Butyrophenone analogues in the carbazole series as potential atypical antipsychotics: Synthesis and determination of affinities at D2, 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors

Article abstract of DOI:10.1016/S0223-5234(00)00109-4

We describe practical and efficient routes for synthesis of 2- aminomethyl-1,2,3,9-tetrahydro-4H-carbazol-4-ones using the Fischer indole synthesis or palladium-catalysed cyclization methodologies, as well as their affinities for D₂, 5-HT(2A) and 5-HT(2C) receptors, and their activity at the 5-HT(2B) receptor. The most active compounds, 4b (QF 2003B) and 4c (QF 2004B), with a pK(i) (5-HT(2A)/D₂) ratio of 1.28 show a potential antipsychotic profile according to Meltzer's classification. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)00109-4

Eur. J. Med. Chem. 35 (2000) 83−95
© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0223523400001094/FLA
83
Original article
Butyrophenone analogues in the carbazole series as potential atypical
antipsychotics: synthesis and determination of affinities at D2, 5-HT2A, 5-HT2B
and 5-HT2C receptors#
Christian F. Masaguera, Enrique Raviñaa*, José Angel Fontenlab, José Breab, Helena Tristánb,
María Isabel Lozab
aDepartamento de Química Orgánica, Laboratorio de Química Farmacéutica, Facultad de Farmacia. Universidad de Santiago de
Compostela, 15706-Santiago de Compostela, Spain
bDepartamento de Farmacología, Facultad de Farmacia. Universidad de Santiago de Compostela, 15706-Santiago de Compostela, Spain
Received 14 June 1999; revised 1 October 1999; accepted 4 October 1999
Abstract – We describe practical and efficient routes for synthesis of 2-aminomethyl-1,2,3,9-tetrahydro-4H-carbazol-4-ones using the Fischer
indole synthesis or palladium-catalysed cyclization methodologies, as well as their affinities for D2, 5-HT2A and 5-HT2C receptors, and their
activity at the 5-HT2B receptor. The most active compounds, 4b (QF 2003B) and 4c (QF 2004B), with a pKi (5-HT2A/D2) ratio of 1.28 show
a potential antipsychotic profile according to Meltzer’s classification. © 2000 Éditions scientifiques et médicales Elsevier SAS
antipsychotics / serotonin 5-HT2 receptors / dopamine D2 receptors
1. Introduction
D2-like family, which includes receptors D2, D3 and D4,
appears to be involved in schizophrenia. The activity of
classical antipsychotics such as haloperidol, and the
intensity of their undesirable side effects (prolactin re-
lease and extrapyramidal symptoms (EPS)), are closely
correlated with their ability to block dopamine receptor
D2. It has also been suggested that receptors D3 [3] and
D4 may be involved in antipsychotic activity (the atypical
antipsychotic clozapine has high affinity for D4), but
clinical assays have shown selective D4 blockers (e.g.
L-745,870 [4], CP-293,019 [5]) to be ineffective as an-
tipsychotics [6].
Schizophrenia is a complex psychological disorder of
unclear aetiology which to some degree affects 0.5–1.5%
of the world’s population. It is widely accepted that the
dopaminergic system plays a key role in schizophrenic
illness. Affected individuals may exhibit a wide spectrum
of behavioural and other symptoms, ranging from social
withdrawal, catatonia and affective flattening of the
personality (‘negative’ symptoms thought to be associ-
ated with dopaminergic hypoactivity in the prefrontal
cortex) to hallucinations, paranoia and disorganized be-
haviour (‘positive’ symptoms thought to be associated
with hyperactive dopaminergic transmission in the me-
solimbic region of the brain) [1, 2].
Haloperidol (figure 1) and other classical butyro-
phenone-based antipsychotics, such as spiperone and
fluanisone, not only have the side effects mentioned
above, but are also ineffective against negative symp-
toms. Clozapine is the prototype of a new group of
‘atypical’ (non-classical) antipsychotics that cause no
EPS and are effective against negative as well as positive
symptoms. This superior activity profile of clozapine and
other atypical antipsychotics, such as risperidone, olan-
zapine and quetiapine, may be due to their blocking not
The cell-borne receptors with which dopamine inter-
acts are broadly classified as D1-like or D2-like. Only the
#Presented in part at the XVth International Symposium on
Medicinal Chemistry, Edinburgh, UK, September, 1998. 21st
paper in the series “Synthesis and CNS activity of conforma-
tionally restricted butyrophenones”. For preceding papers
see [39].
*Correspondence and reprints: qofara@usc.es

84
Figure 1. Structures of some typical and atypical antipsychotics.
only dopamine receptors but also serotonin recep-
tors [7–11].
of atypical antipsychotics [19–21]. Indeed, even the
5-HT2A-selective serotonin antagonist MDL 100,907,
which has no activity at dopamine receptors, has shown
antipsychotic potential in experiments with neurochemi-
cal, electrophysiological and behavioural models [22].
Meltzer also reported [23] that affinities for 5-HT2C
receptors enhance the distinction between typical and
atypical antipsychotic drugs. Clozapine and other atypi-
cal antipsychotics like olanzapine or seroquel have a
higher affinity for 5-HT2C receptors than for D2 recep-
tors [24]. Recently, Reavill et al. have suggested that
5-HT2C receptor antagonism is likely to be the mecha-
nism by which atypical antipsychotic drugs lack
EPS [25]. In addition to that, clozapine exhibits high
affinity at 5-HT2B human receptors (Ki = 7.57 nM [26]);
these receptors, with close operational and molecular
similarity with 5-HT2C receptors [27, 28], were found to
have a low level of expression at the human and rat
CNS [29–32]. However, the presence of these receptors
in rat CNS and their implication in animal models of
Atypical antipsychotics bind to 5-HT2, 5-ht6 and
5-HT7 serotonin receptors [12]. In the case of the last two
recombinant receptors, new supplementary studies are
necessary in order to clarify their role as pharmacological
receptors and their pathophysiological involvements [13,
14]. In any case, 5-HT7 receptors are thought not to be
involved in schizophrenia [15]. 5-HT2 type receptors
have been related with serotonin-mediated mechanisms
not only in schizophrenia but also in anxiety, depression,
anorexia nervosa, migrain, hypertension and other car-
diovascular disorders [16].
