A convenient synthesis by microwave heating and pharmacological evaluation of novel benzoyltriazole and saccharine derivatives as 5-HT1A receptor ligands

Article abstract of DOI:10.1016/S0928-0987(02)00045-3

A series of novel 1,2,3-4-benzoyltriazole and saccharine derivatives were designed and synthesized by microwave heating. They were evaluated on a battery of receptors, including serotonin 5-HT_1A, 5-HT_2A and 5-HT_2C, and the most interesting compounds were further evaluated on dopaminergic D₁, D₂ and adrenergic α₁, α₂ receptors. Conventional and microwave heating of the reactions were compared. Synthesis by microwave heating gave the desired compounds in better yields than those obtained by conventional heating. The overall times for the syntheses were considerably reduced. All compounds displayed moderate affinity for 5-HT_1A receptor. The most interesting compound 33 showed a high affinity (K_i=93 nM) which was combined with no affinity on the other receptors considered.

Full text of DOI:10.1016/S0928-0987(02)00045-3

European Journal of Pharmaceutical Sciences 16 (2002) 15–28
www.elsevier.nl/locate/ejps
A convenient synthesis by microwave heating and pharmacological
evaluation of novel benzoyltriazole and saccharine derivatives as 5-HT1A
receptor ligands
a
a
a
a
Giuseppe Caliendoa , , Ferdinando Fiorino , Elisa Perissutti , Beatrice Severino , Daniela Scolaro ,
*
Stefania Gessib, Elena Cattabrigab, Pier Andrea Boreab, Vincenzo Santagadaa
aDipartimento di Chimica Farmaceutica e Tossicologica di Napoli ‘Federico II’, Via D. Montesano, 49-80131 Napoli, Italy
b
`
Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Universita degli Studi di Ferrara,
Via Fossato di Mortara 17-44100 Ferrara, Italy
Received 3 October 2001; received in revised form 22 March 2002; accepted 8 April 2002
Abstract
A series of novel 1,2,3-4-benzoyltriazole and saccharine derivatives were designed and synthesized by microwave heating. They were
evaluated on a battery of receptors, including serotonin 5-HT1A, 5-HT2A and 5-HT2C, and the most interesting compounds were further
evaluated on dopaminergic D1, D2 and adrenergic a1, a2 receptors. Conventional and microwave heating of the reactions were compared.
Synthesis by microwave heating gave the desired compounds in better yields than those obtained by conventional heating. The overall
times for the syntheses were considerably reduced. All compounds displayed moderate affinity for 5-HT1A receptor. The most interesting
compound 33 showed a high affinity (Ki593 nM) which was combined with no affinity on the other receptors considered.
2002
Elsevier Science B.V. All rights reserved.
Keywords: 5-HT Receptors; Microwave; Medicinal chemistry; Benzoyltriazole; Saccharine
1. Introduction
treatment of anxiety states or depression. For the past
several years we have focused our attention on these
receptor ligands in a search for structural parameters which
determine their affinity and selectivity at 5-HT1A and/or
5-HT2A binding sites (Caliendo et al., 1993, 1995, 1996,
1999, 2000). In particular we studied new arylpiperazine
derivatives and binding studies showed that several of
these compounds had a good affinity for serotoninergic
5-HT1A receptors and low affinity for serotoninergic 5-
HT2A, 5-HT2C, dopaminergic D1, D2 and adrenergic a1,
a2 receptors.
To continue our SAR studies with those class of ligands,
we focused our attention on substitution at the piperazine
moiety (Lopez-Rodriguez et al., 2001) and on the substitu-
tion of the heterocycle ring system (Caliendo et al., 1999;
Malinka et al., 1995) in order to determine whether such
molecular modifications affect the 5-HT1A receptor affinity
and selectivity compared to 5-HT2A and 5-HT2C receptor.
Here we report the synthesis by microwave heating and
affinities for 5-HT1A, 5-HT2A and 5HT2C receptors ob-
tained by radioligand binding studies of new 1- and 2-(4-
R-piperazinylalkyl)-4-benzoyl-1,2,3-triazole (15a–23a and
Serotonin (5-hydroxytryptamine, 5-HT) has been impli-
cated in a variety of physiological and pathophysiological
processes. Many therapeutically useful drugs have 5-HT
receptors as targets (Fletcher et al., 1993; Roth, 1994; Van
Wijngaarden and Soudin, 1997). Within the seven main
classes of 5-HT receptors, 5-HT1A and 5-HT2A subtypes
have been generally accepted as those involved in regula-
tion of mental disorders such as depression, anxiety or
schizophrenia (Fletcher et al., 1993; Albinsson et al.,
1994). Several structurally different compounds possessed
high affinity and selectivity for 5-HT1A receptors (Romero
et al., 1992). Among these some piperazine compounds
such buspirone (Bakish et al., 1998), gepirone (Chojnacka-
Wojcik et al., 1991), or ipsapirone (Malinka et al., 1995)
showed selective agonist or partial agonist activity for
5-HT1A receptors and has proved to be effective for the
*
Corresponding author. Tel.: 139-81-678-648; fax: 139-81-678-649.
E-mail address: caliendo@unina.it (G. Caliendo).
0928-0987/02/$ – see front matter
PII: S0928-0987(02)00045-3
2002 Elsevier Science B.V. All rights reserved.

