Synthesis by microwave irradiation and binding properties of novel 5-HT1A receptor ligands

Article abstract of DOI:10.1016/S0223-5234(01)01287-9

This work reports the synthesis by microwave irradiation and the binding tests on the 5-HT_1A, 5-HT_2A and 5-HT_2C receptors of new substituted piperazines in order to identify selective ligands for 5-HT_1A subtype receptor. Conventional heating and microwave irradiation of the reactions was compared. Synthesis by microwave irradiation gave the desired compounds in better yields than those obtained by conventional heating. The overall times for the syntheses were considerably reduced. Some resulting active compounds (29 and 39) were characterised by a good selectivity profile for the 5-HT_1A subtype receptor. The more active compounds were selected and further evaluated for their binding affinities on D₁, D₂ dopaminergic and α₁, α₂ adrenergic receptors. The compound with higher affinity and selectivity for the 5-HT_1A over all the considered receptors was the 3-{4-[4-(1,2,3,4-tetrahydronaphthyl)-1-piperazinyl]butan}-benzotriazinone (-)29 (5-HT_1A K_i=36 nM, other receptors not active).

Full text of DOI:10.1016/S0223-5234(01)01287-9

Eur. J. Med. Chem. 36 (2001) 873–886
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(01)01287-9/FLA
Original article
Synthesis by microwave irradiation and binding properties of novel 5-HT1A
receptor ligands
Giuseppe Caliendoa,*, Ferdinando Fiorinoa, Elisa Perissuttia, Beatrice Severinoa, Stefania Gessib,
Elena Cattabrigab, Pier Andrea Boreab, Vincenzo Santagadaa
aDipartimento di Chimica Farmaceutica e Tossicologica di Napoli ‘Federico II’, Via D. Montesano, 49-80131 Naples, Italy
bDipartimento di Medicina Clinica e Sperimentale-Sezione di Farmacologia-Universita` degli Studi di Ferrara,
Via Fossato di Mortara, 17-44100 Ferrara, Italy
Received 3 July 2001; revised 23 October 2001; accepted 24 October 2001
Abstract – This work reports the synthesis by microwave irradiation and the binding tests on the 5-HT1A, 5-HT2A and 5-HT2C
receptors of new substituted piperazines in order to identify selective ligands for 5-HT1A subtype receptor. Conventional heating and
microwave irradiation of the reactions was compared. Synthesis by microwave irradiation gave the desired compounds in better
yields than those obtained by conventional heating. The overall times for the syntheses were considerably reduced. Some resulting
active compounds (29 and 39) were characterised by a good selectivity profile for the 5-HT1A subtype receptor. The more active
compounds were selected and further evaluated for their binding affinities on D1, D2 dopaminergic and a1, a2 adrenergic receptors.
The compound with higher affinity and selectivity for the 5-HT1A over all the considered receptors was the 3-{4-[4-(1,2,3,4-tetrahy-
´
dronaphthyl)-1-piperazinyl]butan}-benzotriazinone (−)29 (5-HT1A Ki=36 nM, other receptors not active). © 2001 Editions
scientifiques et me´dicales Elsevier SAS
microwave / serotonin / 5-HT receptors / benzotriazinone
1. Introduction
potentially important event for the treatment of anxi-
ety states. Several structurally different compounds
Serotonin is a neuromediator well known for its
possessed high affinity and selectivity for 5-HT1A re-
implication in mood regulation, anxiety, depression,
ceptors [5]. Among these some piperazine compounds
and insomnia. The discovery of several classes of
such buspirone [6], gepirone [7], or ipsapirone [8]
receptors for serotonin and the search for their impli-
showed selective agonist or partial agonist activity for
cations in the mechanism of mood and anxiety have
5-HT1A receptors and has proved to be effective for
been widely studied [1–4]. One of these receptor sub-
the treatment of anxiety states or depression. The
types, the 5-HT1A one, is found in high concentration
search for new selective 5-HT1A receptor agonists has
in the limbic system in which it is thought to play a
yielded active compounds. During recent years, sero-
role in emotional processes. The activation of the
toninergic 5-HT1A ligands have been suggested as
5-HT1A receptor leads to a number of physiological
useful therapies for anxiety [9, 10] depression [11],
changes that can be easily quantified. The synthesis of
nausea and vomiting [12, 13], Alzheimer’s disease [14],
prostate cancer [15], hypertension, pain [16,17] or
alcoholism [18].
a specific 5-HT1A receptor ligand, the 8-hydroxy-2-(di-
n-propylamino)tetralin (8-OHꢀDPAT), and the study
of its pharmacological properties showed that ago-
In the past, we studied new arylpiperazine deriva-
nists inhibited 5-HT function and release, which is a
tives [19–23] and binding studies showed that several
of these compounds had a good affinity for serotonin-
ergic 5-HT1A receptors and low affinity for serotonin-
* Correspondence and reprints:
E-mail address: caliendo@unina.it (G. Caliendo).

