New potent 5-HT2A receptor ligands containing an N′-cyanopicolinamidine nucleus: Synthesis and in vitro pharmacological evaluation

Article abstract of DOI:10.1016/j.ejmech.2011.11.023

N′-cyanopicolinamidine derivatives, linked to an arylpiperazine moiety, were prepared and their affinity to serotonin 5-HT_1A, 5-HT_2A and 5-HT_2C receptors were evaluated. The combination of structural elements (heterocyclic

Full text of DOI:10.1016/j.ejmech.2011.11.023

European Journal of Medicinal Chemistry 47 (2012) 520e529
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New potent 5-HT2A receptor ligands containing an N0-cyanopicolinamidine
nucleus: Synthesis and in vitro pharmacological evaluation
Ferdinando Fiorino a, Beatrice Severino a, Elisa Magli a, Elisa Perissutti a, Francesco Frecentese a,
Antonella Esposito a, Giuseppina Maria Incisivo a, Antonio Ciano a, Paola Massarelli b, Cristina Nencini b,
,
Vincenzo Santagada a, Giuseppe Caliendo a
*
a Dipartimento di Chimica Farmaceutica e Tossicologica, Università di Napoli “Federico II”, Via D. Montesano, 49-80131 Napoli, Italy
b Dipartimento di Scienze Neurologiche, Neurochirurgiche e del Comportamento e Sezione di Farmacologia “G. Segre”, Università di Siena, Via delle Scotte, 6-53100 Siena, Italy
a r t i c l e i n f o
a b s t r a c t
N0-cyanopicolinamidine derivatives, linked to an arylpiperazine moiety, were prepared and their affinity
to serotonin 5-HT1A, 5-HT2A and 5-HT2C receptors were evaluated. The combination of structural
elements (heterocyclic nucleus, alkyl chain and 4-substituted piperazine) known to be critical for affinity
to 5-HT1A receptors and the proper selection of substituents led to compounds with high specificity and
affinity towards serotoninergic receptors. In binding studies, several molecules showed affinity in
nanomolar and subnanomolar range at 5-HT2A and moderate to no affinity for other relevant receptors
(5-HT1A, 5-HT2C, D1, D2, a1 and a2). N0-cyano-N-(3-(4-(3-chlorophenyl)piperazin-1-yl)propyl)-picolina-
midine (4l) with Ki ¼ 0.000185 nM, was the most active and selective derivative for the 5-HT2A receptor
compared to other serotoninergic, dopaminergic and adrenergic receptors.
Article history:
Received 7 October 2011
Received in revised form
9 November 2011
Accepted 10 November 2011
Available online 20 November 2011
Keywords:
N0-cyanopicolinamidine derivatives
5-HT1A ligands
Ó 2011 Elsevier Masson SAS. All rights reserved.
5-HT2A ligands
Binding assays
1. Introduction
in human hormone refractory prostate cancer tissue [11]. The 5-HT2
receptor family has three known subtypes, 5-HT2A, 5-HT2B, and 5-
Serotonin (5-hydroxytryptamine, 5-HT) [1e4] is involved in
wide-ranging physiological and pathophysiological processes [5].
Pharmacological manipulation of the 5-HT system is believed to
have therapeutic potential, and therefore the subject of intense
research [4].
Among the 14 different serotonin receptors, belonging to the G
protein coupled receptors superfamily [4], 5-HT1A and 5-HT2A
receptors are most frequently considered as important targets for
neuro-biological research and drug development. Activation of
these receptors leads to a number of physiological changes that can
be easily quantified [6e8].
5-HT1A receptor, as a member of the group of G protein coupled
receptors (GPCRs) [9], shows high similarity with other members of
the family. A particular case is represented by the similarity
between 5-HT1AR and a1-adrenoreceptor [10] that show a high
degree of homology (45%) in their amino acid sequence. In addition
to its effects on a wide range of psychiatric disorders, 5-HT1AR is
also involved in the proliferation of human tumour cells (PC3) and
HT2C, with 46e50% sequence homology [4]. Moreover, the 5-
HT2AR and 5-HT2CR transmembrane domains share 80% sequence
homology, suggesting similar pharmacological profiles [12]. Acti-
vation of 5-HT2A receptors stimulates the secretion of various
hormones and influences neuronal plasticity; peripheral 5-HT2A
receptors mediate several processes such as vasoconstriction and
platelet aggregation [4]. The 5-HT2C receptor is involved in physi-
ological functions such as locomotory activity, anxiogenesis and
neuro-endocrine functions, and is implicated in sexual dysfunction
in males [13,14].
On these basis drugs that target 5-HT2 receptors could be useful
for the treatment of several psychiatric disorders, including
depression, anxiety, obsessive-compulsive disorders and schizo-
phrenia [15].
Several chemical classes are already known for their high
affinity towards these receptors and, from a chemical point of view,
they can be subdivided into different classes. Among them one of
the most studied group is that of long-chain arylpiperazines
(LCAPs) [8]. In these derivatives the arylpiperazine moiety, tail of
the molecule, and the heterocyclic head are separated with a spacer
or linker. In the aryl position substituted phenyl is typically located
while the heterocyclic nucleus is an amide or imide. The
* Corresponding author. Tel./fax: þ39 081 678649.
E-mail address: caliendo@unina.it (G. Caliendo).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.11.023

F. Fiorino et al. / European Journal of Medicinal Chemistry 47 (2012) 520e529
521
significance of the respective parts of the LCAP structures on the 5-
HT1A receptor affinity, intrinsic activity, and selectivity has been the
subject of many structureeactivity relationship (SAR) studies
associated with variation of the aromatic systems at the “head” and
“tail” of the structure, with the length of the methylene linker, and
with the replacement of the amide bond with other functions [16].
A huge number of SAR studies revealed the role of individual
fragments, essential for high affinity for 5-HT1A sites and selectivity
for other receptors. However, a limitation of many 5-HT1A receptor
ligands is their undesired high affinity for other receptor subtypes
such as the dopaminergic D2 receptor and a1-adrenoceptor.
In our laboratories, there has been an ongoing effort to develop
more selective serotoninergic ligands [17e26] in order to have
novel pharmacological tools that could improve our knowledge of
the signal transduction mechanism leading to compounds with
high affinity and selectivity.
A previously described study focused on the synthesis and
pharmacological evaluation of a set of arylpiperazine derivatives
containing an N0-cyanoisonicotinamidine nucleus; the binding data
reported in this study identified this original scaffold as an optimal
structural element to enhance 5-HT1A receptor affinity [26].
