New N-n-propyl-substituted 3-aryl- and 3-cyclohexylpiperidines as partial agonists at the D4 dopamine receptor

Article abstract of DOI:10.1021/jm021019a

We have previously reported that compounds dimethyl-substituted on the phenyl ring of N-n-propyl-3-phenylpiperidines (PPEs) have a high (nM) affinity and selectivity toward the D₄ dopamine receptor (D₄ DAR) with m,p-dimethyl PPE (1) having the highest affinity and selectivity. In the present paper we have investigated the role of the methyl substitution by the synthesis of monomethylated (2a-c) and nonmethylated (2d) PPEs followed by the characterization of their biological properties using receptor binding assays. Our findings reveal that the methyl substitution of the phenyl ring is not necessary for a high and selective binding affinity to the D₄ DAR. Moreover, we have also synthesized cyclohexylpiperidines (CHPEs, 3a-d), which all showed higher binding affinities for the D₄ DAR than their aromatic counterparts. These results indicate that a π-π type interaction of the phenyl ring of PPEs with the D₄ DAR might not be essential, whereas a simple hydrophobic attraction between the cyclohexyl substituent of CHPEs and a hypothesized lipophilic pocket of the receptor might be crucial. Furthermore, functional assays indicate that 3d, as well as 1, are partial agonist at the D₄ DAR and therefore might represent new pharmacological tools to investigate the role of D₄ DAR activation in the control of cognitive functions and emotional states in health and disease.

Full text of DOI:10.1021/jm021019a

J . Med. Chem. 2003, 46, 161-168
161
New N-n -P r op yl-Su bstitu ted 3-Ar yl- a n d 3-Cycloh exylp ip er id in es a s P a r tia l
Agon ists a t th e D4 Dop a m in e Recep tor
Marco Macchia,† Luigi Cervetto,*,‡ Gian Carlo Demontis,‡ Biancamaria Longoni,§ Filippo Minutolo,†
Elisabetta Orlandini,† Gabriella Ortore,† Chiara Papi,† Andrea Sbrana,| and Bruno Macchia*,†
Dipartimento di Scienze Farmaceutiche, Universita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy, Dipartimento di Psichiatria,
Neurobiologia, Farmacologia e Biotecnologie, Universita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy, Dipartimento di
Neuroscienze, Universita` di Pisa, Via Roma 67, 56126 Pisa, Italy, and Centro Interdipartimentale di Servizi Informatici per la
Didattica e la Ricerca sul Farmaco, Via Bonanno 6, 56126 Pisa, Italy
Received August 6, 2002
We have previously reported that compounds dimethyl-substituted on the phenyl ring of N-n-
propyl-3-phenylpiperidines (PPEs) have a high (nM) affinity and selectivity toward the D4
dopamine receptor (D4 DAR) with m,p-dimethyl PPE (1) having the highest affinity and
selectivity. In the present paper we have investigated the role of the methyl substitution by
the synthesis of monomethylated (2a -c) and nonmethylated (2d ) PPEs followed by the
characterization of their biological properties using receptor binding assays. Our findings reveal
that the methyl substitution of the phenyl ring is not necessary for a high and selective binding
affinity to the D4 DAR. Moreover, we have also synthesized cyclohexylpiperidines (CHPEs,
3a -d ), which all showed higher binding affinities for the D4 DAR than their aromatic
counterparts. These results indicate that a π-π type interaction of the phenyl ring of PPEs
with the D4 DAR might not be essential, whereas a simple hydrophobic attraction between the
cyclohexyl substituent of CHPEs and a hypothesized lipophilic pocket of the receptor might be
crucial. Furthermore, functional assays indicate that 3d , as well as 1, are partial agonist at
the D4 DAR and therefore might represent new pharmacological tools to investigate the role of
D4 DAR activation in the control of cognitive functions and emotional states in health and
disease.
In tr od u ction
the D4 DAR and ADHD. In addition, allelic variations
of the D4 DAR have been associated with specific
personality traits such as novelty seeking or impulsive,
compulsive, and addictive behaviors, like susceptibility
to drug abuse and compulsory gambling.11 It is relevant
to note that recent investigations at the cellular level
indicate that mice lacking the D4 gene are supersensi-
tive to methamphetamine, cocaine, and alcohol12 and
display cortex hyperexcitability.13 Intriguingly, the psy-
chostimulants amphetamine and methylphenidate, which
block the reuptake of dopamine, thus increasing syn-
aptic dopamine levels,14 are an effective treatment for
70-80% of subjects diagnosed with ADHD and HD. This
observation opens up the possibility that agonists selec-
tive for D4 receptors may have therapeutic value in
ADHD and HD.
Dopaminergic neurons projecting to the prefrontal
cortex and limbic areas play a key role in the control of
cognitive functions1 and emotional states,2 respectively.
Upon release, dopamine interacts with several receptors
(DARs) that belong to the superfamily of G-protein-
coupled receptors (GPCR).3 DARs have been classified
as D1- or D2-like according to their ability to activate or
inhibit adenylyl cyclase, respectively.4 The D2-like sub-
family of DARs includes D2,5 D3,6 and D4 subtypes. D2
7
and D3 receptors are the target of classic and some
atypical neuroleptics, but no conclusive evidence is
presently available from biological and genetic studies
for D4 receptors as the target for atypical neuroleptics.8
Genetic studies have indicated a certain association
between a specific polymorphic variant of the D4 gene,
which translated into a receptor with a blunted response
to dopamine, and an attention deficit with hyperactivity
disorder (ADHD), a disturbance affecting from 3 to 5%
of children.9 Although the association between the D4
polymorphism and ADHD has been controversial, a
recent meta-analysis10 has provided convincing evidence
for the association between several allelic variations of
A general concern is that the altered behavior may
result from a functional imbalance between the meso-
cortical and mesolimbic pathways, and compensation
with stimulants or full agonists of a hypoactive pathway
may lead to excessive stimulation of the other pathway.
A therapeutic approach with partial agonists may solve
the problem of imbalance between different dopamin-
ergic pathways.15,16 By acting as a stimulant in those
brain areas where the dopaminergic projection is hy-
pofunctional, while preventing full activation of other
brain regions that receive the hyperactive or normal
projection, a partial agonist would adjust the balance
between different anatomically and functionally distinct
* Correspondence and reprints (bmacchia@farm.unipi.it, cervetto@
farm.unipi.it, tel +39-050-500209, fax +39-050-40517).
† Dipartimento di Scienze Farmaceutiche, Universita` di Pisa.
‡ Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Bio-
tecnologie, Universita` di Pisa.
§ Dipartimento di Neuroscienze, Universita` di Pisa.
| Centro Interdipartimentale di Servizi Informatici per la Didattica
e la Ricerca sul Farmaco.
