A de novo design probe of a dopamine receptor ligand based on a theoretical approach

Article abstract of DOI:10.1006/bioo.1996.0031

A trial to design de novo a dopamine (DA) receptor ligand was made, taking as the base four structural and electrostatic requirements: (1) a group simulating the interaction of the DA amino group with the TM3 aspartic acid of the receptor, (2) a group that can simulate the interaction of the DA m-hydroxyl group with the TM5 serine of the receptor, (3) a distance between these groups similar to that of the DA anti-coplanar conformer, and (4) a rigid structure keeping the distance between the groups right. After the design 'on paper' of the models of four structures, quantum chemistry calculations were performed to check the properties of the molecules, and then the most encouraging ones were synthesized. None of the compounds synthesized was able to bind D₁- and D₂-dopamine receptor subtypes; this shows that the structural and electrostatic requirements considered in this work are insufficient. In particular, the presence of an arylethylamine moiety seems to be essential for the interaction of a ligand with the DA receptor.

Full text of DOI:10.1006/bioo.1996.0031

BIOORGANIC CHEMISTRY 24, 358–375 (1996)
ARTICLE NO. 0031
A de Novo Design Probe of a Dopamine Receptor Ligand
Based on a Theoretical Approach
M. BAGINSKI,* F. CLAUDI,† G. GIORGIONI,†,1 J. A. FONTENLA,‡ E. ROSA,‡ AND
M. CARDELLINI
†
*Department of Pharmaceutical Technology and Biochemistry, Technical University of Gdansk,
Narutowicza St 11/12, 80-952 Gdansk, Poland; †Department of Chemical Sciences, University of
Camerino, Via S. Agostino 1, 62032 Camerino, Italy; and ‡Department of Pharmacology, Faculty of
Pharmacy, University of Santiago de Compostela, 15706 Santiago de Compostela, Spain
Received March 8, 1996
A trial to design de novo a dopamine (DA) receptor ligand was made, taking as the base
four structural and electrostatic requirements: (1) a group simulating the interaction of the
DA amino group with the TM3 aspartic acid of the receptor, (2) a group that can simulate
the interaction of the DA m-hydroxyl group with the TM5 serine of the receptor, (3) a distance
between these groups similar to that of the DA anti-coplanar conformer, and (4) a rigid
structure keeping the distance between the groups right. After the design ‘‘on paper’’ of the
models of four structures, quantum chemistry calculations were performed to check the
properties of the molecules, and then the most encouraging ones were synthesized. None of
the compounds synthesized was able to bind D1- and D2-dopamine receptor subtypes; this
shows that the structural and electrostatic requirements considered in this work are insufficient.
In particular, the presence of an arylethylamine moiety seems to be essential for the interaction
of a ligand with the DA receptor.
1996 Academic Press, Inc.
INTRODUCTION
The neurotransmitter dopamine (DA) is involved in various central nervous
system (CNS) disorders such as schizophrenia and Parkinson’s disease. Structure–
activity relationship studies on DA agonists and antagonists have attracted consider-
able interest during the last years. The results obtained have been used to discuss
optimal features for DA receptor activation, and various hypothetical DA receptor
models have been suggested. The same studies have given many lead compounds as
well as apomorphine, aminodihydroxytetraline (ADTN), ergoline, and benzazepine
derivatives (1).
Recently, in addition to the structure–activity studies, some attempts have been
made to approach the problem in a more rational way, based on understanding
the molecular mechanism of neurotransmitter–receptor interaction (2). This ap-
proach has been possible since the three-dimensional structure of DA receptors
has been postulated by means of molecular modeling simulations.
Part of those studies concerned conformational analysis and molecular electro-
1 To whom correspondence should be addressed.
358
0045-2068/96 $18.00
Copyright 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.

DESIGN OF DOPAMINE RECEPTOR LIGANDS
359
FIGURE 1
static potential distribution of DA (3–6). Theoretical conformational analysis per-
formed for isolated and solvated DA molecule has revealed that its side chain may
attain various conformations. The energy differences between them are small, but
the anti-conformer with the catechol ring coplanar with the ethylamine (C–C–N)
side chain (Fig. 1) is not energetically favored. This is not in agreement with
experimental studies on conformationally restricted analogs which suggest that the
anti-coplanar conformer is biologically active (7). Therefore, Edvardsen and Dahl
(5) have proposed a ‘‘zipper’’ mechanism for the binding of flexible DA to its
receptor, a suggestion based on molecular dynamics simulations. Molecular electro-
static potential calculations have revealed that the potential and charge distribution
is very dependent on a conformation that can be important for DA-receptor recogni-
tion and DA agonist/antagonist selectivity.
Theoretical molecular modeling calculations have been performed for isolated
DA receptor as well as for receptor–DA interacting structure (8–12). These studies
have been possible since (1) the genes coding for different DA receptors, belonging
to the large G-protein-linked family of receptors, have recently been isolated,
cloned, and decoded (13); (2) the structure of bacteriorhodopsin—a structural
analog of the G-protein-coupled receptors (GPCR)—has been resolved by cryomi-
croscopy study (14). The structural molecular modeling simulations, by homology
to bacteriorhodopsin, together with hydropathicity analysis have revealed that the
DA D2 receptor (similar to other G-protein-coupled receptors) has seven Ͱ-helical
transmembrane segments that form the central core with a putative ligand-binding
site. It has also been found that defined amino acids can be responsible for DA–
receptor interaction. The point mutations of DA receptors also support these data
(15). Generally, it has been postulated that (1) the aspartic acidic residue on the
third transmembrane domain (TM3) interacts with the protonated amino group of
DA, (2) two serine residues on the fifth transmembrane domain (TM5) interact
with the hydroxyl groups of DA, and (3) the aromatic nuclei of a few amino acids
on TM3 and TM6 form the hydrophobic surrounding of DA, which is probably
rearranged during the binding process.
Taking into acocunt the essential aspects of the molecular model of receptor–DA
interaction and the results of DA structural studies, we attempted to design de
novo a DA-receptor ligand. The design was based on four structural and electrostatic
requirements: (1) a group simulating the interaction of the DA amino group with
the TM3 aspartic acid of the receptor, (2) a group which can simulate the interaction

