Synthesis and pharmacological evaluation of potent and highly selective D3 receptor ligands: Inhibition of cocaine-seeking behavior and the role of dopamine D3/D2 receptors

3822

J . Med. Chem. 2003, 46, 3822-3839

Syn th esis a n d P h a r m a cologica l Eva lu a tion of P oten t a n d High ly Selective D₃

Recep tor Liga n d s: In h ibition of Coca in e-Seek in g Beh a vior a n d th e Role of

Dop a m in e D₃/D₂Recep tor s^†

Giuseppe Campiani,*^,¶Stefania Butini,^¶Francesco Trotta,^¶Caterina Fattorusso,^§Bruno Catalanotti,^§

Francesca Aiello, Sandra Gemma,^¶Vito Nacci,^¶Ettore Novellino,^§J ennifer Ann Stark,^|Alfredo Cagnotto,^£

Elena Fumagalli,^£Francesco Carnovali,^£Luigi Cervo,^£and Tiziana Mennini^£

Dipartimento Farmaco Chimico Tecnologico, Universita´ di Siena, via Aldo Moro, 53100 Siena, Italy, Dipartimento di Chimica

delle Sostanze Naturali e Dipartimento di Chimica Farmaceutica e Tossicologica, Universita´ di Napoli “Federico II”, via D.

Montesano, 49, 80131 Napoli, Italy, Dipartimento di Scienze Farmaceutiche, Universita´ della Calabria, 87036 Arcavacata di

Rende, Italy, Cardiff School of Biosciences, Cardiff University, Biomedical Sciences Building, Museum Avenue, P.O. Box 911,

Cardiff CF10 3US, Wales UK, and Istituto di Ricerche Farmacologiche Mario Negri, via Eritrea 62, 20157 Milano, Italy

Received December 17, 2002

The synthesis, pharmacological evaluation, and structure-activity relationships (SARs) of a

series of novel arylalkylpiperazines structurally related to BP897 (3) are described. In binding

studies, the new derivatives were tested against a panel of dopamine, serotonin, and

noradrenaline receptor subtypes. Focusing mainly on dopamine D₃receptors, SAR studies

brought to light a number of structural features required for high receptor affinity and

selectivity. Several heteroaromatic systems were explored for their dopamine receptor affinities,

and combinations of synthesis, biology, and molecular modeling, were used to identify novel

structural leads for the development of potent and selective D₃receptor ligands. Introduction

of an indole ring linked to a dichlorophenylpiperazine system provided two of the most potent

and selective ligands known to date (D₃receptor affinity in the picomolar range). The intrinsic

pharmacological properties of a subset of potent D₃receptor ligands were also assessed in [³⁵S]-

GTPγS binding assays. Evidence from animal studies, in particular, has highlighted the

dopaminergic system’s role in how environmental stimuli induce drug-seeking behavior. We

therefore tested two novel D₃receptor partial agonists and a potent D₃-selective antagonist in

vivo for their effect in the cocaine-seeking behavior induced by reintroduction of cocaine-

associated stimuli after a long period of abstinence, and without any further cocaine. Compound

5g, a nonselective partial D₃receptor agonist with a pharmacological profile similar to 3, and

5p , a potent and selective D₃antagonist, reduced the number of active lever presses induced

by reintroduction of cocaine-associated stimuli. However, 5q, a highly potent and selective D₃

partial agonist, did not have any effect on cocaine-seeking behavior. Although brain uptake

studies are needed to establish whether the compounds achieve brain concentrations comparable

to those active in vitro on the D₃receptor, our experiments suggest that antagonism at D₂

receptors might significantly contribute to the reduction of cocaine craving by partial D₃

agonists.

In tr od u ction

biological basis of drug abuse and dependence, there is

still no effective long-term pharmacological therapy for

cocaine craving and for preventing relapses to abuse,¹

although very recently a candidate drug has been

proposed.⁴Treatments are anyway needed for all phases

of drug abuse, not only for the related problems of

relapse and toxicity.

Drug abuse, and the consequent drug addiction and

dependence, are serious threats to public health. Illness,

crime, domestic violence, reduced productivity, and lost

opportunities are direct consequences. The economic

cost of drug abuse to society is enormous and rising.

There are several reasons, frequently of social origin,

for drug abuse and various causes of relapse. In view

of its potent psychostimulating effects, cocaine (1) is one

of the most abused drugs, and the number of users

among people 12-40 years old who have used cocaine

at least once is extremely high in developed countries.^1-3

Despite huge advances in our understanding of the

Craving is one of the most important factors underly-

ing relapse.^5-7In humans, environmental stimuli that

are reliably associated with the effects of many drugs

of abuse can produce craving and relapse.^8-10In ani-

mals, such cues can induce and maintain drug-seeking

behavior and reinstate drug-seeking after extinction.^11-13

There is evidence that the dopaminergic system is

involved in how environmental stimuli induce drug-

^†In honor of Vito Nacci’s 70th birthday.

* To whom correspondence should be addressed. Tel: 0039-0577-

seeking behavior in rats.^13,14-16Thus D₁and D₂

13

17a-c

234172. Fax: 0039-0577-234333. E-mail: campiani@unisi.it.

¶

receptors antagonists (raclopride, 2) reduced the rein-

statement of drug-seeking behavior induced by cocaine-

associated stimuli. BP897 (3), a D₃partial agonist

lacking reinforcing properties, reduced cocaine-seeking

Universita´ di Siena.

^§Universita´ di Napoli Federico II.

Universita´ della Calabria.

^|Cardiff School of Biosciences, Cardiff University.

£

Istituto di Ricerche Farmacologiche “Mario Negri”.

Published on Web 07/26/2003

Highly Selective D₃Receptor Ligands

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3823

Ch a r t 1

Herein we report the synthesis and the pharmacologi-

cal and biochemical characterization of a new series of

selective and nonselective D₃receptor ligands. From this

series, we initially selected 5g (a nonselective D₃recep-

tor partial agonist), 5p (a highly selective and potent

D₃receptor partial agonist), and 5q (an extremely

potent and selective D₃receptor antagonist) and tested

their effects in the modulation of cocaine-seeking be-

havior induced by presentation of environmental stimuli

associated with and predictive of cocaine availability

following a period of extinction and in the absence of

any further cocaine. Additionally, the rationalization of

the structure-activity relationships by a molecular

modeling study is discussed.

Ch em istr y

The synthesis of compounds 5a -r (Table 1) is de-

scribed in Schemes 2-5. N-Arylpiperazines 7a ,b were

obtained as described in Scheme 1A starting from

piperazine 6 that was N-arylated with a substituted aryl

bromide through a palladium-catalyzed reaction.²⁵The

amine 10 was obtained, according to Scheme 1B, start-

ing from the methoxyphenylpiperazine 8 that was

alkylated with 2-bromoethanol to give the alcohol 9 that

was then transformed into the corresponding amine 10

in a three-step sequence using mesyl and azido inter-

mediates. The synthesis strategy followed to obtain

compounds 5a -h ,l-q is reported in Scheme 2; acids

11a ,c-i or the acid chloride 11b were transformed into

the corresponding hydroxy amides 12a ,c-i and 12b,

respectively, by a standard procedure.^26-28These latter,

after substitution of a halogen for the terminal hydroxy

group (13a -i),^29,30were treated with the arylpiperazine

in the presence of a base to give the desired products

5a -d ,g,h ,l-q, and 14a ,b.³¹Compounds 5e,f were then

obtained after deprotection of the amine of 14a ,b by

catalytic hydrogenation. The synthesis of 5r is shown

in Scheme 3. N-alkylation of the indole nitrogen (15)

by means of 2-bromoacetonitrile (16) followed by treat-

ment of 16 with cobalt boride and sodium borohydride

gave the cyclic lactam 17, which was N-alkylated with

1,4-dibromobutane in the presence of sodium hydride

to yield the bromo-derivative 18.^32-34This was treated

with 1-(2-methoxyphenyl)piperazine in the presence of

a base to give the desired product 5r . Scheme 4

describes the synthesis of 5i. Starting from acid 11a ,

the amide 19 was obtained by reaction with 4-meth-

oxycarbonylpiperidine in the presence of 1,3-dicyclo-

hexylcarbodiimide (DCC) and 1-hydroxybenzotriazole

(HOBT).²⁶Reduction of the ester function (20) and

bromination provided the intermediate 21 that was used

in the next step to obtain the desired product 5i. The

heterocyclic compounds 5j,k were obtained according to

Scheme 5. Starting from the acids 11a and 22, after

transformation to the corresponding amides by means

of thionyl chloride and liquid ammonia, and subsequent

formation of the oxazole ring system (23a ,b), using 1,3-

dichloroacetone,³⁵which were used as alkylating agents,

compounds 5j,k were prepared.

behavior maintained by cocaine in a second-order

schedule in rats.¹⁸Together with the recent finding that

3 reduced cocaine-seeking behavior induced by reintro-

duction of environmental stimuli associated with and

predictive of cocaine availability after a period of

abstinence,^17cthis strongly suggests that compounds

such as 3 could be used for reducing drug craving and

the vulnerability to relapse that are elicited by drug-

associated environmental stimuli. The mechanism un-

derlying this effect is unknown. However, the selective

D₃receptor antagonist SB-277011-A (4)¹⁹also reduced

seeking behavior maintained by cocaine in a second-

order schedule,²⁰suggesting that antagonists at D₃

receptors may be useful in controlling drug-seeking

behavior induced by cocaine-associated cues.

Although 3 was claimed to reduce the response to

cocaine cues through its D₃receptor interaction,¹⁸this

compound is characterized by a multireceptor affinity

profile. In fact, it shows a significant D₂receptor affinity,

together with a potent antagonism at hR_1Aand, to a

lesser extent, hR_2Aadrenoceptors and partial agonist

properties at h5-HT_1Areceptors.²¹Consequently, this

compound may not prove the hypothesis that only D₃

receptors are implicated in mechanisms by which

cocaine produced craving.

These considerations prompted us to develop a project

aimed at identifying novel, selective, and nonselective

D₃/D₂/5-HT_1Aligands (5, Chart 1) to produce compounds

inhibiting cocaine-seeking behavior and to investigate

the role of D₃receptors in cocaine craving. We presented

preliminary partial pharmacological results on one of

the newly developed compounds (the benzofuran ana-

logue 5g) at the XIII Meeting of the Italian Society of

Neuropsychopharmacology (Milan, 9-12 J uly 2002),²²

but while we were preparing this manuscript Gmeiner

and co-workers reported the synthesis, binding studies,

and intrinsic activity of this same analogue,²³and

Tortorella and co-workers reported the identification of

other potent D₃receptor ligands,²⁴structurally related

to our series 5a -r .

P h a r m a cologica l Stu d ies. Resu lts a n d

Discu ssion

1. Bin d in g Assa ys. The binding affinities for D₁, D₂,

D₃, 5-HT_1A, R₁and R₂receptors of compounds 5a -r (as

3824 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18

Ta ble 1. Physical and Chemical Data for Compounds 5a -r

Campiani et al.

a

b

Yields refer to isolated and purified materials. All the compounds were analyzed within (0.4% of the theoretical values.

Highly Selective D₃Receptor Ligands

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3825

Sch em e 1

Sch em e 3

Sch em e 4

Sch em e 2

(a ) Th e Key Role of Differ en t Heter oa r om a tic

System s on D₃Recep tor Affin ity a n d Selectivity.

The naphthalene system of 3 was replaced with differ-

ent heteroaromatic ring scaffolds, leading to extremely

potent D₃receptor ligands. Initially, the result of the

introduction of one or two heterocyclic nitrogen atoms

on the naphthalene ring was evaluated, obtaining the

quinoline analogue 5a and the quinoxaline 5b. When

di- or trihydrochloride salts) are given in Table 2. The

intrinsic activity of a selected subset of compounds at

D₃and 5-HT_1Areceptors was determined in [³⁵S]-GTPγS

binding assays (Figures 1 and 2). The results reported

in Table 2 are summarized as follows.

3826 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18

Campiani et al.

Ta ble 2. Binding Profile of Compounds 5a -r on D_1-3, 5-HT_1A, and Adrenergic Receptors (K_inM ( SD)^a

b

c

d

e

f

g

compd

D₁

D₂

D_3r

5-HT_1A

NT^h

alpha_1NA

alpha_2NA

5a

5b

5c

5d

5e

5f

5g

5h

5i

5j

5k

5l

1350 ( 114

1440 ( 41

1480 ( 146

778 ( 128

NT

83 ( 5

84 ( 2

75 ( 12

59 ( 9

NT

4.8 ( 0.05

11 ( 0.1

0.49 ( 0.01

28 ( 1

NT

3.12 ( 0.31

5.80 ( 0.61

76 ( 12

NT

9.2 ( 0.2

4.3 ( 1.0

1.6 ( 0.5

2.9 ( 0.05

74 ( 6

NT

2565 ( 342

970 ( 71

2676 ( 282

.10 000

NT

1655 ( 461

7750 ( 808

7664 ( 743

>10 000

1567 ( 289

3000

87 ( 19

160 ( 19

244 ( 40

518 ( 193

86 ( 1

NT

333 ( 57

342 ( 53

409 ( 65

>10 000

45 ( 7

61

2.14 ( 0.16

11.8 ( 0.75

NT

36 ( 9

666 ( 106

NT

54 ( 3

76 ( 15

NT

759 ( 59

295 ( 24

41.3 ( 8

NT

5m

5n

5o

5p

5q

5r

7.8 (1.4

NT

0.66 ( 0.03

0.41 ( 0.05

0.18 ( 0.004

0.38( 0.005

0.87 ( 0.16

0.92

1050 ( 241

908 ( 236

>10 000

4.9 ( 1.0

84

629 ( 190

779 ( 181

547 ( 146

8100 ( 1590

33 ( 10

60

1047 ( 148

1887 ( 65

>10 000

89 ( 4

83

BP 897ⁱ

a

Each value is the mean ( SD of three determinations and represents the concentration giving half-maximal inhibition of [³H]-ligand

binding. [³H]-SCH 2339, rat striatum. ^c[³H]-spiperone, rat striatum. [³H]-7-OH-DPAT, Sf9 cells. ^e[³H]-8-OH-DPAT, rat hippocampus.

b

d

^f[³H]-prazosin, rat cortex. [³H]-RX 81002, rat cortex. NT ) not tested. Data from ref 18.

g

h

i

Sch em e 5

tested against dopamine receptors, both compounds

showed a similar low affinity for D₁receptors, an affinity

for D₂receptors in the high nanomolar range, and

nanomolar affinity for the D₃receptor, with the quino-

line analogue 5a being twice as potent (5a vs 5b).

Replacement of the quinoline of 5a by an isoquinoline

system led to a 10-fold increase in D₃receptor affinity

(5c vs 5a ) with a D₁/D₃affinity ratio of 3020 and a D₂/

D₃affinity ratio of 153. This selective D₃receptor ligand

was further tested on 5-HT_1A, R₁and R₂receptors. While

the R₂receptor affinity was similar to that for D₂

receptors, 5c proved to be a potent 5-HT_1Aand R₁ligand.

Introduction of a fused pyrrole ring (5d ) into the

structure of 5b, leading to a pyrroloquinoxaline system,

slightly decreased the D₃receptor affinity, but doubled

the D₁affinity (K_iD1) 778 nM, K_iD3) 28 nM). The

partially saturated 5c counterparts 5e and 5f were less

potent than 5c at D₃receptors (5e and 5f vs 5c), with

twice the stereoselectivity at D₃receptors, indicating a

scarce stereoselective interaction at D₃receptor for both

enantiomers. The naphthalene ring of 3 was also

replaced by bioisosteric ring systems, namely, the

benzofuran and indole. The benzofuran provided a

compound (5g) that was as potent as 3 at D₁, D₂, and

F igu r e 1. [³⁵S]-GTPγS binding to D_3rreceptors expressed in

Sf9 cells was carried out as described in Experimental Section.

Quinpirole, 5g, 5p , 5q, and haloperidol were used at 10 µM

and the values are the mean of three experiments (variability

less than 10%). For statistical analysis Anova and Tukey test

were used. #, P < 0.01 different from basal. §, P < 0.01

different from quinpirole. •, P < 0.01 different from 5g or 5q

alone.