16
G. Caliendo et al. / European Journal of Pharmaceutical Sciences 16 (2002) 15–28
15b–23b) and 2-(4-R-piperazinylalkyl)-1,2-benzisothia-
zolin-3(2H)-one 1,1-dioxide derivatives (26–34).
Conventional heating (oil bath) and microwave heating
of the reactions were compared.
In both lead identification and lead optimisation pro-
cesses there is an acute need for new organic small
molecules. Traditional methods of organic synthesis are
orders of magnitude too slow to satisfy the demand for
these compounds. The fields of combinatorial and auto-
mated medicinal chemistry (i.e. the simultaneous or paral-
lel synthesis of libraries) (Cortese, 1996; Chaiken and
Janda, 1996; Wilson and Czarnik, 1997) have been de-
veloped to meet the increasing requirement of new com-
pounds for drug discovery, within these fields, speed is of
the essence. The application of microwave energy to
organic compounds for conducting 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 re-
actions (Santagada et al., 2001), shortening reaction times
and/or improving yields, as well as to promote new
reactions (Larhed and Hallberg, 2001). In general, the
reasons for rate enhancements by microwave heating are
not fully understood, and some authors have postulated a
specific ‘nonthermal microwave effect’ for those effects
that could not be rationalized as a simple consequence of
superheated solvents and higher reaction temperatures
(Stadler and Oliver Kappe, 2001).
2. Materials and methods
2.1. Chemistry
2.1.1. General procedures
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. Melting points 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
visualized 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 60.4% of the theoretical values. The structure were
verified spectroscopically by proton 1H NMR. Spectra
were recorded on Bruker AM-500 instruments. Chemical
shifts are given as d with references to Me4Si as internal
standard. Mass spectra of the final products were per-
formed on LCQ Thermoquest-ion trap mass spectrometry.
Moreover, when carrying out a reaction in a microwave
oven, the use of a solvent can sometimes be avoided which
is important in order to make the synthesis more en-
vironmentally friendly (‘green chemistry’). The commer-
cially available instrument used herein features a built-in
magnetic stirrer, direct temperature control of the reaction
mixture with the aid of fluoroptic probes and software that
enables online temperature control by regulation of micro-
wave power output (see Section 2).
2.1.2. Synthesis
The synthesis of some intermediates were reported in
Figs. 1 and 2. The synthesis of final compounds 15a–23a,
15b–23b and 26–34 were showed in Figs. 3 and 4. The
synthesis were performed using a microwave oven
(ETHOS 1600, Milestone) especially designed for organic
synthesis. The experimental conditions used in our work
were similar to those used by conventional heating, with
the same amount of starting reagent and volume of solvent.
All reactions were performed in closed vessels. Synthesis
by microwave heating gave the desired compounds in
This article reports the synthesis, by microwave heating,
of new arylpiperazine derivatives and pharmacological
evaluations as selective serotonin receptor ligands.
Fig. 1. Reagents and conditions: (i) SOCl2, toluene, 55 8C, 20 min, 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 Pharmaceutical Sciences 16 (2002) 15–28
17
Fig. 2. Reagents and conditions: (i) SOCl2, toluene, 55 8C, 20 min, mn; (ii) ethyl piperazine-1-carboxylate, K2CO3, NaI, DMF, reflux, 70 min, mn; (iii)
KOH, water, methanol, reflux, 1 h, mn.
Fig. 3. Reagent and conditions: (i) CrO3 /H2SO4; (ii) NaN3, dimethylacetamide, 55 8C, 40 min, mn; (iii) Br(CH2)nCl, K2CO3, n-butanol, reflux, 1 h, mn;
(iv) N-R-substituted-piperazine, K2CO3, NaI, DMF, reflux, 1 h, mn.
Fig. 4. Reagent and conditions: (i) Br(CH2)nCl, K2CO3, DMF, reflux, 20 min, mn; (ii) N-R-piperazine, K2CO3, NaI, DMF, reflux, 1 h, mn.

18
G. Caliendo et al. / European Journal of Pharmaceutical Sciences 16 (2002) 15–28
better yields than those obtained by conventional heating.
The overall times for the syntheses were considerably
reduced.
4-R-substituted-piperazines to obtain final products (Fig.
4). The free bases were converted into their hydrochloride
salts by usual method and purified by crystallization from a
mixture of methyl alcohol and diethyl ether
All the 4-R-substituted piperazines were commercially
available except the 1-(2-thienylmethyl 5-X)-piperazines
(4a,b) and the intermediate 1-(1,2,3,4-tetrahydronaphth-1-
yl)piperazine (8). The synthetic approach employed for the
preparation of 4a,b is summarized in Fig. 1. Treatment of
2-thiophenmethanol (1) with thionyl chloride in toluene
gives the corresponding 2-chloromethyl thiophene 2a. On
the contrary intermediate 2b was commercially available.
The condensation of ethyl piperazine-1-carboxylate with
the corresponding 2a or 2b give, respectively, the inter-
mediates 3a and 3b. Saponification and decarboxylation
provided the desired intermediates 4a and 4b.
The associated physical data of final compounds were
reported in Tables 1 and 2. The parameters of time, power,
temperature used for the microwave heating and the
conditions used by conventional heating were as reported
in Table 3. Analytical purification of each final product
was obtained by chromatography on silica gel column and
further by crystallization from the appropriate solvent. All
new compounds gave satisfactory elemental analyses
1
(C,H,Cl,N,S) and were characterized by H NMR (Tables
4 and 5) and mass spectrometry (LCQ–MS Thermoquest-
ion trap).
Intermediate 1-(1,2,3,4-tetrahydronaphth-1-yl)piperazine
(8) was obtained by using the same conditions, which were
employed for the preparation of compounds 4a,b starting
from 1,2,3,4-tetrahydro-1-naphthol (5) with thionyl chlo-
ride in toluene to give the corresponding 1,2,3,4-tetrahy-
dro-1-chloronaphthyl (6). The condensation of ethyl
piperazine-1-carboxylate with 6, followed by saponifica-
tion and decarboxylation, provided the desired compound
8 (Fig. 2).
2.1.2.1. 2-Chloromethyl thiophene (2a)
Thionylchloride (26 ml, 0.35 mol) was added at 15 8C to
a solution of 2-thiophenmethanol (1) (0.24 mol) in toluene
(200 ml). The mixture was stirred at room temperature for
30 min and then introduced into the reaction vessel and the
desired parameters (microwave power, temperature and
time) were set as reported in Table 3. 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.
The synthesis of final benzoyltriazole derivatives were
obtained by microwave heating as described in Fig. 3.
Oxidation of the phenylethynylcarbinol (9) with chromium
trioxide and concentrated sulphuric acid produced phenyl
ethynyl ketone (10)which was successively treated with
NaN3 in anhydrous dimethylacetamide providing the 4-
benzoyl-1,2,3-triazole (11). Alkylation of 11 in n-butanol
in the presence of potassium carbonate with appropriate
1-bromo-2-chloroethane, 1-bromo-3-chloropropane or 1-
bromo-4-chlorobutane, afforded a mixture of the expected
1-, 2-, 3-chloroalkyl benzotriazole isomers (12–14). The
three isomers were separated by chromatography on a
silica gel column using diethyl ether/n-hexane as eluents.
The faster moving 2-substituted isomers (13) were col-
lected with a higher yield with respect to the slower
moving 1-substituted isomers (12). The yield of inter-
mediate moving 3-substituted isomers (14) were so low
that they were not further considered. Condensation re-
action of the general obtained compounds 12 or 13 with
desired 4-R-substituted-piperazine in DMF in the presence
of K2CO3, NaI, under reflux, give, respectively, the final
1-substituted 15a–23a or 2-substituted compounds 15b–
23b. The free bases were converted into their corre-
sponding hydrochloride salts by usual methods. All the
final products were further purified by crystallization from
a mixture of diethyl ether and ethyl alcohol.
2.1.2.2. 1-(2-Thienylmethyl-5-X)-4-carbetoxypiperazine
(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 3.
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 hydrogen chloride 2 N 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 (yield 92%) as an oil.
1
(3a) H NMR (CDCl3) d 1.23 (t, 3H, J57 Hz), 2.38 (s,
NH), 2.42 (m, 4H), 2.80 (m, 4H), 3.52 (d, 2H, J57.5 Hz),
4.10 (q, 2H, J57 Hz), 6.81 (d, 1H, J57.5 Hz), 6.84 (t, 1H,
J57.5 Hz), 7.12 (t, 1H, J57.5 Hz). Similar 1H NMR
occur in intermediate 3b.
Saccharine derivatives (26–34) were obtained by using
the same general conditions which were employed for the
preparation of 4-benzoyl-1,2,3-triazole derivatives starting
from saccharine nucleus which was alkylated with 1-
bromo-2-chloroethane, 1-bromo-3-chloropropane or 1-
bromo-4-chlorobutane and then condensed with opportune
2.1.2.3. 1-(2-Thienylmethyl-5-X)-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