360
BAGINSKI ET AL.
TABLE 1
Results of AM1 Calculations for DA Moleculea
OH
OH
OH
OH
HO
HO
HO
HO
Conformers
NH2
NH2
NH2
II
NH2
I
III
IV
Torsional angle (degrees)
N11–C10–C9–C5
C10–C9–C5–C4
178
Ϫ2
Ϫ176
167
Ϫ177
Ϫ100
73
Ϫ95
˚
Distance of atoms (A)
O7–N11
O8–N11
7.311
7.903
6.474
7.868
6.700
7.789
6.249
6.610
Net atomic charge (a.u.)
Ϫ0.250
O7
O8
N11
Ϫ0.249
Ϫ0.271
Ϫ0.350
Ϫ0.249
Ϫ0.271
Ϫ0.352
Ϫ0.252
Ϫ0.272
Ϫ0.348
Ϫ0.272
Ϫ0.342
Energy (heat of formation) and relative energy (kcal/mol)
E
Ϫ74.7
Ϫ74.9
Ϫ75.7
Ϫ74.6
⌬E
1.0
0.8
0.0
1.1
Proton affinity of nitrogen atom N11 (kcal/mol)
216.2 216.2 216.2
PA
216.2
a The numbering of atoms is presented in Fig. 1.
of the DA m-hydroxyl group with the TM5 serine of the receptor, (3) the distance
between these groups should be similar, as for the anti-coplanar conformer I of DA
(Table 1), and (4) a rigid structure keeping the distance between the groups right.
In accordance with those requirements the models of four structures were de-
signed ‘‘on paper’’ (Fig. 2). Later quantum chemistry calculations were performed
to check the properties of the molecules, and then the most encouraging ones
were synthesized.
All well-known DA receptor ligands include in their structure an aromatic moiety.
The aromatic ring is considered very important for ligand–receptor binding because
it seems to interact with the aromatic nuclei of two phenylalanine residues on the
DA receptor (8). However, the DA aromatic ring may also be seen as a flat structure
able to penetrate into the receptor pocket surrounded by the aromatic moieties of
the phenylalanine residues. On the other hand, studies developed in the field of ͱ-
adrenergic receptor (G-protein-coupled receptor) show that completely aliphatic
molecules maintain ͱ-blocking properties as well as the relative aromatic com-
pounds (16). Moreover, we have not found any reference to negative biological
test results being obtained by studying nonaromatic compounds as DA receptor
ligands. Therefore, we decided to replace the aromatic ring of DA with a system
of conjugated double bonds to obtain a planar structure. In addition, since a group

DESIGN OF DOPAMINE RECEPTOR LIGANDS
361
F
IG. 2. The atoms were automatically numbered by the Chem3D program.
simulating the phenolic hydroxyl connected to the planar aromatic ring and able
to interact with the serine residue on TM5 is necessary, we have chosen the Ͱ–ͱ
unsaturated carboxylic acid moiety. On the basis of this, we designed structure 1.
Compound 2 was seen as a more complex Ͱ–ͱ unsaturated carboxylic acid which
contains molecule 1 embedded in a more rigid framework. Moreover, the carboxyl
group on compounds 1, 2, and 3 can interact with the serine residue on TM5 as
hydrogen bond donor or acceptor. Structure 4, like DA, bears a phenolic group.
Models 1, 2, and 4 have an amino group very similar to that of DA, and compound
3 contains a nitrogen atom which can simulate the DA amino group.
RESULTS AND DISCUSSION
The quantum chemistry calculations for the four DA conformers shown in Table
1 were performed. Two of them, I and II, are anti-coplanar conformers. The anti-
perpendicular conformer (III) is found in the crystal state and the gauche-perpendic-
ular one (IV) is the most stable conformer found by Edvardsen and Dahl (5) in
molecular dynamics simulations for protonated DA.

362
BAGINSKI ET AL.
TABLE 2
Results of AM1 Calculations for Compound 1 (E Isomer)a
O
OH
HO
O
O
OH
HO
O
Conformers
H
H
H
H
NH2
NH2
NH2
NH2
1a
1b
1c
1d
Torsional angle (degrees)
N9–C5–C4–C3
H22–C5–C4–C3
O10–C8–C7–C1
O11–C8–C7–C1
Ϫ73
171
Ϫ148
Ϫ33
Ϫ73
171
0
179
Ϫ178
Ϫ56
5
Ϫ59
Ϫ149
33
180
Ϫ176
˚
Distance of atoms (A)
O10–N9
O11–N9
5.718
5.800
5.636
5.899
7.054
6.748
6.796
7.068
Net atomic charge (a.u.)
Ϫ0.375
O10
O11
N9
Ϫ0.366
Ϫ0.319
Ϫ0.336
Ϫ0.367
Ϫ0.320
Ϫ0.347
Ϫ0.377
Ϫ0.319
Ϫ0.347
Ϫ0.319
Ϫ0.336
Energy (heat of formation) and relative energy (kcal/mol)
E
Ϫ101.4
Ϫ102.7
Ϫ99.5
Ϫ100.9
⌬E
1.3
0.0
3.2
1.8
Proteon affinity of nitrogen atom N9 (kcal/mol)
215.1 215.1 215.1
PA
215.1
a The numbering of atoms is presented in Fig. 2.
In our study, conformer III was found to have the lowest energy (heat of forma-
tion). This result differs from the studies of Urban et al. (3), who found that the
gauche-perpendicular conformer (our IV) is the most stable one for neutral DA,
but at the same time they did not give energy values for the anti-perpendicular
conformer (our III), so it is difficult to discuss the discrepancy.
It was also found, within our calculations, that the anti-coplanar conformers I
and II lie only approximately 1.0 kcal/mol above conformer III. Generally, since
the differences of energy between conformers are not very big, and conformer I is
postulated as the active one (7), we chose it as the reference for other studied mole-
cules.
The results of calculations performed for compounds 1–4 are presented in Tables
2–5. The carbon atoms C5 of 1 and C10 of 2 (Fig. 2) can have different configurations
with regard to the position of the amino group. These possibilities were accounted
for in the calculations (Tables 2 and 3). The energy difference between the conform-
ers of the same compound sometimes assumes a value of several kilocalories per
mole (especially for compounds 2 and 4). However, it is difficult to analyze this,
since the conformer populations at the receptor site can be different from those in