D₃receptors, but this structural modification improved

the affinity for 5-HT_1A(K_i) 2.14 nM), R₁(K_i) 36 nM),

Highly Selective D₃Receptor Ligands

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3827

4 times less potent than 5g itself at D₂and D₃receptors;

introduction of chlorine atoms at positions 2 and 4 of

the aromatic ring led to the identification of extremely

potent and selective D₃receptor ligands. With respect

to the methoxy derivatives 5c, 5g, and 5h (K_iD3of 0.49,

1.6, and 2.9 nM, with a D₂/D₃affinity ratio of 153, 54,

and 55, respectively), the corresponding 2,4-dichlo-

rophenyl analogues 5n -p (K_iD30.66, 0.41, 0.18 nM, with

D₂/D₃affinity ratio of 518, 998, and >55 000, respec-

tively) showed a better selectivity profile for the D₃

receptor, and 5p could be considered one of the most

potent and selective D₃receptor ligand known to date.

These three compounds were also tested on 5-HT_1A

,

R₁and R₂receptors. While 5n and 5o showed low

affinity for the panel of receptors, 5p showed micromolar

affinity for R₁receptors and negligible affinity for 5-HT_1A

and R₂receptors. Receptor selectivity was improved by

introducing a chlorine (5q) atom at position 5 of the

indole system of 5p . The pattern of receptor affinities

of 5q is similar to that reported for 5p (for D₃receptors,

K_i5q) 0.38 nM vs K_i5p) 0.18 nM), although it has a

lower R₁receptor affinity. In summary, 5p and 5q are

extremely potent and selective D₃receptor ligands.

(d ) In tr in sic P h a r m a cologica l Activities. In [³⁵S]-

GTP-γS binding assays, the reference agonist quinpirole

increased basal binding with EC₅₀80 ( 15 nM, reaching

maximal stimulation at 1-10 µM (80% over basal

value). Compound 5g behaved as a partial agonist at

the cloned D₃receptor expressed on Sf9 cells (Figure

1A). Because of the low efficacy of 5g in enhancing [³⁵S]-

GTP-γS binding a complete dose-response curve could

not be obtained. However, at equimolar saturating

concentrations, it was significantly less active than

quinpirole in increasing [³⁵S]-GTP-γS binding, and its

effect was completely antagonized by haloperidol. In

addition, like haloperidol, it significantly reduced the

enhancing effect of quinpirole.

Similar results were obtained with 5q, which also

behaves as a partial agonist at D₃receptors (Figure 1B),

while 5p was a pure antagonist lacking intrinsic activity

(Figure 1C). Differently from 3, which behaves as a

5-HT_1Areceptor agonist, 5g and 5h behaved as antago-

nists at 5-HT_1Areceptors in the rat hippocampus

membrane, with K_iof 17.5 ( 1.8 and 23.2 ( 2.8 nM,

respectively (Figure 2).

F igu r e 2. Representative inhibition curves of (A) 5g and (B)

5h on agonist-stimulated [³⁵S]-GTPγS binding in rat hippo-

campus. Hippocampal homogenates were incubated in the

presence of 10 µM 5-HT and graded concentrations of 5g and

5h . Each point is the mean of triplicate values, varying by less

than 10%.

and R₂(K_i) 53.6 nM) receptors. The D₃receptor affinity

of the indole analogue 5h (K_i) 2.9 nM) and its D₂/D₃

affinity ratio was similar to that of 5g. However, 5h was

found less potent than 5g on the panel of receptors, with

the exception of D₁receptors.

(b) Mod ifica tion of th e Lin k er . Effect on D₃

Recep tor Affin ity. The selected modifications of the

linker were, in general, detrimental to receptor affinity.

The amide 4-methylene linker of 5g and 3 was modified

by constraining the alkyl chain in a six-membered ring

(5i) or by replacing the amide system with a 4-aminom-

ethyloxazole group (5j,k ). In the case of 5i, the introduc-

tion of the piperidine ring determined a 46-fold drop in

D₃receptor affinity with respect to 5g, while the oxazole

derivative 5j proved to be poorly active at dopamine

receptors. On the contrary, 5k demonstrated a D₂

receptor affinity similar to that of the parent compound

3, showing a dramatic drop in D₃receptor affinity (K_iD2

) 86 nM and K_iD3) 295 nM). It is noteworthy that

compound 5r , designed on the skeleton of 5h , is the only

conformationally constrained analogue showing a D₃

receptor subnanomolar affinity (5r , K_iD3) 0.87 nM) (see

Molecular Modeling section).

(c) Th e In flu en ce of th e Su bstitu en ts on th e

Ar ylp ip er a zin e Rin g System . In the next step of our

SAR study, we evaluated the effect on receptor affinity

and selectivity of halogen atoms or a cyano group on

the phenyl at N-4 of the piperazine ring, in relation to

the 2-methoxyphenylpiperazine scaffold of the previous

analogues.

A cyano group at C-4 of the phenyl ring caused D₃

receptor affinity to drop 26-fold (5l vs 5g), and chlorine

atoms at C-3 and C-4 provided an analogue (5m ) about

The EC₅₀of serotonin for enhancing [³⁵S]-GTP-γS

binding was 523 ( 160 nM, with maximum stimulation

(65% over basal value) at 10-30 µM. The effect of 5-HT

was completely abolished by 10 µM WAY 100635 (data

not shown).

2. Beh a vior a l Effects: Resu lts on 5g, 5p , a n d 5q.

(a ) Coca in e Self-Ad m in istr a tion , Con d ition in g,

Extin ction , a n d Rein sta tem en t P h a ses. In all the

experiments, the separate groups of rats developed

stable cocaine self-administration between 7 and 10

training days with no differences in responding during

the first and second hour of cocaine availability; the

number of lever-pressings during saline dropped gradu-

ally to less than five per session in 10 training days.

The number of days required to meet the training

criterion was the same for the different experimental

groups (mean ( SEM, 17.2 ( 0.6 to 19.3 ( 1.1). No

differences were observed during the final 3 days of the

training phase (P > 0.05, Newman-Keuls test) or

3828 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18

Campiani et al.

F igu r e 3. Effects of (A) 5g, (B) 5p , (C) 5q, and (D) 3 on the number of pressings on the active and inactive levers after

reintroduction of cocaine-associated stimuli. For comparison, the figure also shows the average number of lever pressings after

reintroduction of stimuli associated with no reward. Histograms indicate the mean ( SEM pressings on active and inactive levers

of at least seven rats. 5g, dissolved in sterile saline, 5p and 5q, dissolved in 25% DMSO in sterile 0.9% saline, or vehicles were

given i.p. 30 min before testing. Data for 3 are from ref 17, modified. F_5g(11,83) ) 15.0, P < 0.01; F_5g(11,83) ) 1.5, P > 0.05, for

active and inactive levers respectively, one-way ANOVA for repeated measurements. F_5p(13,78) ) 7.8, P < 0.01 and F_5p(13,78) )

1.6, P > 0.05 for active and inactive levers respectively, one-way ANOVA for repeated measures. F_5q(13,91) ) 7.8, P < 0.01 and

F

_5q(13,91) ) 2.1, P < 0.05 for active and inactive levers respectively, one-way ANOVA for repeated measures. *P < 0.01, different

from saline-associated cue presentation and the three preceding extinction sessions (data not shown, see Results for details),

Neuman-Keuls test. P < 0.01, different from the cocaine-associated cue presentation, Newman-Keuls test.

a

between the first and second daily hour of self-

administration (P > 0.05, mixed factorial or one-way

ANOVA for repeated measurement). The data for the

two daily cocaine sessions were pooled for all subsequent

analyses. During this phase responding on the inactive

lever was minimal in all experimental groups.

In our experiments, rats met the extinction criterion

after an average of 16.0 ( 1.6 sessions. No differences

were found between groups in the mean ( SEM number

of days to extinction, which ranged from 12.0 ( 1.8 to

18.6 ( 1.9 days. Responding on the inactive lever was

again negligible throughout the whole phase.

In all the experiments, the cocaine-associated stimuli

produced immediate recovery of responding that was

significantly higher than after the saline-associated cues

(P < 0.01 Newman-Keuls test) and compared to the

three preceding extinction sessions (data not shown, P

< 0.01 Newman-Keuls test). In all the experiments, the

overall behavioral output after presentation of the

cocaine-associated cues was similar to that during

cocaine self-administration (P > 0.05, Newman-Keuls

test) and significantly different from saline self-admin-

istration (P < 0.01, Newman-Keuls test). The number

of lever pressings during S presentation did not differ

from the three preceding extinction sessions (P > 0.05,

Newman-Keuls test). Since they had to meet the extinc-

tion criterion, lever pressing during the extinction

sessions, preceding the reintroduction of cocaine- and

saline-associated stimuli, did not differ between groups

in all the experiments.

Data are compared to 3 (D). Reintroduction of the cues

always increased the number of pressings on the active

lever [F_5g(11,83) ) 15.0, P < 0.01, F_5p(13,78) ) 7.8, P <

0.01 and F_5q(13,91) ) 8.9, P < 0.01, one-way ANOVA,

P < 0.01 vs saline-associated cues presentation and vs

the three preceding extinction days, data not shown,

Newman-Keuls test] but not on the inactive lever (P >

0.05 vs saline-associated cues presentation and vs the

three preceding extinction days, data not shown, New-

man-Keuls test). Pretreatment with 5g 3 mg/kg, but not

0.3 and 1 mg/kg, significantly reduced the number of

lever pressing induced by presentation of cocaine-

associated cues (P < 0.01 vs vehicle treated group);

moreover, the number of lever responses after 3 mg/kg

5g pretreatment with reintroduction of the drug-associ-

ated stimuli was no longer different from the saline-

associated cues presentation (P > 0.05, Newman-Keuls

test). 5g pretreatment did not affect the number of

inactive lever pressings.

Pretreatment with 0.1-1 mg/kg 5p did not modify

rats’ behavior after presentation of cocaine-associated

stimuli (P < 0.01 vs saline-associated cues presentation

and vs the three preceding extinction days, data not

shown, Newman-Keuls test). However, at 3 mg/kg 5p

partially reduced the number of active lever pressings

induced by reintroduction of the drug-associated stimuli

(P > 0.05 vs saline- and cocaine-associated cues, New-

man-Keuls test). The numbers of responses were no

longer different from those elicited by the saline-

associated stimulus, and not different from those in-

duced by cocaine-associated stimuli. There were no

effects on the number of pressings of the inactive lever.

5q did not modify the number of lever pressings on

the active lever (P < 0.01 vs saline-associated stimuli

presentation and vs the three preceding extinction days,

(b) Effects of 5g, 5p , a n d 5q on Cu e-In d u ced

Rein sta tem en t of Dr u g-Seek in g Beh a vior . Figure

3 shows the effects of 5g (A), 5p (B), and 5q (C), all used

as dihydrochloride salts, on cocaine-seeking behavior

induced by reintroduction of the drug-associated stimuli.

Highly Selective D₃Receptor Ligands

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3829

F igu r e 5. Effects of replacing cocaine (0.25 mg/0.1 mL/

infusion) with 5g (3 mg/mL, 0.1 mL per infusion) or saline (0.1

mL per infusion) on i.v. cocaine self-administration under a

FR1 schedule of reinforcement. Data are the mean ( SEM of

the number of active and inactive lever pressings by six rats

during daily 2 h sessions. The data were analyzed by the one-

way ANOVA for repeated measurements. F_{active lever}(1,10) ) 6.3,

P < 0.05, mixed one-way ANOVA for repeated measurements.

F_{inactive lever}(1,10) ) 4.4, P > 0.05, mixed one-way ANOVA for

repeated measurements. *P < 0.05, vs saline group, Newman-

Keuls test.

F igu r e 4. Effects of 5g or vehicle on self-administration of

two doses of cocaine. Rats were trained to respond for cocaine

(0.125 and 0.5 mg/0.1 mL/infusion) under a fixed ratio 1 (FR1)

schedule of reinforcement. Once baseline was stable, rats

received i.p. 5g, dissolved in 2 mL of sterile saline, or vehicle,

30 min before the 2 h test session. Histograms present the

mean ( SEM number of self-administered infusions during a

2 h test session. The data were analyzed by one-way ANOVA

for repeated measures followed by Dunnett’s test. F_0.125

Active

^lever(2,23) ) 1.8, P > 0.05; F_{0.125 Inactive lever}(2,23) ) 1.8, P > 0.05;

F

_{0.5 Active lever}(2,23) ) 0.6, P > 0.05; F_{0.5 Inactive lever}(2,23) ) 0.4, P

drug-seeking behavior induced by cocaine predictive

cues even after prolonged abstinence. This effect could

not be attributed to drug-induced disruption of behavior

since in no instance were any signs of sedation or motor

disturbance observed that could have interfered with

the behavioral measurements. Also, responses on the

inactive levers were not affected by 5g. It cannot be

excluded that the low rate of responding on the inactive

lever prevented us from detecting some subtle motor

impairment. The fact that 5g reduced rats’ behavior

elicited by cues associated with and predictive of cocaine

availability, with no effect on lever pressing for cocaine,

makes it unlikely that 5g disrupted the rats’ behavior.

> 0.05, one-way ANOVA for repeated measures.

data not shown, Newman-Keuls test). But treatment did

have some effect on the number of inactive lever

pressings [F(13,91) ) 2.1, P < 0.05, one-way ANOVA].

Post-hoc comparison by the Newman-Keuls test, how-

ever, did not reveal any significant effect of 5q at any

dose on reintroduction of cocaine-associated stimuli (P

> 0.05).

(c) Effect of 5g on Coca in e Self-Ad m in istr a tion .

Figure 4 shows the effects of acute pretreatment with

0.3 and 3 mg/kg 5g or vehicle on cocaine self-adminis-

tration under a continuous reinforcement schedule. One-

way ANOVA for repeated measurements found 5g had

no significant effect on either 0.125 or 0.5 mg/0.1 mL/

infusion cocaine [F_{0.125 Active lever}(2,23) ) 1.8, P > 0.05;

This rather supports the idea that nonselective D₃

partial agonists specifically modulate drug-seeking be-

havior induced by cocaine-associated cues,¹⁸as con-

firmed by our recent findings,^17cby which, using the

same experimental procedure as in the present study,

that 3, a nonselective D₃partial agonist, reduced

cocaine-seeking behavior induced by reintroduction of

cocaine-associated stimuli (Figure 3D). Moreover, the

effects of 5g seem to be specific on rats’ behavior elicited

by cues-associated with and predictive of cocaine avail-

ability but not on cocaine primary reinforcing properties.

It also seems unlikely that 5g substituted for cocaine’s

effects, reducing the motivating action of cocaine-

predictive stimuli since 5g, at a dose reducing drug-

seeking behavior (3 mg/kg), did not substitute for

cocaine in the self-administration paradigm. This selec-

tive effect on the behavioral consequences of exposure

to cocaine-associated stimuli may imply dissociable

neural mechanisms underlying responding with condi-

tioned reinforcement and responding for cocaine it-

self.^12,14,36The possibility of antagonism at the 5-HT_1A

receptors being involved in the effect of 5g seems to be

excluded by recent findings that WAY 100625, a selec-

tive 5-HT_1Aantagonist,³⁷was completely inactive on

rats’ behavior elicited by reintroduction of cocaine-

associated stimuli.¹⁷The role of R₁- and R₂-adrenocep-

F_0.125

(2,23) ) 3.3, P > 0.05; F_{0.5 Active lever}(2,23)

Inactive lever

) 0.6, P > 0.05; F_{0.5 Inactive lever}(2,23) ) 0.4, P > 0.05].

(d ) Effects of Rep la cin g Coca in e w ith 5g or

Veh icle on Coca in e Self-Ad m in istr a tion . Figure 5

reports the effects of replacing cocaine with 5g (0.3 mg/

0.1 mL/infusion) or saline (0.1 mL/infusion) on i.v.

cocaine self-administration. The number of infusions

earned by the two groups of rats did not differ during

the 3 days before the cocaine substitution (P > 0.05,

Newman-Keuls test). When cocaine was replaced with

5g or saline treatment had an effect on the number of

active lever pressings [F(1,10) ) 6.3, P < 0.05, mixed

one-way ANOVA for repeated measurements] but not

on the inactive ones [F(1,10) ) 4.4, P > 0.05, mixed one-

way ANOVA for repeated measurements]. Post-hoc

comparisons by the Newman-Keuls test found that 5g

induced a steeper decrease in the number of active lever

pressing on the first and third substitution days (P <

0.05). Resubstituting cocaine for 5g or saline resulted

in prompt reinstatement of drug self-administration in

both groups.

3. Beh a vior a l Stu d ies: Discu ssion . Compound 5g,

a new dopamine D₃partial agonist with 50 times the

selectivity toward D₂receptors, significantly attenuated

3830 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18

Campiani et al.

