylpiperazine triazoles
bearing a fluorosubstituted appendage R, as depicted in Figure 1.
By applying a series of alkyl azides, polyethylene glycol (PEG) de-
rived azides and deoxyfluoroglucoyl azides, the hydrophilicity of
⇑
Corresponding author. Tel.: +49 9131 8544440; fax: +49 9131 8534440.
E-mail address: olaf.prante@uk-erlangen.de (O. Prante).
Figure 1. Structure of the N-arylpiperazine triazole D4 ligand
synthetic route for the click chemistry based synthesis of derivatives of 1.
1 and general
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.026
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2
A. Banerjee et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
Scheme 1. Reagents and conditions: (a) Propargyl bromide, ethyldiisopropylamine,
CHCl3, 0 °C–rt, 24 h, 89%.
the target compound could be varied, aiming at an adequate bal-
ance between potency, D4 receptor selectivity and hydrophilicity.
The most potent and selective candidates from the series are ame-
nable for labeling with 18F, thus providing a lead for the develop-
ment of D4 radioligands for PET imaging studies. Herein, we
report the synthesis and receptor binding data of the new series
of N-arylpiperazine triazoles as fluoro substituted D4R ligands.
The cycloaddition precursor 3 was synthesized by alkylation of
the secondary amine 2 with propargyl bromide using a previously
reported protocol (Scheme 1).8 As a first variation of the fluorine
bearing appendage, we planned to replace the phenyl ring of lead
compound 1 with a fluoroalkyl group. Our initial studies were di-
rected to the target compound 4 with the fluorohexyl side chain.
After conversion of 6-bromohexanol to the corresponding azide
5, cycloaddition with the alkyne 3 gave the triazole 6 which was
reacted with diethylaminosulfur trifluoride (DAST) at ꢀ78 °C to ob-
tain the fluorine substituted product 4 (Scheme 2). To introduce a
polyethyleneglycol-derived side chain, a series of triazole deriva-
tives from type 9 was envisaged. Thus, the bistosylates of diethyl-
ene and triethylene glycol were treated with sodium azide to
obtain the corresponding monoazides 7a and b. These were re-
acted with the central alkyne 3 to obtain the triazoles 8a and b.
Employing tetrabutylammonium fluoride, the structural congeners
Scheme 3. Reagents and conditions: (a) 1, CuSO4ꢁ5H2O, Na-ascorbate, tBuOH, H2O,
CH2Cl2, rt, 16 h, 88% (11a), 94% (11b).
9a and b were formed in 50% yield. Using KHMDS as a base, the
tosylate 8b was coupled with fluoroethanol to obtain the homo-
logue 9c (Scheme 2).
Recently, our group reported on the concomitant fluorination
and glycosylation of peptides using click chemistry with 2-
deoxy-2-[18F]fluoroglucose azide.10 To take advantage from this
method and to suggest the translation of this glycosylation method
to non-peptidic compounds, we aimed at the substitution of the
phenyl ring of 1 by a deoxyfluoroglucosyl moiety. Thus, the alkyne
3 was conjugated with 2-deoxy-2-fluoro-b-
D-glucopyranosyl azide
10a11 and 6-deoxy-6-fluoro-b-
D-glucopyranosyl azide 10b to ob-
tain the respective triazoles 11a and b in 90% yield (Scheme 3).
As a further structural variation of 1, we decided to maintain
the methoxyphenyl ring of the lead compound, while introducing
a fluoroalkoxyphenyl side chain to the triazole (Scheme 4). For this
Scheme 2. Reagents and conditions: (a) NaN3, DMF, 110 °C, 16 h, 96%, (b) 1, CuSO4ꢁ5H2O, Na-ascorbate, tBuOH, H2O, CH2Cl2, rt, 16 h, 97% (4), 87% (8a), 93% (8b). (c) DAST,
CH2Cl2, ꢀ78 °C, 2 h, rt, 16 h, 51%. (d) TsCl, NEt3, CH2Cl2, rt, 16 h, 50–65%. (e) NaN3, CH3CN, 120 °C, 5 h, 45% (7a), 43% (7b). (f) Bu4NF, THF, 80 °C, 1 h, 52% (9a), 50% (9b). (g)
Fluoroethanol, KHMDS, THF, 0 °C–rt, 24 h, 30%.
