Synthesis and in-vitro pharmacological evaluation of new 5-HT1A receptor ligands containing a benzotriazinone nucleus

Article abstract of DOI:10.1002/ardp.200700151

This paper reports the microwave-assisted synthesis and the binding assays on the 5-HT_1A, 5-HT_2A and 5-HT_2C receptors of new benzotriazinone derivatives, in order to identify selective ligands for the 5-HT_1A subtype receptor. Conventional and microwave heating of the reactions were compared. Good yields and short reaction times are the main advantages of our synthetic route. More active compounds were selected and further evaluated for their binding affinities on D₁, D₂ dopaminergic and α₁-, α₂ adrenergic receptors. The 3-(2-(4-(naphthalen-1-yl)piperazin-1-yl)ethyl)benzo[d][1,2,3]triazin-4(3H)- one 5 with K_i = 0.000178 nM was the most active and selective derivative for the 5-HT_1A receptor with respect to other serotonin receptors and the most selective derivative compared to dopaminergic and adrenergic receptors.

Full text of DOI:10.1002/ardp.200700151

20
Arch. Pharm. Chem. Life Sci. 2008, 341, 20–27
Full Paper
Synthesis and In-vitro Pharmacological Evaluation of New
5-HT1A Receptor Ligands Containing a Benzotriazinone
Nucleus
Ferdinando Fiorino1, Beatrice Severino1, Francesca De Angelis1, Elisa Perissutti1,
Francesco Frecentese1, Paola Massarelli2, Giancarlo Bruni2, Elga Collavoli2,
Vincenzo Santagada1, and Giuseppe Caliendo1
1 Dipartimento di Chimica Farmaceutica e Tossicologica, Universitꢀ degli Studi di Napoli ”Federico II”, Napoli,
Italy
2 Dipartimento di Farmacologia ”G. Segre”, Universitꢀ di Siena, Siena, Italy
This paper reports the microwave-assisted synthesis and the binding assays on the 5-HT1A, 5-HT2A
and 5-HT2C receptors of new benzotriazinone derivatives, in order to identify selective ligands
for the 5-HT1A subtype receptor. Conventional and microwave heating of the reactions were com-
pared. Good yields and short reaction times are the main advantages of our synthetic route.
More active compounds were selected and further evaluated for their binding affinities on D1,
D2 dopaminergic and a1, a2 adrenergic receptors. The 3-(2-(4-(naphthalen-1-yl)piperazin-1-yl)e-
thyl)benzo[d][1,2,3]triazin-4(3H)-one 5 with Ki = 0.000178 nM was the most active and selective
derivative for the 5-HT1A receptor with respect to other serotonin receptors and the most selec-
tive derivative compared to dopaminergic and adrenergic receptors.
Keywords: Benzotriazinone / Microwave-assisted synthesis / 5-HT Receptors / Serotonin /
Received: July 17, 2007; accepted: September 5, 2007
DOI 10.1002/ardp.200700151
drug development due to its implication in many physio-
logical processes.
Introduction
5-HT1AR belongs to the G protein-coupled receptors
(GPCRs) [10] and the members of this family possess a sim-
ilar amino acid composition. In particular, the 5-HT1AR
transmembrane amino acid sequence presents 45%
homology with the respective part of the a1-adrenergic
receptor [11].