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G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 873–886
ergic 5-HT2A,5-HT2C, dopaminergic D1, D2 and
adrenergic a1, a2 receptors.
To try to obtain selective 5-HT1A ligands we, there-
fore, decided to introduce some structural modifica-
tion such as the nature of the aromatic heterocycle,
the length of the aliphatic chain (n), and the nature of
the substituents on the piperazine moiety.
This article reports the synthesis, by microwave
irradiation, of new arylpiperazine derivatives and
pharmacological evaluations as selective serotonin re-
ceptor ligands.
Conventional heating (oil bath) and microwave ir-
radiation of the reactions were compared.
In both lead identification and lead optimisation
processes, there is an acute need for new organic
small molecules. Traditional methods of organic syn-
thesis are orders of magnitude too slow to satisfy the
demand for these compounds. The fields of combina-
torial and automated medicinal chemistry (i.e. the
simultaneous or parallel synthesis of libraries) [24–26]
have been developed to meet the increasing require-
ment of new compounds for drug discovery; within
these fields, speed is of the essence. The application of
microwave energy to organic compounds for conduct-
ing synthetic reactions at highly accelerated rates is an
emerging technique. In recent years, microwaves have
become popular among synthetic organic chemists
both to improve classical organic reactions, [27] short-
ening reaction times and/or improving yields, as well
as to promote new reactions [28].
Moreover, when carrying out a reaction in a mi-
crowave oven, the use of a solvent can sometimes be
avoided which is important in order to make the
synthesis more environmentally friendly (‘green chem-
istry’). The commercially available instrument used
herein features a built-in magnetic stirrer, direct tem-
perature control of the reaction mixture with the aid
of fluoroptic probes and software that enables online
temperature control by regulation of microwave
power output (see Section 5).
2. Chemistry
The synthesis of some intermediates 4-X-substi-
tuted piperazines (figures 1 and 2) and final 1,2,3-ben-
zotriazinone (11–30) and 3-hydroxy-1,2,3-benzo-
triazinone (33–42) derivatives (figure 3) were per-
formed using a microwave oven (ETHOS 1600, Mile-
stone) especially designed for organic synthesis. The
experimental conditions used in our work were simi-
lar to those used by conventional heating, with the
same concentration of starting material and volume
of solvent. All reactions were performed in closed
vessels. All the reactions were performed by mi-
crowave program, which was composed by appropri-
ate ramping and holding steps. Identification of the
optimum profile power/time and temperature for ev-
ery considered compound was reported in table III.
All the 4-X-substituted-piperazines were commer-
cially available except the 1-(2-thienylmethyl 5-X)-
piperazines (4a–b) and the intermediates 1-(1,2,3,4-
tetrahydronaphth-1-yl)piperazine (8). The synthetic
approach employed for the preparation of 4a–b is
summarised in figure 1. Treatment of 2-thiophene-
methanol (1) with thionyl chloride in toluene gives the
corresponding 2-chloromethyl thiophene 2a. The
Figure 1. Reagents and conditions: (i) SOCl2, toluene, 55 °C, 20%, mn; (ii) ethyl piperazine-1-carboxylate, K2CO3, NaI, DMF, reflux,
1 h, mn; (iii) KOH, water, methanol, reflux, 1 h, mn.

G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 873–886
875
Figure 2. Reagents and conditions: (i) SOCl2, toluene; (ii) ethyl piperazine-1-carboxylate, K2CO3, NaI, DMF, reflux, 70%, mn; (iii)
KOH, water, methanol, reflux, 1 h, mn.
Figure 3. Reagents and conditions: (i) Br(CH2)nCl, K2CO3, DMF, 30%, mn; (i%) Br(CH2)nCl, NaOH, absolute ethanol, 30%, mn; (ii)
4-X-substituted-piperazine, K2CO3, NaI, DMF, 70%, mn.
intermediate 2b was commercially available. The con-
densation of ethyl piperazine-1-carboxylate with the
corresponding 2a or 2b gives, respectively, the inter-
mediates 3a and 3b. Saponification and decarboxyla-
tion provided the desired intermediates 4a and 4b.
Intermediate 1-(1,2,3,4-tetrahydronaphth-1-yl)-
piperazine (8) was obtained by using the same condi-
tion, which was employed for the preparation of