Meltzer and coworkers [17, 18] have suggested that the
ratio between the pKi’s of antipsychotic agents at 5-HT2A
and D2 receptors reflect an atypical profile, this ratio
appears to be > 1.12 for atypical antipsychotics and <
1.09 for classical antipsychotics.
Recent experimental and clinical studies appear to
confirm the importance of 5-HT2A for the activity profile

85
Figure 2. General structures of some butyrophenone analogues and pKi ratios (5-HT2A/D2) of some potential atypical antipsychotics
in the heterocyclic series.
behaviour, such as has been shown by Duxon et al. [33],
seems a controversial question.
(4a–f), cyclic butyrophenone derivatives in the carbazole
series, as well as the results of studies of the affinities of
the title compounds for D2, 5-HT2A, 5-HT2B and 5-HT2C
receptors. Some preliminary results from this work have
been published in communication form [41]. Some of
these compounds have two butyrophenone pharmacoph-
ores: the semirigid aminoalkyl indolone moiety and the
4-(p-fluorobenzoyl) or the 3-[4-(6-fluoro-1,2-benzisoxa-
zole-3-yl)]piperidine fragments. The 4-(p-fluorobenzo-
yl)piperidine fragment may be considered as a butyrophe-
none pharmacophore constrained in a six membered ring;
this fragment is also an important feature for 5-HT2A
binding [42, 43]. Moreover, the bioisosteric relationships
between benzoyl and 1,2-benzisoxazol moieties are note-
worthy [44, 45].
The development of drugs such as risperidone, oca-
peridone or sertindole which, like clozapine, block both
5-HT2A and D2, has been prompted in part by the finding
that clozapine increases the risk of agranulocytosis [34].
However, since none of these drugs has proven to be as
broadly effective as clozapine, the discovery of effective
antipsychotics with no side effects is still a major research
goal. Likewise, the discovery of new selective pharma-
cological tools acting on the different types of 5-HT2
receptors is essential.
In previous papers [35, 36] we have reported the
synthesis and antipsychotic activity of aminoalkylbenzo-
cycloalkanones (I and II) which are conformationally
restricted butyrophenone analogous of haloperidol, with
the aminobutyl side chain incorporated in a semi-rigid
structure (figure 2). Later, we prepared 5-aminoethyl and
6-aminomethyl-4,5,6,7-tetrahydroindole-4-ones (III and
VI), 4,5,6,7-tetrahydrobenzo[b]thiophen-4-ones (IV and
VII) and 4,5,6,7-tetrahydrobenzo[b]furan-4-ones (V and
VIII) as conformationally constrained butyrophenone
derivatives in the indole, benzothiophene and benzofuran
series, respectively, as putative atypical antipsychotics
[37–39]. Many of such heterocyclic restricted butyrophe-
nones (e.g. QF 0408B [38], QF 0610B [39] and QF
0510B [39], figure 2) show a very favourable 5-HT2A/D2
balance and its cataleptogenic activity is low [40].
As a continuation, within a program of research of new
CNS acting agents, we wish now to report practical and
efficient synthetic strategies for preparing new
2-aminomethyl-1,2,3,9-tetrahydro-4H-carbazol-4-ones
2. Chemistry
For the synthesis of the carbazolones 4a–c (figure 3,
method A) we started from the 1,4-dihydro-3,5-
dimethoxybenzyl alcohol 1, which was readily prepared
from the cheap 3,5-dimethoxy- (or 3,4,5-trimethoxy-)
benzoic acid in 85% yield as previously described [46,
47]. This alcohol has proven to be a versatile synthon in
the preparation of condensed heterocyclic butyrophenone
systems [38, 48]. Application of the Fischer indole meth-
odology [49] with phenylhydrazine in 4% H2SO4 solu-
tion allowed the construction of the desired carbazole
intermediate 2 (figure 3).
Reaction of the resulting 2-hydroxymethyl-1,2,3,9-
tetrahydro-4H-carbazol-4-one 2 with p-toluenesulfonyl
chloride in pyridine afforded the tosylate 3 as a white

86
Figure 3. (a) see [46, 47]; (b) PhNHNH2/4% H2SO4; (c) TsCl/Py; (d) HNRR/NMP; (e) HNRR/DCC/HOBt; (f) LiAlH4/THF;
(g) Li/NH3; (h) PhNR′NH2.
crystalline solid (70%). Nucleophilic displacement of the
tosylate with amines (single or complex, heterocyclic
amines such as piperazines or substituted piperidines, e.g.
p-fluorobenzoylpiperidine, 4-(6-fluoro-1,2-benzisoxazol-
3-yl)piperidine [50]) in N-methyl-2-pyrrolidone (NMP)
provided, after bulb to bulb distillation of NMP, the
amines 4a–c as white crystalline solids with yields
ranging from 55–70% (table I).
prepared from the corresponding 1,4-dihydro-3,5-
dimethoxybenzylamines 7, obtained following our previ-
ously reported methodology [51] for the indole series.
The direct conversion of these amines by the Fischer
indole reaction into a new class of potential atypical
antipsychotics, the 2-aminomethyl-1,2,3,9-tetrahydro-
4H-carbazol-4-ones 4d–f, was achieved by reaction with
phenylhydrazine or N-methyl-N-phenylhydrazine in 4%
H2SO4 solution at reflux temperature, with yields from
60–70% (table I).
Alternatively (figure 3, method B), aminomethyl car-
bazolones with non-aromatic amine fragments have been
Table I. 2-Aminomethyl-1,2,3,9-tetrahydro-4H-carbazol-4-ones 4a–f.
Compound
NRR
R′
Method
A
M.p. (recr. solvent)
229–230 (AcOEt)
Yield (%)
62
4a
H
4b
4c
H
H
A
A
217–219 (AcOET)
55
70
222–223 (iso-PrOH)
4d
4e
NEt2
NEt2
H
H
B
B
184–185 (CH3CN)
207–208 (AcOEt)
66
60
4f
CH3
B
192–193 (acetone)
70

87
Figure 4. (a) BnBr/NaH/Bu4NI; (b) 10% HCl; (c) 2-iodoaniline/pTsOH; (d) Pd(AcO)2/Et3N; (e) H2/Pd.