Several agents are already known for their high affin-
ity toward these receptors and, from a chemical point of
view, they can be subdivided into different classes. The
most studied group is that of long-chain arylpiperazine
(LCAPs) [12, 13] that, for a long time, has drawn great
interest as a source of both neuropsychiatric drugs (bus-
pirone, ziprasidone, aripiprazol) and compounds with a
high therapeutical potential (adatanserin, mazapertine,
flesinoxan, lecozotan, bifeprunox) which among others
exert their action via 5-HT1A and 5-HT2A receptors. Their
diversified receptor-binding profile and intrinsic activity,
depending on either the kind of substituent attached to
the N-4 atom of the piperazine moiety or the nature of an
Serotonin (5-hydroxytryptamine, 5-HT) is one of the most
important targets for medicinal chemistry as it is impli-
cated in numerous physiological and pathophysiological
processes [1–4]. Serotonin receptors may be involved in
impulsivity and alcoholism [5–6] and in the different
phases of sleep [7], sexual behavior, appetite control, ther-
moregulation, and cardiovascular function [8, 9]. In par-
ticular, the 5-HT1A receptor, found in high concentration
in the limbic system where it is thought to play a role in
emotional processes, is a major target for research and
Correspondence: Giuseppe Caliendo, Dipartimento di Chimica Farma-
ceutica e Tossicologica, Universitꢀ degli Studi di Napoli “Federico II”, Via
D. Montesano, 49-80131, Napoli, Italy.
E-mail: caliendo@unina.it
Fax: +39 81 678-649
Abbreviations: 5-hydroxytryptamine (5-HT); G protein-coupled receptors
(GPCRs); of long-chain arylpiperazine (LCAP); structure-activity relation-
ship (SAR) studies
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2008, 341, 20–27
5-HT1A Receptor Ligands Containing Benzotriazinone Nucleus
21
Reagents and conditions: (i) Br(CH2)nCl, K2CO3, DMF, lm; (ii) 4-X-piperazine, K2CO3, NaI, DMF, lm.
Scheme 1. Synthesis route of compounds 1–8.
Table 1. Physiochemical properties of final compounds 3–8.
Compound
n
2
3
2
X
Formulaa)
Mp. (8C )b)
248–250
243–245
246–248
Yield (%)c)
3
4
5
C19H27N5O62HCl
C20H29N5O62HCl
C23H23N5O6HCl
65
87
71
6
7
3
2
C24H25N5O6HCl
C22H22N6O6HCl
251–253
208–210
82
62
8
3
C23H24N6O6HCl
200–202
79
a)
Satisfactory microanalyses obtained: C, H, N values are within l 0.4% of theoretical values.
All compounds were crystallized by ethyl alcohol and diethyl ether.
Yields of the last step are referred to microwave irradiation.
b)
c)
amide or imide terminal fragment, opens possibilities has been the subject of many structure-activity relation-
for the discovery of new potent therapeutic agents [14]. ship (SAR) studies. In particular, much effort has been
The influence of each part of the LCAP structures on the devoted to understand the role of the terminal part in
5-HT1A receptor affinity, intrinsic activity, and selectivity the ligand-receptor interaction and, in consequence, a
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F. Fiorino et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 20–27
great number of many different fragments were used
Physicochemical data of the final compounds are
[15]. However, a limitation of many 5-HT1A receptor reported in Table 1. The parameters of time, power, tem-
ligands in the potential use as drugs or pharmacological perature used for the microwave irradiation, and the con-
tools is their undesired high affinity for other receptor ditions used for the conventional heating are reported in
subtypes. The dopaminergic D2 receptor and a1-adreno- Table 2. Analytical purification of each final product was
ceptor are two examples of receptor sites to which several obtained by chromatography on silica-gel column and
5-HT1A ligands bind with high affinity.
further by crystallization from the appropriate solvent.
Our group has undertaken a research program aimed All new compounds gave satisfactory elemental analyses
at developing new 5-HT1A agents [16–23] with high affin- (C, H, N) and were characterized by mass spectrometry
1
ity and selectivity over other serotoninergic, dopaminer- (Finnigan LCQ–MS ThermoQuest-Ion trap) and H-NMR,
1
gic and adrenergic receptors. In continuation of our performed on the free base of each compound. H-NMR
research program, we have analyzed a new set of deriv- and MS data for all final compounds were consistent
atives where the piperazine-N-alkyl moiety has been with the proposed structures.
linked to a 1,2,3-benzotriazinone fragment (Scheme 1);
this heterocyclic nucleus was already investigated by our
group, in order to develop aryl piperazine derivatives
with high affinity and selectivity for the 5-HT1A receptor
Results and discussion
[21]. Aiming to further explore its influence on the sero- The syntheses of these novel compounds, characterized
toninergic activity, this bicyclic nucleus was linked to by benzotriazinonic nucleus, were successfully improved
not yet studied 4-substituted piperazines (cyclohexyl, with the application of microwave heating to our reac-
naphthyl and quinolyl) via two or three methylene spac- tions; in particular, under controlled microwave irradia-
ing units. All the new compounds were tested for their tion, the reaction times were extensively reduced, from
affinity for 5-HT1A, 5-HT2A and 5-HT2C receptors. Moreover, 2–3 h to 30 min in the syntheses of intermediates (2a
the multireceptor profiles of promising derivatives were and 2b) and from 24 h to 70 min in the obtaining of final
also evaluated in terms of binding affinities for dopami- compounds (Table 2, 3–8).
nergic D1, D2 and a1, a2 adrenoreceptors.