10.1021/jm021019a CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/28/2002

162 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Macchia et al.
Ch a r t 1
Sch em e 1a
a
Reagents and conditions: (a) H2 (1 atm), PtO2 (10 mol %),
concentrated HCl, MeOH, rt, 4 h; (b) H2 (1 atm), PtO2 (50 mol %),
concentrated HCl, MeOH, rt, 24 h; (c) CH3CH2CHO, NaBH3CN,
MeOH, rt, 24 h, then HCl.
dopaminergic pathways. In this regard, the partial
dopamine agonist aripiprazole has recently been pro-
posed as the first member of a new class of antipsychotic
drugs.17
Ch em istr y
Compounds 2a ,c and 3a -d were synthesized as
shown in Scheme 1, whereas compounds 2b and 2d
were prepared as previously reported.18
We have previously reported that a dimethyl substi-
tution on the phenyl ring of 3-phenylpiperidines (PPEs),
studied for several years in view of their dopaminergic
properties,18-25 leads to compounds possessing a high
(nM) affinity toward the D4 DAR.26,27 In particular, the
m,p-dimethyl-substituted derivative 1 proved to be the
most potent and selective compound. Furthermore, some
of these compounds have been reported to activate the
G proteins coupled to these receptors, which is an
indication of their agonist type of activity on D4 DAR.26
Mild hydrogenation of 3-arylpyridines 4a 28 and 4c29
under 1 atm of molecular hydrogen in the presence of
10 mol % of platinum oxide gave aryl piperidines 5a and
5c. Subsequent treatment with proprionic aldehyde in
the presence of NaBH3CN and final salification with
gaseous HCl in Et2O gave N-n-propylated 3-arylpip-
eridines 2a ,c. The synthesis of cyclohexylpiperidines
3a -d started with a more vigorous hydrogenation of
3-arylpyridines 4a ,28 4b,18 4c,29 and 4d ,30 using a higher
amount of catalyst (50 mol % of platinum oxide), which
afforded completely aliphatic intermediates 6a -d . The
same propylation conditions (proprionic aldehyde, NaBH3-
CN) reported above, followed by salification with hy-
drochloric acid, afforded N-n-propylated cyclohexylpip-
eridines 3a -d . It is important to note that the non-
methylated compound 3d is made up of a single dias-
tereoisomer, whereas in the case of 3a and 3b, up to
four diastereoisomers could be obtained; moreover, also
3c can exist as two different diastereoisomeric forms.
Due to the difficult separation of these mixtures,
compounds 3a -c were submitted to biological tests as
unresolved diastereoisomeric mixtures.
It is possible to hypothesize that the aromatic moiety
of these derivatives may interact with a lipophilic pocket
at the level of the active site of the D4 DAR, in line with
the fact that the presence of polar substituents on the
aryl leads to a drop in D4 DAR affinity as for preclamol
(3-PPP),26,27 a 3-(3-hydroxyphenyl)-1-propylpiperidine,
reported as the first autoreceptor-selective agonist.20
Considering that (see above) compounds with an
agonist activity on the D4 DAR may represent a useful
tool for the study and the treatment of the ADHD, we
thought it conceivable to further develop these studies
through the synthesis of new PPE derivatives. In
particular, in this paper, we report the biological evalu-
ation of mono-methyl-substituted (2a -c) and unsubsti-
tuted (2d ) derivatives, to verify the importance of the
presence of the two methyl groups on the phenyl ring
of compound 1 for the affinity of PPE's on the D4 DAR.
We also prepared completely saturated analogues of
compounds 2a-d, namely cyclohexylpiperidines (CHPEs,
3a -d ), with the aim of determining whether the nature
of the interaction of these compounds with the hypoth-
esized lipophilic pocket is of π-π type with an aromatic
receptor moiety or it is simple hydrophobic.
Resu lts
Ra d ioliga n d Bin d in g Assa ys. The affinities of the
PPEs (2a -d ) and CHPEs (3a -d ) for DARs were esti-
mated by means of radioligand competition assays
carried out on bovine retinal and striatal membrane
preparations (Table 1). The antagonists [3H]-SCH23390
and [3H]-YM-09-151-2 were used as specific radioligands
for D1-like and D2-like DARs, respectively.
Bovin e Retin a D1-like Dopam in e Receptor s. PPEs
and CHPEs had monophasic inhibition curves when
tested against D1-like DARs labeled with [3H]-SCH23390.

Partial Agonists at the D4 Dopamine Receptor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 163
Ta ble 1. Binding Affinities of PPE’s and CHPE’s for D1-like and D2-like Dopamine Receptors
Ki (nM)a
compound
D1 (retina)b
D2 (retina)c,d high-affinity
D2 (retina)c low-affinity
D2 (striatum)c
2a
2b
2c
>10 000 (2)
>10 000 (2)
>10 000 (2)
>10 000 (2)
>10 000 (2)
>10 000 (1)
>10 000 (2)
>10 000 (2)
>10 000 (2)
>10 000 (1)
449 ( 99 (3)
42; 124 (2)
44 ( 28 (3)
39 ( 16 (3)
26 ( 9 (3)
5 (1)
26 700 ( 4800 (3)
15 900-11 000 (2)
35 400 ( 1800 (3)
43 700 ( 2300 (3)
43 200 ( 8700 (3)
27 000 (1)
1510; 2270 (2)
nde
13 800; 8400 (2)
10 800; 3600 (2)
420; 1620 (2)
10 000 (1)
2d
3a f
3bf
3cf
4 (1)
64 000 (1)
20 000 (1)
3d
1
4 ( 1 (3)
27 ( 6 (6)
25 ( 8 (3)
24 900 ( 7200 (3)
7 600 ( 1100 (6)
6 300 ( 880 (3)
14 400; 46 300 (2)
8100 ( 1700 (3)
18 300 (1)
PD 168 077
Ki’s for retinal D1 DARs and striatal D2 DARs were obtained from IC50 in eq 1. Ki’s for D2-DARs were obtained from KiH and KiL in
a
eq 2. Kd’s were 2 nM and 0.4 nM for [3H]-SCH23390 and [3H]-YM-09-151-2, respectively; the number of independent experiments is
reported in parentheses. Ki values are expressed as the mean ( standard error of the mean (SEM), for compounds which were tested
three or more times. When two assays were performed, Ki values estimated from each assay are reported separated by a semicolon.
b
d
D1-like receptors labeled with [3H]-SCH23390. c D2-like receptors labeled with [3H]-YM-09-151-2. The high affinity site represents
16%, 18%, 16%, 13%, 16%, 15%, 20%, 20%, 16% of total binding for compounds 1, 2a , 2b, 2c, 2d , 3a , 3b, 3c, 3d . The high affinity site
represents 64% of total binding for PD 168,077 and the difference with compounds 1, 2a -d and 3a -d was statistically significant (P <
0.01, 1-way ANOVA). e Not determined. f Tested as a mixture of diastereomers.