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G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 873–886
compounds 4a–b starting from 1,2,3,4-tetrahydro-1-
naphthol (5) with thionyl chloride in toluene to give
the corresponding 1,2,3,4-tetrahydro-1-chloronaph-
thyl (6). The condensation of ethyl piperazine-1-car-
boxylate with 6, followed by saponification and
decarboxylation, provided desired compound 8 (figure
2). The two enantiomers were obtained as reported in
literature [29] from the (+/−)1-(1,2,3,4-tetrahy-
dronaphthyl) piperazine (8). The synthesis of final
1,2,3-benzotriazinone (11–30) and 3-hydroxy-1,2,3-
benzotriazinone (33–42) derivatives was summarised
in figure 3. The general procedure is as follows: al-
kylation of starting heterocycle 1,2,3-benzotriazin-
4(3H)-one (9) with 1-bromo-2-chloroetane, 1-bromo-
3-chloropropane, or 1-bromo-4-chlorobutane and
K2CO3 in DMF or 3-hydroxy-1,2,3-benzotriazin-
4(3H)-one (31) with 1-bromo-2-chloroetane or 1-
bromo-3-chloropropane in presence of NaOH in
absolute ethanol gave the general corresponding
cloroalkyl benzotriazinone derivatives 10 and 32.
Condensation reaction of the obtained compounds
10 or 32 with desired 4-X-substituted-piperazines was
performed in DMF in the presence of K2CO3, NaI,
under reflux to give, respectively, the final compounds
11–30 and 33–42.
D2) and adrenergic (a1 and a ) receptors. All the
2
compounds were tested as water-soluble, hydrochlo-
ride salts. The following specific radioligands and
tissue sources were used, (a) serotonin 5-HT1A recep-
tors, [3H]-8-OHꢀDPAT, rat cerebral cortex mem-
branes; (b) serotonin 5-HT2A receptors, [3H]ketan-
serin, rat frontal cortex membranes; (c) serotonin
5-HT2C receptors, [3H]mesulergine, rat frontal cortex
membranes; (d) dopamine D1 receptors [3H]SCH-
23390, rat strial membranes; (e) dopamine D2 recep-
tors [3H]spiperone, rat strial membranes; (f) a1
adrenergic receptors [3H]prazosin, rat brain cortex
membranes; (g) a2 adrenergic receptors [3H]-UK, rat
brain cortex membranes. Concentrations required to
inhibit 50% of radioligand specific binding (IC50) were
determined through four independent experiments
with samples in triplicate using seven to nine different
concentrations of the titled compound. Specific bind-
ing, defined as described in the Section 5, represented
more than 75% of total binding in all three assays.
The Ki (nM) values were calculated from the Cheng–
Prusoff equation [30] Ki=IC50/1+(ligand/Kd) and
listed in table IV. (Kd for [3H]-8-OH-DPAT,
[3H]ketanserin, [3H]SCH-23390, [3H]spiperone, [3H]-
Prazosin and [3H]-UK were respectively 0.8, 0.85,
0.20, 0.25, 0.12 and 0.91 nM).
The synthesised products were transformed into the
corresponding hydrochloride salts using dry HCl in
anhydrous diethyl ether. The associated physical data
of final compounds were reported in tables I and II.
The parameters of time, power, temperature used for
the irradiation and conditions used by conventional
heating were as reported in table III. Analytical purifi-
cation of each final product was obtained by chro-
matography on silica gel column and further by
crystallisation from the appropriate solvent. All new
compounds gave satisfactory elemental analyses (C,
4. Results and discussion
Thirty benzotriazinone derivatives (11–30 and 33–
42) were synthesised under microwave irradiation and
were obtained in higher yields (80–94%) and cleaner
reactions than those obtained by conventional heat-
ing. The overall reaction times were dramatically re-
duced from 24 h to 70 min. Analogous results were
obtained for all intermediates (table III). The pre-
pared benzotriazinone derivatives were then evaluated
in vitro, in a preliminary binding screening, for their
affinity for 5-HT1A receptors and their selectivities
were compared with those of two other serotonin
receptors, 5-HT2A and 5-HT2C. Affinity constants (Ki
(nM)) for three subtypes receptors 5-HT1A, 5-HT2A,
5-HT2C, have been determined and compared with
those for ligands 8-OHꢀDPAT, Ketanserine and
Mesulergine. Products showing a Ki values <105 for
at least one of the three examined receptors in the
preliminary binding screening are reported in table
IV, whereas products exhibiting Ki (nM) values >105
for all receptors were considered inactive and were
not reported in the table IV.
1
H, Cl, N, S) and were characterised by H-NMR and
mass spectrometry (LCQ–MS Thermoquest-Ion
1
trap). H-NMR and MS data for all final compounds
were consistent with the proposed structures.
3. Pharmacology
The compounds (11–30) and (33–42) reported in
tables I and II were tested for their in vitro affinity on
serotonin 5-HT1A, 5-HT2A and 5-HT2C receptors by
radioligand binding assays. The more active com-
pounds on serotonin receptors have been selected and
evaluated for their affinity on dopaminergic (D1 and

G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 873–886
Table I. Physicochemical properties of 1,2,3-benzotriazinone derivatives (11–30).
877

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G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 873–886
Table I. (Continued)

G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 873–886
879
Table II. Physicochemical properties of 3-hydroxy-1,2,3-benzotriazinone derivatives (33-42).
In this work, we studied (a) the influence of the
modification of the aromatic heterocycle; (b) the
length of the alkyl side chain (n); (c) the substitutions
on the piperazine moiety (X); and (d) the stereochem-
istry of the tetrahydronaphthyl compounds.
(a) Regarding the modification of the aromatic hete-
rocycle, the replacement of 1,2,3-benzotriazinone
(compounds 11–30) with its analogue 3-hydroxy-
1,2,3-benzotriazinone (compounds 33–42) has led
to the same binding profile. The 5-HT1A affinities
obtained are quite similar.
(b) The influence of the alkyl side chain (n) on the
results reported in table IV showed that generally
highest affinity for 5-HT1A receptors appeared
when n=4 for the serie of 1,2,3-benzotriazinone
and n=3 for the serie of 3-hydroxy-1,2,3-benzo-
triazinone. These results agreed with those ob-
tained for the compound in a previous work [25]

