Design, synthesis, radiolabeling, and in vivo evaluation of carbon-11 labeled N -[2-[4-(3-cyanopyridin-2-yl)piperazin-1-yl]ethyl]-3-methoxybenzamide, a potential positron emission tomography tracer for the dopamine D4 receptors

Article abstract of DOI:10.1021/jm100925m

Here we describe the design, synthesis, and evaluation of physicochemical and pharmacological properties of D₄ dopamine receptor ligands related to N-[2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide (2). Structural features were incorporated to increase affinity for the target receptor, to improve selectivity over D₂ and σ₁ receptors, to enable labeling with carbon-11 or fluorine-18, and to adjust lipophilicity within the range considered optimal for brain penetration and low nonspecific binding. Compounds 7 and 13 showed the overall best characteristics: nanomolar affinity for the D₄ receptor, >100-fold selectivity over D₂ and D₃ dopamine receptors, 5-HT_1A, 5-HT_2A, and 5-HT_2C serotonin receptors and σ₁ receptors, and log-P = 2.37-2.55. Following intraperitoneal administration in mice, both compounds rapidly entered the central nervous system. The methoxy of N-[2-[4-(3-cyanopyridin-2-yl)piperazin-1-yl]ethyl]-3- methoxybenzamide (7) was radiolabeled with carbon-11 and subjected to PET analysis in non-human primate. [¹¹C]7 time-dependently accumulated to saturation in the posterior eye in the region of the retina, a tissue containing a high density of D₄ receptors.

Full text of DOI:10.1021/jm100925m

7344 J. Med. Chem. 2010, 53, 7344–7355
DOI: 10.1021/jm100925m
Design, Synthesis, Radiolabeling, and in Vivo Evaluation of Carbon-11 Labeled
N-[2-[4-(3-Cyanopyridin-2-yl)piperazin-1-yl]ethyl]-3-methoxybenzamide,
a Potential Positron Emission Tomography Tracer for the Dopamine D4 Receptors
Enza Lacivita,† Paola De Giorgio,† Irene T. Lee,‡ Sean I. Rodeheaver,‡ Bryan A. Weiss,‡ Claudia Fracasso,§ Silvio Caccia,§
Francesco Berardi,† Roberto Perrone,† Ming-Rong Zhang, Jun Maeda, Makoto Higuchi, Tetsuya Suhara,
John A. Schetz,‡,^,#,¥,ꢀ and Marcello Leopoldo*,†,ꢀ
†Dipartimento Farmaco-Chimico, Universitaꢀ degli Studi di Bari “A. Moro”, Via Orabona, 4, 70125, Bari, Italy, ‡Department of Pharmacology
and Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, Texas 76107-2699, United States,
§Istituto di Ricerche Farmacologiche “Mario Negri”, Via La Masa 19, 20156 Milan, Italy, Molecular Imaging Center, National Institute of
Radiological Sciences, 9-1, 4-Chome, Anagawa, Inage-ku, 263-8555, Chiba, Japan, ^Department of Psychiatry, Texas College of Osteopathic
Medicine, Fort Worth, Texas 76107, United States, #Department of Health Management and Policy, School of Public Health, Fort Worth,
Texas 76107, United States, and ¥Department of Biology, University of Texas at Arlington, Arlington, Texas 76109, United States.
ꢀ J.A.S. and M.L. contributed equally to this study.
Received July 22, 2010
Here we describe the design, synthesis, and evaluation of physicochemical and pharmacological properties of
D4 dopamine receptor ligands related to N-[2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide
(2). Structural features were incorporated to increase affinity for the target receptor, to improve
selectivity over D2 and σ1 receptors, to enable labeling with carbon-11 or fluorine-18, and to adjust
lipophilicity within the range considered optimal for brain penetration and low nonspecific binding.
Compounds 7 and 13 showed the overall best characteristics: nanomolar affinity for the D4 receptor,
>100-fold selectivity over D2 and D3 dopamine receptors, 5-HT1A, 5-HT2A, and 5-HT2C serotonin
receptors and σ1 receptors, and log P=2.37-2.55. Following intraperitoneal administration in mice, both
compounds rapidly entered the central nervous system. The methoxy of N-[2-[4-(3-cyanopyridin-2-
yl)piperazin-1-yl]ethyl]-3-methoxybenzamide (7) was radiolabeled with carbon-11 and subjected to
PET analysis in non-human primate. [11C]7 time-dependently accumulated to saturation in the posterior
eye in the region of the retina, a tissue containing a high density of D4 receptors.
Introduction
of functional activity (thymidine incorporation instead of cAMP
accumulation).8 Years later, a dose of this same ligand, which
reduced amphetamine-induced increased locomotor activity,
was demonstrated to have atypical antipsychotic potential in
animal models predictive of antipsychotic efficacy in humans.9
This is in contrast to the structurally distinct D4-selective
neutral antagonist PNU-101,387G (sonepiprazole) (Chart 1),
which has no demonstrable antipsychotic activity in humans.10
The D4 receptor has been suggested as a candidate gene
for novelty seeking behavior11-14 and attention deficit hyper-
activity disorder (ADHDa),15-18 although the latter association
or its basis has been questioned.19-21 While certain antagonists
of the D4 receptor reduced hyperactivity in a neonatal 6-hydroxy-
dopamine lesioned rat model of ADHD,22 the hyperactivity
due to neonatal lesioning appears to be mediated by compen-
satory changes in serotonin transporters.23 More recently, the
D4 agonist A-412997 (Chart 1) was shown to improve cogni-
tive function in both a five-trial inhibitory avoidance para-
digm and the social recognition model, which are two animal
models thought to be predictive of anti-ADHD efficacy.24
The D4 receptor has been implicated in the control of penile
tumescence. For instance, D4 selective agonists like ABT-724
The dopamine D4 receptor belongs to the D2-like receptor
family which is characterized by its ability to interact with Gi/o
proteins resulting in inhibition of adenylyl cyclase. Early
interest in the D4 receptor originated from the finding that
the D2-like receptor antagonist clozapine, an atypical anti-
psychotic drug with high antipsychotic efficacy and reduced
extrapyramidal and neuroendocrine side effects, had 10 times
higher affinity for this receptor than for other D2-like receptor
subtypes.1,2 This resulted in an intense search for dopamine
D4 antagonists.3 However, L-745,870 (Chart 1), one of the
first highly D4-selective antagonists to be reported, failed to
show antipsychotic potential in animal models.4 In placebo-
controlled clinical trials, the more bioavailable congener of
1 (L-745,870) named L-750,667 (Chart 1) did not alleviate any
of the symptoms of schizophrenia.5 Instead, there was a trend
toward a worsening of psychotic symptoms. Although these
findings seemed to exclude the D4 receptor as an antipsychotic
drug target, later in vitro studies with 1 provided evidence for
its weak partial agonist activity.6,7 Subsequently, the selective
D4 ligand FAUC 213 (Chart 1), structurally related to 1, was
characterized as a “neutral antagonist” using a different measure
a Abbreviations: ADHD, attention deficit hyperactivity disorder;
PET, positron emission tomography; GPCR, G-protein-coupled recep-
tor; BBB, blood-brain barrier.
*To whom correspondence should be addressed. Phone: þ39 080
5442798. Fax: þ39 080 5442231. E-mail: leopoldo@farmchim.uniba.it.
r
pubs.acs.org/jmc
Published on Web 09/27/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7345
Chart 1. Representative Selective Dopamine D4 Receptor Ligands
(usually in the nanomolar range); selectivity for its target
(>100-fold); ease of labeling with 11C or 18F at high specific
radioactivity; safe administration at low tracer dose; high
blood-brain barrier (BBB) penetrance; inability to serve as
a substrate for efflux transporters; low nonspecific binding;
suitable brain pharmacokinetics in relation to radiolabel half-
life (observable brain uptake and washout kinetics); lack of
troublesome radiometabolites. Of particular importance with
respect to BBB penetration and low nonspecific binding is
tracer lipophilicity. In general, brain penetration by passive
diffusion of molecules across the BBB becomes poor when
the log P is greater than 4.0, molecular weight is larger than
450 Da, and the number of hydrogen bond donors is greater
than 5. However, if molecules are too hydrophilic, their BBB
passage is prevented as well. It has been reported for many
drug classes that the optimum log P value for central nervous
system (CNS) targeted drugs lies between 2.0 and 3.5. More-
over, high lipophilicity attributed to adhesion to proteins and
lipids tends to increase nonspecific binding, an essentially
nonsaturable component of the total tissue uptake of a radio-
ligand. Therefore, it appears that there is an optimal range of
lipophilicity for brain radioligands, wherein brain uptake is
high and nonspecific binding comparatively weak. From data
inthe literature, log P=3.5 appearstobe the acceptable upper
limit of lipophilicity for a PET radioligand.40
Here we describe the synthesis and in vitro characterization
of an improved series of D4 receptor agents derived from our
lead compound 2,41-43 which we rationally designed specifi-
cally for use as a PET radiotracer. By use of our newly
developed selection criteria and assay cascade, one of a dozen
compounds was selected for radiolabeling with carbon-11 and
PET studies in monkey brain.