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A. Banerjee et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
3
Scheme 4. Reagents and conditions: (a) K2CO3, DMF, 70 °C, 12 h, 83% (13a), 98% (13b), 90% (13c). (c) NaN3, CuI, Na-ascorbate, N,N0-Dimethylethylenediamine, EtOH, H2O,
110 °C, 1 h, 97% (14a), 95% (14b), 97% (14c). (c) 1, CuSO4ꢁ5H2O, Na-ascorbate, tBuOH, H2O, CH2Cl2, rt, 16 h, 87% (15a), 90% (15b), 70% (15c). (d) DAST, CH2Cl2, ꢀ78 °C, 2 h, rt,
16 h, 73% (16a), 60% (16c). (e) Fluoroethanol, KHMDS, THF, 0 °C–rt, 24 h, 30%.
Scheme 5. Reagents and conditions: (a) AgOTf, CH2Cl2, rt, 3 h, 67%. (b) 2 N NaOH, EtOH, H2O, 60 °C, 1 h, 85%.
purpose, 4-bromophenol was alkylated by the alkyl bromide or
tosylates 12a–c. After conversion of the arylbromides 13a–c to
the aryl azides 14a–c using an established reaction protocol,12
the products were reacted with the alkyne 3 to yield the triazoles
15a–c (Scheme 4). The alcohols 15a and c were fluorinated with
DAST resulting in 16a and c. The mesylate of the alcohol 15a was
reacted with fluoroethanol to obtain 16d (Scheme 4).
As a final variation, synthesis of a target structure bearing 2-
deoxy-2-fluoroglucose attached to the phenoxy triazole and a pro-
pyl spacer between the glycosyl unit and the aromatic ring (19,
Scheme 5) was intended. Starting from 3,4,6-tri-O-acetyl-2-
[3H]prazosin, respectively. The results of the receptor binding stud-
ies are presented at Table 1. For direct comparison, the receptor
binding data of the lead compound 1 has been also provided at
the end of the table. The receptor binding data clearly indicate that
all test compounds revealed poor affinity to the D1 subtype. None
of the compounds were as potent as the lead compound 1 as far as
the D4R is concerned, however, the D4 subtype selectivity over D2
and D3 receptors of most ligands under study was enhanced when
compared to the reference agent 1 (Table 1).
Replacing the phenyl ring of the triazole with aliphatic moieties
resulted to a loss of D4R affinity. Although the fluorohexyl group of
compound 4 was tolerated by the D4 receptor, the PEG side chains
of 9a–c induced a gradual decrease for D4 affinity with the increase
of the chain length (Table 1). The fluoroglycosyl unit of the test
compounds 11a and b proved to affect D4 receptor binding, as a
significant loss of D4 receptor affinity by a factor of 180 was deter-
mined. The phenyl ring attached to the triazole proved to be effec-
tive for receptor binding since all test compounds containing
phenoxyalkyl side chains (compounds 15b, 16a and d, 19) revealed
single- or double-digit nanomolar affinity for D4R (Table 1), when
the alkyl chain length is not too long. Short chains proved to be
more beneficial, since the compounds containing fluoropropyl
(16a) and fluoroethyl (15b) groups were the most potent ones
within this class. The increase in chain length led to a gradual de-
crease of affinity as seen in the case of the D4 ligands 16c and d and
19. Besides possessing high selectivity over the other dopamine
receptor subtypes, the fluoroalkyl derivatives 16a and 15b
deoxy-2-fluoro-a-D-glucopyranosyl bromide 17, which was syn-
thesized following a procedure of Kovac,13 the alcohol 15a was
subjected to a silver triflate-mediated glycosylation reaction to
yield the product 18 as a mixture of anomers. The deacetylation
of 18 resulted in the final product 19 in a total yield of 85%
(Scheme 5).