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G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 873–886
in which more activity was showed by compounds
obtained for the series 11–30 with the presence of
the tetrahydronaphthyl nucleus (n=4 compounds
( ) 29, (−) 29, (+) 29) and for the series 33–42
with the presence of the pyridine ring (n=3 com-
pound 39). The results showed that the com-
pounds supporting on N-4 of the piperazine
moiety a 2-thienyl methyl-5-X ring did not
present any affinity for the selected receptors ex-
in which the distance between the nitrogen of the
3-hydroxy-1,2,3-benzotriazinone ring and the ni-
trogen of the piperazine ring was n=3. The ana-
logues n=2 for the both series present a low
affinity.
(c) Concerning the influence of the substituent on the
N-4 of piperazine moiety, the highest affinity were
Table III. Conventional heating versus microwave irradiation for intermediates (2a, 3a–b, 4a–b, 6, 7, 8, 10, 32) and final compounds
(11–30 and 33–42).
Compound Conventional heating a
Microwave irradiation
Yield (%)
85
Time (min)
60
Temperature (°C) Yield (%)
Time (min)
Power (W)
Temperature (°C)
2
55
96
5
10
5
5
50
5
5
50
5
5
50
5
80
400
200
80
400
200
80
400
200
80
400
200
80
55
3a
54
24 h
24 h
12 h
12 h
60
reflux
reflux
reflux
reflux
55
80
80
120
110
80
120
110
70
90
80
70
90
3b
63
92
4a
74
95
4b
63
90
5
50
5
400
200
80
80
55
6
87
98
5
10
5
5
60
5
5
50
5
5
20
5
5
60
5
80
20
5
5
60
5
400
200
80
400
200
80
400
200
80
400
200
80
7
63
24 h
12 h
180
reflux
reflux
80
90
80
120
110
70
90
90
8
84
95
10
72–85 b
44–65 b
60–70 b
35–60 b
90–96 b
80–92 b
5
80
120
110
80
120
110
11–30
32
24 h
180
reflux
80–94 b
reflux
400
200
400
200
80
400
200
55
33–42
24 h
85–94 b
80
120
110
The experimental conditions used on microwave irradiation were similar to those used by conventional heating, with the same
amount of starting reagent and volume of solvent. More details on the experimental conditions of the reactions are reported in
Section 5.
a Oil bath.
b Ranging between the reported percentage.

G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 873–886
881
Table IV. Binding affinities of active 1,2,3-benzotriazinone (11–30) and 3-hydroxy-1,2,3-benzotroazinone (33–42) derivatives.
Compound
Receptor affinity Ki (nM) a
5-HT1A [3H]8ꢀOHꢀDPAT
5-HT2A [3H]ketanserin
5-HT2C [3H]mesulergine
12
\105
13009150
\105
\105
13
16009158
40009350
380926
500942
1000990
25009270
\105
350938
680963
60009500
300927
450930
980970
450938
900978
6095
\105
15
\105
\105
(9) 17
(−) 17
(+) 17
19
\105
\105
\105
\105
\105
\105
\105
\105
20
25009260
850990
50009580
\105
\105
21
17009160
40009520
\105
22
23
(9) 25
(–) 25
(+) 25
27
27509250
\105
\105
\105
20009180
\105
50009480
\105
28
\105
\105
(9) 29
(−) 29
(+) 29
30
30009270
\105
3694
10396
\105
\105
30009240
20009220
28009255
\105
\105
330935
\105
34
\105
75009660
35
33009290
650962
13009110
5594
660952
22009200
15009120
\105
37
60009560
20009190
25009240
\105
\105
38
50009520
20009180
\105
39
40
41
\105
\105
42
\105
\105
a Ki value followed by S.E.M. For purpose of comparison, 8-OHꢀDPAT, Ketanserine and Mesulergine binds at 5-HT1A, 5-HT2A
and 5-HT2C receptors with Ki values of 0.80, 0.85 and 1.90 nM, respectively, under these assay conditions.
cept 27 and 28 (n=4) which showed a moderate
affinity but high selectivity versus 5-HT1A recep-
tor subtype. The tested 1,2,3-benzotriazinone
derivatives 11, 14, 16, 18, 24, 26 and the tested
3-hydroxy-1,2,3-benzotriazinone derivatives 33
and 36 were inactive (Ki>105 nM).
Only compounds 12, 20 and 34, supported on the
N-4 piperazine moiety a phenylethyl residue showed
no affinity binding on the 5-HT1A receptor subtype
but exhibited a moderate affinity binding on the 5-
HT2A subtype. The two classes of compounds (11–30)
and (33–42) retained a good and similar selectivity
for 5-HT1A in regard to 5-HT2A and 5-HT2C receptors.
As mentioned in Section 1, the most active com-
pounds of the serie 1,2,3-benzotriazinone (11–30) and
3-hydroxy-1,2,3-benzotriazinone (33–42) on 5-HT1A
(21, 27, 29, 30 and 39), were further evaluated for
their affinity as dopaminergic (D1 and D2) and adren-
ergic (a1 and a2) receptors. As far as the dopaminergic
system was concerned, the D1 and D2 receptor
affinity, all the compounds showed Ki (nM) values of
above 105 except compound 21, which exhibited a Ki
value of 91 nM at the D2 receptor. The affinity for
adrenergic a1 and a2 receptors were quite moderate
(d) Regarding the influence of the stereochemistry of
the tetrahydronaphthyl compounds, the chiral
resolution of the compound 17, 25 and 29 was
performed and enantiomers were tested on the
binding screening. The racemate (+/−) and levo
enantiomers(−) showed affinity for 5-HT1A recep-
tor better than that of their dextro isomers.
The compound with higher affinity and selectivity
for the 5-HT1A over all the considered receptors was
3-{4-[4-(1,2,3,4-tetrahydronaphthyl)-1-piper-
azinyl]butan}-benzotriazinone (−)29 (5-HT1A Ki=36
the
nM, other receptors not active).