(Chart 1) produce erectogenesis in animal models without
nausea.25 However, studies in rats support a role for the D3
receptor, but not the D4 receptor, in the induction of penile
erection by D2-like agonists in rodents.26 Although the D4
receptor is expressed at low levels in the brain, much higher
densities of this receptor are expressed in the retina.27 Dopamine,
via D4 receptors, normally modulates the cascade that couples
light responses to adenylyl cyclase activity in photoreceptor
cells in the retina.28,29
Chemistry
The synthesis of the final compounds is depicted in Scheme 1.
The preparation of the target compounds required the key
amines 18a-f. Among these, 4-(4-chlorophenyl)-1-piperazi-
neethanamine (18a) and 4-(4-methylphenyl)-1-piperazinee-
thanamine (18b) were prepared according to the literature.44
Amines 18c-e were prepared as follows: the appropriate 1-aryl-
piperazine was alkylated with 2-haloethanol to the corre-
sponding alcohol 15c-e. These latter were subsequently con-
densed, under Mitsunobu conditions, with phthalimide to
give 16c-e, which were hydrolyzed with hydrazine hydrate to
afford primary amines 18c-e. An alternative synthetic route
was followed to prepare amine 18f: 1-(5-chloro-2-pyridinyl)-
piperazine45 was first alkylated with chloroacetonitrile and
then treated with borane dimethyl sulfide complex to afford
the desired amine 18f. The final compounds were prepared by
condensing 3-methoxy- or 4-fluorobenzoic acid with amines
18a-f in the presence of 1,10-carbonyldiimidazole.
To better understand dopamine D4 receptor biology in
vivo, a suitable D4 receptor positron emission tomography
(PET) tracer would be a powerful tool allowing noninvasive
D4 receptor imaging. A number of attempts have been made
to identify a D4-selective PET radioligand.30-36 In 2000,
Langer and co-workers reported an attempt to visualize the
dopamine D4 receptor in primate brain with [11C]PB-12
(compound 2, Table 1).32 The radioligand was of little use in
visualizing D4 receptors because it exhibited a very high
background due to nonspecific binding. Similar results were
obtained 2 years later by Zhang and co-workers who prepared
and tested [11C]2 and [11C]YM-50001 (Chart 1) with both
radioligands being unsuitable for D4 receptor imaging with
PET.34 In each case, it was suggested that the high nonspecific
binding of [11C]2 could be due to its relatively high lipophili-
city (ClogP= 3.72).37 More recently, Prante and co-workers
reported on the rational design of a series of pyrazolo[1,5-a]-
pyridine-based dopamine D4 receptor ligands for potential
use as PET ligands. However, the most promising radioligand
of the series, [18F]3 (ClogP=2.9) (Chart 1), was only tested in
rat brain slices by in vitro autoradiography.36 Thus, to date,
there are no verified examples of a D4-selective PET tracer.
An adequate PET tracer for neuroreceptor visualization must
fulfill many criteria:38,39 high affinity for the target receptor
Results and Discussion
Lipophilicity Evaluation. The pivotal role of PET tracer
lipophilicity is well recognized, and it has been reviewed in
depth by Waterhouse.40 Lipophilicity can be evaluated in
various theoretical and experimental ways. The most com-
mon experimental lipophilicity measurement involves parti-
tioning of a compound between octanol and aqueous solution
(log P). The log P refers to partitioning of the neutral mole-
cule species and log D7.4 the partitioning of all species present
in solution at a given pH, which accounts for solubility effects
associated with ionization. When lipophilicity is expressed
as log P or log D7.4, compounds that seem most effective for

7346 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Lacivita et al.
Table 1. Lipophilicity Values and Binding Affinities at Dopamine D4 Receptors of the Target Benzamides
imaging have log Por log D7.4 of <3.5. On such a basis, we have
modified our reference compound 2 and designed compounds
4-14 which showed computer estimated values of lipophi-
licity below the guideline value (3.5). Then, the experimental
log P, log D7.4, and pKa values of 4-14 were determined
by potentiometric titrations (Table 1). Considering the pKa
values of target compounds, it can be deduced that the
percentage of protonated species at physiological pH is not
very high, and this accounts for the similarities between log P
and log D7.4 values. Experimental log P values were, how-
ever, different from the calculated values. The largest differ-
ence (1.05 log units) was shown in the case of compound 7
(ClogP=1.50 vs log P=2.55), whereas the smallest (0.26 log
units) was for 12 (ClogP = 2.81 vs log P = 2.37). While the
explanation of such differences is beyond the scope of this
study, these data confirm that the lipophilicity of a molecule
is the result of all intermolecular solute-solvent interactions in
both aqueous and organic solvents and not only the sum of
the contribution of isolated fragments. In fact, the difference
between log P values and ClogP can, and in our case does,
vary among structurally related compounds. For example,
compounds 5 and 11 have a difference between the calculated
andexperimental logP of0.37and 0.93, respectively; for 7 and
13, the difference was 1.05 and 0.43, respectively. In spite of
these discrepancies, all the experimental logP values were
below the guideline value with the exception of those for 9 and
11, which had logP values of 3.75 and 3.79, respectively.
Therefore, in those cases when lipophilicity must fall within a
narrow range and ClogP values are borderline, an experi-
mental determination is prudent.
Rational Design and Structure-Affinity Relationships. All
the target compounds were designed taking into account the
structural requirements for an adequate PET tracer as detailed
in the Introduction. Compounds 4-8 have a methoxy group

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7347
Scheme 1a
Table 2. Displacement of Specifically Bound Radioligand from Cloned
Dopamine D2L and σ1 Receptors by a Single High Concentration of
Compound (1 μM)a
% displacement ( SEM
compd
D2L
σ1
2
7.8 ( 4.2
7.6 ( 3.7
84 ( 3.0
0 ( 2.8
22 ( 7.2
7.2 ( 8.4
13 ( 4.2
27 ( 15
4
5
6
7
4.4 ( 1.3
5.8 ( 0.95
48 ( 10
29 ( 4.6
89 ( 5.2
0 ( 0.58
32 ( 8.4
43 ( 2.1
8.3 ( 6.2
15 ( 8.4
29 ( 3.2
21 ( 4.6
21 ( 2.9
4.6 ( 2.1
1.0 ( 3.7
33 ( 1.9
8
9
10
11
12
13
14
a Nonspecific binding is defined by the following: 5 μM (þ)-butacla-
mol displaced 100% of [3H]MSP from D2L receptors, and 5 μM BD1063
displaced 100% of [3H]-(þ)-pentazocine from σ1 receptors.
affinity than the 3-methoxy analogue 5. Also, the rank order
of binding affinity among the derivatives was practically
unchanged with the sole exception of 3-cyano-2-pyridinyl 13,
which had slightly less affinity than the 1,2-benzisoxazol-3-yl
11. All in all, the proposed structural modifications led to a
range of D4 ligands possessing affinities higher than the lead
compound 2. Among them, 11 and 13 demonstrated subna-
nomolar Ki values. Subsequently, all compounds were evalu-
ated at 1 μM for their ability to displace [3H]methylspiperone
from the rat cloned D2L dopamine receptor (Table 2). Strong
displacement was observed for compounds 5 and 11, indicat-
ing that they have significant affinity for the D2L receptor.
Moderate displacement (40-50%) was observed for 9, 10,
13, and 14, whereas 2, 4, 6-8, and 12, had practically no
effect on the specific binding of the radioligand to the
D2L receptor. Because some compounds touted as being
D4-selective actually have high affinity for the σ1 receptor,48
all compounds were evaluated at 1 μM for their ability to
displace specifically bound [3H]-(þ)-pentazocine from the
human cloned σ1 receptor (Table 2). Little or no displacement
(<40%) was observed for all the tested compounds, indicat-
ing that they have little or no affinity for the σ1 receptor.
Taken together, these data indicate that compounds 7 and 13
have high selectivity for the D4 receptor over D2L and σ1 recep-
tors. Therefore, both compounds were evaluated further for
potential interactions with selected other G-protein-coupled
receptors (GPCRs) and for their functional properties at the
D4 receptor. While 7 and 13 had a similar binding profile for
D3, CB1, and 5-HT2C receptors, 7 had significantly less radio-
ligand displacing activity at 5-HT1A and 5-HT2A receptors
than 13 (Table 3). Both 7 and 13 were able to stimulate D4
receptors measured as a decrease in forskolin-stimulated
levels of cAMP accumulation (Figure 1), indicating that they
have agonist properties.
a Reagents: (A) 2-chloroethanol or 2-bromoethanol, K2CO3; (B) phtha-
limide, Ph3P, DEAD; (C) (i) hydrazine hydrate, (ii) conc HCl; (D) chloro-
acetonitrile, K2CO3;(E) borane-methyl sufide complex; (F) 3-methoxy- or
4-fluorobenzoic acid, CDI.
that can be easily accessed in the [11C] radiolabeled form via
the corresponding phenol derivative. Compounds 9-14
possess the 4-fluorobenzamide group which can be obtained
in the [18F] radiolabeled form via the corresponding 4-nitro-
benzamide or 4-tributyltinphenyl analogue. The structural
modifications of 2 were also guided by the previous structure-
affinity relationship studies. Thus, in the first group of com-
pounds the N-[2-(4-arylpiperazin-1-yl)ethyl]-3-methoxyben-
zamide structure was left unchanged (compounds 4-8, Table 1).