Ta ble 3. Calculated Energy Differences, Expressed in kcal/

mol, between the Syn and Anti Conformers of the

Nitrogen-Containing Aromatic System of 5a , 5c, and 5h

∆E_syn-anti(kcal/mol)

compd

MM ꢀ ) 1

MM ꢀ ) 80*r

AM1

5a

5c

5h

8.88

8.85

-4.28

0.87

0.03

a

-2.06

a

∆E_syn-anticannot be measured for the absence of the syn

conformer (see text).

F igu r e 6. x, y, w, and z indicate the atoms defining the torsion

angle τ in the general structure of compounds 5a ,c,h .

tors in drug-seeking behavior elicited by presentation

of cocaine-associated stimuli is not known.

A partial reduction of cocaine-seeking behavior was

also observed with the selective D₃antagonist 5p . In

no instance were any effects observed that could have

interfered with the measurement of rats’ behavior. This

result is in agreement with a recent report that 4, a

potent and selective D₃antagonist¹⁹that crosses the

blood-brain barrier, reduced seeking behavior main-

tained by cocaine in a second-order schedule.²⁰

However, differently from 5g, the highly potent and

selective partial agonist 5q did not have any effect

against cocaine-seeking behavior. Although brain up-

take studies are needed to establish whether the

compounds achieve brain concentrations comparable to

those active in vitro on D₃receptors, these results,

together with previous findings,¹⁷suggest that antago-

nism at D₂receptors might significantly contribute to

the activity of partial D₃agonists, like 5g and 3, in

reducing cocaine craving.

4. Molecu la r Mod elin g Stu d ies. Th e Design of 5r .

The aim of this investigation was to identify the

conformational features of the structures of our leads

able to improve D₃receptor affinity maintaining a

significant D₂receptor interaction, to design potential

therapeutic agents against cocaine craving. We analyzed

F igu r e 7. Comparison of the heteroarylcarboxamide moieties

of 5a (yellow), 5c (green), and 5h (orange) in their anti

conformation. Superimposition was obtained by fitting: (i) the

amide nitrogen, (ii) the carbonyl carbon, (iii) the aromatic C-1

carbon, (iv) the aromatic nitrogen. Heteroatoms are colored

by atom type (N blue, O red).

Table 3). On the contrary, 5h showed a conformational

preference for the syn orientation of τ when MM

calculations were performed in a vacuum (Table 3).

MM conformers of 5a , 5c, and 5h have been used as

starting structures for a full AM1 semiempirical geom-

etry optimization. The results obtained confirmed the

lower potential energy of the syn conformer of 5h (Table

3), and evidenced a conformational instability of the syn

conformers of 5a and 5c. In particular, during AM1

calculation, the anti conformers maintained their start-

ing geometry, while the syn conformers rotated to τ )

-161° and τ ) -115° (5a and 5c, respectively), indicat-

ing a propensity to turn into the anti conformers. This

conformational behavior can be explained by the elec-

trostatic repulsion occurring between the aromatic

nitrogen and the carbonyl group of 5a and 5c, and, to a

lesser extent, by the steric overlap between the amide

hydrogen and the hydrogen at C3 (5a ) and C4 (5c) of

the heteroaromatic ring. On the contrary, the indole NH

group of compound 5h tends to assume a syn orientation

with respect to the carbonyl group since, in this case,

the syn conformer presents less dipole repulsion and,

therefore, it is energetically favored. The strong influ-

ence of electrostatic interactions in the conformational

behavior of 5a , 5c, and 5h is confirmed by the different

results obtained varying the dielectric constant in MM

calculations, and using AM1 semiempirical method

(Table 3). Since a low dielectric medium more closely

resembles the interior of a protein, it is likely that 5a

and 5c bind to D₃receptor assuming an anti conforma-

tion of τ, and 5h assuming a syn conformation.

compounds 5a (D₃, K_i) 4.8 nM, D₂, K_i) 83 nM, K_iD2

Ki_D3) 17), 5c (D₃, K_i) 0.49 nM, D₂, K_i) 75 nM, K_iD2

/

K_iD3) 153), and 5h (D₃, K_i) 2.9 nM, D₂, K_i) 160 nM,

_iD2/K_iD3) 55). Since the structures of these compounds

K

differ solely in the nature of the nitrogen-containing

heteroaromatic system, this study focused on the con-

formational analysis of the heteroaromatic carboxamide

moiety by using molecular mechanics (MM) and semiem-

pirical (MOPAC, AM1) calculations.

As expected, MM energy minimizations indicated the

trans form of the amide bond as the energetically

favored, and AM1 semiempirical calculations confirmed

these results (data not shown). To investigate the

orientation of the heteroaromatic rings, with respect to

the trans amide bond, we analyzed the rotation around

the torsion angle τ (5a ,c,h , Figure 6). It has to be

underlined that the conjugation effect between the

amide bond and the heteroaromatic moiety limits the

rotation about the considered angle. Accordingly, MM

energy minimizations identified two different minima

corresponding to τ ∼ 0° and τ ∼ 180°, herein called the

syn and anti conformers, respectively. In the case of 5a

and 5c the anti conformers resulted to be energetically

In Figure 7 is reported the superimposition of the

heteroaromatic carboxamide moieties of 5a , 5c, and 5h ,

considered in their anti conformations. It is noteworthy

the extra volume occupied by 5a (D₃, K_i) 4.8 nM) with

favored, with an increased value of ∆E (E_syn- E_anti

)

when the calculations were carried out in a vacuum (ꢀ

) 1) rather than in an aqueous environment (ꢀ ) 80,

Highly Selective D₃Receptor Ligands

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3831

respect to 5c (D₃, K_i) 0.49 nM); considering their close

structural similarity, we supposed that the unfavorable

orientation of the bicyclic system of 5a is responsible

for its lower D₃receptor affinity. On the other hand,

the anti conformer of 5h (D₃, K_i) 2.9 nM) showed a

good overlap with the heteroaromatic carboxamide

moiety of 5c (Figure 7), but resulted to be energetically

disfavored with respect to the syn conformer (Table 3),

in which the nitrogen of the indole ring did not fit the

nitrogen of 5c. On this basis, to improve D₃receptor

affinity, we designed an ethylene bridge between the

indole and the amide nitrogen of 5h to constrain τ in

the anti orientation (5r ). Accordingly, compound 5r

showed an increased D₃receptor affinity with respect

to 5h (K_i5r) 0.87 nM vs K_i5h) 2.9 nM), very similar to

that of 5c (K_i) 0.49 nM) while showing a better D₂/D₃

may be essential for its activity. This seems to apply

only for partial D₃agonists, since compound 4, a

selective brain penetrating D₃receptor antagonist,¹⁹

reduced drug-seeking behavior in rats,²⁰and similar

though partial, results were obtained in the present

study with 5p , a potent and selective D₃receptor

antagonist. On these basis, through a molecular model-

ing study, we designed the conformationally constrained

analogue 5r , characterized by a similar binding profile

to 5g, with a 2-fold higher affinity at D₂and D₃

receptors, thus being a lead structure for the generation

of a new series of D₂/D₃receptor ligands.

In conclusion, the results suggest that 5g, a partial

agonist at D₃receptors with significant affinity for D₂

receptors, and 5p , a potent and selective D₃antagonist,

could be useful in the pursuit of medications for drug-

seeking behavior induced by environmental stimuli

associated with cocaine.

receptor affinity ratio (5c K_iD2/K_iD3) 153 vs 5r K_iD2

/

K_iD3) 52). With respect to 5h , the constrained analogue

5r showed a 3-fold higher affinity at D₃receptors, with

D₁/D₃and D₂/D₃affinity ratios of 1801 and 52, respec-

tively (for 5h : D₁/D₃and D₂/D₃affinity ratios of 334 and

55, respectively). While the affinity for 5-HT_1Aand R₂

receptors was similar to 5h , 5r was found to be 3- and

20-fold more potent than 5h at D₂and R₁receptors,

respectively. With respect to 5g (a compound active

against cocaine craving), 5r showed the same D₁/D₃and

D₂/D₃affinity ratios and a similar pattern of R₁, R₂, and

5-HT_1Areceptor affinity. Consequently, 5r represents

an interesting lead for the generation of a new series of

D₂/D₃receptor ligands potentially active against cocaine

craving, and it is a candidate for future in vivo studies.

Exp er im en ta l Section

Melting points were determined using an Electrothermal

8103 apparatus. IR spectra were taken with Perkin-Elmer 398

and FT 1600 spectrophotometers. ¹H NMR spectra were

recorded on a Bruker 200 MHz spectrometer with TMS as

internal standard; the value of chemical shifts (δ) are given

in ppm and coupling constants (J ) in Hertz (Hz). All reactions

were carried out in an argon atmosphere. GC-MS were

performed on a Saturn 3 (Varian) or Saturn 2000 (Varian) GC-

MS System using a Chrompack DB5 capillary column (30 m

× 0.25 mm i.d.; 0.25 µm film thickness). Mass spectra were

recorded using a VG 70-250S spectrometer. ESI-MS and

APCI-MS spectra were taken by a LCQDeca-Thermofinnigan

spectrometer. Optical rotations were recorded on a Perkin-

Elmer model 343 polarimeter at the sodium D line at 20 °C.

Elemental analyses were done on

a Perkin-Elmer 240C

Con clu sion s

elemental analyzer and the results were within 0.4% of the

theoretical values, unless otherwise noted. Yields refer to

purified products and are not optimized. For testing, com-

pounds 5a -r were transformed into the corresponding hydro-

chloride salts by a standard procedure.

A series of novel arylalkylpiperazines was synthesized

and evaluated in D₁, D₂, D₃, 5-HT_1A, R₁and R₂receptor

binding assays. SAR studies delineated a number of

structural features required for high D₃receptor affinity

and selectivity. Several heteroaromatic systems were

explored for their dopamine, serotonin, and noradrena-

line receptor affinities, and novel structural leads were

identified for the development of potent and selective

D₃receptor ligands. An indole ring coupled to a 2,4-

dichlorophenylpiperazine ring system led to two of the

most potent and selective D₃receptor ligands known to

date (5p and 5q).

We also assessed intrinsic pharmacological properties

of a subset of potent D₃receptor ligands were also

assessed in [³⁵S]-GTPγS binding assays. Since it was

recently suggested that in vivo activity of 3 could not

be attributed just to a mediated response, we investi-

gated the effects of 5g, a partial D₃agonist and 5-HT_1A

antagonist with low receptor selectivity, 5q, a partial

D₃receptor agonist with extremely high D₃receptor

affinity and selectivity, and 5p , a highly selective and

very potent D₃receptor antagonist, on cocaine-seeking

behavior induced by reintroduction of cocaine-associated

stimuli after a long period of abstinence and in the

absence of any further cocaine. While 5g had a phar-

macological profile similar to 3, 5q had no effect on

cocaine-seeking behavior. Although brain uptake studies

are needed to establish whether the compounds achieve

brain concentrations comparable to those active in vitro,

these results, though only preliminary, do suggest that

the D₂component of the receptor binding profile of 5g

1-(2,4-Dich lor op h en yl)p ip er a zin e (7a ). A mixture of

1-bromo-2,4-dichlorobenzene (1.0 g, 4.4 mmol), piperazine (1.14

g, 13.3 mmol), sodium tert-butoxide (0.59 g, 6.2 mmol), tris-

(dibenzylideneacetone)dipalladium-(0) (10.13 mg, 0.01 mmol),

and BINAP (20.7 mg, 0.03 mmol) in anhydrous toluene (20

mL) was heated to 80 °C under argon. After the sample was

stirred for 2 h, the mixture was allowed to cool to room

temperature, taken up in ethyl ether (30 mL), filtered, and

concentrated. The crude product was then purified by flash

chromatography (20% methanol in chloroform) to give 0.65 g

(65% yield) of 7a as a yellow oil; ¹H NMR (CDCl₃) δ 2.60 (br s,

1H), 3.14 (m, 8H), 6.87 (d, 1H, J ) 8.7 Hz), 7.09 (dd, 1H, J )

8.5, 2.3 Hz), 7.26 (d, 1H, J ) 2.5 Hz). GC-MS m/z 230 [M]⁺,

188, 172, 149 (100). Anal. (C₁₀H₁₂Cl₂N₂) C, H, N.

1-(4-Cya n op h en yl)p ip er a zin e (7b). Starting from 4-bro-

mobenzonitrile (1.2 g, 6.6 mmol), the title compound was

obtained following the above-described procedure as a yellow

oil (90% yield): IR (CHCl₃) 2218 cm^-1; ¹H NMR (CDCl₃) δ 1.69

(br s, 1H), 2.99 (m, 4H), 3.26 (m, 4H), 6.83 (m, 2H), 7.47 (m,

2H). Anal. (C₁₁H₁₃N₃) C, H, N.

1-(2-Hydr oxyeth yl)-4-(2-m eth oxyph en yl)piper azin e (9).

2-Bromoethanol (0.32 mL, 4.6 mmol) was added to a vigorous

stirred mixture of 1-(2-methoxyphenyl)piperazine hydrochlo-

ride (1.0 g, 4.4 mmol) and anhydrous potassium carbonate

(1.50 g, 10.9 mmol) in dry acetonitrile (10 mL), and then the

suspension was refluxed for 10 h under argon. After that time,

the suspension was filtered and concentrated. The crude

product was chromatographed (20% methanol in chloroform)

1

to give 9 (0.34 g, 34% yield) as a yellow oil; H NMR (CDCl₃)

δ 2.58 (t, 2H, J ) 5.4 Hz), 2.70 (m, 4H), 2.95 (br s, 1H), 3.09

(m, 4H), 3.65 (t, 2H, J ) 5.4 Hz), 5.83 (s, 3H), 6.94 (m, 4H).

Anal. (C₁₃H₂₀N₂O₂) C, H, N.

3832 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18

Campiani et al.

1-(2-Am in oeth yl)-4-(2-m eth oxyp h en yl)p ip er a zin e (10).

1-(2-Methanesulfonylethyl)-4-(2-methoxyphenyl)piperazine. A

solution of methanesulfonyl chloride (0.41 mL, 0.54 mmol) in

dry dichloromethane (2 mL) was added dropwise to a cooled

solution (0 °C) of 9 (127.0 mg, 0.54 mmol) and triethylamine

(0.11 mL, 0.80 mmol) in dry dichloromethane (5 mL) under

argon. The resulting solution was warmed to room tempera-

ture and stirred for 12 h. After quenching of the sample with

water (7 mL), the mixture was extracted with ethyl acetate.

The organic layers were dried and evaporated. Chromatogra-

phy of the crude product (10% methanol in chloroform) gave

118.0 mg of the methanesulfonyl derivative (70% yield) as an

amorphous solid; ¹H NMR (CDCl₃) δ 2.78 (m, 6H), 3.02 (s, 3H),

3.10 (m, 4H), 3.64 (t, 2H, J ) 6.9 Hz), 3.85 (s, 3H), 6.93 (m,

4H). Anal. (C₁₄H₂₂N₂O₄S) C, H, N.

mmol), the title compound was prepared following the proce-

dure described for 12a . The product 12d was obtained as

1

colorless prisms (93% yield): mp (methanol) 108-109 °C; H

NMR (CDCl₃) δ 1.67 (m, 4H), 3.52 (q, 2H, J ) 11.5, 5.6) 3.72

(t, 2H, J ) 5.8 Hz), 6.65 (br s, 1H), 6.82 (s, 1H), 7.11 (d, 1H, J

) 8.0 Hz), 7.29 (m, 1H), 7.39 (d, 1H, J ) 7.7 Hz), 7.59 (d, 1H,

J ) 7.8 Hz), 9.25 (br s, 1H). Anal. (C₁₃H₁₆N₂O₂) C, H, N.

N-[1-(4-Hyd r oxy)bu tyl]qu in olin e-2-ca r boxa m id e (12e).

Starting from 2-quinolinecarboxylic acid 11e (0.5 g, 2.88

mmol), the title compound was prepared following the proce-

dure described for 12a . The product 12e was obtained as

1

colorless prisms (99% yield): mp (methanol) 131-132 °C; H

NMR (CDCl₃) δ 1.59 (m, 4H), 3.45 (m, 2H), 3.58 (m, 2H), 7.50

(m, 3H), 7.91 (d, 1H, J ) 8.3 Hz), 8.13 (m, 2H), 8.34 (br s, 1H).

Anal. (C₁₄H₁₆N₂O₂) C, H, N.

1-(2-Azid oeth yl)-4-(2-m eth oxyp h en yl)p ip er a zin e. To a

solution of the above-described methanesulfonyl-intermediate

(1.0 g, 3.2 mmol) in dimethyl sulfoxide (15 mL), sodium azide

(248.0 mg, 3.81 mmol) was added and the mixture was heated

to 45 °C with stirring for 15 h. After cooling of the sample to

room temperature, the solution was quenched with water (20

mL) and the mixture was extracted with diethyl ether. The

organic layers were dried and evaporated. The residue was

chromatographed (ethyl acetate) to give the azido-derivative

N-[1-(4-Hydr oxy)bu tyl]isoqu in olin e-3-car boxam ide (12f).