The obtained six new arylpiperazine derivatives 3–8
were evaluated for activity and selectivity. Introduction
of a 1,2,3-benzotriazinonic nucleus as terminal part,
Chemistry
The synthesis of final compounds 3–8, reported in slight modifications concerning the alkyl spacer chain
Scheme 1, was performed using a microwave oven length (two or three units), and the introduction of new
(ETHOS 1600, Milestone Inc.) especially designed for rings on the N-4 piperazine moiety are depicted in
organic synthesis. The experimental conditions used in Scheme 1 and were performed in an effort to obtain com-
our work were similar to those used by conventional pounds with high affinity and selectivity for 5-HT1AR over
heating, with the same amount of starting reagent and other serotoninergic receptors, as well as dopaminergic
volume of solvent. Synthesis by microwave irradiation and adrenergic receptors. As anticipated, two of these
gave the desired compounds in better yields than those compounds are potent 5-HT1A receptor ligands. In fact,
obtained by conventional heating. The overall times for they showed nanomolar or even subnanomolar 5-HT1A
the synthesis were considerably reduced. All the 4-X- receptor affinities (Table 3). Besides the outstanding 5-
piperazines (Scheme 1) employed for the preparation of HT1A receptor affinity of compound 5 (Ki = 0.000178 nM),
compounds 3–8 are commercially available.
Ki values were clustered in a relatively narrow range from
Alkylation of the starting heterocycle 1,2,3-benzotria- 7.79 nM for 6 to 105 nM (compound 4). Compounds 7, 8,
zin-4(3H)-one with 1-bromo-2-chloroethane or 1-bromo-3- and 3 where less active showing Ki values of 370, 317, and
chloropropane, in presence of K2CO3 in DMF, gave the cor- 5090 nM, respectively.
responding chloroalkyl benzotriazinone derivatives 2a
Concerning the most active compounds 5 and 6, the
and 2b. The obtained intermediates were condensed with derivative with piperazinylethyl chain linked to the ben-
the desired 4-X-piperazine in DMF in the presence of zotriazinonic nucleus 5 showed high and preferential
K2CO3, and NaI, under reflux to give the final compounds affinity for the 5-HT1AR, relative to the compound in
3–8.
which the spacer is one atom longer 6. The influence of
All the final compounds were transformed into the the alkyl chain length, observed for our compounds, is in
corresponding hydrochloride salts using dry gaseous HCl accordance with recently reported structure affinity rela-
in anhydrous diethyl ether.
tionships (SARs) of new models of arylpiperazines [23].
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Arch. Pharm. Chem. Life Sci. 2008, 341, 20–27
5-HT1A Receptor Ligands Containing Benzotriazinone Nucleus
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Table 2. Conventional heating versus microwave irradiation for intermediates 2a, 2b, and the final compounds 3–8.
Compd.
Conventional heatinga)
Microwave irradiationb)
Yield (%)
Time (min)
Temp. (8C)
Yield (%)
Time (min)
Power (Watt)
Temp. (8C)
2a–2b
3–8
72–85
120–180
80
90–96
5
5
20
5
200
80
400
80
90
90
80
22–48
24h
reflux
62–87
80
60
5
400
200
120
110
a)
Oil bath.
b)
The experimental conditions used on microwave irradiation were similar to those used by conventional heating, with the same
amounts of starting reagent and volume of solvent.
Table 3. Affinities of compounds 3–8 for 5-HT1A, 5-HT2A, and 5-HT2C receptors.
Compound
Substituent
X
Receptor affinity Ki l SD (nM)
n
2
3
5-HT1A
5-HT2A
5-HT2C
[3H]8OH-DPAT
[3H]ketanserin
[3H]mesulergine
3
4
5090 l 203
105 l 6
A104
A104
A104
A104
5
6
7
8
2
3
2
3
0.000178 l 0.00003
7.79 l 0.06
5.29 l 0.3
127 l 17
A104
A104
370 l 4.7
315 l 137
1630 l 270
A104
317 l 70
5670 l 336
For purpose of comparison, 8-OH-DPAT, ketanserin, and mesulergine binds 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.
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F. Fiorino et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 20–27
Table 4. Affinities of compounds 5 and 6 for D1, D2, a1, and a2 receptors.