Also, we have studied intramolecular palladium-
catalysed cyclization of the N-(2-iodophenyl)enaminone
of compound 1 to afford the corresponding carbazolone
(figure 4). Iida et al. [52] reported the palladium-
catalysed cyclization of the N-(2-bromophenyl)enami-
none of dimedone to give the tetrahydrocarbazolone
derivative, but in low yield. However, the cyclization was
successfully achieved by Sakamoto et al. using the
N-(2-iodophenyl)enaminone and Pd(OAc)2 as cata-
lyst [53]. The application of this methodology to the
O-benzyl ether of alcohol 1 allows us to obtain the
tetrahydrocarbazolone derivative 11 in good yield.
The 5-benzyloxymethyl-3-(2-iodophenyl)amino-2-cy-
clohexen-1-one was synthesized by dehydroxy-con-
densation of 2-iodoaniline with 5-benzyloxymethyl-1,3-
cyclohexanedione 9, obtained in turn by benzylation and
subsequent acidic hydrolysis of enol-ether groups of
alcohol 1 (figure 4). The palladium-catalysed carbazole-
cyclization was carried out using Pd(OAc)2 and triethyl-
amine in DMF, with 87% yield. The benzyl-protecting
group was quantitatively removed by hydrogenolysis
with palladium on carbon in ethanol to give the desired
intermediate 2.
3. Results and discussion
The dopamine D2 and serotonin 5-HT2A and 5-HT2C
receptor affinities, and the 5-HT2A and 5-HT2B antago-
nistic affinities of compounds 4a–4f are shown in table II.
Figure 5 shows the radioligand–competition binding
curves recorded with compound 4b and [3H]ketanserine
(5-HT2A receptors, figure 5A) or [3H]mesulergine (5-
HT2C receptors, figure 5C), and the dose–response curve
for 4b (5-HT2B receptors, figure 5B).
Table II. Inhibition constants (pKi) at D2, 5-HT2A and 5-HT2C receptors, and antagonist activity (pA2) at 5-HT2A and 5-HT2C receptors of
compounds 4a–f.
Compound
pKi ± SEMa
pA2 ± SEMb
pKi ratio ± SEM
D2
5-HT2A
5-HT2C
5-HT2A
5-HT2B
5-HT2A/D2
4a (QF 2006B)
4b (QF 2003B)
4c (QF 2004B)
4d (QF 2002B)
4e (QF 2014B)
4f (QF 2001B)
Haloperidol
5.81 ± 0.33
6.25 ± 0.24
6.85 ± 0.21
< 5
< 5
< 5
6.20 ± 0.61
8.04 ± 0.80
8.80 ± 0.88
< 5
< 5
< 5
5.92 ± 0.06
6.23 ± 0.09
7.24 ± 0.06
n.t.c
6.50 ± 0.20
9.20 ± 0.27
9.50 ± 0.32
n.t.
n.t.
n.t.
6.80 ± 0.05
7.22 ± 0.23
6.35 ± 0.10
n.t.
n.t.
n.t.
1.07 ± 0.05
1.29 ± 0.10
1.28 ± 0.10
–
–
–
0.91
1.23
n.t.
n.t.
8.48 ± 0.12
6.58 ± 0.05
7.70 ± 0.22
8.12 ± 0.07
5.60d
–
–
Clozapine
8.20d
8.62 ± 0.05
6.77 ± 0.20
a Binding affinity (striatal membranes: D2 [3H]spiperone; rat frontal cortex: 5-HT2A [3H]ketanserin; bovine choroid plexus: 5-HT2C
[3H]mesulergine). b Antagonism of serotonin at 5-HT2A (rat aorta) and 5-HT2B (rat stomach fundus) receptors. c Not tested. dData from
reference [25].

88
Compounds 4a–c inhibited the binding of [3H]-
spiperone to D2 receptors with pKi values ranging from
5.81–6.85, and the binding of [3H]-ketanserine to 5-HT2A
receptors with pKi values from 6.20–8.80. Compounds
4d–f, bearing simple amines, did not show affinity for
either D2 or 5-HT2A receptors. The most active com-
pounds, 4b (QF 2003B) and 4c (QF 2004B), exhibited a
high affinity for 5-HT2A receptors, while the pKi value for
the D2 receptor was lower. Both of them show a pKi
(5-HT2A/D2) ratio of 1.28. In keeping with the hypoth-
eses suggested by Meltzer et al. [17, 18] regarding the
combination of 5-HT2A-blocking and D2-blocking activi-
ties, the compounds 4b and c are thought to have an
atypical antipsychotic profile from Meltzer’s, pKi ratio >
1.12.
The p-fluorobenzoyl derivative 4b has a pKi (5-HT2A
/
D2) ratio for compound 4b (1.28), higher than that for
haloperidol (0.91), risperidone (1.20) or clozapine (1.23).
This compound also exhibited a pA2 value of 9.2 in
suppressing serotonin-induced contractions in rat aorta
ring stripped of endothelium [54]. According to Meltzer’s
classification 4b shows a profile of an atypical antipsy-
chotic.
The replacement of the benzoylpiperidine fragment of
4b by a 1,2-benzisoxazolyl-piperidine moiety (compound
4c) increased the affinity for both D2 and 5-HT2A
receptors, with pKi values of 6.85 and 8.80, respectively.
The affinity for 5-HT2A receptors (pKi and pA2) is higher
than that exhibited by clozapine.
The introduction of o-methoxyphenylpiperazine or
amines with non-aromatic fragments does not improve its
affinity for 5-HT2A and D2 receptors.
Compounds 4a–c did not show a significant difference
of affinity between 5-HT2B and 5-HT2C receptors. The
compounds 4b and 4c showed higher affinity at 5-HT2A
receptors than at 5-HT2B receptors: the p-fluoro-
benzoylpiperidine derivative 4b exhibits a 100-fold
higher antagonist activity (Ki vs. Kb) at 5-HT2A receptors
than at 5-HT2B receptors. This selectivity increases up to
1 000-fold in the benzisoxazolylpiperidine derivative 4c.
Both 4b and 4c display higher affinity (Ki) at 5-HT2A than
at 5-HT2C receptors, about 60-fold for 4b and 35-fold for
4c.
The presence of an o-methoxyphenylpiperazine
strongly modifies this selectivity, conserving 5-HT2B
activity, slightly decreasing 5-HT2C affinity and dramati-
cally decreasing 5-HT2A affinity and activity.
Figure 5. A) Inhibition by 4b of [3H]ketanserin-binding by
5-HT2A receptors in rat frontal cortex membrane preparations.
