New serotonin 5-HT2A, 5-HT2B, and 5-HT2C receptor antagonists: Synthesis, pharmacology, 3D-QSAR, and molecular modeling of (aminoalkyl)benzo and heterocycloalkanones

Article abstract of DOI:10.1021/jm011014y

A series of 52 conformationally constrained butyrophenones have been synthesized and pharmacologically tested as antagonists at 5-HT_2A, 5-HT_2B, and 5-HT_2C serotonin receptors, useful for dissecting the role of each 5-HT

Full text of DOI:10.1021/jm011014y

54
J . Med. Chem. 2002, 45, 54-71
New Ser oton in 5-HT2A, 5-HT2B, a n d 5-HT2C Recep tor An ta gon ists: Syn th esis,
P h a r m a cology, 3D-QSAR, a n d Molecu la r Mod elin g of (Am in oa lk yl)ben zo a n d
Heter ocycloa lk a n on es
J ose´ Brea,† J ordi Rodrigo,‡ Antonio Carrieri,§ Ferran Sanz,‡ M. Isabel Cadavid,† Mar´ıa J . Enguix,†
Mar´ıa Villazo´n,† Guadalupe Mengod,| Yolanda Caro, Christian F. Masaguer, Enrique Ravin˜a,
Nuria B. Centeno,‡ Angelo Carotti,§ and M. Isabel Loza*,†
Departamento de Farmacologı´a, Facultad de Farmacia, Universidad de Santiago de Compostela,
E-15782, Santiago de Compostela, Spain, Research Group on Biomedical Informatics, IMIM, Universitat Pompeu Fabra,
C/ Drive Aiguader 80, E-08003, Barcelona, Spain, Dipartimento Farmacochimico, University of Bari, Via Orabona, 4,
I-70126, Bari, Italy, Departamento de Neuroquı´mica, Instituto de Investigaciones Biome´dicas de Barcelona, CSIC-IDIBAPS,
C/ Rossello´, 161 Bis, E-08036, Barcelona, Spain, and Departamento de Qu´ımica Orga´nica, Laboratorio de Quı´mica
Farmace´utica, Facultad de Farmacia, Universidad de Santiago de Compostela, E-15782, Santiago de Compostela, Spain
Received August 6, 2001
A series of 52 conformationally constrained butyrophenones have been synthesized and
pharmacologically tested as antagonists at 5-HT2A, 5-HT2B, and 5-HT2C serotonin receptors,
useful for dissecting the role of each 5-HT2 subtype in pathophysiology. These compounds were
also a consistent set for the identification of structural features relevant to receptor recognition
and subtype discrimination. Six compounds were found highly active (pKi > 8.76) and selective
at the 5-HT2A receptor vs 5-HT2B and/or 5-HT2C receptors. Piperidine fragments confer high
affinity at the 5-HT2A receptor subtype, with benzofuranone- and thiotetralonepiperidine as
the most selective derivatives over 5-HT2C and 5-HT2B receptors, respectively; Ki 2A/2C and/or
KB 2A/2B ratios greater than 100 were obtained. Compounds showing a more pronounced
selectivity at 5-HT2A/5-HT2C than at 5-HT2A/5-HT2B bear 6-fluorobenzisoxazolyl- and p-
fluorobenzoylpiperidine moieties containing one methylene bridging the basic piperidine to
the alkanone moiety. An ethylene bridge between the alkanone and the amino moieties led to
ligands with higher affinities for the 5-HT2B receptor. Significant selectivity at the 5-HT2B
receptor vs 5-HT2C was observed with 1-1[(1-oxo-1,2,3,4-tetrahydro-3-naphthyl)methyl]-4-[3-
(p-fluorobenzoyl)propyl]piperazine (more than 100-fold higher). Although piperidine fragments
also confer higher affinity at 5-HT2C receptors, only piperazine-containing ligands were selective
over 5-HT2A. Moderate selectivity was observed at 5-HT2C vs 5-HT2B (10-fold) with some
compounds bearing a 4-[3-(6-fluorobenzisoxazolyl)]piperidine moiety in its structure. Molecular
determinants for antagonists acting at 5-HT2A receptors were identified by 3D-QSAR (GRID-
GOLPE) studies. Docking simulations at 5-HT2A and 5-HT2C receptors suggest a binding site
for the studied type of antagonists (between transmembrane helices 2, 3, and 7) different to
that of the natural agonist serotonin (between 3, 5, and 6).
In tr od u ction
Three 5-HT2 receptor subtypes (5-HT2A, 5-HT2B, and
5-HT2C) show considerable homology at genetic, struc-
tural, and functional levels.3,6,7 All are G-protein coupled
receptors (GPCRs) and have been initially associated
with activation of phospholipase C via Gq-protein. In
addition, a variety of other G-protein coupling and
effector mechanisms has been described for 5-HT2
receptor subtypes: 5-HT2A receptors can activate phos-
pholipase A2 via G11-protein,8 and they can also mediate
decreased cGMP levels, enhancing neurotransmitter
Serotonin (5-HT), a major neurotransmitter found in
the central nervous system (CNS), is also present in
many peripheral tissues.1-3 Its numerous biological
functions are mediated by a variety of serotonin recep-
tors.3,4 The interaction with these different serotonin
receptors constitutes the mechanism of action of many
drugs. In particular, type 2 serotonin receptors (5-HT2)
mediate the actions of several drugs used in treating
diseases such as schizophrenia, feeding disorders, per-
ception, depression, migraines (prophylactically), hy-
pertension, anxiety, hallucinations, and gastrointestinal
dysfunctions.5,6
release from glutamate terminals;9 5-HT2B can bind
10,11
G11
and increase nitric oxide synthase activity via
PDZ-dependent activation;12 5-HT2C receptors can medi-
ate increased cGMP levels, when coupled to Go and/or
G11 via phospholipase A2.13
* To whom correspondence should be addressed. Tel: +34-981-547-
139. Fax: +34-981-594-595. E-mail: ffmabel@usc.es.
† Departamento de Farmacolog´ıa, Universidad de Santiago de
Compostela.
5-HT2A receptors have been found at high density in
the cerebral cortex,14 mostly in pyramidal cells, and also
in interneuronal regions.14-16 They are also present, at
lower density, in the hippocampus, striatum, and other
cerebral regions,14,15 in platelets, and in vascular and
uterine smooth muscles.3,7,17,18
‡ Research Group on Biomedical Informatics, Universitat Pompeu
Fabra.
§ Dipartimento Farmacochimico, University of Bari.
| Departamento de Neuroqu´ımica, Instituto de Investigaciones
Biome´dicas de Barcelona.
Departamento de Qu´ımica Orga´nica, Universidad de Santiago de
Compostela.
10.1021/jm011014y CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/08/2001

New Serotonin Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 55
Ch a r t 1
5-HT2C receptors, first described by Pazos et al.19 in
the choroid plexus, are distributed with varying density
in several areas of the brain, including the cortex, basal
ganglia, hippocampus, and hypothalamus.20,21 They
have not been found in peripheral tissues.
classical antipsychotics.31 Clozapine, as well as other
atypical antipsychotics, binds not only to 5-HT2 recep-
32,33
tors (5-HT2A, 5-HT2B, and 5-HT2C
)
but also, to a
different extent, to a number of other GPCRs, including
5-HT1A, 5-HT6, 5-HT7, D1, D2, D4, H1, R1, R2, M1, M2,
and M4.2,34-41 Despite this lack of specificity, the in-
volvement of 5-HT2 receptors in the pharmacological
profile of atypical antipsychotics is supported by many
biological, pharmacological, and clinical studies.42,43
5-HT2B receptors were originally described as
22
23
5-HT1-like and 5-HT2F and were found to be opera-
tionally and functionally similar to 5-HT2A and 5-HT2C
Subsequent studies showed that in contrast with 5-HT2A
.
,
Initially, the 5-HT2 subtype indicated as relevant in
5-HT2B is blocked only weakly by ketanserin, and
operational and structural differences with the 5-HT2C
subtype have been described.3,24 Cloning23,25 allowed
their confident classification as a new subtype of 5-HT2
44-51
schizophrenia by most studies has been 5-HT2A
.
However, now it seems possible that some of the effects
of atypical antipsychotics that have been attributed to
their blocking of 5-HT2A may instead be due to the
blockade of 5-HT2B and/or 5-HT2C. In particular, affinity
for 5-HT2C is an important feature in discriminating
classical from atypical antipsychotics.52 Clozapine, olan-
zapine, seroquel, and other atypical antipsychotics have
indeed greater affinity for 5-HT2C (and in addition for
5-HT2A) than for D2 receptors.53 The blockade of 5-HT2C
has been indicated as responsible for relatively mild
extrapyramidal side effects observed for atypical anti-
psychotics, because 5-HT2C rather than 5-HT2A blockade,
can prevent the extrapyramidal side effects induced by
haloperidol.54 On the other hand, 5-HT2C antagonists,
by enhancing dopamine release in the cortex, would
efficiently counteract the hypofrontality that contributes
to the deficit symptoms of schizophrenia.55,56
receptors, 5-HT2B.
26 5-HT2B mRNA is widely distributed
in peripheral tissues, having been found in heart,
skeletal, and vascular muscles, adipose tissue, the
intestine, ovary, uterus, testis, liver, kidney, lung,
pancreas, trachea, spleen, thymus, thyroid, and in
prostate and salivary glands of mammals.6,18,27-29 In the
CNS, they have been found in the retina and spinal cord
and at lower levels in a variety of brain areas.6,29
Immunochemical studies also suggested their presence
in the CNS,30 although their real presence, distribution,
and functions in the brain are still controversial.
Clozapine and other “atypical antipsychotics” have
been shown to be effective against the negative symp-
toms of schizophrenia. The blockade of 5-HT2 receptors
has been pointed to as the origin of the distinctive
pharmacological profile of these drugs, causing an inci-
dence of tardive diskinaesia and other extrapyramidal
side effects that are lower than those found with
A deeper understanding of the roles of 5-HT2A
,
5-HT2B, and 5-HT2C receptors in schizophrenia and in
the pharmacology of antipsychotics, as in other CNS and

56 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Ta ble 1. Structures and Codes of Studied Compounds
Brea et al.
peripheral disorders (anxiety, depression, hypertension,
etc.), has been hampered by a scarcity of truly selective
ligands able to distinguish sharply among these receptor
subtypes, especially as regards to differentiation be-
tween 5-HT2B and 5-HT2C receptors.
The comparative physiological and pharmacological
behaviors of 5-HT2 receptors depend on their structural
features. All of the GPCRs share a transmembrane
bundle of seven R-helices57 that in the case of the
serotonin and other aminergic receptors, includes the
binding site.58 Structural information on a single GPCR,
bovine rhodopsin, is now available. This information
was first obtained at low resolution59 and recently at
higher resolution.57 On the basis of the rhodopsin
structural information57,59 and of additional data coming
from molecular biology experiments,60-63 increasingly
accurate models of GPCRs are being produced.60,61,63-65
These models can be useful for understanding ligand
binding and receptor activation features.
Among the reported selective ligands,66 besides ket-
anserin, spiperone, MDL100907, and other 5-HT2A
selective ligands, some 5-HT2B/5-HT2C selective ligands
have also been described. A selection of them and their
pKi values at 5-HT2A/5-HT2B/5-HT2C receptors (in pa-

New Serotonin Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 57
Sch em e 1a
rentheses) are (Chart 1), pyridylcarbamoylindolines
SB-206553 (1) (6.3/8.5(pA2)/8.3)67 and SB-221284 (2)
(6.4/7.9/8.6);68 bisarylcarbamoylindolines SB-228357 (3)
(6.9/8.0/9.0) and SB243213 (4) (6.8/7.0/9.0);69 benzene-
sulfonamido-substituted valerophenone RS-102221
(5) (6.2/6.5/8.7);70 naphthylpyrimidine RS-127445 (6)
(6.3/9.5/6.4);71 indolonaphthyridine SDZ SER-082 (7)
(6.2/7.3/7.8);72 benzo[e]isoindolone and benzo[h]isoquino-
linone derivatives 8 and 9, respectively, both with a
pKi ) 8.0 at the 5-HT2C receptor;73 and other types of
compounds.74,75
The chemical heterogeneity of the aforementioned
selective ligands, their different affinities for other
GPCRs, and their different pharmacokinetic profile
prevent an unequivocal attribution of their biological
effects at the CNS (especially those related with an
antipsychotic profile) to a likely selective interaction
with a certain 5-HT2 receptor subtype.
In previous papers,64,76-84 we have reported different
compounds potentially useful to study the structural
and pharmacological features of the three 5-HT2 recep-
tor subtypes within a single chemical family, those of
the butyrophenones.
a
Reagents: (i) N-(2-Pyridyl)piperazine, DCC/HOBt. (ii) (1)
(CH2OH)2/p-TsOH, (2) LiAlH4, (3) HCl.
Sch em e 2a
The present paper reports the study of a great number
of butyrophenones with a large molecular diversity in
their alkanone and/or amino moieties (Table 1). To
properly explore structure-activity relationships (SAR)
and structure-selectivity relationships (SSR), typical
N1-aryl (heteroaryl) substituents, as well as the longer
N1-p-fluorobenzoylpropyl and p-fluorobenzoyl-butyl sub-
stituents, were chosen for the piperazine moiety, whereas
N-substitution at the piperidine ring was limited to the
benzoyl, p-fluorobenzoyl, and 6-fluorobenzisoxazol-3-yl
substituents.
In particular, the synthesis and pharmacological
profile of the novel butyrophenones are reported here
along with new pharmacological data obtained for
compounds previously reported by us. Moreover, three-
dimensional 3D-QSAR (GRID/GOLPE) models obtained
from the study of structural and biological data of the
extended set of compounds as well as molecular models
arising from docking simulations of selected ligands on
5-HT2A and 5-HT2C receptors are presented and dis-
cussed in this paper.
a
Reagents: (i) 4-(6-Fluorobenzisoxazol-3-yl)piperidine, NMP.
presented in Table 2. All of the compounds of the series
have been designed on the basis of the union of two
fragments that share an amino group (see Table 1). The
amino moieties a , b, c, and d were selected in order to
explore slight electronic variations in the phenyl-piper-
azine structure. Structures e and f were considered to
evaluate the elongation of the corresponding ketanserin
moiety (g), with the additional difference of considering
piperazine instead of piperidine; g and h were consid-
ered because they were the corresponding fragments of
ketanserin and risperidone, respectively.
The preparation of compound 10c was carried out
following the same synthetic strategy reported for
compounds 10a and 10g (Scheme 1).64 The 3-oxo-
indane-1-carboxylic acid64 was condensed with N-(2-
pyridyl)piperazine in the presence of 1,3-dicyclohexyl-
carbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt)
to give the corresponding amide 25. The ketone group
of this compound was protected by ethylene glycol and
p-toluenesulfonic acid (p-TsOH), the amide group was
reduced to amine with LiAlH4, and the ethylene ketal
was cleaved in acidic medium to afford the amine 10c
in 65% yield from 3-oxo-indane-1-carboxylic acid.
Compound 11h was synthesized (Scheme 2) from 3-(p-
toluenesulfonyloxymethyl)-1,2,3,4-tetrahydronaphtha-
len-1-one85 via nucleophilic displacement of the tosylate
with 4-(6-fluorobenzisoxazol-3-yl)piperidine in N-meth-
yl-2-pyrrolidone (NMP) and provided, after bulb to bulb
distillation of NMP, the target compound 11h in 70%
yield.
Resu lts a n d Discu ssion
A large series of conformationally constrained buty-
rophenones have been synthesized and pharmacologi-
cally tested as antagonists of the 5-HT2A, 5-HT2B, and
5-HT2C serotonin receptors as useful tools for dissecting
the pathophysiological role of each 5-HT2 subtype and
for identifying structural features relevant to receptor
recognition and subtype discrimination. A partial study
of a limited number (28) of these compounds was
reported in our preceding paper, which included the
determination of 5-HT2A and 5-HT2C activities for 28 and
17 compounds, respectively.64 Now, the set of com-
pounds has been enlarged by studying 24 new com-
pounds. The pharmacological activities (affinity and
antagonistic activity) of the resulting 52 compounds
were determined for both 5-HT2A and 5-HT2C receptors.
Moreover, the affinity at the 5-HT2B receptor was
measured for 43 ligands. The whole set of compounds
studied is reported in Table 1, and their affinities are
Compound 12a was prepared according to our proce-
dure used in the synthesis of compound 10c (Scheme
3). By reaction of (1-oxo-2-indanyl)acetic acid77 with
N-(o-methoxyphenyl)piperazine, the amide 26 was ob-
tained in 85% yield. Ketalization of the carbonyl group
with ethylene glycol and p-TsOH, reduction of the amide