The title compound was prepared starting from 3-isoquinoli-

necarboxylic acid 11f (100.0 mg, 0.57 mmol) and following the

procedure as described to obtain 12a . 12f was obtained as

1

colorless prisms (96% yield): mp (methanol) 126-127 °C; H

NMR (CDCl₃) δ 1.74 (m, 4H), 3.57 (q, 2H, J ) 12.7, 6.3 Hz),

3.73 (t, 2H, J ) 5.9 Hz), 7.40 (m, 2H), 7.75 (m, 2H), 8.39 (br s,

1H), 8.57 (s, 1H), 9.14 (s, 1H). Anal. (C₁₄H₁₆N₂O₂) C, H, N.

(S)-(-)-N-[4-(1-Hyd r oxy)bu tyl]-2-(ben zyloxyca r bon yl)-

1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r boxa m id e (12g). The

title compound was prepared starting from (S)-(-)-2-(benzyl-

oxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

11g (190.0 mg, 0.61 mmol) and following the procedure as

described to obtain 12a . 12g was obtained as a yellow oil (97%

yield); ¹H NMR (CDCl₃) δ 1.23 (m, 4H), 1.83 (br s, 1H), 3.02-

3.44 (m, 6H), 4.61-4.78 (m, 3H), 5.20 (s, 2H), 6.20 (br s, 1H),

7.18 (m, 4H), 7.34 (m, 5H); ESI-MS m/z 405 [M + Na]⁺; ESI-

MS/MS of [M + Na]⁺361 (100), 269, 253, 230, 175. Anal.

1

(94% yield) as a yellow oil; H NMR (CDCl₃) δ 2.72 (m, 6H),

3.12 (m, 4H), 3.40 (t, 2H, J ) 5.8), 3.85 (s, 3H), 6.93 (m, 4H);

GC-MS m/z 262 [M + H]⁺, 219, 205 (100), 190, 175, 162, 150,

134, 121. Anal. (C₁₃H₁₉N₅O) C, H, N.

The azido-derivative (0.7 g, 2.7 mmol) was dissolved in dry

methanol (10 mL) in the presence of dry triethylamine (1.12

mL, 8.0 mmol) and then 1,3-propanedithiol (0.81 mL, 8.0

mmol) was added. The solution was stirred at room temper-

ature for 3 days and the white precipitate formed was filtered

off and the solution was concentrated. The oily residue was

recrystallized to give 620.0 mg of 10 (98% yield) as an

amorphous solid that was used in the next step without further

purification. For an analytical sample: mp (methanol) 76-77

°C; ¹H NMR (DMSO-d₆) δ 2.17 (t, 2H, J ) 6.3 Hz), 2.35 (t, 2H,

J ) 2.3 Hz), 2.48 (m, 4H), 2.92 (m, 4H), 3.73 (s, 3H), 6.88 (m,

4H). Anal. (C₁₃H₂₁N₃O) C, H, N.

N-[1-(4-Hyd r oxy)bu tyl]ben zo[b]fu r a n -2-ca r boxa m id e

(12a ). To a solution of 2-benzofurancarboxylic acid 11a (0.50

g, 3.08 mmol) in dry dichloromethane, 1-hydroxybenzotriazole

hydrate (0.46 g, 3.4 mmol) and 1,3-dicyclohexylcarbodiimide

(0.7 g, 3.4 mmol) were added at 0 °C under argon; the

suspension was warmed to room temperature and stirred for

1 h. Then 4-amino-1-butanol (0.28 mL, 3.08 mmol) was added

and the mixture was stirred overnight at room temperature.

The resulting suspension was filtered through Celite, washed

with chloroform, and the filtrate evaporated. The crude product

was purified by flash chromatography (10% methanol in

chloroform) to give 0.7 g (97% yield) of 12a as colorless

prisms: mp (methanol) 95-96 °C; ¹H NMR (CDCl₃) δ 1.67 (m,

4H), 2.14 (br s, 1H), 3.53 (m, 2H), 3.73 (m, 2H), 6.89 (br s,

(C₂₂H₂₆N₂O₄) C, H, N. [R] ²⁰-3.43 (c 2.04, CHCl₃).

D

(R)-(+)-N-[4-(1-Hyd r oxy)bu tyl]-2-(ben zyloxyca r bon yl)-

1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r boxa m id e (12h ). The

title compound was prepared starting from (R)-(+)-2-(benzyl-

oxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

11h (250.0 mg, 0.80 mmol) and following the procedure as

described to obtain 12a . 12h was obtained as a yellow oil (98%

yield); ¹H NMR (CDCl₃) δ 1.23 (m, 4H), 1.83 (br s, 1H), 3.02-

3.44 (m, 6H), 4.61-4.78 (m, 3H), 5.20 (s, 2H), 6.20 (br s, 1H),

7.18 (m, 4H), 7.34 (m, 5H). Anal. (C₂₂H₂₆N₂O₄) C, H, N. [R] ²⁰

D

+3.43 (c 3.26, CHCl₃).

N-[1-(4-H yd r oxy)b u t yl]-5-ch lor oin d ole-2-ca r b oxa m -

id e (12i). Starting from 5-chloroindole-2-carboxylic acid 11i

(300.0 mg, 1.53 mmol), the title compound was prepared

following the procedure described for 12a . 12i was obtained

as colorless prisms (90% yield): mp (methanol) 102-103 °C;

¹H NMR (CD₃OD) δ 1.61 (m, 4H), 3.40 (t, 2H, J ) 6.6 Hz),

3.59 (t, 2H, J ) 5.8 Hz), 6.95 (s, 1H), 7.14 (dd, 1H, J ) 8.8, 1.9

Hz), 7.34 (d, 1H, J ) 8.7 Hz), 7.53 (d, 1H, J ) 1.7 Hz); GC-MS

m/z 266 [M]⁺, 194, 178 (100), 150, 123, 114, 88, 70. Anal.

(C₁₃H₁₅ClN₂O₂) C, H, N.

N-[1-(4-Br om o)b u t yl]b en zo[b]fu r a n e-2-ca r b oxa m id e

(13a ). To a stirred solution of 12a (0.50 g, 2.14 mmol) in

acetonitrile (25 mL), triphenylphosphine (0.86 g, 3.22 mmol)

and carbon tetrabromide (1.06 g, 3.22 mmol) were added at

room temperature. After 2 h, the mixture was quenched with

15% NaOH and extracted with ethyl acetate. The organic

layers were dried and evaporated. The residue was chromato-

graphed (20% n-hexane in ethyl acetate) to afford 0.58 g (91%

yield) of 13a as colorless prisms: mp (ethyl acetate) 65-66

°C; ¹H NMR (CDCl₃) δ 1.67 (m, 4H), 3.37 (m, 4H), 7.36 (m,

4H), 7.63 (d, 1H, J ) 7.7 Hz); GC-MS m/z 297 [M + H]⁺, 216

(100), 202, 188, 174, 161, 145, 118, 89. Anal. (C₁₃H₁₄BrNO₂)

C, H, N.

1H), 7.25-7.48 (m, 4H), 7.63 (d, 1H, J ) 7.7 Hz). Anal. (C₁₃H₁₅

NO₃) C, H, N.

-

N-[1-(4-Hydr oxy)bu tyl]qu in oxalin e-2-car boxam ide (12b).

Starting from quinoxaline-2-carboxylic acid chloride 11b (166.0

mg, 0.86 mmol), the title compound was prepared following

the above-described procedure, and it was obtained as colorless

prisms (40% yield): mp (methanol) 138-139 °C; ¹H NMR

(CDCl₃) δ 1.55 (m, 4H), 3.53 (m, 2H), 3.63 (t, 2H, J ) 6.5 Hz),

7.24 (s, 1H), 7.84 (m, 2H), 8.14 (m, 3H), 9.64 (br s, 1H). Anal.

(C₁₃H₁₅N₃O₂) C, H, N.

N-[1-(4-H yd r oxy)b u t yl]p yr r olo[1,2-a ]q u in oxa lin e-4-

ca r boxa m id e (12c). The title compound was prepared start-

ing from pyrrolo[1,2-a]quinoxaline-4-carboxylic acid 11c (100.0

mg, 0.47 mmol) and following the procedure as described for

12a . 12c was obtained as a yellow oil (47% yield); ¹H NMR

(CDCl₃) δ 1.68 (m, 4H), 2.45 (br s, 1H), 3.62 (m, 2H), 3,93 (m,

3H), 6.91 (m, 1H), 7.45 (m, 2H), 7.79 (m, 4H) 8.21 (br s, 1H).

Anal. (C₁₆H₁₇N₃O₂) C, H, N.

N-[1-(4-Br om o)bu tyl]qu in oxalin e-2-car boxam ide (13b).

The title compound was prepared starting from 12b (84.0 mg,

0.34 mmol) and following the above-described procedure. 13b

1

was obtained as a yellow oil (38% yield); H NMR (CDCl₃) δ

1.93 (m, 4H), 3.48 (t, 2H, J ) 5.9 Hz), 3.61 (m, 2H, J ) 6.3

N-[1-(4-H yd r oxy)b u t yl]in d ole-2-ca r b oxa m id e (12d ).

Starting from 2-indolecarboxylic acid 11d (150.0 mg, 0.93

Hz), 7.83 (m, 2H), 8.09 (m, 3H), 9.52 (br s, 1H). Anal. (C₁₃H₁₄

BrN₃O) C, H, N.

-

Highly Selective D₃Receptor Ligands

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3833

N-[1-(4-Br om o)bu tyl]p yr r olo[1,2-a ]qu in oxa lin e-4-ca r -

boxa m id e (13c). To a solution of 12c (60.0 mg, 0.22 mmol)

in acetonitrile (3 mL), triphenylphosphine (86.0 mg, 0.32

mmol), and carbon tetrabromide (106 mg, 0.32 mmol) were

added under vigorous stirring at room temperature. After 5

h, the mixture was quenched with 15% NaOH and extracted

with ethyl acetate. The organic layers were dried and evapo-

rated. The residue was chromatographed (50% n-hexane in

ethyl acetate) to afford 29.0 mg (38% yield) of 13c as a yellow

oil; ¹H NMR (CDCl₃) δ 1.94 (m, 4H), 3.51 (m, 4H), 6.91 (m,

The methanesulfonyl intermediate (340.0 mg, 0.99 mmol)

was suspended in dry tetrahydrofuran (5 mL) and lithium

iodide (200.0 mg, 1.48 mmol) was added in portions. The

suspension was stirred under argon in an ultrasound bath at

40 °C. After stirring of the sample for 5 h, the solvent was

removed under reduced pressure, water was added, and the

mixture was extracted with ethyl acetate. The organic layers

were collected, dried, concentrated and the crude product was

chromatographed (10% methanol in chloroform) to give 0.37

g of pure 13i (99% yield) as colorless prisms: mp (methanol)

124-125 °C; ¹H NMR (acetone-d₆) δ 1.60 (m, 2H), 1.79 (m,

2H), 3.19 (m, 2H), 3.31 (m, 2H), 6.86 (s, 1H), 7.19 (m, 1H),

7.40 (m, 2H), 7.75 (m, 1H). Anal. (C₁₃H₁₄ClIN₂O) C, H, N.

1H), 7.52 (m, 2H), 7.83 (m, 4H), 8.21 (br s, 1H). Anal. (C₁₆H₁₆

BrN₃O) C, H, N.

-

N-[1-(4-Br om o)bu tyl]in d ole-2-ca r boxa m id e (13d ). The

title compound was prepared starting from 12d (170.0 mg, 0.73

mmol) and following the procedure described to obtain 13a .

13d was obtained as colorless prisms (84% yield): mp (ethyl

acetate) 133-134 °C; ¹H NMR (CDCl₃) δ 1.96 (m, 4H), 3.56

(m, 4H), 7.28 (m, 5H), 7.60 (d, 1H, J ) 7.6 Hz), 9.80 (br s, 1H).

Anal. (C₁₃H₁₅BrN₂O) C, H, N.

N-[1-(4-Br om o)bu tyl]qu in olin e-2-ca r boxa m id e (13e).

The title compound was prepared starting from 12e (0.7 g, 2.86

mmol) and following the procedure described to obtain 13a .

13e was obtained as a yellow oil (57% yield); ¹H NMR (CDCl₃)

δ 1.60 (m, 4H), 3.16 (t, 2H, J ) 6.7 Hz), 3.29 (m, 2H), 7.29 (d,

1H, J ) 7.5 Hz), 7.50 (m, 2H), 7.83 (d, 1H, J ) 8.4 Hz), 8.06

(m, 2H), 8.27 (m, 1H). Anal. (C₁₄H₁₅BrN₂O) C, H, N.

(S)-(-)-N-[4-[4-(2-Meth oxyph en yl)piper azin -1-yl]bu tyl]-

2-(ben zyloxyca r bon yl)-1,2,3,4-tetr a h yd r oisoqu in olin e-3-

ca r boxa m id e (14a ). Starting from 13g (180.0 mg, 0.40 mmol)

the title compound was prepared following the procedure

described for 5a and was obtained as a colorless oil (80% yield);

¹H NMR (acetone-d₆) δ 1.30 (m, 4H), 2.22 (m, 2H), 2.45 (m,

4H), 2.95 (m, 4H), 3.13 (m, 4H), 3.79 (s, 3H), 4.64 (br s, 3H),

5.16 (s, 2H), 6.84 (m, 4H), 7.16 (s, 5H), 7.34 (m, 4H); ESI-MS

m/z 579 [M + Na]⁺557 [M + H]⁺; ESI-MS/MS of [M + Na]⁺

535 (100), 443. Anal. (C₃₃H₄₀N₄O₄) C, H, N. [R] ²⁰-5.15 (c

D

0.97, CHCl₃).

(R)-(+)-N-[4-[4-(2-Meth oxyph en yl)piper azin -1-yl]bu tyl]-

2-(ben zyloxyca r bon yl)-1,2,3,4-tetr a h yd r oisoqu in olin e-3-

ca r boxa m id e (14b). Starting from 13h (150.0 mg, 0.34 mmol)

the title compound was obtained following the procedure

described for 14a and was obtained as a colorless oil (91%

yield); ¹H NMR (acetone-d₆) δ 1.30 (m, 4H), 2.22 (m, 2H), 2.45

(m, 4H), 2.95 (m, 4H), 3.13 (m, 4H), 3.79 (s, 3H), 4.64 (br s,

3H), 5.16 (s, 2H), 6.84 (m, 4H), 7.16 (s, 5H), 7.34 (m, 4H). Anal.

N-[1-(4-Br om o)bu tyl]isoqu in olin e-3-car boxam ide (13f).

To a solution of 12f (140.0 mg, 0.57 mmol) in acetonitrile (10

mL), triphenylphosphine (225.0 mg, 0.86 mmol) and carbon

tetrabromide (285.0 mg, 0.86 mmol) were added under vigor-

ous stirring at room temperature. After 2 h, the mixture was

quenched with 15% NaOH and extracted with ethyl acetate.

The organic layers were dried and evaporated. The residue

was chromatographed (30% n-hexane in ethyl acetate) to give

130.0 mg of 13f (75% yield) as a yellow solid: mp (ethyl

(C₃₃H₄₀N₄O₄) C, H, N. [R] ²⁰+5.15 (c 0.97, CHCl₃).

D

1-(Cya n om eth yl)in d ole-2-ca r boxylic Acid Eth yl Ester

(16). A mixture of sodium hydride (60% in mineral oil) (190.0

mg, 7.94 mmol) and ethyl indole-2-carboxylate (15, 1.0 g, 5.29

mmol) in N,N-dimethylformamide (4.6 mL), was stirred at

room temperature for 0.5 h and to this bromoacetonitrile (0.74

mL, 10.60 mmol) in N,N-dimethylformamide (1 mL) was

added. The reaction mixture was then maintained at 60-65

°C for 0.5 h, and stirred for 5-6 h further at room temperature,

left overnight and decomposed with ice. The separated solid

was recrystallized from ethanol to give 16 (90% yield) as

1

acetate) 72-73 °C; H NMR (CDCl₃) δ 2.06 (m, 4H), 3.48 (m,

4H), 7.66 (m, 2H), 7.93 (m, 2H), 8.36 (br s, 1H), 8.55 (s, 1H),

9.08 (s, 1H). Anal. (C₁₄H₁₅BrN₂O) C, H, N.