G. Caliendo et al. / European Journal of Pharmaceutical Sciences 16 (2002) 15–28
19
Table 1
Physicochemical properties of 4-benzoyl-1,2,3-triazole derivatives (15a–23a) and (15b–23b)
R
n
Formulaa
MW
1-Substituted 4-benzoyl-1,2,3-triazoles
2-Substituted 4-benzoyl-1,2,3-triazoles
Compoundb
m.p. (8C)
Yieldc (%)
Compoundb
m.p. (8C)
Yieldc (%)
2
2
C20H22N6O?HCl
C19H21N7O?HCl
398.89
399.88
15a
16a
178–180
203–205
80
79
15b
16b
200–202
238–240
90
85
2
3
3
3
3
3
4
C25H29N5O?HCl
C21H24N6O?HCl
C20H23N7O?HCl
C26H31N5O?HCl
C23H24F3N5O?HCl
C21H24ClN5OS?HCl
C23H24F3N5O?HCl
452.00
412.92
413.91
466.02
479.93
466.43
493.96
17a
18a
19a
20a
21a
22a
23a
206–208
228–230
200–202
227–229
184–186
239–241
182–184
70
78
72
71
72
70
73
17b
18b
19b
20b
21b
22b
23b
191–193
213–214
202–204
197–199
168–172
219–220
162–164
84
92
85
85
86
82
88
a Satisfactory microanalyses obtained: C, H, N, Cl, F, S values are within 60.4% of the theoretical ones.
b All compounds were crystallized by methyl alcohol and diethyl ether.
c Yield related to last step are referred to microwave irradiation.

20
G. Caliendo et al. / European Journal of Pharmaceutical Sciences 16 (2002) 15–28
Table 2
Physicochemical properties of 1,2-benzisothiazolin-3(2H)-one 1,1-dioxide derivatives (26–34)
Compounda
R
n
Formulab
MW
m.p. (8C)
Yieldc (%)
26
2
C23H27N3O3S?HCl
462.00
224–225
85
27
28
2
2
C18H21N3O3S2?HCl
427.96
475.91
242–243
240–242
80
87
C20H20F3N3O3S?HCl
29
3
C24H29N3O3S?HCl
476.03
213–215
89
30
31
3
3
C19H23N3O3S2?HCl
441.99
489.94
233–235
220–222
82
94
C21H22F3N3O3S?HCl
32
33
34
4
4
4
C25H31N3O3S?HCl
C20H25N3O3S2?HCl
C22H24F3N3O3S?HCl
490.06
456.02
503.97
222–224
197–199
111–112
92
95
90
a All compounds were crystallized by methyl alcohol and diethyl ether.
b Satisfactory microanalyses obtained: C, H, N, Cl, F, S values are within 60.4% of the theoretical ones.
c Yield related to last step are referred to microwave irradiation.

G. Caliendo et al. / European Journal of Pharmaceutical Sciences 16 (2002) 15–28
21
Table 3
Conventional heating versus microwave irradiation for intermediates (2a, 3a,b, 4a,b, 6–8, 10–13, 25) and final compounds (15a–23a, 15b–23b, 26–34)a
Compound
Conventional heatingb
Microwave irradiation
Yield (%)
Time (h)
Temp. (8C)
Yield (%)
Time (min)
Power (W)
Temp. (8C)
5
10
5
80
400
200
80
2a
3a
3b
4a
4b
6
85
1
24
24
12
12
1
55
96
55
80
5
54
63
74
63
87
63
Reflux
Reflux
Reflux
Reflux
55
80
92
95
90
98
90
50
5
5
400
200
80
120
110
80
50
5
5
400
200
80
120
110
80
50
5
5
400
200
80
110
110
80
50
5
5
400
200
80
110
110
10
5
5
400
200
80
55
80
7
24
Reflux
60
5
5
400
200
80
120
110
80
8
84
85
95
–
Reflux
95
50
5
400
200
120
110
10c
11
7
4
–
–
5
–
–
80
12
25
98
30
5
5
400
200
80
55
80
12
60–70d
65–80d
30–38d
42–62d
51–71d
55–72d
20
20
24
24
2
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
70–80d
75–90d
70–80d
82–95d
80–92d
80–95d
50
5
5
400
200
80
120
110
80
13
50
5
5
400
200
80
120
110
80
15a–23a
15b–23b
25
50
5
5
400
200
80
120
110
80
50
5
5
400
200
80
120
110
80
10
5
5
400
200
80
120
110
80
26–34
24
50
5
400
200
120
110
a 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 2.
b Oil bath.
c Compound obtained by only conventional synthesis.
d Ranging between the reported percentage.