The modification was limited to the aryl group linked to the
piperazine ring. In particular, replacement of -Cl in 2 with
-CH3 led to 4 which has only 2-fold lower affinity for the D4
receptor than 2. On the other hand, replacement of -Cl in
2 with -CN gave 6 which had considerably less affinity than
2 (Ki=63.95 nM). Replacement of the phenyl ring in 2 with a
2-pyridyl gave 8 which demonstrated only 2-fold less affinity
than 2. In a previous study we found that N-[4-[4-(1,2-
benzisoxazol-3-yl)piperazin-1-yl]butyl]-3-methoxybenzamide
displayed high affinity (Ki =7.58 nM) for the D4 receptor.46
Thus, we wanted to evaluate if a shorter alkyl chain (ethyl vs
butyl) increased affinity for the D4 receptor. Compound 5
displayed higher affinity (Ki=1.93 nM) than either the butyl
homologue cited above and 2. The replacement of 4-chloro-
phenyl group in 2 by the 3-cyano-2-pyridinyl one (7) was
undertaken because a number of D4 ligands reported in the
literature were characterized by such a group.47 This replace-
ment was particularly beneficial because 7 had improved
affinity for the D4 receptor (Ki = 1.52 nM). A second set of
derivatives were designed by replacing the 3-methoxybenza-
mide of 2, 4-8 with a 4-fluorobenzamide (compounds 9-14,
Table 1). The 4-fluorobenzamides 9-14 possessed higher D4
affinities than their 3-methoxy counterparts. The most notable
increase in affinity was observed in the case of 1,2-benzisoxazol-
3-yl derivative 11 which has approximately 6-fold higher
Considering that a PET tracer needs to have high affinity
for the target receptor and, in particular, it is preferable that the
Bmax clearly exceeds the Kd of the ligand (ideally Bmax/Kd>10),49
one could ask if compounds 7 and 13 meet this requirement.
D4 receptor binding sites have previously been determined
indirectly by the use of [3H]NGD-94-1,50 [3H]PNU-101958,51
and [3H]nemonapride.52 However, these studies have delivered
controversial data on the distribution and concentration of
D4 receptor in the human and rat brain. For example, using
[3H]nemonapride in combination with 1, raclopride, sulpiride,

7348 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Lacivita et al.
Table 3. Displacement of Specifically Bound Radioligand by 7 and 13
from Cloned Human Dopamine D3 Receptor, Cloned Serotonin Receptor
Subtypes, and the CB1 Cannabinoid Receptor by a Single High
Concentration of Compound (1 μM)a
Table 4. Mean ( SD (n = 3) Maximum Plasma and Brain Concentra-
tions after ip Dosing of 10 mg/kg of Compounds 7 (after 30 min) and
13 (after 15 min) in Micea
concentration
% displacement ( SEM
compd
plasma (μg/mL)
brain (μg/g)
receptor
7
13
7
13
0.37 ( 0.21 (0.22 ( 0.12)
0.13 ( 0.10 (<0.1)
0.14 ( 0.02 (<0.1)
0.47 ( 0.20 (<0.1)
D3
5.1 ( 1.1
9.3 ( 5.9
24 ( 4.7
3.2 ( 3.8
6.5 ( 16
7.2 ( 3.2
29 ( 4.7
53 ( 12
0 ( 4.1
5-HT1A
5-HT2A
5-HT2C
CB1
a Shown in parentheses is the N-dealkylated metabolite 1-(6-cyano-2-
pyridyl)piperazine.
9.9 ( 0.1
Disposition Studies in Mice with Compounds 7 and 13. Mice
were given an intraperitoneal dose of the 4-fluorobenzamide
13 and the 3-methoxybenzamide 7 and were sacrificed at various
times thereafter to obtain basic information on the concen-
trations of unchanged compound achieved in brain and their
relationship with plasma concentrations. The presence of
1-(6-cyano-2-pyridyl)piperazine was also monitored in par-
allel because many N-substituted 1-arylpiperazines are repor-
ted to undergo metabolic N-dealkylation.54 As shown in
Table 4, compound 13 rapidly reached the systemic circula-
tion with maximal plasma concentrations (Cmax) 15 min after
dosing (i.e., the first sampling time). However, these plasma
concentrations were low and variable (0.13 ( 0.10 μg/mL)
and rapidly fell below the limit of quantitation after 30 min
(i.e., less than 0.1 μg/mL, using 0.2 mL of mouse plasma).
Compound 7 peaked slightly later, yielding a higher mean
Cmax (0.37 ( 0.21 μg/mL at 30 min), and these concentrations
were consistently detected only up to 60 min after dosing. Thus,
in both cases the data did not permit adequate determination
of a terminal elimination phase and the calculation of con-
ventional pharmacokinetic parameters. This behavior may
be partly due to a large presystemic biotransformation after
intraperitoneal dosing because 1-arylpiperazine derivatives
are generally extensively biotransformed before reaching the
systemic circulation, and their clearance is almost entirely
due to hepatic metabolism.54 Concentrations of 1-(6-cyano-
2-pyridyl)piperazine, however, were low for 7 (0.10-0.2 μg/mL)
or not detectable for 13 (<0.1 μg/mL) within 30-60 min of
intraperitoneal dosing (10 mg/kg). This suggests that N-deal-
kylation of the aliphatic side chain may have contributed to
but did not account for the first-pass effect and clearance of 7
and 13 in mice. Brain uptake was rapid for both derivatives:
quantifiable levels were consistently evident up to 30 min for
7 and 60 min in the case of 13. Mean brain Cmax averaged
0.47 ( 0.20 μg/g for the 4-fluorobenzamide 13 (at 15 min post
dose) and 0.14 ( 0.02 μg/g for the 3-methoxybenzamide 7
(at 30 min post dose), giving rise, at these times, to a mean
brain-to-plasma concentration ratio higher for the more
lipophilic fluorobenzamide derivative. While an accurate
determination of whole brain exposure and brain-to-plasma
distribution ratios was not possible, rough estimates based
on the limits of quantification further suggested that the
fluorobenzamide is concentrated in brain tissue of mice more
than its methoxybenzamide analogue. In both cases the brain
concentrations of the dealkylated metabolite 1-(6-cyano-2-
pyridyl)piperazine were below the limit of detection within
180 min of dosing (about 0.1 μg/g, using approximately 200 mg
of brain tissue).
a Nonspecific binding is defined by the following: 5 μM 7-OH-
DPAT displaced 100% of [3H]MSP from D3 receptors;
5
μM
NAN-190 displaced 100% of [3H]-MPPF from 5-HT1A receptors;
5 μM mianserin displaced 100% of [3H]MSP from 5-HT2A receptors;
5 μM mianserin displaced 100% of [3H]mesulergine from 5-HT2C
receptors; 10 μM O-2050 displaced 100% of [3H]CP55,940 from rat
brain CB1 receptors.
Figure 1. Compounds 7 and 13 are agonists of the cloned D4 do-
pamine receptor as determined by Gi/o-mediated changes in intra-
cellular cAMP. The D4-selective antagonist spiperone, the D4-
selective agonist PD168,077, the D2-like agonist (-)-quinpirole,
and dopamine are shown for comparison.
and a σ receptor antagonist, a low density of D4 receptors
was observed (Bmax=8.9 fmol (mg of protein)-1) only in the
3
hippocampus.52 In contrast, Primus and co-workers used
[3H]NGD-94-1 and reported Bmax values ranging from 8.9 to
28.9 fmol (mg of protein)-1 in hippocampal, cortical, and
3
limbic regions.50 Importantly, these values originate from
homogenized tissue and do not necessarily reflect the tissue
heterogeneity to be encountered in the intact brain in vivo.53
Therefore, the concentration of D4 receptor sites determined
by indirect methods should be viewed with some caution,
making the lack of precise Bmax values for the D4 receptor in
the brain difficult to estimate the affinity value to be targeted
when developing a D4 PET tracer. Since compounds 7 and 13
had the best combination of lipophilicity, high affinity (Ki in
the low nanomolar range), and selectivity for the D4 receptor
of the ligands listed in Table 1, they were selected for further
testing in order to assess their potential of use in vivo as PET
tracers.