Receptor affinity Ki l SD (nM)
Compound
D1
D2
a1
a2
[3H]SCH-23390
[3H]spiperone
[3H]prazosin
[3H]yohimbine
5
6
A104
A104
A104
A104
112 l 47
562 l 170
1880 l 107
3600 l 458
Moreover, regarding the influence of the substituent ent on the N-4 of the piperazine moiety [23]. Moreover,
on the N-4 of the piperazine moiety, the naphthyl group although the exceptionally high affinity of compound 5
for compounds 5 and 6 conferred the highest affinity for is probably not due to its ability to form a hydrogen bond
the 5-HT1A receptor. Instead, the cyclohexyl or quinolyl between a substituent and Asn-386 of the receptor (like
moiety seems to show an unfavorable affinity profile the OCH3 substituent), but rather solvent accessibility
compared to 5 and 6. In fact, only compound 4 showed a and hydrophobic interaction with the receptor are deci-
quite moderate affinity for the 5-HT1A receptor with Ki val- sive. In fact, Zlatovic et al. [24] have recently reported that
ues of 105 nM.
some arylpiperazine, such as naphthylpiperazine, can
The 5-HT2A and 5-HT2C receptor affinities of the tested interact directly with the hydrophobic part of the 5-HT1A
compounds were always lower than those observed for 5- receptor binding site. In particular the hydrophobic part
HT1A receptors and ranged from 5.29 (5) to A104 nM (for 3, of the binding site in the 5-HT1A receptor, formed by Trp
4, 6, 7, and 8) for 5-HT2A and from 5670 (8) to A104 nM (for 358, Phe 361, and Tyr 390, is significant for the stabiliza-
3–7) for 5-HT2C receptors. The most 5-HT1A active com- tion of the ligand-receptor complex. Finally, these
pound, 3-(2-(4-(naphthalen-1-yl)piperazin-1-yl)ethyl)ben-
aspects, correlated to the hydrophobic interaction with
zo[d][1,2,3]triazin-4(3H)-one 5 (Ki = 0.000178 nM), was also the receptor, can be useful to clarify also the smaller
the most selective derivative with respect to the seroto- affinity correlated to the cyclohexyl or quinolyl deriv-
nin receptors 5-HT2A and 5-HT2C.
atives, where a non-aromatic ring or the more hydro-
Additionally, the affinity of the most active com- philic quinolyl moiety are less favorable to the hydropho-
pounds 5 and 6 on several other receptors (a1 and a2 adre- bic interaction with the receptor.
nergic and D1 and D2 dopaminergic receptors) was exam-
ined in order to verify their selectivity. Results are sum- The NMR spectral data were provided by Centro di Ricerca
marized in Table 4. The two derivatives proved highly Interdipartimentale di Analisi Strumentale, Universitꢀ degli
selective against dopaminergic receptors with Ki values Studi di Napoli “Federico II”. The assistance of the staff has
of above 104 nM. Regarding a1 and a2 adrenergic recep- been gratefully appreciated.
tors, only compound 5 showed quite moderate affinity
(112 nM). The high selectivity towards a1 receptors, exhib-
ited by compound 5, is very interesting considering that
the amino acid sequence of the transmembrane part of 5-
HT1AR is highly homologous to that of the a1 adrenergic
receptor.
The authors have declared no conflict of interest.
Experimental
Synthesis – general procedures
These results support further on the choice of the
1,2,3-benzotriazinonic scaffold for the design and prepa-
ration of serotoninergic ligands endowed with high 5-
HT1A affinity. The high difference in affinity observed
between 5 and 6 demonstrates again that the alkyl chain
length represents a critical structural feature in deter-
mining 5-HT1A receptor affinity and selectivity. In fact, in
contrast to all other substituents generally reported in
literature, the naphthyl derivative exhibits a higher
affinity for the shorter chain compared to the longer
chain. This aspect was demonstrated by a molecular mod-
eling study, which could be due, as recently reported in
literature, to the very hydrophobic nature of the substitu-
Synthesis was performed using a microwave oven ETHOS 1600
(Milestone Inc.). All reactions were performed in standard pyrex
glassware with a reflux condenser fitted through the roof of the
microwave cavity and were performed by microwave program
which was composed by appropriate temperature ramping and
holding steps. The temperature of the stirred reaction mixture
was monitored directly by a microwave-transparent fluoroptic
probe inserted into the solution.
All reactions were followed by thin layer chromatography car-
ried out on Merck silica gel 60 F254 plates (Merck, Darmstadt, Ger-
many) with fluorescent indicator and the plates were visualized
with UV light (254 nm). Preparative chromatographic purifica-
tions were performed using silica gel column (Kieselgel 60). Melt-
ing points were determined using a Kofler hot-stage apparatus
(C. Reichert, Vienna, Austria) and are uncorrected. The struc-
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5-HT1A Receptor Ligands Containing Benzotriazinone Nucleus
25
1
tures were verified spectroscopically by H-NMR performed on
the free base of each compound. Spectra were recorded on
Bruker AMX-500 instruments (Bruker Bioscience, Billerica, MA,
USA). Chemical shifts are given as d with references to Me4Si. The
following abbreviations are used to describe peak patterns when
appropriate: s (singlet), d (doublet), t (triplet), m (multiplet). Mass
spectra of the final products were performed on LCQ Thermo-
Quest Ion trap mass spectrometry (Finnigan, Germany). Where
analyses are indicated only by the symbols of the elements,
results obtained are within l 0.4% of the theoretical values.