22
G. Caliendo et al. / European Journal of Pharmaceutical Sciences 16 (2002) 15–28
Table 4
1H NMR data of 4-benzoyl-1,2,3-triazole derivatives (15a–23a) and (15b–23b)
Compound
1-Substituted 4-benzoyl-1,2,3-triazoles
Compound
2-Substituted 4-benzoyl-1,2,3-triazoles
Solvent
1H NMR (d )
Solvent
1H NMR (d )
15a
DMSO-d6
3.35 (m, 4H, 2CH2-pip), 3.73 (t, J57.5 Hz,
2H, CH2N-pip), 3.89 (m, 4H, 2CH2-pip),
5.17 (t, J57.5 Hz, 2H, CH2N-triazole),
6.94–8.34 (mm, 9H, ArH), 9.15 (s, 1H, H-triaz)
15b
DMSO-d6
3.20 (m, 4H, 2CH2-pip), 3.74 (t, J57.5 Hz,
2H, CH2N-pip), 3.92 (m, 4H, 2CH2-pip),
5.26 (t, J57.5 Hz, 2H, CH2N-triaz),
7.00–8.29 (mm, 9H, ArH), 8.62 (s, 1H, H-triaz)
16a
17a
DMSO-d6
3.23 (m, 4H, 2CH2-pip), 3.70 (t, J57.5 Hz,
2H, CH2N-pip), 3.88 (m, 4H, 2CH2-pip),
5.17 (t, J57.5 Hz, 2H, CH2N-triaz),
16b
17b
DMSO-d6
3.15 (m, 4H, 2CH2-pip), 3.62 (t, J57.5 Hz,
2H, CH2N-pip), 3.92 (m, 4H, 2CH2-pip),
5.20 (t, J57.5 Hz, 2H, CH2N-triaz),
6.86–8.56 (mm, 8H, ArH), 9.14 (s, 1H, H-triaz)
6.93–8.22 (mm, 8H, ArH), 8.60 (s, 1H, H-triaz)
CDCl3
1.92–2.00 (m, 3H, CHN-pip and CH2-Tetrahy),
2.36 (t, J57.5 Hz, 2H, CH2N-pip),
2.58–2.74 (m, 8H, 4CH2-pip),
CDCl3
1.92–2.00 (m, 3H, CHN-pip and CH2-Tetrahy),
2.36 (t, J57.5 Hz, 2H, CH2N-pip),
2.58–2.74 (m, 8H, 4CH2-pip),
2.89–3.26 (m, 4H, 2CH2-Tetrahy),
2.89–3.26 (m, 4H, 2CH2-Tetrahy),
4.54 (t, J57.5 Hz, 2H, CH2N-triaz),
6.95–8.28 (mm, 9H, ArH), (s, 1H, H-triaz)
4.54 (t, J57.5 Hz, 2H, CH2N-triaz),
6.95–8.28 (mm, 9H, ArH), 9.16 (s, 1H, H-triaz)
18a
19a
20a
DMSO-d6
DMSO-d6
CDCl3
2.48 (m, 2H, CH2CH2CH2), 3.17 (m, 4H, 2CH2-pip),
3.22 (t, J57.5 Hz, 2H, CH2N-pip),
18b
19b
20b
DMSO-d6
DMSO-d6
CDCl3
2.49 (m, 2H, CH2CH2CH2), 3.18 (m, 4H,
2CH2-pip), 3.25 (t, J57.5 Hz, 2H, CH2N-pip),
3.90 (m, 4H, 2CH2-pip), 5.03 (t, J57.5 Hz,
2H, CH2N-triaz), 6.95–8.18 (mm, 9H, ArH),
8.61 (s, 1H, H-triaz).
3.82 (m, 4H, 2CH2-pip), 5.28 (t, J57.5 Hz,
2H, CH2N-triaz), 6.90–8.35 (mm, 9H, ArH),
9.11 (s, 1H, H-triaz)
2.55 (m, 2H, CH2CH2CH2), 3.12 (m, 4H, 2CH2-pip),
3.24 (t, J57.5 Hz, 2H, CH2N-pip),
2.56 (m, 2H, CH2CH2CH2), 3.13 (m, 4H,
2CH2-pip), 3.27 (t, J57.5 Hz, 2H, CH2N-pip),
3.52–3.67 (m, 4H, 2CH2-pip), 4.83 (t, J57.5 Hz,
2H, CH2N-triaz), 6.85–8.28 (mm, 8H, ArH),
8.54 (s, 1H, H-triaz)
3.52–3.91 (m, 4H, 2CH2-pip), 4.76 (t, J57.5 Hz,
2H, CH2N-triaz), 6.86–8.55 (mm, 8H, ArH),
9.09 (s, 1H, H-triaz)
1.59 (q, J57.5 Hz, 2H, CH2CH2CH2),
1.93–2.08 (m, 3H, CHN-pip and CH2-Tetrahy),
2.22 (t, J57.5 Hz, 2H, CH2N-pip),
2.47–2.61 (m, 8H, 4CH2-pip), 2.67–2.76 (m, 4H,
2CH2-Tetrahy), 3.87 (t, J57.5 Hz, 2H,
CH2N-triaz), 6.93–8.27 (mm, 9H, ArH),
9.19 (s, 1H, H-triaz)
1.59 (q, J57.5 Hz, 2H, CH2CH2CH2),
1.93–2.08 (m, 3H, CHN-pip and CH2-Tetrahy),
2.22 (t, J57.5 Hz, 2H, CH2N-pip),
2.47–2.61 (m, 8H, 4CH2-pip),
2.67–2.76 (m, 4H, 2CH2-Tetrahy), 3.87 (t, J57.5 Hz,
2H, CH2N-triaz), 6.93–8.27 (mm, 9H, ArH),
9.19 (s, 1H, H-triaz).
21a
22a
DMSO-d6
DMSO-d6
2.57 (m, 2H, CH2CH2CH2), 3.19 (m, 4H,
2CH2-pip), 3.37 (t, J57.5 Hz, 2H, CH2N-pip),
3.67–4.08 (m, 4H, 2CH2-pip), 4.75 (t, J57.5 Hz,
2H, CH2N-triaz), 7.24–8.35 (mm, 9H, ArH),
9.12 (s, 1H, H-triaz)
21b
22b
DMSO-d6
DMSO-d6
2.58 (m, 2H, CH2CH2CH2), 3.21 (m, 4H,
2CH2-pip), 3.36 (t, J57.5 Hz, 2H, CH2N-pip),
3.66–4.05 (m, 4H, 2CH2-pip), 4.80 (t, J57.5 Hz,
2H, CH2N-triaz), 7.24–8.29 (mm, 9H, ArH),
8.56 (s, 1H, H-triaz).
2.48 (m, 2H, CH2CH2CH2), 2.61 (m, 4H,
2CH2-pip), 3.28 (t, J57.5 Hz, 2H,
2.51 (m, 2H, CH2CH2CH2), 2.61 (m, 4H,
2CH2-pip), 3.31 (t, J57.5 Hz, 2H,
CH2CH2CH2N-pip), 3.32–3.80 (m, 4H, 2CH2-pip),
4.54 (s, 2H, CH2N-pip), 4.76 (t, J57.5 Hz, 2H,
CH2N-triaz), 7.27–8.39 (mm, 7H, ArH),
9.13 (s, 1H, H-triaz)
CH2CH2CH2N-pip), 3.35–3.80 (m, 4H,
2CH2-pip), 4.50 (s, 2H, CH2N-pip),
4.83 (t, J57.5 Hz, 2H, CH2N-triaz),
7.27–8.32 (mm, 7H, ArH), 8.60 (s, 1H, H-triaz)
23a
DMSO-d6
1.89–2.08 (m, 4H, CH2CH2CH2CH2),
23b
DMSO-d6
1.94–2.15 (m, 4H, CH2CH2CH2CH2),
3.19 (m, 4H, 2CH2-pip), 3.39 (t, J57.5 Hz,
2H, CH2N-pip), 3.62–4.05 (m, 4H, 2CH2-pip),
4.65 (t, J57.5 Hz, 2H, CH2N-triaz),
3.23 (m, 4H, 2CH2-pip), 3.42 (t, J57.5 Hz,
2H, CH2N-pip), 3.64–4.07 (m, 4H, 2CH2-pip),
4.77 (t, J57.5 Hz, 2H, CH2N-triaz),
7.24–8.35 (mm, 9H, ArH), 9.11 (s, 1H, H-triaz)
7.30–8.32 (mm, 9H, ArH), 8.58 (s, 1H, H-triaz).