These data indicated that both 7 and 13 can reach the
brain and have fast uptake kinetics ideal for a PET tracer.
Because of the relative ease of radiolabeling, compound 7
was selected as a PET D4 tracer candidate.
Radiosynthesis of [11C]7. Derivative 19, the desmethyl
precursor for 11C-radiolabeling, was prepared as shown in

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7349
Figure 2. Time-activity curves of [11C]7 in seven CNS regions for
90 min in rhesus monkey.
Figure 3. PET imaging in rhesus monkey with [11C]7 reveals time
dependent and saturable binding that is most intense in the posterior
regions of the eye. Importantly, there is an intense signal in the
region of the retina with little or no background signal in other parts
of the CNS. Note different scales used to capture data for retina and
brain. Mean images of [11C]7 from 30 to 90 min after ligand injection
are shown.
Scheme 2a
has been reported that both barriers display very similar
properties.55 Moreover, the lack of a persistent signal in
other CNS structures indicates that [11C]7 undergoes a rapid
wash-out from the tissues in which it is not significantly
bound to the D4 receptor. It also demonstrates that [11C]7
has very low nonspecific binding or off-target binding char-
acteristics. This behavior suggests that labeling of lower
density brain regions would require a D4 ligand having an in
vitro affinity higher than that of 7 (Ki = 1.52 nM).
a Reagents: (A) 3-hydroxybenzoic acid, CDI; (B) [11C]CH3I, NaOH.
Scheme 2. 3-Hydroxybenzoic acid was condensed with 4-(3-
cyanopyridin-2-yl)-1-piperazinoethanamine (18e) to give benz-
amide 19 in 40% yield. For the radiolabeling 19 was reacted
with [11C]CH3I in DMF and 0.5 N NaOH to afford [11C]7 in
an incorporation yield of 40.5 ( 10.8% (n = 4, determined
by HPLC). Purification by semipreparative reverse-phase
HPLC afforded [11C]7 (>99%). No significant peak of unreac-
ted 19 was seen on the HPLC chromatogram of the final
radioactive product. The retention time of [11C]7 on the ana-
lytical reverse-phase HPLC system was 6.2 min. The total
synthesis time was about 28 min from the end of bombard-
ment. The specific radioactivity at time of injection of [11C]7
was 2885 GBq/μmol, corresponding to a total injected dose
of 150.3 MBq (52 pmol, 18 ng) in the monkey experiments.
PET Studies in Rhesus Monkey. After intravenous injec-
tion of [11C]7 in a male rhesus monkey, the uptake of [11C]7 in
all brain regions peaked at 45 s and then the radioactivity
rapidly declined, suggestive of no specific binding in these
regions (Figure 2). About 3.1% of the injected radioactivity
reached the brain at peak time. In contrast, radioactivity was
markedly higher in the retina compared to the brain regions
examined (Figure 3). The ratio of retinal radioactivity to the
cerebral radioactivity was more than 6-fold at the end of the
scanning period.
Conclusions
We report here on a systematic strategy for discovering
ligands suitable as PET radiotracers for imaging of dopamine
D4 the receptor in the CNS. Starting from our high-affinity
ligand 2 and guided in part by previous structure-activity
relationship studies from our laboratories and other labora-
tories, wedeveloped an assay cascade leading tothedesignofa
set of compounds possessing chemical features amenable to
carbon-11 or fluorine-18 labeling and calculated logP values
within the range that is considered optimal for a radiotracer.
The log P values of the target compounds were determined
experimentally confirming that almost all the compounds
possessed the desired lipophilicity. Affinity screening of com-
pounds at the dopamine D4 receptor as well as at selected other
off-target receptors (dopamine D2, dopamine D3, serotonin
5-HT1A, 5-HT2A, 5-HT2C, σ1, and cannabinoid CB1) guided
our selection of two potent and selective ligands, namely,
N-[2-[4-(3-cyanopyridin-2-yl)piperazin-1-yl]ethyl]-3-methoxy-
benzamide (7) and N-[2-[4-(3-cyanopyridin-2-yl)piperazin-
1-yl]ethyl]-4-fluorobenzamide (13). Compounds 7and 13 showed
Ki values of 1.52 and 0.93 nM, respectively, and>100-fold
selectivity over the off-target receptors. When tested in dis-
position studies in mice to evaluate their BBB penetrance and
first-pass metabolism characteristics, both 7 and 13 rapidly
entered the brain and, importantly in this context, were also
rapidly cleared from the brain. Carbon-11 radiolabeling of
the phenol precursor 19 resulted in the 3-methoxybenzamide
While [11C]7 did not accumulate in any region of the brain
that has been reported to express dopamine D4 receptors, it
accumulated to saturable levels in the posterior eye in the
region of retina. This is consistent with the reported high
density of dopamine D4 receptors (Bmax = 134 fmol (mg of
3
protein)-1) determined by in situ autoradiography with the
D4-selective ligand [125I]L-750,667.27 The presence of the
radioligand in the region of the retina suggests that the
compound was able to cross the blood-retina barrier, and
therefore, it also has the potential to cross the BBB because it

7350 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Lacivita et al.
derivative [11C]7. [11C]7 was injected into rhesus monkey, and
brain penetrance as well as its fast washout was confirmed in
this non-human primate. Despite there being no labeling of
dopamine D4 receptor in the brain regions examined, [11C]7
did accumulate in a time-dependent and saturable fashion in
the posterior eye in the region of the retina, a CNS tissue rich
in dopamine D4 receptors. While more questions may need to
be answered and further improvements could be made, we
believe that 7 represents a significant step forward in the
development of a CNS PET tracer selective for the dopamine
D4 receptor, having allowed for the first time CNS imaging of
D4 receptor in a structure with the highest known density of
this receptor subtype. Importantly, the strategy we worked
out appears to serve as a suitable paradigm for developing
additional D4-selective PET tracers which would have higher
affinitypresumablymaking them suitablefor imaging in brain
regions having a very low density of the D4 receptor. Finally,
because of its high affinity and specificity, compound 7 in the
tritiated form may have utility as a radioligand for studying
the distribution and density of the dopamine D4 receptor
protein in ex vivo or in vitro studies.
ESIþ-MS m/z 248.2 (MHþ). ESIþ-MS/MS m/z 161.2 (100),
114.4 (22).
4-[4-(2-Hydroxyethyl)-1-piperazinyl]benzonitrile (15d). A stirred
mixture of 4-piperazinobenzonitrile (0.37 g, 2.0 mmol), 2-chloro-
ethanol (0.17 mL, 2.4 mmol), and K2CO3 (0.35 g, 2.5 mmol) in
acetonitrile was refluxed overnight. After cooling, the mixture was
evaporated to dryness and H2O (20 mL) was added to the residue.
The aqueous phase was extracted with CH2Cl2 (2 ꢀ 20 mL). The
collected organic layers were dried over Na2SO4 and evaporated
under reduced pressure. The crude residue was chromatographed
(CHCl3/CH3OH, 19:1 as eluent) to yield pure 15d as a white solid
1
(0.30 g, 64% yield). H NMR (CDCl3): δ 1.71 (br s, 1H, D2O
exchanged), 2.61 (t, 2H, J=5.5 Hz), 2.65 (app t, 4H), 3.34 (app t,
4H), 3.67 (t, 2H, J = 5.5 Hz), 6.83-6.88 (m, 2H), 7.47-7.52
(m, 2H). GC-MS m/z 232 (Mþ þ 1, 5), 231 (Mþ, 17), 200 (100),
157 (24), 129 (24).
3-Cyano-2-[4-(2-hydroxyethyl)-1-piperazinyl]pyridine (15e). Title
compound was prepared from 1-(3-cyano-2-pyridyl)piperazine and
2-chloroethanol following the same procedure described above for
15d. Pure 15e was obtained by column chromatography (CHCl3/
1
CH3OH, 19:1, as eluent) as a yellow oil in 83% yield. H NMR
(CDCl3): δ 2.51 (br s, 1H, D2O exchanged), 2.75 (t, 2H, J=5.3 Hz),
2.83 (app t, 4H), 3.76 (t, 2H, J=5.3 Hz), 3.83 (app t, 4H), 6.80 (q,
1H, J=7.4, 7.7 Hz), 7.79 (dd, 1H, J=1.9, 7.7 Hz), 8.34-8.37 (m,
1H). GC-MS m/z233 (Mþ þ 1, 1), 232 (Mþ, 5), 201 (100), 172 (47),
146 (46), 100 (40).
Experimental Section
Chemistry. The purity of the tested compounds 2, 4-14 has
been assessed by RP-HPLC and combustion analysis. All
compounds showed g95% purity. Column chromatography
was performed with 1:30 Merck silica gel 60A (63-200 μm) as
the stationary phase. Melting points were determined in open
capillaries on a Gallenkamp electrothermal apparatus. Elemen-
tal analyses (C, H, N) were performed on Eurovector Euro EA
3000 analyzer; the analytical results were within (0.4% of the
theoretical values for the formula given. 1H NMR spectra were
recorded at 300 MHz on a Varian Mercury-VX spectrometer.