In continuation of our research program, we designed a new set
of derivatives where the piperazine-N-alkyl moiety has been linked
to a novel original N0-cyanopicolinamidine fragment as terminal
part of LCAPs (Scheme 1); this choice was made considering that it
could be interesting to investigate the correlation between the
nitrogen position in the pyridine ring and binding affinity/selec-
tivity. The N0-cyanopicolinamidine scaffold was linked via three
methylene spacing units to piperazines substituted in position 4
with aliphatic and/or aromatic substitutes. The alkyl chain length
(three units) and the substitutes on the N-4 of the piperazine
moiety were the same employed in our previous study [26]. This
choice was done in order to obtain a complete and comparative
structure-affinity and structure-selectivity relationship study
between the N0-cyanoisonicotinamidine derivatives and the N0-
cyanopicolinamidine derivatives. All the new compounds were
tested for their affinity for 5-HT1A, 5-HT2A and 5-HT2C receptors and
the multireceptor profiles of promising derivatives were also
evaluated in terms of binding affinities for dopaminergic (D1, D2)
The general procedure is as follows: methyl-N-cyano-2-
pyridinecarboximidate (2) was prepared starting from 2-
cyanopyridine (1) by base-catalyzed conversion in anhydrous
methanol followed by treatment with cyanamide in aqueous
phosphate buffer (NaH2PO4$2H2O/Na2HPO4 ¼ 4:1). The conver-
sion of methyl-N-cyano-2-pyridinecarboximidate (2) to N-(3-
bromopropyl)-N0-cyanopicolinamidine (3) was obtained by
reaction of (2) with 3-bromopropylamine$HBr and triethyl-
amine in anhydrous methanol. Subsequent condensation of
compound (3) with the desired 4-X-substituted piperazine,
performed in CH3CN in the presence of K2CO3 and NaI, under
reflux, provided the final compounds 4aeq. 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 anal-
yses and were characterized by 1H NMR and mass spectrometry
(API 2000 Applied Biosystem). 1H NMR and MS data for all final
compounds were consistent with the proposed structures. As
already reported in literature, due to tautomeric equilibrium
about sp2 carbon, cyanoamidine derivatives may exist as
a mixture of two tautomers (I and II in Fig. 1). The NMR spectra
of 4aeq confirmed that all final compounds exist predominantly
as the cyanoimino form (I). Moreover, regarding the configura-
tional determination of geometrical isomers (E/Z) on the ami-
dine bond C]N, the N-cyanoamidine derivatives 4aeq exhibited
only one set of relevant 1H NMR signals, implying the existence
of either a single geometric form or, more probably, a fast
equilibrium of two isomeric forms as already described in
literature [27].
3. Results and discussion
Several of the reported derivatives were potent 5-HT2A receptor
ligands, in fact, they showed nanomolar or even subnanomolar 5-
HT2A receptor affinities (Table 1). Besides the outstanding 5-HT2A
receptor affinity of compound 4l (Ki ¼ 0.000185 nM, Fig. 3), other
interesting Ki values were those of compounds 4m (0.00078 nM,
Fig. 4), 4d (0.00157 nM, Fig. 2), 4n (0.0161 nM, Fig. 5) and 4h
(0.867 nM) while compounds 4o, 4i, 4p, 4q, 4g and 4f were less
active with Ki values of 1.12, 27, 51.8, 21.5, 904 and 2730 nM,
respectively. Other derivatives showed Ki values of above 104 nM or
no affinity.
and adrenergic (a1, a2) receptors.
2. Chemistry
Derivatives 4aeq are generally characterized by a binding
affinity/selectivity profile that is surprisingly shifted towards 5-
HT2A receptors subtypes. Moreover the introduction of non
The synthetic strategy employed for the preparation of
the target compounds (Table 1) is summarized in Scheme 1.
NCN
NCN
N
N
N
CN
NH(CH2)3Br
OCH3
i
ii
(3)
(1)
(2)
iii
NCN
N
N
N
H
N
X
(4a-q)
Scheme 1. Reagents and conditions: (i) CH3ONa, H2NCN, anhydrous MeOH; (ii) Br(CH2)3NH2$HBr, TEA, anhydrous MeOH; (iii) 4-X-substituted piperazine, K2CO3, NaI, CH3CN,
70 ꢀC, 4 h.

522
F. Fiorino et al. / European Journal of Medicinal Chemistry 47 (2012) 520e529
Table 1
Affinities of compounds 4aeq for 5-HT1A, 5-HT2A and 5-HT2C receptors.
CN
N
N
N
H
N
N
X
Receptor affinity Ki ꢁ SD (nM)
Compd
X
5-HT1A [3H]8OH-DPAT
5-HT2A [3H]ketanserin
5-HT2C [3H]mesulergine
4a
No affinity
No affinity
>104
4b
4c
No affinity
No affinity
No affinity
No affinity
389 ꢁ 17.7
597 ꢁ 8.8
OCH3
OCH2CH3
4d
4e
4f
4.68 ꢁ 0.389
0.00157 ꢁ 0.000106
>104
1.61 ꢁ 0.458
No affinity
No affinity
0.0842 ꢁ 0.00088
17.2 ꢁ 1.36
OCH3
H3CH2CO
2730 ꢁ 143
NC
4g
4h
4i
>104
904 ꢁ 10.2
0.867 ꢁ 0.0308
27 ꢁ 0.97
28.8 ꢁ 2.67
29.7 ꢁ 2.14
No affinity
H3C
CH3
30 ꢁ 1.79
170 ꢁ 11.7
Cl
Cl
4l
6.67 ꢁ 0.070
1.31 ꢁ 0.263
0.000185 ꢁ 0.0000168
0.000778 ꢁ 0.0000331
3.28 ꢁ 0.223
1.81 ꢁ 0.178
4m
N
4n
4o
4p
3080 ꢁ 154
30.6 ꢁ 2.20
>104
0.0161 ꢁ 0.00120
1.12 ꢁ 0.178
22.5 ꢁ 1.74
>104
N
N
O
O
51.8 ꢁ 0.27
No affinity
O
4q
>104
21.5 ꢁ 0.21
No affinity
O
For purpose of comparison, 8-OH-DPAT, ketanserine and mesulergine bind 5-HT1A, 5-HT2A and 5-HT2C receptors with values of 0.80, 0.85 and 1.90 nM, respectively, under
these assay conditions.

F. Fiorino et al. / European Journal of Medicinal Chemistry 47 (2012) 520e529
523
CN
CN
HN
Compound 4l
N
N
N
N
H
N
N
N
N
N
X
X
I
II
Fig. 1. Tautomeric equilibrium about sp2carbon of the cyanoamidine derivatives.
aromatic or larger substituents on the piperazine nitrogen, also in
this series of derivatives, caused a dramatic decrease of the receptor
affinity.