3-(2-(4-(Naphthalen-1-yl)piperazin-1-
yl)ethyl)benzo[d][1,2,3]triazin-4(3H)-one 5
1H-NMR (CDCl3) d 2.88 (m, 4H), 3.05 (t, 2H, J = 6.8 Hz, CH2-N-pip),
3.10 (m, 4H), 4.71 (t, 2H, J = 6.9 Hz, CH2-N-benz), 7.06 (d, 1H, J =
8.1 Hz, ArH), 7.39 (t, 1H, J = 7.6 Hz, ArH), 7.46 (m, 2H, ArH), 7.53
(d, 1H, J = 8.1 Hz, ArH), 7.82 (m, 2H, ArH), 7.97 (t, 1H, J = 7.6 Hz,
ArH), 8.19 (m, 2H, ArH), 8.38 (d, 1H, J = 8.1 Hz, ArH). ESI-MS: 386.2
[M + H]+.
3-(3-(4-(Naphthalen-1-yl)piperazin-1-
yl)propyl)benzo[d][1,2,3]triazin-4(3H)-one 6
1H-NMR (CDCl3) d 2.21 (q, 2H, J = 6.9 Hz, -CH2-), 2.65 (t, 2H, J =
6.8 Hz, CH2-N-pip), 2.70 (m, 4H), 2.99 (m, 4H), 4.62 (t, 2H, J =
6.9 Hz, CH2-N-benz), 6.96 (d, 1H, J = 8.1 Hz, ArH), 7.37 (t, 1H, J =
7.6 Hz, ArH), 7.45 (m, 2H, ArH), 7.52 (d, 1H, J = 8.1 Hz, ArH), 7.81
(m, 2H, ArH), 7.96 (t, 1H, J = 7.6 Hz, ArH), 8.16 (m, 2H, ArH), 8.37
(d, 1H, J = 8.1 Hz, ArH). ESI-MS: 400.2 [M + H]+.
General procedure for preparation of 3-(n-chloroalkyl)-
1,2,3-benzotriazin-4(3H)-one 2a, 2b
A mixture of 1-bromo-2-chloroethane or 1-bromo-3-chloropro-
pane (0.09 mol), 1,2,3-benzotriazin-4H-one 1 (0.03 mol), K2CO3
(0.05 mol) in DMF (70 mL) was introduced into the reaction ves-
sel and the desired parameters (microwave power, temperature,
and time) were set as reported in Table 2. After cooling, the mix-
ture 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 chroma-
tographed on silica gel column (diethyl ether/methanol, 9 : 1 v/
v) to give compounds 2a and 2b as solids (obtained yields: 2a
90%; 2b 96%). 1H-NMR spectra for all intermediates were consis-
tent with the proposed structures.
3-(2-(4-(Quinolyn-8-yl)piperazin-1-
yl)ethyl)benzo[d][1,2,3]triazin-4(3H)-one 7
1H-NMR (CDCl3) d 2.93 (m, 4H), 3.02 (t, 2H, J = 6.8 Hz, CH2-N-pip),
3.37 (m, 4H), 4.69 (t, 2H, J = 6.9 Hz, CH2-N-benz), 7.10 (t, 1H, J =
7.6 Hz, ArH), 7.35 (m, 2H,), 7.42 (d, 1H, J = 8.1 Hz, ArH), 7.80 (t,
1H, J = 7.6 Hz, ArH), 7.94 (t, 1H, J = 7.6 Hz, ArH), 8.08 (d, 1H, J =
7.6 Hz, ArH), 8.15 (d, 1H, J = 8.1 Hz, ArH), 8.36 (d, 1H, J = 7.6 Hz,
ArH), 8.85 (d, 1H, J = 7.6 Hz, ArH). ESI-MS: 387.0 [M + H]+.