All spectra were recorded on free bases. All chemical shift values
are reported in ppm (δ). Recording of mass spectra was done on
an HP6890-5973 MSD gas chromatograph/mass spectrometer;
only significant m/z peaks, with their percentage of relative inten-
sity in parentheses, are reported. ESIþ-MS/MS analysis were
performed with an Agilent 1100 series LC-MSD trap system VL
workstation. All spectra were in accordance with the assigned
structures. RP-HPLC analysis was performed on a Perkin-Elmer
series 200 LC instrument using a Phenomenex Gemini RP-18
column, (250 mm ꢀ 4.6 mm, 5 μm particle size) and equipped
with a Perkin-Elmer 785A UV/vis detector setting of λ=254 nm.
Compounds 2 and 4-14 were eluted with CH3OH/H2O/Et3N,
4:1:0.01, v/v at a flow rate of 1 mL/min. When necessary, a standard
procedure was used to transform final compounds into their
hydrochloride salts. The following compounds were synthesized
according to published procedures: 1-(4-chloro-2-pyridinyl)-
piperazine,45 1-(3-cyano-2-pyridinyl)piperazine,56 4-(4-chloro-
phenyl)-1-piperazinoethanamine (18a),44 4-(4-methylphenyl)-
1-piperazinoethanamine (18b).44 The NMR and MS spectra
of compounds 2, 4, and 8 have been reported earlier.42,46
3-[4-(2-Hydroxyethyl)-1-piperazinyl]-1,2-benzisoxazole (15c).
A solution of 3-(1-piperazinyl)-1,2-benzisoxazole (0.6 g, 2.96
mmol) in dioxane (15 mL) was treated with anhydrous K2CO3
(1.43 g, 10.3 mmol) and KI (0.04 g, 0.2 mmol). After addition of
2-bromoethanol (1.2 mL, 17.3 mmol) the mixture was refluxed
overnight. The solvent was distilled off, and the residue was
partitioned between H2O (20 mL) and CHCl3 (20 mL). The
organic phase was separated, dried over anhydrous Na2SO4,
and concentrated under reduced pressure. The crude residue was
chromatographed (CHCl3/MeOH 19:1, as eluent) to afford the
pure alcohol as a white solid (0.56 g, 77% yield). 1H NMR
(CDCl3): δ 2.10 (br s, 1H, D2O exchanged), 2.64 (t, 2H, J=5.5
Hz), 2.73 (app t, 4H), 3.60 (app t, 4H), 3.68 (t, 2H, J=5.5 Hz),
7.20-7.24 (m, 1H), 7.44-7.49 (m, 2H), 7.69 (d, 1H, J=8.0 Hz).
General Procedure for Preparation of Compounds 16c-e.
Diethyl azodicarboxylate (2.25 mmol) was added to a stirred
solution containing the appropriate alcohol 15c-e (1.5 mmol),
triphenylphosphine (1.5 mmol), and phthalimide (2.25 mmol) in
anhydrous THF (20 mL). The resulting mixture was stirred at
room temperature under anhydrous condition until the alcohol
disappeared (TLC). Then the mixture was partitioned between
AcOEt (20 mL) and H2O (20 mL). The organic layer was sepa-
rated, dried (Na2SO4), and concentrated in vacuo. The crude
residue was chromatographed as detailed below to afford pure
compounds as pale yellow solids in quantitative yield.
2-[2-[4-(1,2-Benzisoxazol-3-yl)-1-piperazinyl]ethyl]-1H-isoindole-
1,3(2H)-dione (16c). Eluted with CHCl3/AcOEt, 1:1. 1H NMR
(CDCl3): δ 2.69-2.74 (m, 6H), 3.50 (app t, 4H), 3.86 (t, 2H, J=
6.4 Hz), 7.20 (dtd, 1H, J=1.6, 6.3, 8.1 Hz), 7.43-7.49 (m, 2H),
7.66-7.74 (m, 3H), 7.83-7.85 (m, 2H). ESIþ-MS m/z 377.1
(MHþ). ESIþ-MS/MS m/z 174.2 (100), 147.3 (24).
2-[2-[4-(4-Cyanophenyl)-1-piperazinyl]ethyl]-1H-isoindole-1,3-
(2H)-dione (16d). Eluted with CHCl3/AcOEt, 9:1. 1H NMR
(CDCl3): δ 2.64 (app t, 4H), 2.69 (t, 2H, J=6.3 Hz), 3.24 (app t,
4H), 3.85 (t, 2H, J=6.3Hz), 6.80-6.84(m,2H), 7.45-7.48 (m, 2H),
7.71-7.73 (m, 2H), 7.83-7.85 (m, 2H). GC-MS m/z 361 (Mþ þ
1, 1), 360 (Mþ, 1), 200 (100), 157 (17).
2-[2-[4-(3-Cyanopyridin-2-yl)-1-piperazinyl]ethyl]-1H-isoindole-
1,3(2H)-dione (16e). Eluted with CHCl3/AcOEt, 1:1. 1H NMR
(CDCl3): δ 2.66 (app t, 4H), 2.69 (t, 2H, J=6.3 Hz), 3.66 (app t,
4H), 3.85 (t, 2H, J=6.3 Hz), 6.71 (q, 1H, J=7.7 Hz), 7.70-7.75
(m, 3H), 7.82-7.86 (m, 2H), 8.31 (dd, 2H, J=1.9, 5.0 Hz). GC-
MS m/z 362 (Mþ þ 1, 1), 361 (Mþ, 4), 242 (19), 201 (100), 172 (19).
4-(5-Chloropyridin-2-yl)piperazinoacetonitrile (17). A stirred
mixture of 1-(5-chloropyridin-2-yl)piperazine (2.25 g, 11.4 mmol),
chloroacetonitrile (0.60 mL, 9.5 mmol), and an excess of K2CO3
in acetonitrile (50 mL) was refluxed overnight. After cooling, the
mixture was evaporated to dryness. Then H2O was added to the
residue. The aqueous phase was extracted with CH2Cl2 (2ꢀ30 mL),
and the collected organic layers were dried over Na2SO4 and
evaporated under reduced pressure. The crude residue was chro-
matographed (CHCl3/AcOEt, 9:1, as eluent) to give pure 17 as a
1
pale yellow semisolid (2.0 g, 90% yield). H NMR (CDCl3):
δ 2.69 (app t, 4H), 3.55-3.59(m, 6H), 6.59(dd, 1H,J=0.5, 9.1 Hz),
7.43 (dd, 1H, J=2.5, 9.1 Hz), 8.11 (dd, 1H, J=0.5, 2.5 Hz). GC-
MS m/z238 (Mþ þ 2, 9), 236 (Mþ, 27), 143 (33), 141 (100), 113 (21).
General Procedure for Preparation of Amines 18c-e. Phtha-
limide derivative 16c-e (1.5 mmol) was mixed with an excess of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7351
hydrazine hydrate in EtOH (20 mL) and refluxed for 8 h. Then
the mixture was cooled on ice bath and acidified with concen-
trated HCl. The mixture was refluxed for 1 h. Then the mixture
was cooled and filtered. The filtrate was concentrated under
reduced pressure, and the residue was partitioned between 10%
aqueous NaOH and CHCl3. The separated organic layer was
dried over Na2SO4 and concentrated in vacuo to give the pure
amine in 70-80% yield.
N-[2-[4-(3-Cyanopyridin-2-yl)piperazin-1-yl]ethyl]-3-methoxy-
benzamide (7). Eluted with CHCl3/AcOEt, 1:1. 1H NMR
(CDCl3): δ 2.67-2.71 (m, 6H), 3.60 (q, 2H, J = 5.3 Hz), 3.76
(app t, 4H), 3.85 (s, 3H), 6.77 (dd, 1H, J=4.7, 7.7 Hz), 6.85 (br s,
1H, D2O exchanged), 7.02-7.05 (m, 1H), 7.26-7.38 (m, 3H),
7.77 (dd, 1H, J = 1.1, 8.7 Hz), 8.35 (dd, 1H, J = 1.1, 3.7 Hz).
GC-MS m/z 366 (Mþ þ 1, 1), 365 (Mþ, 2), 201 (100), 146 (18).
The hydrochloride salt melted at 180 ꢀC dec (from MeOH/
4-(1,2-Benzisoxazol-3-yl)-1-piperazinoethanamine (18c). 1H
NMR (CDCl3): δ 1.72 (br s, 2H, D2O exchanged), 2.50 (t, 2H,
J=6.0 Hz), 2.65 (app t, 4H), 2.83 (t, 2H, J=6.0 Hz), 3.58 (app t,
4H), 7.20 (dtd, 1H, J=1.6, 6.2, 8.0 Hz), 7.42-7.50 (m, 2H), 7.68
(d, 1H, J=8.3 Hz). ESIþ-MS m/z 247.2 (MHþ). ESIþ-MS/MS
m/z 230.2 (100).