(S)-(-)-N-[1-(4-Br om o)b u t yl]-2-(b en zyloxyca r b on yl)-

1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r boxa m id e (13g). To

a solution of 12g (230.0 mg, 0.60 mmol) in acetonitrile (3 mL),

triphenylphosphine (237.0 mg, 0.90 mmol) and carbon tetra-

bromide (300.0 mg, 0.90 mmol) were added under vigorous

stirring at room temperature. After 5 h, the mixture was

quenched with 15% NaOH and extracted with ethyl acetate.

The organic layers were dried and evaporated. The residue

was chromatographed (20% n-hexane in ethyl acetate) to afford

180.0 mg (67% yield) of 13g as a colorless oil; ¹H NMR (CDCl₃)

δ 1.27 (m, 4H), 3.15 (m, 6H), 4.35 (br s, 3H), 5.21 (s, 2H), 5.87

(br s, 1H), 7.19 (s, 4H), 7.35 (s, 5H). Anal. (C₂₂H₂₅BrN₂O₃) C,

H, N. [R]_D-0.96 (c 4.14, CHCl₃).

(R)-(+)-N-[1-(4-Br om o)b u t yl]-2-(b en zyloxyca r b on yl)-

1,2,3,4-tetr ah ydr oisoqu in olin e-3-car boxam ide (13h ). Start-

ing from 12h (250.0 mg, 0.65 mmol), the title compound was

prepared following the above-described procedure and obtained

as a colorless oil (65% yield); ¹H NMR (CDCl₃) δ 1.27 (m, 4H),

3.15 (m, 6H), 4.35 (br s, 3H), 5.21 (s, 2H), 5.87 (br s, 1H), 7.19

(s, 4H), 7.35 (s, 5H). Anal. (C₂₂H₂₅BrN₂O₃) C, H, N. [R]_D+0.96

(c 1.43, CHCl₃).

N-[1-(4-Iodo)bu tyl]-5-ch lor oin dole-2-car boxam ide (13i).

N-[1-(4-Methanesulfonyl)butyl]-5-chloroindole-2-carboxam-

ide. To a stirred and cooled (0 °C) solution of 12i (270.0 mg,

1.01 mmol) and triethylamine (0.18 mL, 1.31 mmol) in dry

N,N-dimethylformamide (4 mL), methanesulfonyl chloride

(0.85 mL, 1.10 mmol) was added dropwise. The solution was

left at 0 °C under argon overnight. Water was added to the

formed suspension and was extracted with chloroform. The

organic layers were dried and concentrated and the residue

was purified by chromatography (10% methanol in chloroform)

to afford the methanesulfonyl-derivative (97% yield) as a

yellow amorphous solid; ¹H NMR (acetone-d₆) δ 1.77 (m, 4H),

3.05 (s, 3H), 3.51 (m, 2H), 4.27 (m, 2H), 7.02 (s, 1H), 7.19 (m,

1H), 7.55 (m, 2H), 7.90 (br s, 1H), 10.88 (br s, 1H). Anal.

(C₁₄H₁₇ClN₂O₄S) C, H, N.

1

colorless prisms: mp (ethanol) 83-84 °C; H NMR (CDCl₃) δ

1.42 (t, 3H, J ) 7.3 Hz), 4.41 (q, 2H, J ) 14.2, 7.2 Hz), 5,60 (s,

2H), 7.37 (m, 4H), 7.71 (d, 1H, J ) 7.9 Hz); GC-MS m/z 228

[M]⁺(100), 199, 182, 154, 128, 115, 101, 89, 77. Anal.

(C₁₃H₁₂N₂O₂) C, H, N.

1,2,3,4-Tetr ah ydr opyr azin o[1,2-a ]in dole-1(2H)-on e (17).

To a warm solution (60 °C) of 16 (200.0 mg, 0.87 mmol) in dry

methanol (8 mL) under argon, freshly prepared cobalt boride

(450.0 mg, 3.50 mmol), was added under stirring. Sodium

borohydride (166.0 mg, 4.38 mmol) was cautiously added

portionwise and the mixture was refluxed for 3 h. The mixture

was cooled, and the solvent removed under reduced pressure

then water was added and the mixture was extracted with

ethyl acetate. The organic layers were dried, evaporated and

the crude product was purified by chromatography (10%

methanol in chloroform) to give 17 (68% yield) as colorless

prisms: mp (methanol) 261-265 °C (dec); ¹H NMR (CDCl₃) δ

3.82 (m, 2H), 4.27 (m, 2H), 6.65 (br s, 1H), 7.23 (m, 4H), 7.72

(d, 1H, J ) 8.0 Hz); APCI-MS m/z 187 [M + H]⁺; APCI-MS/

MS of [M + H]⁺159 (100), 144. Anal. (C₁₁H₁₀N₂O) C, H, N.

N-[1-(4-Br om o)bu tyl]-1,2,3,4-tetr a h yd r op yr a zin o[1,2-a ]-

in d ole-1(2H)-on e (18). To a suspension of 17 (130.0 mg, 0.69)

in anhydrous N,N-dimethylformamide (1 mL) sodium hydride

(60% in mineral oil) (20.0 mg, 0.83 mmol) was added. After

stirring of the sample for 1 h at 60 °C under argon, a solution

of 1,4-dibromobutane (0.41 mL, 3.47 mmol) in anhydrous N,N-

dimethylformamide (0.5 mL) was added dropwise. The mixture

was refluxed under argon at 110 °C for 3 h. Then the solvent

was evaporated under reduced pressure, and the residue was

resuspended in water and extracted with dichloromethane.

The combined organic layers were dried evaporated and the

3834 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18

Campiani et al.

residue was chromatographed (30% ethyl acetate in n-hexane)

to give 18 as a yellow solid (41% yield): mp (ethyl acetate)

101-102 °C; H NMR (CDCl₃) δ 1.85 (m, 4H), 3.67 (m, 4H),

and concentrated and the crude product was chromatographed

(10% methanol in chloroform) to give 0.6 g of 5a (94% yield)

as a yellow oil; ¹H NMR (CDCl₃) δ 1.29 (m, 4H), 2.04 (m, 2H),

2.22 (m, 4H), 2.67 (m, 4H), 3.18 (m, 2H), 3.42 (s, 3H), 6.50 (m,

4H), 7.18 (m, 1H), 7.34 (m, 1H), 7.44 (d, 1H, J ) 7.8 Hz), 7.58

(s, 1H), 7.72 (d, 1H, J ) 8.2 Hz), 7.90 (s, 1H), 8.12 (br s, 1H).

Anal. (C₂₅H₃₀N₄O₂) C, H, N.

N-[4-[4-(2-Met h oxyp h en yl)p ip er a zin -1-yl]b u t yl]q u in -

oxa lin e-2-ca r boxa m id e (5b). Starting from 13b (40.0 mg,

0.13 mmol), the title compound was prepared following the

above-described procedure and was obtained as a yellow oil

(91% yield); ¹H NMR (CDCl₃) δ 1.63 (m, 4H), 2.40 (m, 2H),

2.58 (m, 4H), 3.00 (m, 4H), 3.50 (m, 2 H), 3.75 (s, 3H), 6.81

(m, 4H), 7.75 (m, 2H), 8.08 (m, 3H), 9.52 (br s, 1H). Anal.

(C₂₄H₂₉N₅O₂) C, H, N.

1

3.81 (m, 2H), 4.29 (m, 2H), 7.20 (m, 4H), 7.70 (d, 1H, J ) 8.0

Hz); APCI-MS m/z 321 [M + H]⁺, 241, 227, 199 (100), 187,

159, 144, 117. Anal. (C₁₅H₁₇BrN₂O) C, H, N.

(Ben zo[b]fu r a n -2-yl)-[(4-m eth oxyca r bon yl)p ip er id in -

1-yl]m eth a n on e (19). Starting from 2-benzofurancarboxylic

acid 11a (0.5 g, 3.1 mmol) and using methyl 4-piperidinecar-

boxylate (0.44 g, 3.1 mmol), the title compound was prepared

following the procedure described for 12a . Compound 19 was

obtained as colorless prisms (70% yield): mp (methanol) 88-

89 °C; ¹H NMR (CDCl₃) δ 1.67 (m, 4H), 2.53 (m, 1H), 3.17 (m,

2H), 3.66 (s, 3H), 4.34 (m, 2H), 7.40 (m, 5H). Anal. (C₁₆H₁₇

NO₄) C, H, N.

-

N-[4-[4-(2-Meth oxyph en yl)piper azin -1-yl]bu tyl]isoqu in -

olin e-3-ca r boxa m id e (5c). Starting from 13f (60.0 mg, 0.20

mmol), the title compound was prepared following the above-

described procedure and was obtained as colorless prisms (97%

(Ben zo[b]fu r a n -2-yl)-[4-(h yd r oxym et h yl)p ip er id in -1-

yl]m eth a n on e (20). To a solution of 19 (180.0 mg, 0.63 mmol)

in methanol (5 mL), sodium borohydride (238.0 mg, 6.3 mmol)

was added in portions and the mixture was refluxed for 2 h.

Then 0.5 N HCl (2 mL) was added to decompose the excess of

hydride and the solvent was removed under reduced pressure.

The residue was extracted with ethyl acetate, dried, concen-

trated and chromatographed (50% ethyl acetate in n-hexane)

to give 100.0 mg of 20 (61% yield) as colorless prisms: mp

(ethyl acetate) 91-92 °C; ¹H NMR (CDCl₃) δ 1.19 (m, 4H), 1.78

(m, 2H), 2.99 (m, 2H), 3.66 (m, 2H), 4.58 (m, 2H), 7.41 (m,

5H). Anal. (C₁₅H₁₇NO₃) C, H, N.

1

yield): mp (methanol) 132-133 °C; H NMR (CDCl₃) δ 1.69

(m, 4H), 2.48 (t, 2H, J ) 6.7 Hz), 2.67 (m, 4H), 3.11 (m, 4H),

3.57 (q, 2H, J ) 12.3, 6.2 Hz), 3.84 (s, 3H), 6.96 (m, 4H), 7.72

(m, 2H), 8.00 (m, 2H), 8.34 (br s, 1H), 8.60 (s, 1H), 9.14 (s,

1H); ¹³C NMR (CD₃OD) δ 22.3, 27.2, 40.3, 50.4, 51.5, 56.7, 57.6,

114.3, 122.0, 122.6, 125.2, 129.5, 130.1, 130.3, 132.2, 133.7,

135.5, 138.7, 139.6, 149.7, 150.2, 153.6, 161.4. Anal. (C₂₅H₃₀N₄O₂)

C, H, N.

N-[4-[4-(2-Meth oxyph en yl)piper azin -1-yl]bu tyl]pyr r olo-

[1,2-a ]qu in oxa lin e-4-ca r boxa m id e (5d ). Starting from 13c

(38.0 mg, 0.11 mmol), the title compound was prepared

following the procedure described for 5a and was obtained as

a yellow oil (95% yield); ¹H NMR (CDCl₃) δ 1.73 (m, 4H), 2.49

(t, 2H, J ) 6.7 Hz), 2.68 (m, 4H), 3.09 (m, 4H), 3.55 (m, 2H),

3.85 (s, 3H), 6.89 (m, 4H), 7.54 (m, 2H), 7.93 (m, 4H), 8.22 (m,

1H). Anal. (C₂₇H₃₁N₅O₂) C, H, N.

(Ben zo[b]fu r a n -2-yl)-[4-(br om om eth yl)p ip er id in -1-yl]-

m eth a n on e (21). Starting from 20 (100.0 mg, 0.39 mmol), the

title compound was prepared following the procedure described

for 13a . Compound 21 was obtained as colorless prisms (49%

1

yield): mp (ethyl acetate) 63-64 °C; H NMR (CDCl₃) δ 1.33

(m, 4H), 1.93 (m, 3H), 3.03 (m, 2H), 3.31 (d, 2H, J ) 5.9 Hz),

4.58 (m, 2H), 7.26 (m, 3H), 7.49 (d, 1H, J ) 8.3 Hz), 7.61 (d,

1H, J ) 7.6 Hz). Anal. (C₁₅H₁₆BrNO₂) C, H, N.

(S)-(-)-N-[4-[4-(2-Meth oxyph en yl)piper azin -1-yl]bu tyl]-

1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r boxa m id e (5e). To a

methanol/ethyl acetate solution (1:1, 2 mL) under N₂in a thick-

walled Parr test tube 10% Pd/C (62.0 mg) was added. To the

resulting suspension a solution of 14a (170.0 mg, 0.30 mmol)

in methanol (0.5 mL) was added. The suspension was shaken

for 3 h in a Parr hydrogenator under 40 psi of H₂. The mixture

was filtered through Celite, and the Celite was washed with

methanol. Then the filtrate was concentrated, and the result-

ing crude product was chromatographed (10% methanol in

chloroform) to afford 107.0 mg of 5e (84% yield) as colorless

2-(Ben zo[b]fu r a n -2-yl)-4-(ch lor om et h yl)-1,3-oxa zole

(23a ). To a solution of acid 11a (301.6 mg, 1.86 mmol) in

anhydrous benzene (3 mL), thionyl chloride (0.67 mL, 9.1

mmol) dissolved in anhydrous benzene (1 mL) was added

dropwise. The reaction mixture was heated at reflux for 2 h

under argon. After cooling the solvent was removed under

vacuum and the residue was dissolved in anhydrous tetrahy-

drofuran (2 mL) and added to liquid ammonia at -50 °C. After

1 h of stirring, the reaction mixture was warmed to room

temperature the solvent was removed, the corresponding

amide was obtained in quantitative yield as a white amorphous

solid and was used in the next step without purification. A

sample was recrystallized from ethyl acetate: mp 158-159

1

prisms mp (methanol) 150-155 °C (dec); H NMR (acetone-

d₆) δ 1.53 (m, 4H), 2.35 (m, 2H), 2.53 (m, 4H), 2.77 (dd, 2H, J

) 16.4, 10.1 Hz), 3.04 (m, 4H), 3.26 (m, 2H), 3.44 (dd, 1H, J )

9.9, 4.9 Hz), 3.79 (s, 3H), 3.95 (s, 2H), 6.86 (m, 5H), 7.02 (br s,

1H), 7.08 (m, 4H), 7.45 (br s, 1H); ¹³C NMR (CD₃OD) δ 22.0,

27.3, 31.0, 39.2, 45.5, 50.0, 52.0, 56.2, 56.5, 57.5, 114.0, 121.5,

122.5, 127.7, 128.6, 128.8, 129.2, 129.3, 130.1, 131.8, 135.4,

153.7, 169.8. ESI-MS m/z 867 [2M + Na]⁺(100), 445 [M +

Na]⁺, 423 [M + H]⁺. Anal. (C₂₅H₃₄N₄O₂) C, H, N. [R]²⁰_D-57.14

(c 1.68, CHCl₃).

1

°C; H NMR (DMSO-d₆) δ 7.33 (m, 2H), 7.53 (s, 1H), 7.59 (d,

1H, J ) 8.1 Hz), 7.72 (d, 1H, J ) 7.6 Hz), 8.09 (br s, 2 H). The

amide (300.0 mg, 1.86 mmol) and 1,3-dichloroacetone (709.0

mg, 5.6 mmol) were fused at 130 °C under argon. After fusion

the mixture was stirred for 1 h. Then water was added and

the mixture was extracted with dichloromethane. The organic

layers were dried and evaporated and the residue was chro-

matographed (dichloromethane) to afford 25a (54% yield) as

an amorphous white solid; ¹H NMR (CDCl₃) δ 4.66 (s, 2H),

7.34 (m, 3H), 7.73 (m, 3H); GC-MS m/z 233 [M]⁺(100), 198,

170, 143, 130, 115. Anal. (C₁₂H₈ClNO₂) C, H, N.

(R)-(+)-N-[4-[4-(2-Meth oxyph en yl)piper azin -1-yl]bu tyl]-

1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r boxa m id e (5f). Start-

ing from 14b (160.0 mg, 0.28 mmol), the title compound was

obtained following the procedure described for 5e. 5f was

obtained as a white solid (81% yield): mp (methanol) 150-

155 °C (dec); ¹H NMR (acetone-d₆) δ 1.53 (m, 4H), 2.35 (m,

2H), 2.53 (m, 4H), 2.77 (dd, 2H, J ) 16.4, 10.1 Hz), 3.04 (m,

4H), 3.26 (m, 2H), 3.44 (dd, 1H, J ) 9.9, 4.9 Hz), 3.79 (s, 3H),

3.95 (s, 2H), 6.86 (m, 5H), 7.02 (br s, 1H), 7.08 (m, 4H), 7.45

(br s, 1H).¹³C NMR (CD₃OD) δ 15.4, 22.0, 27.3, 30.9, 39.3, 45.6,

50.2, 52.1, 56.5, 56.5, 57.5, 66.9, 114.1, 121.5, 122.5, 127.6,

128.6, 128.9, 129.2, 129.3, 130.1, 131.9, 135.4, 153.7, 169.7.