General procedure for preparation of 3-[2 or 3-(4-X-
3-(3-(4-(Quinolyn-8-yl)piperazin-1-
piperazin-1-yl)-alkyl]-1,2,3-benzotriazin-4(3H)-one 3–8
A mixture of appropriate 3-(n-chloroalkyl)-1,2,3-benzotriazin-
4(3H)-one 2a or 2b (0.03 mol), 4-X-piperazine (0.03 mol) and NaI
(0.05 mol), 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 2.
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
on anhydrous Na2SO4, concentrated, and chromatographed on
silica gel column (diethyl ether/ethanol 9 : 1 v/v) to give the final
compounds 3–8 as white crystalline solids, (yield ranging
between 65–84%).
yl)propyl)benzo[d][1,2,3]triazin-4(3H)-one 8
1H-NMR (CDCl3) d 2.20 (q, 2H, J = 6.9 Hz, -CH2-), 2.64 (t, 2H, J =
6.8 Hz, CH2-N-pip), 2.77 (m, 4H), 3.30 (m, 4H), 4.60 (t, 2H, J =
6.9 Hz, CH2-N-benz), 7.05 (t, 1H, J = 7.6 Hz, ArH), 7.34 (m, 2H), 7.40
(d, 1H, J = 8.1 Hz, ArH), 7.79 (t, 1H, J = 7.6 Hz, ArH), 7.93 (t, 1H, J =
7.6 Hz, ArH), 8.08 (d, 1H, J = 7.6 Hz, ArH), 8.14 (d, 1H, J = 8.1 Hz,
ArH), 8.35 (d, 1H, J = 7.6 Hz, ArH), 8.84 (d, 1H, J = 7.6 Hz, ArH). ESI-
MS: 401.1 [M + H]+.
Hydrochloride salts: general procedure
The hydrochloride salts were prepared by adding HCl ethereal
solution to an ethanolic solution of the free bases. All derivatives
were recrystallized by diethyl ether and ethyl alcohol, formulae
and melting points are reported in Table 1. The final compounds
were obtained as white crystals.
3-(2-(4-Cyclohexylpiperazin-1-yl)ethyl)benzo[d][1,2,3]-
triazin-4(3H)-one 3
Pharmacology – general procedures
1H-NMR (CDCl3) d 1.05–1.86 (m, 11H, cyclohexyl), 2.56 (m, 4H),
2.61 (m, 4H), 2.89 (t, 2H, J = 6.8 Hz, CH2-N-pip), 4.62 (t, 2H, J =
6.9 Hz, CH2-N-benz), 7.80 (t, 1H, J = 7.6 Hz, ArH), 7.95 (t, 1H, J =
7.6 Hz, ArH), 8.15 (d, 1H, J = 8.1 Hz, ArH), 8.35 (d, 1H, J = 8.1 Hz,
ArH). ESI-MS: 342.1 [M + H]+.
The newly synthesized compounds were tested for in-vitro affin-
ity for serotonin 5-HT1A, 5-HT2A, and 5-HT2C receptors by radioli-
gand binding assays. The more active compounds on serotonin
receptors have been selected and evaluated for their affinity for
dopaminergic (D1 and D2) and adrenergic (a1 and a 2) receptors.
All the compounds were dissolved in ethanol or in 5% DMSO.
The following specific radioligands and tissue sources were
used: (a) serotonin 5-HT1A receptor, [3H]-8-OH-DPAT, rat brain cor-
tex, (b) serotonin 5-HT2A receptor, [3H]ketanserin, rat brain cor-
tex, (c) serotonin 5-HT2C receptor, [3H]mesulergine, rat brain cor-
tex, (d) dopamine D1 receptor [3H]SCH-23390, rat striatum, (e)
dopamine D2 receptor [3H]spiperone, rat striatum, (f) a1 adrener-
gic receptor [3H]prazosin, rat brain cortex, and (g) a2 adrenergic
receptor [3H]yohimbine, rat brain cortex. Non-specific binding
3-(3-(4-Cyclohexylpiperazin-1-
yl)propyl)benzo[d][1,2,3]triazin-4(3H)-one 4
1H-NMR (CDCl3) d 1.04–1.78 (m, 11H, cyclohexyl), 2.10 (q, 2H, J =
6.9 Hz, -CH2-), 2.12 (m, 4H), 2.44 (m, 4H), 2.49 (t, 2H, J = 6.8 Hz,
CH2-N-pip), 4.55 (t, 2H, J = 6.9 Hz, CH2-N-benz), 7.80 (t, 1H, J =
7.6 Hz, ArH), 7.94 (t, 1H, J = 7.6 Hz, ArH), 8.14 (d, 1H, J = 8.1 Hz,
ArH), 8.35 (d, 1H, J = 8.1 Hz, ArH). ESI-MS: 356.1 [M + H]+.