Concerning the influence of the N-4 substituent of the pipera-
zine moiety, the 3-chlorophenyl group (4l), and the 3-
trifluoromethylphenyl group (4m), conferred the highest affinity
and selectivity for 5-HT2A receptor. The presence of
a 2-
methoxyphenyl, pyrimidinyl or 2,3-dimethylphenyl group on the
N-4 of the piperazine moiety, led also to compounds which
exhibited high affinity for 5-HT2A receptor (4d Ki ¼ 0.00157 nM;
4n Ki ¼ 0.0161 nM and 4h Ki ¼ 0.867 nM). Non aromatic moiety as
well as 2-ethoxyphenyl and 2-cyanophenyl groups furnished
compounds characterized by a high decrease in binding affinity
compared to the other N-4 substitutes at the piperazine ring.
The 5-HT1A and 5-HT2C receptor affinities of the tested
compounds were always lower than those observed for 5-HT2A
receptors except for compounds 4e and 4g that showed higher
affinities towards 5-HT1A (Ki ¼ 0.0842 nM) and 5-HT2C receptors
(Ki ¼ 28.8 nM), respectively. N-(3-(4-(3-chlorophenyl)piperazin-
1-yl)propyl)-N0-cyanopicolinamidine (4l) (Ki ¼ 0.000185 nM)
showed not only the highest affinity but also one of the best
selectivity profiles with respect to the other serotoninergic
receptors studied.
Fig. 3. Binding curve of compound 4l.
Additionally, the affinity of the most active compounds (4d, 4e,
4f, 4h, 4l, 4m, 4n, 4o, 4p and 4q) on several other receptors (a1 and
a2 adrenergic and D1 and D2 dopaminergic receptors) was
Compound 4m
Compound 4d
Fig. 4. Binding curve of compound 4m.
Fig. 2. Binding curve of compound 4d.

524
F. Fiorino et al. / European Journal of Medicinal Chemistry 47 (2012) 520e529
A comparative structure-affinity and structure-selectivity rela-
Compound 4n
tionship study between the N0-cyanoisonicotinamidine derivatives
and the N0-cyanopicolinamidine derivatives emphasizes the high
selectivity afforded by the discussed N0-cyanopicolinamidine scaf-
fold to shift the binding affinity towards 5-HT2A receptors subtypes.
Moreover this study confirmed that the propyl chain associated to
the appropriate aromatic ring substitution plays a critical role not
only in determining 5-HT1A receptor affinity and selectivity but it is
also useful to determinate a favourable 5-HT2A receptor affinity/
selectivity profile.
The outstanding affinity of 4l, supporting the 3-chlorophenyl
group, and 4m, supporting
a
very hydrophobic 3-
trifluoromethylphenyl group, could be explained, as already re-
ported by Parker et al. [28], in terms of correlation between
lipophilicity and binding affinity towards 5-HT2A receptors that
appear decisive compared to all other compounds. Moreover, the
interesting affinity/selectivity profile of 4d, 4n, and 4o, sup-
porting a 2-methoxyphenyl, a pyrimidinyl and a pyridin-2-yl
group, respectively, are in accordance with data already re-
ported in literature where, as general trend, the presence of an
ether oxygen (4d) or a nitrogen (4n and 4o) acts as hydrogen
bond acceptors from key residues such as threonine and serine in
the 5-HT2A receptor binding pocket [29].
The main feature of these new derivatives was represented by
the interest in the N0-cyanopicolinamidine fragment as a molecular
scaffold that plays a critical role in determining 5-HT2A receptor
affinity and selectivity, evolved from our previous work [26] with
the N0-cyanoisonicotinamidine scaffold. This result could be due to
the different position of nitrogen into the aromatic system of the
N0-cyanopicolinamidine nucleus compared to the N0-cyanoisoni-
cotinamidine scaffold and could be explained considering also the
topography of the 5-HT2A receptor binding site. Molecular model-
ling of the 5-HT2A receptor, together with detailed site-directed
Fig. 5. Binding curve of compound 4n.
mutagenesis studies, suggests
a hydrophobic binding pocket
surrounding the ligands, with aromatic residues such as Trp-151,
Phe-243, Phe-244, Trp336, Phe-339, Phe-340, Trp-367 and Tyr-
370. Polar moieties may interact with Ser-159, Thr-160, Ser-239,
Ser-242 and Asn-343 from the 5-HT2A binding site [5] and
protonated amine, like piperazine moiety, undergoes an electro-
static binding interaction with the conserved aspartate (Asp-159)
residue in transmembrane helix 3 (TM3) [30]. Moreover human 5-
HT2A receptor and rat receptor (object of our study) are highly
homologous in their amino acid sequences. Both contain Ser-239,
but one of the three differences in the TMD sequence is seen at
position 242 in fifth TMD, where the human receptor expresses
a serine and the rat receptor expresses an alanine residue. The Ser-
239 and Ser-242 in the human receptor are predicted to act as
a hydrogen-bonding site for example for serotonin 5-hydroxyl
group and indole NH respectively [31]. However, as already re-
ported [32], H-bond of the indole NH to the side chains of Ser-159
or Thr-160 may also be possible since Ser-242 is mutated into Ala in
the rat 5-HT2AR. On the basis of these considerations we support
the idea that, similarly to the indolic NH [31], the nitrogen lone pair
into the aromatic system of the N0-cyanopicolinamidine nucleus
can form H-bond with the side chains of Ser-159 or Thr-160.
Therefore, the different position of nitrogen into the aromatic
systems of the N0-cyanopicolinamidine and N0-cyanoisonicotina-
midine nuclei can explain the different bent to form favourable H-
bond with Ser-159 or Thr-160 and, consequently, the different
affinity/selectivity binding profile of N0-cyanopicolinamidine
derivatives towards 5-HT2A receptors and N0-cyanoisonicotinami-
dine derivatives towards 5-HT1A receptors.
examined in order to verify the selectivity of these compounds.
Results are summarized in Table 2. All the compounds proved
highly selective against dopaminergic receptors with Ki values of
above 104 nM except for compound 4f, which exhibited Ki value of
52.4 nM on D1 receptor and compound 4m, which exhibited Ki
value of 83.7 nM on D2 receptor. Regarding a1 and a2 adrenergic
receptors, only compound 4d showed quite moderate affinity
(105 nM and 245 nM respectively), while compound 4o showed
quite moderate affinity only towards a1 receptors (130 nM); these
data are very interesting considering the high degree of homology
existing between these two receptors and demonstrate that these
compounds possess a very good binding profile, preferring 5-
HT2ARs over all other evaluated receptors.
Table 2
Affinities of compounds 4d, 4e, 4f, 4h, 4l, 4m, 4n, 4o, 4p and 4q for D1, D2, a1 and a2
receptors.