4-(Ch lor om eth yl)-2-(2-n a p h th yl)-1,3-oxa zole (23b). The

title compound was obtained following the procedure described

for 23a . The compound 23b was obtained as an amorphous

1

white solid (37% yield); H NMR (CDCl₃) δ 4.60 (s, 2H), 7.54

(m, 2H), 7.74 (s, 1H), 7.91 (m, 4H), 8.10 (d, 1H, J ) 8.8 Hz),

8.53 (s, 1H); GC-MS m/z 243 [M]⁺(100), 208, 180, 153, 139,

127. Anal. (C₁₄H₁₀ClNO) C, H, N.

N-[4-[4-(2-Meth oxyp h en yl)p ip er a zin -1-yl]bu tyl]qu in o-

lin e-2-ca r boxa m id e (5a ). To a solution of 13e (482.0 mg, 1.57

mmol) in dry acetonitrile (5 mL) under argon, 1-(2-methoxy-

phenyl)piperazine (207.0 mg, 1.57 mmol) and triethylamine

(0.35 mL, 2.54 mmol) were added; the solution was refluxed

overnight with stirring. The solvent was removed under

reduced pressure, water was added, and the mixture was

extracted with dichloromethane. The organic layers were dried

Anal. (C₂₅H₃₄N₄O₂) C, H, N. [R] ²⁰+57.14 (c 0.39, CHCl₃).

D

N-[4-[4-(2-Meth oxyp h en yl)p ip er a zin -1-yl]bu tyl]ben zo-

[b]fu r a n -2-ca r boxa m id e (5g). Starting from 13a (0.5 g, 1.69

mmol) the title compound was prepared following the proce-

dure described for 5a and was obtained as colorless prisms

1

(60% yield): mp (methanol) 120-121 °C; H NMR (CDCl₃) δ

Highly Selective D₃Receptor Ligands

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3835

1.67 (m, 4H), 2.48 (m, 2H), 2.65 (m, 4H), 3.12 (m, 4H), 3.56

(m, 2H), 3.85 (s, 3H), 6.90 (m, 4H), 7.36 (m, 4H), 7.63 (d, 1H,

J ) 7.7 Hz). Anal. (C₂₄H₂₉N₃O₃) C, H, N.

amine (44 µL, 0.31 mmol) were added, and the solution was

refluxed overnight under stirring. The workup was the same

followed for 5a . The product 5n was obtained as colorless

prisms (72% yield): mp (methanol) 127-128 °C; ¹H NMR

(CDCl₃) δ 1.71 (m, 4H), 2.50 (m, 2H), 2.64 (m, 4H), 3.05 (m,

4H), 3.55 (m, 2H), 6.94 (d, 1H, J ) 8.8 Hz), 7.16 (dd, 1H, J )

8.5, 2.3 Hz), 7.34 (d, 1H, J ) 2.1 Hz), 7.73 (m, 2H), 8.00 (m,

N-[4-[4-(2-Meth oxyp h en yl)p ip er a zin -1-yl]bu tyl]in d ole-

2-ca r boxa m id e (5h ). Starting from 13d (35.0 mg, 0.12 mmol)

the title compound was prepared following the procedure

described for 5a and was obtained as a yellow oil (48% yield);

¹H NMR (CDCl₃) δ 1.74 (m, 4H), 2.52 (m, 2H), 2.68 (m, 4H),

3.09 (m, 4H), 3.62 (m, 2H), 3.90 (s, 3H), 6.97 (m, 4H), 7.20 (m,

3H), 7.40 (d, 1H, J ) 8.0 Hz), 7.56 (d, 1H, J ) 8.0 Hz), 10.43

(br s, 1H). Anal. (C₂₄H₃₀N₄O₂) C, H, N.

2H), 8.33 (br s, 1H), 8.60 (s, 1H), 9.13 (s, 1H). Anal. (C₂₄H₂₆

Cl₂N₄O) C, H, N.

-

N-[4-[4-(2,4-Dich lor op h en yl)p ip er a zin -1-yl]bu tyl]ben -

zo[b]fu r a n -2-ca r boxa m id e (5o). Starting from 13a (300.0

mg, 1.01 mmol) the title compound was prepared following the

above-described procedure. Compound 5o was obtained as

(Ben zo[b]fu r a n -2-yl)-[4-[[4-(2-m et h oxyp h en yl)p ip er -

a zin om eth yl]p ip er id in -1-yl]m eth a n on e (5i). Starting from

21 (63.0 mg, 0.19 mmol) the title compound was prepared

following the procedure described for 5a . The product 5i was

1

colorless prisms (68% yield): mp (methanol) 112-113 °C; H

NMR (CDCl₃) δ 1.59 (m, 4H), 2.44 (t, 2H, J ) 6.6 Hz), 2.61

(m, 4H), 3.02 (m, 4H), 3.50 (q, 2H, J ) 12.2, 6.2 Hz), 6.88 (d,

1H, J ) 8.7 Hz), 7.08 (m, 2H), 7.32 (m, 4H), 7.62 (d, 1H, J )

7.5 Hz); ¹³C NMR (CDCl₃) δ 24.3, 27.5, 39.2, 51.1, 53.2, 57.8,

76.6, 77.8, 110.1, 111.6, 121.0, 122.6, 123.6, 126.7, 127.5, 127.6,

1

obtained as a yellow oil (60% yield); H NMR (CDCl₃) δ 1.25

(m, 2H), 1.89 (m, 3H), 2.27 (d, 2H, J ) 6.4 Hz), 2.62 (m, 4H),

3.08 (m, 6H), 3.85 (s, 3H), 4.57 (m, 2H), 6.83 (m, 4H), 7.32 (m,

3H), 7.49 (d, 1H, J ) 8.1 Hz), 7.61 (d, 1H, J ) 7.6 Hz). Anal.

(C₂₆H₃₁N₃O₃) C, H, N.

128.0, 129.3, 130.2, 148.0, 149.0, 154.6, 158.8. Anal. (C₂₃H₂₅

Cl₂N₃O₂) C, H, N.

-

N-[[2-(Ben zo[b]fu r a n -2-yl)-1,3-oxa zol-4-yl]m eth yl]-2-[4-

(2-m eth oxyp h en yl)p ip er a zin -1-yl]-1-eth a n a m in e (5j). The

amine 10 (82.0 mg, 0.35 mmol), Na₂CO₃(37.0 mg. 0.35 mmol)

and KI (57.7 mg, 0.35 mmol) were suspended in 1-butanol (2

mL). To the suspension under vigorous stirring under argon,

a solution of 23a (57.0 mg, 0.23 mmol) in 1-butanol (1 mL)

was added and the resulting mixture was refluxed for 20 h.

Then the mixture was filtered, the filtrate was evaporated and

the residue was purified by chromatography (ethyl acetate)

to afford 5j (18% yield) as a yellow oil; ¹H NMR (CDCl₃) δ 2.22

(m, 2H), 2.63 (m, 2H), 2.70 (m, 4H), 3.15 (m, 4H), 3.70 (s, 2H),

3.85 (s, 3H), 6.92 (m, 4H), 7.33 (m, 3H), 7.65 (m, 3H). Anal.

(C₂₅H₂₈N₄O₃) C, H, N.

N-[4-[4-(2,4-Dich lor oph en yl)piper azin -1-yl]bu tyl]in dole-

2-ca r boxa m id e (5p ). Starting from 13d (141.0 mg, 0.48

mmol), the title compound was prepared following the above-

described procedure as a yellow oil (65% yield); ¹H NMR

(CDCl₃) δ 1.68 (m, 4H), 2.49 (t, 2H, J ) 6.8 Hz), 2.63 (m, 4H),

3.04 (m, 4H), 3.55 (m, 2H), 6.67 (m, 1H), 6.86 (m, 2H), 6.90 (s,

1H), 7.16 (m, 2H), 7.27 (m, 1H), 7.33 (m, 1H), 7.45 (d, 1H, J )

8.2 Hz), 7.64 (d, 1H, J ) 8.0 Hz), 9.97 (br s, 1H). Anal. (C₂₃H₂₆

Cl₂N₄O) C, H, N.

-

N-[4-[4-(2,4-Dich lor op h en yl)p ip er a zin -1-yl]b u t yl]-5-

ch lor oin d ole-2-ca r boxa m id e (5q). Starting from 13i (27.0

mg, 0.11 mmol), the title compound was prepared following

1

N-[[2-(2-Naph th yl)-1,3-oxazol-4-yl]m eth yl]-2-[4-(2-m eth -

oxyp h en yl)p ip er a zin -1-yl]-1-eth a n a m in e (5k ). Following

a procedure as described for 5j, the title compound was

the above-described procedure as a yellow oil (73% yield); H

NMR (CDCl₃) δ 1.69 (m, 4H), 2.47 (t, 2H, J ) 6.5 Hz), 2.63

(m, 4H), 3.03 (m, 4H), 3.53 (m, 2H) 6.56 (br s, 1H), 6.75 (d,

1H, J ) 1.4 Hz) 6.90 (d, 1H, J ) 8.3 Hz), 7.19 (m, 2H), 7.37 (d,

1H, J ) 8.2 Hz), 7.59 (d, 1H, J ) 1.3 Hz), 9.66 (br s, 1H). Anal.

(C₂₃H₂₅Cl₃N₄O) C, H, N.

1

obtained as a yellow oil (16% yield); H NMR (CDCl₃) δ 2.21

(m, 2H), 2.64 (m, 2H), 2.81 (m, 4H), 3.15 (m, 4H), 3.66 (s, 2H),

3.85 (s, 3H), 6.90 (m, 4H), 7.57 (m, 2H), 7.67 (s, 1H), 7.88 (m,

3H), 8.15 (d, 1H, J ) 8.0 Hz), 8.56 (s, 1H). Anal. (C₂₇H₃₀N₄O₂)

C, H, N.

N-[4-[4-(2-Meth oxyph en yl)piper a zin -1-yl]bu tyl]-1,2,3,4-

tetr a h yd r op yr a zin o[1,2-a ]in d ole-1(2H)-on e (5r ). To a so-

lution of 18 (80.0 mg, 0.25 mmol) in 6 mL dry acetonitrile

under argon, 1-(2-methoxyphenyl)piperazine (48.0 mg, 0.25

mmol) and triethylamine (56 µL, 0.40 mmol) were added; the

solution was refluxed overnight under stirring. The solvent

was removed under reduced pressure, water was added and

the mixture was extracted with dichloromethane. The organic

layers were dried and concentrated and the crude product was

chromatographed (10% methanol in chloroform) to give 100.0

mg of 5r (93% yield) as yellow oil; ¹H NMR (acetone-d₆) δ 1.68

(m, 4H), 2.49 (m, 2H), 2.63 (m, 4H), 3.10 (m, 4H), 3.66 (m,

2H), 3.79 (m, 2H), 3.85 (s, 3H), 4.27 (m, 2H), 6.91 (m, 4H),

7.14 (m, 1H), 7.31 (m, 3H), 7.70 (d, 1H, J ) 8.1 Hz); ¹³C NMR

(CD₃OD) δ 22.2, 25.6, 41.2, 46.5, 47.4, 50.4, 51.3, 56.8, 57.6,

111.1, 114.3, 121.8, 122.1, 122.6, 123.3, 125.8, 127.4, 128.6,

129.8, 130.7, 133.1, 138.1, 153.6, 162.3. Anal. (C₂₆H₃₂N₄O₂) C,

H, N.

N-[4-[4-(4-Cya n op h en yl)p ip er a zin -1-yl]bu tyl]ben zo[b]-

fu r a n -2-ca r boxa m id e (5l). To a solution of 13a (120.0 mg,

0.40 mmol) in dry acetonitrile 6 mL under argon, 7b (76.0 mg,

0.40 mmol) and triethylamine (92 µL, 0.65 mmol) were added

and the solution was refluxed overnight with stirring. The

solvent was removed under reduced pressure, water was added

and the mixture was extracted with dichloromethane. The

organic layers were dried and concentrated and the crude

product was chromatographed (10% methanol in chloroform)

to give 130.0 mg of 5l (65% yield) as colorless prisms: mp

(methanol) 146-148 °C; ¹H NMR (CDCl₃) δ 1.64 (m, 4H), 2.44

(t, 2H, J ) 6.6 Hz), 2.57 (m, 4H), 3.32 (m, 4H), 3.51 (q, 2H, J

) 12.3, 6.2 Hz), 6.82 (m, 2H), 6.90 (br s, 1H), 7.34 (m, 6H),

7.65 (d, 1H, J ) 7.6 Hz); ¹³C NMR (CD₃OD) δ 22.4, 27.5, 39.3,

45.9, 52.7, 57.7, 103.5, 111.4, 111.5, 112.9, 113.0, 116.6, 120.5,

123.9, 125.1, 128.4, 128.9, 134.8, 153.8. ESI-MS m/z 425 [M +

Na]⁺, 403 [M + H]⁺; ESI-MS/MS of [M + H]⁺242, 216 (100),

173. Anal. (C₂₄H₂₆N₄O₂) C, H, N.

Biologica l Stu d ies

N-[4-[4-(3,4-Dich lor op h en yl)p ip er a zin -1-yl]bu tyl]ben -

zo[b]fu r a n -2-ca r boxa m id e (5m ). To a solution of 13a (50.0

mg, 0.17 mmol) in dry acetonitrile (3 mL) under argon, 1-(3,4-

dichlorophenyl)piperazine (39.0 mg, 0.17 mmol) and triethyl-

amine (38 µL, 0.27 mmol) were added; the solution was

refluxed overnight under stirring. The workup was same as

that for 5a . Pure 5m was obtained as colorless prisms (56%

1. An im a ls. Procedures involving animals and their care

were conducted in conformity with the institutional guidelines

that are in compliance 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).

1

yield): mp (methanol) 121-122 °C; H NMR (CDCl₃) δ 1.69

(m, 4H), 2.47 (m, 2H), 2.60 (m, 4H), 3.20 (m, 4H), 3.52 (m,

2H), 6.82 (m, 3H), 7.39 (m, 4H), 7.66 (d, 1H, J ) 7.5 Hz). Anal.

(C₂₃H₂₅Cl₂N₃O₂) C, H, N.

N-[4-[4-(2,4-Dich lor op h en yl)p ip er a zin -1-yl]b u t yl]iso-

qu in olin e-3-ca r boxa m id e (5n ). To a solution of 13f (60.0

mg, 0.19 mmol) in dry acetonitrile (5 mL) under argon, 1-(2,4-

dichlorophenyl)piperazine (45.3 mg, 0.19 mmol) and triethyl-

Male Sprague-Dawley CD-COBS rats (Charles River, Italy)

were used, weighing 150 g (for binding assays) or 250-275 g

(for in vivo studies) at the beginning of the experiments. They

were housed individually at constant room temperature (21

( 1 °C) and relative humidity (60%) under an inverted light/

dark schedule (light 8:00 p.m. - 8:00 a.m.) with food and water

ad libitum. Animals were allowed to adapt to laboratory

3836 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18

Campiani et al.

conditions for at least two weeks and were handled for 5 min

per day during this period.

min at 37 °C without [³⁵S]-GTPγS and then for 45 min at 37

°C with [³⁵S]-GTPγS.

For evaluation of [³⁵S]-GTPγS binding to D_3rreceptors⁴³the

Sf9 cells were resuspended in Hepes 25 mM, pH 8, containing

100 mM NaCl, 1 mM EDTA, 3 mM MgCl₂, 0.5 mM DTT,

0.003% digitonine, 10 µM GDP, with a glass/Teflon potter. The

binding assay was carried out in a final incubation volume of

120 µL, consisting of 100 µL of membrane suspension (about

15 µg of protein/sample), 10 µL of [³⁵S]-GTPγS (final concen-

tration 1 nM) and 10 µL of displacing agent or solvent.

Nonspecific binding was obtained in the presence of 10 µM

GTPγS; samples were preincubated for 15 min at 30 °C

without [³⁵S]-GTPγS and then for 30 min at 30 °C with [³⁵S]-

GTPγS.

2. Bin d in g Assa ys. Rats were killed by decapitation and

their brains were quickly removed. The various brain regions

were dissected (striatum for D₁and D₂, cortex for R₁- and R₂-

adrenoceptors, hippocampus for 5-HT_1A) and stored at -80 °C

until use.