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26
F. Fiorino et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 20–27
was determined as described in the Experimental (Section 4),
and specific binding as the difference between total and non-spe-
cific binding. Blank experiments were carried out to determine
the effect of 5% DMSO on the binding and no effects were
observed. Competition experiments were analyzed by the “Easy
Fit” program [25] to obtain the concentration of unlabeled drug
that caused 50% inhibition of ligand binding (IC50), with six con-
centrations of test compounds, each performed in triplicate.
The IC50 values obtained were used to calculate apparent inhibi-
tion constants (Ki) by the method of Cheng and Prussoff [26]
from the following equation: Ki = IC50/(1 + S/KD), where S repre-
sents the concentration of the hot ligand used and KD its recep-
tor-dissociation constant (KD values, obtained by Scatchard anal-
ysis [27], were calculated for each labeled ligand).
define non-specific binding, and 100 nM spiperone was added to
all tubes to block binding to 5-HT2A receptors. Tubes were incu-
bated for 15 min at 378C, filtered on Schleicher and Schuell
(Keene, NH, USA) glass fibre filters presoaked in polyethylene
imine, and washed with 10 mL of ice-cold buffer. Filters were
counted at an efficiency of 50%.
D1 Dopaminergic binding assay
The binding assay for D1 dopaminergic receptors was that
described by Billard et al. [30]. Corpora striata were homogenized
in 30 vol. (w/v) ice-cold 50 mM Tris-HCl buffer (pH 7.7 at 258C)
using a Polytron PT10 (setting 5 for 20 s). Homogenates were cen-
trifuged twice for 10 min at 50000 g with resuspension of the
pellet in fresh buffer. The final pellet was resuspended in 50 mM
ice-cold Tris-HCl containing 120 mM NaCl, 5 mM KCl, 2 mM
CaCl2, 1 mM MgCl2, 0.1% ascorbic acid and 10 lM pargyline
(pH 7.1 at 378C). Each assay tube contained 50 lL [3H]SCH-23390
(85.0 Ci/mmol, Perkin Elmer Life Sciences) to achieve a final con-
centration of 0.4 nM, and 900 lL resuspended membranes (3 mg
fresh tissue). The tubes were incubated for 15 min at 378C 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 7.7 at 258C). The radioactivity bound to the filters was meas-
ured by a liquid scintillation counter. Specific [3H]SCH-23390
binding was defined as the difference between binding in the
absence or in the presence of 0.1 lM piflutixol.
5-HT1A binding assay
Radioligand-binding assays were performed following a pub-
lished procedure [28]. Cerebral cortex from male Sprague-Daw-
ley rats (180–220 g) was homogenized in 20 volumes of ice-cold
Tris-HCl buffer (50 mM, pH 7.7 at 228C) with a Polytron PT10,
Brinkmann Instruments (setting 5 for 15 sec), and the homoge-
nate was centrifuged at 50000 g for 10 min at 08C. The resulting
pellet was then resuspended in the same buffer, incubated for
10 min at 378C, and centrifuged at 50000 g for 10 min. The final
pellet was resuspended in 80 volumes of the Tris-HCl buffer con-
taining 10 lM pargyline, 4 mM CaCl2, and 0.1% ascorbate. To
each assay tube was added the following: 0.1 mL of the drug dilu-
tion (0.1 mL of distilled water if no competing drug was added),
0.1 mL of [3H]-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]-8-OH-
DPAT) (170.0 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA,
USA) in the same buffer as above to achieve a final assay concen-
tration of 0.1 nM, and 0.8 mL of resuspended membranes. The
tubes were incubated for 30 min at 378C, and the incubations
were terminated by vacuum filtration through Whatman GF/B
filters (Brandel Biomedical Research and Laboratories Inc., Gai-
thersburg, MD, USA). 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 spectrometer (Packard TRI-
CARBm 2000CA – Packard BioScience s. r. l., Pero, Milan, Italy).
Specific [3H]-8-OH-DPAT binding was defined as the difference
between binding in the absence and presence of 5-HT (10 lM).
