2-{4-[3-(4-Aryl/heteroaryl-1-piperazinyl)propoxy]phenyl}-2H-benzotriaz-oles and their N-oxides as ligands for serotonin and dopamine receptors

Article abstract of DOI:10.1016/S0014-827X(99)00046-4

A small set of 2-{4-[3-(4-aryl/heteroaryl-piperazinyl)propoxy]phenyl}-2H-benzotriazoles and corresponding N-oxides were prepared. The synthesized compounds were able to bind on some serotonin (5-HT(1A), 5-HT(2A)) and dopamine (D₂, D₃

Full text of DOI:10.1016/S0014-827X(99)00046-4

www.elsevier.nl/locate/farmac
Il Farmaco 54 (1999) 402–410
2
-{4-[3-(4-Aryl/heteroaryl-1-piperazinyl)propoxy]phenyl}-2H-
benzotriazoles and their N-oxides as ligands for serotonin and
ꢀ
dopamine receptors
a,
b
b
c
Anna Sparatore *, Mara Goegan , Alfredo Cagnotto , Fabio Sparatore
a
Istituto di Chimica Farmaceutica e Tossicologica, Uni6ersit a` di Milano, Viale Abruzzi 42, I-20131 Milan, Italy
b
Istituto di Ricerche Farmacologiche ‘Mario Negri’, Via Eritrea 62, I-20157 Milan, Italy
c
Dipartimento di Scienze Farmaceutiche, Uni6ersit a` di Geno6a, Viale Benedetto XV 3, I-16132 Genoa, Italy
Received 23 November 1998; accepted 24 March 1999
Abstract
A small set of 2-{4-[3-(4-aryl/heteroaryl-piperazinyl)propoxy]phenyl}-2H-benzotriazoles and corresponding N-oxides were
prepared. The synthesized compounds were able to bind on some serotonin (5-HT1A, 5-HT2A) and dopamine (D , D ) receptors,
2
3
while displaying poor or no affinity for 5-HT1B, 5-HT2C, 5-HT , and 5-HT subtypes. The strong contribution of the N-oxide
3
4
function for the binding on 5-HT1A, D2 and D3 receptors is noteworthy. For 2-{4-[3-[4-(2-methoxyphenyl)-1-piper-
azinyl]propoxy]phenyl}-2H-benzotriazol-1-oxide (4b), the binding constants (K ) were 11.9 (5-HT ) and 10.5 nM (D ). In a
i
1A
3
general pharmacological screening, the 2-{4-[3-(4-phenyl-1-piperazinyl)propoxy]phenyl}-2H-benzotriazole (3a) exhibited only very
weak activities, with the exception of protecting mice from cyanide-induced hypoxia. © 1999 Elsevier Science S.A. All rights
reserved.
Keywords: Benzotriazoles; Benzotriazole-N-oxides; Serotonin receptor ligands; Dopamine receptor ligands
1
. Introduction
the bleeding time in mice. The same compound and its
N-oxide protected mice from potassium cyanide hy-
poxia and, most importantly, exerted good anti-hyper-
cholesterolemic action (see Scheme 1).
Pursuing the study of basic derivatives of 2-(4-
hydroxyphenyl)benzotriazole, we have now prepared a
set of 2-{4-[3-(4-aryl/heteroaryl-1-piperazinyl)propoxy]-
phenyl}benzotriazoles and corresponding N-oxides of
formulas 3 and 4, which should exhibit affinity for
serotonin receptors, beside other possible activities
In a recent note [1] we described the synthesis and the
pharmacological investigation of a set of 2-[4-(dialkyl-
aminoalkoxy)phenyl]benzotriazoles 1 and of the corre-
sponding N-oxides 2. Among the observed activities, it
is worthy to recall the following. At concentrations
ranging from 3 to 10 mM, all tested compounds
strongly inhibited the guinea pig ileum contractions
induced either electrically or by means of several ago-
nists; of particular interest was the antagonism to
leukotriene D4. The 2-[4-(dimethylaminopropoxy)-
phenyl]benzotriazole inhibited platelet aggregation in-
(
Scheme 2).