Receptor affinity Ki ꢁ SD (nM)
Compd
D1
D2
a1
a2
[3H]SCH-23390
[3H]spiperone
[3H]prazosin
[3H]yohimbine
4d
4e
4f
4h
4l
4m
4n
4o
4p
4q
>104
>104
105 ꢁ 28.1
245 ꢁ 37.7
No affinity
571 ꢁ 38.4
318 ꢁ 7.8
1360 ꢁ 77
>104
>104
>104
>104
52.4 ꢁ 3.57
>104
152 ꢁ 7.4
729 ꢁ 9.3
174 ꢁ 19.2
770 ꢁ 15.9
>104
130 ꢁ 11.9
No affinity
No affinity
>104
>104
381 ꢁ 33
>104
>104
83.7 ꢁ 3.23
>104
>104
No affinity
>104
>104
>104
In fact, compound 4e (Ki value of 0.0842 nM) supporting p-
methoxy-phenylpiperazine moiety, was the only derivative that
showed a favourable affinity/selectivity binding profile towards 5-
No affinity
No affinity
No affinity
No affinity
964 ꢁ 67.0
>104

F. Fiorino et al. / European Journal of Medicinal Chemistry 47 (2012) 520e529
525
HT1A receptors. This result, apparently clashing with the low
5.1.2. Methyl N-cyano-2-pyridinecarboximidate (2)
affinity and selectivity of the previously reported 4-methoxy-phe-
nylpiperazine derivatives [25,26] could probably be attributed to
favourable steric interaction of the methoxy group (4e) with the
surrounding receptor residues that leads to the formation of
a hydrogen bond with Asn-386 associated to the presence of the N0-
cyanopicolinamidine scaffold.
Finally the interesting 5-HT2C receptor affinities of some deriv-
atives (4d Ki ¼ 1.61 nM; 4h Ki ¼ 29.7 nM; 4l Ki ¼ 3.28 nM; 4m
Ki ¼ 1.81 nM; 4n Ki ¼ 22.5 nM) are lower than those observed for 5-
HT2AR and comparable to those observed for 5-HT1AR, determining
for these derivatives a bad affinity/selectivity profile towards the 5-
HT2C receptor. Moreover, also in this series, the derivatives char-
acterized by the presence of an aliphatic group (4a, 4b and 4c) on
the N-4 position led to an unfavourable affinity/selectivity profile,
confirming, as already reported [24e26], that a non aromatic
moiety is less favourable to the hydrophobic interaction with the
receptor, determining the formation of weaker complexes with the
receptor.
A
mixture of 2-cyanopyridine (1) (10 g, 0.096 mol) and
NaOMe (0.26 g, 0.0048 mol) in anhydrous methanol (180 ml)
was stirred overnight at room temperature. AcOH (0.32 g,
0.0053 mol) was then added with stirring to neutralize the
reaction solution and the solution was evaporated in vacuo.
Diethyl ether (150 ml) was added to the residue and the resultant
precipitate was filtered off. The filtrate was evaporated to give
crude methyl-2-pyridinecarboximidate (7.0 g) as an oil. Crude
compound was then added to a mixture of NH2CN (4.33 g,
0.103 mol), NaH2PO4$2H2O (32.14 g) and Na2HPO4 (7.30 g) in
water (55 ml). After vigorous stirring for 4 h at room tempera-
ture, the reaction mixture was extracted several times with
CH2Cl2. The combined organic layers were dried on anhydrous
Na2SO4 and concentrated in vacuo to yield crude methyl-N-
cyano-2-pyridinecarboximidate (2) (8.4 g), which was used
directly in the following reaction. 1H NMR (400 MHz, CDCl3)
d:
4.16 (s, 3H); 7.63 (dd, 1H, J ¼ 9.4, 7.3); 7.94 (d, 1H, J ¼ 3.4); 7.98
(dd, 1H, J ¼ 7.3, 2.4); 8.83 (ddd, 1H, J ¼ 9.4, 3.4, 2.4). 13C NMR
(400 MHz, CDCl3)
d: 46.35; 116.90; 123.64; 126.80; 136.25;
148.13; 149.15, 153.10.
4. Conclusion
5.1.3. N-(3-Bromopropyl)-N0-cyanopicolinamidine (3)
We have described the synthesis of a new series of arylpiper-
azines as 5-HT2A ligands (4aeq), containing a novel heterocyclic
fragment. Some of the described compounds showed high in vitro
affinity and selectivity towards 5-HT2A receptors. Compound 4l was
the most potent (Ki ¼ 0.000185 nM) and selective derivative for 5-
HT2A receptor with respect to the other serotonin, dopaminergic
and adrenergic receptors, and the affinity/selectivity profile of
compound 4m (Ki ¼ 0.000778 nM) supporting on the N-4 of the
piperazine moiety the 3-trifluoromethylphenyl group appears
interesting besides. The binding data presented in this study
identified the N0-cyanopicolinamidine nucleus as an optimal
structural element to shift the affinity/selectivity profile towards 5-
HT2A receptor binding, compared to the N0-cyanoisonicotinamidine
scaffold that furnished a series of derivatives with good 5-HT1A
affinity and selectivity.
To a solution of methyl N-cyano-2-pyridinecarboximidate (2)
(8 g,
0.050 mol)
in
anhydrous
MeOH
(120 mL),
3-
bromopropylamine$HBr (11.93 g, 0.055 mol) and triethylamine
(5.56 g 0.055 mol) were successively added and the reaction
mixture was stirred at room temperature for 18 h. After evapora-
tion, the residue was dissolved in CHCl3. The solution was washed
with water, dried on anhydrous Na2SO4 and concentrated in vacuo.
The residue was purified by column chromatography (diethyl
ether/ethanol 9:1 (v/v)). The combined and evaporated product
fractions were crystallized from diethyl ether /hexane, yielding
8.4 g (63 %) of the desired product as a brown solid: mp: 73e74 ꢀC;
1H NMR (400 MHz, CDCl3)
d
: 2.15 (qt, 2H, J ¼ 6.2); 2.75 (t, 2H,
J ¼ 6.2); 3.83 (q, 2H, J ¼ 6.2); 7.47 (t, 1H, J ¼ 7.3); 7.86 (t, 1H, J ¼ 7.3);
8.5 (d, 1H, J ¼ 7.3); 8.64 (d, 1H, J ¼ 7.3); 9.62 (s, 1H). 13C NMR
(400 MHz, CDCl3) d: 27.33; 38.70; 53.91; 117.20; 122.54; 125.66;
135.10; 148.23; 150.70, 154.20.
5. Experimental
5.1.4. General procedure for the condensation of 4-substituted
arylpiperazines with derivative 3
5.1. Synthesis
A mixture of N-(3-bromopropyl)-N0-cyanopicolinamidine (3)
(0.006 mol) and NaI (0.009 mol) in acetonitrile was stirred under
reflux for 30 min. Then the appropriate 4-substituted arylpiper-
azine (0.06 mol) and anhydrous K2CO3 (0.009 mol) were added. The
reaction mixture was stirred under reflux for 4 h. After cooling, the
mixture was filtered, concentrated to dryness and the residue was
dissolved in water (50 mL). The solution was extracted several
times with CH2Cl2. The combined organic layers were dried on
anhydrous Na2SO4 and the solvent removed under vacuum. The
crude mixture was purified by silica gel column chromatography
using diethyl ether/methanol 8:2 (v/v) as eluent. The crude prod-
ucts were recrystallized from diethyl ether.