B) Inhibition by 4b of serotonin-induced contractions of rat
stomach fundus (points show the average of 2–3 replicate
experiments; vertical bars indicate SEM). C) Inhibition by 4b
of [3H]mesulergine-binding by 5-HT2C receptors in bovine
choroid plexus membrane preparations. Graphs A and C show
duplicate and triplicate, respectively, points from a single
experiment; a total of two replicate experiments were perfor-
med in each case.
4. Conclusions
We have developed a practical and efficient 4 (for
amines with non-aromatic fragments) or 5 (for amines

89
with aromatic cycles) step synthesis for new derivatives
in the carbazole series as atypical antipsychotics from
cheap and readily available starting materials using Fis-
cher indole methodology. Also, we have studied and
successfully applied the palladium-catalysed cyclization
as an alternative route for the preparation of carbazole
intermediates. The promising affinity for both D2 and
5-HT2A receptors shown by compound 4c (QF 2004B),
its high Meltzer ratio (1.28) and its good 5-HT2 selectiv-
ity has prompted us to choose this compound for further
development.
3 151, 2 932, 1 629, 1 611 cm–1. 1H-NMR (DMSO-d6): δ
2.26–2.43 (m, 3H), 2.77 (dd, 1H, J1 = 8.9 Hz, J2 = 16.5
Hz), 2.99–3.05 (m, 1H), 3.40–3.51 (m, 2H), 4.70 (br. s,
1H), 7.09–7.17 (m, 2H), 7.34–7.40 (m, 1H), 7.91–7.94
(m, 1H), 11.82 (s, 1H). MS (EI, m/z): 215 (M+, 67%).
Anal. C13H13NO2 (C, H, N).
5.1.2. 2-(p-Toluenesulfonyl)oxymethyl-1,2,3,9-
tetrahydro- 4H-carbazol-4-one 3
To a solution of carbazolone 2 (0.35 g, 1.63 mmol) and
a catalytic amount of dimethylaminopyridine in dry
pyridine (7 mL) at 0 °C, p-toluenesulfonyl chloride
(0.56 g, 2.93 mmol) was added. The mixture was stirred
at 0 °C for 24 h and water (20 mL) was added. The
precipitate was collected by filtration, washed with water
and dried to give 0.54 g (89% yield) of a white solid, m.p.
215–216 °C (CH3CN). IR (KBr) νmax: 3 228, 1 625,
5. Experimental protocols
5.1. Chemistry
Melting points were determined with a Kofler hot stage
instrument or a Gallenkamp capillary melting point
apparatus and are uncorrected. Infrared spectra were
recorded with a Perkin Elmer 1600 FTIR spectrophoto-
meter; the main bands are given in cm–1. Proton NMR
spectra (1H-NMR) were recorded with a Brucker WM
AMX (300 MHz); chemical shifts are recorded in parts
per million (δ) downfield from tetramethylsilane (TMS).
Mass spectra were performed on Kratos MS-50 or Varian
Mat-711 mass spectrometers in fast atom bombardment
(FAB) mode (with 2-hydroxyethyl disulphide 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 moni-
tored by thin layer chromatography (TLC) on Merck 60
GF254 chromatogram sheets using iodine vapour and/or
UV light for detection; unless otherwise stated the puri-
fied compounds each showed a single spot. Elemental
analyses were performed on a Perkin Elmer 240B appa-
ratus at the Microanalyses Service of the University of
Santiago; unless otherwise stated all reported values are
within ± 0.4% of the theoretical compositions.
1
1 610 cm–1. H-NMR (DMSO-d6) δ 2.25–2.38 (m, 2H),
2.41 (s, 3H), 2.59–2.68 (m, 1H), 2.78 (d, 1H, J1 = 10.2
Hz, J2 = 16.4 Hz), 3.02 (dd, 1H, J1 = 4.3 Hz, J2 = 16.4
Hz), 4.07–4.16 (m, 2H), 7.08–7.18 (m, 2H), 7.37–7.40
(m, 1H), 7.48 (d, 2H, J = 8.2 Hz), 7.81 (d, 2H, J = 8.3
Hz), 7.88–7.91 (m, 1H), 11.87 (s, 1H). MS (EI, m/z): 369
(M+, 11%). Anal. C20H19NO4S (C, H, N).
5.1.3. 1-Benzyloxymethyl-3,5-dimethoxy-
1,4-dihydrobenzene 8
To a 60% suspension of NaH in mineral oil (0.17 g,
7.1 mmol) in dry THF (10 mL), a solution of 1,4-dihydro-
3,5-dimethoxybenzyl alcohol 1 (1.0 g, 5.9 mmol) in dry
THF (10 mL) was added dropwise. Then a catalytic
amount of tetrabutylammonium iodide and benzyl bro-
mide (0.83 mL, 7.1 mmol) were added. The mixture was
stirred at room temperature under argon for 36 h and
methanol (4 mL) was added. The mixture was stirred for
30 min and then partitioned between AcOEt (40 mL) and
water (40 mL). The organic phase was dried (Na2SO4),
filtered and concentrated in vacuo to give a yellow oil
which was purified by column chromatography (silica
gel, eluent: AcOEt/hexane 1:15) to afford 1.15 g (75%
yield) of a colourless oil. IR (KBr) νmax: 3 029, 2 846,
1 694, 1 661, 1 599 cm–1. 1H-NMR (CDCl3) δ 2.80–2.83
(m, 2H), 3.29–3.31 (m, 1H), 3.35–3.37 (m, 2H), 3.59 (s,
6H), 4.58 (s, 2H), 4.71–4.72 (m, 2H), 7.29–7.39 (m, 5H).
Anal. C16H20O3 (C, H).
5.1.1. 2-Hydroxymethyl-1,2,3,9-tetrahydro-
4H-carbazol-4-one 2
A solution of phenylhydrazine (5 mL, 3.52 mmol) in
4% H2SO4 (5 mL) was added dropwise to a solution of
1,4-dihydro-3,5-dimethoxybenzyl alcohol 1 [46, 47]
(0.6 g, 3.5 mmol) in 4% H2SO4 (5 mL) at 40 °C. The
resulting solution was refluxed under Argon for 4 h. After
cooling, the solution was basified with an aqueous 25%
NH3 solution and extracted with AcOEt (3 × 15 mL). The
organic layer was dried, filtered and concentrated to give
a brown solid which was purified by column chromatog-
raphy (silica gel, eluent: AcOEt): 0.34 g (45% yield) of a
5.1.4. 5-Benzyloxymethyl-1,3-cyclohexanedione 9
A solution of 8 (1 g, 3.8 mmol) and 10% HCl (3.5 mL)
in THF (35 mL) was stirred at room temperature for 12 h.