Their mean inhibition constants were higher than 10
µM (Table 1).
Bovin e Retin a D2-lik e Dop a m in e Recep tor s. The
PPEs and CHPEs had biphasic inhibition curves when
tested against DARs labeled with [3H-]YM-09-151-2, as
shown in Figure 1A for 2a and 2d , and in Figure 1B for
3a and 3d as representative compounds.
Among the PPEs, compound 2a , characterized by the
presence of a methyl group in the ortho position of the
phenyl ring possesses an affinity 10-fold lower (higher
KiH) than those of m- and p-monomethyl-substituted
derivatives (2b,c) and of the unsubstituted derivative
2d , whose values are similar to that of m,p-dimethyl-
substituted derivative 1.
All CHPEs 3a -d proved to possess a significant
affinity. The 2′-monomethyl substituted derivative 3a
showed a KiH about 20-fold lower than its aromatic
counterpart 2a . Also in this series, the best affinities
were found with the unsubstituted derivative 3d and
3′- and 4′-monomethyl-substituted derivatives (3b ,c).
Table 1 reports the high- and low-affinity inhibition
constants (KiH and KiL), respectively, for PPE and
CHPEs.
Bovin e Str ia tu m D2-lik e Dop a m in e Recep tor s.
All the PPEs and CHPEs had monophasic inhibition
curves with Ki in the micromolar range when tested
against the D2 striatal receptors labeled with [3H-]YM-
F igu r e 1. Competition curves of PPE and CHPE for retinal
D4 DARs are biphasic. Examples of typical biphasic competi-
09-151-2.
tion experiments between [3H]-YM-09-151-2 and o-methyl
F u n ction a l Assa ys for Agon ist Activity. To assess
substituted (open squares, compounds 2a and 3a ) or nonsub-
the agonist properties of PPEs and CHPEs, two different
stituted (open circles, compounds 2d and 3d ) derivatives of
PPE and CHPE for retinal DARs are illustrated in panels A
functional assays were carried out on the m,p-dimethyl-
and B, respectively. The ordinate is the [3H]-YM-09-151-2
specifically bound at each drug concentration, expressed as a
percentage of the quantity bound in the absence of the
competitor. Average high-affinity constants are reported in in
Table 1 as KiH. The continuous curves through the data points
were drawn in accordance with a two-site model (see eq 2 in
Experimental Section), using parameters that minimize the
sum of squares (SS) between experimental data and the
theoretical curve (eq 3). For all the compounds, the F-test (see
Experimental Section), suggests that the two-site model
provides a significantly better fit to data than the one-site
model, with P < 0.01.
substituted derivative 1, the completely aliphatic un-
substituted derivative 3d , and the reference compound
PD 168,077.
Effects of Gu a n in e Nu cleotid e An a logu es on th e
Bin d in g Affin ity for D4 DARs. The presence of a high
affinity component in the displacement curves of PPEs
and CHPEs indicates functional heterogeneity of D4
DARs in retinal membrane preparations. To test the
hypothesis that PPEs and CHPEs recognize with a high
affinity D4 DARs coupled to a GTP binding protein, we
carried out competition binding assays in the presence
or absence of guanosine 5′-[â,γ-imido]triphosphate (GMP-
PNP), a non-hydrolyzable analogue of GTP. As shown
in Figure 2 for 1, 3d , and PD 168,077, GMP-PNP
abolished the high affinity state of D4 DARs, and the
competition assays are best described by monophasic

164 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Macchia et al.
F igu r e 3. Effects of 1, 3d , and PD 168 077 on melatonin
synthesis and release in the retina. Data plot of the average
value and the sem of the ratio between melatonin measured
in the extracellular medium upon synthesis and release in
response to treatment compared with basal levels (see meth-
ods). CNTR, FRSK, and PD indicate control, forskolin, and PD
168 077, respectively. Note the ordinate axis break between
0.2 and 0.7. The number of independent replicates (n) is
reported inside each column.
of PD 168,077 was reduced and became similar to that
of 1 and 3d alone.
F igu r e 2. Effects of GTP analogue GMP-PNP on high-affinity
interaction of 1, 3d , and PD 168 077 with D4-DARs. (A)
Datapoints are average values, with their standard error of
the mean, from two independent competition assays for 1 in
the absence (open circles) and in the presence (filled circles)
of 200 µM GMP-PNP. (B) Datapoints are average values, with
their standard error of the mean, from 3 independent competi-
tion assays for 3d in the absence (open circles) and in the
presence (filled circles) of 200 µM GMP-PNP. (C) Datapoints
are average values, with their standard error of the mean, from
two independent competition assays for PD 168 077 (PD)in the
absence (open circles) and in the presence (filled circles) of 200
µM GMP-PNP. For 1, 3d , and PD, in the absence of GMP-
PNP the two-site model provides a better fit than the one-site
model (P < 0.01). In the presence of GMP-PNP, the two-site
model is not significantly better than the one-site model (P <
0.1).
Discu ssion a n d Con clu sion s
We have previously reported26 that among the di-
methyl-substituted PPEs, compound 1, in which the two
methyl groups are in the meta,para positions, had the
best affinity and selectivity for the D4 DAR, whereas
the o-methyl substitution, occurring in four other pos-
sible regioisomers, caused a decrese in the binding
affinity.
The present results for PPEs 2a -d show that the
o-monomethyl derivative 2a possesses a significantly
lower affinity than its meta and para analogues 2b,c,
thus confirming the detrimental effect of a methyl
moiety in this position.
We hypothesized that the aromatic moiety of these
derivatives may interact with a lipophilic pocket at the
level of the binding site of the D4 DAR. This is consistent
with the decreased affinity for the D4 DAR of 3-PPP,
which has a polar hydroxy moiety on the phenyl ring.
curves with Ki values close to KiL of control experiments.
Mela ton in Syn th esis Assa ys. Compounds 1, 3d ,
and PD 168,077 were evaluated for their agonist prop-
erties at the D4 DARs by a functional test that measures
the suppression of melatonin synthesis by retinal pho-
toreceptors.
The fact that compound 2d , which lacks methyl
substituents, had a similar affinity profile to that of
monomethyl-substituted PPEs 2b ,c and dimethyl-
substituted PPE 1 indicates that the presence of the
methyl substituents in the meta and/or para positions
of the phenyl ring may not play a crucial role for the
interaction with the hypothesized lipophilic binding
pocket. On the contrary, the methyl substituent in the
ortho position might hinder the locking of the aromatic
moiety of PPEs within the binding pocket.