5.1.1. General procedures
All reagents and substituted piperazines were commercial
products purchased from Aldrich. Melting points were deter-
mined using a Kofler hot-stage apparatus and are uncorrected. 1H
NMR and 13C NMR spectra were recorded on Varian Mercury Plus
400 MHz instrument. Unless otherwise stated, all spectra were
recorded in CDCl3. Chemical shifts are reported in ppm using
Me4Si as internal standard. The following abbreviations are used
to describe peak patterns when appropriate:
s (singlet),
d (doublet), t (triplet), m (multiplet), q (quartet), qt (quintet), dd
(double doublet), ddd (double dd), br s (broad singlet). Mass
spectra of the final products were performed on API 2000
Applied Biosystem mass spectrometer. Where analyses are indi-
cated only by the symbols of the elements, results obtained are
within ꢁ0.4% of the theoretical values. All reactions were fol-
lowed by TLC, carried out on Merck silica gel 60 F254 plates with
fluorescent indicator and the plates were visualized with UV light
(254 nm). Preparative chromatographic purifications were per-
formed using silica gel column (Kieselgel 60). Solutions were
dried over Na2SO4 and concentrated with Buchi rotary evapo-
rator at low pressure.
5.1.5. N-(3-(4-allylpiperazin-1-yl)propyl)-N0-cyanopicolinamidine
(4a)
From 3 and 1-allylpiperazine.
Yield: 32%; mp 93e94 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.83 (m,
2H); 2.32 (br s, 4H, 2CH2 pip.); 2.51 (br s, 4H, 2CH2 pip.); 2.55 (t, 2H,
N1eCH2, J ¼ 5.5); 3.00 (d, 2H, J ¼ 6.6); 3.81 (br s, 2H, N-CH2); 5.14
(dd, 2H); 5.81 (m, 1H,); 7.48 (t, 1H, J ¼ 7.3); 7.87 (t, 1H, J ¼ 7.7); 8.50
(d, 1H, J ¼ 8.0); 8.64 (d, 1H, J ¼ 4.4); 9.75 (s, 1H). 13C NMR (400 MHz,
CDCl3) d: 24.36; 43.29; 52.96; 53.60; 57.91; 62.11; 117.55; 118.30;
123.54; 126.86; 135.14; 137.89; 148.24; 148.90, 151.80.

526
F. Fiorino et al. / European Journal of Medicinal Chemistry 47 (2012) 520e529
ESI-MS: 313.3 [M þ H]þ; 335.2 [M þ Na]þ.
Yield: 20%; mp 118e120 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.88
Anal. (C17H24N6), C, H, N.
(m, 2H); 2.65 (t, 2H, N1eCH2, J ¼ 5.1); 2.73 (br s, 4H, 2CH2 pip.); 3.30
(br s, 4H, 2CH2 pip.); 3.87 (br s, 2H, NeCH2); 6.98 (d, 1H, J ¼ 7.1);
7.02 (t, 1H, J ¼ 7.1); 7.44e7.56 (m, 3H); 7.58 (d, 1H, J ¼ 7.3); 7.86 (t,
1H, J ¼ 7.7); 8.53 (br s, 1H); 9.78 (s, 1H).
5.1.6. N0-cyano-N-(3-(4-(2-methoxyethyl)piperazin-1-yl)propyl)
picolinamidine (4b)
From 3 and 1-(2-methoxyethyl)piperazine.
ESI-MS: 374.4 [M þ H]þ; 396.0 [M þ Na]þ; 412.0 [M þ K]þ.
Anal. (C21H23N7), C, H, N.
Yield: 16%; mp 60e61 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.83 (m,
2H); 2.28 (br s, 4H, 2CH2 pip.); 2.38 (t, 2H, N1eCH2, J ¼ 5.4); 2.55 (br
s, 4H, 2CH2 pip.); 2.57 (t, 2H N4eCH2, J ¼ 5.5); 3.35 (s, 3H, OCH3);
3.50 (t, 2H, CH2eO, J ¼ 5.5); 3.81 (br s, 2H, NeCH2,); 7.47 (t, 1H,
J ¼ 7.3); 7.86 (t, 1H, J ¼ 7.7); 8.50 (d, 1H, J ¼ 8.0); 8.64 (d, 1H, J ¼ 4.4);
9.80 (s, 1H).
5.1.12. N0-cyano-N-(3-(4-(2,3-dimethylphenyl)piperazin-1-yl)
propyl)picolinamidine (4h)
From 3 and 1-(2,3-dimethylphenyl)piperazine.
Yield: 15%; mp 119e120 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.89
ESI-MS: 331.1 [M þ H]þ.
(m, 2H); 2.21 (s, 3H, CH3); 2.27 (s, 3H, CH3); 2.38 (br s, 4H, 2CH2
pip.); 2.65 (br s, 4H, 2CH2 pip.); 2.95 (t, 2H, N1eCH2, J ¼ 5.1); 3.87
(br s, 2H, NeCH2); 6.87 (d, 1H, J ¼ 7.7); 6.91 (d, 1H, J ¼ 7.7); 7.09 (t,
1H, J ¼ 7.7); 7.44 (t, 1H, J ¼ 7.3); 7.86 (t, 1H, J ¼ 7.7); 8.51 (d, 1H,
J ¼ 8.0); 8.62 (d, 1H, J ¼ 4.4); 10.01 (s, 1H).
Anal. (C17H26N6O), C, H, N.
5.1.7. N0-cyano-N-(3-(4-(2-ethoxyethyl)piperazin-1-yl)propyl)
picolinamidine (4c)
From 3 and 1-(2-ethoxyethyl)piperazine.
ESI-MS: 377.9 [M þ H]þ; 399.0 [M þ Na]þ; 415.1 [M þ K]þ.
Anal. (C22H28N6), C, H, N.
Yield: 56%; mp 195e197 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.17 (t,
3H, J ¼ 6.9); 1.76 (br s, 4H, 2CH2 pip.); 1.81 (qt, 2H, J ¼ 5.8); 2.55 (t,
2H, N1eCH2, J ¼ 6.2); 2.56 (br s, 4H, 2CH2 pip.); 2.58 (t, 2H, N4eCH2,
J ¼ 6.2); 3.47 (q, 2H, OeCH2, J ¼ 6.9); 3.52 (t, 2H, CH2eO, J ¼ 6.2);
3.81 (br s, 2H, NeCH2); 7.46 (t, 1H, J ¼ 7.3); 7.86 (t, 1H, J ¼ 7.7); 8.49
(d, 1H, J ¼ 8.0); 8.64 (d, 1H, J ¼ 4.4); 9.80 (s, 1H).