Then the solvent was removed in vacuo and the residue
was dissolved in CH2Cl2, dried (Na2SO4), filtered and
concentrated under reduced pressure to give 0.71 g (80%
yield) of a white solid, m.p. 96–98 °C (AcOEt/hexane).
white solid, m.p. 210–211 °C (EtOAc). IR (KBr): νmax
=

90
IR (KBr) νmax = 2 856, 1 592, 1 532 cm–1. 1H-NMR
(CDCl3) δ 2.40–2.46 (m, 1H), 2.61 (dd, 2H, J1 = 6.1 Hz,
J2 = 15.7 Hz), 2.70 (dd, 2H, J1 = 5.6 Hz, J2 = 15.7 Hz),
3.25 (d, 1H, J = 18.0 Hz), 3.34 (d, 1H, J = 18.0 Hz), 3.49
(d, 2H, J = 3.6 Hz), 4.44 (s, 2H), 7.22–7.37 (m, 5H).
Anal. C14H16O3 (C, H).
5.1.8. General procedure for the preparation
of the 2-aminomethyl-1,2,3,9-tetrahydro-4H-
carbazol-4-ones 4a–c
A solution of the tosylate 3 (130 mg, 0.35 mmol) and
the amine (0.70 mmol) in (1-methyl)-2-pyrrolidinone
(3mL) was stirred at 85 °C for 48 h. The solvent was
evaporated in vacuo, and the residue was dissolved in
AcOEt and washed with water. The organic phase was
dried, filtered and concentrated at reduced pressure to
afford a solid which was purified by column chromatog-
raphy (silica gel, eluent: AcOEt) to give the desired
amine.
5.1.5. 5-Benzyloxymethyl-3-(2-iodophenyl)amino-
2-cyclohexen-1-one 10
A mixture of 2-iodoaniline (0.48 g, 2.2 mmol), 9
(0.5 g, 2.1 mmol) and a catalytic amount of p-toluene-
sufonic acid in benzene (25 mL) was refluxed for 24 h
using a Dean-Stark apparatus for azeotropic removal of
water. The solution was washed with water (25 mL),
dried (Na2SO4) and concentrated in vacuo. The residue
was purified by column chromatography (eluent: AcOEt/
hexane 2:1) to give 0.74 g (79% yield) of a slighty yellow
5.1.8.1. 2-[4-(o-Methoxyphenyl)piperazinyl]methyl-
1,2,3,4-tetrahydrocarbazol-4-one 4a
62% yield, m.p. 228–230 °C (AcOEt). IR (KBr) νmax
=
1
3 218, 2 811, 1 625, 1 610 cm–1. H-NMR (CDCl3) δ
2.38 (dd, 1H, J1 = 9.7 Hz, J2 = 15.8 Hz), 2.46–2.51 (m,
2H), 2.55–2.71 (m, 6H), 2.73–2.82 (m, 1H), 3.09 (br. s,
4H), 3.17 (dd, 1H, J1 = 3.6 Hz, J2 = 16.3 Hz), 3.86 (s,
3H), 6.85–7.04 (m, 4H), 7.21–7.28 (m, 2H), 7.33–7.36
(m, 1H), 8.21–8.24 (m, 1H), 8.71 (s, 1H). MS (EI, m/z):
389 (M+, 35%). Anal. C24H27N3O2 (C, H, N).
1
oil. IR (film) νmax = 3 285, 2 970, 1 603 cm–1. H-NMR
(CDCl3) δ 2.18–2.28 (m, 1H), 2.44 (d, 1H, J = 16.2 Hz),
2.58 (br. s, 3H), 3.42–3.56 (m, 2H), 4.55 (s, 2H), 5.38 (s,
1H), 5.96 (br. s, 1H), 6.89–6.96 (m, 1H), 7.32–7.35 (m,
7H), 7.85 (d, 1H, J = 7.9 Hz).
5.1.8.2. 2-[4-(p-Fluorobenzoyl)piperidinyl]methyl-
1,2,3,4-tetrahydrocarbazol-4-one 4b
5.1.6. 2-Benzyloxymethyl-1,2,3,9-tetrahydro-
4H-carbazol- 4-one 11
55% yield, m.p. 217–219 °C. IR (KBr): νmax = 3 220,
1
2 930, 1 677, 1 627, 1 610 cm–1. H-NMR (CDCl3) δ
A solution of 10 (440 mg, 1 mmol), Pd(OAc)2 (12 mg,
0.05 mmol), Et3N (170 µL, 1.2 mmol) in DMF (1.5 mL)
was heated in a sealed tube at 120 °C for 2 h. After
removing the solvent in vacuo, the residue was dissolved
in CH2Cl2 (30 mL) and washed with water (30 mL). The
organic layer was dried (Na2SO4), filtered and concen-
trated to give a brown solid which was chromatographed
on a silica gel column (eluent: AcOEt/hexane 1:2) to give
270 mg (87% yield) of a white solid, m.p. 186–188 °C
(AcOEt). IR (KBr) νmax = 3 179, 2 930, 1 629,
1.75–1.86 (m, 4H), 1.96–2.17 (m, 2H), 2.24–2.43 (m,
3H), 2.50–2.57 (m, 1H), 2.60–2.82 (m, 2H), 2.89–3.04
(m, 2H), 3.15–3.28 (m, 2H), 7.14 (t, 2H, J = 8.6 Hz),
7.20–7.27 (m, 2H), 7.31–7.37 (m, 1H), 7.97 (dd, 2H, J1 =
5.4 Hz, J2 = 8.8 Hz), 8.18–8.21 (m, 1H), 8.87 (s, 1H). MS
(EI, m/z): 404 (M+, 1%). Anal. C25H25FN2O2 (C, H, N).