DESIGN OF DOPAMINE RECEPTOR LIGANDS
363
TABLE 3
Results of AM1 Calculations for Compound 2a
O
OH
HO
O
O
OH
HO
O
Conformers
H
H
H
H
NH2
NH2
NH2
NH2
2a
2b
2c
2d
Torsional angle (degrees)
N12–C10–C4–C3
H18–C10–C4–C3
O13–C11–C2–C3
O14–C11–C2–C3
Ϫ110
135
160
Ϫ110
135
Ϫ10
171
143
Ϫ97
159
146
Ϫ93
Ϫ12
168
Ϫ21
Ϫ22
˚
Distance of atoms (A)
O13–N12
O14–N12
6.939
6.272
6.332
6.952
7.255
6.447
6.623
7.227
Net atomic charge (a.u.)
Ϫ0.373
O13
O14
N12
Ϫ0.370
Ϫ0.316
Ϫ0.334
Ϫ0.371
Ϫ0.315
Ϫ0.345
Ϫ0.373
Ϫ0.320
Ϫ0.349
Ϫ0.320
Ϫ0.334
Energy (heat of formation) and relative energy (kcal/mol)
E
Ϫ81.1
Ϫ82.0
Ϫ77.4
Ϫ78.6
⌬E
0.9
0.9
4.6
3.4
Proteon affinity of nitrogen atom N12 (kcal/mol)
218.4 218.4 218.4
PA
218.4
a The numbering of atoms is presented in Fig. 2.
the gas phase. It also means that the low energetic conformer in the gas phase need
not be the active one. The same problem regards the DA molecule.
When the distances between functional groups (or rather atoms) are considered,
compound 1 (conformers 1c and 1d), compound 2 (conformers 2c and 2d and, to
some extent, 2a and 2b), and compound 4 (conformer 4a) are the best simulators
of the DA molecule. The values of net atomic charge for oxygen atoms in the
compounds with carboxyl groups (1–3) are obviously more negative than for those
in DA, so stronger electrostatic interaction of these groups with the serine residue
on the DA receptor can be expected. The values of net atomic charge for the
nitrogen atom are very similar for all compounds (except for compound 3), and
comparable with those for the DA molecule. The value for proton affinity of the
nitrogen atom, which can be used to estimate to some extent the possibility of
forming a hydrogen bond, is better for compounds 1–3 than for the DA molecule.
The amino group (i.e., the nitrogen atom) in these compounds can form a similar
or stronger ionic bond with aspartic acid residue on the DA receptor.
Taking into account the distance requirement, which may be very important for
a possible interaction of the functional groups with the DA receptor, compound 3
(Fig. 2, Table 4) was excluded from further studies. The results of molecular model-
ing calculations for the other structures 1, 2, and 4 were encouraging, so it was

364
BAGINSKI ET AL.
TABLE 4
Results of AM1 Calculations for Compound 3a
HO
O
HO
O
Conformers
N
N
3a
3b
Torsional angle (degrees)
O16–C15–C1–C6
O17–C15–C1–C6
0
180
180
0
˚
Distance of atoms (A)
O16–N14
O17–N14
5.157
6.468
6.432
4.954
Net atomic charge (a.u.)
Ϫ0.361
O16
O17
N14
Ϫ0.368
Ϫ0.313
Ϫ0.138
Ϫ0.319
Ϫ0.137
Energy (heat of formation) and relative energy (kcal/mol)
E
Ϫ21.2
Ϫ21.3
⌬E
0.1
Proton affinity of nitrogen atom N14 (kcal/mol)
218.2
0.0
PA
218.2
a The numbering of atoms is presented in Fig. 2.
decided to synthesize these compounds. Meanwhile, however, we had found some
reports (17) about dopaminergic activity of 2-amino-5-hydroxyphenalene 4 deriva-
tives, and thus only compounds 1 and 2 were synthesized. Since the hydroxyl of
the carboxylic acid group is more acidic than phenolic hydroxyl, we also synthesized
the ester and amide derivatives to evaluate the influence of acidity on the ligand–
receptor interaction.
The synthetic approach to 3-aminocyclohexylidene acetic acid 1 (Scheme 1)
started from commercially available 2-cyclohexen-1-one, which was reacted with
azidotrimethylsilane and ethylene glycol to give 3-azidocyclohexanone ethylene
ketal 5. This compound was reduced by lithium aluminum hydride to the 3-amino-
cyclohexanone ethylene ketal 6, which was treated with acetic anhydride to give
acetamide 7. After ketal cleavage, 3-acetamidocyclohexanone 8 was reacted with
triethylphosphonacetate to obtain the 3-N-acetylaminocyclohexylidenacetic acid
ethyl ester 9 as E–Z isomers mixture. Single isomers were separated by recrystalliza-
tion; the exact structures were identified by NMR analysis observing the NOE
(nuclear Overhouser effect) between the hydrogen on C7 and those on C6 (E
isomer) (numbering in Fig. 2) or on C2 (Z isomer). E isomer 9 was hydrolyzed to
target compound 1. Then the amino group was protected with a buthyloxycarbonyl
(Boc) group. The N-Boc-amino acid 10 was treated with ethyl chloroformate, trieth-
ylamine and ammonia to give the corresponding amide 11, or with potassium

DESIGN OF DOPAMINE RECEPTOR LIGANDS
365
TABLE 5
Results of AM1 Calculations for Compound 4a
HO
HO
Conformers
H2N
H
H
NH2
4a
4b
Torsional angle (degrees)
O15–C13–C12–C3
H25–C13–C12–C3
176
60
70
Ϫ169
˚
Distance of atoms (A)
O11–N15
7.314
Net atomic charge (a.u.)
Ϫ0.252
6.287
O11
N15
Ϫ0.254
Ϫ0.343
Ϫ0.335
Energy (heat of formation) and relative energy (kcal/mol)
E
Ϫ13.6
Proton affinity of nitrogen atom N15 (kcal/mol)
212.9
Ϫ10.0
212.9
PA
a The numbering of atoms is presented in Fig. 2.
carbonate and iodomethane to give ester 13. The Boc group cleavage gave, respec-
tively, the 3-aminocyclohexylidenacetamide 12 and the 3-aminocyclohexylidenacetic
acid methyl ester 14.
Scheme 2 shows the synthetic approach to 5-amino-5,6,7,8-tetrahydro-1-naphtha-
lenecarboxylic acid 2. 5-Oxo-5,6,7,8-tetrahydro-1-naphthalenecarboxylic acid 15
(18–20) was reacted with hydroxylamine to give the corresponding oxime 16. This
was hydrogenated over Pd/C in trifluoroacetic acid to amine 2. The amino group
was protected with a Boc group, and the N-Boc-amino acid 19 was treated with
N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), and ammonia to
give the corresponding amide 20. The Boc group cleavage gave the 5-amino-5,6,7,8-
tetrahydro-1-naphthalenecarboxamide 21. Treatment of amino acid 2 with thionyl
chloride and methanol gave the 5-amino-5,6,7,8-tetrahydro-1-naphthalenecarbox-
ylic acid ethyl ester 18.
To evaluate the affinities for D1 and D2 DA receptors, compounds 1, 2, 12, 14,
18, and 21 and the reference compound were tested in in vitro radioligand competi-
tion assays on rat striatal membrane using [3H]SCH23390 and [3H]spiperone as
radioligands Table 6.
Although some molecular parameters calculated by theoretical methods for com-
pounds 1 and 2 agree with the reference DA conformer I (claimed to be active),
no compounds show any significant affinity to the DA receptor. These results suggest
that an aromatic ring seems to be necessary for the ligand–receptor interaction.
Moreover, the Ͱ–ͱ unsaturated carboxylic moiety cannot be considered a bioisost-