Et2O). Anal. (C20H23N5O2 2HCl) C, H, N.
3
N-[2-[4-(4-Chlorophenyl)piperazin-1-yl]ethyl]-4-fluorobenz-
amide (9). Eluted with CHCl3/MeOH, 19:1. 1H NMR (CDCl3):
δ 2.72-2.76 (m, 6H), 3.23 (app t, 4H), 3.62 (q, 2H, J=5.3 Hz),
6.81-6.87 (m, 2H), 6.99 (br s, 1H, D2O exchanged), 7.07-7.15
(m, 2H), 7.18-7.24 (m, 2H), 7.79-7.85 (m, 2H). GC-MS m/z
363 (Mþ þ 2, 3), 361 (Mþ, 8),211(32),209(100),166(23).Mp180-
181 ꢀC (from CHCl3/n-hexane). Anal. (C19H21FClN3O) C, H, N.
N-[2-[4-(4-Methylphenyl)piperazin-1-yl]ethyl]-4-fluorobenz-
amide (10). Eluted with CHCl3/MeOH, 19:1. 1H NMR (CDCl3):
δ 2.27 (s, 3H), 2.71-2.76 (m, 6H), 3.22 (app t, 4H), 3.62 (q, 2H,
J=5.5 Hz), 6.83-6.87 (m, 2H), 7.00 (br s, 1H, D2O exchanged),
7.01-7.14 (m, 4H), 7.79-7.85 (m, 2H). GC-MS m/z 342 (Mþ þ
1, 3), 341 (Mþ, 13), 189 (100), 123 (22). Mp 156-158 ꢀC (from
CHCl3/n-hexane). Anal. (C20H24FN3O) C, H, N.
4-(4-Cyanophenyl)-1-piperazinoethanamine (18d). 1H NMR
(CDCl3): δ 1.83 (br s, 2H, D2O exchanged), 2.47 (t, 2H, J =
6.0 Hz), 2.58 (app t, 4H), 2.82 (t, 2H, J = 6.3 Hz), 3.32 (app t,
4H), 6.82-6.88 (m, 2H), 7.45-7.50 (m, 2H). GC-MS m/z 231
(Mþ þ 1, 1), 230 (Mþ, 3), 200 (100), 157 (33), 70 (34).
4-(3-Cyanopyridin-2-yl)-1-piperazinoethanamine (18e). 1H NMR
(CDCl3):δ1.75 (br s, 2H, D2O exchanged), 2.48 (t, 2H, J=6.1Hz),
2.59 (app t, 4H), 2.82 (t, 2H, J = 6.3 Hz), 3.73 (app t, 4H), 6.73
(q, 1H, J=7.4, 7.7 Hz), 7.75 (dd, 1H, J=1.9, 7.6 Hz), 8.32 (dd, 1H,
J=1.9, 4.7 Hz). GC-MS m/z 232 (Mþ þ 1, 1), 231 (Mþ, 1), 201
(100), 172 (37), 146 (36).
N-[2-[4-(1,2-Benzisoxazol-3-yl)piperazin-1-yl]ethyl]-4-fluoro-
benzamide (11). Eluted with CHCl3/MeOH, 98:2. 1H NMR
(CDCl3): δ 2.74-2.82 (m, 6H), 3.61-3.68 (m, 6H), 6.93 (br s, 1H,
D2O exchanged), 7.03-7.15 (m, 2H), 7.19-7.24 (m, 1H), 7.44-
7.53 (m, 2H), 7.67 (d, 1H, J=8.0 Hz) 7.80-7.87 (m, 2H). ESIþ-MS
m/z 369.1 (MHþ). ESIþ-MS/MS m/z 166.2 (100). Mp 138-
140 ꢀC (from CHCl3/n-hexane). Anal. (C20H21FN4O2) C, H, N.
N-[2-[4-(4-Cyanophenyl)piperazin-1-yl]ethyl]-4-fluorobenz-
amide (12). Eluted with CHCl3/MeOH, 19:1. 1H NMR (CDCl3):
δ 2.70-2.72 (m, 6H), 3.39 (app t, 4H), 3.59-3.65 (m, 2H),
6.84-6.89 (m, 2H), 6.93 (br s, 1H, D2O exchanged), 7.07-7.15
(m, 2H), 7.48-7.52 (m, 2H), 7.78-7.83 (m, 2H). GC-MS m/z
353 (Mþ þ 1, 1), 351 (Mþ, 2), 200 (100), 157 (20). Mp 193-194 ꢀC
(from CHCl3/n-hexane). Anal. (C20H21FN4O) C, H, N.
N-[2-[4-(3-Cyanopyridin-2-yl)piperazin-1-yl]ethyl]-4-fluorobenz-
amide (13). Eluted with CHCl3/MeOH, 19:1. 1H NMR (CDCl3):
δ 2.78 (br s, 6H), 3.62-3.67 (m, 2H), 3.82 (app t, 4H), 6.80 (q,
1H, J=7.4, 7.7 Hz), 6.93 (br s, 1H, D2O exchanged), 7.08-7.16
(m, 2H), 7.79 (dd, 1H, J=1.9, 7.4 Hz), 7.82-7.87 (m, 2H), 8.36
(dd, 1H, J = 1.9, 5.0 Hz). GC-MS m/z 354 (Mþ þ 1, 1), 353
(Mþ, 1), 201 (100), 123 (22). Mp 155-156 ꢀC (from CHCl3/
n-hexane). Anal. (C19H20FN5O) C, H, N.
N-[2-[4-(5-Chloropyridin-2-yl)piperazin-1-yl]ethyl]-4-fluorobenz-
amide (14). Eluted with CHCl3/MeOH, 19:1. 1H NMR (CDCl3):
δ 2.65-2.72 (m, 6H), 3.56-3.64 (m, 6H), 6.59 (d, 1H, J=9.1 Hz),
6.93 (br s, 1H, D2O exchanged), 7.07-7.15 (m, 2H), 7.43 (dd,
1H, J=2.8, 9.1 Hz), 7.78-7.84 (m, 2H), 8.11 (d, 1H, J=2.5 Hz).
GC-MS m/z 364 (Mþ þ 2, 1), 362 (Mþ, 3), 210 (100), 181 (34),
155 (40), 123 (52). Mp 173-175 ꢀC (from CHCl3/n-hexane).
Anal. (C18H20FClN4O) C, H, N.
N-[2-[4-(3-Cyanopyridin-2-yl)piperazin-1-yl]ethyl]-3-hydroxy-
benzamide (19). Eluted with AcOEt. 1H NMR (CDCl3): δ 2.67-
2.71 (m, 6H), 3.56-3.62 (m, 2H), 3.76 (app t, 4H), 6.75-6.79 (m,
1H), 6.96 (br s, 1H, D2O exchanged), 6.97-6.99 (m, 1H), 7.18-
7.24 (m, 2H), 7.48 (m, 1H), 7.78 (dd, 1H, J=1.9, 7.4 Hz), 8.35
(dd, 1H, J=1.9, 4.7 Hz). ESIþ-MS m/z 352.1 (MHþ). ESIþ-MS/
MS m/z 164.2 (100), 121.2 (15).
4-(5-Chloropyridin-2-yl)piperazineethanamine (18f). Borane-
methyl sulfide complex as 10.0 M BH3 in excess methyl sulfide
(1.6 mL, 16 mmol) was dropped into an ice cooled solution of
nitrile 17 (5.1 mmol) in anhydrous THF (10 mL), under stirring.
After being refluxed for 4 h, the reaction mixture was cooled at
-10 ꢀC and MeOH was added dropwise very carefully until gas
evolution ceased. The mixture was treated with 3 N HCl (20 mL)
and was refluxed for 1 h. After cooling, the mixture was alkalized
with 3 N NaOH and extracted with CH2Cl2 (2 ꢀ 30 mL). The
collected organic layers were dried over Na2SO4 and the solvent
was evaporated under reduced pressure to give the pure amine as
a white semisolid (64% yield). 1H NMR (CDCl3): δ 1.73 (br s,
2H, D2O exchanged), 2.46 (t, 2H, J=6.1 Hz), 2.54 (app t, 4H),
2.82 (app t, 2H), 3.50 (app t, 4H), 6.57 (d, 1H, J=9.1 Hz), 7.40
(dd, 1H, J=2.5, 9.1 Hz), 8.09 (d, 1H, J=2.5 Hz). GC-MS m/z
242 (Mþ þ 2, 1), 240 (Mþ, 4), 212 (33), 210 (100), 181 (57), 155
(62), 112 (33).
General Procedure for Preparation of Compounds 5-7, 9-14,
19. A mixture of the appropriate benzoic acid (0.48 mmol) and
1,10-carbonyldiimidazole (0.50 mmol) in 10 mL of anhydrous
THF was stirred for 8 h. A solution of amine 18a-f (0.48 mmol)
in anhydrous THF (10 mL) was added, and then the mixture was
stirred until the benzoic acid disappeared (TLC). The reaction
mixture was partitioned between AcOEt (20 mL) and H2O (20 mL).