Tissues were homogenized in about 50 vol of ice-cold Tris

HCl 50 mM, pH 7.4 using an Ultra-Turrax TP-1810 (2 × 20 s)

and centrifuged (Beckman Avanti J -25) at 50000g for 10 min

at 4 °C. The pellets were resuspended in the same volume of

fresh buffer, incubated at 37 °C for 10 min, and centrifuged

again as before. The pellets were then washed once by

resuspension in fresh buffer, centrifuged again at 50000g for

10 min, resuspended just before the binding assay in the

appropriate incubation buffer (for D₁and D₂receptors: Tris

HCl, 50 mM, pH 7.4 containing 10 µM pargyline, 0.1% ascorbic

acid, 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂.

For 5-HT_1Areceptors: Tris HCl, 50 mM, pH 7.7 containing 10

µM pargyline, 4 mM CaCl₂for [³H]-8-OH-DPAT binding or Tris

HCl, 50 mM, pH 7.4 containing 4 mM MgCl₂, 160 mM NaCl,

0.3 mM EGTA, 300 µM GDP for [³⁵S]-GTPγS binding. For R₁-

adrenoceptors: Tris HCl, 50 mM, pH 7.4 containing 10 µM

pargyline, 0.1% ascorbic acid. For R₂-adrenoceptors: Tris HCl,

50 mM, pH 7.5.

After incubation, samples were filtered through Whatman

GF/B glass fiber filters using a Brandel apparatus (mod.

M-48R) and washed three times with 4 mL of ice-cold buffer.

The radioactivity trapped on the filters was counted in 4 mL

of “Ultima Gold MV” (Packard) in an DSA 1409 (Wallac) liquid

scintillation counter, with a counting efficiency of 50% for [³H]

or 90% for [³⁵S]. For inhibition experiments, the drugs were

added to the binding mixture at 7-9 different concentrations.

Inhibition curves were analyzed using the “Allfit” program⁴⁴

running on an IBM AT-PC. The K_ivalues were derived from

the IC₅₀values using the Cheng and Prusoff equation.⁴⁵

3. Beh a vior a l Stu d ies. The apparatus, surgical procedure,

behavioral training, and testing were identical to those previ-

ously described in detail.¹⁷Animals were trained and tested

using standard rodent operant test chambers (MED Associates

Inc., St. Albans, VT) containing two stainless steel levers. In

half the chambers the right-hand lever was designated as the

active lever, and in the others, the left-hand one.

[³H]-SCH 23390 (sa 75.5 Ci/mmol, NEN) binding³⁸to D₁

receptors was assayed in a final incubation volume of 0.5 mL,

consisting of 0.25 mL of membrane suspension (2 mg of tissue/

sample), 0.25 mL of [³H]-ligand (0.4 nM) and 10 µL of

displacing agent or solvent. Nonspecific binding was obtained

in the presence of 10 µM (-)-cis-flupentixol; samples were

incubated for 15 min at 37 °C. [³H]-Spiperone (sa 16.5 Ci/mmol,

All training and testing was conducted during the dark

phase of the light/dark cycle at approximately the same time

each day. To facilitate acquisition of cocaine self-administra-

tion, before surgery the rats were trained to press a lever for

food pellets on a fixed ratio 1 (FR-1) schedule. As soon as this

behavior was mastered, rats returned to an unrestricted diet

and a chronic jugular catheter was implanted.

NEN) binding³⁸to D₂receptors was assayed in

a final

incubation volume of 1 mL, consisting of 0.5 mL of membrane

suspension (1 mg tissue/sample) 0.5 mL of [³H]-ligand (0.2 nM)

and 20 µL of displacing agent or solvent. Nonspecific binding

was obtained with of 100 µM (-)-sulpiride; samples were

incubated for 15 min at 37 °C. [³H]-7-OH-DPAT (sa 139 Ci/

mmol, Amersham) binding³⁹to D₃receptors was assayed in a

final incubation volume of 1 mL, consisting of 0.5 mL of

membrane suspension (about 12 µg prot./sample of rat cloned

dopamine receptor subtype 3 in Sf9 cells (Signal Screen),

resuspended in Tris HCl, 50 mM, pH 7.4 containing 5 mM

EDTA, 5 mM MgCl₂, 5 mM KCl, 1.5 mM CaCl₂, 120 mM NaCl,

0.5 mL of [³H]-ligand (0.7 nM) and 20 µL of displacing agent

or solvent. Nonspecific binding was obtained in the presence

of 1 µM dopamine; samples were incubated for 60 min at 25

°C. [³H]-Prazosin (sa 83.8 Ci/mmol, NEN) binding⁴⁰to R₁-

adrenoceptors was assayed in a final incubation volume of 1

mL, consisting of 0.5 mL of membrane suspension (10 mg of

tissue/sample) 0.5 mL of [³H]-ligand (0.2 nM) and 20 µL of

displacing agent or solvent. Nonspecific binding was obtained

with 1 µM prazosin; samples were incubated for 30 min at 25

°C. [³H]-RX 821002 (sa 49 Ci/mmol, Amersham) binding⁴¹to

R₂-adrenoceptors was assayed in a final incubation volume of

1 mL, consisting of 0.5 mL of membrane suspension (16 mg of

tissue/sample) 0.5 of [³H]-ligand (1 nM) and 20 µL of displacing

agent or solvent. Nonspecific binding was obtained with 10

µM clonidine; samples were incubated for 30 min at 25 °C.

[³H]-8-OH-DPAT (sa 120 Ci/mmol, NEN) binding⁴⁰to 5-HT_1A

receptors was assayed in a final incubation volume of 0.5 mL,

consisting of 0.25 mL of membrane suspension (10 mg tissue/

sample), 0.25 mL of [³H]-ligand (1 nM) and 10 µL of displacing

agent or solvent. Nonspecific binding was obtained in the

presence of 1 µM serotonin; samples were incubated for 30 min

at 25 °C.

Catheters were made in-house using guide cannulae (C313G

5UP, Plastic One), silicon tubing (0.30 × 0.60 and 0.64 × 1.19

mm, i.d. × o.d. respectively, Degania Silicone Ltd., Israel),

dental cement (Paladur, Heraus Kulzer GmbH, Wehrheim/

Ts., Germany) and silicon rubber (Elastosil E43, Wacker-

Chemie GmbH, Mu¨nchen, Germany) according to Caine and

co-workers⁴⁶with few modifications. They were implanted in

rats deeply anaesthetised with equithesin [3.0 mL/kg intrap-

eritoneally (i.p.)] with the proximal end reaching the heart

through the right jugular vein and continuing subcutaneously

(s.c.) over the right shoulder, exiting dorsally between the

scapulae. Catheters were kept patent by daily intravenous

(i.v.) infusions of 0.1 mL heparinised (30 units/mL) sterile 0.9%

saline before and after each self-administration session.

4. Coca in e Self-Ad m in istr a tion a n d Con d ition in g. Rats

were trained in the operant cages to self-administer cocaine

i.v. (MacFarlan-Smith, Edinburg, UK), 0.25 mg/0.1 mL/infu-

sion, under a FR-1 for 2 h per day. As soon as they developed

stable levels of daily cocaine intake, rats were subjected to a

discriminative learning regimen that comprised three daily 1-h

sessions, separated by 1 h home cage rest, when either cocaine

or saline was available as the only infusion solution, in an

unpredictable sequence. Each training day included one saline

and two cocaine sessions presented in random order.

Sessions were started by extension of the levers and

concurrent presentation of the respective discriminative stimuli

that lasted throughout the session. The discriminative stimu-

lus associated with cocaine (S^D+) consisted of a white noise

(20 dB above background), and the S^D-, i.e., the stimulus

predictive of vehicle solution, consisted of illumination of the

house light. To prevent accidental overdosing, after each

infusion of cocaine the lever remained inactive for 20-s time-

out (TO), signaled by the cue light above the active lever

coming on. The TO after each infusion of saline was instead

signaled by an intermittent tone (7 kHz, 70 dB). The S^Ds were

[³⁵S]-GTPγS (sa 1064 Ci/mmol, Amersham) binding to

5-HT_1Aserotonin receptors⁴²was assayed in a final incubation

volume of 1.04 mL, consisting of 1 mL of membrane suspension

from rat hippocampus (200 µg of protein/sample), 20 µL of [³⁵S]-

GTPγS (final concentration 0.1 nM), and 20 µL of displacing

agent or solvent. Nonspecific binding was obtained in the

presence of 10 µM GTPγS, samples were preincubated for 20

Highly Selective D₃Receptor Ligands

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3837

not turned off during the TO periods. Responses on the inactive

lever (whenever it was presented) were recorded but had no

programmed consequences.

This training was conducted daily until cocaine-reinforced

responding stabilized ((10% over three consecutive training

days) and the rats almost stopped responding during saline

sessions (e5 responses during each of three successive ses-

sions).

9. Effects of Rep la cin g Coca in e w ith 5g or Veh icle on

Coca in e i.v. Self-Ad m in istr a tion . To study whether 5g has

intrinsic rewarding properties sufficient to maintain drug-

taking behavior we evaluated in animals with a daily history

of cocaine i.v. self-administration whether 5g (given i.v. at the

dose of 3 mg/mL) could substitute for i.v. cocaine. In this

experiment rats first acquired i.v. 0.25 mg/0.1 mL cocaine self-

administration under continuous reinforcement. When base-

line rates of cocaine self-administration were stable (three

consecutive days with the same number of responses ( 10%),

5g 3 mg/mL or saline were substituted for cocaine and self-

administration was allowed to continue for a further 7 days,

after which, cocaine at the training dose was substituted for

5g or saline.

10. Sta tistica l An a lysis. Data are expressed as mean (

SEM of the active and inactive lever-pressings during the self-

administration, extinction, and reinstatement phases. First of

all in each experiment the number of cocaine infusions earned

in the two separate 1 h sessions were analyzed by mixed

factorial ANOVA or one-way ANOVA for repeated measure-

ments. Since no differences were observed in the first and

second hour of cocaine self-administration, these data were

pooled for statistical analysis. The number of lever-pressings

during the last 3 days of extinction, before any or between the

different reinstatement test sessions also did not differ because

they had to meet the extinction criterion, so they too were

pooled for statistical analysis.

Differences between the mean numbers of the last three

cocaine and saline self-administration sessions, the preceding

three extinction sessions, and the reinstatement sessions were

analyzed by mixed factorial ANOVA or one-way ANOVA for

repeated measurements. When a significant effect was found,

post-hoc comparison was done by the Newman-Keuls test.

To evaluate the effects of 5g on cocaine self-administered

under fixed ratio schedule the number of reinforcers earned

during the 2 h session was analyzed by one way ANOVA for

repeated measurements followed by the Newman-Keuls post-

hoc comparison. The ability of 5g and vehicle to substitute for

cocaine in the i.v. self-administration procedure was analyzed

by mixed one-way ANOVA for repeated measurements.

11. Molecu la r Mod elin g. Molecular modeling was run on

a Silicon Graphics Indigo2 R10000 workstation. Structures of

compounds 5a , 5c, 5h , and 5r were built using the Builder

module in Insight2000 (Accelrys, San Diego). Atomic potentials

and charges were assigned using the cff91 force field.⁴⁷All the

compounds were considered protonated on the alkyl-piperazine

nitrogen. The piperazine ring of 5a , 5c, and 5h was assumed

in a chair conformation, since, as already reported,⁴⁸it is

present in all arylpiperazines found in the Cambridge Crystal-

lographic Structural Database, suggesting it is energetically

preferred, as compared to the boat conformation. On the other

hand, the butyl chain was assumed in an extended conforma-

tion to minimize intramolecular interactions which would

affect the evaluation of the energy contribution of the car-

boxamide moiety.

The starting conformations were geometrically optimized

(Discover module, Accelrys, San Diego) using the cff91 force

field in a vacuum and in an aqueous environment (ꢀ ) 1 and

ꢀ ) 80*r, respectively). Energy minimizations were done with

a conjugate gradient⁴⁹as minimization algorithm until the

maximum RMS derivative was less than 0.001 kcal/mol. This

protocol was applied to all the molecular mechanic (MM)

calculations.

12. An a lysis of Or ien ta tion of th e Heter oa r ylca r boxa -

m id e Gr ou p of Com p ou n d s 5a , 5c, a n d 5h . For each

compound, conformations were generated by rotating the

torsion angle τ (as defined in Results and Discussion) by 30°

within a 0-359° range, while the amide bond was rotated 180°

within the same range. All the conformations were subjected

to the MM energy minimization protocols, leading to four

conformers for each molecule, both in a vacuum and in an

aqueous environment. MM conformers of 5a , 5c, and 5h were

subjected to a full geometry optimization using the quantum

mechanical method AM1 in the Mopac 6.0 package⁵⁰in Ampac/

5. Extin ction . Beginning 1 day after meeting the self-

administration training criterion each rat was placed on

extinction conditions until the end of the experiment. Sessions

began by extension of both the active and inactive levers

without either the cocaine- or the saline-associated stimuli.

Responses on the previously active lever resulted in activation

of the syringe pump motor but had no other programmed

consequences; responses on the inactive lever were also

recorded but had no programmed consequences. One daily 1

h extinction session was conducted, until a criterion of e5

responses per session over three consecutive days was met.

6. Rein sta tem en t. Reinstatement tests began 1 day after

individual animals met the extinction criterion. Tests lasted

1 h and involved exposing the rats to the cocaine- or saline-

associated stimuli under conditions identical to the self-

administration phase, except that cocaine or saline were not

available. After the initial reinstatement test session rats were

further placed on extinction conditions until retested. Test

sessions for each animal were separated by at least 3 days in

which rates of responding under extinction conditions re-

mained at the criterion. To control for order effects, different

drug doses and the vehicles were administered in a random

sequence across the reinstatement test sessions.

7. Effects of 5g, 5p , a n d 5q on Cu e-In d u ced Rein sta te-

m en t of Dr u g-Seek in g Beh a vior . To determine whether 5g,

5p , and 5q affected rats’ behavior induced by presentation of

cocaine-associated stimuli after extinction and in the absence

of any further cocaine, three independent groups of rats were

prepared for testing. In the first experiment a group of eight

rats was evaluated to see whether 0.3, 1, and 3 mg/kg 5g

affected the behavior induced by cocaine-associated cues. The

compound, dissolved in 2 mL of sterile saline, or vehicle, was

given i.p. 30 min before testing. In two other separate groups

of seven rats 5p and 5q, dissolved in 25% DMSO in a final

volume of 2 mL of sterile saline, or vehicle, were given i.p. 30

min before testing, at doses from 0.1 to 3 mg/kg. The test

sessions in the presence of the stimuli associated with cocaine

or saline were arranged in a random sequence.

8. Effect of 5g on Coca in e Self-Ad m in istr a tion . To

evaluate the effect of 5g on the reinforcing properties of cocaine

two groups of six rats were initially trained to press a lever of

an operant box for food reinforcement, as described above. As

soon as this behavior was mastered rats underwent chronic

jugular catheter implantation.

After a five-day recovery, animals were allowed to self-

administer cocaine hydrochloride, dissolved in sterile saline,

for 2 h daily sessions on a FR1 schedule with 20 s TO. These

sessions began by extension of both the active and inactive

levers and terminated with their retraction at the end of the

sessions. An active lever press resulted in a 6 s infusion of

cocaine HCl (0.25 mg/0.1 mL) and illumination of the stimulus

light above the active lever, which remained on for 20 s,

signaling the TO period during which pressing a lever would

not result in cocaine infusion. Inactive lever pressings were

recorded but had no programmed consequences. Rats received

a 2 h self-administration session 7 days a week. Once the

animals had established a stable pattern of cocaine intake (the

total number of drug infusions per session stabilized to within

10% for three consecutive days), they were randomly assigned

to two experimental groups to self-administer two doses of

cocaine (0.125 and 0.5 mg/0.1 mL/infusion). After 3 days of

stable baseline, the effects of 30 min pretreatment with 0.3

and 3 mg/kg 5g or vehicle on cocaine self-administration was

assessed. The different dosages of 5g and vehicle were given

to rats in a counterbalanced order. The test sessions for each

animal were separated by at least 3 days of stable baseline.

3838 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18

Campiani et al.

(18) Pilla, M.; Perachon, S.; Sautel, F.; Garrido, F.; Mann, A.;

Wermuth, C. G.; Schwartz, J . C.; Everitt, B. J .; Sokoloff, P.

Selective inhibition of cocaine-seeking behavior by a partial D₃

receptor agonist. Nature 1999, 400, 371-375.