5.1.13. N-(3-(4-(2-chlorophenyl)piperazin-1-yl)propyl)-N0-
cyanopicolinamidine (4i)
From 3 and 1-(2-chlorophenyl)piperazine.
Yield: 26%; mp 86e88 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.88 (m,
ESI-MS: 345.4 [M þ H]þ; 367.1 [M þ Na]þ; 383.0 [M þ K]þ.
Anal. (C18H28N6O), C, H, N.
2H); 2.65 (t, 2H, N1eCH2, J ¼ 5.1); 2.71 (br s, 4H, 2CH2 pip.); 3.14 (br
s, 4H, 2CH2 pip.); 3.87 (br s, 2H, NeCH2); 6.97 (d, 1H, J ¼ 8.0); 7.01(t,
1H, J ¼ 8.0); 7.26 (t, 1H, J ¼ 8.0); 7.36 (d, 1H, J ¼ 8.0); 7.44 (t, 1H,
J ¼ 7.3); 7.86 (t, 1H, J ¼ 7.7); 8.53 (d, 1H, J ¼ 8.0); 8.57 (d, 1H, J ¼ 4.4);
9.94 (s, 1H).
5.1.8. N0-cyano-N-(3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)
picolinamidine (4d)
From 3 and 1-(2-methoxyphenyl)piperazine.
ESI-MS: 383.2 [M þ H]þ.
Yield: 20%; mp 113e116 ꢀC. 1H NMR (400 MHz, CDCl3)
d
: 1.67 (m,
Anal. (C20H23ClN6), C, H, N.
2H); 1.87 (qt, 2H, eCH2e, J ¼ 5.5); 2.65 (t, 2H, N1eCH2, J ¼ 5.1); 2.72
(br s, 4H, 2CH2 pip.); 3.15 (br s, 4H, 2CH2 pip.); 3.87 (s, 3H, OCH3);
6.87 (d, 1H, J ¼ 7.4); 6.89 (d, 1H, J ¼ 7.4); 6.93 (t, 1H, J ¼ 7.4); 7.01 (t,
1H, J ¼ 7.4); 7.41 (t, 1H, J ¼ 7.3); 7.84 (t, 1H, J ¼ 7.7); 8.51 (d, 1H,
J ¼ 8.0); 8.55 (d, 1H, J ¼ 4.4); 10.06 (s, 1H).
5.1.14. N-(3-(4-(3-chlorophenyl)piperazin-1-yl)propyl)-N0-
cyanopicolinamidine (4l)
From 3 and 1-(3-chlorophenyl)piperazine.
Yield: 30%; mp 148e149 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.89
ESI-MS: 379.4 [M þ H]þ; 401.6 [M þ Na]þ; 417.5 [M þ K]þ.
Anal. (C21H26N6O), C, H, N.
(m, 2H); 2.63 (br s, 4H, 2CH2 pip.); 2.66 (t, 2H, N1eCH2, J ¼ 5.1); 3.25
(br s, 4H, 2CH2 pip.); 3.87 (br s, 2H, NeCH2); 6.77 (d, 1H, J ¼ 8.0);
6.82 (d, 1H, J ¼ 8.0); 6.87 (s, 1H); 7.16 (t, 1H, J ¼ 8.0); 7.39 (t, 1H,
J ¼ 7.3); 7.83 (t, 1H, J ¼ 7.7); 8.39 (d, 1H, J ¼ 4.4); 8.51 (d, 1H, J ¼ 8.0);
9.96 (s, 1H).
5.1.9. N0-cyano-N-(3-(4-(4-methoxyphenyl)piperazin-1-yl)propyl)
picolinamidine (4e)
From 3 and 1-(4-methoxyphenyl)piperazine.
ESI-MS: 383.5 [M þ H]þ.
Yield: 15%; mp 106e108 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 1.90
Anal. (C20H23ClN6), C, H, N.
(m, 2H); 2.63 (br s, 4H, 2CH2 pip.); 2.66 (t, 2H, N1eCH2, J ¼ 5.1); 3.16
(br s, 4H, 2CH2 pip.); 3.78 (s, 3H, OCH3); 3.88 (br s, 2H, NeCH2); 6.84
(d, 2H, J ¼ 8.9); 6.88 (d, 2H, J ¼ 8.9); 7.39 (t, 1H, J ¼ 7.3); 7.83 (t, 1H,
J ¼ 7.7); 8.44 (d, 1H, J ¼ 4.4); 8.51 (d, 1H, J ¼ 8.0); 9.97 (s, 1H).
ESI-MS: 379.9 [M þ H]þ; 401.3 [M þ Na]þ; 417.3 [M þ K]þ.
Anal. (C21H26N6O), C, H, N.
5.1.15. N0-cyano-N-(3-(4-(3-(trifluoromethyl)phenyl)piperazin-1-
yl)propyl)picolinamidine (4m)
From 3 and 1-(3-(trifluoromethyl)phenyl)piperazine.
Yield: 24%; mp 115e116 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.90 (m,
2H); 2.64 (br s, 4H, 2CH2 pip.); 2.67 (t, 2H, N1eCH2, J ¼ 5.1); 3.29 (br
s, 4H, 2CH2 pip.); 3.89 (br s, 2H, NeCH2); 7.06 (d, 1H, J ¼ 8.0); 7.1 (s,
1H); 7.11 (d, 1H, J ¼ 8.0); 7.35 (t, 1H, J ¼ 8.0); 7.39 (t, 1H, J ¼ 7.3); 7.84
(t, 1H, J ¼ 7.7); 8.38 (d, 1H, J ¼ 4.4); 8.51 (d, 1H, J ¼ 8.0); 9.91 (s, 1H).
ESI-MS: 417.6 [M þ H]þ; 439.4 [M þ Na]þ; 455.2 [M þ K]þ.
Anal. (C21H23F3N6), C, H, N.
5.1.10. N0-cyano-N-(3-(4-(2-ethoxyphenyl)piperazin-1-yl)propyl)
picolinamidine (4f)
From 3 and 1-(2-ethoxyphenyl)piperazine.
Yield: 14%; mp 122e125 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.43 (t,
3H, J ¼ 6.9); 1.88 (m, 2H); 2.64 (t, 2H, N1eCH2, J ¼ 5.1); 2.71 (br s,
4H, 2CH2 pip.); 3.17 (br s, 4H, 2CH2 pip.); 3.86 (br s, 2H, NeCH2);
4.04 (q, 2H, OCH2, J ¼ 6.9); 6.85 (d, 1H, J ¼ 7.7); 6.89 (d, 1H, J ¼ 7.7);
6.91 (t, 1H, J ¼ 7.7); 6.96 (t, 1H, J ¼ 7.7); 7.41 (t, 1H, J ¼ 7.3); 7.85 (t,
1H, J ¼ 7.7); 8.50 (d, 1H, J ¼ 8.0); 8.55 (d, 1H, J ¼ 4.4); 10.10 (s, 1H).