The separated organic layer was washed with a saturated aqueous
solution of Na2CO3 (20 mL), dried (Na2SO4), and concentrated
in vacuo. The crude residue was chromatographed as detailed
below to afford the pure arylcarboxamide in 40-50% yield.
N-[2-[4-(1,2-Benzisoxazol-3-yl)piperazin-1-yl]ethyl]-3-methoxy-
benzamide (5). Eluted with CHCl3/MeOH, 98:2. 1H NMR
(CDCl3): δ 2.73 (t, 2H, J = 5.5 Hz), 2.77 (app t, 4H), 3.60-
3.65 (m, 6H), 3.84 (s, 3H), 6.95 (br s, 1H, D2O exchanged), 7.03
(dt, 1H, J=2.5, 6.9 Hz), 7.20-7.22 (m, 2H), 7.23-7.26 (m, 1H),
7.29-7.34 (m, 1H), 7.37-7.52 (m, 2H), 7.66-7.69 (m, 1H).
ESIþ-MS m/z 381.0 (MHþ). ESIþ-MS/MS m/z 178.2 (100). The
hydrochloride salt melted at 202-204 ꢀC (from MeOH/Et2O).
Anal. (C21H24N4O3 HCl H2O) C, H, N.
Lipophilicity Data. Lipophilicity data of compounds 2 and
4-13 were obtained by the pH metric technique using a GlpKa
apparatus (Sirius Analytical Instruments Ltd., Forrest Row,
East Sussex, United Kingdom) as described elsewhere.57 The
low aqueous solubility of the investigated compounds required
pKa measurements to be performed in the presence of methanol
as cosolvent. Three separate 20 mL semiaqueous solutions of
approximately 5 ꢀ 10-5 M, in 20-50% w/w of MeOH, were
initially acidified with 0.5 M HCl to pH 3.5. The solutions were
then titrated with 0.5 M KOH to pH 11. The initial estimates of
3
3
N-[2-[4-(4-Cyanophenyl)piperazin-1-yl]ethyl]-3-methoxybenz-
amide (6). Eluted with CHCl3/MeOH, 19:1. 1H NMR (CDCl3):
δ 2.66-2.70 (m, 6H), 3.36 (app t, 4H), 3.60 (q, 2H, J=5.5 Hz),
3.84 (s, 3H), 6.79 (br s, 1H, D2O exchanged), 6.84-6.89 (m, 2H),
7.03 (dq, 1H, J=1.1, 1.4, 8.0 Hz), 7.26-7.38 (m, 3H), 7.47-7.52
(m, 2H). GC-MS m/z 364 (Mþ, 2), 213 (16), 200 (100), 157 (21).
Mp 174-175 ꢀC (from CHCl3/n-hexane). Anal. (C21H24N4O2)
C, H, N.

7352 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Lacivita et al.
Table 5. Radioligand and Drugs Used to Define Nonspecific Binding for Each of the Receptor Subtypes Testeda
drug for defining
nonspecific binding
receptor
radioligand
binding buffer
50 mM Tris, pH 7.4 at 25 ꢀC
binding conditions
90 min at 25 ꢀC
90 min at 25 ꢀC
90 min at 25 ꢀC
90 min at 25 ꢀC
90 min at 25 ꢀC
90 min at 25 ꢀC
5-HT1A [3H]MPPF
5-HT2A [3H]MSP
5-HT2C [3H]mesulergine
NAN-190
mianserin
mianserin
50 mM Tris, pH 7.4 at 25 ꢀC
50 mM Tris, pH 7.4 at 25 ꢀC
50 mM Tris, pH 7.4 at 25 ꢀC
50 mM Tris, pH 7.4 at 25 ꢀC
50 mM Tris, pH 7.4 at 25 ꢀC
50 mM Tris, pH 8.0 at 37 ꢀC
D2
D3
D4
σ1
[3H]MSP
(þ)-butaclamol
7-OH-DPAT
(þ)-butaclamol
BD1063
[3H]MSP
[3H]MSP
[3H]-(þ)-pentazocine
180 min at 37 ꢀC with shaking
CB1
[3H]CP55,940
O-2050
50 mM Tris, pH 7.4 at 30 ꢀC, 2.5 mM EDTA, 5 mM MgCl2, 180 min at 30 ꢀC with shaking
5 mg/mL fatty acid free BSA
a [3H]MPPF is 4-(200-methoxy)phenyl-1-[200-(N-2-pyridinyl)-p-fluorobenzamido]ethylpiperazine (Perkin Elmer, St. Louis, MO, NET-1109, 80 Ci/
mmol). [3H]MSP is [3H]methylspiperone (Perkin Elmer, St. Louis, MO, NET-856, 84 Ci/mmol). [3H]mesulergine (GE Healthcare Life Sciences,
Piscataway, NJ, TRK845, 80 Ci/mmol). [3H]-(þ)-pentazocine (Perkin Elmer, St. Louis, MO, NET1056, 29 Ci/mmol). [3H]CP55,940 (Perkin Elmer,
St. Louis, MO, NET1051, 180 Ci/mmol). Note that all radioligands were tested at approximately 0.5 pM, except for [3H]-(þ)-pentazocine and
[3H]CP55,940, which were tested at 1 and 2 nM, respectively. A concentration of 5 μM was used to define nonspecific binding for all receptors.
the psKa values, which are the apparent ionization constants in
the mixed solvent, were obtained by Bjerrum plots. These values
were then refined by a weighted nonlinear least-squares proce-
dure (Refinement Pro 1.0 software) to create a multiset, where
the refined values were extrapolated to zero cosolvent concen-
tration using the Yasuda-Shedlovsky equation.58 To obtained
log P data, at least three separate titrations were performed on
each compound, of approximately 5 ꢀ 10-5 M, using various
n-octanol/water ratios (from 0.005 to 1). The biphasic solutions
were initially acidified to pH 3.5 with 0.5 M HCl and then
titrated with 0.5 M KOH to pH 11. The obtained data were
refined as described above. The log P values were obtained by
the multiset approach, as described elsewhere.57 All titrations
were carried out at 25 ( 0.1 ꢀC under an inert nitrogen gas atmo-
sphere to exclude CO2.
5-HT1A, 5-HT2A, and 5-HT2C receptors because all these re-
ceptor subtypes are absent in HEK293 cells; i.e., HEK293 cells
lack specific binding for the radioligands used to characterize
each of the receptor subtypes (data not shown). HEK293 cell
lines stably expressing high levels of D2, D4, 5-HT1A, 5-HT2A, or
5-HT2C receptors were created as described by us previously.60-62
The expression levels of the different receptor subtypes in indi-
vidual clonal lines were determined by radioligand saturation
isotherm binding utilizing standard rapid filtration techniques
and cell membrane preparations as described by us previously.63
Cloned human σ1 receptors were stably expressed in human
MCF-7 cells as described by us previously, because MCF-7 cells
lack specific binding for [3H]-(þ)-pentazocine, the radioligand
used to characterize σ1 receptor binding potential.48 The mem-
branes from these σ1-expressing MCF-7 cells were prepared as
described previously.48 With the exception of membranes con-
taining D3 receptors, all membranes isolated from clonal cell
lines were placed in their respective binding buffers (Table 5) and
kept on ice until use that same day. Frozen membranes from
CHO-K1 cells containing the human dopamine D3 receptor
were purchased from Perkin-Elmer Life Science.
Whole rat brains with the cerebellum and brain stem removed
were used as the tissue source for rat cannabinoid CB1 receptors.
Briefly, two to three stripped and frozen Sprague-Dawley rat
brains (Pel Freeze Bio, 56005-2) were allowed to slow-thaw by
incubating on ice in 30 mL of cold 20 mM Tris, pH 7.4, at 2 ꢀC
and 1:1000 v/v diluted protease inhibitor cocktail (Sigma-Aldrich,
P8340). Thawed brains were homogenized with 10 strokes in a
Dounce glass-glass homogenizer and the suspension was cen-
trifuged for 10 min at 1000g (Sorvall Legend RT). The resulting
supernatant was transferred to a new tube and centrifuged for
1 h at 4 ꢀC at 25000g (Sorvall RC-5). The supernatant was
discarded and the pellet resuspended in 5 mL of cold 20 mM
Tris, pH 7.4, at 2 ꢀC and rehomogenized with four strokes. The
homogenate was separated into 1 mL aliquots in cryotubes and
stored in liquid nitrogen until use at a 1:5 v/v dilution in binding
buffer (see Table 5).