5.1.8.3. 2-[4-(6-Fluorobenzisoxazol-3-yl)piperidinyl]-
methyl-1,2,3,4-tetrahydrocarbazol-4-one 4c
1
1 584 cm–1. H-NMR (CDCl3) δ 2.48 (dd, 1H, J1 = 11.1
70% yield, m.p. 222–223 °C (iso-PrOH). IR (KBr)
νmax = 3 159, 2 946, 1 627, 1 608 cm–1. 1H-NMR
(DMSO-d6) δ 1.78–2.05 (m, 4H), 2.09–2.20 (m, 2H),
2.26 (dd, 1H, J1 = 10.5 Hz, J2 = 16.3 Hz), 2.34–2.53 (m,
4H), 2.72 (dd, 1H, J1 = 9.5 Hz, J2 = 16.6 Hz), 2.90–3.00
(m, 2H), 3.07–3.16 (m, 2H), 7.09–7.17 (m, 2H), 7.25 (dt,
J1 = 2.1 Hz, J2 = 9.1 Hz), 2.34–2.41 (m, 1H), 7.65 (dd,
1H, J1 = 2.1 Hz, J2 = 9.1 Hz), 7.91–8.00 (m, 2H), 11.81
(s, 1H). Anal. C25H24FN3O2 (C, H, N).
Hz, J2 = 16.4 Hz), 2.61–2.77 (m, 2H), 2.90 (dd, 1H, J1 =
9.6 Hz, J2 = 16.5 Hz), 3.12 (dd, 1H, J1 = 4.8 Hz, J2 = 16.5
Hz), 3.47–3.60 (m, 2H), 4.53 (dd, 2H, J1 = 2.1 Hz, J2 =
13.7 Hz), 7.20–7.36 (m, 8H), 8.19–8.22 (m, 1H), 8.58 (s,
1H). Anal. C20H19NO2 (C, H, N).
5.1.7. Removal of the benzyl protecting group: 2-
hydroxymethyl-1,2,3,9-tetrahydro-4H-carbazol-4-one 2
A mixture of carbazolone 11 (220 mg, 0.72 mmol) and
10% Pd on charcoal (25 mg) in ethanol (20 mL) was
stirred under H2 at 45 °C for 48 h. After cooling, the
mixture was filtered through Celite and the solvent was
evaporated under reduced pressure to give 150 mg (97%)
of carbazolone 2 as a white solid.
5.1.9. General procedure for the synthesis of the
3,5-dimethoxybenzamides 5a and 5b
A solution of 3,5-dimethoxybenzoic acid (1.00 g,
5 mmol), the amine (5 mmol) and 1-hydroxy-
benzotriazole (0.74 g, 5 mmol) in DMF (10 mL) was
allowed to stir at 0 °C under argon atmosphere. N,

91
N≠-Dicyclohexylcarbodiimide (1.13 g, 5 mmol) was
added and the mixture stirred for 30 min at 0 °C and 20 h
at room temperature. The precipitate was filtered off and
the solution was concentrated in vacuo. The residue was
dissolved in CH2Cl2 and washed with 5% HCl and 5%
NaHCO3. The organic layer was dried, filtered and
concentrated at reduced pressure. The crude compound
was purified by column chromatography (silica gel,
eluent: AcOEt) to afford the pure amide.
5.1.11. General procedure for the synthesis of the
3,5-dimethoxy-1,4-dihydrobenzylamines 7a and 7b
A solution of the amine 6a or 6b (6.45 mmol) in a
mixture of tert-butanol (45 mL) and anhydrous ether
(45 mL) was added dropwise to liquid NH3 (30 mL) to
–35 °C. When the addition was complete, Li (1.00 g,
150 mmol) was added portionwise, the mixture was
allowed to warm to room temperature and was stirred for
4 h at that temperature. The reaction mixture was
quenched by adding of NH4Cl (6.10 g) and the excess of
NH3 allowed to evaporate by stirring at room tempera-
ture. The resulting white solid was dissolved in water
(40 mL), the alcoholic phase was separated and the
aqueous phase was extracted with CH2Cl2. The combined
organic layers were concentrated under reduced pressure
to give the desired amine.
5.1.9.1. N,N-Diethyl-3,5-dimethoxybenzamide 5a
1
95% yield. IR (film): νmax = 2 936, 1 676 cm–1. H-
NMR (CDCl3) δ 1.09 (s, 3H); 1.20 (s, 3H); 3.23 (5.a.,
2H); 3.48 (5.a., 2H); 6.43 (1H, t, J = 2.2 Hz); 6.45 (2H,
d, J = 2.2 Hz).
5.1.9.2. N-(3,5-Dimethoxybenzoyl)morpholine 5b
5.1.11.1. N,N-Diethyl-3,5-dimethoxy-1,4-
dihydrobenzylamine 7a
98% yield, m.p. 67–69 °C. IR (KBr) νmax = 2 971,
1 633 cm–1. 1H-NMR (CDCl3) δ 3.46–3.56 (2× s. a., 8H);
3.70 (s, 6H); 6.49 (d, 1H, J = 2.2 Hz); 6.51 (d, 2H, J = 2.2
Hz).
99% yield. IR (film): νmax = 2 926, 1 694, 1 663 cm–1.
1H-NMR (CDCl3) δ 1.02 (t, 6H, J = 7.1 Hz); 2.29 (d, 2H,
J = 7.1 Hz); 2.54 (q, 4H, J = 7.1 Hz); 2.77 (dd, 2H, J1 =
1.0 Hz, J2 = 6.8 Hz); 3.01–3.08 (m, 1H); 3.56 (s, 6H);
4.71–4.72 (m, 2H).
5.1.10. General procedure for the synthesis of the
3,5-dimethoxybenzylamines 6a and 6b
5.1.11.2. N,N-(3,5-Dimethoxy-1,4-
To a suspension of LiAlH4 (0.93 g, 25 mmol) in THF
(50 mL) at 0 °C, was added dropwise a solution of the
amide 5a or 5b (5 mmol) in THF (50 mL) under argon.