Actually, the aryl/heteroarylpiperazine moiety is a
well known template for affinity to serotonin receptors
2], but affinity for dopamine and adrenergic a recep-
duced by tromboxane A , PAF and ADP and increased
2
[
1
tors can also be expected [3–5]. Since the structure–ac-
tivity relationships are still rather loosely defined, the
prepared compounds were now tested as ligands for
both serotonin and dopamine receptors. Affinity for a1
adrenergic receptors will be tested later. However, com-
pound 3a was subjected to a large pharmacological
ꢀ
Presented at the ‘XIII Conv. Naz. -Div. Chim. Farm. S.C.I.’,
Paestum, Italy, Sept. 23–27, 1996.
*
E-mail address: sparator@imiucca.csi.unimi.it (A. Sparatore)
Corresponding author. Fax: +39-022-9514197.
0014-827X/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(99)00046-4

A. Sparatore et al. / Il Farmaco 54 (1999) 402–410
403
screening (with about 60 in vitro and in vivo assays) in
order to explore how the pharmacological profile of
compounds of structures 1 and 2 would change just
after the replacement of the dialkylamino head with the
simplest arylpiperazinyl moiety.
The UV spectra were quite close to those of other
2-(4-alkoxyphenyl) derivatives of benzotriazole and
benzotriazole-1-oxide [1] (umax: 223sh, 248, 258sh, 317
nm); the presence of the aryl/heteroarylpiperazine
residue only produced an intensification of the absorp-
tion maximum around 243–248 nm.
2. Chemistry
3
. Experimental
Compounds of formulas 3 and 4 were obtained by
reacting 2-(4-hydroxyphenyl)benzotriazole (and corre-
sponding N-oxide) with the required 3-(aryl/hetero-
arylpiperazinyl)propyl chlorides in ethanolic sodium
hydroxide solution.
The 2-(4-hydroxyphenyl)benzotriazole and the corre-
sponding N-oxide were prepared as already described
Melting points were determined by the capillary
method on a B u¨ chi apparatus and are uncorrected.
The elemental analyses were performed at the Micro-
analytical Laboratory of the Dipartimento di Scienze
Farmaceutiche of Genoa University and the analytical
results for the indicated elements were within 90.3%
of the calculated values.
[
1], while the 3-(tert-amino)propylchlorides were ob-
tained by refluxing the dichloromethane solution of
-bromo-3-chloropropane and aryl/heteroarylpiper-
UV spectra were recorded with a Perkin–Elmer
1
1
Lambda 16 spectrophotometer, H NMR spectra were
azine in a ratio of 1:2.
taken on a Bruker AC 200 spectrometer, using CDCl3
as solvent.
In the case of 2-methoxyphenylpiperazine, the use of
toluene solution and a ratio bromochloropropane/
piperazine of 1.5:1 was found to be more satisfactory.
The structures of the final compounds 3a–d and
3.1. 3-[4-(Aryl/heteroaryl)piperazin-1-yl]propyl
4a–d were supported by the elemental analytical results
chlorides
and by spectral data.
1
The H NMR spectra did not exhibit any unusual
To a boiling solution of aryl/heteroarylpiperazine (10
mmol) in 7 ml of dichloromethane, 772 mg (5 mmol) of
1-bromo-3-chloropropane was added dropwise. The
mixture was further refluxed for about 2 h monitoring
the reaction progression through TLC on silica (eluent:
dichloromethane, methanol and conc. aqueous ammo-
nia, 20:3:0.5 v/v). After cooling, the solvent was thor-
oughly removed under reduced pressure and the residue
was taken up with dry ether to separate the piperazine
hydrobromide.
feature, thus only the spectra of compounds 3b and 4b
are described as examples: 3b (CDCl ): l 8.27 and 7.07,
J=9.1 Hz (2 pseudo doublets, 2H+2H, p-phenylene),
.93, J=6.59 and 3.07 Hz and 7.41, J=6.57 and 3.09
Hz (2 pseudo dd, 2H+2H, benzotriazole), 7.03–6.80
m, 4H, 2-methoxyphenyl), 4.14, J=6.26 Hz (t, 2H,
O–CH –), 3.87 (s, 3H, OCH ), 3.13 and 2.70 (2
pseudo s, 4H+4H, 4CH piperazine), 2.64, J=7.32 Hz
t, 2H, C–CH –N), 2.07, J=6.73 Hz (quint, 2H, –C–
3
7
(
–
2
3
2
(
2
CH –C–). 4b (CDCl ): l 8.05 and 7.09, J=9.16 Hz (2
pseudo doublets, 2H+2H, p-phenylene), 7.79, J=8.20
The ether solution was evaporated to dryness and the
residue chromatographed on silica using dichloro-
methane with an increasing amount of methanol (2–
10%) as eluent.