(19) Reavill, C.; Taylor, S. G.; Wood, M. D.; Ashmeade, T.; Austin,

N. E.; Avenell, K. Y.; Boyfield, I.; Branch, C. L.; Cilia, J .;

Coldwell, M. C.; Hadley, M. S.; Hunter, A. J .; J effrey, P.; J ewitt,

F.; J ohnson, C. N.; J ones, D. N. C.; Medhurst, A. D. Middlemiss,

D. N.; Nash, D. J .; Riley, G. J .; Routledge, C.; Stemp, G.; Thewlis,

K. M.; Trail, B.; Vong, A. K. K.; Hagan, J . J . Pharmacological

actions of a novel, high-affinity, and selective human dopamine

D₃receptor antagonist, SB-277011-A. J . Pharmacol. Exp. Ther.

2000, 294, 1154-1165.

(20) Di Ciano, P.; Underwood, R.; Hagan, J .; Everitt, B. J . Attenu-

ation of drug-seeking by a selective D₃receptor antagonist. Soc.

Neurosci. Abstr. 2001, 27, 647-649.

(21) Cussac, D.; Newman-Tancredi, A.; Audinot, V.; Nicolas, J . P.;

Boutin, J . A.; Gobert, A.; Millan, M. J . The novel dopamine D₃

receptor partial agonist, BP897, is a potent ligand at diverse

adrenergic and serotoninergic receptors. Soc. Neurosci. Abstr.

2000, 26, 808-809.

Mopac module of Insight2000. The AM1 method was preferred

to the PM3 method because of its lower degree of pyramidal-

ization of the conjugated nitrogens (see ref 50). The MMOK

keyword was used to constrain the amide nitrogen to be

planar. The NOTHIEL option was used to disable the Thiel’s

FSTMIN technique in the BFGS routine, so gradient minimi-

zation could be done. GNORM value was set to 0.5. To reach

full geometry optimization we increased the criteria for

terminating all optimizations by a factor of 100, using the

keyword PRECISE.

Ack n ow led gm en t. We thank MURST, Rome, Italy

(PRIN 01) and Regione Campania, Italy (Legge regio-

nale 31-12-94 n.41, annualita´ 1999 Area di interesse

C.3) for financial support. The authors thank Mrs. J udy

Baggott for manuscript editing.

Refer en ces

(22) These results were partially presented at the XIII° Meeting of

the Italian Society of Neuropsychopharmacology: Novel frontieres

in neuropsychopharmacology: from genome to therapy, Milan,

9-12 J uly 2002; Carnovali, F.; Campiani, G.; Butini, S.; Aiello,

F.; Trotta, F.; Stark, J .; Cagnotto, A.; Mennini, T.; Cervo, L.

Controllo dei comportamenti di ricerca della cocaina dovuti agli

stimoli precedentemente associati nei ratti: c’e’ coinvolgimento

dei recettori D3?, P-41, 402.

(23) Bettinetti, L.; Shlotter, K.; Hu¨bner, H.; Gmeiner, P. Interactive

SAR studies: rational discovery of super-potent and highly

selective dopamine D₃receptor antagonists and partial agonists.

J . Med. Chem. 2002, 45, 4594-4597.

(24) Leopoldo, M.; Berardi, F.; Colabufo, N. A.; De Giorgio, P.;

Lacivita, E.; Perrone, R.; Tororella, V. Structure-affinity rela-

tionship study on N-[4-(4-Arylpiperazin-1-yl)butyl]arylcarboxa-

mides as potent and selective dopamine D₃receptor ligands. J .

Med. Chem. 2002, 45, 5727-5735.

(25) Wolfe, J . P.; Buchwald, S. L. Scope and limitations of the Pd/

BINAP-catalyzed amination of aryl bromides. J . Org. Chem.

2000, 65, 1144-1157.

(26) Crescenza, A.; Botta, M.; Corelli, F.; Santini, A.; Tafi, A. Cyclic

dipeptides. Synthesis of methyl (R)-6-[(tert-butoxycarbonyl)-

amino]-4,5,6,7-tetrahydro-2-methyl-5-oxo-1,4-thiazepine-3-car-

boxylate and its hexahydro analogues: elaboration of a novel

ACE/NEP inhibitor. J . Org. Chem. 1999, 64, 3019-3025.

(27) Nagarajan, K.; Ranga Rao, V.; Venkateswarlu, A. Condensed

heterotricycles: pyrrolo[1,2-a]quinoxaline derivatives. Indian J .

Chem. 1972, 10, 344-350.

(28) Tarver, J . E., J r.; Pfizenmayer, A. J .; J oullie´, M. M. Total

syntheses of conformationally constrained didemnin B ana-

logues. Replacements of N,O-dimethyltyrosine with L-1,2,3,4-

tetrahydroisoquinoline and L-1,2,3,4-tetrahydro-7-methoxyiso-

quinoline. J . Org. Chem. 2001, 66, 7575-7587.

(1) Carrol, F. I.; Howell, L. L.; Kuhar, M. J . Pharmacotherapies for

treatment of cocaine abuse: preclinical aspects. J . Med. Chem.

1999, 42, 2721-2736.

(2) Substance Abuse and Mental Health Services Administration

National Household Survey on Drug Abuse: Main Findings,

1997; Department of Health and Human Services: Washington,

DC, 1998.

(3) Schuster, C. R.; Kuhar, M. J . Pharmacological Aspects of Drug

Dependence: Toward an Integrated Neurobehavioral Approach.

Springer-Verlag: New York, 1996; Vol. 118.

(4) Woolverton, W. L.; Ranaldi R.; Wang, Z.; Ordway G. A.; Paul I.

A.; Petukhov P.; Kozikowski, A. Reinforcing strength of a novel

dopamine transporter ligand: pharmacodynamic and pharma-

cokinetic mechanisms. J . Pharmacol. Exp. Ther. 2002, 303, 211-

217.

(5) Gawin, F. H.; Kleber, H. D. Abstinence symptomatology and

psychiatric diagnosis in cocaine abusers. Clinical observations.

Arch. Gen. Psychiatry 1986, 43, 107-113.

(6) Rohsenow, D. J .; Niaura, R. S.; Childress, A. R.; Abrams, D. B.;

Monti, P. M. Cue reactivity in addictive behaviors: theoretical

and treatment implications. Int. J . Addict. 1990, 25, 957-

993.

(7) Gawin, F. H. Cocaine addiction: psychology and neurophysiol-

ogy. Science 1991, 251, 1580-1586.

(8) Wikler, A. Dynamics of drug dependence. Arch. Gen. Psychiatry

1973, 28, 611-616.

(9) O’Brien, C. P.; Childress, A. R.; McLellan, A. T.; Ehrman, R. A

learning model of addiction. Res. Publ. Assoc. Res. Nerv. Ment.

Dis. 1992, 70, 157-177.

(10) O’Brien, C. P.; McLellan, A. T. Myths about the treatment of

addiction. Lancet 1996, 347, 237-240.

(11) De Wit, H.; Stewart, J . Reinstatement of cocaine-reinforced

responding in the rat. Psychopharmacology 1981, 75, 134-

143.

(29) Verheyden, J . P. H.; Moffatt, J . G. Halo sugar nucleosides.

Reactions for the chlorination and bromination of nucleoside

hydroxyl groups. J . Org. Chem. 1972, 37, 2289-2299.

(30) McKillop, A.; Taylor, R. J . K.; Watson, R. J .; Lewis, N. An

improved procedure for the preparation of the Garner aldehyde

and its use for the synthesis of N-protected 1-halo-2-(R)-amino-

3-butenes. Synthesis 1994, 1, 31-33.

(31) Lopez-Rodriguez, M. L.; Morcillo, M. J .; Fernandez, E.; Porras,

E.; Orensanz, L.; Beneytez, M. E.; Manzanares, J .; Fuentes, J .

A. Synthesis and structure-activity relationships of a new model

of arylpiperazines. Study of the psychochemical influence of the

pharmacophore on 5-HT_1A/R₁-adrenergic receptor affinity: syn-

thesis of a new derivative with mixed 5-HT_1A/D₂antagonist

properties. J . Med. Chem. 2001, 44, 186-197.

(32) Rajur, S. B.; Merwade A. Y.; Flendi S. B.; Basanagoudar I. D.

Synthesis of 1,2,3,4-tetrahydropyrazino[1,2-a]indoles and ethyl

1-(2-amino-ethyl)indole-2-carboxylates. Indian J . Chem. 1989,

28B, 1065-1068.

(33) Heinzman, S. W.; Ganem, B. Mechanism of sodium borohydride-

cobaltous boride reductions. J . Am. Chem. Soc. 1982, 104, 6801-

6802.

(12) Meil, W. M.; See, R. E. Lesions of the basolateral amygdala

abolish the ability of drug associated cues to reinstate responding

during withdrawal from self-administered cocaine. Behav. Brain

Res. 1997, 87, 139-148.

(13) Weiss, F.; Maldonado-Vlaar, C. S.; Parsons, L. H.; Kerr, T. M.;

Smith, D. L.; Ben-Shahar, O. Control of cocaine-seeking behavior

by drug-associated stimuli in rats: effects on recovery of

extinguished operant-responding and extracellular dopamine

levels in amygdala and nucleus accumbens. Proc. Natl. Acad.

Sci. U.S.A. 2000, 97, 4321-4326.

(14) Whitelaw, R. B.; Markou, A.; Robbins, T. W.; Everitt, B. J .

Excitotoxic lesions of the basolateral amygdala impair the

acquisition of cocaine-seeking behavior under a second schedule

of reinforcement. Psychopharmacology 1996, 127, 213-224.

(15) Di Ciano, P.; Blaha, C. D.; Phillips, A. G. Conditioned changes

in dopamine oxidation currents in the nucleus accumbens of rat

by stimuli paired with self-administration or yoked-administra-

tion of D-amphetamine. Eur. J . Neurosci. 1998, 10, 1121-

1127.

(16) Ito, R.; Dalley, J . W.; Howes, S. R.; Robbins, T. W.; Everitt, B.

J . Dissociation in conditioned dopamine release in the nucleus

accumbens core and shell in response to cocaine cues and during

cocaine-seeking behavior in rats. J . Neurosci. 2000, 20, 7849-

7495.

(34) Anzini, M.; Corelli, F.; Cagnotto, A.; Skorupska, M. 5,6-Dihydro-

5-{4-[4-(2-methoxyphenyl)-1-piperazinyl]butyl}-4H-pyrrolo[1,2-

a][1,4]benzodiazepine-4,6-dione and related compounds as new

5-HT_1Areceptor ligands. Med. Chem. Res. 1993, 3, 249-256.

(35) Einsiedel, J .; Thomas, C.; Hu¨bner, H.; Gmeiner, P. Phenylox-

azoles and phenylthiazoles as benzamide bioisosteres: synthesis

and dopamine receptor binding profiles. Bioorg. Med. Chem. Lett.

2000, 10, 2041-2044.

(17) Spealman, R. D. Antagonism of behavioural effects of cocaine

by selective dopamine receptor blockers. Psychopharmacology

1990, 101, 142-145. (b) Howell, L. L.; Byrd, L. D. Enhanced

sensitivity to the behavioural effects of cocaine by selective

dopamine receptor blockers. J . Pharmacol. Exp. Ther. 1992, 262,

907-915. (c) Cervo, L.; Carnovali, F.; Stark, J . A.; Mennini, T.

Cocaine-seeking behavior in response to drug-associated stimuli

in rats: involvement of D₃and D₂dopamine receptors. Neuro-

psychopharmacology 2003, 28, 1150-1159.

(36) Everitt, B. J .; Wolf, M. E. Psychomotor stimulant addiction: a

neural systems perspective. J . Neurosci. 2002, 22, 3312-

3320.

Highly Selective D₃Receptor Ligands

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3839

(37) Fletcher, A.; Forster, E. A.; Bill, D. J .; Brown, G.; Cliffe, I. A.;

Hartley, J . E.; J ones, D. E.; McLenachan, A.; Stanhope, K. J .;

Critchley, D. J .; Childs, K. J .; Middlefell, V. C.; Lanfumey, L.;

Corradetti R.; Laporte, A. M.; Gozlan, H.; Hamon, M.; Dourish,

C. T. Electrophysiological, biochemical, neurohormonal and

behavioural studies with WAY-100635, a potent, selective and

silent 5-HT_1Areceptor antagonist. Behav. Brain Res. 1996, 73,

337-353.

(43) Pregenzer, J . F.; Alberts, G. L.; Im W. B. Agonist-induced [³⁵S]-

GTPγS binding in the membranes of Spodoptera frugiperda

insect cells expressing the human D₃dopamine receptor. Neu-

rosci. Lett. 1997, 226, 91-94.

(44) De Lean, K. W.; Munson, P. J .; Rodbard, D. Simultaneous

analysis of families of sigmoidal curves: application to bioassay,

radioligand assay and physiological dose-response curves. Am.

J . Physiol. 1978, 235, E97-E102.

(38) Campiani, G.; Nacci, V.; Bechelli, S.; Ciani, S.; Garofalo, A.;

Fiorini, I.; Wikstrom, H. V.; de Boer, P.; Tepper, P. G.; Cagnotto,

A.; Mennini, T. New antipsychotic agents with serotonin and

dopamine antagonist properties based on a pyrrolo[2,1-b][1,3]-

benzothiazepine structure. J . Med. Chem. 1998, 41, 3763-3772.

(39) Claudi, F.; Di Stefano, A.; Napolitani, F.; Cingolani, G. M.;

Giorgioni, G.; Fontenla, J . A.; Montenegro, G. Y.; Rivas, M. E.;

Rosa, E.; Michelotto, B.; Orlando, G.; Brunetti, L. Binding and

preliminary evaluation of 5-hydroxy- and 10-hydroxy-2,3,12,12a-

tatrahydro-1H-[1]benzoxepino[2,3,4-ij]isoquinoline as dopamine

receptors ligands. J . Med. Chem. 2000, 43, 599-608.

(45) Cheng, Y.; Prusoff, W. H. Relationship between the inhibition

costant (Ki) and the concentration of inhibition which causes

50% inhibition (IC₅₀) of an enzymatic reaction. Biochem. Phar-

macol. 1973, 22, 3099-3108.

(46) Caine, S. B.; Lintz, R.; Koob, G. F. Intravenous drug self-

administration techniques in animals. In Behavioral Neuro-

science: A Practical Approach; Sahgal, E. A., Ed; Oxford IRL

Press: Oxford, 1993; pp 117-143.

(47) Maple, J . R.; Diniur, U.; Hagler, A. T. Derivation of force fields

for molecular mechanics and dynamics from ab initio energy

surfaces. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5350-5354.

(48) Nilsson, J .; Wikstro¨m, H. V.; Smilde, A.; Glase, S.; Pugsley, T.;

Cruciani, G.; Pastor, M.; Clementi, S. GRID/GOLPE 3D Quan-

(40) Modica, M.; Santagati, M.; Russo, F.; Selvaggini, C.; Cagnotto,

A.; Mennini, T. High affinity and selectivity of [[(arylpiperazi-

nyl)alkyl]thio]thieno[2,3-d]pyrimidinone derivatives for the 5-HT_1A

receptor. Synthesis and structure-affinity relationships. Eur. J .

Med. Chem. 2000, 35(7-8), 677-689.

titative Structure-Activity Relationship Study on

a Set of

Benzamides and Naphthamides, with Affinity for the Dopamine

D₃Receptor Subtype. J . Med. Chem. 1997, 40, 833-840.

(49) Fletcher, R. Unconstrained Optimization. In Practical Methods

of Optimization; J ohn Wiley & Sons: New York, 1980; Vol. 1.

(50) (a) Stewart, J . J . P. MOPAC: A Semiempirical Molecular Orbital

Program J . Comput.-Aided Mol. Des. 1990, 4, 1-105. (b)

Stewart, J . J . P. MOPAC MANUAL, 6th ed.; 1990.

(41) Grijalba, B.; Callado, L. F.; Meana, J . J .; Garcia-Sevilla, J . A.;

Pazos, A. Alpha2-Adrenoceptor subtypes in the human brain:

a

pharmacological delineation of [³H]RX-821002 binding to

membrane and tissue sections. Eur. J . Pharm. 1996, 310, 83-

93.

(42) Alper, R. H.; Nelson, D. L. Characterization of 5-HT_1Areceptor-

mediated [³⁵S]-GTPγS binding in rat hippocampal membranes.

Eur. J . Pharmacol. 1998, 343, 303-312.

J M0211220

Article Doi

Synthesis and pharmacological evaluation of potent and highly selective D3 receptor ligands: Inhibition of cocaine-seeking behavior and the role of dopamine D3/D2 receptors

DOI: 10.1021/jm0211220

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm0211220

Full text of DOI:10.1021/jm0211220

Products guided by the article

R&D Labs maybe for 600709-82-8

Relevant to this article

Hot Product