366
BAGINSKI ET AL.
S
CHEME. 1. (a) (CH3)3SiN3 , SiCl4 , HOCH2CH2OH; (b) LiAlH4 ; (c) (CH3CO)2O; (d) CH3COOH;
(e) (C2H5O)3POCH2COOC2H5 , NaH; (f) 2 NaOH, 6 HCl; (g) (t-BuOCO)2O, (C2H5)3N; (h)
ClCOOC2H5 , (C2H5)3N, NH3 ; (i) HCl; (j) CH3I, K2CO3 ; (k) HCl.
N
N

DESIGN OF DOPAMINE RECEPTOR LIGANDS
367
S
CHEME. 2. (a) NH2OH и HCl, pyridine; (b) H2 , Pd/C, CF3COOH; (c) SOCl2 ; (d) CH3OH; (e) (t-
BuOCO)2O, N(C2H5)3 ; (f) NHS, DCC, NH3 ; (g) HCl.
ere of the hydroxyphenyl group. Although compound 2 contains an aromatic group,
it lacks affinity to the DA receptors. This shows that the position of the aromatic
ring is also very important, and is in agreement with the suggestion that a distance
equivalent to the spacing of two methylene groups in an extended conformation
between the phenyl ring and amino group of DA is determinant for dopaminergic
activity (21). However, in view of our results and the previous considerations, it is
difficult to say whether the presence of the aromatic ring is needed because of
sterical requirements or, rather because of electrostatic interactions. The latter can
be described as aromatic–aromatic or aromatic–polar group interactions and may
be important for ligand DA receptor binding. Aromatic interactions are postulated
as an ordinary type of interaction inside proteins as well as between receptor and
ligand (22, 23).
Since the main compounds do not interact with DA receptor, the influence of

368
BAGINSKI ET AL.
TABLE 6
pKi Values and 95% Confidence Intervals for the New and Reference Drugs Assayed
D1
D2
Dopamine
Apomorphine
SCH23390
6.38 (6.04–6.72)
6.74 (6.63–6.85)
9.13 (9.09–9.17)
7.28 (7.26–7.31)
8.10 (7.94–8.26)
7.66(7.53–7.80)
6.94 (6.78–7.11)
NT
6.14 (5.95–6.33)
7.26 (6.97–7.56)
NTa
SKF38393
NT
NT
NT
(ϩ)-Butaclamol
cis-(Z)-flupentixol
Chlorpromazine
Spiperone
Sulpiride
Compounds: 1, 2, 12, 14, 18, and 21
NT
8.96 (8.87–9.05)
6.55 (6.30–6.79)
Inactive
NT
Inactive
a Not tested.
hydroxyl acidity cannot be discussed here. In conclusion, the results show that (1)
the structural and electrostatic requirements considered in this work are insufficient
to design a dopaminergic ligand; (2) the presence of an aromatic ring separated by
two methylenes from the amino group in an extended conformation seems to be
an essential prerequisite for the interaction between a ligand and the DA receptors.
Our study has also revealed that more structural data about DA receptor are
needed to understand the requirements for binding of DA to its receptor.
EXPERIMENTAL
Molecular Modeling
Molecular modeling calculations were performed by semiempirical quantum
chemistry program AM1 (24) within the MOPAC 6.0 packet (25). The AM1 input
structure for the DA molecule was taken from X-ray data (26). The structures of
molecules 1–4 were built into program Chem3D2 using standard values of bond
lengths, bond angles, and torsional angles. For all four molecules, the calculations
were performed for the different isomers with regard to the positions of the amino
and carboxyl groups. The geometry within calculations was fully optimized. The
studies for all molecules were performed in vacuo.
Proton affinity (PA) was determined by employing the heat of formation ac-
cording to
PA ϭ ⌬H0f (B) ϩ ⌬Hf0(Hϩ) Ϫ ⌬H0f (BHϩ),
where ⌬H0f (B) and ⌬H0f (BHϩ) are calculated by AM1 heats of formation for the
2 Chem3D Molecular Modelling and Analysis is a trademark of Cambridge Scientific Computing Inc.,
875 Massachusetts Avenue, Sixth Floor, Cambridge, MA 02139. It contains Allinger’s MM2 program
(QCPE 395).