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G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 873–886
only in two cases (21 and 27). In particular,
compound 21 showed for only a1 receptor a Ki value
of 212 nM; compound 27 showed moderate Ki values
both for a1 and a2 receptor, respectively, of 240 and
375 nM. These preliminary structure–activity
relationship studies showed that new structural
modification led to new derivatives characterised by
minor affinity and a high selectivity for 5-HT1A
receptor.
(microwave power, temperature and time) were set as
reported in table III. The mixture was cooled, washed
twice with ice-water, dried, and evaporated to give 2a as
an oil (yield 96%), which was used without further
purification in the next step.
5.1.2. Ethyl 4-[(5-X-thien-2-yl) methyl]-
piperazine-1-carboxylate (3a–b)
A mixture of 2-chloromethyl-5-X-thiophene (2a or 2b)
(0.65 mol), ethyl piperazine-1-carboxylate (0.79 mol),
potassium carbonate (1.43 mol), and sodium iodide
(0.06 mol) in 600 mL of DMF was introduced into the
reaction vessel and the desired parameters (microwave
power, temperature and time) were set as reported in
table III. After cooling, the mixture was filtered and the
solvent was evaporated off. The residual oil was taken
up with ice water, and the product was extracted three
times with ethyl acetate. A solution of 2 N hydrogen
chloride was then added. The hydrochloride formed was
filtered off. The base was displaced from its salt with
sodium carbonate in a dichloromethane–water mixture.
The organic layer was separated, dried, and evaporated
under vacuum to give respectively 3a (yield 80%) or 3b
5. Experimental protocols
5.1. Chemistry
Synthesis was performed using a microwave oven
ETHOS 1600, Milestone. The experiments were carried
out at atmospheric pressure in standard Pyrex glassware
with a reflux condenser fitted through the roof of the
microwave cavity. The temperature of the stirred
reaction mixture was monitored directly by
a
microwave-transparent fluoroptic probe inserted into
the solution. Melting points (m.p.) were determined
using a Kofler hot-stage apparatus and are uncorrected.
Thin layer chromatography was performed on Merck
Silica Gel 60 plates with fluorescent indicator and the
plates were visualised with UV light (254 nm). Kieselgel
60 was used for column chromatography. Where
analyses are indicated only by the symbols of the
elements, results obtained are within 0.4% of the
theoretical values. The structure was verified spectro-
1
(yield 92%) as an oil; (3a) H-NMR (CDCl3) l 1.23 (t,
3H, J=7 Hz), 2.38 (s, NH), 2.42 (m, 4H), 2.80 (m, 4H),
3.52 (d, 2H, J=7.5 Hz), 4.10 (q, 2H, J=7Hz), 6.81 (d,
1H, J=7.5 Hz), 6.84 (t, 1H, J=7.5 Hz), 7.12 (t, 1H,
1
J=7.5 Hz). Similar H-NMR occur in intermediate 3b.
5.1.3. 1-[(5-Thien-2-yl) methyl]piperazine (4a–b)
Potassium hydroxide (5.36 mol) was slowly added
into the reaction vessel containing a solution of 1-(2-
thienylmethyl-5-X)-4-carbetoxypiperazine (3a or 3b)
(0.46 mol), water (100 mL), and methanol (400 mL).
The desired parameters (microwave power, temperature
and time) were set as reported in table III. The reaction
was monitored by thin layer chromatography. After the
starting material had disappeared, the mixture was
cooled, filtered, and extracted with dichloromethane.
The organic layer was separated, dried, and evaporated
to give respectively 4a (yield 95%) or 4b (yield 90%) as
1
scopically by proton H-NMR. Spectra were recorded
on Bruker AM-500 instruments. Chemical shifts are
given as l with references to Me4Si as internal standard.
Mass spectra of the final products were performed on
LCQ thermoquest-ion trap mass spectrometry. Optical
rotation (h) of the pure enantiomers was measured by a
Perkin–Elmer 141 optical activity polarimeter. An-
hydrous Na2SO4 was used as a drying agent for organic
extraction throughout. Analytical data, m.p. and
crystallisation solvents are reported in tables I and II.
Compounds (+) 8 and (−) 8 were obtained from (+/−)8
using the same procedure as reported in [29].
1
an oil; (4a) H-NMR(CDCl3) l 2.41(s, NH), 2.48 (m,
4H), 2.84 (m, 4H), 3.60 (d, 2H, J=7.5 Hz), 6.83 (d, 1H,
J=7.5 Hz), 6.86 (t, 1H, J=7.5 Hz), 7.15 (t, 1H, J=7.5
1
5.1.1. 2-Chloromethyl thiophene (2a)
Hz). Similar H-NMR occur in intermediate 4b.
Thionylchloride (26 mL, 0.35 mol) was added at
15 °C to a solution of 2-thiophenmethanol (1) (0.24
mol) in toluene (200 mL). The mixture was stirred at
room temperature (r.t.) for 30 min and then introduced
into the reaction vessel and the desired parameters
5.1.4. 1-Chloro-1,2,3,4-tetrahydronaphthalene (6)
Thionylchloride (26 mL, 0.50 mol) was added at
15 °C to a solution of 1,2,3,4-tetrahydro-1-naphthol (5)
(0.36 mol) in toluene (300 mL). The mixture was stirred