Radiosynthesis of [11C]7. Carbon-11 was produced by 14N(p,
R)11C nuclear reaction using CYPRIS HM-18 cyclotron (Sumitomo
Heavy Industry Co. Ltd., Tokyo, Japan). [11C]CH3I with high
specific activity was produced by the single-pass I2 method as
described previously.59 By the nuclear reaction and a successive
hot atom process, [11C]CH4 (total radioactivity: 44 GBq) was
produced in the target chamber at an initial pressure of 15 bar
with 18 MeV protons (14.2 Mev on target). [11C]CH4 was passed
through a heated I2 column once and converted to [11C]CH3I,
which was collected in a reaction vessel containing a 300 μL of
anhydrous DMF solution of desmethyl precursor 19 (1 mg) and
NaOH (10 μL, 0.5 N) at -15 to -20 ꢀC. After radioactivity
reached a plateau, the reaction mixture was heated at 70 ꢀC for
3 min. The HPLC solvent was added to the reaction vessel to
terminate the reaction. The radioactive mixture was applied to
an HPLC purification system (SHISEIDO CAPCELL PAK
C18 UG80 column, 10 mm ꢀ 250 mm; mobile phase CH3OH/
H2O/triethylamine, 6/4/0.1%; flow rate, 5 mL/min; λ= 254 nm).
The fraction corresponding to [11C]7 was collected in a flask
containing 100 μL of ascorbic acid (25%) and evaporated to
dryness under a vacuum. The residue was dissolved in 3 mL of
saline (pH 7.4) for animal experiments. At the end of synthesis,
210-440 MBq (n=4) of [11C]7 was obtained as an intravenous
injectable solution. The radiochemical purity and specific activ-
ity of [11C]7 were assayed by analytical HPLC (SHISEIDO
CAPCELL PAK C18 UG80 column, 10 mmꢀ250 mm; mobile
phase CH3OH/H2O/triethylamine, 6/4/0.1%; flow rate, 5 mL/min;
λ = 254 nm). The identity of [11C]7 was confirmed by co-
injection with an authentic nonradioactive sample. The amount
of carrier in the final product solution was measured by the same
analytical HPLC. The radiochemical purity and specific activity
was>99% and 2770-3890 GBq/μmol (n= 4), respectively.
Biological Methods. 1. Preparation of Crude Membranes for
Binding Assays. A human embryonic kidney cell line (HEK293)
was employed as the host cell for expressing individually the
cloned rat dopamine D2 and D4 receptors and human serotonin
2. Radioligand Binding Assays. Compounds were tested for
their ability to compete with radioligands specifically bound to
membranes from cells or tissues expressing the dopamine D2 or
D3 or D4 receptors, serotonin 5-HT1A, 5-HT2A, or 5-HT2C recep-
tors, σ1 receptors, and cannabinoid CB1 receptors. The radio-
ligands and drugs used to define nonspecific binding to each
receptor system are shown in Table 5. All binding reactions were
allowed to reach equilibrium prior to rapid filtration. Glass
tubes were used in all binding reactions except those for canna-
binoid receptors which utilized silanized glass tubes. Binding
reactions were rapidly terminated by filtration through GF/C
filters pretreated with 0.5% polyethyleneimine, or GF/B filter
pretreated with 1% polyethyleneimine in the case of cannabinoid
receptors, and washing with 3 ꢀ 3 mL of ice-cold (0-2 ꢀC)

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7353
binding buffer at the pH at that temperature. Radioactivity
bound to filters was quantified in a scintillation counter. Membrane
protein concentrations were determined using the bicinchonic
acid protein reagent (BCA) and a bovine serum albumin stan-
dard curve. Membranes protein concentrations varied from
0.01 to 0.05 mg/mL. Binding affinities (Ki) were determined by
competition binding with fixed concentrations of the radioli-
gands described in Table 5. With the exception of 6, 7, and 12,
which were dissolved in DMF, all compounds were solubilized
in DMSO at concentrations ranging from 1 to 10 mM. These
nonaqueous solution stocks were then diluted at least 1:1000 v/v
in the final assay solution.
3. Calculations and Data Analysis. All points were run in
triplicate. Each experiment was repeated three to four times, and
averaged values were reported with their standard deviation or
standard error. The inhibition constant (Ki) values were calculated
from IC50 values using the Cheng-Prusoff equation: Ki=IC50/
(1 þ [ligand]/KD).64 In cases where the displacement was>20%
but less than 100% at the highest concentration of inhibitor, the
bottom of the curve fit was set to zero so that accurate IC50 values
could be calculated. A 95% confidence interval was employed
for all curve-fitting procedures using Graphpad’s Prism soft-
ware, version 4.0.
PET camera (Hamamatsu Photonics K.K., Japan) designed for
laboratory animals, which provides 31 transaxial slices 3.6 mm
(center-to-center) apart, a 33.1 cm field of view, and spatial
resolution of 2.6 mm full width at half-maximum.65 The monkey
was anesthetized with ketamine at 10 mg/kg im (Ketalar,
Sankyo Co. Ltd., Japan) with a head fixation device to ensure
accuracy of repositioning.66 Following transmission scans for
attenuation correction using a 68Ge-68Ga source for 20 min, a
dynamic emission scan in 3D acquisition mode was immediately
performed after intravenous injection of [11C]7 (38.5 ( MBq;
specific radioactivity, 2.9 TBq/μmol at injection) for 90 min
(0.5 minꢀ6 frames, 1 minꢀ7 frames, 2 minꢀ20 frames, 5 minꢀ
8 frames). Dynamic emission scan images were reconstructed
with filtered back-projection using a 4.0 mm Colsher filter.
Volumes of interest (VOIs) were placed on the hippocampus,
entorhinal cortex, and striatum using PMOD image analysis
software (PMOD Group, Zurich, Switzerland) with reference to
the magnetic resonance (MR) image of the monkey brain. The
MR image was acquired with a 1.5 T gyroscan S15/ACS2
(Philips Electronic, Eindhoven, The Netherlands) by means
of a three-dimensional T1-weighted spin-echo sequence. The
tracer uptake in each VOI was estimated as percent of injected
dose per unit volume (% ID/mL).
4. Preliminary Disposition Studies. Male CD1 mice weighing
25-30 g (Charles River, Italy) were administered compounds 7
or 13 (10 mg/kg ip, dissolved in ethanol/PEG 40/saline
(10:40:50, v/v) and were sacrificed by decapitation at 15, 30,
60, 120, and 240 min after dosing to determine the plasma and
brain concentrations of the parent compounds and their poten-
tial metabolite 1-(6-cyano-2-pyridyl)piperazine. Procedures in-
volving animals and their care were conducted in conformity
with the institutional guidelines that are in complicance with
national (D.L. n. 116, G.U., Suppl. 40, 18 Febbraio 1992, Circolare
No. 8, G.U., 14 Luglio 1994) and international laws and policies
(EEC Council Directive 86/609, OJ L 358, 1, Dec 12, 1987; Guide
for the Care and Use of Laboratory Animals, U.S. National
Research Council, 1996). Concentrations of the selected compounds
were determined by HPLC with UV detection (λ = 264 nm).
Briefly, to 0.2 mL of plasma, 0.01 mL of 1 M NaOH, 0.03 mL of
the internal standard 1-(2-thiazolyl)piperazine (10 μg/mL), and
5 mL of dicloromethane were added. After being shaken,
samples were centrifuged, and to the organic extract 0.15 mL
of mobile phase was added. After further shaking and centrifu-
gation, 0.1 mL of the mobile phase was injected onto the HPLC
system. Brains were homogenized in distilled water (1 g/10 mL),
and an amount of 2 mL of the homogenate was processed as
above. Chromatography was performed on a reversed-phase
column XTerra RP 18 (4.6 mm ꢀ 150 mm, 5 μm particle size).
The mobile phase was CH3CN/ 0.005 M KH2PO4 (pH 3.5)
(5:95, v/v) (solvent A) and CH3CN/ 0.005 M KH2PO4 (pH 3.5)
(70:30, v/v) (solvent B). The HPLC system was set up to operate
at a flow rate of 1 mL/min, following a simple gradient: step 1,
from the initial condition (100% solvent A) to 20% solvent A
(80% B) in 12 min; step 2, from 20% to 100% solvent A (from
80% to 0% of solvent B) in 2 min. The total run time was 18 min.
The retention times were 8.7 min for 7, 8.8 for 13, 4.5 min for
1-(6-cyano-2-pyridyl)piperazine, and 7.7 min for internal standard.
The limit of quantification was about 100 ng/mL or ng/g, using
0.2 mL of plasma or about 200 mg of tissue. At these concentra-
tions, the coefficients of variation (CV) were generally between
10% and 15%, and all higher concentrations gave CV between
5% and 10% for all compounds in both tissues.
Acknowledgment. This study was supported by the Nati-
onal Institutes of Health [Grants R01-MH063162 and R01-
MH063162-06S1 (J.A.S.)] and the National Science Founda-
tion [DBI-0649889 (J.A.S.)].
Supporting Information Available: Elemental analysis data of
target compounds and time-activity data. This material is
available free of charge via the Internet at http://pubs.acs.org.
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