D2 Dopaminergic binding assay
The procedure used in the radioligand binding assay was
reported in detail by Creese et al. [31]. Corpora striata were
homogenized in 30 vol. (w/v) ice-cold 50 mM Tris-HCl buffer
(pH 7.7 at 258C) using a Polytron PT10 (setting 5 for 20 s). Homo-
genates were centrifuged twice for 10 min at 50000 g with resus-
pension of the pellet in fresh buffer. The final pellet was resus-
pended in 50 mM ice-cold Tris-HCl containing 120 mM NaCl,
5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 0.1% ascorbic acid, and
10 lM pargyline (pH 7.1 at 378C). Each assay tube contained
50 lL [3H]spiperone (15.7 Ci/mmol, Perkin Elmer Life Sciences) to
achieve a final concentration of 0.4 nM and 900 lL resuspended
membranes (3 mg fresh tissue). The tubes were incubated for
15 min at 378C and the incubation was terminated by rapid fil-
tration 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 7.7 at 258C). The radioactivity bound to the
filters was measured by a liquid scintillation counter. Specific
[3H]spiperone binding was defined as the difference between
binding in the absence or in the presence of 1 lM (+)-butaclamol.
Binding assays of 5-HT2A and 5-HT2C
Radioligand-binding assays were performed as previously
reported by Herndon et al. [29]. Briefly, frontal cortical regions of
male Sprague-Dawley rats (180–220 g) were dissected on ice and
homogenized (1 : 10 w/v) in ice-cold buffer solution (50 mM Tris
HCl, 0.5 mM EDTA, and 10 mM MgCl2 at pH 7.4) with a Polytron
PT10 (setting 5 for 15 sec) and centrifuged at 3000 g for 15 min.
The pellet was resuspended in buffer (1 : 30 w/v), incubated at
378C for 15 min and then centrifuged twice more at 3000 g for
10 min (with resuspension between centrifugations). The final
pellet was resuspended in buffer that also contained 0.1% ascor-
bate and 10– 5 M pargyline.
Assays were performed in triplicate in a 2.0 mL volume con-
taining 5 mg wet weight of tissue and 0.4 nM [3H]ketanserin
hydrochloride (88.0 Ci/mmol; Perkin Elmer Life Sciences) for 5-
HT2A receptor assays, and 10 mg wet weight of tissue and 1 nM
[3H]mesulergine (87.0 Ci/mmol; Amersham Biosciences Europe
GmbH, Freiburg, Germany) for 5-HT2C receptor assays. Cinan-
serin (1.0 lM) was used to define non-specific binding in the 5-
HT2A assay. In the 5-HT2C assays, mianserin (1.0 lM) was used to
a1 Adrenergic binding assay
The procedure used in the radioligand binding assay has been
reported in detail by Greengrass and Brenner [32]. Brain cortex
was homogenized in 30 vol. (w/v) ice-cold 50 mM Tris-HCl buffer,
(pH 7.2 at 258C) using a Polytron PT10 (setting 5 for 20 s). Homo-
genates were centrifuged twice for 10 min at 50000 g with resus-
pension of the pellet in fresh buffer. The final pellet was resus-
pended in 50 mM ice-cold Tris-HCl, (pH 7.4 at 258C). Each assay
tube contained 50 lL drug solution, 50 lL [3H]prazosine (80.5 Ci/
mmol, Perkin Elmer Life Sciences) to achieve a final concentra-
tion of 0.4 nM, and 900 lL resuspended membranes (10 mg fresh
tissue). The tubes were incubated for 30 min at 258C and the
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Arch. Pharm. Chem. Life Sci. 2008, 341, 20–27
5-HT1A Receptor Ligands Containing Benzotriazinone Nucleus
27
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 7.2 at 258C). The radioactivity bound to the filters was meas-
ured by a liquid scintillation counter. Specific [3H]prazosin bind-
ing was defined as the difference between binding in the
absence or in the presence of 10 lM phentolamine.
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The procedure used in the radioligand binding assay was
reported in detail by Perry and U'Prichard [33]. Brain cortex was
homogenized in 30 vol. (w/v) ice-cold 5 mM Tris-HCl, 5 mM EDTA
buffer (pH 7.3 at 258C) using a Polytron PT10 (setting 5 for 20 s).
Homogenates were centrifuged three times for 10 min at 50000
g with resuspension of the pellet in fresh buffer. The final pellet
was resuspended in 50 mM ice-cold Tris-HCl, 0.5 mM EDTA
(pH 7.5 at 258C). Each assay tube contained 50 lL drug solution,
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to achieve a final concentration of 1 nM, and 900 lL resus-
pended membranes (10 mg fresh tissue). The tubes were incu-
bated for 30 min at 258C 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, 0.5 mM EDTA buffer (pH 7.5 at 258C). The
radioactivity bound to the filters was measured by a liquid scin-
tillation counter. Specific [3H]yohimbine binding was defined as
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