DESIGN OF DOPAMINE RECEPTOR LIGANDS
369
free and protonated molecules, respectively, and ⌬H0f (Hϩ) is the experimental value
(367.2 kcal/mol) (27) of the heat of formation for Hϩ. Proton affinity was calculated
only for the most stable conformer of each compound.
Methods
Melting points were determined on a Buchi 510 apparatus and are uncorrected.
Microanalyses were performed on a 1106 Carlo Erba CHN analyzer, and the results
1
were within Ϯ0.4% of the calculated values. H NMR spectra were recorded on a
Varian VXR 200-MHz spectrometer. Chemical shifts are reported in parts per
million (ͳ) downfield from the internal standard tetramethylsilane (Me4Si). The IR
spectra were run on a Perkin-Elmer Model 297 spectrometer as nujol mulls or
liquid films. The identity of all new compounds was confirmed by both elemental
analysis and NMR data; homogeneity was confirmed by TLC on silica gel Machery–
Nagel Alugram SilG UV/254. Solutions were routinely dried over anhydrous
Na2SO4 prior to evaporation. Chromatographic purifications were accomplished on
Merck-60 silica gel columns 70–230 mesh ASTM from Merck with a reported
solvent.
Syntheses
3-Aminocyclohexylidenacetic acid hydrochloride 1. A suspension of 3-N-acetyl-
aminocyclohexylidenacetic acid ethyl ester 9 (0.5 g, 2.2 mmol) in 2
was warmed to reflux overnight. The solution was cooled in an ice bath and acidified
with 6 HCl. Water was evaporated at 30ЊC in vacuo and the solid residue was
N
NaOH (5 ml)
N
dried on P2O5 and triturated in CHCl3 . The suspension was filtered and the solid
washed with cold anhydrous ethanol. The filtrate was evaporated to dryness and
the white solid residue (0.21 g) was recrystallized from anhydrous ethanol/anhy-
1
drous Et2O. MP 223ЊC. Yield 50%. IR (nujol) 1670 (CuO), 1625 (CuO) cmϪ .
NMR (DMSO-d6) ͳ: 12.20 (s, 1H, COOH); 8,20 (s, 3H, NHϩ3 ); 5.65 (s, 1H, CHuC);
3.46 (m, 2H, H-cyclohex); 3.10 (m, 1H, N-CH); 2.27 (t, 1H, H-cyclohex); 1.90
(m, 3H, H-cyclohex); 1.53 (m, 1H, H-cyclohex); 1.35 (m, 1H, H-cyclohex). Anal.
(C8H13NO2 и HCl) C, H, N.
5-Amino-5,6,7,8-tetrahydro-1-naphthalenecarboxylic acid trifluoroacetate 2. A sus-
pension of 5-hydroxymino-5,6,7,8-tetrahydro-1-naphthalenecarboxylic acid 16
(2.8 g, 13.6 mmol) and 10% Pd/C (1.4 g) in trifluoroacetic acid (100 ml) was hydroge-
nated at 30 psi of hydrogen in a Parr shaker. Hydrogenation was stopped when
the theoretical amount of hydrogen had been taken up. The reaction mixture was
filtered on celite and the solvent was removed in vacuo. The residue was triturated
in anhydrous Et2O. The solid was filtered and recrystallized from ethyl acetate. MP
1
193–195ЊC. Yield 63%. IR (nujol) 1685 (CuO) cmϪ . NMR (DMSO-d6) ͳ: 13.10
(s, 1H, COOH); 8.35 (s, 3H, NHϩ3 ); 7.78 (d, 1H, ArH); 7.68 (d, 1H, ArH); 7.38 (t,
1H, ArH); 4.30 (m, 1H, CH–N); 3.00 (m, 2H, CH2); 1.92 (m, 4H, 2CH2). Anal.
(C11H13NO2 и CF3COOH) C, H, N.
3-Azidocyclohexanone ethylene ketal 5. To a solution containing 2-cyclohexen-
1-one (0.96 ml, 10 mmol) and anhydrous ethylene glycol (1.11 ml, 20 mmole) in
anhydrous CH2Cl2 (15 ml), azidotrimethylsilane (3 ml, 22 mmol) and tetrachlorosi-

370
BAGINSKI ET AL.
lane (0.1 ml of a solution 1
M
in dichloromethane) were added. The reaction mixture
was warmed to reflux for 3 h and, after cooling, was filtered on silica gel. After
solvent evaporation an oily residue was obtained (1.8 g) that was not further purified.
1
Yield 98%. IR 2080 (N3) cmϪ . NMR (CDCl3) ͳ: 3.90 (m, 4H, 2CH2O); 3.45 (m,
1H, CH–N); 1.95 (m, 2H, H-cyclohex); 1.46 (m, 6H, H-cyclohex). Anal. (C8H13N3O2)
C, H, N.
3-Aminocyclohexanone ethylene ketal 6. In a well-dried three-neck flask, lithium
aluminum hydride (2.28 g, 60 mmol) was suspended in anhydrous Et2O (100 ml),
and then a solution of 3-azidocyclohexanone ethylene ketal 5 (5.4 g, 30 mmol) in
anhydrous Et2O (100 ml) was added dropwise. The reaction mixture was warmed
to 65ЊC for 5 h. After cooling in an ice bath, the excess LiAlH4 was quenched by
successive dropwise additions of 2.28 ml of water, 2.28 ml 15% NaOH, and 6.84 ml
of water. The solution was filtered. The solvent evaporation under reduced pressure
gave an oily residue (2.98 g). Yield 63%. NMR (CDCl3) ͳ: 3.86 (m, 4H, 2CH2O);
2.86 (m, 1H, CH–N); 1.35 (m, 10H, H-cyclohex and NH2). Part of the residue was
dissolved in anhydrous Et2O and added to a saturated solution of oxalic acid
in anhydrous ethanol. The solution was evaporated to dryness, and the residue
recrystallized from anhydrous ethanol/anhydrous Et2O: mp 182–184ЊC. Anal.
(C8H15NO2 и HCl) C, H, N.
3-N-Acetylaminocyclohexanone ethylene ketal 7. A solution of 3-aminocyclohexa-
none ethylene ketal 6 (1 g, 6.3 mmol) in acetic anhydride (10 ml) was warmed at
140ЊC for 30 min. The solvent was distilled under reduced pressure and the yellow
oily residue (0.95 g) triturated in Et2O. The white solid obtained was filtered and
recrystallized from ethyl acetate. MP 112–115ЊC. Yield 76%; IR (nujol) 1620 (CuO)
1
cmϪ ; NMR (CDCl3) ͳ: 6.36 (d, 1H, NH); 4.25 (m, 1H, NCH); 3.97 (s, 4H, 2CH2O);
1.97 (1, 3H,CH3); 1.88 (dd, 1H, H-cyclohex); 1.63 (m, 7H, H-cyclohex). Anal.
(C10H17NO3) C, H, N.
3-N-Acetylaminocyclohexanone 8. A solution of 3-N-acetylaminocyclohexanone
ethylene ketal 7 (4 g, 20 mmol) in acetic acid (50 ml of 80% solution in water) was
warmed at 65ЊC for 1.5 h. The reaction mixture was concentrated under reduced
pressure and basified with a saturated solution of Na2CO3 . The basic solution was
extracted with CHCl3 . The collected organic layers were evaporated to dryness.
The residue (2.55 g) was recrystallized from benzene. MP 86–87ЊC. Yield 82%. IR
1
(nujol) 1690 (CuO), 1625 (CuO) cmϪ . NMR (CDCl3) ͳ: 5,45 (d, 1H, NH); 4,28
(m, 1H, N-CH) 2,70 (dd, 1H, H-cyclohex); 2,35 (m, 3H, H-cyclohex); 1,97 (s, 3H,
CH3); 1,85 (m, 4H, H-cyclohex). Anal. (C8H13NO2) C, H, N.
3-N-Acetylaminocyclohexylidenacetic acid ethyl ester 9. To a suspension of NaH
(0.29 g, 7.3 mmole 60% dispersion in mineral oil) in anhydrous benzene (20 ml)
under nitrogen atmosphere, triethylphosphonacetate (1.46 ml, 7.3 mmol) was added
dropwise over a 1-h period. During this period, the temperature was maintained
at 30–35ЊC. The reaction mixture was stirred for 1 h at room temperature; then
3-N-acetylaminocyclohexanone 8 (1 g, 6.4 mmol) was added dropwise over a 1-h
period, maintaining the reaction temperature at 20–30ЊC. The slurry was stirred
for 1 h at room temperature and then warmed at 65ЊC for 2 h. After cooling, water
(25 ml) was added and the organic layer was separated. The water solution was
extracted three times with CHCl3 . The collected organic phases were dried. The