The resulting mixture was stirred at room temperature for
24 h. Excess of LiAlH4 was decomposed at 0 °C by the
successive dropwise addition of water (1.5 mL), 10%
NaOH (1.5 mL) and water (6 mL). The insoluble salts
were removed by filtration and washed with AcOEt. The
organic portions were combined, and the solvent was
removed under reduced pressure to afford the free base.
dihydrobenzyl)morpholine 7b
95%, yield, m.p. 158–160 °C. IR (KBr) νmax = 2 939,
1
2 802, 1 693, 1 660 cm–1. H-NMR (CDCl3) δ 2.25 (d,
2H, J = 7.3 Hz); 2.46 (t, 4H, J = 4.6 Hz); 2.78 (dd, 2H,
J1 = 0.9 Hz, J2 = 6.4 Hz); 3.11–3.18 (m, 1H); 3.57 (s,
6H); 3.72 (t, 4H, J = 4.6 Hz); 4.69 (dd, 2H, J1 = 1.4 Hz,
J2 = 2.0 Hz).
5.1.12. General procedure for the preparation of
the 2-aminomethyl-1,2,3,9-tetrahydro-4H-
carbazol-4-ones 4d–f
A solution of phenylhydrazine hydrochloride (0.39 g,
1.6 mmol) and the amine 7a or 7b (1.8 mmol) in 4%
H2SO4 (12 mL) was heated at reflux temperature for 3 h.
The resulting solution was basified with 25% NH3 and
extracted with AcOEt (3 × 15 mL). The organic layer was
dried (Na2SO4), filtered and concentrated to give a brown
solid which was purified by column chromatography
(silica gel, eluent: CH2Cl2/MeOH, 9:1) to give the
carbazolone as a white solid.
5.1.10.1. N,N-Diethyl-3,5-dimethoxybenzylamine 6a
84% yield, b.p. 95–100 °C/0.1 mm Hg. IR (film): νmax
= 2 968, 1 596 cm–1. 1H-NMR (CDCl3) δ 1.04 (t, 6H, J =
7.1 Hz); 2.52 (q, 4H, J = 7.1); 3.51 (s, 2H); 3.78 (s, 6H);
6.34 (t, 1H, J = 2.3 Hz); 6.53 (d, 2H, J = 2.3 Hz).
Hydrochloride salt: white needles, m.p. 169–170 °C (iso-
PrOH). Anal. C13H21NO2 HCl (C, H, N).
5.1.10.2. N-(3,5-Dimethoxybenzyl)morpholine 6b
88% yield. IR (film): νmax = 2 955, 2 805, 1 597,
1 460 cm–1. 1H-NMR (CDCl3): δ 2.47 (t, 3H, J = 4.6 Hz);
3.46 (s, 2H); 3.71–3.74 (m, 4H); 3.79 (s, 6H); 6.36 (t, 1H,
J = 2.3 Hz); 6.51 (d, 2H, J = 2.3 Hz). Hydrochloride salt:
m.p. 188–189 °C (iso-PrOH). Anal. C13H19NO3 HCl (C,
H, N).
5.1.12.1. 2-Diethylaminomethyl-1,2,3,4-
tetrahydrocarbazol-4-one 4d
66% yield, m.p. 184–185 °C (CH3CN). IR (KBr): νmax
= 3 182, 2 966, 1 628, 1 617 cm–1. 1H-NMR (DMSO-d6)
δ 0.92 (t, 6H, J = 7.1 Hz), 2.18–2.27 (m, 1H), 2.33–2.53
(m, 8H), 2.69 (dd, 1H, J1 = 7.9 Hz, J2 = 16.8 Hz),

92
3.04–3.10 (m, 1H), 7.08–7.17 (m, 2H), 7.35–7.39 (m,
1H), 7.91–7.94 (m, 1H), 11.79 (s, 1H). Anal. C17H22N2O
(C, H, N).
performed in duplicate. Incubations (15 min at 37 °C)
were stopped by rapid vacuum filtration through GF-52
glass fibre filters (Schleicher and Schuell) in a Brandel
M-30 cell harvester. The filters were rinsed three times
with 3 mL of ice-cold 50 mM Tris-HCl buffer (pH 7.4)
and radioactivity was determined by liquid scintillation
counting in a Beckman LS-6000LL apparatus (counting
efficiency approximately 50%). Competition analyses
were carried out with the aid of the Prism program
(GraphPad); Ki values were calculated from the Cheng-
Prusoff equation [55]: Ki = IC50/(1 + D/Kd), where D is
the concentration and Kd the apparent dissociation con-
stant of the ligand.
5.1.12.2. 2-Morpholinylmethyl-1,2,3,4-
tetrahydrocarbazol-4-one 4e
60% yield, m.p. 207–208 °C (AcOEt). IR (KBr) νmax
=
3 177, 2 956, 1 618 cm–1. 1H-NMR (CDCl3) δ 2.30–2.50
(m, 7H), 2.55–2.65 (m, 1H), 2.67–2.79 (m, 2H), 3.14 (dd,
1H, J1 = 4.4 Hz, J2 = 16.3 Hz), 3.70 (t, 4H, J1 = 4.7 Hz),
7.19–7.25 (m, 2H), 7.31–7.36 (m, 1H), 8.19–8.22 (m,
1H), 8.90 (s, 1H). MS (EI, m/z): 284 (M+, 1%). Anal.
C17H20N2O2.
5.1.12.3. 2-Diethylaminomethyl-9-methyl-
1,2,3,4-tetrahydrocarbazol-4-one 4f
5.2.2. 5-HT2A receptor binding assays
Male Sprague-Dawley rats (200–250 g) were asphyxi-
ated with CO2 and decapitated. The frontal cortex,
containing 5-HT2A receptors [56, 57], was dissected free
on ice, frozen on dry ice and stored at –70 °C until use
(generally less than 1 week later). All membrane prepa-
ration 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 (Brinkmann
Instruments, Westbury, NY). The homogenate was cen-
trifuged twice at 4 °C (900 g for 10 min followed by
40 000 g for 30 min). The supernatant was discarded and
the pellet 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 40 000 g.
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 buffer or the compound under test, 50 µL of
masking ligand solution (1 µM methysergide) as re-
quired, 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 (pre-soaked in 3% polyethylenylimine) in a
Brandel cell harvester (Gaithersburg, MD) followed by
washing with ice-cold Tris-HCl buffer (pH 6.6) to remove
unbound radioligand. The radioactivity retained on filters
was determined by liquid scintillation counting in a beta
counter (Beckman, LS-1800).
This compound was prepared following the same
procedure but using N-methyl-N-phenylhydrazine as
starting material; yield 70%; m.p. 192–193 °C (acetone).