58 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Brea et al.
Ta ble 2. Binding and Functional Data of the Studied Ligands at 5-HT2 Receptors
5-HT2A
5-HT2B
5-HT2C
compd
pKi
pA2
pA2
pKi
KB2A/KB2Ba
Ki2A/Ki2Ca
10a
10c
10e
10g
11a
11e
6.05 ( 0.21
<5b
6.21 ( 0.1
ndc
7.20 ( 0.2
8.13 ( 0.09
6.48 ( 0.50
7.27 ( 0.70
7.04 ( 0.2
6.35 ( 0.03
<5
6.44 ( 0.07
6.07 ( 0.03
<5
0.15
nd
501
30
nd
589
0.41
0.027
692
42
5.5
1778
7.34 ( 0.1
7.58 ( 0.14
6.29 ( 0.60
7.75 ( 0.60
6.94 ( 0.20d
8.80 ( 0.80
8.57 ( 0.03
6.71 ( 0.14
6.91 ( 0.60
7.17 ( 0.15d
8.60 ( 0.80
7.27 ( 0.10
8.42 ( 0.3
7.39 ( 0.70
7.14 ( 0.70
8.11 ( 0.80
7.88 ( 0.70
6.55 ( 0.80
6.04 ( 0.60
8.06 ( 0.01
8.37 ( 0.80
7.04 ( 0.70
5.82 ( 0.06
5.75 ( 0.5
6.97 ( 0.13
7.24 ( 0.12
7.93 ( 0.02
7.94 ( 0.05
8.33 ( 0.11
7.37 (0.15
8.15 ( 0.13
8.76 ( 0.2
6.84 ( 0.12
6.76 ( 0.11
6.54 ( 0.13
8.84 ( 0.17
8.56 ( 0.20
7.95 ( 0.09
8.17 ( 0.18
<5
6.66 ( 0.17
5.96 ( 0.07
5.55 ( 0.36
<5
nd
<5
<5e
11g
11h
12a
12e
7.86 ( 0.86
nd
6.8 ( 0.6
7.30 ( 0.70
7.2 ( 0.30
nd
nd
6.63 ( 0.14
6.98 ( 0.10
6.55 ( 0.20
5.21 ( 0.07
<5e
4.6
nd
nd
148
39
1.5
50
7.5 ( 0.25
0.63
12g
13e
13g
14a
14e
14g
15g
16a
16e
16g
16h
17a
18a
18c
18e
18g
(-)18h
(+)18h
(()18h
19e
19g
19h
20a
20c
20e
20g
20h
21g
21h
22a
22b
22c
22d
22e
22f
8.12 ( 0.11
nd
6.43 ( 0.23
5.14 ( 0.2
6.12 ( 0.4
7.13 ( 0.48
6.8 ( 0.20
7.15 ( 0.21
7.40 ( 0.28
6.9 ( 0.3
nd
6.78 ( 0.07
5.71 ( 0.32
6.08 ( 0.09
6.63 ( 0.02
5.48 ( 0.14
6.65 ( 0.01
6.30 ( 0.05
5.44 ( 0.03
6.63 ( 0.05
7.45 ( 0.01
6.42 ( 0.02
6.65 ( 0.04
6.04 ( 0.06
<5
5.40 ( 0.12
6.03 ( 0.04
6.95 ( 0.10
6.84 ( 0.07
6.69 ( 0.12
<5
49
nd
66
36
219
5.8
46
29
38
13
0.26
4.1
89
2.5
0.60
18
8.12 ( 0.75
6.72 ( 0.06
7.35 ( 0.11
7.47 ( 0.51
6.75 ( 0.60
nd
nd
nd
nd
7.50 ( 0.70
5.90 ( 0.01
6.40 ( 0.05
7.79 ( 0.06
7.82 ( 0.16
nd
100
0.39
3.6
2.1
0.22
nd
nd
nd
nd
nd
nd
nd
nd
6.78 ( 0.13
5.18 ( 0.18
5.60 ( 0.7
6.88 ( 0.35
nd
0.13
17
155
8.7
37
16
nd
nd
9.13 ( 0.7
7.63 ( 0.08
8.92 ( 0.04
nd
6.95 ( 0.02
6.31 ( 0.50
6.83 ( 0.2
9.02 ( 0.04
9.87 ( 0.40
nd
nd
nd
nd
nd
nd
nd
nd
6.24 ( 0.3
<5
7.06 ( 0.13
nd
7.19 ( 0.16
7.01 ( 0.07
6.80 ( 0.07
6.35 ( 0.13
6.23 ( 0.29
6.48 ( 0.1
nd
776
1349
72
44
741
59
6.38 ( 0.06
7.06 ( 0.06
7.09 ( 0.17
5.77 ( 0.10
6.40 ( 0.7
6.48 ( 0.06
6.90 ( 0.05
6.45 ( 0.11
7.16 ( 0.08
<5
nd
50
0.58
0.20
1.0
468
4365
nd
nd
nd
nd
nd
nd
nd
nd
3.7
22
0.56
9.8
1.4
229
46
31
10
6.19 ( 0.20
<5
1.0
1445
0.028
1.0
33
25
616
2951
7.66 ( 0.05
5.08 ( 0.10
<5
<5
5.46 ( 0.03
6.01 ( 0.24
6.36 ( 0.12
6.23 ( 0.08
6.71 ( 0.57
6.61 ( 0.38
6.64 ( 0.02
<5
6.02 ( 0.03
5.90 ( 0.04
7.29 ( 0.20
7.97 ( 0.03
8.26 ( 0.12d
6.72 ( 0.09
6.45 ( 0.01
7.71 ( 0.17
8.97 ( 0.09
9.14 ( 0.11d
6.20 ( 0.61
8.04 ( 0.80
8.80 ( 0.88
8.93 ( 0.15d
7.28 ( 0.03
8.12 ( 0.07
8.29 ( 0.04
9.51 ( 0.03
<5
<5
22g
22h
7.28 ( 0.07
7.95 ( 0.07
<5
<5
5.40 ( 0.52e
6.24 ( 0.03
<5
23a
23c
23 g
23h
6.30 ( 0.12
6.17 ( 0.06
7.58 ( 0.20
9.25 ( 0.05
6.68 ( 0.23
<5
6.63 ( 0.19
6.30 ( 0.01
0.42
47
8.9
3.0
89
18
65
6.46 ( 0.03
7.16 ( 0.01
7.46 ( 0.30e
5.92 ( 0.06
6.23 ( 0.09
7.24 ( 0.06
7.56 ( 0.26e
5.34 ( 0.03
7.80 ( 0.1
7.36 ( 0.1
7.38 ( 0.03
891
24a
24g
24h
6.50 ( 0.20
9.20 ( 0.27
9.50 ( 0.32
6.80 ( 0.05
7.22 ( 0.23
6.35 ( 0.10
0.50
96
1412
1.9
65
36
haloperidol
clozapine
ketanserin
r isp er id on e
9.16
8.87 ( 0.11
6.77 ( 0.2
a
b
Ki and KB ratios. Values <5 were arbitrarily substituted by 4.5 to allow the calculations of Ki and KB ratios. c nd ) not determined.
Data obtained from human 5-HT2A receptors transfected into CHO cells. e Data obtained from human 5-HT2C receptors transfected into
d
HeLa cells.
group with LiAlH4, and subsequent ketone deprotection
in acidic medium produced the desired amine 12a , in
70% yield. The preparation of compound 20c was carried
out following the same route: (4-oxo-4,5,6,7-tetrahydro-
benzo[b]thiophen-5-yl)acetic acid64 was condensed with
N-(2-pyridyl)piperazine in the presence of DCC/HOBt
to give the amide 27. The ketonic group was protected
with ethylene glycol and p-TsOH, the amide group was
reduced to amine with LiAlH4, and the ethylene ketal
was cleaved in acidic medium to afford the amine 20c
in 70% yield from (4-oxo-4,5,6,7-tetrahydrobenzo[b]-
thiophen-5-yl)acetic acid.

New Serotonin Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 59
Sch em e 3a
a
Reagents: (i) N-(2-Pyridyl)piperazine, DCC/HOBt. (ii) (1) (CH2OH)2/p-TsOH, (2) LiAlH4, (3) HCl.
Sch em e 4a
a
Reagents: (i) N-(2-Pyridyl)piperazine, KI, Na2CO3. (ii) PPA.
Sch em e 5a
a
Reagents: (i) (1) BrCH2CO2Et/LDA, (2) KOH. (ii) HNRR, DCC/
HOBt. (iii) (1) (CH2OH)2/p-TsOH, (2) LiAlH4, (3) HCl.
The preparation of compound 18c was carried out
following the synthetic strategy shown in Scheme 4.
Methyl γ-bromo-â-(2-thienyl)butyrate64 underwent nu-
cleophilic substitution with N-(2-pyridyl)piperazine in
basic methyl isobutyl ketone to give the â-aminoester
28 in 95% yield. Finally, acid-catalyzed ring closure with
polyphosphoric acid (PPA) afforded compound 18c in
40% yield.
Compounds 23a , 23c, 23g, and 23h were prepared
by amide-linking the two moieties, protecting nonamide
carbonyls as ketals, reducing the amide carbonyl, and
deprotecting (Scheme 5). Alkylation of 4,5,6,7-tetrahy-
drobenzo[b]furan-4-one with lithium diisopropylamide
at -70 °C, followed by quenching with ethyl bromoac-
etate, gave ethyl ester that was then hydrolyzed to the
thienocycloalkanone acetic acid 30 in 50% overall yield.
The amides 30-33 were prepared in good yields by
direct acid-amine coupling, with carboxylate activation
by DCC in the presence of HOBt. Ketalization of
carbonyl groups with ethylene glycol and p-TsOH or
pyridinium tosylate in anhydrous toluene, with azeo-
tropic distillation of water in a Dean-Stark apparatus,
F igu r e 1. Binding competition experiments; a representative
experiment is shown with compound 24g at (A) 5-HT2A binding
sites in rat frontal cortex, the radioligand used was [3H]-
ketanserin (b), nonspecific binding was measured with 10-6
M metisergide (9), and the assay was performed with triplicate
points and (B) 5-HT2C binding sites in bovine choroid plexus,
the radioligand used was [3H]mesulergine (b), nonspecific
binding was measured with 10-6 M mianserine (9), and the
assay was performed with duplicate points. A total of three
experiments was carried out in each case.
provided the corresponding ethylene ketals, which were
reduced with LiAlH4 without further purification. Fi-
nally, deketalization to restore carbonyl groups in acidic
medium gave the amines 23a , 23c, 23g, and 23h .
Figure 1 shows binding competition experiments
carried out with [3H]-ketanserin and [3H]-mesulergine
in rat cortex and bovine choroids plexus (5-HT2A and
5-HT2C, respectively), for ligand 24g. All of the active
compounds showed competition concentration-response
curves of specific radioligand binding against increasing

60 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Brea et al.
Data in Table 2 show widely distributed 5-HT2A pKi
values (4.47 log units, if 4.5 is arbitrarily assigned to
values <5, n ) 52) and less spread 5-HT2C pKi values
(2.95 log units, if 4.5 is assigned to values <5, n ) 52).
The 5-HT2B pA2 values are also moderately spread (3.00
log units, if again 4.5 is assigned to values <5, n ) 43).
A preliminary data analysis shows no significant
pairwise correlations among the activities at the three
receptors subtypes, suggesting different binding modes
and/or different binding sites. The antagonist activities
toward 5-HT2A expressed as pA2 were generally even
higher than the corresponding binding affinities (pKi),
indicative of an efficient transductor-effector amplifica-
tion of the signal in functional assays, making these
compounds of potential pharmacological interest. There
is a good correlation between pKi and pA2 data (r2
)
0.723) for the 5-HT2A receptor. This result suggests a
notable similarity between binding and functional re-
sponse in central and peripheral 5-HT2A receptors.
The highest 5-HT2A affinities were observed for cy-
cloalkanones bearing piperidine fragments (19 com-
pounds: 23h , 20g, 11g, 24h , 19h , 12g, 11h , 20h , 13g,
16h , 18h , 21h , 19g, 14g, 16g, 24g, 22h , 21g, and 15g,
all with pKi > 7.8), with 23h as the most potent
compound (pKi ) 8.97), whereas most of the lowest
affinities corresponded to piperazine derivatives (13
compounds: 23c, 11a , 24a , 10a , 16e, 22e, 22f, 18a , 18c,
22c, 10c, 22a , and 22d , all with pKi < 6.5).
As far as the binding to 5-HT2C receptor subtype is
concerned, highest affinities were generally obtained for
piperidine derivatives (16g, 24h , 21h , 23h , 20a , 19h ,
11h , and 20h , pKi g 6.9), with 16g as the most potent
compound (pKi ) 7.45), whereas lowest affinities cor-
responded to piperazine derivatives (14e, 16a , 18e, 12e,
22g, 22h , 22e, 22f, 22a , 22d , 18c, 11e, 22b, 19e, 10e,
and 23c, pKi < 5.5). Ligands carrying the aminometh-
ylbenzofuranone moiety showed pKi values lower than
5 (22g, 22h , 22e, 22f, 22a , 22d , and 22b) with the
exception of the 2-pyridyl-4-piperazine derivative 22c
(pKi ) 6.64).
F igu r e 2. Functional assays; an example is shown with
compound 13g at (A) 5-HT2A receptors in smooth muscle of
rat thoracic aorta, cumulative concentration-response curves
to 5-HT in the absence (b) and in the presence (9) of 10-7
M
For 5-HT2B receptor ligands, a less clear picture came
from the analysis of their pA2 values, since they
represent activities involving indirect measures, and
consequently, transducer-effector coupling can induce
some interferences, especially when there are no com-
pounds with really high potency. Indeed, the highest
and lowest pA2 values were displayed by compounds 12e
(pA2 ) 7.50) and 11e (pA2 ) 4.50), respectively, both
sharing the same piperazine fragment and having
relatively similar cycloalkanone moieties. Another strong
and unexpected effect was the dramatic drop in activity,
from 7.50 to 5.14, resulting from the introduction of a
fluorine atom in the cycloalkanone moiety (13e) in
comparison with the highest affinity ligand 12e. Inter-
estingly, eight of the top 10 high affinity ligands bear
an aminoethylcycloalkanone moiety, whereas four out
of five bottom low affinity ligands present an amino-
methyl moiety.
13g and (B) 5-HT2B receptors in rat stomach fundus, cumula-
tive concentration-response curves to 5-HT in the absence (b)
and in the presence (9) of 10-5 M 13g. Points are the mean of
four experiments in each case, vertical bars show sem.
concentrations of ligands, the slopes not being signifi-
cantly different from unity at the 5% level of statistical
significance.
Figure 2 shows 5-HT concentration-response curves
in rat aorta (5-HT2A receptor) and rat stomach fundus
(5-HT2B receptor) for compound 13g. All of the active
compounds concentration dependently displaced these
curves to the right in a parallel way without depression
of their maximum, which is the typical behavior of
competitive antagonists.
The numerous compounds, as well as the good spread
of the biological data obtained from the present data
set, permit a detailed and robust SAR analysis for the
three serotonin 5-HT2 receptors, also at the 3D quan-
titative level. To avoid the loss of meaningful informa-
tion from the SSR analysis, affinity values <5 were
arbitrarily fixed at 4.50 and these arbitrary values
were used to calculate the Ki and KB ratios reported in
Table 2.
Compounds with high selectivity for the 5-HT2A
receptor subtype (vs 5-HT2B and 5-HT2C) were ob-
tained: compounds 22h , 11e, 22b, 19e, 10e, 22g, 20g,
13g, and 11g all displayed 5-HT2A/5-HT2C Ki ratios
higher than 100, the most selective compound being the
benzofuranone-piperidine derivative 22h that shows a