G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 873–886
883
at r.t. for 30 min and then introduced into the reaction
5.1.7. 3-(n-Chloroalkyl)-1,2,3-benzotriazin-4(3H)-one
(10)
vessel and the desired parameters (microwave power,
temperature and time) were set as reported in table III.
The mixture was cooled, washed twice with ice water,
dried, and evaporated to give 6 as an oil (yield 98%),
which was used without further purification in the next
step.
A mixture of 1-bromo-2-chloroethane or 1-bromo-3-
chloropropane or 1-bromo-4-chlorobutane (0.09 mol),
1,2,3-benzotriazin-4H-one (9) (0.03 mol), K2CO3 (0.05
mol) in DMF (70 mL) was introduced into the reaction
vessel and the desired parameters (microwave power,
temperature and time) were set as reported in table III.
After cooling the mixture was concentrated to dryness
and the residue was dissolved in water (50 mL); the
solution was extracted several times with CH2Cl2. The
organic phase was dried, concentrated and chro-
matographed by column of silica gel (ether/methanol,
9:1 v/v) to give the general compound 10 as a solid
5.1.5. Ethyl 4-(1,2,3,4-tetrahydro-1-naphth-1-yl)-
piperazine-1-carboxylate (7)
A mixture of 1,2,3,4-tetrahydro-1-chloronaphthyl (6)
(0.85 mol), ethyl piperazine-1-carboxylate (1.0 mol),
potassium carbonate (1.87 mol), and sodium iodide
(0.06 mol) in 600 mL of DMF was introduced into the
reaction vessel and the desired parameters (microwave
power, temperature and time) were set as reported in
table III. After cooling, the mixture was filtered and the
solvent was evaporated off. The residual oil was taken
up with ice water, and the product was extracted three
times with ethyl acetate. A solution of 2 N hydrogen
chloride was then added. The hydrochloride formed was
filtered off. The base was displaced from its salt with
sodium carbonate in a dichloromethane–water mixture.
The organic layer was separated, dried, and evaporated
1
(yields ranging between 90 and 96%). H-NMR spectra
for all intermediates were consistent with the proposed
structures.
5.1.8. General procedure for compounds 11–30
The synthesis of 3-{2-[4-(benzyl)-1-piperazinyl]ethyl]}-
benzotriazinone (11) is typical; a mixture of 11 (0.03
mol), N-benzylpiperazine (0.03 mol) and NaI (0.05
mol), K2CO3 (0.05 mol) in DMF (50 mL) was intro-
duced into the reaction vessel and the desired parame-
ters (microwave power, temperature and time) were set
as reported in table III. After cooling, the mixture was
concentrated to dryness and the residue was dissolved in
water (50 mL). The solution was extracted several times
with CH2Cl2. The organic phase was dried, concentrated
and chromatographed by column of silica gel (ether–
ethanol 9:1 v/v) to give the final compound 11 as a white
1
under vacuum to give 7 as an oil (yield 90%); H-NMR
(CDCl3) l 1.25 (t, 3H, J=7 Hz), 1.60–2.10 (m, 4H),
2.31–2.90 (m, 6H), 3.50 (t, 4H, J=6 Hz), 3.82 (t, 1H,
J=7 Hz), 4.15 (q, 2H, J=7 Hz), 7.00–7.75 (m, 4H).
5.1.6. ( ) 1-(1,2,3,4-Tetrahydronaphth-1-yl) piperazine
(8)
1
Potassium hydroxide (7.00 mol) was slowly added
into the reaction vessel containing a solution of ethyl
4-(1,2,3,4-tetrahydronaphth-1-yl)piperazine-1-carboxyl-
ate (7) (0.60 mol), water (100 mL), and methanol (400
mL). The desired parameters (microwave power, tem-
perature and time) were set as reported in table III. The
reaction was monitored by thin layer chromatography.
After the starting material had disappeared, the mixture
was cooled, filtered, and extracted with dichloro-
methane. The organic layer was separated, dried, and
evaporated. The residual oil was taken up with ethanol,
and oxalic acid (0.57 mol) dissolved in ethanol was
added. The oxalate formed was filtered off, and the base
was displaced from its salt with sodium carbonate in a
dichloromethane–water mixture. The organic layer was
dried, and the solvent was evaporated under vacuum to
crystalline solid (yield 92%). H-NMR (CDCl3) l 2.40
(m, 4H), 2.70 (m, 4H), 2.90 (t, 2H, J=6.8 Hz), 3.49 (s,
2H), ?4.60 (t, 2H, J=6.8 Hz), 7.19–7.29 (m, 5H), 7.80
(t, 1H, J=7.6 Hz), 7.95 (t, 1H, J=7.6 Hz), 8.19 (d, 1H,
1
J=8.1 Hz), 8.37 (d, 1H, J=7.9 Hz). H-NMR spectra
for all final compounds were consistent with proposed
structures.
5.1.9. 3-(n-Chloroalkoxy-1,2,3-benzotriazin-4(3H)-one
(32)
A mixture of 1-bromo-2-chloroethane or 1-bromo-3-
chloropropane (0.023 mol), 3-hydroxy-1,2,3-benzotri-
azin-4H-one (31) (0.023 mol), NaOH (0.023 mol) in
absolute ethanol (50 mL) was introduced into the reac-
tion vessel and the desired parameters (microwave
power, temperature and time) were set as reported in
table III. After cooling, the mixture was concentrated to
dryness and the residue was dissolved in water (40 mL).
The solution was extracted several times with CH2Cl2.
1
give an oil 8 (yield 95%); H-NMR (CDCl3) l 1.50–2.10
(m, 4H), 2.00 (s, NH), 2.41–2.80 (m, 6H), 2.90 (t, 4H,
J=6 Hz), 3,75 (t, 1H, J=7 Hz), 7.02–7.80 (m, 4H).