As shown in Figure 3 Forskolin (FRSK) increased the
synthesis of melatonin and the selective D4 DAR agonist
PD 168,077, at the concentration of 0.2 µM, completely
blocked the FRSK-induced increase of melatonin syn-
thesis. Compounds 1 and 3d , at the same concentration
of 0.2 µM, suppressed FRSK-induced increase of mela-
tonin synthesis, but to a smaller extent than PD
168 077. Note that a 5000-fold increase in concentration
did not enhance the extent of suppression. These results
may indicate that compounds 1 and 3d are partial
agonists at the D4 DARs.
The interaction of these compounds with the hypoth-
esized lipophilic pocket could have been of π-π type
with an aromatic receptor moiety or of a simple hydro-
phobic nature. Completely aliphatic CHPEs 3a -d gen-
erally showed an affinity for the D4 DAR even higher
than their corresponding aromatic PPE analogues: this
result is consistent with a non π-π type hydrophobic
interaction.
To assess the possibility that 1 and 3d are partial
agonists at the D4 DAR, we measured the effect of 0.2
µM 1 or 3d on the suppression of FRSK-induced
melatonin release by 0.2 µM PD 168,077. As shown in
Figure 3, in the presence of either 1 or 3d the efficacy

Partial Agonists at the D4 Dopamine Receptor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 165
In the aliphatic series of CHPEs 3a -d the affinity of
the 2′-monomethyl substituted compound (3a ) was not
so different from those of 3b-d .
activation followed by adenylyl cyclase inhibition. An
intriguing property of 3d , as well as of 1, is their partial
agonism, which may provide new pharmacological tools
to investigate the role of D4 DAR activation in the
control of cognitive functions and emotional states in
health and disease.
Among all the new PPEs and CHPEs herein reported,
completely aliphatic compounds 3b-d showed the best
biological properties in terms of affinity and selectivity
for the D4 DAR. However, it is important to note that
compounds 3a -c were tested as mixtures of diastere-
omers. Therefore, estimates of their binding affinities
are useful only for making a crude comparison. Con-
sidering the structural simplicity of the nonmethylated
compound 3d , which, unlike 3b and 3c, can exist only
as a single diastereoisomer, we chose this compound,
together with PPE 1, for further functional evaluation.
The observation of high- and low-affinity components
in the displacement curves of PPE and CHPE (see
Figure 1), which is typical of the interaction of agonists
with receptors that couple to GTP binding proteins, may
indicate that PPE and CHPE are agonists. To evaluate
this hypothesis we carried out competition binding
assays in the presence of the nonhydrolyzable GTP
analogue GMP-PNP.
The results illustrated in Figure 2 are consistent with
the idea that 1, 3d , and the reference compound PD
168 077 are agonists which, upon binding to the D4
DARs, promote GDP/GTP exchange by the G-protein R
subunit. The R subunit with bound GMP-PNP has low
affinity for the receptor, causing the dissociation of the
ternary complex Râγ. The change of receptor conforma-
tion following dissociation from the R-subunit is char-
acterized by a state of low affinity for agonists, and the
competition curves became monophasic, with a single
binding component with a low affinity.
The observation that for PPE and CHPE the high-
affinity site was less than 30% of total binding, lower
than the 60% estimated for PD 168 077, which is a
selective agonist at the D4 DARs, may indicate that at
the functional level PPE and CHPE are agonists at the
retinal D4 DAR, although with a lower efficacy than the
agonist PD 168 077. To test this hypothesis, the ability
of 1 and 3d to inhibit the forskolin-induced synthesis
of melatonin was compared with that of PD 168 077.
Data in Figure 3 indicate that 1 and 3d have a lower
efficacy than PD 168 077 in inhibiting the synthesis of
melatonin induced by FRSK. Coapplication of PD 168 077
with either 1 or 3d brought about a reduction of PD
168,077 efficacy in blocking FRSK-induced melatonin
synthesis. These results indicate that compounds 1 and
3d are partial agonists at the D4 DAR.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Kofler
hot-stage apparatus and are uncorrected. NMR spectra were
obtained with
a Varian Gemini 200 MHz spectrometer.
Chemical shifts (δ) are reported in parts per million (ppm)
downfield from tetramethylsilane and referenced from solvent
references. Electron impact (EI) mass spectra were obtained
on a HP-5988A mass spectrometer at 70 eV. The elemental
compositions of the compounds agreed to within (0.4% of the
calculated value. Chromatographic separations were per-
formed on silica gel columns by flash (Kieselgel 40, 0.040-
0.063 mm; Merck) or gravity column (Kieselgel 60, 0.063-
0.200 mm; Merck) chromatography. Reactions were followed
by thin-layer chromatography (TLC) on Merck aluminum silica
gel (60 F254) sheets that were visualized under a UV lamp (254
nm). Evaporation was performed in vacuo (rotating evapora-
tor). Sodium sulfate was always used as the drying agent.
3-Arylpyridines 4a ,28 4b,18 4c,29 4d ,30 and N-n-propyl-3-arylpi-
peridines 2b,d ,18 were prepared as reported in the literature.
P r ep a r a tion of 3-Ar ylp ip er id in e Hyd r och lor id es 5a ,c.
Gen er a l P r oced u r e. A solution of the appropriate 3-arylpy-
ridine (4a 28 or 4c18) (4.9 mmol) in methanol (50 mL) and 37%
aqueous HCl (1.5 mL), in the presence of 0.10 g (0.44 mmol)
of platinum dioxide, was submitted to hydrogenation under 1
atm of molecular hydrogen for 4 h at room temperature. The
mixture was filtered through a Celite pad and the filtrate was
concentrated under a vacuum. The residue was dissolved in
diethyl ether and treated with gaseous HCl. The precipitate
was collected by filtration and recrystallized from EtOH/Et2O.
3-(2-Meth ylp h en yl)p ip er id in e h yd r och lor id e (5a ): 0.64
g (3.0 mmol, 62% yield); mp 155-157 °C; 1H NMR (D2O) δ
1.55-2.12 (m, 4H), 2.35 (s, 3H), 2.58-3.68 (m, 5H), 7.02-7.55
(m, 4H) MS (EI, 70 eV) m/e 175 (M+ - HCl). Anal. C12H17N‚
HCl (C, H, N).
3-(4-m eth ylp h en yl)p ip er id in e h yd r och lor id e (5c): 0.77
g (3.6 mmol, 75% yield); mp 166-168 °C; 1H NMR (D2O) δ
1.44-2.18 (m, 4H), 2.31 (s, 3H), 2.71-3.69 (m, 5H), 7.20-7.24
(m, 4H); MS (EI, 70 eV) m/e 175 (M+ - HCl). Anal. C12H17N‚
HCl (C, H, N).