The title compound was eluted as first fraction (50–
66% yield), followed by 1,3-bis-(4-aryl-1-piper-
azinyl)propane. Using dichloromethane plus methanol
(10%) and conc. aqueous ammonia (1%) the unreacted
arylpiperazine was recovered.
2
3
Hz (pseudo t, 2H, H and H benzotriazole N-oxide),
5
6
7
7
.52–7.30 (m, 2H, H and H benzotriazole N-oxide),
.04–6.80 (m, 4H, 2-methoxyphenyl), 4.14, J=6.27 Hz
4 7
(
t, 2H, –O–CH –), 3.87 (s, 3H, OCH ), 3.12 and 2.70
2
3
(
2 pseudo s, 4H+4H, 4CH piperazine), 2.63, J=7.28
2
Hz (t, 2H, C–CH –N); 2.06, J=6.64 Hz (quint, 2H,
2
–
C–CH –C–).
2
Scheme 1.

404
A. Sparatore et al. / Il Farmaco 54 (1999) 402–410
Scheme 2.
4
. Pharmacology
3.2. 3-[4-(2-Methoxyphenyl)piperazin-1-yl]propyl
chloride
4.1. Binding assays
To a boiling solution of 1-bromo-3-chloropropane
3.52 g, 22.4 mmol) in 12 ml of toluene, a solution of
Experiments were performed on compounds 3a–d
and 4a–d to establish their ability to displace specific
(
1
4
-(2-methoxyphenyl)piperazine (2.86 g, 14.9 mmol) in
.5 ml of toluene was added dropwise.
radioligands from serotonin (5-HT , 5-HT , 5-HT ,
1A
1B
2A
5
-HT , 5-HT , 5-HT ) and dopamine (D , D )
2C 3 4 2 3
The mixture was refluxed for about 80 min, monitor-
receptors.
ing
dichloromethane, methanol, conc. aqueous ammonia;
0:3:0.3 v/v). The reaction mixture was cooled in ice
the
reaction
through
TLC
on
silica
Male CRL:CD(SD)BR-COBS rats (125–150 g,
Charles River, Calco, Italy) and male Cri:(HA)BR al-
bino guinea pigs (180–200 g, Charles River) were killed
by decapitation and their brains were rapidly dissected
into the various areas (rat hippocampus for 5-HT ; rat
(
2
and the 1-(2-methoxyphenyl)piperazine hydrobromide
was filtered and washed with toluene and then with dry
ether.
The solution was evaporated to dryness under re-
duced pressure leaving 2.06 g (calculated 2 g) of an oily
product which, on cooling in a freezer, crystallizes (m.p.
1A
striatum for 5-HT1B and D ; rat cortex for 5-HT ,
2
2A
5
-HT , 5-HT ; rat olfactory tubercle for D ; guinea pig
2C 3 3
striatum for 5-HT ). Tissues were homogenized in
4
about 50 volumes of ice-cold Tris–HCl, 50 mM, pH 7.4
or Hepes-HCl 50 mM, pH 7.4, for 5-HT4 and D3
47–49°C). The TLC showed the presence of only a very
(
slight amount of the 1,3-bis-(4-substituted-piper-
azinyl)propane, therefore the compound was used for
the next step without further purification.
receptors) using an Ultra Turrax TP-1810 (2×20 s),
and homogenates were centrifuged at 50 000 g for 10
min (Beckman Avanti J-25 centrifuge). Each pellet was
resuspended in the same volume of fresh buffer, incu-
bated at 37°C for 10 min and centrifuged again at
50 000 g for 10 min. The pellet was then washed once
by resuspension in fresh buffer and centrifuged as
before.