G. Caliendo et al. / European Journal of Pharmaceutical Sciences 16 (2002) 15–28
23
Table 5
1H NMR data of 1,2-benzisothiazolin-3(2H)-one 1,1-dioxide derivatives (26–34)
Compound
Solvent
CDCl3
1H NMR (d )
26
1.95–2.10 (m, 3H, CHN-pip and CH2-Tetrahy), 2.32 (t, J57.5 Hz,
2H, CH2N-pip), 2.53–2.71 (m, 8H, 4CH2-pip), 2.87–2.96 (m, 4H,
2CH2-Tetrahy), 3.87 (t, J57.5 Hz, 2H, CH2N-benz),
6.93–8.04 (mm, 8H, ArH)
27
28
29
CDCl3
CDCl3
CDCl3
2.48 (m, 4H, 2CH2-pip), 2.70 (t, J57.5 Hz, 2H, CH2CH2N-pip),
3.34 (m, 4H, 2CH2-pip), 3.80 (s, 2H, CH2N-pip), 4.13 (t, J57.5 Hz,
2H, CH2N-benz), 6.90–7.96 (mm, 7H, ArH)
2.69 (m, 4H, 2CH2-pip), 2.84 (t, J57.5 Hz, 2H, CH2N-pip),
3.23 (m, 4H, 2CH2-pip), 3.97 (t, J57.5 Hz, 2H, CH2N-benz),
7.04–8.07 (mm, 8H, ArH)
1.68 (q, J57.5 Hz, 2H, CH2CH2CH2), 1.90–2.00 (m, 3H,
CHN-pip and CH2-Tetrahy), 2.04 (t, J57.5 Hz, 2H, CH2N-pip),
2.47–2.61 (m, 8H, 4CH2-pip), 2.67–2.76 (m, 4H, 2CH2-Tetrahy),
3.86 (t, J57.5 Hz, 2H, CH2N-benz), 7.03–8.06 (mm, 8H, ArH)
30
31
32
CDCl3
CDCl3
CDCl3
2.02 (q, J57.5 Hz, 2H, CH2CH2CH2), 2.46–2.48 (m, 10H,
4CH2-pip and CH2CH2CH2N-pip), 3.70 (s, 2H, CH2N-pip),
3.84 (t, J57.5 Hz, 2H, CH2N-benz), 6.90–8.06 (mm, 7H, ArH)
2.41 (m, 2H, CH2CH2CH2), 3.26 (m, 4H, 2CH2-pip),
3.68 (t, J57.5 Hz, 2H, CH2N-pip), 3.99 (m, 4H, 2CH2-pip),
4.10 (t, J57.5 Hz, 2H, CH2N-triaz), 7.28–8.51 (mm, 8H, ArH)
1.64 (q, J57.5 Hz, 2H, CH2CH2CH2CH2), 1.67 (t, J57 Hz,
1H, CHN-pip), 1.88 (q, J57.5 Hz, 2H, CH2CH2CH2CH2),
1.97 (q, J57 Hz, 2H, CH2-Tetrahy), 2.41 (t, J57.5 Hz, 2H,
CH2N-pip), 2.50–2.67 (m, 8H, 4CH2-pip), 2.68–2.76 (m,
4H, 2CH2-Tetrahy), 3.80 (t, J57.5 Hz, 2H, CH2N-benz),
7.03–8.07 (mm, 8H, ArH)
33
34
CDCl3
CDCl3
1.59 (q, J57.5 Hz, 2H, CH2CH2CH2CH2), 1.84 (q, J57.5 Hz,
2H, CH2CH2CH2CH2), 2.39 (t, J57.5 Hz, 2H,
CH2CH2CH2CH2N-pip), 2.51 (m, 8H, 4CH2-pip) 3.72 (s, 2H,
CH2N-pip), 3.80 (t, J57.5 Hz, 2H, CH2N-benz),
6.91–8.06 (mm, 7H, ArH)
1.68 (q, J57.5 Hz, 2H, CH2CH2CH2CH2), 1.92 (q, J57.5 Hz,
2H, CH2CH2CH2CH2), 2.43 (t, J57.5 Hz, 2H, CH2N-pip),
2.63 (m, 4H, 2CH2-pip), 3.22 (m, 4H, 2CH2-pip),
3.83 (t, J57.5 Hz, 2H, CH2N-benz), 6.98–8.15 (mm, 8H, ArH)
parameters (microwave power, temperature and time) were
set as reported in Table 3. 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%)
2.1.2.4. 1,2,3,4-Tetrahydro-1-chloronaphtyl (6)
Thionylchloride (26 ml, 0.50 mol) was added at 15 8C to
a solution of 1,2,3,4-tetrahydro-1-naphtol (5) (0.36 mol) in
toluene (300 ml). The mixture was stirred at room
temperature for 30 min and then introduced into the
reaction vessel and the desired parameters (microwave
power, temperature and time) were set as reported in Table
3. 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.
1
or 4b (yield 90%) as an oil. (4a) H NMR(CDCl3) d
2.41(s, NH), 2.48 (m, 4H), 2.84 (m, 4H), 3.60 (d, 2H,
J57.5 Hz), 6.83 (d, 1H, J57.5 Hz), 6.86 (t, 1H, J57.5
1
Hz), 7.15 (t, 1H, J57.5 Hz). Similar H NMR occur in
intermediate 4b.