New Serotonin Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 61
pKi ) 7.97 at the 5-HT2A receptor and a Ki 2A/2C ratio of
2951. 5-HT2A/5-HT2B selectivity was studied based on
antagonistic potency (pA2), because this was the only
pharmacological parameter that was determined for
both receptors. Good selectivity (higher than 100) for
5-HT2A over 5-HT2B was found for compounds 20h , 24h ,
19e, 23h , 18h , 11e, 10e, 20g, 18e, and 13g, and the
highest selectivity (KB ratio ) 4365) was associated to
the thiotetralone-piperidine ligand 20h that also pos-
sesses an outstanding affinity for the 5-HT2A receptor
subtype (pA2 ) 9.87). A comparative analysis of the
5-HT2A/5-HT2C (Ki) vs 5-HT2A/5-HT2B (KB) activity ratios
indicates that compounds 11e, 19e, 10e, 20g, 13g, and
12g exhibit a high selectivity for the 5-HT2A receptor
vs both the 5-HT2B and the 5-HT2C receptors. In
contrast, three compounds show 5-HT2A/5-HT2C selec-
isoxazol-3-yl or p-fluorobenzoyl-piperidine moieties as
well as a single methylene bridging the basic piperidine
and the alkanone moiety.
To gain more insight into the main interactions
underlying selective binding of the studied series of
compounds to the three receptor subtypes, also at a
quantitative and 3D level, the same data set found in
Table 2 was analyzed by means of 3D-QSAR and
docking studies. Affinity and activity data (pKi and pA2)
lower than 5 were not used in the 3D-QSAR studies.
3D-QSAR approaches rely on advanced statistical
methods to correlate the dependent variable (usually the
biological activity) with 3D chemical descriptors, such
as molecular interaction potentials, which are interac-
tion energies of the considered molecules with proper
molecular probes. In the past, the majority of such
studies have been carried out by means of comparative
molecular field analysis (CoMFA) developed by Cramer
more than 10 years ago.86 In the past few years, the
so-called GRID/GOLPE87 approach has also been used
as a successful improvement over CoMFA. Although
CoMFA and GRID/GOLPE rely on the same principles
and similar statistical algorithms, GRID offers an
excellent set of molecular probes for the computation
of interaction potentials, and GOLPE provides, besides
a classical PLS method,88 different useful tools for data
pretreatment, variable selection, and results interpreta-
tion. This often leads to more meaningful results and
limits the risk of deriving over fitted models.
tivities that are more pronounced than those of 5-HT2A
/
5-HT2B: 22h (2951 vs 22), 22g (616 vs 3.7), and 11g
(148 vs 4.6). These three compounds are 6-fluorobenz-
isoxazol-3-yl and p-fluorobenzoylpiperidine derivatives
with one methylene bridging the basic piperidine to the
alkanone moiety. These structural features may be
useful in designing high affinity 5-HT2A ligands with
higher selectivity vs 5-HT2C than 5-HT2B. 5-HT2A/5-HT2B
selectivities greater than 5-HT2A/5-HT2C resulted for
compounds 20h (4365 vs 46), 24h (1412 vs 36), and 23h
(891 vs 65), all bearing 6-fluorobenzisoxazol-3-yl piper-
idine moieties but linked through different bridges to
varying alkanones.
It is worth noting that all of the 5-HT2C selective
ligands bear a substituted piperazine as the basic
moiety. 5-HT2C/5-HT2A selectivity was observed only for
very few compounds (18a , 20a , 10a , 16e, 22c, and 10c),
the last two presenting the lowest 5-HT2A/5-HT2C Ki
ratios (0.028 and 0.027), corresponding to 5-HT2C af-
finities (37- and 36-fold higher than 5-HT2A, respec-
tively). Unfortunately, this selectivity is associated to
a relatively low affinity for the 5-HT2C receptor subtype
(6.64 and 6.07, respectively). Moderate selectivity for
5-HT2C receptor vs 5-HT2B (10-fold) was observed in
some fluorobenzisoxazo-3-yl-piperidine ligands.
In our previous study of a subset of the present series
of compounds,64 we performed a CoMFA analysis lead-
ing to the identification, at the 3D level, of the main
molecular determinants for a high 5-HT2A receptor
binding affinity, within the limited considered series.
In that study, we found that the steric, lipophilic, and
electrostatic properties of the ligands play an essential
role in the binding to the 5-HT2A receptor.
We selected the GRID/GOLPE approach for our new
3D-QSAR studies because its aforementioned objective
advantages in comparison with the classical CoMFA
approach. Indeed, the atom probes available in GRID89
for the calculation of the molecular interaction fields
are much more numerous than those implemented in
CoMFA and most of them better resemble the functional
groups of amino acids constituting the putative binding
site of enzymes and receptors. For the present study,
Only a few ligands, 20c, 10a , and 18a , elicited a small
but significant selectivity for 5-HT2B over 5-HT2A recep-
tor, with 7.6-, 6.7-, and 5.01-fold preference, respectively.
All of these ligands showed relatively good affinities and
carry a substituted piperazine moiety. Important selec-
tivity at the 5-HT2B receptor over 5-HT2C was observed
for compound 12e, which is a p-fluoro-benzoyl-propyl-
piperazine derivative.
+
3
sp3
we chose four probes (hydroxyl, Csp , N
, DRY),
according to the nature of the amino acids forming the
antagonist binding site of the 5-HT2 receptors as it is
discussed in the docking simulation section. Another
advantage of GRID/GOLPE is that it includes advanced
mathematical tools such as variable pretreatment,
D-Optimal, and FFD variable selection methods as well
as smart region definition (SRD).90 This allows one to
obtain more robust and easily interpretable 3D-QSAR
models.
The results of the GRID/GOLPE analyses of the three
different binding data sets (5-HT2A pKi, 5-HT2B pA2, and
5-HT2C pKi), carried out using the four selected probes,
are summarized in Table 3. Models with high statistical
coefficients were obtained only in the analysis of the
5-HT2A binding data whereas the analysis of the 5-HT2B
and 5-HT2C data yielded poorer models. It is noteworthy
that for 5-HT2A, the four different probes gave PLS
The salient results emerging from the previous SAR
and SSR analyses may be summarized as follows: (i)
Compounds containing a piperidine fragment exhibit
high affinity for the 5-HT2A receptor, while those having
benzofuranone or thiotetralone-piperidine moieties are
the most selective derivatives for 5-HT2A in comparison
to 5-HT2C and 5-HT2B, respectively. (ii) Although the
piperidine fragment is associated with higher affinities
for the 5HT2C receptors than the piperazine one, only
piperazine-containing ligands were selective for 5-HT2C
in comparison to 5-HT2A. (iii) An ethylene bridge be-
tween the alkanone and the amino moieties led to
ligands with the highest affinities for the 5-HT2B recep-
tor. (iv) Compounds showing a 5-HT2A/5-HT2C selectivity
greater than the 5-HT2A/5-HT2B one bear 6-fluorobenz-

62 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Brea et al.
Ta ble 3. Statistical Results of the GRID/GOLPE Analyses on
5-HT2A-C Ligands
derivatives. Red polyhedra represent zones where the
receptor poorly tolerates the presence of even small
groups, such as the methoxy group of the low activity
compound 18a . On the other hand, a small but signifi-
cant green polyhedron appears close to the carbonyl
oxygen and the sulfur atom of the hetero-fused cycloal-
kanone moiety of the highly active ligands 20g and 20h ,
indicating a favorable zone for hydrogen bonding or
receptor
5-HT2A
probeb
nc
vard
ONC
q2 e
r2 f
hydroxyl (O1) 49a 1695
4
4
4
4
3
3
1
1
1
1
1
4
0.815 0.883
0.828 0.887
0.830 0.891
0.842 0.932
0.136 0.529
0.177 0.517
0.247 0.403
0.371 0.541
0.378 0.489
0.399 0.519
0.353 0.481
0.341 0.866
N+ sp3 (N3+)
C sp3 (C3)
DRY
49a 1696
49a 1536
49a 1893
5-HT2B
5-HT2C
hydroxyl (O1) 38
2075
2000
1273
1637
963
1013
1441
1983
N+ sp3 (N3+)
C sp3 (C3)
DRY
38
38
38
+
sp3
3
dipole-dipole interaction. The other probes, Csp , N
,
and DRY, gave PLS coefficient contour maps fully
compatible with that previously developed with CoMFA
by using steric, electrostatic, and lipophilic (MLP91
calculations) fields. One strong outlier (compound 22b)
was omitted from the analysis. Its elimination can be
justified, at least in part, considering its unique chemi-
cal structure; it is the only ligand bearing an unsubsti-
tuted phenyl ring attached to the piperazine ring.
hydroxyl (O1) 41
N+ sp3 (N3+)
C sp3 (C3)
DRY
41
41
41
a
b
Compound 22b was excluded from the analysis. The letters
in parentheses refer to the GRID probe code. c n ) number of data
d
points. var ) number of variables remaining after pretreatment.
e q2 ) squared cross-validated correlation coefficient. f r2 ) squared
correlation coefficient.
The lack of significant PLS models for the 5-HT2B
receptor subtype could be partially ascribed to the
nature of the biological data, which are functional and
not binding experiments. In other words, the functional
response is inherently a very complex event, most likely
unrelated to the classical physicochemical properties
commonly used to describe receptor-ligand interactions.
Moreover, the spread and distribution of affinity data
for the 5-HT2B receptor subtype were not as good as for
5-HT2A. Because of these intrinsic limitations, no fur-
ther effort was made to improve the statistics of the
PLS models for the 5-HT2B receptor subtype shown in
Table 3.
F igu r e 3. GRID/GOLPE PLS coefficient contour map for
5-HT2A receptor ligands (O1 probe; contour levels: 0.005 green
and -0.005 red; for color code, see text). Compounds 20g
(QF0601B), 20h (QF0610B), and 18a (QF0607B) are displayed
to aid interpretation.
Also, in the modeling of 5-HT2C binding affinity, we
obtained PLS models with modest fitting and quite
unsatisfactory predictive power (r2 e 0.87; q2 e 0.34)
(see Table 3). A possible reason for this lack of predictive
power could be ascribed to the small variability of the
5-HT2C data in comparison with that of the 5-HT2A. On
the other hand, it has to be pointed out that the same
ligand alignment was used for all of the GRID/GOLPE
analyses (see the Experimental Section). This could be
a source of inaccuracy of the models obtained for two of
the receptors, since the comparative docking simulations
described below show significantly different binding
geometries of the two considered ligands in the models
of 5-HT2A and 5-HT2C receptors. In any case, poorer
statistics were obtained by testing alternative ligand
superpositions (data not shown).
models with similar statistical figures (see Table 3).
These findings support the hypothesis that the binding
process of our ligands to the 5-HT2A receptor is influ-
enced by a combination of steric, electrostatic, hydro-
phobic, and hydrogen bonding interactions as assessed
+
3
sp3
by Csp , N
, DRY, and hydroxyl probes, respectively.
Moreover, it is rather interesting to observe that the
present statistical results are highly comparable with
those found in our previous 3D-QSAR study conducted
with CoMFA on a reduced set of ligands.64 The impor-
tance of the steric, electrostatic, and lipophilic effects
in the modulation of the binding process was clearly
highlighted in both studies. As an important comple-
ment to previous CoMFA results, GRID/GOLPE allowed
us to elucidate the role of hydrogen bonding in the
receptor binding of our ligands, by using the hydroxyl
probe provided by GRID.
From site-directed mutagenesis studies, it has been
postulated that hydroxyl groups of some serine residues
present in the third transmembrane R-helix region of
the serotoninergic receptor family may form important
hydrogen bonds with the ligands.61 This hypothesis was
fully supported by our GRID/GOLPE results. As can be
seen from the graphical representations of the PLS
coefficients in Figure 3, there are regions around the
ligands where the different substituents of our deriva-
tives could generate favorable (green) or unfavorable
(red) interactions with the hydroxyl probe. Green poly-
hedra appear in the region surrounding the fluoro atoms
of the highly active compounds 20g and 20h , suggesting
a possible favorable hydrogen bond interaction for both
To gain insight on the molecular features governing
the binding process of the studied series of antagonists,
we performed computational simulations of the docking
of two of the studied compounds (20c and 20h ) into
models of the rat 5-HT2A and human 5-HT2C receptors
(the species were selected in agreement with the bio-
logical data). Compounds 20c and 20h were selected
because despite having the structural differences con-
centrated in a single molecular fragment (the amino
moiety), they exhibit completely different pharmacologi-
cal profiles. Thus, while compound 20h shows one of
the highest 5-HT2A affinities (the highest pA2 in func-
tional assays) and a relatively high 5-HT2C affinity,
compound 20c has a relatively low 5-HT2A affinity and
an even worse 5-HT2C one. A second important argu-
ment for the selection of compounds 20c and 20h was
their relatively low number of conformational degrees
of freedom.

New Serotonin Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 63
F igu r e 4. Models of the docking of 20h (QF0610B) (top) and 20c (QF0604B) (bottom) into the 5-HT2A (left) and 5-HT2C (right)
receptors.
The models of the receptors were built following the
protocol indicated in the corresponding experimental
section, and they were successfully assessed in different
ways: (a) We did not find significant contradictions
between our models and the topology of the recently
published crystal structure of rhodopsin57 (for instance,
both have the same relative positions of the highly
conserved residues, including the network of hydrogen
bonds between transmembrane helices 1, 2, and 7
(TMH1, TMH2, and TMH7), which involves the highly
conserved Asn of TMH1). (b) A quality assessment
performed using the Procheck software92 resulted in
excellent quality parameters and plots. (c) Molecular
dynamics simulations using AMBER 4.1, during 1 ns
at 310 K, did not produce any significant distortion in
the model. (d) An automatic exploration of the docking
of the natural agonist, serotonin, carried out with the
QXP program93 placed this agonist in a pocket between
TMH3, TMH5, and TMH6, yielding experimentally
known interactions with Asp15563 and Ser15961 in both
receptors and with Ser239 in 5-HT2A and Ser219 in
94-96
5-HT2C
.
The docking of the two antagonists in the models of
the two receptors (rat 5-HT2A and human 5-HT2C) was

64 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Brea et al.
bundle in agreement with the two aforementioned
binding positions. The dimensions of such entrances and
the space between the helices make more feasible
binding positions parallel to the helices for long and
relatively rigid molecules as ketanserin and the present
ones, in agreement with the positions found in our
docking experiments. The different binding modes of
agonists and antagonists obtained in the present simu-
lations could explain, at least in part, their divergent
pharmacological behaviors.
In conclusion, structural modification of conforma-
tionally constrained butyrophenones generated ligands
that exhibit high affinity and selectivity for the different
5-HT2 receptors. Among such compounds, 11e, 19e, 10e,
20g, 13g, and 12g emerged as 5HT2A selective ligands,
while 22c turned out to be a selective 5HT2C ligand, and
10a and 18a were identified as slightly selective 5-HT2B
ligands. These compounds may constitute interesting
pharmacological tools for a deeper understanding of the
role played by each 5-HT2 receptor subtype in schizo-
phrenia and other CNS disorders.
As far as the molecular modeling is concerned, the
3D-QSAR analysis allowed the identification of the main
molecular determinants for a high affinity binding at
the 5-HT2A and, to a lesser extent, at the 5-HT2C
receptors.
Some interesting structural features, determining
5-HT2 subtype selectivity, were also found. In particular,
piperidine-containing derivatives showed a higher se-
lectivity toward 5-HT2A and 5-HT2C receptors as com-
pared to the corresponding piperazine derivatives. Some
of the latter derivatives displayed some selectivity for
5-HT2C and, although at a lower level, for the 5-HT2B
receptor subtype. Moreover, the highest affinity ligands
at the 5-HT2B receptor were characterized by the pres-
ence of a two-carbon ethylene bridge between the amino
and the alkanone moieties.
F igu r e 5. Comparison of the docking positions in the 5-HT2A
receptor obtained for an agonist (serotonin) and an antagonist
(20c (QF0604B)).
automatically explored by means of 300 runs of the
MCDOCK module of the QXP program. Figure 4 shows
the lowest energy docking complexes (one per molecule
and per receptor) found along the docking simulations.
It has to be pointed out that in all of the docking
complexes obtained, the antagonists were always au-
tomatically located between helices TMH2, TMH3, and
TMH7, exhibiting an extended conformation approxi-
mately parallel to the axes of the helices. Hydrogen bond
interactions with three residues in TMH3 (Cys148,
Asp155, and Ser159) were obtained in all of the cases.
Hydrophobic interactions with residues in TMH2 and
TMH7 also stabilized the complexes. The performed
docking simulations performed were unable to explain
the pKi differences between the considered ligands and
the receptors being considered. This limitation could be
caused by imperfections in the models of the receptors
or in the QXP docking software. It has to be pointed
out that even slight inexactnesses in geometric param-
eters provoke notable changes in the energy values
resulting from molecular modeling computations.
The resulting docking positions of the antagonists
studied are totally different from those previously
described for the natural agonist 5-HT, and they are
similar to those obtained by other authors when simu-
lating the docking of different 5-HT2C antagonists in the
corresponding receptor model.66 It has to be pointed out
that even initially placing the antagonists in the binding
pocket between TMH3, TMH5, and TMH6, with the
amino group of the ligand in front of Asp155 of TMH3,
QXP explorations yield docking positions in the pocket
between TMH2, TMH3, and TMH7, as those shown in
Figure 4. An opposite experiment that consisted in
placing an agonist (5-HT) between TMH2, TMH3, and
TMH7 and carrying out a QXP exploration yielded the
above-described docking position of serotonin between
TMH3, TMH5, and TMH6. Figure 5 illustrates the
different binding positions of an agonist (5-HT) and an
antagonist (20c) obtained when simulating their dock-
ing into the 5-HT2A receptor. There are experimental
indications (mutations of the serines in TMH5) that
antagonists structurally related to the present series
(i.e., ketanserin) do not directly interact with TMH5.61,94
On the other hand, the packing of the extracellular loops
described in the recent experimental structure of rho-
dopsin57 shows two entrances into the transmembrane
Finally, a docking simulation study of some ligands
at 5-HT2A and 5-HT2C receptor models, provided results
consistent with 3D-QSAR findings and suggested an
interesting hypothesis for the antagonist binding mode.
Exp er im en ta l Section
Ch em istr y. Melting points were determined using a Kofler
hot stage instrument or a Gallenkamp capillary melting point
apparatus and are uncorrected. Infrared spectra were recorded
by means of a Perkin-Elmer 1600 Fourier transform infrared
spectrophotometer; the main absorption bands are given in
1
cm-1. H spectra were recorded with a Bruker WM AMX (300
MHz); chemical shifts are recorded in parts per million (δ)
downfield from tetramethylsilane. Mass spectra were per-
formed on Kratos MS-50 or Varian Mat-711 mass spectrom-
eters in fast atom bombardment (FAB) mode (with 2-hydroxy-
ethyl disulfide as matrix) or by electron impact (EI). Flash
column chromatography was performed using Kieselgel 60
(60-200 mesh, E. Merck AG, Darmstadt, Germany). Reactions
were monitored by thin-layer chromatography on Merck 60
GF254 chromatogram sheets using iodine vapor and/or UV light
for detection; unless otherwise stated, each purified compound
showed a single spot. Elemental combustion analyses were
performed on a Perkin-Elmer 240B apparatus at the Micro-
analysis Service of the Universidad de Santiago de Compos-
tela; unless otherwise stated, all reported values are within
(0.4% of the theoretical compositions. Solvents were purified
as per Vogel97 by distillation over the drying agent under an
argon atmosphere and were used immediately. Unless other-
wise stated, hydrochlorides were prepared by dropwise addi-
tion, with cooling, of a saturated solution of HCl in anhydrous