3.3. 2-{4-[3-(4-Aryl/heteroaryl-piperazin-1-yl)-
propoxy]phenyl}-2H-benzotriazoles and corresponding
N-oxides (3a–d, 4a–d)
To
a
solution of 10 mmol of 2-(4-hydroxy-
Just before the binding assays [6–8] the pellet was
resuspended in the appropriate incubation buffer (final
phenyl)benzotriazole (or corresponding N-oxide) in 11
ml of ethanol, 410 mg (ꢀ10 mmol) of sodium hydrox-
ide was added. The solution was boiled under reflux
and under a stream of nitrogen and 10.8 mmol of
-[4-(aryl/heteroaryl)piperazin-1-yl]propyl chloride was
added. The mixture was refluxed for about 2 h monitor-
ing the reaction through TLC on silica
dichloromethane, methanol, conc. ammonia; 20:3:0.3
volume: 0.5 ml for 5-HT1A and 5-HT receptors; 1 ml
3
for the others): 50 mM Tris–HCl, pH 7.7 for 5-HT2A
receptors, with the addition of 10 mM pargyline for the
3
other receptors and further containing 4 mM CaCl for
2
5-HT , 4 mM CaCl and 0.1% ascorbic acid for 5-
1A
2
HT ; 4 mM CaCl , 0.1% ascorbic acid and 0.1 mM
1B
2
(
spiperone for 5-HT ; 25 mM Tris–HCl, pH 7.4+10
2C
v/v). After cooling, the ethanol was removed under
reduced pressure, water was added and the mixture was
basified with 2 N NaOH and extracted with ethyl
acetate.
mM pargyline for 5-HT receptors; 50 mM Hepes-HCl,
3
pH 7.4+10 mM pargyline for 5-HT ; 50 mM Tris–
4
HCl, pH 7.4 containing 10 mM pargyline, 120 mM
NaCl, 5 mM KCl, 2 mM CaCl , 1 mM MgCl and
2
2
After drying with sodium sulfate, the solvent was
evaporated and the solid residue was washed with a
mixture of dry ether/petroleum ether. The obtained
compounds were crystallized as indicated in Table 1.
For the pharmacological assays, the free bases were
converted into the hydrochlorides by means of 1 N
ethanolic HCl; the solution was evaporated to dryness
and the residue washed several times with dry ether.
0.1% ascorbic acid for D receptors; 50 mM Hepes Na,
2
pH 7.5 containing 1 mM EDTA, 0.1% albumine,
0.005% ascorbic acid and 200 nM eliprodil for D3
receptors.
The following incubation conditions were used.
3
5-HT : [ H]-8-OH-DPAT (sp. act.127 Ci/mmol),
1A
final concentration 1 nM, 30 min, 25°C (non specific
binding: 5-HT 1 mM).

A. Sparatore et al. / Il Farmaco 54 (1999) 402–410
405
3
5
-HT : [ H]-serotonin (sp. act.30.0 Ci/mmol), final
4.2. General pharmacological screening
1
B
concentration 2 nM, 30 min, 25°C (non specific
binding: 5-HT 10 mM).
A broad pharmacological screening was performed
on compound 3a by MDS Panlabs Inc., Bothell, WA,
USA.
3
5
-HT : [ H]-ketanserin (sp. act. 80.9 Ci/mmol), final
2
A
concentration 0.7 nM, 15 min, 37°C (non specific
This screening consisted of the determination of the
maximum tolerated dose (p.os and i.p.), with simulta-
neous behavioural examination (Irwin test), and in 34
primary in vivo tests (using a suitable m.t.d. fraction,
depending on test type) and 26 in vitro tests.
The procedures for these tests have been described
already [1,11–16].
binding: methysergide 1 mM).
3
5
-HT : [ H]-mesulergine (sp. act. 82.0 Ci/mmol),
2
C
final concentration 1 nM, 30 min, 37°C (non specific
binding: mesulergine 10 mM).
3
5
-HT : [ H]-zacopride (sp. act. 85.0 Ci/mmol), final
3
concentration 0.4 nM, 30 min, 25°C (non specific
binding: GR38032 1 mM).
3
5
-HT4: [ H]-GR113808 (sp. act. 84.0 Ci/mmol), final
concentration 0.1 nM, 30 min, 37°C (non specific
5
. Results and discussion
binding: 5-HT 10 mM).
3
D : [ H]-spiperone (sp. act. 15.0 Ci/mmol), final con-
2
The results of radioligand binding experiments are
centration 0.2 nM, 15 min, 37°C (non specific bind-
summarized in Tables 2 and 3.
ing: (−)sulpiride 100 mM).
3
From Table 2 it is evident that the tested compounds
displayed quite different degrees of affinity for the six
serotonin receptor subtypes investigated. Actually,
moderate to good affinity was exhibited for 5-HT1A and
D : [ H]-7-OH-DPAT sp. act. 52 Ci/mmol), final
3
concentration 0.7 nM, 60 min, 25°C (non specific
binding: dopamine 1 mM).