P r ep a r a tion of 3-Cycloh exylp ip er id in e Hyd r och lo-
r id es 6a -d . Gen er a l P r oced u r e. A solution of the appropri-
ate arylpyridine hydrochloride 4a ,28 4b,18 4c,29 or 4d ,30 (4.9
mmol) in methanol (50 mL), in the presence of 0.54 g (2.4
mmol) of platinum dioxide, was submitted to hydrogenation
under 1 atm of molecular hydrogen for 24 h at room temper-
ature. The mixture was filtered through a Celite pad, and the
filtrate was concentrated under a vacuum. The residue was
dissolved in diethyl ether and treated with gaseous HCl. The
precipitate was collected by filtration and recrystallized from
MeOH/Et2O.
It is important to note that we previously found that
the two enantiomers of PPE 1 have a similar binding
profile for the D4-DAR;27 furthermore, both enantiomers
have biphasic competition curves, which are converted
to monophasic in the presence of GMP-PNP (unpub-
lished). These observations suggest that the partial
agonism found for racemic 1 may not be ascribed to
different agonist/antagonist properties of the two enan-
tiomers. Considering the overall similarity between 1
and 3d in the binding and functional profile, as well as
in the molecular structure, the same conclusion may
apply also to racemic 3d .
3-(2-Meth ylcycloh exyl)p ip er id in e h yd r och lor id e (6a ):
0.75 g (3.5 mmol, 71% yield); mp 162-164 °C; 1H NMR (D2O)
δ 0.83 (d, 3H, J ) 7.2 Hz), 1.08-2.04 (m, 15H), 2.59 (t, 1H, J
) 12.3 Hz), 2.84 (td, 1H, J ) 12.5, 2.6 Hz), 3.21-3.48 (m, 2H);
MS (EI, 70 eV) m/e 181 (M+ - HCl). Anal. C12H23N‚HCl (C, H,
N).
3-(3-Meth ylcycloh exyl)p ip er id in e h yd r och lor id e (6b):
0.53 g (2.4 mmol, 50% yield); mp 150-152 °C; 1H NMR (D2O)
δ 0.83 (d, 3H, J ) 6.8 Hz), 0.92-1.94 (m, 15H), 2.71 (t, 1H, J
) 12.3 Hz), 2.83 (td, 1H, J ) 12.6, 3.2 Hz), 3.29-3.39 (m, 2H);
MS (EI, 70 eV) m/e 181 (M+ - HCl). Anal. C12H23N‚HCl (C, H,
N).
3-(4-Meth ylcycloh exyl)p ip er id in e h yd r och lor id e (6c):
0.88 g (4.0 mmol, 82% yield); mp 143-145 °C; 1H NMR (D2O)
δ 0.82 (d, 3H, J ) 6.4 Hz, minor diastereomer), 0.87 (d, 3H, J
) 7.0 Hz, major diastereomer), 0.93-1.96 (m, 15H), 2.66 (t,
In conclusion, we have reported here compound 3d
as the first completely aliphatic compound able to
selectively bind the D4 DARs and promote G-protein

166 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Macchia et al.
1H, J ) 12.1 Hz), 2.84 (td, 1H, J ) 12.5, 2.7 Hz), 3.20-3.48
(m, 2H); MS (EI, 70 eV) m/e 181 (M+ - HCl). Anal. C12H23N‚
HCl (C, H, N).
duplicate test tubes contained a final volume of 1 mL: 0.5 mL
of membranes, 0.1 mL of drug, and 0.1 mL of tracer. For [3H]-
YM-09-151-2 binding, duplicate test tubes contained a final
volume of 2 and 1.5 mL, for striatum and retina, respectively:
0.5 mL of membranes, either 0.15 or 0.2 mL of tracer, and
drug for retina or striatum, respectively. The affinity of [3H]-
SCH23390 and [3H]-YM-09-151-2 was measured by saturation
experiments in similar conditions, and the values were 2 and
0.4 nM, respectively. Nonspecific binding was assessed by 2
mM DA.
At the end of the incubation period, the radioactivity bound
to the receptor was separated from the free ligand by rapid
filtration under a vacuum, using a 30-well filtration apparatus
(Brandel). Filters were counted by liquid scintillation spec-
troscopy (LS-1600, Packard, Canberra Co.) and converted from
cpm to dpm.
3-Cycloh exylp ip er id in e h yd r och lor id e (6d ): 0.65 g (3.2
mmol, 66% yield); mp 160-162 °C; 1H NMR (D2O) δ 1.06-
2.01 (m, 16H), 2.60-2.93 (m, 2H), 3.18-3.62 (m, 2H); MS (EI,
70 eV) m/e 167 (M+ - HCl). Anal. C11H21N‚HCl (C, H, N).
P r ep a r a tion of N-n -P r op yl-3-a r ylp ip er id in e Hyd r o-
ch lor id es 2a ,c a n d N-n -P r op yl-3-cycloh exylp ip er id in e
Hyd r och lor id es 3a -d . Gen er a l P r oced u r e. A solution of
the appropriate arylpiperidine hydrochloride 5a ,c or cyclo-
hexylpiperidine hydrochloride 6a -d (0.94 mmol) in methanol
(10 mL) was treated at room temperature with propionic
aldehyde (0.13 g, 2.2 mmol) and NaBH3CN (0.16 g, 2.3 mmol).
The mixture was stirred at room temperature for 24 h, and
then the reaction was quenched with water (50 mL). The
resulting mixture was partially concentrated under a vacuum
(to remove most of the methanol), and the remaining aqueous
suspension was extracted with ethyl acetate. The organic
phase was dried over sodium sulfate and concentrated under
a vacuum. The residue was dissolved in Et2O and treated with
gaseous HCl. The precipitate was collected by suction filtration
and recrystallized from MeOH/Et2O.
N-n -P r op yl-3-(2-m eth ylp h en yl)p ip er id in e h yd r och lo-
r id e (2a ): 0.040 g (0.16 mmol, 17% yield); mp 179-181 °C;
1H NMR (D2O) δ 0.92 (t, 3H, J ) 7.2 Hz),1.63-2.18 (m, 6H),
2.35 (s, 3H), 2.90-3.69 (m, 7H), 7.15-7.33 (m, 4H); MS (EI,
70 eV) m/e 217 (M+ - HCl). Anal. C15H23N‚HCl (C, H, N).
N-n -P r op yl-3-(4-m eth ylp h en yl)p ip er id in e h yd r och lo-
r id e (2c): 0.078 g (0.31 mmol, 33% yield); mp 187-189 °C; 1H
NMR (D2O) δ 1.00 (t, 3H, J ) 7.2 Hz), 1.51-2.19 (m, 6H), 2.31
(s, 3H), 2.52-3.68 (m, 7H), 7.05-7.13 (m, 4H); MS (EI, 70 eV)
m/e 217 (M+ - HCl). Anal. C15H23N‚HCl (C, H, N).