New Serotonin Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 65
1
ether to a solution of the amine in anhydrous ether or absolute
ethanol/ether.
1669, 1635, 1592. H NMR (CDCl3): δ 1.97-2.08 (m, 1H, H6
benzothiophene); 2.28-2.43 (m, 1H, H6 benzothiophene); 2.34
(dd, 1H, J ) 17.3, 7.5, HCH-CONRR); 3.07-3.22 (m, 4H, 2H7
and H5 benzothiophene, -HCH-CONRR); 3.50-3.83 (m, 8H,
piperazine); 6.61-6.65 (m, 2H, H3 and H5 pyridine); 7.03 (d,
1H, J ) 5.2, H2 benzothiophene); 7.34 (d, 1H, J ) 5.2, H3
benzothiophene); 7.45-7.50 (m, 1H, H4 pyridine); 8.16 (dd, 1H,
J ) 4.9, 1.6, H6 pyridine). MS (FAB, m/z): 356 (MH+). Anal.
(C19H21N3O2S) C, H, N.
1-[(4-Oxo-4,5,6,7-tetr a h yd r oben zo[b]fu r a n -5-yl)a cetyl]-
4-(o-m eth oxyp h en yl)p ip er a zin e (30). Compound 30 was
prepared from (4-oxo-4,5,6,7-tetrahydrobenzo[b]furan-5-yl)-
acetic acid using dimethylformamide (DMF) as solvent, yield
4-Oxo-4,5,6,7-tetr a h yd r oben zo[b]fu r a n -5-yla cetic Acid
(29). To a stirred mixture of diisopropylamine (2.98 mL, 0.021
mol) in dry tetrahydrofuran (THF, 200 mL) at -20 °C, a 2.5
M solution of n-BuLi in hexane (8.53 mL, 0.021 mol) was
added. The mixture was stirred for 30 min at -20 °C and for
another 30 min at -70 °C. After this time, a solution of 4,5,6,7-
tetrahydrobenzo[b]furan-4-one (2.86 g, 0.021 mol) in dry THF
(60 mL) was added dropwise. After the solution was stirred
for 1 h at -70 °C, ethyl bromoacetate (2.0 mL, 0.021 mol) was
added, and the mixture was stirred for 30 min at -70 °C and
then for 18 h at room temperature. The solvent was removed
in vacuo, and the residue was dissolved in AcOEt and washed
with water, 5% NaHCO3, and 5% HCl. The organic extracts
were dried (Na2SO4) and concentrated to give a residue that
was purified by column chromatography (CH2Cl2) to yield ethyl
4-oxo-4,5,6,7-tetrahydrobenzo[b]furan-5-yl acetate (3.22 g, 69%)
as a pale yellow oil. To a solution of this ester (2.0 g, 9 mmol)
in methanol (10 mL), a 10% solution of NaOH in methanol
(10.8 mL, 27 mmol) was added. The mixture was stirred at
reflux temperature for 2 h. After this time, the solvent was
removed under reduced pressure; the residue was dissolved
in water, washed with CH2Cl2, and acidified with concentrated
HCl. The solid precipitate was collected by filtration and
crystallized from H2O to give the acid 29 (1.31 g, 75%) as white
crystals of mp 103-104 °C. IR: 3100, 1732, 1641, 1582. 1H
NMR (DMSO-d6): δ 1.97 (dq, 1H, J ) 5.7, 1.3, 1H6); 2.18-
2.20 (m, 1H, 1H6); 2.34 (dd, 1H, J ) 16.5, 6.6, -HCH-COOH);
2.66 (dd, 1H, J ) 16.5, 5.4 Hz, -HCH-COOH); 2.74-2.93 (m,
3H, H5, 2H7); 6.64 (d, 1H, J ) 2, H3); 7.66 (d, 1H, J ) 1.8, H2);
12.12 (s, 1H, -OH). MS (EI, m/z): 194 (M+). Anal. (C10H10O4)
C, H.
Gen er a l P r oced u r e for Syn th esis of Ketoa m id es 25-
27 a n d 30-33. A solution of the amine (10 mmol), HOBt (1.35
g, 10 mmol), and the corresponding carboxylic acid (10 mmol)
in anhydrous CH2Cl2 (25 mL) was stirred under argon at room
temperature for 15 min and then cooled to 0 °C. At this
temperature, DCC (2.06 g, 10 mmol) was added and the
reaction mixture was kept at 0-5 °C for 1 h and then allowed
to reach room temperature and left overnight. The precipitated
1,3-dicyclohexylurea was filtered off, and the filtrate was
washed several times with 5% NaHCO3 and water, dried (Na2-
SO4), and condensed to dryness. The oily residue was purified
by flash chromatography (AcOEt/hexane) to give the amide
as a white crystalline solid. The synthesis of each particular
amide is described below.
1
85%, mp 121-122 °C (cyclohexane). IR: 2927, 1661, 1645. H
NMR (CDCl3): δ 1.94-2.02 (m, 1H, 1H6 benzofuran); 2.26-
2.34 (dd, 1H, J ) 16.2, 8.3, -HCH-CONRR); 2.40-2.47 (m,
1H, 1H6 benzofuran); 2.93-3.02 (m, 3H, 2H7 and H5 benzofu-
ran); 3.03-3.09 (m, 4H, -CON(CH2-CH2)2N-); 3.22 (dd, 1H,
J ) 16.2, 3.6, -HCH-CONRR); 3.71-3.74 (m, 4H, -CON-
(CH2-CH2)2N-); 3.88 (s, 3H, -OCH3); 6.66 (d, 1H, J ) 2, H3
benzofuran); 6.87-7.01 (m, 4H, Ph); 7.32 (d, 1H, J ) 1.9, H3
benzofuran). MS (EI, m/z): 368 (M+). Anal. (C21H24N2O4) C,
H, N.
1-[(4-Oxo-4,5,6,7-tetr a h yd r oben zo[b]fu r a n -5-yl)a cetyl]-
4-(2-p yr id yl)p ip er a zin e (31). Compound 31 was prepared
from (4-oxo-4,5,6,7-tetrahydrobenzo[b]furan-5-yl)acetic acid
using DMF as solvent, yield 90%, mp 122-123 °C (cyclohex-
1
ane). IR: 2920, 1678, 1623, 1595. H NMR (CDCl3): δ 1.91-
2.05 (m, 1H, 1H6 benzofuran); 2.31 (dd, 1H, J ) 16.1, 7.9,
-HCH-CONRR); 2.35-2.44 (m, 1H, 1H6 benzofuran); 2.93-
3.23 (m, 4H, H5 and 2H7 benzofuran, -HCH-CONRR); 3.50-
3.81 (m, 8H, piperazine); 6.66 (d, 1H, J ) 1.9, H3 benzofuran);
6.64-6.68 (m, 2H, H3 and H5 pyridine); 7.31 (d, 1H, J ) 1.9,
H2 benzofuran); 7.47-7.53 (m, 1H, H4 pyridine); 8.18-8.20
(m, 1H, H6 pyridine). MS (FAB, m/z): 340 (MH+). Anal. (C19
-
H
21N3O3) C, H, N.
1-[(4-Oxo-4,5,6,7-tetr a h yd r oben zo[b]fu r a n -5-yl)a cetyl]-
4-(p -flu or ob en zoyl)p ip er id in e (32). Compound 32 was
prepared from (4-oxo-4,5,6,7-tetrahydrobenzo[b]furan-5-yl)-
acetic acid using DMF as solvent, yield 80%, mp 124-125 °C
1
(cyclohexane). IR: 2920, 1671, 1636, 1595. H NMR (CDCl3):
δ 1.66-1.86 (m, 4H, -N(CH2-CH2)2CH-); 1.89-1.98 (m, 2H,
2H6 benzofuran); 2.24 (dd, 1H, J ) 16.1, 8.1, -HCH-CO-
NRR); 2.29-2.44 (m, 1H, -N(HCH-CH2)2CH-); 2.85-3.00 (m,
3H, 2H7 and H5 benzofuran); 3.09-3.26 (m, 2H, -HCH-CO-
NRR, -N(HCH-CH2)2CH-); 3.44-3.49 (m, 1H, RRCH-CO-
Ph); 4.01-4.06 (m, 1H, -N(HCH-CH2)2CH-); 4.54-4.61 (m,
1H, -N(HCH-CH2)2CH-); 6.65 (d, 1H, J ) 1.8, H3 benzofu-
ran); 7.15 (t, 2H, J ) 8.5, H3 and H5 Ph); 7.31 (d, 1H, J ) 1.9,
H2 benzofuran); 7.97 (dd, 2H, J ) 8.6, 5.4, H2 and H6 Ph). MS
(FAB, m/z): 384 (MH+). Anal. (C22H22FNO4) C, H, N.
1-[(4-Oxo-4,5,6,7-tetr a h yd r oben zo[b]fu r a n -5-yl)a cetyl]-
4-(6-flu or ob en zisoxa zol-3-yl)p ip er id in e (33). Compound
33 was prepared from (4-oxo-4,5,6,7-tetrahydrobenzo[b]furan-
5-yl)acetic acid using DMF as solvent, yield 80%, mp 141-
143 °C (cyclohexane). IR: 2921, 1670, 1641, 1517. 1H NMR
(CDCl3): δ 1.93-2.16 (m, 5H, 1H6 benzofuran, -N(CH2-CH2)2-
CH-); 2.32 (dd, 1H, J ) 16.1, 7.8, -HCH-CONRR); 2.39-
2.93 (m, 2H, 1H6 benzofuran, -N(HCH-CH2)2CH-); 2.94-
3.38 (m, 6H, H5, 2H7, -HCH-CONRR, -N(HCH-CH2)2CH-,
-N(CH2-CH2)2CH-); 4.11 (m, 1H, -N(HCH-CH2)2CH-);
4.60-4.73 (m, 1H, -N(HCH-CH2)2CH-); 6.66 (d, 1H, J ) 1.9,
H3 benzofuran); 7.05-7.11 (m, 1H, H5 benzisoxazole); 7.26 (dd,
1H, J ) 5.1, 2.1, H7 benzisoxazole); 7.31 (d, 1H, J ) 1.9, H2
benzofuran); 7.70 (dd, 1H, J ) 8.7, 5.1, H4 benzisoxazole). MS
(FAB, m/z): 397 (MH+). Anal. (C22H21FN2O4) C, H, N.
1-[(1-Oxo-in d a n -3-yl)ca r b on yl]-4-(2-p yr id yl)p ip e r a -
zin e (25). Compound 25 was prepared from 3-oxo-indane-1-
carboxylic acid, yield 87%, mp 175-177 °C (i-PrOH). IR: 1714,
1644. 1H NMR (CDCl3): δ 2.87 (dd, 1H, J ) 18.5, 7.5, H2
indanone); 2.98 (dd, 1H, J ) 18.5, 4.1, H2 indanone); 3.55-
3.91 (m, 8H, piperazine); 4.59 (dd, 1H, J ) 7.4, 4.2, H3
indanone); 6.64-6.71 (m, 2H, H3 and H5 pyridine); 7.28 (t, 1H,
J ) 7.3, H6 indanone); 7.39-7.42 (m, 1H, H4 pyridine); 7.50-
7.61 (m, 2H, H4 and H5 indanone); 7.71 (t, 1H, J ) 8.3, H7
indanone); 8.16 (dd, 1H, J ) 4.9, 1.5, H6 pyridine). MS (FAB,
m/z): 322 (MH+). Anal. (C19H19N3O2) C, H, N.
1-[(1-Oxoin d a n -2-yl)a cetyl]-4-(o-m eth oxyp h en yl)p ip er -
a zin e (26). Compound 26 was prepared from (1-oxo-2-indan-
yl)acetic acid, yield 85%, mp 138-139 °C (EtOH). IR: 1702,
1
1644, 1600. H NMR (CDCl3): δ 2.69 (dd, 1H, J ) 17.2, 9.2,
HCH-CONRR); 2.95 (dd, 1H, J ) 17.0, 4.2, H3 indanone);
3.03-3.14 (m, 2H, H2 indanone, HCH-CONRR); 3.18-3.28
(m, 4H, -CON(CH2-CH2)2N-); 3.53 (dd, 1H, J ) 17.0, 7.4,
H3 indanone); 3.76-3.87 (m, 4H, -CON(CH2-CH2)2N-); 3.91
(s, 3H, -OCH3); 6.92-7.18 (m, 4H, Ph); 7.38 (d, 1H, J ) 7.4,
H6 indanone); 7.47 (d, 1H, J ) 7.3, H4 indanone); 7.60 (dt, 1H,
J ) 7.4, 1.1, H5 indanone); 7.78 (d, 1H, J ) 7.6, H7 indanone).
MS (FAB, m/z): 365 (MH+). Anal. (C22H24N2O3) C, H, N.
Gen er a l P r oced u r e for Syn th esis of Am in ok eton es
10c, 12a , 20c, 23a , 23c, 23g, a n d 23h . A stirred solution of
the amide (8.2 mmol), ethylene glycol (45 mL, 0.82 mol), and
p-TsOH (50 mg) in anhydrous toluene (75 mL) was refluxed
in a Dean-Stark apparatus for 4 days with azeotropic distil-
lation of water. After the solution was cooled, the toluene
solution was washed several times with 10% Na2CO3 and
water and dried (Na2SO4), and the solvent was removed under
1-[(4-Oxo-4,5,6,7-tetr a h yd r oben zo[b]th iop h en -5-yl)a c-
etyl]-4-(2-p yr id yl)p ip er a zin e (27). Compound 27 was pre-
pared from (4-oxo-4,5,6,7-tetrahydrobenzo[b]thiophen-5-yl)-
acetic acid, yield 85%, mp 135-137 °C (MeOH-ether). IR:

66 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Brea et al.
reduced pressure. The resulting crude ethylene ketal was
dissolved in anhydrous ether (30 mL) and added dropwise
under argon to a stirred suspension of LiAlH4 (1.18 g, 31 mmol)
in anhydrous ether (60 mL). The reaction mixture was heated
under reflux for 8 h, cooled to 0 °C in an ice-bath, and then
quenched by sequential dropwise addition of H2O (1.5 mL),
NaOH 10% (3 mL), and H2O (4 mL). The coarse precipitate
formed was filtered out and thoroughly washed with ether.
The combined filtrates were treated with 10% HCl and heated
under reflux for 1-2 h. On cooling, the aqueous phase was
made alkaline with 10% NaOH and extracted with CH2Cl2
three times, the combined organic extracts were dried (Na2-
SO4), and the solvent was removed in vacuo. The residue was
dissolved in anhydrous ether, and ether-saturated HCl gas was
cautiously added to the resulting solution. The white precipi-
tate formed was recovered and kept overnight in a vacuum
desiccator. Each particular aminoketone is described below.
2.28 (m, 1H, H6 benzofuran); 2.43-2.51 (m, 3H, H5 benzofuran,
-CH2-NRR), 2.52-2.58 (m, 4H, -N(CH2-CH2)2N-); 2.81-
2.93 (m, 2H, H7 benzofuran); 3.49 (t, 4H, J ) 5.1, -N(CH2-
CH2)2N-); 6.56-6.60 (m, 2H, H3 and H5 pyridine); 6.62 (d, 1H,
J ) 1.9, H3 benzofuran); 7.27 (d, 1H, J ) 1.8, H2 benzofuran);
7.42-7.48 (m, 1H, H4 pyridine); 8.16 (d, 1H, J ) 8.0, H6
pyridine). MS (FAB, m/z): 326 (MH+). Anal. (C19H23N3O2) C,
H, N. Hydrochloride: mp 197-198 °C (i-PrOH).
1-[(4-Oxo-4,5,6,7-tetr a h yd r oben zo[b]fu r a n -5-yl)eth yl]-
4-(p-flu or oben zoyl)p ip er id in e (23g). Yield, 85%, mp 112-
113 °C (i-PrOH). IR: 2953, 1683, 159. 1H NMR (CDCl3): δ
1.52-1.64 (m, 1H, -HCH-CH2-NRR); 1.84-1.97 (m, 5H,
-N(CH2-CH2)2CH-, -HCH-CH2-NRR); 1.97-2.41 (m, 4H,
-N(HCH-CH2)2CH-, 2H6 benzofuran); 2.46-2.49 (m, 3H,
-CH2-NRR, H5 benzofuran); 2.89-3.01 (m, 4H, -N(HCH-
CH2)2CH-, 2H7 benzofuran); 3.14-3.20 (m, 1H, -N(HCH-
CH2)2CH-); 6.65 (d, 1H, J ) 1.9, H3 benzofuran); 7.12 (t, 2H,
J ) 8.6, H3 and H5 Ph); 7.30 (d, 1H, J ) 1.9, H2 benzofuran);
7.93 (dd, 2H, J ) 8.7, 5.6, H2 and H6 Ph). MS (FAB, m/z): 370
(MH+). Anal. (C22H24FNO3) C, H, N. Hydrochloride: mp 275-
276 °C (i-PrOH). Anal. (C22H24FNO3‚HCl) C, H, N.
1-[(4-Oxo-4,5,6,7-tetr a h yd r oben zo[b]fu r a n -5-yl)eth yl]-
4-(6-flu or oben zisoxa zol-3-yl)p ip er id in e (23h ). Yield, 85%,
mp 124-125 °C (i-PrOH). IR: 2920, 1676, 1610. 1H NMR
(CDCl3): δ 1.57-1.70 (m, 1H, -HCH-CH2-NRR); 1.94-2.48
(m, 9H, -N(HCH-CH2)2CH-, -HCH-CH2-NRR, 2H6 ben-
zofuran); 2.50-2.66 (m, 3H, -CH2-NRR, H5 benzofuran);
2.83-2.98 (m, 4H, 2H7 benzofuran, -N(HCH-CH2)2CH-);
3.03-3.12 (m, 1H, -N(HCH-CH2)2CH-); 6.65 (d, 1H, J ) 1.9,
H3 benzofuran); 7.04 (dt, 1H, J ) 8.8, 2.1, H5 benzisoxazole);
7.07-7.27 (m, 1H, H7 benzisoxazole); 7.31 (d, 1H, J ) 1.9, H2
benzofuran); 7.69 (dd, 1H, J ) 8.7, 5.0, H4 benzisoxazole). MS
(FAB, m/z): 383 (MH+). Anal. (C22H23FN2O3) C, H, N. Hydro-
chloride: mp 257-258 °C (i-PrOH).
1-[(1-Oxo-1,2,3,4-t et r a h yd r o-3-n a p h t h yl)m et h yl]-4-(6-
flu or oben zisoxa zol-3-yl)p ip er id in e (11h ). A solution of
3-(p-toluenesulfonyloxymethyl)-1,2,3,4-tetrahydronaphthalen-
1-one85 (0.24 g, 0.7 mmol) and 4-(6-fluorobenzisoxazol-3-yl)-
piperidine (0.55 g, 1.4 mmol) in 1-methyl-2-pyrrolidone (NMP,
7 mL) was stirred at 85 °C for 24 h. The solvent was
evaporated under reduced pressure, and the residue was
dissolved in CH2Cl2 and washed with water. The organic layer
was dried (Na2SO4), filtered, and concentrated under vacuo
to give an oil, which was purified by chromatography (silica
gel, EtOAc/hexane 1:1) to give the amine 11h (0.14 g, 51%) as
a yellowish oil. IR: 2941, 1684, 1616. 1H NMR (CDCl3): δ
2.04-2.26 (m, 6H, -CH2-N(CH2-CH2)2CH-); 2.33-2.50 (m,
4H, -CH2-N(CH2-CH2)2CH-); 2.74-2.90 (m, 2H, H3 and H4
tetralone); 3.00-3.20 (m, 4H, 2H2 and H4 tetralone, -N(CH2-
CH2)2CH-); 7.07 (dt, 1H, J ) 8.9, 2.1, H5 benzisoxazole); 7.25
(dd, 1H, J ) 8.7, 2.1, H7 benzisoxazole); 7.29-7.34 (m, 2H, H5
and H7 tetralone); 7.49 (dt, 1H, J ) 7.4, 1.4, H6 tetralone);
7.60-7.73 (m, 1H, H4 benzisoxazole); 8.03 (d, 1H, J ) 7.8, H8
tetralone). MS (EI, m/z): 378 (M+). Hydrochloride: mp 248-
250 °C (i-PrOH). Anal. (C23H23FN2O2‚HCl‚0.75H2O) C, H, N.
1-[(1-Oxoin d a n -3-yl)m et h yl]-4-(2-p yr id yl)p ip er a zin e
(10c). Yield, 85%, mp 175-177 °C (AcOEt). IR: 2945, 2825,
1
1717. H NMR (CDCl3): δ 2.50 (dd, 1H, J ) 12.4, 8.5, HCH-
NRR); 2.60 (dd, 1H, J ) 19.2, 3.0, H2 indanone); 2.59-2.67
(m, 4H, -CH2-N(CH2CH2)2-N-); 2.72 (dd, 1H, J ) 12.4, 6.7,
-HCH-NRR); 2.85 (dd, 1H, J ) 19.3, 7.5, H2 indanone); 3.51-
3.61 (m, 4H, -N(CH2CH2)2-N-); 3.60-3.64 (m, 1H, H3
indanone); 6.62-6.69 (m, 2H, H3 and H5 pyridine); 7.38 (t, 1H,
J ) 7.3, H6 indanone); 7.45-7.51 (m, 1H, H4 pyridine); 7.60
(t, 1H, J ) 7.4, H5 indanone); 7.67 (d, 1H, J ) 7.6, H4
indanone); 7.75 (d, 1H, J ) 7.6, H7 indanone); 8.19 (dd, 1H,
J ) 4.8, 1.5, H6 pyridine). MS (FAB, m/z): 308 (MH+). Anal.
(C19H21N3O) C, H, N. Hydrochloride: mp 230-232 °C (i-PrOH).
1-[(1-Oxoin d a n -2-yl)eth yl]-4-(o-m eth oxyp h en yl)p ip er -
a zin e (12a ). Yield, 70%. IR: 2938, 1715, 1608. 1H NMR
(CDCl3): δ 1.80-1.86 (m, 1H, HCH-CH2-NRR); 2.18-2.23
(m, 1H, HCH-CH2-NRR); 2.61 (t, 2H, J ) 7.4, -CH2-NRR);
2.69-2.80 (m, 5H, H2 indanone, -CH2-N(CH2-CH2)2N-);
2.88 (dd, 1H, J ) 17.1, 4.0, H3 indanone); 3.03-3.14 (m, 4H,
-CH2-N(CH2-CH2)2N-); 3.37 (dd, 1H, J ) 17.0, 8.0, H3
indanone); 3.87 (s, 3H, -OCH3); 6.84-7.02 (m, 4H, Ph); 7.36
(t, 1H, J ) 7.4, H6 indanone); 7.47 (d, 1H, J ) 7.5, H4
indanone); 7.58 (t, 1H, J ) 7.7, H5 indanone); 7.76 (d, 1H, J )
7.6, H7 indanone). MS (FAB, m/z): 351 (MH+). Hydrochlo-
ride: mp 215-216 °C (MeOH-ether). Anal. (C22H26N2O2‚2HCl)
C, H, N.
1-[2-(4-Oxo-4,5,6,7-tetr a h yd r oben zo[b]th iop h en -5-yl)-
eth yl]-4-(2-p yr id yl)p ip er a zin e (20c). Yield, 82%. IR: 2822,
1
1667, 1593. H NMR (CDCl3): δ 1.62-1.73 (m, 1H, -HCH-
CH2-N<); 1.98-2.09 (m, 1H, H6 benzothiophene); 2.17-2.38
(m, 2H, H6 benzothiophene, -HCH-CH2-N<); 2.53 (t, 2H,
J ) 7.8, -CH2-N<); 2.51-2.55 (m, 1H, H5 benzothiophene);
2.59 (t, 4H, J ) 5.1, -N(CH2-CH2)2N-Ar); 2.99-3.16 (m, 2H,
H7 benzothiophene); 3.53 (t, 4H, J ) 5.1, N(CH2-CH2)2N-
Ar); 6.59-6.65 (m, 2H, H3 and H5 pyridine); 7.05 (d, 1H, J )
5.3, H2 benzothiophene); 7.37 (d, 1H, J ) 5.3, H3 ben-
zothiophene); 7.43-7.49 (m, 1H, H4 pyridine); 8.17 (dd, 1H,
J
) 4.8, 1.3, H6 pyridine). MS (FAB, m/z): 342 (MH+).
Hydrochloride: mp 275-277 °C (MeOH-ether). Anal. (C19H23
N3OS‚3HCl) C, H, N.
-
Meth yl γ-[4-(2-P yr id yl)p ip er a zin -1-yl]-â-(2-th en yl)bu -
tyr a te (28). To a solution of N-(2-pyridyl)piperazine (0.82 g,
5 mmol) and methyl γ-bromo-â-thenylbutyrate (1.38 g, 5 mmol)
in MIK (35 mL) were added K2CO3 (2.5 g) and KI (0.045 g).
This mixture was refluxed for 12 h. Inorganic salts were then
filtered out, and the solvent was removed under reduced
pressure. The resulting oil was dissolved in CH2Cl2, washed
several times with water, and dried (Na2SO4). The CH2Cl2 was
removed under reduced pressure, and the residue was purified
by column chromatography (AcOEt/hexane, 1:1) to afford the
desired aminomethyl ester 28 (1.7 g, 95%) as an oil. IR: 1733,
1593. 1H NMR (CDCl3): δ 2.21-2.36 (m, 4H, -CH2-N(HCH-
CH2)2N-Ar); 2.44-2.61 (m, 5H, -CH2-COO, >CH-CH2-
N(HCH-CH2)2N-Ar); 2.87-2.93 (m, 2H, thiophene-CH2-);
3.49 (t, 4H, J ) 4.6, N(CH2-CH2)2N-Ar); 3.64 (s, 3H, -CO2-
CH3); 6.58-6.64 (m, 2H, H3 and H5 pyridine); 6.79 (d, 1H, J )
2.9, H3 thiophene); 6.92 (dd, 1H, J ) 5.1, 3.4, H4 thiophene);
7.14 (dd, 1H, J ) 5.2, 1.0, H5 thiophene); 7.46 (dd, 1H, J )
8.6, 5.2, H4 pyridine); 8.17 (dd, 1H, J ) 4.8, 1.7, H6 pyridine).
1-[(4-Oxo-4,5,6,7-tetr a h yd r oben zo[b]fu r a n -5-yl)eth yl]-
4-(o-m eth oxyph en yl)piper azin e (23a). Yield, 89%, mp 124-
125 °C (i-PrOH). IR: 2935, 1674, 1596. 1H NMR (CDCl3): δ
1.62-1.65 (m, 1H, -HCH-CH2NRR); 2.01 2.22 (m, 1H, HCH-
CH2NRR); 2.22-2.45 (m, 2H, 2H6 benzofuran); 2.47-2.61 (m,
2H; -CH2-NRR); 2.63-2.71 (m, 4H, -N(CH2-CH2)2N-);
2.88-2.93 (m, 3H, 2H7 and H5 benzofuran); 3.10-3.19 (m, 4H,
-N(CH2-CH2)2N-); 3.85 (s, 3H, -OCH3); 6.65 (d, 1H, J ) 2
Hz, H3 benzofuran); 6.83-7.01 (m, 4H, Ph); 7.31 (d, 1H, J ) 2
Hz, H2 benzofuran). MS (FAB, m/z): 355 (MH+). Hydrochlo-
ride: mp 223-224 °C (MeOH). Anal. (C21H26N2O3‚2HCl)
C, H, N.
1-[(4-Oxo-4,5,6,7-tetr a h yd r oben zo[b]fu r a n -5-yl)eth yl]-
4-(2-p yr id yl)p ip er a zin e (23c). Yield, 80%, mp 72-73 °C (i-
PrOH). IR: 2953, 2830, 1672, 1592. 1H NMR (CDCl3): δ 1.55-
1.62 (m, 1H, -HCH-CH2-NRR); 1.90-1.98 (m, 1H, H6
benzofuran); 2.13-2.20 (m, 1H, -HCH-CH2-NRR); 2.23-