Incubation was stopped by rapid filtration under
vacuum through GF/B (in 0.5% polyethylenimine for
5
-HT2A receptors, while only very poor or no affinity
was observed for 5-HT , 5-HT , 5-HT , and 5-HT
4
1
B
2C
3
5
-HT receptors) or GF/C (in 0.1% albumine only for
3
receptors, with IC50 ranging from 1.8 to more than 10
mM. Thus, the investigated compounds exhibited, on
the whole, a good selectivity for 5-HT1 and 5-HT
receptors versus the other subtypes. This is particularly
interesting for 5-HT2A and 5-HT2C receptors, which
share about 80% amino acid homology within the seven
transmembrane domains [17]. The most selective com-
pounds in this regard are 4a, followed by 3b and 3c,
which inhibit [ H]ketanserin binding to 5-HT recep-
tors at about 10
tration tested (10
binding to 5-HT2C receptors.
The nature of the aryl/heteroaryl moiety on the
piperazine nitrogen and the presence or absence of the
D3 receptors) filters (Schleicher and Schuell), which
were then washed with 12 ml (4×3 times) of ice-cold
Tris–HCl, 50 mM, pH 7.4 or Hepes-HCl, 50 mM, pH
.4, using a Brandel M-48R cell harvester. Dried filters
were immersed in vials containing 4 ml of Filter Count
Packard) for the measurement of trapped radioactivity
A
2A
7
(
with a LKB 1214 RACKBETA liquid scintillation spec-
trometer with a counting efficiency of about 60%.
Dose–inhibition curves were analyzed by the ‘Allfit’
program [9] to obtain the concentration of the unla-
beled drug that caused 50% inhibition of ligand bind-
ing. The K values were derived from the IC values
3
2A
−
7
M while, until the highest concen-
M), not affecting [ H]mesulergine
−
5
3
i
50
according the method of Cheng and Prusoff [10].
Table 1
2
2
-{4-[3-(4-Aryl/heteroarylpiperazin-1-yl)propoxy]phenyl}-2H-benzotriazoles (3a–d) and 2-{4-[3-(4-aryl/heteroarylpiperazin-1-yl)propoxy]phenyl}-
H-benzotriazole-1-oxides (4a–d)
Comp
Formula a
M.p. (°C) free base
Crystall. solvent b
Yield (%)
M.p. (°C) hydrochloride
3
3
3
3
4
4
4
4
a
b
c
d
a
b
c
C25H27N O
167–168
134–135
127–128
152–152.5
140–141
127–128
132–133
160–161
A
B
B
B
B
B
B
B
52
71
57
55
65
51
67
47
252–255
236–238
238–239
242–244
235–236
203–205
212–213
227–228
5
C26H29N O2
5
C24H26N O
6
C23H25N O
7
C25H27N O2
7
C26H29N O3
5
C24H26N O2
6
d
C23H25N O2
7
a
Analytical results for C,H,N were within 90.3% of the calculated values.
A, dry ether; B, ethyl acetate.
b

Table 2
Affinity of compounds 3a–d, 4a–d and reference drugs on serotonin receptors: IC50 nM9S.D. (K nM9S.D.)