24
G. Caliendo et al. / European Journal of Pharmaceutical Sciences 16 (2002) 15–28
2.1.2.5. Ethyl-4-(1,2,3,4-tetrahydro-1-naphtyl)piperazine-
1-carboxylate (7)
2.1.2.8. 4-Benzoyl-1H-1,2,3-triazole (11)
To a stirred and heated solution of NaN3 (0.10 mol) in
dimethylacetamide anhydrous (80 ml) was slowly added
phenyl ethynyl ketone 10 (0.10 mol) dissolved in di-
methylacetamide anhydrous (80 ml). The reaction mixture
was introduced into the reaction vessel and the desired
parameters (microwave power, temperature and time) were
set as reported in Table 3. After stirring evaporation of the
solvent under reduced pressure gave a liquid residue which
was diluted with water. The aqueous layer was acidified to
pH 5 with 10% HCl and extracted with ether (33200 ml).
The combined organic layers were dried over anhydrous
Na2SO4. After evaporation of the solvent the solid residue
was purified by crystallization from ethyl alcohol to give
11 (yield 98%). The physical data are in agreement with
those given in Caliendo et al. (1999).
A mixture of 1,2,3,4-tetrahydro-1-chloronaphtyl (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 3.
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 7 as
an oil (yield 90%), 1H NMR (CDCl3) d 1.25 (t, 3H, J57
Hz), 1.6–2.1 (m, 4H), 2.3–2.9 (m, 6H), 3.5 (t, 4H, J56
Hz), 3.8 (t, 1H, J57 Hz), 4.15 (q, 2H, J57 Hz), 7–7.75
(m, 4H).
2.1.2.9. General procedure for preparation of 1- and
2-(chloroalkyl)-4-benzoyl-1,2,3-triazole derivatives (12
and 13)
To a stirred solution of 4-benzoyl-1,2,3-triazole (11)
(0.10 mol) and appropriate 1-bromo-2-chloroethane, 1-
bromo-3-chloropropane or 1-bromo-4-chlorobutane (0.15
mol), in butan-2-one (80 ml) was added anhydrous K2CO3
(0.30 mol). The mixture was introduced into the reaction
vessel and the desired parameters (microwave power,
temperature and time) were set as reported in Table 3.
After cooling the solvent was removed under reduced
pressure. The residue was diluted with H2O and extracted
several times with CHCl3. Evaporation of the solvent in
vacuo provided the crude residue consisting of the ex-
pected triazole isomers. The mixture isomers was sepa-
rated and purified using a silica-gel column chromatog-
raphy (diethyl ether/n-hexane 95:5 as eluent) to give
1-isomer (12) (yield ranging between 80 and 87%) and
2-isomer (13) (yield ranging between 90 and 94%).
1H NMR spectra for all intermediates were consistent
with the proposed structures.
2.1.2.6. (6) 1-(1,2,3,4-Tetrahydronapht-1-yl)piperazine
(8)
Potassium hydroxide (7.00 mol) was slowly added into
the reaction vessel containing a solution of ethyl 4-
(1,2,3,4-tetrahydro-1-naphtyl)piperazine-1-carboxylate (7)
(0.60 mol), water (100 ml), and methanol (400 ml). The
desired parameters (microwave power, temperature and
time) were set as reported in Table 3. 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. 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 or-
ganic layer was dried, and the solvent was evaporated
under vacuum to give an oil 8 (yield 95%), 1H
NMR(CDCl3) d 1.5–2.1 (m, 4H), 2.0 (s, NH), 2.4–2.8 (m,
6H), 2.9 (t, 4H, J56 Hz), 3,75 (t, 1H, J57 Hz), 7–7.8 (m,
4H).
2.1.2.10. General procedure for preparation of 1- and
2-[2-, 3- or 4-[4-(R)1-piperazin-yl]alkyl](4-benzoyl)-
1,2,3-triazoles (15a–23a) and (15b–23b)
A mixture of appropriate 1- or 2-(chloroalkyl)-4-ben-
zoyl-1,2,3-triazole (10) or (11) (0.006 mol) and NaI (0.01
mol) in DMF (50 ml) was stirred under reflux for 30 min.
Then appropriate 4-R-substituted piperazine (0.006 mol)
and anhydrous K2CO3 (0.01) were added. The obtained
reaction mixture was introduced into the reaction vessel
and the desired parameters (microwave power, temperature
and time) were set as reported in Table 3. 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
(diethyl ether/ethanol, 9:1 v/v) to give 1-isomers (15a–
23a) or 2-isomers (15b–23b) in the relative yields pro-
2.1.2.7. Phenyl ethynyl ketone (10)
A solution of chromium trioxide (0.10 mol) in water (30
ml) and concentrated sulphuric acid (8.5 ml) was slowly
added to a stirred and cooled solution of propyl-
ethynylcarbinol 9 (0.15 mol) in acetone (50 ml). The
reaction mixture was carried out 4 8C in an atmosphere of
nitrogen. After stirring for 7 h, water was added to dissolve
the precipitated chromium salts and the product was
extracted with chloroform. Evaporation of the organic
solution gave a yellow solid which was recrystallized from
aqueous methanol to give 16.6 g (85%) of 10 as pale
yellow needles. The physical data are in agreement with
those given in Caliendo et al. (1999).