New Serotonin Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 67
MS (EI, m/z): 359 (M+). Hydrochloride: mp 137-139 °C
(MeOH-ether). Anal. (C19H25N3O2S‚3HCl) C, H, N.
homogenized with 10 volumes of 0.32 M sucrose in a Polytron
homogenizer (Brinkmann Instruments, Westbury, NY). The
homogenate was centrifuged twice at 4 °C (900g for 10 min
followed by 40 000g for 30 min). The supernatant was dis-
carded, and the pellet was resuspended in Tris-HCl buffer (pH
7.5) in a Teflon/glass homogenizer (10 strokes by hand). The
homogenate was incubated at 37 °C for 15 min to remove
endogenous 5-HT and centrifuged for 30 min at 40 000g. The
final pellet was resuspended in Tris-HCl buffer of pH 7.5.
Competition at [3H]mesulergine binding sites was determined
by a protocol analogous to that described above for the 5-HT2A
binding assay, using a final [3H]mesulergine concentration of
2 nM, and 1 µM mianserine as nonspecific masking ligand.
The mixtures were incubated for 1 h at room temperature.
Membranes were harvested on Whatman GF/B filter (pre-
soaked in 3% polyethylenimine). The radioactivity retained on
filters was determined 12 h later, by liquid scintillation
counting in a beta counter (Beckman, LS-6000LL). IC50 and
Ki values were calculated as for rat 5-HT2A binding sites.
1-[(4-Oxo-4,5,6,7-t et r a h yd r ob en zo[b]t h iop h en -6-yl)-
m et h yl]-4-(2-p yr id yl)p ip er a zin e (18c). To 34 g of PPA
(Fluka), stirring under argon at 90 °C, was slowly added crude
methyl γ-[4-(2-pyridyl)piperazin-1-yl]-â-(2-thenyl)butyrate 28
(1 g, 2.8 mmol), after which the temperature was increased to
130 °C. After 5 h, the reaction mixture was poured into ice-
water, made alkaline with 5 N NaOH, and extracted with CH2-
Cl2. The organic phase was washed several times with water
to neutral pH and dried (Na2SO4), and the CH2Cl2 was
removed in vacuo to afford the amine 18c (0.36 g, 40%) as an
oil. IR: 2927, 1669, 1594. 1H NMR (CDCl3): δ 2.32 (dd, 1H,
J ) 16.1, 11.1, H5 benzothiophene); 2.25-2.75 (m, 7H, H6
benzothiophene, -CH2-N(CH2-CH2)2N-Ar); 2.74 (dd, 1H,
J ) 17.0, 5.4, H7 benzothiophene); 2.75 (dd, 1H, J ) 16.1, 15.1,
H5 benzothiophene); 3.28 (dd, 1H, J ) 16.5, 3.9, H7 ben-
zothiophene); 3.53 (t, 4H, J ) 5.1, N(CH2-CH2)2N-Ar); 6.59-
6.65 (m, 2H, H3 and H5 pyridine); 7.07 (d, 1H, J ) 5.3, H2
benzothiophene); 7.38 (d, 1H, J ) 5.3, H3 benzothiophene);
7.47 (dd, 1H, J ) 8.6, 5.1, H4 pyridine); 8.18 (dd, 1H, J ) 4.9,
1.2, H6 pyridine). MS (FAB, m/z): 328 (MH+). Hydrochloride:
mp 144-146 °C (MeOH-ether). Anal. (C18H21N3OS‚3HCl)
C, H, N.
An ta gon ism of Ser oton in a t 5-HT2A Recep tor s fr om
Ra t Aor ta . Antagonism of serotonin at 5-HT2A receptors22,99
was assayed using denuded thoracic aorta from male (250-
350 g) Sprague-Dawley rats, anesthetized by CO2, and killed
by decapitation. The descending aorta was removed, cleaned,
stripped of endothelium, and cut into rings 4 mm in length79
that were then mounted under a tension of 2 g in an organ
bath containing 20 mL of Krebs solution of the following
composition (mM): NaCl, 119; KCl, 4.7; MgSO4‚7H2O, 1.2;
CaCl2‚2H2O, 1.5; KH2PO4, 1.2; NaHCO3, 25; glucose, 11.
Chlorpheniramine (1 µM) was added to block the uptake of
serotonin.100,101 The bath solution was maintained at 37 °C and
aerated with carbogen (95% O2, 5% CO2). Before the addition
of drugs, the tissue strips were equilibrated for 1 h under a 2
g load. Isometric contractions were recorded during cumulative
addition of serotonin using a Grass FTO3C transducer and a
Grass 7D polygraph. Following the equilibration period, the
rings were sensitized by the addition of 10 µM 5-HT for 8 min.
Equilibration periods (60 min)102 were then alternated with
the construction of cumulative 5-HT concentration-response
curves, as per Van Rossum103 (from 30 nM to 100 µM). Two
control runs giving identical curves were followed by test runs
with ketanserin or the new compounds, which were added to
the bath solution 20 min before the end of the preceding
equilibration period. 5-HT concentration-effect curves were
fitted to the following equation using the Kaleidagraph
program (Synergy Software, Reading, PA): E ) Emax[A]s/
EC50 + [A]s; where Emax, [A], and s represent the maximum
response, agonist concentration, and curve slope, respectively.
EC50 is the concentration of agonist that produces 50% of the
maximal response. Values of EC50 are given as mean ( sem
(standard error mean). Antagonist potency was measured with
a one dose method103 in terms of pA2 (-log concentration of
antagonist required to maintain a constant response when
agonist concentration is doubled), using the following equa-
tion: pA2) log((EC50′/EC50) - 1)/C; where EC50 and EC50′ are
the concentrations of the agonist whose effect is 50% of the
maximal effect, in the absence and in the presence of C
concentration of the antagonist, respectively.
P h a r m a cology. Binding and functional assays were carried
out for most of the compounds prepared and plotted as shown
in Figures 1 and 2.
R a t 5-HT2A Bin d in g Sit e Assa ys. Male 200-250
g
Sprague-Dawley rats were anaesthetized with CO2 and killed
by decapitation. The frontal cortex, containing 5-HT2A recep-
tors,22,15 was dissected free on ice, frozen on dry ice, and stored
at -70 °C until use (generally less than 1 week later).
Membrane preparation procedures were carried out at 4 °C.
The tissue was thawed on ice and homogenized with 10
volumes of 0.32 M sucrose in a Polytron homogenizer (Brink-
mann Instruments, Westbury, NY). The homogenate was
centrifuged twice at 4 °C (900g for 10 min followed by 40 000g
for 30 min). The supernatant was discarded, and the pellet
was resuspended in Tris-HCl buffer (pH 8.07) in a Teflon/glass
homogenizer (10 strokes by hand). The homogenate was
incubated at 37 °C for 15 min to remove endogenous 5-HT and
centrifuged for 30 min at 40 000g. The final pellet was
resuspended in Tris-HCl buffer of pH 8.07 containing 4 mM
CaCl2 and 0.1% ascorbic acid. Competition at [3H]ketanserin
binding sites was assayed in triplicate in assay mixtures
consisting of 750 µL of membrane homogenate, 50 µL of [3H]-
ketanserin, 50 µL of either the buffer or the compound under
test, 50 µL of nonspecific masking ligand solution (1 µM
methysergide) when required, and buffer to a final volume of
1 mL. Mixtures were incubated for 30 min at 37 °C. The assay
was terminated by rapid filtration through Whatman GF/C
filter strips (presoaked in 3% polyethylenimine) in a Brandel
cell harvester (Gaithersburg, MD) followed by washing with
ice-cold Tris-HCl buffer (pH 6.6) to remove unbound radioli-
gand. The radioactivity retained on filters was determined 3
h later, by liquid scintillation counting in a beta counter
(Beckman, LS-6000LL).
The nonlinear curve-fitting program Kaleidagraph (Synergy
Software, Reading, PA) was used to fit the equation E )
Emax - [Emax - Emin/(1 + (IC50/C)n)], where Emax and Emin are
dpm at the beginning and the end of the competition experi-
ment, respectively; IC50 is the drug concentration required to
inhibit binding by 50%; C is the concentration of the inhibitor;
and n is the slope of the sigmoidal decay. Nonspecific binding
was determined independently in the presence of unlabeled 1
µM methysergide. Ki values were calculated from the Cheng-
Prusoff equation:98 Ki ) IC50/(1 + D/Kd), where D is the
concentration and Kd is the apparent dissociation constant of
the ligand.
Bovin e 5-HT2C Bin d in g Site Assa ys. Bovine choroid
plexus containing 5-HT2C receptors19 was dissected free on ice,
frozen on dry ice, and stored at -70 °C until use (generally
less than 1 week later). Membrane preparation procedures
were carried out at 4 °C. The tissue was thawed on ice and
An t a gon ism of Ser ot on in a t 5-HT2B R ecep t or s fr om
Ra t Stom a ch F u n d u s. Antagonism of serotonin at 5-HT2B
receptors was assayed using rat stomach fundus from male
(250-300 g) Sprague-Dawley rats, anesthetized by CO2 and
killed by decapitation. The stomach was dissected free from
the abdomen and immersed in modified Krebs solution of the
following composition (mM): NaCl, 118; KCl, 4.7; MgSO4‚
7H2O, 1.2; CaCl2‚2H2O, 2.5; KH2PO4, 1.18; NaHCO3, 25;
glucose, 11. Strips of stomach fundus were prepared by Vane’s
method105 and mounted in organ baths containing 10 mL of
the same Krebs solution as above, maintained at 37 °C with
aeration using carbogen (95% O2, 5% CO2). Before the addition
of drugs, the tissue strips were equilibrated for 1 h under a 1
g load. Isometric contractions were recorded during cumulative
addition of serotonin using a Grass FTO3C transducer and a
Grass 7D polygraph. Then, a protocol analogous to that

68 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Brea et al.
described above for 5-HT2A receptors was followed. Concentra-
tion-response curves for 5-HT were constructed as per Van
Rossum103 over the concentration range of 0.01 nM-10 µM.
of carbonyl and methoxy groups, the isoxazolyl and pyridyl
nitrogens, the charged amino group, and the carbonyl of the
cycloalkanone moiety were chosen as key fitting elements for
ligand superposition.64 The molecular interaction potentials
were calculated with GRID using four different atom probes,
namely the sp3 carbon (C1), the charged sp3 nitrogen (N3+),
the alcoholic hydroxyl (sp3 oxygen, O1), and the hydrophobic
probe (DRY) to measure steric, electrostatic, hydrogen bonding,
and hydrophobic interactions, respectively. All of the GRID
calculations were performed in a box with dimensions equal
to 30 Å × 20 Å × 25 Å using a grid spacing of 1 Å.
Cell Cu ltu r e. Chinese hamster ovary cells (CHO-FA4 cells)
that stably express the cloned human 5-HT2A serotonin recep-
tor were kindly provided by Dr. Kelly Berg and Dr. William
P. Clarke (University of Texas Health Science Center, San
Antonio, Texas). Cells were maintained in standard tissue
culture plates (150 mm diameter) in Dulbecco’s modified
Eagle’s medium (DMEM) F-12 medium (Gibco BRL, Paisley,
U.K.) supplemented with 10% fetal calf serum (FCS, Gibco
BRL, Paisley, U.K.), 1% L-glutamine (BIOSTAR, Alcorco´n,
Spain), and 300 µg/mL hygromycin (Gibco BRL, Paisley, U.K.).
HeLa cells were transfected to stably express the cloned
human 5-HT2C serotonin receptors. Cells were maintained in
standard tissue culture plates (150 mm diameter) in DMEM
(BIOSTAR, Alcorco´n, Spain), supplemented with 10% FCS
(BIOSTAR, Alcorco´n, Spain), 1% L-glutamine (BIOSTAR,
Alcorco´n, Spain), and 300 µg/mL geneticine (Gibco BRL,
Paisley, U.K.).
Hu m a n 5-HT2A Recep tor Bin d in g Assa ys. Prior to
harvesting, stably transfected cells were grown for 24 h in
DMEM F-12 medium supplemented with 10% dialyzed FCS
(Gibco BRL, Paisley, U.K.) to prevent regulatory effects of
5-HT, which is present in regular FCS. The culture medium
was aspirated, and cells were washed twice with ice-cold
phosphate-buffered saline (PBS), scraped off the culture plate
in PBS, and pelleted by centrifugation at 1000g for 10 min at
4 °C. Cell pellets were homogenized with a Polytron homog-
enizer in 50 mM Tris-HCl, pH 7.5, at 4 °C. The homogenate
was centrifuged at 24 000g for 30 min at 4 °C, and the
resulting pellet was resuspended in assay buffer (50 mM Tris-
HCl, pH 7.5). Competition at [3H]ketanserin binding sites was
assayed in duplicate in assay mixtures containing 750 µL of
membrane homogenate, 50 µL of [3H]ketanserin, 50 µL of
either the buffer or the compound under test, 50 µL of non-
specific masking ligand solution (1 µM methysergide) when
required, and buffer to a final volume of 1 mL. Mixtures were
incubated for 30 min at 37 °C. The assay was terminated by
rapid filtration through Whatman GF/C filter strips (presoaked
in 3% polyethylenimine) in a Brandel cell harvester (Gaith-
ersburg, MD) followed by washing with ice-cold Tris-HCl buffer
(pH 6.6) to remove unbound radioligand. The radioactivity
retained on filters was determined 3 h later, by liquid
scintillation counting in a beta counter (Beckman, LS-6000LL).
IC50 and Ki values were calculated as for rat 5-HT2A binding
sites.
Hu m a n 5-HT2C Recep tor Bin d in g Assa ys. Prior to
harvesting, stably transfected cells were grown for 24 h in
DMEM medium supplemented with 10% dialyzed FCS (Gibco
BRL). Cell membranes were obtained as for the human 5-HT2A
binding assay. Competition at [3H]mesulergine binding sites
was determined by a protocol analogous to that described
above for the bovine 5-HT2C binding assay.
Dr u gs. 5-Hydroxytryptamine‚HCl, mianserine, and methy-
sergide and all other drugs and chemicals were reagent grade
products from Sigma-R. B. I. (Alcobendas, Spain). Aqueous
solutions of all drugs as their hydrochlorides were prepared
daily using distilled water. All drug concentrations mentioned
above are final molar concentrations. [3H]Ketanserin (60.08
Ci/mmol) and [3H]mesulergine (76 Ci/mmol) were obtained
from DuPont NEN (Boston, MA) and Amersham (England),
respectively.
Molecu la r Mod elin g. GRID-GOLP E. The ligands were
built in the most extended conformations using standard bond
distances and angles as defined in SYBYL 6.5.106 The ligand
geometries were then optimized by energy minimization using
the parameter set included in the MOPAC (version 6.0)107 suite
of programs, with the PM3 Hamiltonian. The molecular
alignment was carried out with low energy conformers, fol-
lowing a protocol described in a previous publication of some
of the present authors64 and taking into account the pharma-
cophoric model developed by Ho¨ltje for 5-HT2A antagonists.108
The centroid(s) of the aromatic system(s), the oxygen atoms
The resulting probe-target interaction energies for each
compound were unfolded to produce one-dimensional vector
variables for each compound, which were assembled in the so-
called X matrix. This matrix was pretreated by first using a
cutoff of 5 kcal/mol to produce a more symmetrical distribution
of energy values and then zeroing small variable values and
removing variables with small standard deviation, using
appropriate cutoffs. In addition, variables taking only two,
three, or four values and presenting a skewed distribution
were also removed. The SRD (number of seeds ) 1500, critical
distance ) 1.0 Å, collapsing cutoff ) 2.0 Å) was applied
followed by three consecutive FFD variables selection runs.
PLS cross-validated analyses were performed with the leave-
one-out procedure. In all cases, the optimal number of com-
ponent (ONC) was chosen by monitoring the changes in the
fitting (r2) and predictive (q2) ability of the model upon addition
of a new latent variable. Because in many cases models with
the highest q2 value had a relatively large number of compo-
nents, the associated risk of overfitting was avoided by
selecting, from a plot of q2 vs the number of components, the
ONC as the one at which the curve starts to flatten.
Dock in g. Models of the transmembrane R-helices bundle
of both receptor subtypes (5-HT2A and 5-HT2C) were built
following a procedure analogous to others previously de-
scribed.109,110 First, on the basis of the transmembrane se-
quences taken from the Swissprot database,111 each helix was
separately built with the Biopolymer module of Insight II112
using constant values of angles φ (-44°) and ι (-59°). Then,
each helix was submitted to a molecular mechanics geometrical
optimization using the AMBER 4.1 program.113 Once opti-
mized, the seven helices were packed in agreement with the
electron density maps of bovine rhodopsin.59 To place the
helices in their appropriate relative rotational position, we
considered their hydrophobicity profiles as well as experimen-
tal information on the relative position of particular residues,
for example, the known hydrogen bond between an Asp in
TMH2 and an Asn in TMH7.60 Then, the geometry of the
resulting transmembrane bundle was geometrically optimized
using again the AMBER 4.1 program.
Ack n ow led gm en t. We are grateful to Dr. W. P.
Clarke and Dr. K. Berg for kindly providing CHO-FA4
cells and to Manuel Pastor for helpful discussions. The
Spanish authors gratefully acknowledge the support by
CICYT (SAF94-0593-C04, SAF95-1081, SAF96-0336,
and SAF1998-0148-C04), Xunta de Galicia (XUGA
20319B97 and PGIDT00PXI20310PR), and CESCA
grants. Y.C. was aided with a predoctoral grant from
Ministerio de Educacio´n y Cultura. The Italian authors
gratefully acknowledge the support by CNR and MURST
(Rome).
Refer en ces
(1) Martin, G. R.; Humphrey, P. P. Receptors for 5-Hydroxy-
tryptamine: Current Perspectives on Classification and Nomen-
clature. Neuropharmacology 1994, 33, 261-273.
(2) Roth, B. L.; Craigo, S. C.; Choudhary, M. S.; Uluer, A.; Monsma-
FJ , J .; Shen, Y.; Meltzer, H. Y.; Sibley, D. R. Binding of Typical
and Atypical Antipsychotic Agents to 5-Hydroxytryptamine-6
and 5-Hydroxytryptamine-7 Receptors. J . Pharmacol. Exp. Ther.
1994, 268, 1403-1410.