i
Comp. or
reference
drug
5-HT1A [3H]8OH-DPAT 5-HT1B [3H]serotonin rat 5-HT2A [3H]ketanserin
5-HT2C [3H]mesulergin
rat cortex
5-HT3 [3H]zacopride
rat cortex
5-HT4 [3H]GR 113808
guinea pig striatum
Selectivity K 5-
i
rat hippocampus
striatum
rat cortex
HT2A/K 5-HT
i
1A
3
3
3
3
4
4
4
4
a
b
c
d
a
b
c
57289540 (34319323)
73.4915.2 (40.998.4) 28679811 (15019424) 193965 (107936)
19929369 (9909185) \10 000
\10 000
320968.7 (160934)
21.595.39 (11.993.4) 18759123 (982964)
11549313 (6439174) \10 000
156918.7 (86.9910.4) \10 000
1.490.25 (0.7690.13) 4.190.6 (2.0990.31)
137912.6 (84.5997.6)
\10 000
\10 000
\10 000
\10 000
\10 000
0.03
2.62
0.31
816392442 (654091956) \10 000
557948 (310926)
10199198 (5689110)
699591736 (36649909) 155926 (86.6914.6)
\10 000
\10 000
\10 000
\10 000
\10 000
\10 000
\10 000
\10 000
0.54
7569157 (421987)
740291040 (41269579) 660391539 (529091233) \10 000
30439697 (17079391) \10 000
23669365 (18959292)
921292908 (614191938) 35.38
\10 000
86579756 (42229368) \10 000
101923 (67.3915.2)
6.41
19.64
/
d
Serotonin
Methisergide
Mesulergine
Quipazine
4.590.6 (2.5090.34)
6.491.7 (4.0391.07)
5
4
4.490.41 (2.0190.18)
(
1
9
9
9
)
402
410

A. Sparatore et al. / Il Farmaco 54 (1999) 402–410
407
Table 3
Affinity of compounds 3a–d, 4a–d and reference drugs on dopamine receptors: IC50 nM9S.D. (K nM9S.D.)
i
Comp. or reference drug
D2 [3H]spiperone rat striatum
D3 [3H]7OH-DPAT rat olfactory tubercle
Selectivity K D /K D
i 2 i 3
a
3
3
3
3
4
4
4
4
a
b
c
d
a
b
c
\10 000
369978.6 (270.3957.6)
83.495.6 (42.992.9)
18969220.5 (13109152)
n.d. b
89918 (47.999.7)
2091.4 (10.590.7)
262.6948.5 (181.4933.5)
246.9942.8 (127.1922.1)
\27.1
387960 (172926)
41249177 (1832978)
\10 000
11619203 (476983)
298955 (132924)
\10 000
4.01
1.40
9.94
12.57
a
\38.1
d
963192410 (428091071)
287957 (127924)
33.67
(−)Sulpiride
Dopamine
42.395 (20.792.4)
a
Calculated on IC50 ratio.
b
−5
and 10−7 M, is respectively 61 and 0.
%
Inhibition of specific binding at 10
N-oxide function exert an important modulatory role
on the affinity of different compounds for 5-HT1A and
The binding constants (K ) relative to 2-[3-(4-
i
phenylpiperazin-1-yl)propyl]benzotriazole and to its 2-
methoxyphenyl analogue, reported by Caliendo et al.
[21] and by Mokrosz et al. [22,23], were obtained using
human recombinant receptors and animal tissue prepa-
rations, respectively, and therefore they are not directly
comparable.
Nevertheless, they clearly indicate how the phenyl
residue is more suitable than the 2-methoxyphenyl one
for binding on 5-HT2A receptor and vice-versa for what
concerns the 5-HT1A one, as already observed for our
compounds.
5-HT2A receptors.
As already observed by many authors for other kinds
of arylpiperazine ligands, the 2-methoxyphenyl residue
appeared as the most suitable for binding to 5-HT1A
receptors. However, the strong positive influence of the
N-oxidation of the benzotriazole ring on the affinity on
this receptor must be pointed out, even though it did
not homogeneously affect all compounds. Actually, the
N-oxidation enhanced the affinity of 2-pyridyl deriva-
tive 3c only 1.5 times, but improved the affinity of the
pyrimidyl derivative by more than 100 times.
Even more important is the observation that the
absolute values of the binding constants of the two
compounds described by Caliendo et al. and by
Compound 4b, containing the 2-methoxyphenyl
residue and the N-oxide function, was definitively the
most active one with K =11.9 nM.
Mokrosz et al. were quite comparable with the K of
i
i
Concerning the 5-HT2A receptor, the phenyl and, to a
minor extent, the 2-methoxyphenyl residues were most
suitable for binding.
The N-oxidation of benzotriazole ring exerted a neg-
ative influence on the affinity, with a particularly delete-
rious effect on the 2-pyridyl derivative.
our corresponding compounds 3a and 3b, suggesting
that the oxyphenyl moiety present in the latter, al-
though increasing the overall size of the molecules and
the distance between the benzotriazole ring and the
basic head, did not hinder a valid binding to both
5-HT1A and 5-HT2A receptors and actually may con-
tribute to it.