1
IR (KBr): νmax = 2 967, 2 792, 1 636 cm–1. H-NMR
(CDCl3) δ 1.01 (t, 6H, J = 7.1 Hz), 2.31 (dd, 1H, J1 = 9.5
Hz, J2 = 16.0 Hz), 2.41–2.69 (m, 9H), 3.15 (dd, 1H, J1 =
3.1 Hz, J2 = 16.0 Hz), 3.72 (s, 3H), 7.24–7.33 (m, 3H),
8.23–8.26 (m, 1H). MS (FAB, m/z): 285 (M+ + 1, 100%).
Anal. C18H24N2O (C, H, N).
5.2. Pharmacology
5.2.1. D2 receptor binding assays
Male Sprague-Dawley rats were killed by decapitation
and their brains were rapidly removed and dissected on
an ice-cold plate. Striatal membrane preparations were
obtained by homogenization (Polytron homogenizer, set-
ting 6.10 s) in 50 mM Tris-HCl (pH 7.7 at 25 °C; about
100 µL per mg of tissue) containing 5 mM EDTA; the
homogenates were centrifuged (49 000 g for 15 min at
4 °C, Sorvall RC-26 plus), resuspended in 50 mM Tris-
HCl buffer (pH 7.4 at 25 °C) and centrifuged again (same
conditions), and the final pellets were stored at –80 °C
pending use. Just before binding assays, the pellets were
resuspended (1.25 mg original wet weight per 750 µL for
D2 assays, 1.00 mg per 750 µL for D1) in 50 mM Tris-
HCl buffer (pH 7.4 at 25 °C) containing 120 mM NaCl,
5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2. For D2
binding assays, 750 µL aliquots of striatal membrane
preparation were added to ice-cold tubes containing (a)
100 µL of [3H]spiperone, (b) 50 µL of ketanserin (final
concentration 50 nM) to block 5-HT2A receptors, and
either (c) 100 µL of buffer (for total binding assay) or (d)
100 µL of sulpiride (final concentration 10 µM) to allow
quantification of nonspecific binding by [3H]spiperone, or
(e) 100 µL of the compounds to be tested. The final assay
volume was thus 1 mL in all cases. All assays were
The non-linear curve-fitting program Kaleidagraph
(Synergy Software, Reading, PA) was used to fit the
equation E = Emax – [Emax – Emin/(1 + (IC50/C)n)], where
E
max and Emin are dpm at the beginning and the end of the
competition experiment, respectively, IC50 is the drug
concentration required to inhibit binding by 50%, C is the

93
concentration of the inhibitor and n is the slope of the
decay. Non-specific binding was determined indepen-
dently in the presence of unlabelled methysergide. IC50
and pKi values were calculated as for D2 receptors.
5.2.5. Antagonism of serotonin at 5-HT2B receptors
from rat stomach fundus
Male Sprague-Dawley rats (250–300 g) were killed by
cervical dislocation. 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 method [64] and mounted in organ
baths containing 10 mL of the same Krebs solution as
above, maintained at 37 °C with aeration using oxygen,
carbon dioxide (95% O2, 5%CO2). Before 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 transducer
FTO3C and a Grass polygraph 7D.
Concentration–response curves for serotonin were con-
structed as per Van Rossum. [65]. In the initial control
runs, stable contractions were achieved over the concen-
tration range of 0.01 nM–10 µM. Following the initial
control run, each tissue strip was run alternately with and
without antagonist. Between runs, the tissues were
washed and allowed to rest for 60 min. Antagonist
potency was measured according to Mackay [66] in terms
of pA2 (–log concentration of antagonist required to
maintain a constant response when the agonist concen-
tration is doubled).
5.2.3. 5-HT2C receptor binding assays
Bovine choroid plexus containing 5-HT2C recep-
tors [58] was treated as described in the previous assay. A
suspension of the resulting pellet in the same buffer was
stored on ice while not being manipulated. 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 mM mianserine as a 5-HT2A
receptor masking ligand. The mixtures were incubated for
1 h at room temperature. Membranes were harvested on
Whatman GF/B filter paper. Non-specific binding was
determined in the presence of unlabelled mianserine. IC50
and pKi values were calculated as for D2 and 5-HT2A
receptors.
5.2.4. Antagonism of serotonin at 5-HT2A receptors
from rat aorta
Antagonism of serotonin at 5-HT2A receptors [13, 14,
56] was assayed using thoracic aorta from male Sprague-
Dawley rats (250–350 g) killed by cervical dislocation.
The descending aorta was removed, cleaned, stripped of
endothelium and cut into rings 4 mm in length [59] that
were then mounted under a tension of 2 g in a
CELASTER-IOS 1 computerized 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. Clorpheniramine (1 µM) was added to block uptake
of serotonin [60, 61]. The bath solution was maintained at
37 °C and aerated with oxygen and carbon dioxide, (95%
O2, 5% CO2). Isometric contraction force was monitored
via a CPOL 0–25 g transducer. Following equilibration
for 60 min under a load of 2 g, the rings were sensitized
by addition of 10 µM 5-HT for 8 min. Equilibration
periods (60 min) [62] were then alternated with the con-
struction of cumulative 5-HT concentration–effect curves
(from 30 nM to 100 µM). Two control runs giving
identical curves were followed by test runs with ket-
anserin or the new compounds which were added to the
bath solution 20 min before the end of the preceding
equilibration period. Antagonist potency was measured,
following Arunlaksana and Schild [63], in terms of pA2
(–log concentration of antagonist required to maintain a
constant response when agonist concentration is
doubled).
5-Hydroxytryptamine HCl was supplied by Sigma, and
mianserine and methysergide by R.B.I. Aqueous solu-
tions of all drugs as their hydrochlorides were prepared
daily using distilled water. All drug concentrations men-
tioned above are final molar concentrations in the tissue
bath. [3H]ketanserin (60.08 Ci/mmol) and [3H]me-
sulergine (76 Ci/mmol) were obtained from DuPont NEN
(Boston, MA) and Amersham (UK), respectively. All
other drugs and chemicals were reagent grade products
from Sigma (St Louis, MO).
Acknowledgements
This research was supported by the Spanish Comisión
Interministerial de Ciencia y Tecnología (CYCIT) under
grant SAF 95-1081 and by the Xunta de Galicia under
grant XUGA 20319 B97. Thanks are given to Emilia
Rivas for her assistance in dopamine binding determina-
tions.
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