New Serotonin Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 69
(3) Hoyer, D.; Clarke, D. E.; Fozard, J . R.; Hartig, P. R.; Martin, G.
R.; Mylecharane, E. J .; Saxena, P. R.; Humphrey, P. P. Inter-
national Union of Pharmacology Classification of Receptors for
5-Hydroxytryptamine (Serotonin). Pharmacol. Rev. 1994, 46,
157-203.
(4) Uphouse, L. Multiple Serotonin Receptors: too Many, not
Enough, or J ust the Right Number? Neurosci. Biobehav. Rev.
1997, 21, 679-698.
(23) Kursar, J . D.; Nelson, D. L.; Wainscott, D. B.; Cohen, M. L.; Baez,
M. Molecular Cloning, Functional Expression, and Pharmaco-
logical Characterization of a Novel Serotonin Receptor (5-
Hydroxytryptamine2F) from Rat Stomach Fundus. Mol. Phar-
macol. 1992, 42, 549-557.
(24) Cohen, M. L.; Fludzinski, L. A. Contractile Serotonergic Receptor
in Rat Stomach Fundus. J . Pharmacol. Exp. Ther. 1987, 243,
264-269.
(25) Foguet, M.; Hoyer, D.; Pardo, L. A.; Parekh, A.; Kluxen, F. W.;
Kalkman, H. O.; Stuhmer, W.; Lubbert, H. Cloning and Func-
tional Characterization of the Rat Stomach Fundus Serotonin
Receptor. EMBO J . 1992, 11, 3481-3487.
(26) Baxter, G.; Kennett, G.; Blaney, F.; Blackburn, T. 5-HT2 Receptor
Subtypes: a Family Re-United? Trends Pharmacol. Sci. 1995,
16, 105-110.
(5) Cowen, P. J . Serotonin Receptor Subtypes: Implications for
Psychopharmacology. Br. J . Psychiatry Suppl. 1991, 7-14.
(6) Roth, B. L.; Willins, D. L.; Kristiansen, K.; Kroeze, W. K.
5-Hydroxytryptamine2-Family Receptors (5-Hydroxytryptamine2A
,
5-Hydroxytryptamine2B, 5-Hydroxytryptamine2C): Where Struc-
ture Meets Function. Pharmacol. Ther. 1998, 79, 231-257.
(7) Martin, G. R. 5-Hydroxytryptamine Receptors. The IUPHAR
Compendium of Receptor Characterization and Classification;
1998; pp 167-185.
(8) Felder, C. C.; Kanterman, R. Y.; Ma, A. L.; Axelrod, J . Serotonin
Stimulates Phospholipase A2 and the Release of Arachidonic
Acid in Hippocampal Neurons by a Type 2 Serotonin Receptor
that is Independent of Inositolphospholipid Hydrolysis. Proc.
Natl. Acad. Sci. U.S.A. 1990, 87, 2187-2191.
(9) Thellung, S.; Barzizza, A.; Maura, G.; Raiteri, M. Serotonergic
Inhibition of The Mossy Fibre-Granule Cell Glutamate Trans-
mission in Rat Cerebellar Slices. Naunyn-Schmiedebergs Arch.
Pharmacol. 1993, 348, 347-351.
(10) Loric, S.; Maroteaux, L.; Kellermann, O.; Launay, J . M. Func-
tional Serotonin-2B Receptors are Expressed by a Teratocarci-
noma-Derived Cell Line During Serotoninergic Differentiation.
Mol. Pharmacol. 1995, 47, 458-466.
(11) Tournois, C.; Mutel, V.; Manivet, P.; Launay, J . M.; Kellermann,
O. Cross-Talk Between 5-Hydroxytryptamine Receptors in a
Serotonergic Cell Line. Involvement of Arachidonic Acid Me-
tabolism. J . Biol. Chem. 1998, 273, 17498-17503.
(12) Manivet, P.; Mouillet, R. S.; Callebert, J .; Nebigil, C. G.;
Maroteaux, L.; Hosoda, S.; Kellermann, O.; Launay, J . M. PDZ-
Dependent Activation of Nitric-Oxide Synthases by the Serotonin
2B Receptor. J . Biol. Chem. 2000, 275, 9324-9331.
(13) Kaufman, M. J .; Hartig, P. R.; Hoffman, B. J . Serotonin 5-HT2C
Receptor Stimulates Cyclic GMP Formation in Choroid Plexus.
J . Neurochem. 1995, 64, 199-205.
(14) Pazos, A.; Cortes, R.; Palacios, J . M. Quantitative Autoradio-
graphic Mapping of Serotonin Receptors in the Rat Brain. II.
Serotonin-2 Receptors. Brain Res. 1985, 346, 231-249.
(15) Roth, B. L.; McLean, S.; Zhu, X. Z.; Chuang, D. M. Characteriza-
tion of Two [3H]Ketanserin Recognition Sites in Rat Striatum.
J . Neurochem. 1987, 49, 1833-1838.
(16) Willins, D. L.; Deutch, A. Y.; Roth, B. L. Serotonin 5-HT2A
Receptors are Expressed on Pyramidal Cells and Interneurons
in the Rat Cortex. Synapse 1997, 27, 79-82.
(17) Maayani, S.; Wilkinson, C. W.; Stollak, J . S. 5-Hydroxy-
tryptamine Receptor in Rabbit Aorta: Characterization by
Butyrophenone Analogues. J . Pharmacol. Exp. Ther. 1984, 229,
346-350.
(18) Ullmer, C.; Schmuck, K.; Kalkman, H. O.; Lubbert, H. Expres-
sion of Serotonin Receptor mRNAs in Blood Vessels. FEBS Lett.
1995, 370, 215-221.
(19) Pazos, A.; Hoyer, D.; Palacios, J . M. The Binding of Serotonergic
Ligands to the Porcine Choroid Plexus: Characterization of a
New Type of Serotonin Recognition Site. Eur. J . Pharmacol.
1984, 106, 539-546.
(20) Pazos, A.; Probst, A.; Palacios, J . M. Serotonin Receptors in the
Human Brain-III. Autoradiographic Mapping of Serotonin-1
Receptors. Neuroscience 1987, 21, 97-122.
(21) (a) Molineaux, S. M.; J essell, T. M.; Axel, R.; J ulius, D. 5-HT1C
Receptor is a Prominent Serotonin Receptor Subtype in the
Central Nervous System. Proc. Natl. Acad. Sci. U.S.A. 1989, 86,
6793-6797. (b) Pompeiano, M.; Palacios, J . M.; Mengod, G.
Distribution of the serotonin 5-HT2 receptor family mRNAs:
comparison between 5-HT2A and 5-HT2C receptors. Brain Res.
Mol. Brain Res. 1994, 23, 163-178.
(22) (a) Bradley, P. B.; Engel, G.; Feniuk, W.; Fozard, J . R.; Hum-
phrey, P. P.; Middlemiss, D. N.; Mylecharane, E. J .; Richardson,
B. P.; Saxena, P. R. Proposals for the Classification and
Nomenclature of Functional Receptors for 5-Hydroxytryptamine.
Neuropharmacology 1986, 25, 563-576. (b) Mengod, G.; Pom-
peiano, M.; Martinez-Mir, M. I.; Palacios, J . M. Localization of
the mRNA for the 5-HT2 receptor by in situ hybridization
histochemistry. Correlation with the distribution of receptor
sites. Brain Res. 1990, 524, 139-143. (c) Lopez-Gimenez, J . F.;
Mengod, G.; Palacios, J . M.; Vilaro, M. T. Selective visualization
of rat brain 5-HT2A receptors by autoradiography with [3H]MDL
100, 907. Naunyn-Schmiedebergs Arch. Pharmacol. 1997, 356,
446-454.
(27) Schmuck, K.; Ullmer, C.; Engels, P.; Lubbert, H. Cloning and
Functional Characterization of the Human 5-HT2B Serotonin
Receptor. FEBS Lett. 1994, 342, 85-90.
(28) Bonhaus, D. W.; Bach, C.; DeSouza, A.; Salazar, F. H.; Matsuoka,
B. D.; Zuppan, P.; Chan, H. W.; Eglen, R. M. The Pharmacology
and Distribution of Human 5-Hydroxytryptamine2B (5-HT2B
)
Receptor Gene Products: Comparison with 5-HT2A and 5-HT2C
Receptors. Br. J . Pharmacol. 1995, 115, 622-628.
(29) Kursar, J . D.; Nelson, D. L.; Wainscott, D. B.; Baez, M. Molecular
Cloning, Functional Expression, and mRNA Tissue Distribution
of the Human 5-Hydroxytryptamine2B Receptor. Mol. Pharmacol.
1994, 46, 227-234.
(30) Duxon, M. S.; Flanigan, T. P.; Reavley, A. C.; Baxter, G. S.;
Blackburn, T. P.; Fone, K. C. Evidence for Expression of the
5-Hydroxytryptamine-2B Receptor Protein in the Rat Central
Nervous System. Neuroscience 1997, 76, 323-329.
(31) Meltzer, H. Y. An Overview of the Mechanism of Action of
Clozapine. J . Clin. Psychiatry 1994, 55 (Suppl. B), 47-52.
(32) Wainscott, D. B.; Lucaites, V. L.; Kursar, J . D.; Baez, M.; Nelson,
D. L. Pharmacologic Characterization of the Human 5-Hydroxy-
tryptamine2B Receptor: Evidence for Species Differences. J .
Pharmacol. Exp. Ther. 1996, 276, 720-727.
(33) Barker, E. L.; Westphal, R. S.; Schmidt, D.; Sanders, B. E.
Constitutively Active 5-Hydroxytryptamine2C Receptors Reveal
Novel Inverse Agonist Activity of Receptor Ligands. J . Biol.
Chem. 1994, 269, 11687-11690.
(34) Ward, R. P.; Hamblin, M. W.; Lachowicz, J . E.; Hoffman, B. J .;
Sibley, D. R.; Dorsa, D. M. Localization of Serotonin Subtype 6
Receptor Messenger RNA In The Rat Brain by In Situ Hybrid-
ization Histochemistry. Neuroscience 1995, 64, 1105-1111.
(35) Monsma-FJ , J .; Shen, Y.; Ward, R. P.; Hamblin, M. W.; Sibley,
D. R. Cloning and Expression of a Novel Serotonin Receptor with
High Affinity for Tricyclic Psychotropic Drugs. Mol. Pharmacol.
1993, 43, 320-327.
(36) Blake, T. J .; Tillery, C. E.; Reynolds, G. P. Antipsychotic Drug
Affinities at alpha2-Adrenoceptor Subtypes in Post-Mortem
Human Brain. J . Psychopharmacol. (London) 1998, 12, 151-
154.
(37) Bymaster, F. P.; Nelson, D. L.; DeLapp, N. W.; Falcone, J . F.;
Eckols, K.; Truex, L. L.; Foreman, M. M.; Lucaites, V. L.;
Calligaro, D. O. Antagonism by Olanzapine of Dopamine D1,
Serotonin(2), Muscarinic, Histamine H-1 and alpha(1)-Adrenergic
Receptors In Vitro. Schizophr. Res. 1999, 37, 107-122.
(38) Michal, P.; Lys´ıkova, M.; El-Fakahany, E. E.; Tucek, S. Clozapine
interaction with the M2 and M4 subtypes of Muscarinic Recep-
tors. Eur. J . Pharmacol. 1999, 376, 119-125.
(39) Seeman, P.; Corbett, R.; Van-Tol, H. H. Atypical Neuroleptics
have Low Affinity for Dopamine D2 Receptors or are Selective
for D4 Receptors. Neuropsychopharmacology 1997, 16, 93-110.
(40) Creese, I.; Burt, D. R.; Snyder, S. H. Dopamine Receptor Binding
Predicts Clinical and Pharmacological Potencies of Antischizo-
phrenic Drugs. Science 1976, 192, 481-483.
(41) Seeman, P.; Van-Tol, H. H. Dopamine Receptor Pharmacology.
Trends Pharmacol. Sci. 1994, 15, 264-270.
(42) Horacek, J . Novel Antipsychotics and Extrapyramidal Side
Effects. Theory and Reality. Pharmacopsychiatry 2000, 33
(Suppl.), 134-142.
(43) Aghajanian, G. K.; Marek, G. J . Serotonin Model of Schizophre-
nia: Emerging Role of Glutamate Mechanisms. Brain Res. Rev.
2000, 31, 302-312.
(44) Green, M. F.; Marshall-BD, J .; Wirshing, W. C.; Ames, D.;
Marder, S. R.; McGurk, S.; Kern, R. S.; Mintz, J . Does Risperi-
done Improve Verbal Working Memory in Treatment-Resistant
Schizophrenia? Am. J . Psychiatry 1997, 154, 799-804.
(45) Sipes, T. E.; Geyer, M. A. DOI Disrupts Prepulse Inhibition of
Startle in Rats Via 5-HT2A Receptors in the Ventral Pallidum.
Brain Res. 1997, 761, 97-104.
(46) Gleason, S. D.; Shannon, H. E. Blockade of Phencyclidine-
Induced Hyperlocomotion by Olanzapine, Clozapine and Sero-
tonin Receptor Subtype Selective Antagonists in Mice. Psycho-
pharmacology 1997, 129, 79-84.