Since such substantial changes introduced in the
molecular structure could have affected the importance
of the basic arylpiperazine head for the binding on
serotonin receptors, the previously described [1] com-
pounds of structures 1 and 2 were subjected to binding
Thus, for this receptor the most active compound
was the simple phenyl–piperazinylpropoxyphenylben-
zotriazole 3a, with K =84.59 nM.
i
As a consequence of the positive or negative influ-
ence of N-oxidation on the affinity of 5-HT1A and
5-HT2A receptors, respectively, compound 4b exhibited
a good selectivity for 5-HT1A versus 5-HT2A receptor
experiments on 5-HT1A and 5-HT receptors and were
3
with a ratio 5-HT /5-HT1A=35, while compound 3a
found completely unable to displace the specific radio-
ligands up to the highest concentration tested (10 mM).
The results of Table 3 show that the tested com-
pounds exhibited also variable degrees of affinity for
dopamine D and D receptors, with a general higher
2
A
was a very selective 5-HT2A ligand. On the other hand,
compounds 3b and 4a appear interesting for their bal-
anced and fairly high affinity for both receptors.
In the last few years, a number of 1/2-[v-(4-arylpiper-
azin-1-yl)alkyl]benzotriazoles were described [5,18–23]
and some of them were found to be good ligands for
2
3
activity for the D subtype. Apart from the case of
3
2-pyridyl derivative 3c, the ratio of affinity D /D (K
2
3
i
5-HT1A and/or 5-HT2A receptors.
ratio) ranged from 4 to more than 38, indicating a
These compounds are different from ours because of
moderate to good selectivity for the D receptor.
3
the absence of the oxyphenyl moiety between the ben-
zotriazole nitrogen and the polymethylene chain.
The phenyl- and 2-methoxyphenyl substituted com-
pounds generally displayed a much higher affinity for

408
A. Sparatore et al. / Il Farmaco 54 (1999) 402–410
both types of receptors than the 2-pyridyl and 2-pyrim-
idyl derivatives.
these receptors, but had higher affinity for the first and
last of them. On the other hand, compound 3a was
characterized by a reasonably good affinity for the
5-HT2A and D3 receptors and good selectivity with
It is worth noting that the N-oxides, with the excep-
tion of the 2-pyridyl derivative, always showed a higher
affinity for the dopamine receptors considered; once
more, the enhancing effect of the N-oxidation did not
homogeneously influence all compounds (Table 4).
It should be noted that compounds 3b and 4b were
respect to the 5-HT1A and D receptors.
2
Therefore, these compounds deserve further investi-
gation in order to define their agonistic or antagonistic
activity and consequently, to study their potential as
anxiolytic, antipsychotic or antidepressive agents, possi-
bly devoid of the usual neurological or autonomic
side-effects.
Actually, the related dialkylaminoalkoxyphenylben-
zotriazoles and their N-oxides (1 and 2), previously
described [1], displayed a number of biological activities
that could be somewhat troublesome for a psychophar-
macological agent.
comparable to (−)sulpiride (K =127 nM) for displac-
i
3
ing [ H]spiperone from the D2 receptor, while com-
pounds 3b and 4a–b were comparable to dopamine
3
itself (K =20.7 nM) for displacing [ H]7-OH-DPAT
i
from the D receptor.
3
The N-oxide 4b, containing the 2-methoxyphenyl
residue, was rather potent at the D3 receptor with
K =10.5 nM, also having a moderate affinity for the
i
D receptor (K ratio D /D =12.6).
Therefore, the simplest compound 3a, endowed with
only moderate affinities for serotonin and dopamine
receptors, was subjected to a general pharmacological
screening in order to explore how the exchange of the
aliphatic basic head of 1 and 2 with the arylpiperazinyl
one would affect the pharmacological profile, besides
giving rise to the observed affinity for serotonin and
dopamine receptors.
2
i
2
3
On the whole, the behaviour of these compounds
versus D and D receptors recalls that of the isosteric
2
3
benzimidazole derivatives described by Wright et al.
24].
Among
[
them,
2-{4-[3-(4-phenylpiperazin-1-
yl)propoxy]phenyl}benzimidazole was the most potent
and selective, exhibiting a K for the D receptor of 1.5
i
3
nM and a D /D ratio of 271. The 2-methoxyphenyl
The results of the general pharmacological screening
indicated that compound 3a is well tolerated up to a
dose of 300 mg/kg p.os and up to 100 mg/kg i.p. in
mice, without any sign of central or autonomic effect
during the 72 h of observation after administration.