DESIGN OF DOPAMINE RECEPTOR LIGANDS
371
solvent evaporation gave the product (1.34 g, yield 82%) as a mixture of E–Z
isomers. Pure E isomer was obtained by recrystallizing the crude product three
1
times from benzene. MP 174–76ЊC. IR (nujol) 1705 (CuO), 1625 (CuO) cmϪ .
NMR (CDCl3) ͳ: 5.65 (s, 1H, CHuC); 5,42 (d, 1H, NH); 4,15 (q, 2H, CH2); 4.05
(m, 1H, N-CH) 3.10 (m, 1H, H-cyclohex); 2.53 (m, 2H, H-cyclohex); 2.05 (m, 1H,
H-cyclohex); 1.97 (s, 3H, CH3); 1.90 (m, 1H, H-cyclohex); 1.85 (m, 1H, H-cyclohex);
1.57 (m, 2H, H-cyclohex); 1.27 (t, 3H, CH3). Anal. (C12H19NO3) C, H, N.
To the mother liquor Et2O was added and the Z isomer precipitated was filtered
and recrystallized from benzene. MP 139–143ЊC. IR (nujol) 1705 (CuO), 1625
1
(CuO) cmϪ . NMR (CDCl3) ͳ: 5.75 (s, 1H, CHuC); 5.58 (d, 1H, NH); 4.15 (q,
2H, CH2); 4.05 (m, 1H, N-CH) 3.12 (dd, 1H, H-cyclohex); 2.61 (m, 1H, H-cyclohex);
2.20 (m, 2H, H-cyclohex); 1.97 (s, 3H, CH3); 1.85 (m, 1H, H-cyclohex); 1.65 (m,
1H, H-cyclohex); 1.57 (m, 2H, H-cyclohex); 1.27 (t, 3H, CH3). Anal. (C12H19NO3)
C, H, N.
3-N-(ter-Butoxycarbonyl)aminocyclohexylidenacetic acid 10. To a stirred solution
containing 3-N-acetylaminocyclohexylidenacetic acid 9 (0.75 g, 3.9 mmol) and trieth-
ylamine (2.36 ml, 16.9 mmol) in methanol (25 ml), di-t-butyldicarbonate (3.4 g, 15.6
mmol) was added over a 1-h period. The reaction mixture was warmed at 50ЊC for
3 h. The solvent was evaporated and water (20 ml) added to the residue. The
solution was acidified at pH 2 with KHSO4 and immediately extracted three times
with ethyl acetate. The collected organic layers were dried and evaporated to
dryness. The oily residue (0.95 g) was purified by flash chromatography using a
mixture of cyclohexane–ethyl acetate (8 : 2) as eluant. After solvent evaporation of
pure fractions the residue was recrystallized from CHCl3–petroleum ether. MP
1
135–36ЊC. Yield 60%. IR (nujol) 1675 (CuO), 1625 (CuO) cmϪ . NMR (CDCl3)
ͳ: 9.50 (bs, 1H, COOH); 5.65 (s, 1H, CHuC); 4.60 (d, 1H, NH); 3.72 (m, 1H,
H-cyclohex); 3.15 (m, 1H, H-cyclohex); 2.50 (m, 2H, H-cyclohex); 2.09 (m, 1H,
H-cyclohex); 1.87 (m, 2H, H-cyclohex); 1.48 (m, 11H, H-cyclohex, 3CH3). Anal.
(C13H21NO4) C, H, N.
3-N-(ter-Butoxycarbonyl)aminocyclohexylidenacetamide 11. To a solution of
3-N-ter-butoxycarbonylaminocyclohexylidenacetic acid 10 (0.12 g, 0.47 mmol) and
triethylamine (0.186 ml, 1.41 mmol) in anhydrous ethyl acetate (15 ml), a solution
of ethyl chloroformate (0.045 ml, 0.47 mmol) in anhydrous ethyl acetate (5 ml) was
added dropwise under nitrogen atmosphere. The reaction mixture was stirred at
room temperature for 1 h and then filtered. Ammonia was bubbled for 10 min
into the clear solution. The flask was corked and stirring was maintained at room
temperature for 1 h. The solvent was removed in vacuo, and the solid residue
(0.11 g) was triturated in petroleum ether, filtered, and recrystallized from ethyl
acetate/cyclohexane. MP 170–171ЊC. Yield 75%. IR (nujol) 1665 (CuO), 1640
1
(CuO) cmϪ . NMR (CDCl3) ͳ: 5.65 (s, 1H, CHuC); 5.42 (bs, 2H, NH2); 4.55 (d,
1H, NH); 3.70 (m, 1H, H-cyclohex); 3.20 (m, 1H, H-cyclohex); 2.50 (m, 2H,
H-cyclohex); 2.02 (m, 1H, H-cyclohex); 1.83 (m, 2H, H-cyclohex); 1.68 (m, 1H,
H-cyclohex); 1.48 (m, 10H, H-cyclohex and 3CH3). Anal. (C13H22N2O3) C, H, N.
3-Aminocyclohexylidenacetamide hydrochloride 12. Anhydrous HCl was bubbled
for 10 min into a solution of 3-(N-ter-butoxycarbonyl)aminocyclohexylidenaceta-
mide 11 (0.09 g, 0.35 mmol) in CHCl3 (10 ml). The flask was corked and the reaction