ESI-MS: 393.5 [M þ H]þ; 415.2 [M þ Na]þ.
5.1.16. N0-cyano-N-(3-(4-(pyrimidin-2-yl)piperazin-1-yl)propyl)
picolinamidine (4n)
From 3 and 1-(pyrimidin-2-yl)piperazine.
Yield: 27%; mp 110e111 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.89 (m,
2H); 2.55 (br s, 4H, 2CH2 pip.); 2.61 (t, 2H, N1eCH2, J ¼ 5.1); 3.58 (br
s, 4H, 2CH2 pip.); 3.90 (br s, 2H, NeCH2); 6.49 (t,1H, J ¼ 4.4); 7.49 (d,
2H, J ¼ 4.4); 7.43 (t, 1H, J ¼ 7.3); 7.85 (t, 1H, J ¼ 7.7); 8.30 (d, 1H,
J ¼ 4.4); 8.52 (br s, 1H); 9.99 (s, 1H).
Anal. (C22H28N6O), C, H, N.
5.1.11. N0-cyano-N-(3-(4-(2-cyanophenyl)piperazin-1-yl)propyl)
picolinamidine (4g)
ESI-MS: 351.0 [M þ H]þ; 373.2 [M þ Na]þ; 389.0 [M þ K]þ.
Anal. (C18H22N8), C, H, N.
From 3 and 2-(piperazin-1-yl)benzonitrile.

F. Fiorino et al. / European Journal of Medicinal Chemistry 47 (2012) 520e529
527
5.1.17. N0-cyano-N-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)
5.2.2. 5-HT1A binding assay
picolinamidine (4o)
Radioligand binding assays were performed following a pub-
lished procedure [36]. Cerebral cortex from male Sprague-Dawley
rats (180e220 g) was homogenized in 20 volumes of ice-cold
TriseHCl buffer (50 mM, pH 7.7 at 22 ꢀC) with a Polytron PT10,
Brinkmann Instruments (setting 5 for 15 s), and the homogenate
was centrifuged at 50,000 g for 10 min. The resulting pellet was
then resuspended in the same buffer, incubated for 10 min at 37 ꢀC,
and centrifuged at 50,000 g for 10 min. The final pellet was resus-
From 3 and 1-(pyridin-2-yl)piperazine.
Yield: 16%; mp 90e92 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.91 (m,
2H); 2.62 (br s, 4H, 2CH2 pip.); 2.72 (t, 2H, N1eCH2, J ¼ 5.1); 3.61 (br
s, 4H, 2CH2 pip.); 3.88 (br s, 2H, N-CH2); 6.64 (d, 1H, J ¼ 8.7); 7.39
(m, 2H); 7.48 (t, 1H, J ¼ 8.7); 7.83 (t, 1H, J ¼ 7.3); 8.21 (br s, 1H); 8.42
(d, 1H, J ¼ 4.4); 8.51 (d, 1H, J ¼ 8.0); 10.01 (s, 1H).
ESI-MS: 350.0 [M þ H]þ; 372.2 [M þ Na]þ; 388.2 [M þ K]þ.
Anal. (C19H23N7), C, H, N.
pended in 80 volumes of the TriseHCl buffer containing 10 mM
pargyline, 4 mM CaCl2, and 0.1% ascorbate. To each assay tube was
added the following: 0.1 mL of the drug dilution (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 concentration of 0.1 nM, and 0.8 mL of
resuspended membranes. The tubes were incubated for 30 min at
37 ꢀC, and the incubations were terminated by vacuum filtration
through Whatman GF/B filters (Brandel Biomedical Research and
Laboratories Inc., Gaithersburg, MD, USA). The filters were washed
twice with 5 mL of ice-cold TriseHCl buffer, and the radioactivity
bound to the filters was measured by liquid scintillation spec-
trometer (Packard TRI-CARBÒ 2000CA e 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-
5.1.18. N-(3-(4-((benzo[d][1,3]dioxol-5-yl)methyl)piperazin-1-yl)
propyl)-N0-cyanopicolinamidine (4p)
From 3 and 1-((benzo[d][1,3]dioxol-6-yl)methyl)piperazine.
Yield: 63%; mp 104e105 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.82
(m, 2H); 2.48 (br s, 4H, 2CH2 pip.); 2.49 (br s, 4H, 2CH2 pip.); 2.54 (t,
2H, N1eCH2, J ¼ 5.1); 3.44 (s, 2H, CH2); 3.80 (br s, 2H, NeCH2); 5.93
(s, 2H, OCH2O); 6.73 (m, 1H, J ¼ 7.6); 6.83 (m, 2H); 7.51 (t, 1H,
J ¼ 7.3); 7.87 (t, 1H, J ¼ 7.7); 8.51 (m, 2H); 9.92 (s, 1H).
ESI-MS: 407.3 [M þ H]þ; 429.1 [M þ Na]þ; 445.2 [M þ K]þ.
Anal. (C22H26N6O2), C, H, N.
5.1.19. N-(3-(4-(2-furoyl)piperazin-1-yl)propyl)-N0-
cyanopicolinamidine (4q)
From 3 and 1-(2-furoyl)piperazine.
Yield: 29%; mp 102e104 ꢀC. 1H NMR (400 MHz, CDCl3)
d
: 1.88
HT (10 mM).
(m, 2H); 2.57 (br s, 4H, 2CH2 pip.); 2.61 (t, 2H, N1eCH2, J ¼ 5.1); 3.89
(br s, 4H, 2CH2 pip.); 3.92 (br s, 2H, NeCH2); 6.48 (d, 1H, J ¼ 3.2);
7.01 (d, 1H, J ¼ 3.2); 7.48 (m, 2H); 7.87 (t, 1H, J ¼ 7.7); 8.54 (m, 2H);
9.81 (s, 1H).
5.2.3. 5-HT2A and 5-HT2C binding assays
Radioligand binding assays were performed as previously re-
ported by Herndon et al. [37]. Briefly, frontal cortical regions of
male Sprague-Dawley rats (180e220 g) were dissected on ice and
homogenized (1:10 w/v) in ice-cold buffer solution (50 mM
TriseHCl, 0.5 mM EDTA, and 10 mM MgCl2 at pH 7.4) with a Poly-
tron PT10 (setting 5 for 15 s) and centrifuged at 3000 g for 15 min.
The pellet was resuspended in buffer (1:30 w/v), incubated at 37 ꢀC
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% ascorbate and
10ꢂ5 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, Boston,
MA, USA) 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) for 5-HT2C receptor assays. Cinanserin
ESI-MS: 367.2 [M þ H]þ; 389.3 [M þ Na]þ; 405.1 [M þ K]þ.