Specific binding was obtained by subtracting nonspecific
binding from totals and normalized to specific binding in the
absence of drugs. Normalized data from 10-12 concentrations
were fitted by either a single-site model (eq 1)
[L]n
[L]n + ICn50
B(L) ) 100 - 100 ×
(1)
or a two-site model, (eq 2)
[L]
[L]
B([L]) ) 100 - BH
×
- (100 - BH)‚
(2)
[L] + KH
[L] + KL
by nonparametric fitting using a modified Lowenberg-Mar-
quardt algorithm implemented in the data analysis program
Microcal Origin, version 6.0 (Microcal Software, Inc., Northamp-
ton, MA 01060). The acceptance of a two-site model versus a
single-site model was performed in accordance with the criteria
described by Munson and Rodbard.31 Briefly, the statistical
significance of a two-site versus a one-site model was assessed
by the F-test, in accordance with the relation:
N-n -P r op yl-3-(2-m eth ylcycloh exyl)p ip er id in e h yd r o-
ch lor id e (3a ): 0.117 g (0.45 mmol, 48% yield); mp 169-171
°C; 1H NMR (D2O) δ 0.86 (d, 3H, J ) 6.8 Hz), 0.90 (t, 3H, J )
7.2 Hz), 1.08-2.46 (m, 19H), 2.92-3.16 (m, 2H), 3.49-3.67 (m,
2H); MS (EI, 70 eV) m/e 223 (M+ - HCl). Anal. C15H29N‚HCl
(C, H, N).
N-n -P r op yl-3-(3-m eth ylcycloh exyl)p ip er id in e h yd r o-
ch lor id e (3b): 0.085 g (0.33 mmol, 35% yield); mp 162-164
°C; 1H NMR (D2O) δ 0.81 (d, 3H, J ) 6.6 Hz), 0.94 (t, 3H, J )
7.3 Hz), 1.12-2.37 (m, 19H), 2.60-3.12 (m, 2H), 3.35-3.68 (m,
2H); MS (EI, 70 eV) m/e 223 (M+ - HCl). Anal. C15H29N‚HCl
(C, H, N).
(SS2 - SS1)
SS1
(df2 - df1)
F )
(3)
df1
where SS1 and SS2 are the sum of squares for the vertical
distance between experimental data and the computed curve
for the one-site and two-site models, respectively; for the 12-
point competition curves, the degrees of freedom (dfl and df2)
are 9 and 8 for the one-site and two-site models, respectively.
The probability of finding an F value equal or higher by
chance was taken from the tabulated values of the F distribu-
tion with (df2 - df1) and df1 degrees of freedom. Ki values
were obtained from IC50 by the Cheng-Prusoff equation:32
N-n -P r op yl-3-(4-m eth ylcycloh exyl)p ip er id in e h yd r o-
ch lor id e (3c): 0.109 g (0.42 mmol, 45% yield); mp 174-176
1
°C; H NMR (D2O) δ 0.80-0.98 (m, 6H), 1.09-2.32 (m, 19H),
2.59-2.97 (m, 2H), 3.24-3.45 (m, 2H); MS (EI, 70 eV) m/e 223
(M+ - HCl). Anal. C15H29N‚HCl (C, H, N).
N-n -P r opyl-3-cycloh exylpiper idin e h ydr och lor ide (3d):
0.035 g (0.14 mmol, 15% yield); mp 188-190 °C; 1H NMR (D2O)
δ 0.99 (d, 3H, J ) 7.3 Hz), 1.05-1.24 (m, 6H), 1.64-2.00 (m,
12H), 2.22-2.57 (m, 2H), 2.83-2.94 (m, 2H), 3.40-3.56 (m,
2H); MS (EI, 70 eV) m/e 209 (M+ - HCl). Anal. C14H27N‚HCl
(C, H, N).
Ra d ioliga n d Bin d in g. Radioreceptor binding studies with
[3H]-SCH23390 (a D1-like receptor antagonist, 80 Ci/mmol;
New England Nuclear, Boston, MA) and [3H]-YM-09-151-2 (a
D2-like receptor antagonist, 81-87 Ci/mmol; New England
Nuclear, Boston, MA) were performed in membrane prepara-
tions from bovine retina and striatum. Striatum was homo-
genized in a saline solution (1:20 w/v) containing in mM: Tris,
50; EDTA, 1; CaCl2, 1.5; MgCl2, 4; KCl, 5; NaCl, 120; pH 7.4.
The homogenate was centrifuged at 48 000 g for 20 min at 4
°C, resuspended 1:20, recentrifuged, and resuspended at a final
dilution of about 1.25 mg of original wet tissue/mL of saline
(1:800 w/v) for use in the binding assay.
IC50
Ki )
(4)
[L]
Kd
1 +
(
)
where IC50 is the drug concentration inhibiting 50% of specific
binding. Kd values were 2 and 0.4 nM for [3H]-SCH23390 and
[3H]-YM-09-151-2, respectively.
Data in Figure 2 display the results of experiments carried
out in the absence (control) and in the presence of 100-200
µM of guanosine 5′-[â,γ-imido]triphosphate (GMP-PNP), an
analogue of GTP that is resistant to the intrinsic GTPase
activity of the R-subunit of GTP binding protein. The use of
this nonhydrolyzable analogue of GTP aims to stabilize the
R-subunit of G-proteins in a form separated from the âγ
subunits and the receptor as well. The receptor dissociated
from the R-subunit has a low affinity for agonists; therefore,
the conversion of a competition curve from biphasic to monopha-
sic is an indication that the competing compound acts like an
agonist, promoting GDP-GMP-PNP exchange. The ensuing
Retinas were treated similarly to striata except that the last
resuspension for use in the binding assay was at about 4 mg
of original wet tissue/mL of saline (1:250 w/v). For competition
experiments membranes were incubated at 30 °C in the
presence of 0.1 nM [3H]-YM-09-151-2 or 0.5 nM [3H]-SCH23390
for 60 or 20 min, respectively. For [3H]-SCH23390 binding,

Partial Agonists at the D4 Dopamine Receptor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 167
(5) Bunzow, J . R.; van Tol, H. H. M.; Grandy, D. K.; Albert, P.; Salon,
J .; Christie, M.; Machida, C. A.; Neve, K. A.; Civelli O. Cloning
and expression of a rat D2 dopamine receptor cDNA. Nature
1988, 336, 783-787.
(6) Sokoloff, P.; Giros, B.; Martres, M. P.; Bouthenet, M. L.;
Schwartz, J . C. Molecular cloning and characterization of a novel
dopamine receptor (D3) as a target for neuroleptics. Nature 1990,
347, 146-150.