884
G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 873–886
The organic phase was dried, concentrated and chro-
matographed by column of silica gel (ether–methanol,
9:1 v/v) to give the general compound 32 as a solid
(yields ranging between 80 and 94%).1H-NMR spectra
for all intermediates were consistent with the proposed
structures.
defined as the difference between binding in the absence
and presence of 5-HT (1 mM).
5.2.2. 5-HT2A and 5-HT2C binding assays
Radioligand binding assays were performed as previ-
ously reported by Claudi et al. [32]. Briefly, frontal
cortical regions of male Wistar rats (150–200 g, Charles
River) were dissected on ice, homogenised in ice-cold
buffer solution (50 mM Tris–HCl, pH 7.7, at 25 °C)
and centrifuged at 40 000×g for 10 min. The superna-
tant was discarded and the pellet was resuspended in the
same volume of Tris–HCl buffer and incubated at
37 °C for 10 min prior to a second centrifugation.
5.1.10. General procedure for compounds 33–42
The synthesis of 3-{2-[4-(benzyl)-1-piperazinyl]-
ethyloxy]}benzotriazinone (33) is typical; a mixture of
32 (0.03 mol), N-benzylpiperazine (0.03 mol), NaI
(0.010 mol) and K2CO3 (0.05 mol) in DMF (50 mL) was
introduced into the reaction vessel and the desired
parameters (microwave power, temperature and time)
were set as reported in table III. After cooling, the
mixture was concentrated to dryness and the residue
was dissolved in water (50 mL). The solution was ex-
tracted several times with CH2Cl2. The organic phase
was dried, concentrated and chromatographed by
column of silica gel (ether–ethanol 9:1 v/v) to give the
final compound 33 as a white crystalline solid (yield
Binding experiments with [3H]ketanserin (76 Ci mmol−1
;
New England Nuclear) and [3H]mesulergine (75.8 Ci
mmol−1; Amersham) were performed in 250 mL of
buffer, which contained 1 nmol [3H]ketanserin or
[3H]mesulergine, membranes from 10 mg (wet weight) of
tissue and the compounds to be tested. After 30 min of
incubation at 25 °C, separation of bound from free
radioligand was performed by rapid filtration through
Whatman GF/B glass fibre filters, which were washed
three times with ice-cold buffer, dried and counted in 5
mL of Aquassure (Packard, Downers Grove, USA).
Non specific binding was measured in the presence of 10
mM 5-HT for 5-HT2A sites and 10 mM cinanserin for
5-HT2C sites with specific binding defined as the total
binding minus the non-specific binding.
1
92%) H-NMR (CDCl3) l 2.33 (m, 4H), 2.56 (m, 4H),
2.91 (t, 2H, J=6.8 Hz), 3.39 (s, 2H), 4.57 (t, 2H, J=6.8
Hz), 7.23–7.30 (m, 5H), 7.97 (t, 1H, J=7.6 Hz), 7.81 (t,
1H, J=7.6 Hz), 8.20 (d, 1H, J=8.1 Hz), 8.38 (d, 1H,
1
J=7.9 Hz). H-NMR spectra for all final compounds
(33–42) were consistent with the proposed structures.
5.2. Pharmacology
5.2.3. D1 and D2 dopaminergic binding assay
5.2.1. 5-HT1A binding assay
The binding assay for D1 dopaminergic receptors was
that described by Trampus et al. [33]. Corpora striate
were homogenised in 100 vol. (w/v) ice cold 50 mM
Tris–HCl buffer (pH 7.4 at 25 °C) using a Polytron
PT10 (setting 5 for 20 s). Homogenates were centrifuged
twice for 10 min at 20 000×g with resuspension of the
pellet in fresh buffer. The final pellet was resuspended in
50 mM ice cold Tris–HCl pH 7.4. containing (mM): 120
NaCl, 5 KCl, 2 CaCl2 and 1 MgCl2. Each assay tube
contained [3H]SCH-23390 and [3H]spiperone to achieve
a final concentration of 1 nM, and resuspended mem-
branes (3 mg fresh tissue). The tubes were incubated for
45 min at 25 °C and the incubation was terminated by
rapid filtration under vacuum through Whatman GF/B
glass fibre filters. The filters were washed three times
with 5 mL ice-cold 50 mM Tris–HCl buffer (pH 7.4 at
25 °C).The radioactivity bound to the filters was mea-
sured by a liquid scintillation counter. Specific [3H]SCH-
23390 and [3H]spiperone binding was defined as the
difference between binding in the absence or in the
Radioligand binding assay was performed following a
published procedure [31]. Cerebral cortex from male
Wistar rats (150–200 g) was homogenised in 20 volumes
of ice-cold Tris–HCl buffer (50 mM, pH 7.7) with a
Brinkmann Polytron (setting 5 for 15 s), and the ho-
mogenate was centrifuged at 48 000×g for 10 min. The
resulting pellet was then resuspended in the same buffer,
incubated for 10 min at 37 °C, and centrifuged at
48 000×g for 10 min. The final pellet was resuspended (8
mg wet weight) in 250 mL of 50 mM Tris–HCl, pH
7.7 containing
1
nM [3H]-8-OHꢀDPAT ([3H]-8-
OHꢀDPAT), membranes from 8 mg (wet weight) of
tissue and the compounds to be tested. The tubes were
incubated for 30 min at 25 °C, and the incubations were
terminated by vacuum filtration through Whatman GF/
B filters. The filters were washed twice with 5 mL of
ice-cold Tris–HCl buffer, and the radioactivity bound
to the filters was measured by liquid scintillation spec-
trometry. Specific [3H]-8-OHꢀDPAT binding was