Anal. (C19H22N6O2), C, H, N.
5.2. In vitro receptor binding
5.2.1. General procedures
The compounds dissolved in ethanol or in 5% DMSO were
tested for in vitro affinity for 5-HT1A, 5-HT2A and 5-HT2C recep-
tors by radioligand binding assays. The compounds showing the
highest affinity towards serotonin receptors were selected and
evaluated for their affinity for dopaminergic (D1 and D2) and
adrenergic (a1 and a2) receptors. All the following specific
radioligands and tissue sources were used: (a) serotonin 5-HT1A
receptor, [3H]-8-OH-DPAT, rat brain cortex; (b) serotonin 5-HT2A
receptor, [3H]ketanserin, rat brain cortex; (c) serotonin 5-HT2C
receptor, [3H]mesulergine, rat brain cortex; (d) dopamine D1
receptor, [3H]SCH-23390, rat striatum; (e) dopamine D2 receptor,
[3H]spiperone, rat striatum; (f) a1 adrenergic receptor, [3H]pra-
zosin, rat brain cortex; (g) a2 adrenergic receptor, [3H]yohim-
bine, rat brain cortex.
Non-specific binding was determined as described in
Supplementary material, and specific binding as the difference
between total and non-specific 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 [33] to obtain the concentration of
unlabelled drug that caused 50% inhibition of ligand binding (IC50),
with six concentrations of tested compounds, each performed in
triplicate. The IC50 values obtained were used to calculate apparent
inhibition constants (Ki) by the method of Cheng and Prusoff [34],
from the following equation: Ki ¼ IC50/(1 þ S/KD) where S repre-
sents the concentration of the hot ligand used and KD its receptor
dissociation constant (KD values, obtained by Scatchard analysis
[35], were calculated for each labelled ligand).
(1.0
mM) was used to define non-specific binding in the 5-HT2A
assay. In the 5-HT2C assays, mianserin (1.0
mM) was used to define
nonspecific-binding, and 100 nM spiperone was added to all tubes
to block binding to 5-HT2A receptors. Tubes were incubated for
15 min at 37 ꢀC, filtered on Schliecher 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%.
5.2.4. D1 dopaminergic binding assay
The binding assay for D1 dopaminergic receptors was that
described by Billard et al. [38]. Corpora striata were homogenized
in 30 volumes (w/v) ice-cold 50 mM TriseHCl buffer (pH 7.7 at
25 ꢀC) using a Polytron PT10 (setting 5 for 20 s). Homogenates were
centrifuged twice for 10 min at 50,000 g with resuspension of the
pellet in fresh buffer. The final pellet was resuspended in 50 mM
ice-cold TriseHCl containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2,
1 mM MgCl2, 0.1% ascorbic acid and 10
37 ꢀC). Each assay tube contained 50 L [3H]SCH-23390 (85.0 Ci/
mmol, Perkin Elmer Life Sciences, Boston, MA, USA) to achieve
final concentration of 0.4 nM, and 900 resuspended
mM pargyline (pH 7.1 at
m
a
mL

528
F. Fiorino et al. / European Journal of Medicinal Chemistry 47 (2012) 520e529
membranes (3 mg fresh tissue). The tubes were incubated for
15 min at 37 ꢀC and the incubation was terminated by rapid
filtration under vacuum through Whatman GF/B glass fibre filters.
The filters were washed three times with 5 mL ice-cold 50 mM
TriseHCl buffer (pH 7.7 at 25 C). The radioactivity bound to the
filters was measured by a liquid scintillation counter. Specific [3H]
SCH-23390 binding was defined as the difference between binding
TriseHCl, 0.5 mM EDTA buffer (pH 7.5 at 25 ꢀC). The radioactivity
bound to the filters was measured by a liquid scintillation counter.
Specific [3H]yohimbine binding was defined as the difference
between binding in the absence or in the presence of 10
phentolamine.
mM
Acknowledgements
in the absence or in the presence of 0.1 mM piflutixol.
The NMR spectral data were provided by Centro di Ricerca
Interdipartimentale di Analisi Strumentale, Università degli Studi di
Napoli “Federico II”. The assistance of the staff is gratefully
appreciated.
5.2.5. D2 dopaminergic binding assay
The procedure used in the radioligand binding assay was re-
ported in detail by Creese et al. [39] Corpora striata were homog-
enized in 30 vol. (w/v) ice-cold 50 mM TriseHCl buffer (pH 7.7 at
25 ꢀC) using a Polytron PT10 (setting 5 for 20 s). Homogenates were
centrifuged twice for 10 min at 50,000 g with resuspension of the
pellet in fresh buffer. The final pellet was resuspended in 50 mM
ice-cold TriseHCl containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2,
Appendix. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.11.023.
1 mM MgCl2, 0.1% ascorbic acid and 10
37 ꢀC). Each assay tube contained 50 L [3H]spiperone (15.7 Ci/
mmol, Perkin Elmer Life Sciences, Boston, MA, USA) to achieve
final concentration of 0.4 nM, and 900 resuspended
membranes (3 mg fresh tissue). The tubes were incubated for
15 min at 37 ꢀC and the incubation was terminated by rapid
filtration under vacuum through Whatman GF/B glass fibre filters.
The filters were washed three times with 5 mL ice-cold 50 mM
TriseHCl buffer (pH 7.7 at 25 ꢀC). The radioactivity bound to the
filters was measured by a liquid scintillation counter. Specific [3H]
spiperone binding was defined as the difference between binding
mM pargyline (pH 7.1 at
m
References
[1] H.G. Baumgarten, M. Gother, Serotoninergic neurons and 5-HT receptors in
the CNS, In: Handb. Exp. Pharm., vol. 129. Springer-Verlag, Berlin, 1997.
[2] G.R. Martin, R.M. Eglen, D. Hoyer, M.W. Hamblin, F. Yocca, Advances in
serotonin receptor research; molecular biology, signal transmission, and
therapeutics, Ann. N.Y. Acad. Sci. (1998) New York.
a
mL
[3] N.M. Barnes, T. Sharp, A review of central 5-HT receptors and their function,
Neuropharmacology 38 (1999) 1083e1152.
[4] D. Hoyer, J.P. Hannon, G.R. Martin, Molecular, pharmacological and functional
diversity of 5-HT receptors, Pharmacol. Biochem. Behav. 71 (2002) 533e554.
[5] A.R. Blaazer, P. Smid, C.G. Kruse, Structureeactivity relationships of phenyl-
alkylamines as agonist ligands for 5-HT2A receptors, Chem. Med. Chem. 3
(2008) 1299e1309.
in the absence or in the presence of 1
mM (þ)-butaclamol.
[6] B. