4-[ω-[4-Arylpiperazin-1-yl]alkoxy]phenylimidazo[1,2-a]pyridine derivatives: Fluorescent high-affinity dopamine D3 receptor ligands as potential probes for receptor visualization

Article abstract of DOI:10.1021/jm070721+

Sixteen long-chain arylpiperazines bearing the fluorescent moiety 2-phenylimidazo[1,2-a]pyridine were synthesized as fluorescent dopamine D ₃ receptors ligands (385 nM < K_i < 0.72 nM). The most potent D₃ compounds 15a and

Full text of DOI:10.1021/jm070721+

J. Med. Chem. 2007, 50, 5043-5047
5043
4-[ω-[4-Arylpiperazin-1-yl]alkoxy]phenyl)imidazo[1,2-a]pyridine Derivatives: Fluorescent
High-Affinity Dopamine D3 Receptor Ligands as Potential Probes for Receptor Visualization
Marcello Leopoldo,* Enza Lacivita, Elena Passafiume, Marialessandra Contino, Nicola A. Colabufo, Francesco Berardi, and
Roberto Perrone
Dipartimento Farmaco-Chimico, UniVersita` degli Studi di Bari, Via Orabona, 4, 70125 Bari, Italy
ReceiVed June 20, 2007
Sixteen long-chain arylpiperazines bearing the fluorescent moiety 2-phenylimidazo[1,2-a]pyridine were
synthesized as fluorescent dopamine D3 receptors ligands (385 nM < Ki < 0.72 nM). The most potent D3
compounds 15a and 19a (Ki ) 1.6 and 0.72 nM, respectively) showed good Stokes shift and high quantum
yield in ethanol (Φ ) 0.74 and 0.66, respectively). In the first attempt, 15a was unable to visualize D3
receptors expressed in CHO cells by epifluorescence microscopy.
Chart 1. Structures of Fluorophores and Reference Dopamine
D3 Agents
Fluorescent high-affinity ligands represent a class of widely
applicable tools. The spatial precision offered by fluorescent
visualization overcomes the diverse localization and low
concentration of receptor molecules. For example, fluorescent
ligands have allowed the localization of R1-adrenoceptors,1
dopamine transporter,2 adenosine A1 receptors,3 and peripheral-
type benzodiazepine receptors,4 the study of time course 5-HT3
receptor cluster formation,5 the real-time visual tracking of µ-
and δ-opioid receptor-ligand complexes internalization and
trafficking,6 and ligand-regulated somatostatin receptor oligo-
merization.7 Moreover, fluorescent ligand binding has been
proposed as an alternative to radioligand binding, although some
analytical issues have been encountered.8-10 Fluorescent ligands
can give also information on the biophysical characteristics of
the ligand binding site because some fluorophores show
quantum yield depending on the lipophilicity or pH of the
environment.11,12
G-protein-coupled receptors (GPCRs) represent the largest
family of cell-surface receptors mediating cellular communica-
tion and are a major target for drugs in current clinical use.13
The endogenous ligands for GPCRs are a diverse range of
hormones, transmitters, autocrine factors, and even photons. In
each case, however, the receptor transduces the binding of ligand
to an intracellular signal via activation of a heterotrimeric
guanosine triphosphate binding protein (G-protein). As a
consequence of this, a range of downstream intracellular signals
are activated, resulting in short-term effects (e.g., changes in
cellular calcium levels) and long-term effects (e.g., gene
transcription). Dopamine is one such ligand for which there are
five characterized GPCRs (D1-5 receptors).
The D3 receptor is responsible for mediating the physiological
effects of dopamine in diverse tissues such as the brain and
kidney and signals via G-proteins of the Gi/o family to inhibit
adenylate cyclase, modulates ion flow through potassium and
calcium channels, and activates kinases, most notably mitogen-
activated protein kinase. From the beginning, attention has been
attracted to the restricted distribution of the D3 receptor in the
brain (islands of Calleja, ventral striatum/nucleus accumbens,
dentate gyrus, and striate cortex), seemingly related to functions
of dopamine associated with the limbic brain.14 Initial pharma-
cological studies have investigated D3 receptors as potential
therapeutic targets for the treatment of Parkinson’s disease
because it was evident that the anti-Parkinsonian (motor) effects
of dopaminergic agonists were due to activation of D2 and/or
D3 receptors.15 Recent in vitro, in vivo, and clinical observations
have provided compelling evidence that dopamine D3 receptors
play a major role in the expression of the neuroprotective and
neurorestorative actions of dopaminergic agonists. Therefore,
the recruitment of dopamine D3 receptors would appear to be a
promising strategy for the development of more effective agents
for preventing the degeneration and to be promising for the
restoration of the function of dopaminergic neurons in Parkin-
son’s disease.16 Accumulating evidence indicates that D3
receptor antagonists appear highly promising for attenuating
cocaine reward and relapse in preclinical models of addiction,17
for reducing alcohol craving and relapse behavior,18 and for
decreasing nicotine-seeking behavior and nicotine relapse in
rodents.19
During the past decade, we20-22 and other research groups23
have extensively studied D3 receptor ligands with the N-[4-(4-
arylpiperazin-1-yl)alkyl]arylcarboxamide structure (general for-
mula I, Chart 1). The most relevant structural features for high
D3 receptor affinity were (i) a 2,3-dichlorophenyl or a 2-meth-
oxyphenyl moiety linked to the N-1 of the piperazine ring, (ii)
an arylcarboxamide moiety originating from an aromatic bicyclic
carboxylic acid, and (iii) an intermediate alkyl chain of four
methylene units. As a part of our efforts in this field, we have
identified some dopamine D3 agents as potential positron
emission tomography radioligands.24,25 Now we want to extend
our interest in molecular imaging probes to fluorescent D3
receptor ligands.
Fluorescent ligands have been prepared by tagging a ligand
with a fluorophore such as fluorescein,5 BODIPY,6 coumarin,9
and dansyl26 into an area of the structure that would have
minimal influence on receptor binding. However, this structural
* To whom correspondence should be addressed. Phone: +39-080-
5442798. Fax: +39-080-5442231. E-mail: leopoldo@farmchim.uniba.it.
10.1021/jm070721+ CCC: $37.00 © 2007 American Chemical Society
Published on Web 09/12/2007

5044 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Brief Articles
Scheme 1a
Table 1. Fluorescence Properties of Fluorophores 1a-c and 3-5 in
Ethanol
excitation λmax
emission λmax
compd
R
X
(nm)
(nm)
Φ
1aa
1ba
1ca
3
4
5
H
H
7-CH3
6-CH3
7-CH3
8-CH3
OCH3
H
H
OCH3
OCH3
OCH3
325
313
315
330
325
325
381
374
378
380
385
375
0.55
0.50
0.56
0.68
0.43
0.53
a Reagents: (A) Na2CO3, EtOH.
dichlorophenyl)piperazinyl counterparts of 15a-22a (derivatives
15b-22b, Table 2).
a Data taken from ref 29.
Chemistry
modification can change lipophilicity, affinity, and selectivity
or even change a competitive ligand into a noncompetitive
one.27,28
The new fluorophores 3-5 have been prepared from the
reaction of the appropriate methyl-2-aminopyridine with 2-bromo-
1-(4-methoxyphenyl)ethanone (Scheme 1). The target com-
pounds were synthesized according to Scheme 2. The key
intermediates 6-9 were prepared by condensing the appropriate
2-aminopyridine with 2-bromo-1-(4-hydroxyphenyl)ethanone.
The final compounds 15a,b-18a,b with a three-methylene
intermediate chain were prepared from the reaction of phenols
6-9 with the derivatives 10a,b, which were prepared by
alkylating the appropriate 1-arylpiperazine with 1-bromo-3-
chloropropane. To obtain the compounds with a four-methylene
intermediate chain, derivatives 6-9 were alkylated with 1-bromo-
4-chlorobutane to give 11-14, which were reacted with the
appropriate arylpiperazine to give the final compounds 19a,b-
22a,b.
For our purpose, we envisaged the possibility of including a
fluorescent core into a framework endowed with affinity for
the dopamine D3 receptor. In this way, it should be possible
overcome the above-mentioned shortcomings and allow access
to an entire series of fluorescent ligands with the possibility of
optimizing the fluorescence properties and receptor affinity at
the same time.
We have selected as fluorescent moiety compound 1a (Chart
1), reported for the first time by Tomoda et al.,29 which is
characterized by the 2-phenylimidazo[1,2-a]pyridine moiety.
Derivative 1a presented an oxygen that can be easily function-
alized to afford potential D3 receptor ligands structurally related
to the D3 receptor ligands reported by Wright and co-workers,
exemplified by 2 (Chart 1).30 Moreover, Tomoda demonstrated
that the presence of an electron-donating group on the imidazo-
[1,2-a]pyridine moiety enhanced the fluorescent properties of
this class of compounds. In particular, the 7-methyl derivative
1c showed higher quantum yield than the unsubstituted deriva-
tive 1b (Table 1). Therefore, by combination of the substitution
patterns of 1a and 1c, new fluorophores 3-5 were prepared
that showed quantum yields similar to or higher than the
quantum yield of fluorophore 1a (Table 1). On the basis of the
such observations, the potential fluorescent D3 ligands 15a-
18a (Table 2) formally derived from 2 were designed by
replacing the benzimidazole ring with the imidazo[1,2-a]pyridine
substructure. The higher homologues 19a-22a (Table 2) were
also prepared. Finally, we have also evaluated the 1-(2,3-
Results and Discussion
The affinity values for dopamine D3 and D2 receptors and
fluorescent properties of the target compounds are displayed in
Table 2. The 1-(2-methoxyphenyl)piperazine derivatives 15a-
18a, which were formally derived from compound 2, displayed
moderate to high D3 receptor affinities (Ki ranging from 176 to
1.6 nM). In particular, the replacement of the benzimidazole in
ω-position of the propyl chain of 2 with imidazo[1,2-a]pyridine
did not change the affinity for the D3 receptor (2, Ki ) 1.7 nM;
15a, Ki ) 1.6 nM). On the other hand, the presence of a methyl
substituent on the imidazo[1,2-a]pyridine moiety (derivatives
16a-18a) was detrimental for D3 affinity. We next elongated
the intermediate propyl chain because in other classes of D3
Table 2. Binding Affinities of Ligands 15a,b-22a,b for Dopamine D3 and D2 Receptors and Their Fluorescence Properties in Ethanol
Ki ( SEM,a nM
compd
15a
15b
16a
16b
17a
17b
18a
18b
19a
19b
20a
20b
21a
21b
22a
R
n
Ar
D3
D2
excitation λmax (nm)
emission λmax (nm)
Φ
H
H
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
2-OCH3-Ph
2,3-di-Cl-Ph
2-OCH3-Ph
2,3-di-Cl-Ph
2-OCH3-Ph
2,3-di-Cl-Ph
2-OCH3-Ph
2,3-di-Cl-Ph
2-OCH3-Ph
2,3-di-Cl-Ph
2-OCH3-Ph
2,3-di-Cl-Ph
2-OCH3-Ph
2,3-di-Cl-Ph
2-OCH3-Ph
2,3-di-Cl-Ph
1.6 ( 0.2
156 ( 30
176 ( 15
28.9 ( 3.7
71 ( 8.0
8.0 ( 0.5
50 ( 3.5
225 ( 12
0.72 ( 0.10
38 ( 5
146 ( 20
8.0 ( 0.25
40 ( 4.5
60 ( 8.0
385 ( 20
54.4 ( 1.8
16.0 ( 2.5
49 ( 5
325
330
323
325
330
325
330
330
335
327
325
325
327
325
325
322
381
380
400
380
387
387
380
380
380
380
380
382
386
387
380
380
0.74
0.58
0.83
0.59
0.72
0.57
0.77
0.67
0.66
0.56
0.61
0.61
0.68
0.58
0.77
0.59
44 ( 2.5
78 ( 5.5
31%b
6-CH3
6-CH3
7-CH3
7-CH3
8-CH3
8-CH3
H
78 ( 6
83 ( 4
49 ( 5.3
125 ( 16
179 ( 20
68 ( 4
47 ( 2.0
149 ( 20
52 ( 3.5
111 ( 10
159 ( 25
560 ( 25
2.6 ( 0.2
H
6-CH3
6-CH3
7-CH3
7-CH3
8-CH3
8-CH3
22b
haloperidol
a The values are the mean ( SEM from three independent experiments in triplicate. b Full Ki not obtained. Percentage of inhibition measured at 10 µM.

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 5045
Scheme 2a
a Reagents: (A) Na2CO3, EtOH; (B) 1-bromo-3-chloropropane, Et3N; (C); 1-bromo-4-chlorobutane, KOH; (D) 18-crown-6, KOH; (E) 1-(2-
methoxyphenyl)piperazine or 1-(2,3-dichlorophenyl)piperazine.
ligands20-22 a butyl linker was preferred (compounds 19a-22a).
Compounds 19a-21a showed slightly higher D3 affinity values
than their lower homologues 15a-17a. In particular, 19a
displayed remarkable D3 receptor affinity (Ki ) 0.72 nM). Only
compound 22a (n ) 4) was less potent than 18a (n ) 3). Next,
the 2-methoxyphenyl ring of derivatives 15a-22a was replaced
with a 2,3-dichlorophenyl ring (compounds 15b-22b) because
this substitution pattern is frequently shared by structures
possessing a high D3 receptor affinity. Surprisingly, this
modification on the unsubstituted derivatives 15a and 19a
caused a loss in affinity for the D3 receptor (15b and 19b,
respectively). On the other hand, the same structural modifica-
tion was tolerated by the methyl substituted imidazo[1,2-a]-
pyridine derivatives. In fact, 1-(2,3-dichlorophenyl)piperazine
derivatives 16b-18b and 20b-22b were at least as potent as
their 1-(2-methoxyphenyl)piperazine counterparts except for
18b. Among the 2,3-dichlorophenyl derivatives, 17b and 20b
showed D3 receptor affinity in the nanomolar range (Ki ) 8
nM in both cases). Taken together, affinity data for D3 receptors
of target compounds 15a,b-22a,b did not seem to be deter-
mined by only one of the structural features considered here
for modification.
The D2 receptor affinities of the target compounds do not
differ greatly from those for D3 receptor. In most cases the D3/
D2 Ki ratios, or vice versa, were e10. The only relevant
exceptions were represented by 15a and 19a, which exhibited
30- and 248-fold selectivity over the D2 receptor.
As far as the fluorescent properties of 15a,b-22a,b are
concerned, we observe that the structural modifications effected
on the original fluorophores 1a and 3-5 were well tolerated.
In fact, all final compounds were fluorescent in ethanol, showing
an acceptable difference of excitation to emission maximal
wavelengths (Stokes shift). The excitation wavelengths varied
from 322 to 335 nm, whereas emission wavelengths varied from
380 to 400 nm. In particular, the most potent D3 ligands 15a
and 19a showed a Stokes shift of 56 and 45 nm, respectively.
Moreover, the structural modifications performed on the original
fluorophores 1a and 3-5 led to an enhancement of quantum
yields (Φ) in ethanol except for 16b and 20a,b (Table 2). In
particular, the Φ value enhancement was more evident for the
Figure 1. Emission spectra of 15a in (A) chloroform, (B) PBS buffer,
and (C) ethanol.
1-(2-methoxyphenyl)piperazine derivatives. Moreover, com-
pounds with n ) 3 (15a,b-18a,b) showed Φ values equal to
or higher than their homologues with n ) 4 (19a,b-22a,b).
On the basis of Φ values in ethanol and affinity values for
the D3 receptor, 15a was selected for a preliminary attempt to
visualize D3 receptors in CHO cells by epifluorescence micro-
scopy. For this purpose, cells were incubated with different
concentrations of 15a in cell culture medium and were
subsequently processed for epifluorescence microscopy analysis.
Unfortunately, cell autofluorescence background was observed
and we did not detect fluorescent labeling by 15a at 3-1000
nM. This result prompted us to evaluate whether fluorescence
of 15a was affected by environment polarity. Actually, a loss
in emission intensity of 15a in CHCl3 or PBS buffer was found
(Figure 1) and this might explain, at least in part, the result
from the epifluorescence microscopy experiment.
Conclusions
We have reported here the synthesis of a series of fluorescent
ligands of dopamine D3 receptor designed on the basis of the
structure of the 1-(2-methoxyphenyl)piperazine derivative 2 and
of the fluorescent compounds 1a and 3-5. High-affinity ligands
for the human D3 receptor were obtained. The most potent D3
ligands 15a and 19a (Ki ) 1.6 and 0.72 nM, respectively)
showed good Stokes shift and high quantum yields in ethanol
(Φ ) 0.74 and 0.66, respectively). Compound 15a was unable

5046 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Brief Articles
22a,b. This material is available free of charge via the Internet at
http://pubs.acs.org.
to visualize D3 receptors expressed in CHO cells in a preliminary
experiment by epifluorescence microscopy. However, the
fluorescent properties of the ligands presented here do not
exclude their use in other fluorescence techniques, such as two-
photon fluorescence microscopy. This sensitive technique allows
visualization of fluorescent probes in living cells by two-near-
infrared-photon excitation, thus avoiding excitation wavelengths
typical of one-photon fluorescence microscopy (300-560 nm)
that cause damage to the substrates and cell autofluorescence.31
References
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Experimental Section
General Procedure for the Synthesis of 15a,b-18a,b. A
mixture of phenol 6-9 (1.0 mmol), the alkylating agent 10a,b (1.0
mmol), powdered KOH (10 mmol), and 18-crown-6 (1.0 mmol) in
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(4-[3-[4-(2-Methoxyphenyl)piperazin-1-yl]propoxy]phenyl)-
imidazo[1,2-a]pyridine (15a). Yield, 75%. ESI+/MS m/z 443
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(from CH3OH/Et2O). Anal. (C27H30N4O2‚4HCl‚H2O) C, H, N.
General Procedure for the Synthesis of Compounds 19a,b-
22a,b. A stirred mixture of alkylating agent 11-14 (1.0 mmol),
1-(2-methoxyphenyl)piperazine or 1-(2,3-dichlorophenyl)piperazine
(1.2 mmol) and a slight excess of K2CO3 in CH3CN was refluxed
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afford the target compounds.
(4-[3-[4-(2-Methoxyphenyl)piperazin-1-yl]butoxy]phenyl)imi-
dazo[1,2-a]pyridine (19a). Yield, 74%. ESI+/MS m/z 457 (MH+).
ESI+/MS/MS m/z 247 (100). 1H NMR (CDCl3): δ 1.76-1.90 (m,
4H), 2.53 (app t, 2H), 2.71 (br s, 4H), 3.13 (br s, 4H), 3.86 (s, 3H),
4.05 (t, 2H, J ) 6.2 Hz), 6.76 (t, 1H, J ) 6.6 Hz), 6.84-7.03 (m,
6H), 7.15 (app t, 1H), 7.60 (d, 1H, J ) 8.8 Hz), 7.78 (s, 1H), 7.85-
7.90 (m, 2H), 8.10 (d, 1H, J ) 6.9 Hz). Mp 128-130 °C (from
CHCl3/n-hexane). Anal. (C28H32N4O2) C, H, N.
Fluorescence Spectroscopy. Emission and excitation spectra of
15a,b-22a,b were recorded as detailed in Supporting Information.
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285 nm; Φ ) 0.37),29 according to Demas et al.32
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Acknowledgment. The authors thank Professor Roberto
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Supporting Information Available: Procedure for the synthe-
ses and spectral data of 3-9, 10b, 11-14; spectral data of 15b,
16a,b-18a,b, 19b, 20a,b-22a,b; biological methods and statistical
analysis; excitation and emission spectra of 15a and 19a in ethanol;
experimental procedures for fluorescence spectroscopy and fluo-
rescent labeling of CHO cells; elemental analysis data of 15a,b-

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