G. Caliendo et al. / European Journal of Pharmaceutical Sciences 16 (2002) 15–28
25
1
vided in Table 1, while H NMR were reported in Table 4.
from male Wistar rats (150–200 g) was homogenized 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
homogenate was centrifuged at 48 0003g for 10 min. The
resulting pellet was then resuspended in the same buffer,
incubated for 10 min at 37 8C, and centrifuged at 48 0003
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-hydroxy-2-(di-n-propylamino)tetralin ([3H]-
8-OH-DPAT), membranes from 8 mg (wet weight) of
tissue and the compounds to be tested. The tubes were
incubated for 30 min at 25 8C, 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 spectrometry. Specific
[3H]-8-OH-DPAT binding was defined as the difference
between binding in the absence and presence of 5-HT (1
mM). Ki values were calculated from the Cheng–Prusoff
equation (Cheng and Prusoff, 1973): Ki 5 IC50 /1 1
(ligand/Kd), where Kd for [3H]-8-OH-DPAT was 0.8 nM.
Spectral data of the title compounds (15a and 15b) refer to
the hydrochloride salts.
2.1.2.11. General procedure for preparation of 2-(chloro-
alkyl)-1,2-benzisothiazolin-3(2H)-one 1,1-dioxide deriva-
tives (25)
A solution of appropriate 1-bromo-2-chloroethane, 1-
bromo-3-chloropropane or 1-bromo-4-chlorobutane (0.25
mol) in DMF (30 ml) was added dropwise to a stirred
solution of saccharine (24) (0.1 mol) and anhydrous
K2CO3 (0.15 mol) in DMF (50 ml). The obtained mixture
was introduced into the reaction vessel and the desired
parameters (microwave power, temperature and time) were
set as reported in Table 3. After cooling the mixture was
diluted with water and extracted several times with CHCl3.
The combined organic layers were dried and concentrated
in vacuo. The residue was chromatographed by column of
silica gel (ethyl acetate/n-hexane, 9:1 v/v) to give the
general compound 25 as solid (yields ranging between 50
1
and 71%). H NMR spectra for all intermediates were
consistent with the proposed structures.
2.2.1.2. 5-HT2A and 5-HT2C binding assay
2.1.2.12. General procedure for preparation of 2-[2, 3, or
4[4-(R)-1-piperazin-yl]alkyl]-1,2-benzisothiazolin-3(2H)-
one 1,1-dioxide (26–34)
Radioligand binding assays were performed as previous-
ly reported by Claudi et al. (1999). Briefly, frontal cortical
regions of male Wistar rats (150–200 g, Charles River)
were dissected on ice, homogenized in ice-cold buffer
solution (50 mM Tris–HCl, pH 7.7, at 25 8C) and cen-
trifuged at 40 0003g for 10 min. The supernatant was
discarded and the pellet was resuspended in the same
volume of Tris–HCl buffer and incubated at 37 8C for 10
min prior to a second centrifugation. Binding experiments
with [3H]ketanserin (76 Ci/mmol, New England Nuclear)
and [3H]mesulergine (75.8 Ci/mmol, 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 8C, separation of bound
from free radioligand was performed by rapid filtration
through Whatman GF/B glass fiber 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. Ki values were
calculated from the Cheng–Prusoff equation (Cheng and
Prusoff, 1973): Ki 5 IC50 /1 1 (ligand/Kd), where Kd 5
0.85 nM for [3H]ketanserin and Kd 51.9 nM for
[3H]mesulergine.
A mixture of appropriate N-(chloroalkyl)-benzensul-
fimide (25) (0.10 mol) and NaI (0.16 mol) in DMF (50
ml) was stirred under reflux for 30 min. Then the appro-
priate 4-R-substituted piperazine (0.10 mol) and anhydrous
K2CO3 (0.16 mol) were added. The obtained reaction
mixture was introduced into the reaction vessel and the
desired parameters (microwave power, temperature and
time) were set as reported in Table 3. After cooling, the
mixture was filtered, diluted with water (50 ml) and
extracted several times with CHCl3. The organic phase
was dried, concentrated in vacuo and the residue was
purified on a silica-gel column chromatography (diethyl
ether/ethanol, 9.5:0.5 v/v) to give the final compounds
26–34 in the relative yield provided in Table 2. 1H NMR
data are reported in Table 5.
2.1.2.13. Hydrochloride salts: general procedure
The hydrochloride salts were prepared by adding an HCl
ethereal solution to ethanolic solution of free bases.
Recrystallization solvent, formulae and melting points
were reported in Tables 1 and 2. They were obtained as
white crystals.
2.2. Pharmacology
2.2.1. General methods
2.2.1.3. D1 and D2 dopaminergic binding assay
The binding assay for D1 dopaminergic receptors was
described by Trampus et al. (1991). Corpora striatae were
homogenized in 100 vol (w/v) ice cold 50 mM Tris–HCl
buffer (pH 7.4 at 25 8C) using a Polytron PT10 (setting 5
2.2.1.1. 5-HT1A binding assay
Radioligand binding assay was performed following a
published procedure (Dalpiaz et al., 1996) Cerebral cortex
.