(7) van Tol, H. H. M.; Bunzow, J . R.; Guan, H. C.; Sunahara, R. K.;
Seeman, P.; Niznik, H. B.; Civelli, O. Cloning of the gene for a
human dopamine D4 receptor with high affinity for the antip-
sychotic Clozapine. Nature 1991, 350, 610-614.
(8) Rowley, M.; Bristow, L. J .; Hutson, P. H. Current and Novel
Approaches to the Drug Treatment of Schizophrenia J . Med.
Chem. 2001, 44, 477-501.
(9) LaHoste, G. J .; Swanson, J . M.; Wigal, S. B.; Glabe, C.; Wigal,
T.; King, N.; Kennedy, J . L. Dopamine D4 receptor gene poly-
morphism is associated with attention deficit hyperactivity
disorder. Mol. Psych. 1996, 1, 121-124.
(10) Faraone, S. V.; Doyle, A. E.; Mick, E.; Biederman, J . Meta-
analysis of the association between the 7-repeat allele of the
dopamine D(4) receptor gene and attention deficit hyperactivity
disorder. Am. J . Psych. 1991, 158, 1052-1057.
(11) Comings, D. E.; Gonzalez, N.; Wu, S.; Gade, R.; Muhleman, D.;
Saucier, G.; J ohnson, P.; Verde, R.; Rosenthal, R. J .; Lesieur,
H. R.; Rugle, L. J .; Miller, W. B.; MacMurray, J . P. Studies of
the 48 bp repeat polymorphism of the DRD4 gene in impulsive,
compulsive, addictive behaviors: Tourette syndrome, ADHD,
pathological gambling, and substance abuse. Am. J . Med. Genet.
1999, 88, 358-368.
(12) Rubinstein, M.; Phillips, T. J .; Bunzow, J . R.; Falzone, T. L.;
Dziewczapolski, G.; Zhang, G.; Fang, Y.; Larson, J . L.; McDou-
gall, J . A.; Chester, J . A.; Saez, C.; Pugsley, T. A.; Gershanik,
O.; Low, M. J .; Grandy, D. K. Mice lacking dopamine D4
receptors are supersensitive to ethanol, cocaine, and metham-
phetamine. Cell 1997, 90, 991-1001.
dissociation of the receptor from the R-subunit stabilizes the
state of the receptor characterized by a low affinity for
agonists.
Mela ton in Syn th esis. Tests for the agonist activity of
compounds were carried out by exploiting the reported inhibi-
tion of melatonin synthesis by the mammalian retina in
response to D4 DAR activation.33 Guinea-pig rather than
bovine retinas were used, to improve tissue viability by
avoiding the hypoxia associated with the transfer of tissue
from the slaughterhouse to the lab (1 h). Tissue viability is
critical because the synthesis of melatonin depends on activa-
tion by cAMP of transcription of the serotonin N-acetyl
transferase (NAT) gene (unpublished). Note that retinal DARs
have similar characteristics in bovine and guinea-pig tissue.34
Adult albino guinea-pigs (250-400 g) obtained from a local
supplier (Stefano Morini S.a.S., S. Polo d′Enza, Italy) were kept
on a 12:12 light:dark cycle, and reared in accordance with the
rules of the local Animal Welfare Committee for the Care and
Use of Laboratory Animals. On the day of the experiment, the
animal was dark-adapted for 1 h and then anaesthetized by
an initial intraperitoneal injection of 35 mg kg-1 pentothal
sodium (Gellini S.p.A., Aprilia, Italy). After enough anaesthetic
had been provided to fully suppress corneal reflexes, the eye
was quickly (6 min) enucleated in dim red light and the animal
killed by an intraperitoneal lethal dose of anaesthetic (350 mg
kg-1 pentothal sodium). For each experimental session, from
two to three adult male guinea-pigs (200-400 g) were used.
After enucleation, the anterior pole was discarded and the
posterior pole (eyecup) was bathed in 250 µL of Locke’s
solution, the composition of which was (mM) NaCl, 140; KCl,
3.6; CaCl2, 1.2; MgCl2, 2.4; glucose, 10; Hepes, 10; pH 7.6 with
NaOH). Each eyecup with 250 µL of Locke’s solution was
positioned in a custom-made chamber obtained by cutting the
bottom of a 12 × 75 mm polystyrene tube; the chambers were
positioned in a light-proof container and incubated at 35 °C
in a thermostated water-shaking bath for the duration of the
experiment. Owing to its lipophilic nature, melatonin diffuses
across the plasma membrane of photoreceptor cells; a change
in melatonin synthesis will thus translate into a concentration
change in the medium where the eyecup is incubated. Starting
at 13:00 h, 50 µL of incubating medium was collected every
30 min, followed by the addition of 50 µL of fresh Locke’s
solution. For each retina, the first five samples were pooled
and the melatonin content of this 250 µL (1st interval) was
used to obtain a baseline for normalization between retinas
and treatment. Ten more 50 µL samples were collected from
15:30 to 20:00 h and pooled in groups of five samples, which
are indicated as 2nd and 3rd interval. Compounds were added
at 15:30 at 5× concentrations, and care was taken to add
subsequent aliquots containing the desired compound(s) at the
final concentration. Data in Figure 3 report the ratio of
melatonin measured in the 3rd to that in the 1st interval.
Melatonin in the samples was measured using a commercial
immunoassay kit (ICN, Italy), in accordance with the manu-
facturer’s instruction. Briefly, samples were desalted by three
elutions with water through C18 columns, and melatonin was
finally eluted from columns by methanol. Samples were then
dried under nitrogen and reconstituted in 50 µL of H2O.
Samples were assayed by a competition enzyme-linked im-
munosorbent assay (ELISA), and the photometric measure-
ment at 405 nm was converted to absolute melatonin concen-
tration by a calibration curve that used authentic melatonin.
(13) Rubinstein, M.; Cepeda, C.; Hurst R. S.; Flores-Hernandez, J .;
Ariano, M. A.; Falzone, T. L.; Kozell, L. B.; Meshul, C. K.;
Bunzow, J . R.; Low, M. J .; Levine, M. S.; Grandy, D. K.
Dopamine D4 receptor-deficient mice display cortical hyperex-
citability. J Neurosci. 2001 21, 3756-3763.
(14) During, M. J .; Bean, A. J .; Roth, R. H. Effects of CNS stimulants
on the in vivo release of the colocalized transmitters, dopamine
and neurotensin, from rat prefrontal cortex. Neurosci. Lett. 1992,
140, 129-133.
(15) Kosten, T. R.; George, T. P.; Kosten, T. A. The potential of
dopamine agonists in drug addiction. Expert Opin. Investig.
Drugs 2002, 4, 491-499.
(16) Tamminga, C. A. Partial dopamine agonists in the treatment of
psychosis. J . Neural Transm. 2002, 109, 411-420.