The final products 4, 9a–c, 11a and b, 15b, 16a, c and d and 19
were evaluated in vitro for their abilities to displace [3H]spiperone
14
from the cloned human dopamine receptors D2long, D2short
,
D315
and D4.416 being stably expressed in Chinese hamster ovary (CHO)
cells.17 The D1 affinities were determined by employing porcine
striatal membrane preparations and the D1 selective antagonist
[3H]SCH23390. In addition the receptor affinity profile was ex-
tended by the related biogenic amine receptors 5-HT1A, 5-HT2
and
a1 that were determined applying porcine cortical membranes
and the selective radioligands [3H]WAY100635, [3H]ketanserin and
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4
A. Banerjee et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
Table 1
Receptor binding affinities of the fluorinated target compounds to the human dopamine receptor subtypes D2long, D2short, D3 and D4, the porcine D1 receptor, 5-HT1A, 5-HT2 and
a
1 receptors.
Compd
Kia,b (nM)
[3H]WAY600135
[3H]SCH23390
D1
[3H]spiperone
D2long
[3H]ketanserin
5-HT2
[3H]prazosin
cLogPc
D2short
D3
D4.4
5-HT1A
a1
4
9a
9b
9c
11a
11b
15b
16a
16c
16d
19
26000
21000
30000
60000
54000
77000
45000d
38000d
43000
3600
460
560
750
800
20d
23
54
240
870
770
12000
18000
420
420
560
6400
110
210
490
1300
490
2500
120
130
120d
260
210
3.14
1.50
1.37
1.23
-0.03
0.19
3.93
4.15
3.54
4.06
2.45
3.45
1600
7700
8300
23000
24000
1600
340
2700
440
4100
120
1700
5100
7600
17000
25000
17000d
2800
200
27000
23000
18000
38000
25000
10000
13000
8200
3400
4700
42000
51000
18000
520
2900
770
1500
65
150
680
500
340
14d
5.1d
100
16
2900
5700
340
480
9000
16000
27000
32
2.7
650d
7800d
1
a
Ki-values were determined in two to four independent experiments each done in triplicate.
All standard deviations ( SD) are within <40% of the appropriate Ki values.
The cLogP values are obtained using the software ChemDraw (PerkinElmer).
Standard deviations ( SEM) are within <25% of the appropriate Ki values.
b
c
d
displayed good selectivity of 100 over the serotonin receptor 5-
HT1A. The calculated LogP values revealed a wide range of variation
(Table 1) showing that lipophilicity of the ligands can be fine-tuned
just by varying the N-substitution of the triazolyl moiety.
Moreover, we studied and compared the intrinsic activities of
the most potent D4R ligands 15b and 16a by measuring the rate
of [3H]thymidine incorporation into growing CHO cells stably
expressing D4.4 receptor.18 Using quinpirole as a reference full ago-
nist, the results of the mitogenesis assay of the fluoroethoxy
substituted test compound 15b indicate weak partial agonism
(27%) at a potency of 63 nM. Very interestingly, the corresponding
fluoropropoxy analogue 16a revealed neutral antagonism.
Starting from compound 1 as the lead structure, we synthesized
a series of triazolyl derivatives, displaying high and preferential
affinity for the D4 receptor. Within the series of triazolyl D4R li-
gands, both affinity and selectivity could be tuned by altering the
appendage attached to one of the triazole nitrogen atoms.
Although none of them was as potent as the lead compound, the
D4 subtype selectivity was improved. Due to their D4 binding
affinity in the low nanomolar range and good selectivity profiles,
the target compounds 16a and 15b are suitable candidates for
studying their 18F-labeled analogs as D4R imaging agents for PET.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.09.
026.
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Acknowledgements
This research was funded by the Deutsche Forschungsgemeins-
chaft (DFG) through Grant PR 677/2-3 and partly through Grant PR
677/5-1.
Please cite this article in press as: Banerjee, A.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.09.026