26
G. Caliendo et al. / European Journal of Pharmaceutical Sciences 16 (2002) 15–28
for 20 s). Homogenates were centrifuged twice for 10 min
at 20 0003g 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 membranes (3 mg fresh tissue).
The tubes were incubated for 45 min at 25 8C and the
incubation was terminated by rapid filtration under vacuum
through Whatman GF/B glass fiber filters. The filters were
washed three times with 5 ml ice-cold 50 mM Tris–HCl
buffer (pH 7.4 at 25 8C).The radioactivity bound to the
filters was measured 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 presence of 10 mM SCH-23390 and 1 mM (1)-
butaclamol, respectively. Ki values were calculated from
the Cheng–Prusoff equation (Cheng and Prusoff, 1973):
Ki 5 IC50 /1 1 (ligand/Kd), where Kd 50.20 nM for
[3H]SCH-23390 and Kd 50.25 nM for [3H]spiperone.
5 mg fresh tissue and the compounds to be tested. The
tubes were incubated for 1 h at 25 8C and the incubation
was terminated by rapid filtration under vacuum through
Whatman GF/B glass fiber filters. The filters were washed
three times with 5 ml ice-cold 50 mM Tris–HCl, 0.5 mM
EDTA buffer (pH 7.5 at 25 8C). The radioactivity bound to
the filters was measured by a liquid scintillation counter.
Specific [3H]UK binding was defined as the difference
between binding in the absence or in the presence of 10
mM phentolamine. Ki values were calculated from the
Cheng–Prusoff equation (Cheng and Prusoff, 1973): Ki 5
IC50 /1 1 (ligand/Kd), where Kd 50.91 nM for [3H]UK.
3. Results and discussion
Affinity for 5-HT receptors including 5-HT1A, 5-HT2A,
5-HT2C subtypes were determined. Ki (nM) values listed in
Table 6 reveal that the majority of the tested 4-benzoyl-
1,2,3-triazole derivatives showed a moderate to low affini-
ty for 5-HT1A receptors, lower than reference compounds.
Compounds which showed a Ki (nM) values .105 for all
considered receptors were not reported in Table 6. The
above results suggest that both the character of new
substituents on the piperazine ring and the nature and
position of the side chain on the 4-benzoyl-1,2,3-triazole
ring, have only a minor effect on the 5-HT1A affinity. The
only exception was found for compounds 23a and 23b
2.2.1.4. a1-Adrenergic binding assay
The procedure used in the radioligand binding assay has
been reported in detail by Varani et al. (1995). Brain cortex
was homogenized in 30 vol (w/v) ice-cold 50 mM Tris–
HCl buffer, (pH 7.4 at 25 8C) using a Polytron PT10
(setting 5 for 20 s). Homogenates were centrifuged twice
for 20 min at 40 0003g 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 8C)
containing [3H]prazosin to achieve a final concentration 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 8C and the incubation was terminated by rapid
filtration under vacuum through Whatman GF/B glass
fiber filters. The filters were washed three times with 5 ml
ice-cold 50 mM Tris–HCl, buffer (pH 7.4 at 25 8C). 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. Ki values were
calculated from the Cheng–Prusoff equation (Cheng and
Prusoff, 1973): Ki 5 IC50 /1 1 (ligand/Kd), where Kd 5
0.12 nM for [3H]prazosin.
Table 6
Receptor affinities (Ki) of active 4-benzoyl-1,2,3-triazole and 1,2-ben-
zisothiazolin-3(2H)-one 1,1-dioxide derivatives
Compound
Receptor affinity Ki (nM)a
5-HT1A
5-HT2A
5-HT2C
[3H]8-OH-DPAT
[3H]ketanserin
[3H]mesulergine
Benzoyltriazole 1-substituted
18a
20a
21a
23a
26006240
17006150
30006250
600650
.105
.105
.105
.105
.105
42006300
.105
60006500
Benzoyltriazole 2-substituted
18b
20b
21b
22b
23b
14006130
900685
13006110
13006115
285625
27506250
.105
.105
.105
.105
.105
35006220
50006480
.105
55006450
Saccharine derivatives
2.2.1.5. a2-Adrenergic binding assay
29
30
31
32
33
34
300625
42506390
.105
700665
21006215
.105
.105
Binding assays were performed essentially according to
Varani et al. (1999). Brain cortex was homogenized in 30
vol (w/v) ice-cold 50 mM Tris–HCl, using a polytron
PT10 (setting 5 for 20 s). Homogenates were centrifuged
for 20 min at 40 0003g with resuspension of the pellet in
fresh buffer. The final pellet was resuspended in 50 mM
ice-cold Tris–HCl, 0.5 mM EDTA, 10 mM MgCl2 (pH 7.5
at 25 8C). Each assay tube contained [3H]UK to achieve a
final concentration of 1 nM, resuspended membranes from
1000698
400635
166614
9368
.105
.105
42506390
.105
10068
22506210
.105
8-OH-DPAT
Ketanserine
Mesulergine
0.80
–
–
–
0.85
–
–
–
1.90
a Ki value followed by S.E.M.

G. Caliendo et al. / European Journal of Pharmaceutical Sciences 16 (2002) 15–28
27
where the introduction of the m-trifluorophenyl ring sig-
nificantly increased the binding affinity for 5-HT1A re-
ceptors (Ki 5600 and 285 nM, respectively). Almost all the
investigated compounds displayed a low or no affinity for
5-HT2A and 5-HT2C receptors.
5-HT1A affinity. It seems a good idea to focus further
studies on saccharine derivatives using preceding sub-
stituents (Caliendo et al., 1999) on the piperazine ring that
displayed the highest serotoninergic affinity.
The most interesting compounds were saccharine deriva-
tives (26–34). In fact the results reported in Table 6
indicate that several of the considered compounds showed
moderate to high affinity on the 5-HT1A subtype receptors
except compounds with n52 which were all inactive (26,
27 and 28). The structures endowed with highest 5-HT1A
affinity (32, 33 and 34) showed Ki values of 166, 93 and
100 nM, respectively. All these derivatives have a buthyl
(CH2)4 connecting group between the N-saccharine nu-
cleus and N-piperazine ring.
Acknowledgements
This work was supported by a grant from Regione
Campania ai sensi della L.R. 31 dicembre 1994, no. 41,
art. 3, 18 comma. The NMR spectral data were provided by
Centro di Ricerca Interdipartimentale di Analisi Strumen-
`
tale, Universita degli Studi di Napoli ‘Federico II’. The
assistance of the staff is gratefully appreciated.
In particular, compound 33 had the highest binding
affinity and showed the most favourable affinity–selectivi-
ty profile for the 5-HT1A receptor (both 5-HT2A /5-HT1A
and 5-HT2C /5-HT1A Ki ratio were .1075). As mentioned
in Section 1, the most active compounds on 5-HT1A
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