G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 873–886
885
presence of 10 mM SCH-23390 and 1 mM (+)-bu-
taclamol, respectively.
Acknowledgements
This work was supported by a grant from Regione
Campania ai sensi della L. R. 31 dicembre 1994, No. 41,
art. 3, 1 comma. The NMR spectral data were provided
by Centro di Ricerca Interdipartimentale di Analisi
Strumentale, Universita` degli Studi di Napoli ‘Federico
II’. The assistance of the staff is gratefully appreciated.
5.2.4. h1 adrenergic binding assay
The procedure used in the radioligand binding assay
has been reported in detail by Varani et al. [34]. Brain
cortex was homogenised in 30 vol. (w/v) ice-cold 50 mM
Tris–HCl buffer, (pH 7.4 at 25 °C) using a Polytron
PT10 (setting 5 for 20 s). Homogenates were centrifuged
twice for 20 min at 40 000×g with resuspension of the
pellet in fresh buffer. The final pellet was resuspended in
50 mM ice-cold Tris–HCl, 140 mM NaCl (pH 7.4 at
25 °C) containing [3H]prazosin to achieve a final con-
centration of 1 nM, resuspended membranes from 5 mg
fresh tissue and the compounds to be tested. The tubes
were incubated for 1 h at 25 °C and the incubation was
terminated by rapid filtration under vacuum through
Whatman GF/B glass fibre filters. The filters were
washed three times with 5 mL ice-cold 50 mM Tris–
HCl, buffer (pH 7.4 at 25 °C). The radioactivity bound
to the filters was measured by a liquid scintillation
counter. Specific [3H]prazosin binding was defined as the
difference between binding in the absence or in the
presence of 10 mM phentolamine.
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