(17) Burris, K. D.; Molski, T. F.; Xu C.; Ryan, E.; Tottori, K.; Kikuchi,
T.; Yocca, F. D.; Molinoff, P. B. Aripiprazole, a novel antipsy-
chotic, is a high-affinity partial agonist at human dopamine D2
receptors. J . Pharmacol. Exp. Ther. 2002, 302, 381-389.
(18) Hacksell, U.; Arvidsson, L. E.; Svensson, U.; Nilsson, J . L. G.;
Sanchez, D.; Wikstro¨m, H.; Lindberg, P.; Hjorth, S.; Carlsson,
A. 3-Phenylpiperines. Central Dopamine-Autoreceptor Stimulat-
ing Activity. J . Med. Chem. 1981, 24, 1475-1482.
(19) Wikstro¨m, H. Centrally Acting Dopamine D2 Receptor Ligands:
Agonist. Prog. Med. Chem. 1992, 29, 185-215.
(20) Astra. Preclamol 3-PPP. Drugs Future. 1987, 12, 88-89.
(21) Wikstro¨m, H.; Sanchez, D.; Lindberg, P.; Hacksell, U.; Arvidsson,
L. E.; J ohansson, A. M.; Thorberg, S. O.; Nilsson, J . L. G.;
Svensson, K.; Hjoth, S.; Clark, D.; Carlsson, A. Resolved 3-(3-
Hydroxyphenyl)-N-n-Propylpiperidine and Its Analogues: Cen-
tral Dopamine Receptor Activity. J . Med. Chem. 1984, 27, 1030-
1036.
(22) Liljefors, T.; Wikstro¨m, H. A Molecular Mechanics Approach to
the Understanding of Presynaptic Selectivity for Centrally
Acting Dopamine Receptor Agonists of Phenylpiperidine Series.
J . Med. Chem. 1986, 29, 1896-1904.
(23) Liljefors, T.; Bøgesø, K.; Hyttel, J .; Wikstro¨m, H.; Svensson, K.;
Carlsson, A. Pre- and Postsynaptic Dopaminergic Activities of
Indolizidine and Quinolizidine Derivatives of 3-(3-Hydroxyphen-
yl)-N-(n-propyl)piperidine (3-PPP). Further Developments of a
Dopamine Receptor Model. J . Med. Chem. 1990, 33, 1015-1022.
(24) Sonesson, C.; Lin, C. H.; Hansson, L.; Waters, N.; Svensson, K.;
Carlsson, A.; Smith, M. W.; Wikstro¨m, H. Substituted (S)-
Phenylpiperidines and Rigid Congeners as Preferential Dopam-
ine Autoreceptor Antagonists: Synthesis and Structure-Activity
Relationships. J . Med. Chem. 1994, 37, 2735-2753.
Refer en ces
(1) Brozoski, T. J .; Brown R. M.; Rosvold, H. E.; Goldman, P. S.
Cognitive deficit caused by regional depletion of dopamine in
prefrontal cortex of rhesus monkey. Science 1979, 205, 929-
932.
(2) Spanagel, R.; Weiss, F. The dopamine hypothesis of reward: past
and current status. Trends Neurosci. 1999, 22 (11), 521-527.
(3) Dohlman, H. G.; Thorner, J .; Caron, M. G.; Lefkowitz, R. J .
Model systems for the study of seven-transmembrane-segment
receptors. Annu. Rev. Biochem. 1991, 60, 653-688.
(4) Kebabian, J . W.; Calne, D. B. Multiple Receptors for Dopamine.
Nature 1979, 277, 93-96.
(25) Hansson, L. O.; Waters, N.; Holm, S.; Sonesson, C. On the
Quantitative Structure-Activity Relationships of Meta-Substi-
tuted (S)-Phenylpiperidines, a Class of Preferential Dopamine
D
2 Autoreceptor Ligands: Modeling of Dopamine Synthesis and
Release in Vivo by Means of Partial Least Squares Regression.
J . Med. Chem. 1995, 38, 3121-3131.

168 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Macchia et al.
(26) Cervetto, L.; Demontis, G. C.; Giannaccini, G.; Longoni, B.;
Macchia, B.; Macchia, M.; Martinelli, A.; Orlandini E. N-n-
Propyl-Substituted 3-(Dimethylphenyl)-Piperidines Display Novel
Discriminative Properties between Dopamine Receptor Sub-
types: Synthesis and Receptor Binding studies. J . Med. Chem.
1998, 41, 4933-4938.
(27) Macchia, B.; Cervetto, L.; Demontis, G. C.; Domiano, P.; Longoni,
B.; Macchia, M.; Minutolo, F.; Orlandini, E.; Ortore, G.; Papi C.
Synthesis and dopaminergic properties of the two enantiomers
of 3-(3,4-dimethylphenyl)-1-propylpiperidine, a potent and selec-
tive dopamine D4 receptor ligand. Bioorg. Med. Chem. Lett.
2001, 11, 223-225.
magnoli, F.; Rossello, A. Synthesis and R-Adrenergic and I1-
Imidazoline Activity of 3-Phenylpiperidines Dimethyl-Substi-
tuted on the Phenyl Ring. Eur. J . Med. Chem. 1998, 33, 911-
919.
(31) Munson, P. J .; Rodbard, D. Ligand: a Versatile Computerized
Approach for Characterization of Ligand-Binding System. Anal.
Biochem. 1980, 107, 220-239.
(32) Cheng, Y.; Prusoff, W. H. Relationship between the Inhibition
Constant (Ki) and the Concentration of Inhibitor which Causes
50% Inhibition (IC50) of an Enzymatic Reaction. Biochem.
Pharmacol. 1973, 22, 3099-3108.
(33) Tosini, L.; Dirden, J . C. Dopamine inhibits melatonin release
in the mammalian retina: in vitro evidence. Neuroscience Lett.
2000, 286, 119-122.
(28) Aliprantis, A. O.; Canary, J . W. Observation of Catalytic
Intermediates in the Suzuki Reaction by Electrospray Mass
Spectrometry. J . Am. Chem. Soc. 1994, 116, 6985-6986.
(29) Benniston, A. C. Building off the back of Chelators: Synthesis
of 3,3′′-bis(4-methylphenyl)-2,2′:6′,2′′-Terpyridine. Tetrahedron
Lett. 1997, 38, 8279-8282.
(34) Watling, K. J .; Iversen, L. L. Comparison of the binding of [3H]-
spiperone and [3H]-domperidone in homogenates of mammalian
retina and caudate nucleus. J . Neurochem. 1981, 37, 1130-1143.
(30) Macchia, B.; Calderone, V.; Giannaccini, G.; Lucacchini, A.;
Macchia, M.; Martinelli, A.; Martinotti, E.; Orlandini, E.; Ro-
J M021019A