372
BAGINSKI ET AL.
mixture stirred at room temperature for 1 h. The solvent was removed in vacuo,
and the solid residue (0.05 g) recrystallized from anhydrous ethanol/anhydrous
1
Et2O. MP 211–213ЊC. Yield 75%. IR (nujol) 1660 (CuO) cmϪ . NMR (D2O) ͳ:
5.68 (s, 1H, CHuC); 3.27 (m, 1H, H-cyclohex); 2.98 (m, 1H, H-cyclohex); 2.57 (m,
1H, H-cyclohex); 2.22 (m, 1H, H-cyclohex); 2.02 (m, 2H, H-cyclohex); 1.83 (m, 1H,
H-cyclohex); 1.48 (m, 2H, H-cyclohex). Anal. (C8H15N2O.HCl) C, H, N.
3-N-(ter-butoxycarbonyl)aminocyclohexylidenacetic acid methyl ester 13. To a
solution of 3-N-(ter-butoxycarbonyl)aminocyclohexylidenacetic acid 10 (0.5 g, 1.95
mmol) in acetone, K2CO3 (0.55 g, 4 mmol) and iodomethane (0.5 ml, 8.3 mmol)
were added. The reaction mixture was warmed to reflux for 8 h. The solvent was
removed in vacuo and the residue purified by column chromatography using ethyl
acetate–n-hexane (2 : 8) as eluant. The collected pure fractions were evaporated to
dryness and the oily residue (0.5 g) recrystallized from n-hexane. MP 83–85ЊC.
1
Yield 91%. Ir (nujol) 1690 (CuO); 1635 (CuO) cmϪ . NMR (CDCl3) ͳ: 5.67 (s,
1H, CHuC); 4.50 (bs, 1H, NH); 3.70 (m, 4H, H-cyclohex and CH3); 3.15 (m, 1H,
H-cyclohex); 2.50 (m, 2H, H-cyclohex); 1.93 (m, 4H, H-cyclohex); 1.48 (m, 10H,
H-cyclohex, 3CH3). Anal. (C14H23NO4) C, H, N.
3-Aminocyclohexylidenacetic acid methyl ester hydrochloride 14. Anhydrous HCl
was bubbled for 10 min into a solution of 3-N-(ter-butoxycarbonyl)aminocyclohex-
ylidenacetic acid methyl ester 13 (0.22 g, 0.81 mmol) in CHCl3 (10 ml). The flask
was corked and the reaction mixture stirred at room temperature for 1 h. The
solvent was removed in vacuo and the solid residue (0.15 g) recrystallized from
anhydrous ethanol/anhydrous Et2O. MP 150–152ЊC. Yield 90%. IR (nujol) 1700
1
(CuO) cmϪ . NMR (CDCl3) ͳ: 8.53 (bs, 3H, NHϩ3 ); 5.75 (s, 1H, CHuC); 3.67 (s,
3H, CH3); 3.58 (m, 1H, H-cyclohex); 3.30 (m, 1H, H-cyclohex); 2.70 (m, 1H,
H-cyclohex); 2.48 (m, 1H, H-cyclohex); 2.10 (m, 3H, H-cyclohex); 1.60 (m, 2H,
H-cyclohex). Anal. (C9H15NO2 и HCl) C, H, N.
5-Hydroxyamino-5,6,7,8-tetrahydro-1-naphthalenecarboxylic acid 16. 5-Oxo-
5,6,7,8-tetrahydro-1-naphthalenecarboxylic acid 15 (1 g, 5.2 mmol) was added to a
solution of hydroxylamine hydrochloride (1 g, 1.44 mmol) and anhydrous pyridine
(2 ml) in anhydrous ethanol. The mixture was refluxed for 2 h. The solution was
evaporated in vacuo and the residue dissolved in water and acidified with 6
N
HCl. The solid precipitate was filtered and recrystallized from ethyl acetate. MP
1
241–242ЊC. Yield 94%. IR (nujol) 1690 (CuO) cmϪ . NMR (DMSO-d6) ͳ: 13.00
(s, 1H, COOH); 11.20 (s, 1H, OH); 8.1 (d, 1H, ArH); 7.75 (d, 1H, ArH); 7.25 (t,
1H, ArH); 3.00 (t, 2H, CH2); 2.70 (t, 2H, CH2); 1.70 (m, 2H, CH2). Anal. (C11H11NO3)
C, H, N.
5-Amino-5,6,7,8-tetrahydro-1-naphthalenecarboxylic acid methyl ester hydrochlo-
ride 18. A solution of 5-amino-5,6,7,8-tetrahydro-1-naphthalenecarboxylic acid
trifluoroacetate 2 (2 g, 6 mmol) in freshly distilled thionyl chloride (15 ml) was
warmed at 75ЊC for 2 h. The reaction mixture was evaporated to dryness and the
solid residue triturated in anhydrous Et2O. The filtration gave the desired acyl
1
chloride 17: MP 189–193ЊC. IR (nujol) 1755 (CuO) cmϪ . NMR (DMSO-d6) ͳ:
8.60 (bs, 3H, NHϩ3 ); 7.80 (t, 2H, ArH); 7.39 (t, 1H, ArH); 4.48 (m, 1H, CHN); 3.00
(m, 2H, CH2); 1.92 (m, 4H, 2CH2).
The acyl chloride 17 was dissolved in methanol (20 ml) and the solution refluxed