70 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Brea et al.
(47) Okuyama, S.; Chaki, S.; Kawashima, N.; Suzuki, Y.; Ogawa, S.;
Kumagai, T.; Nakazato, A.; Nagamine, M.; Kamaguchi, K.;
Tomisawa, K. The Atypical Antipsychotic Profile of NRA0045,
a Novel Dopamine D4 and 5-Hydroxytryptamine2A Receptor
Antagonist, in Rats. Br. J . Pharmacol. 1997, 121, 515-525.
(48) Ereshefsky, L.; Riesenman, C.; True, J . E.; J avors, M. Serotonin-
Mediated Increase in Cytosolic [Ca++] in Platelets of Risperi-
done-Treated Schizophrenia Patients. Psychopharmacol. Bull.
1996, 32, 101-106.
(49) Schmidt, C. J . Development of a Selective 5-HT2A Receptor
Antagonist for the Treatment of Schizophrenia. Presented at
IBS’s International Conference on Serotonin Receptors. Targets
for New Therapeutic Agents, 1996.
(66) Forbes, I. T.; Dabbs, S.; Duckworth, D. M.; Ham, P.; J ones, G.
E.; King, F. D.; Saunders, D. V.; Blaney, F. E.; Naylor, C. B.;
Baxter, G. S.; Blackburn, T. P.; Kennett, G. A.; Wood, M. D.
Synthesis, Biological Activity, and Molecular Modeling of Selec-
tive 5-HT(2C/2B) Receptor Antagonists. J . Med. Chem. 1996, 39,
4966-4977.
(67) Kennett, G. A.; Wood, M. D.; Bright, F.; Cilia, J .; Piper, D. C.;
Gager, T.; Thomas, D.; Baxter, G. S.; Forbes, I. T.; Ham, P.;
Blackburn, T. P. In Vitro And In Vivo Profile of SB 206553, a
Potent 5-HT2C/5-HT2B Receptor Antagonist with Anxiolytic-Like
Properties. Br. J . Pharmacol. 1996, 117, 427-434.
(68) Bromidge, S. M.; Dabbs, S.; Davies, D. T.; Duckworth, D. M.;
Forbes, I. T.; Ham, P.; J ones, G. E.; King, F. D.; Saunders, D.
V.; Starr, S.; Thewlis, K. M.; Wyman, P. A.; Blaney, F. E.; Naylor,
C. B.; Bailey, F.; Blackburn, T. P.; Holland, V.; Kennett, G. A.;
Riley, G. J .; Wood, M. D. Novel and selective 5-HT2C/2B receptor
antagonists as potential anxiolytic agents: Synthesis, quantita-
tive structure-activity relationships, and molecular modeling
of substituted 1-(3-pyridylcarbamoyl)indolines. J . Med. Chem.
1998, 41, 1598-1612.
(69) Bromidge, S. M.; Dabbs, S.; Davies, D. T.; Davies, S.; Duckworth,
D. M.; Forbes, I. T.; Gaster, L. M.; Ham, P.; J ones, G. E.; King,
F. D.; Mulholland, K. R.; Saunders, D. V.; Wyman, P. A.; Blaney,
F. E.; Clarke, S. E.; Blackburn, T. P.; Holland, V.; Kennett, G.
A.; Lightowler, S.; Middlemiss, D. N.; Trail, B.; Riley, G. J .;
Wood, M. D. Biarylcarbamoylindolines are Novel and Selective
5-HT2C Receptor Inverse Agonists: Identification of 5-Methyl-
1-[[2-[(2-Methyl-3-Pyridyl)Oxy]-5-Pyridyl]Carbamoyl]-6-Trifluo-
romethylindoline (SB-243213) as a Potential Antidepressant/
Anxiolytic Agent. J . Med. Chem. 2000, 43, 1123-1134.
(70) Weinhardt, K. K.; Bonhaus, D. W.; De Souza, A. Some Benzene-
sulfonamido-Substituted Valerophenones that are Selective
Antagonists for the 5-HT2C Receptor. Bioorg. Med. Chem. Lett.
1996, 6, 2692.
(71) Bonhaus, D. W.; Flippin, L. A.; Greenhouse, R. J .; J aime, S.;
Rocha, C.; Dawson, M.; Van Natta, K.; Chang, L. K.; Pulido-
Rios, T.; Webber, A.; Leung, E.; Eglen, R. M.; Martin, G. R. RS-
127445: a Selective, High Affinity, Orally Bioavailable 5-HT2B
Receptor Antagonist. Br. J . Pharmacol. 1999, 127, 1075-1082.
(72) Nozulak, J .; Kalkman, H. O.; Floersheim, P.; Hoyer, D.; Schoeff-
ter, P.; Buerki, H. R. (+)-cis-4,5,7a,8,9,10,11,11a-octahydro-7H-
10-methylindolo[1,7-bc] [2,6]naphthyridine: a 5-HT2C/2B Re-
ceptor Antagonist with Low 5-HT2A Receptor Affinity. J . Med.
Chem. 1995, 38, 28-33.
(73) Bo¨s, M.; Stadler, H.; Wichmann, J .; J enck, F.; Martin, J . R.;
Moreau, J . L.; Sleight, A. J . Synthesis of O-methylasparvenone-
derived Serotonin-Receptor Antagonists. Helv. Chim. Acta 1998,
91, 525-538.
(74) Claudi, F.; Scoccia, L.; Giorgioni, G.; Accorroni, B.; Marucci, G.;
Gessi, S.; Siniscalchi, A.; Borea, P. A. 4-(4-fluorobenzoyl)-1-[2-
(4-iodo-2,5-dimethoxyphenyl)ethyl]piperidine and its Deriva-
tives: Synthesis and Affinity at 5-HT2A, 5-HT2B and 5-HT2C
Serotonin Receptors. Eur. J . Med. Chem. 1999, 34, 843-852.
(75) Forbes, I. T.; Ham, P.; Booth, D. H.; Martin, R. T.; Thompson,
M.; Baxter, G. S.; Blackburn, T. P.; Glen, A.; Kennett, G. A.;
Wood, M. D. 5-Methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahy-
dropyrrolo[2,3-f]indole: a Novel 5-HT2C/5-HT2B Receptor An-
tagonist with Improved Affinity, Selectivity, and Oral Activity.
J . Med. Chem. 1995, 38, 2524-2530.
(76) Cortizo, L.; Santana, L.; Ravin˜a, E.; Orallo, F.; Fontenla, J . A.;
Castro, E.; Calleja, J . M.; de Ceballos, M. L. Synthesis and
Antidopaminergic Activity of Some 3-(aminomethyl)tetralones
as Analogues of Butyrophenone. J . Med. Chem. 1991, 34, 2242-
2247.
(50) Padich, R. A.; McCloskey, T. C.; Kehne, J . H. 5-HT Modulation
of Auditory and Visual Sensorimotor Gating: II. Effects of the
5-HT2A Antagonist MDL 100, 907 on Disruption of Sound and
Light Prepulse Inhibition Produced by 5-HT Agonists in Wistar
Rats. Psychopharmacology 1996, 124, 107-116.
(51) Martin, P.; Waters, N.; Carlsson, A.; Carlsson, M. L. The
Apparent Antipsychotic Action of the 5-HT2A Receptor Antago-
nist M100907 in a Mouse Model of Schizophrenia is Counter-
acted by Ritanserin. J . Neural Transm. 1997, 104, 561-564.
(52) Herrick-Davis, K.; Grinde, E.; Teitler, M. Inverse agonist activity
of atypical antipsychotic drugs at human 5-hydroxytryptamine2C
receptors. J . Pharmacol. Exp. Ther. 2000, 295, 226-232.
(53) Meltzer, H. Y.; McGurk, S. R. The Effects of Clozapine, Risperi-
done, and Olanzapine on Cognitive Function in Schizophrenia.
Schizophr. Bull. 1999, 25, 233-255.
(54) Reavill, C.; Kettle, A.; Holland, V.; Riley, G.; Blackburn, T. P.
Attenuation of Haloperidol-Induced Catalepsy by
a 5-HT2C
Receptor Antagonist. Br. J . Pharmacol. 1999, 126, 572-574.
(55) Knable, M. B.; Weinberger, D. R. Dopamine, the Prefrontal
Cortex and Schizophrenia. J . Psychopharmacol. 1997, 11, 123-
131.
(56) Cussac, D.; Newman, T. A.; Nicolas, J . P.; Boutin, J . A.; Millan,
M. J . Antagonist Properties of the Novel Antipsychotic, S16924,
at Cloned, Human Serotonin 5-HT2C Receptors:
a Parallel
Phosphatidylinositol and Calcium Accumulation Comparison
with Clozapine and Haloperidol. Naunyn-Schmiedebergs Arch.
Pharmacol. 2000, 361, 549-554.
(57) Palczewski, K.; Kumasaka, T.; Hori, T.; Behnke, C. A.; Mo-
toshima, H.; Fox, B. A.; Le Trong, I.; Teller, D. C.; Okada, T.;
Stenkamp, R. E.; Yamamoto, M.; Miyano, M. Crystal Structure
of Rhodopsin: a G Protein-Coupled Receptor. Science 2000, 289,
739-745.
(58) Wang, C. D.; Gallaher, T. K.; Shih, J . C. Site-Directed Mutagen-
esis of the Serotonin 5-Hydroxytryptamine2 Receptor: Identifi-
cation of Amino Acids Necessary for Ligand Binding and
Receptor Activation. Mol. Pharmacol. 1993, 43, 931-940.
(59) Baldwin, J . M.; Schertler, G. F.; Unger, V. M. An Alpha-carbon
Template for the transmembrane Helices in the Rhodopsin
Family of G-Protein-Coupled Receptors. J . Mol. Biol. 1997, 272,
144-164.
(60) Sealfon, S. C.; Chi, L.; Ebersole, B. J .; Rodic, V.; Zhang, D.;
Ballesteros, J . A.; Weinstein, H. Related Contribution of Specific
Helix 2 And 7 Residues to Conformational Activation of the
Serotonin 5-HT2A Receptor. J . Biol. Chem. 1995, 270, 16683-
16688.
(61) Almaula, N.; Ebersole, B. J .; Zhang, D.; Weinstein, H.; Sealfon,
S. C. Mapping The Binding Site Pocket of the Serotonin
5-Hydroxytryptamine2A Receptor. Ser3.36(159) Provides a Sec-
ond Interaction Site for the Protonated Amine of Serotonin but
not of Lysergic Acid Diethylamide or Bufotenin. J . Biol. Chem.
1996, 271, 14672-14675.
(62) Kristiansen, K.; Dahl, S. G.; Edvardsen, Ø. A Database of
Mutants and Effects of Site-Directed Mutagenesis Experiments
on G-Protein Coupled Receptors. Proteins: Struct., Funct., Genet.
1996, 26, 81-94.
(63) Kristiansen, K.; Kroeze, W. K.; Willins, D. L.; Gelber, E. I.;
Savage, J . E.; Glennon, R. A.; Roth, B. L. A Highly Conserved
Aspartic Acid (Asp-155) Anchors the Terminal Amine Moiety of
Tryptamines and Is Involved in Membrane Targeting of the
5-HT2A Serotonin Receptor but does not Participate in Activation
via a “Salt-Bridge Disruption” Mechanism. J . Pharmacol. Exp.
Ther. 2000, 293, 735-746.
(64) Ravin˜a, E.; Negreira, J .; Cid, J .; Masaguer, C. F.; Rosa, E.; Rivas,
M. E.; Fontenla, J . A.; Loza, M. I.; Tristan, H.; Cadavid, M. I.;
Sanz, F.; Lozoya, E.; Carotti, A.; Carrieri, A.; Conformationally
Constrained Butyrophenones with Mixed Dopaminergic (D2) and
Serotoninergic (5-HT2A, 5-HT2C) Affinities: Synthesis, Phar-
macology, 3D-QSAR, and Molecular Modeling of (Aminoalkyl)-
benzo- and -thienocycloalkanones as Putative Atypical Anti-
psychotics. J . Med. Chem. 1999, 42, 2774-2797.
(65) Konvicka, K.; Guarnieri, F.; Ballesteros, J . A.; Weinstein, H. A
proposed Structure for Transmembrane Segment 7 of G Protein
Coupled Receptors Incorporating an Asn-Pro/Asp-Pro Motif.
Biophys. J . 1998, 75, 601-611.
(77) Fontenla, J . A.; Osuna, J .; Rosa, E.; Castro, M. E.; Ferreiro, T.;
Loza, M. I.; Calleja, J . M.; Sanz, F.; Rodriguez, J .; Ravin˜a, E.;
Fueyo, J .; Masaguer, C. F.; Vidal, A.; de Ceballos, M. L.
Synthesis and Atypical Antipsychotic Profile of some 2-(2-
piperidinoethyl)benzocycloalkanones as Analogues of Butyrophe-
none. J . Med. Chem. 1994, 37, 2564-2573.
(78) Loza, M. I.; Verde, I.; Castro, M. E.; Orallo, F.; Fontenla, J . A.;
Calleja, J . M.; Ravin˜a, E.; Cortizo, L.; de Ceballos, M. L. 5-HT2
Antagonist Activity of 2-Aminomethyltetralones. Bioorg. Med.
Chem. Lett. 1991, 1, 717-720.
(79) Loza, M. I.; Ferreiro, T. G.; Sanz, F.; Lozoya, E.; Rodriguez, J .;
Manaut, F.; Verde, I.; Castro, E.; Fontenla, J . A.; Cadavid, I.;
Honrubia, A.; Fueyo, J .; Ravin˜a, E. Antiserotoninergic Activity
of 2-aminoethylbenzocyclanones in Rat Aorta: Structure-Activ-
ity Relationships. J . Pharm. Sci. 1993, 82, 513-517.
(80) Ravin˜a, E.; Fueyo, J .; Masaguer, C. F.; Negreira, J .; Cid, J .; Loza,
I.; Honrubia, A.; Tristan, H.; Ferreiro, T.; Fontenla, J . A.; Rosa,
E.; Calleja, J . M.; de Ceballos, M. L. Synthesis And Affinities
For Dopamine (D2) and 5-Hydroxytryptamine (5-HT2A) Receptors
of 1-(benzoylpropyl)-4-(1-oxocycloalkyl-2-ethyl)-piperazines as
Cyclic Butyrophenone Derivatives. Chem. Pharm. Bull. 1996,
44, 534-541.

New Serotonin Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 71
(81) Masaguer, C. F.; Ravin˜a, E.; Fontenla, J . A.; Brea, J .; Tristan,
H.; Loza, M. I. Butyrophenone Analogues in the Carbazole Series
as Potential Atypical Antipsychotics: Synthesis and Determi-
nation of Affinities at D2, 5-HT2A, 5-HT2B and 5-HT2C Receptors.
Eur. J . Med. Chem. 2000, 35, 83-95.
(82) Masaguer, C. F.; Casariego, I.; Ravin˜a, E. Conformationally
Restricted Butyrophenones with Mixed Dopaminergic (D2) and
Serotoninergic (5-HT2A) affinities. Synthesis of 5-aminoethyl- and
6-aminomethyl-4-oxotetrahydroindoles as Potential Atypical An-
tipsychotics. Chem. Pharm. Bull. 1999, 47, 621-632.
(83) Masaguer, C. F.; Formoso, E.; Ravin˜a, E.; Tristan, H.; Loza, M.
I.; Rivas, E.; Fontenla, J . A. Butyrophenone Analogues in the
Carbazole Series: Synthesis and Determination of Affinities at
D2 and 5-HT2A Receptors. Bioorg. Med. Chem. Lett. 1998, 8,
3571-3576.
(84) Ravina, E.; Casariego, I.; Masaguer, C. F.; Fontenla, J . A.;
Montenegro, G. Y.; Rivas, M. E.; Loza, M. I.; Enguix, M. J .;
Villazon, M.; Cadavid, M. I.; Demontis, G. C. Conformationally
Constrained Butyrophenones with Affinity for Dopamine (D1,
D2, D4) and Serotonin (5-HT2A, 5-HT2B, 5-HT2C) Receptors.
Synthesis of aminomethylbenzoy[b]furanones, and their Evalu-
tion as Antipsychotics. J . Med. Chem. 2000, 43, 4678-4693.
(85) J ulia, S.; Bonnet, Y. Etude des ce´tones avec noyau cyclopropane
I. Les benzo- et naphtho- bicyclo- (0,1,4) hepte´nones. B. Soc.
Chim. Fr. 1957, 1340-1347.
(86) Cramer, R. D.; Patterson, D.; Bunce, J . D. Comparative Molec-
ular Field Analysis (COMFA). 1. Effects of Shape on Binding of
Steroids to Carrier Protein. J . Am. Chem. Soc. 1988, 110, 5959-
5967.
(87) Baroni, M.; Constantino, G.; Cruciani, G.; Valigi, R.; Clementi,
S. Generating Optimal Linear PLS Estimation (GOLPE): An
Advanced Chemometric Tool for Handling 3D QSAR Problems.
Quant. Struct.-Act. Relat. 1993, 12, 9-20.
(88) Dunn, W. J ., III; Wold, S.; Edlund, U.; Hellberg, S.; Gasteiger,
J . Multivariate Structure-Activity Relationships Between Data
from a Battery of Biological Tests and an Ensemble of Chemical
Descriptors: The PLS Methodol. Quant. Struct.-Act. Relat. 1984,
3, 131-137.
(89) Goodford, P. J . A Computational Procedure for Determining
Energetically Favorable Binding Sites on Biologically Important
Macromolecules. J . Med. Chem. 1985, 28, 849-857.
(95) Wang, C. D.; Gallaher, T. K.; Shih, J . C. Site-Directed Mutagen-
esis of the Serotonin 5-Hydroxytryptamine2 Receptor: Identi-
fication of Amino Acids Necessary for Ligand Binding and
Receptor Activation. Mol. Pharmacol. 1993, 43, 931-940.
(96) Almaula, N.; Ebersole, B. J .; Ballesteros, J . A.; Weinstein, H.;
Sealfon, S. C. Contribution of a Helix 5 Locus to Selectivity of
Hallucinogenic and Nonhallucinogenic Ligands for the Human
5-Hydroxytryptamine2A and 5-Hydroxytryptamine2C Receptors:
Direct and Indirect Effects on Ligand Affinity Mediated by the
Same Locus. Mol. Pharmacol. 1996, 50, 34-42.
(97) Vogel, A. I. Vogel’s Textbook of Practical Organic Chemistry, 5th
ed.; Longman: Harlow, U.K., 1989.
(98) Cheng, Y.; Prusoff, W. H. Relationship Between the Inhibition
Constant (K1) and the Concentration of Inhibitor which Causes
50 Per Cent Inhibition (I50) of an Enzymatic Reaction. Biochem.
Pharmacol. 1973, 22, 3099-3108.
(99) Hoyer, D.; Martin, G. R. Classification and Nomenclature of
5-HT Receptors: A Comment on Current Issues. Behav. Brain
Res. 1996, 73, 263-268.
(100) Gruetter, C. A.; Lemke, S. M.; Anestis, D. K.; Szarek, J . L.;
Valentovic, M. A. Potentiation of 5-Hydroxytryptamine-Induced
Contraction in Rat Aorta by Chlorpheniramine, Citalopram and
Fluoxetine. Eur. J . Pharmacol. 1992, 217, 109-118.
(101) Fukuda, S.; Su, C.; Lee, T. J . Mechanisms of Extraneuronal
Serotonin Uptake in the Rat Aorta. J . Pharmacol. Exp. Ther.
1986, 239, 264-269.
(102) Cohen, M. L.; Fuller, R. W.; Wiley, K. S. Evidence for 5-HT2
Receptors Mediating Contraction in Vascular Smooth Muscle.
J . Pharmacol. Exp. Ther. 1981, 218, 421-425.
(103) Van Rossum, J . M. Cumulative dose-response curves II. Tech-
nique for the making of dose-response curves in the isolated
organs and the evaluation of drug parameters. Arch. Intern. Med.
1963, 143-299.
(104) MacKay, D. How Should Values of pA2 and Affinity Constants
for Pharmacological Competitive Antagonists be Estimated? J .
Pharm. Pharmacol. 1978, 30, 312-313.
(105) Vane, J . R. A sensitive method for the assay of 5-hydroxy-
tryptamine. Br. J . Pharmacol. 1957, 12, 344-349.
(106) SYBYL 6.5. Tripos Association, St. Louis, MO.
(107) Stewart, J . J . P. MOPAC: a semiempirical orbital program. J .
Comput.-Aided Mol. Des. 1990, 4, 1-103.
(108) Holtje, H. D.; Folkers, G. Molecular Modeling: Basic Principles
and Applications; VCH: Weinheim, 1997.
(90) Pastor, M.; Cruciani, G.; Clementi, S. Smart Region Definition:
a New Way to Improve the Predective Ability and Interpret-
ability of Three-Dimensional Quantitative Structure-Activity
Relationships. J . Med. Chem. 1997, 40, 1455-1464.
(91) Gaillard, P.; Carrupt, P. A.; Testa, B.; Boudon, A. Molecular
Lipophilicity Pontential, a Tool in 3D QSAR: Method and
Applications. J . Comput.-Aided Mol. Des. 1994, 8, 83-96.
(92) Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J .
(109) Ballesteros, J . A.; Weinstein, H. Integrated Methods for the
Construction of Three-Dimensional Models and Computacional
Probing of Structure-Function Relations in G Protein Coupled
Receptors. Methods Neurosci. 1995, 25, 366-428.
(110) Carrieri, A.; Centeno, N. B.; Rodrigo, J .; Sanz, F.; Carotti, A.
Theoretical evidence of a salt bridge disruption as the initiating
process for the R1d-adrenergic receptor activation: a molecular
dynamics and docking study. Proteins 2001, 43, 382-394.
(111) Attwood, T. K.; Beck, M. E.; Flower, D. R.; Scordis, P.; Selley, J .
N. The PRINTS Protein Fingerprint Database in its Fifth Year.
Nucleic Acids Res. 1998, 26, 304-308.
M. PROCHECK:
a Program to Check the Stereochemical
Quality of Protein Structures. J . Appl. Crystallogr. 1993, 26,
283-291.
(93) McMartin, C.; Bohacek, R. S. QXP: Powerful Rapid Computer
Algorithms for Structure-Based Drug Design. J . Comput.-Aided
Mol. Des. 1997, 11, 333-344.
(112) Insight II, 98.0. Molecular Simulation Inc.: San Diego, 1998.
(113) Pearlman, D. A.; Case, D. A.; Caldwell, J . W.; Ross, W. S.;
Cheatham, T. E., III; Ferguson, D. M.; Seibel, G. L.; Singh, U.
C.; Weiner, P. K.; Kollman, P. A. AMBER, version 4.1; 1995.
(94) J ohnson, M. P.; Wainscott, D. B.; Lucaites, V. L.; Baez, M.;
Nelson, D. L. Mutations of Transmembrane IV and V Serines
Indicate that all Tryptamines do not Bind to the Rat 5-HT2A
Receptor in the Same Manner. Mol. Brain Res. 1997, 49, 1-6.
J M011014Y