Results of in vivo tests aimed to show specific activity
on CNS (analgesic and anticonvulsive activity, 5-
methoxy-N,N-dimethyltriptamine potentiation, apo-
morfine and oxotremorine antagonism, tetrabenazine
hypothermia antagonism) performed at doses in the
range 30–100 mg/kg p.os or 10–30 mg/kg i.p. were
completely negative, apart from a slight reduction of
phenylquinone-induced writhings (−10%).
2
3
analogue showed a K for the D receptor of 1.7 nM
i
3
and a D /D ratio of 5.2. Thus, the benzimidazole ring
2
3
seems somewhat more suitable for binding to dopamine
receptors than the benzotriazole nucleus.
Obviously the comparison of our K data with those
i
of Wright et al. should be made with great caution,
since the latter were obtained using cloned human D2L
and D receptors transfected into CHO-K1 cells, while
3
we used receptor preparation from rat striatum and rat
olfactory tubercle; moreover, Wright et al. used
3
[
H]spiperone as a ligand for both D and D receptors,
2 3
3
while we used [ H]spiperone for the former receptor
and [ H]7OH-DPAT for the latter.
3
Concerning the cardiovascular apparatus, compound
3
a, at the dose of 100 mg/kg p.os, exerted only a
From the global results of the radioligand binding
experiments, we may conclude that some of the synthe-
sized compounds appeared endowed with peculiar in-
teresting binding profiles on serotonine 5-HT1A and
modest (−4%) reduction of blood pressure in sponta-
neously hypertensive rats during the 4 h period of
observation, associated with a moderate reduction
(
−7%) of heart rate. At the same dose, no protection
5
-HT2A and dopamine D and D receptors. Particu-
2 3
against chloroform induced arrhythmia was seen in
mice.
larly, compounds 3b and 4b exhibited affinity for all
Table 4
Further negative responses were observed in the fol-
lowing tests for anti-inflammatory, anti-allergic and hy-
poglicemic activities, protection from ethanol induced
gastric ulcers (100 mg/kg; p.os in rats); saluretic and
diuretic activities, protection from stress induced gastric
ulcers (30 mg/kg; p. os in rats); reduction of pentagas-
trin induced gastric acidity (10 mg/kg, i.p. in rats);
Enhancing or lowering effect of N-oxidation on affinity on different
receptors (K ratio: benzotriazoles/ benzotriazole N-oxides)
i
Benzotriazole/benzotriazole
N-oxide
5HT1A 5HT2A D2
D3
a
a
3a/4a
3b/4b
3c/4c
3d/4d
21.4 0.98
3.4 0.25
\8.6
1.3
5.64
4.08
7.22
serotonin induced bradycardia=5-HT antagonism (10
3
1.5 0.07
B0.4
\1
a
a
mg/kg; i.p. in mice); antihypercholesterolemic activity
\64
0.33
(
300 mg/kg; p.os in mice). The lack of the hypocholes-
a
Calculated on IC50 ratio.
terolemic activity, so strongly present in the dimethyl-

A. Sparatore et al. / Il Farmaco 54 (1999) 402–410
409
aminopropoxyphenylbenzotriazoles 1 is worth noting.
Concerning the in vitro assays on isolated guinea pig
left and right atria, ileum and trachea, rat portal vein
and vas deferens, compound 3a failed to show any
activity apart from a modest positive inotropic activity
moved most of their pharmacological activities; the
only one maintained and somehow improved was the
ability to protect mice from cyanide-induced hypoxia.
(
+11%) on guinea pig left atria and a moderate antag-
Acknowledgements
onism to calcium (−35%) and neurokinin NK1
(
−29%) induced guinea pig ileum contractions.
Financial support from the CNR and from the Min-
istero dell’Universit a` e della Ricerca Scientifica e Tec-
nologica is gratefully acknowledged. The authors also
thank Dr Tiziana Mennini (Istituto di Ricerche Farma-
cologiche ‘Mario Negri’, Milan, Italy) for her
collaboration.
Compound 3a produced these responses at the con-
centration of 45 mg/ml (100 mM), thus clearly differing
from the dialkylaminoalkoxyphenylbenzotriazoles (1
and 2) formerly described [1], which, at the same con-
centration were active in most of the performed assays
and gave still significant responses in many of them at
3
–10 mM concentrations. Compound 3a was also lack-
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