DESIGN OF DOPAMINE RECEPTOR LIGANDS
373
for 1.5 h. The solvent was removed in vacuo and the oily residue was triturated in
anhydrous Et2O. The solid was filtered and recrystallized from anhydrous methanol/
1
anhydrous Et2O. MP 215–217ЊC. Yield 76%. IR (nujol) 1700 (CuO) cmϪ . NMR
(DMSO-d6) ͳ: 8.58 (bs, 3H, NHϩ3 ); 7.82 (d, 1H, ArH); 7.75 (d, 1H, ArH); 7.40 (t,
1H, ArH); 4.50 (m, 1H, CHN); 3.83 (s, 3H, CH3); 2.97 (m, 2H, CH2); 1.90 (m, 4H,
2CH2). Anal. (C12H15NO2 и HCl): C, H, N.
5-[N-(t-Butoxycarbonyl)amino]-5,6,7,8-tetrahydro-1-naphthalenecarboxylic acid
19. To a solution of 5-amino-5,6,7,8-tetrahydro-1-naphthalenecarboxylic acid
trifluoracetate 2 (2 g, 6.6 mmol) and triethylamine (4 ml, 28 mmol) in methanol
(10 ml), di-t-butyldicarbonate (5.76 g, 26.4 mmol) was added portionwise over a
1-h period. The reaction mixture was warmed at 50ЊC for 3 h. After solvent evapora-
tion, the residue was dissolved in water and acidified with solid KHSO4 up to pH
2. The solution was immediately extracted with ethyl acetate. The organic layer
was dried, filtered, and evaporated to dryness. The oily residue was recrystallized
from ethyl acetate. MP 207ЊC. Yield 83%. IR (nujol) 1680 (CuO), 1650 (CuO)
1
cmϪ . NMR (DMSO-d6) ͳ: 12.80 (s, 1H, COOH); 7.60 (d, 1H, ArH); 7.30 (m, 3H,
NH, 2ArH); 4.68 (m, 1H, CHUN); 2.90 (m, 2H, CH2); 1.82 (m, 2H, CH2); 1.62 (m,
2H, CH2); 1.40 (s, 9H, 3CH3). Anal. (C16H21NO4) C, H, N.
5-[N-(t-Butoxycarbonyl)amino]-5,6,7,8-tetrahydro-1-naphthalenecarboxamide20.
To a solution of 5-[N-(t-butoxycarbonyl)amino]-5,6,7,8-tetrahydro-1-naphtha-
lenecarboxylic acid 19 (0.4 g, 1.37 mmol) and N-hydroxysuccinimide (NHS) (0.19 g,
1.64 mmol) in CHCl3 (20 ml), dicyclohexylcarbodiimide (DCC) (0.33 g, 0.0016
mmol) was added. The reaction mixture was stirred at room temperature for 2.5 h.
After filtration, ammonia was bubbled into the clear solution for 10 min. The flask
was corked and the reaction mixture stirred at room temperature for 1 h. After
filtration, the clear solution was evaporated to dryness and the oily residue recrystal-
lized from ethyl acetate. MP 207ЊC. Yield 99%. IR (nujol) 1665 (CuO), 1650
1
(CuO) cmϪ . NMR (DMSO-d6) ͳ: 7.68 (d, 1H, ArH); 7.25 (m, 5H, NHCO, CONH2
and 2ArH); 4.65 (m, 1H, CH–N); 2.77 (m, 2H, CH2); 1.88 (m, 2H, CH2); 1.68 (m,
2H, CH2); 1.45 (s, 9H, 3CH3). Anal. (C16H22N2O3) C, H, N.
5-Amino-5,6,7,8-tetrahydro-1-naphthalene carboxamide hydrochloride 21. Anhy-
drous HCl was bubbled for 10 min into a solution of 5-[N-(t-butoxycarbonyl)amino]-
5,6,7,8-tetrahydro-1-naphthalene carboxaminde 20 (0.3 g, 1 mmol) in CHCl3 (25
ml). The flask was corked and the reaction mixture stirred at room temperature
for 1 h. The solid was filtered and recrystallized from anhydrous methanol/anhy-
drous Et2O. MP 288–290ЊC. Yield 88%. IR (nujol) 1665 (CuO), 1650 (CuO)
1
cmϪ . NMR (DMSO-d6) ͳ: 8.60 (s, 3H, NHϩ3 ); 7.77 (s, 1H, NHCO); 7.65 (dd, 1H,
ArH); 7.45 (s, 1H, NHCO); 7.30 (m, 2H, ArH); 4.45 (m, 1H, CHUN); 2.99–2.68
(m, 2H, CH2); 2.15–1.65 (m, 4H, 2CH2). Anal. (C11H14N2O и HCl) C, H, N.
Pharmacology
Male Sprague–Dawley rats (300–350 g body weight) were obtained from the
breeding facilities at the University of Santiago. Rats were killed by decapitation,
and brains were rapidly removed and dissected on an ice-cold plate.
[3H]Spiperone (95 Ci/mmol) and [3H]SCH23390 (81 Ci/mmol) were purchased

374
BAGINSKI ET AL.
from Amersham International (England), unlabeled spiperone и HCl, R(ϩ)-
SCH23390 и HCl, SKF38393 и HCl, (ϩ)butaclamol и HCl, cis(z)-flupentixol и 2HCl,
and R(Ϫ)-apomorphine и HCl from Research Biochemicals Inc. and dopa-
mine и HCl, chlorpromazine и HCl, and sulpiride и HCl from Sigma. Reference drugs
or new compounds were stored in 1 m
M
solutions at Ϫ20ЊC, and diluted to the
required concentration on ice immediately before binding assays.
Striatal membrane preparations were obtained by homogenization (Polytron
homogenizer, setting 6 for 10 s) of tissue in 50 m
about 100 Ȑl/mg of tissue) containing 5 m EDTA. Homogenates were centrifuged
(31,000g for 15 min at 4ЊC, Beckman J2-MI), then resuspended in 50 m Tris–HCl
M
Tris–HCl (pH 8.7 at 25ЊC,
M
M
buffer (pH 7.4 at 25ЊC), and then centrifuged again (same conditions). Final pellets
were stored at Ϫ80ЊC until assay.
Just before the binding assay, pellets were resuspended (1.25 mg original wet
weight per 750 Ȑl for D2 binding assays, 1.00 mg per 750 Ȑl for D1 binding assays)
in 50 m
2 m CaCl2 , and 1 m
preparations were added to ice-cold tubes containing (a) 100 Ȑl of [3H]spiperone
plus 50 Ȑl of ketanserin (final concentration 50 n to block 5-HT2A receptors), and
either (b) 100 Ȑl of buffer (total binding) or (c) 100 Ȑl of sulpiride (final concentra-
tion 10 Ȑ
to allow quantification of specific binding by [3H]spiperone), or (d)
100 Ȑl of new or reference drug (concentrations between 1 n and 3 Ȑ ). For D1
M
Tris–HCl buffer (pH 7.4 at 25ЊC) containing 120 m
M
NaCl, 5 m
M
KCl,
M
M
MgCl2 . For D2 binding assays, aliquots of striatal membrane
M
M
M
M
binding assays, the same procedure was followed except that (a) was 100 Ȑl of
[3H]SCH23390 plus 50 Ȑl of buffer, and (c) was 100 Ȑl of nonradiolabeled SCH23390
(final concentration 1 Ȑ
M
to allow quantification of specific binding by
[3H]SCH23390). The final assay volume was, thus, in all cases 1 ml. All assays were
performed in duplicate.
Incubations (15 min at 37ЊC) were stopped by rapid filtration under vacuum
through GF-52 glass-fibers filters (Schlelcher and Schuell) in a Brandel (M-30) cell
harvester. Filters were rinsed three times with 3 ml of ice-cold 50 m
M
Tris–HCl
buffer (pH 7.4). Radioactivity was determined by liquid scintillation counting in a
Beckman LS-6000LL apparatus (counting efficiency approximately 50%).
Competition analyses were carried out with the aid of the Prism program
(GraphPad), and Ki values were calculated as Ki ϭ IC50/(1 ϩ L/KD), where L is
the concentration and KD the apparent dissociation constant of the ligand.
ACKNOWLEDGMENTS
This work was supported by Consiglio Nazionale delle Ricerche (CNR, Rome), and in part by the
Xunta of Galicia (Spain), through Project Grant XUGA 20308B94 and through a personal grant to
E. Rosa.
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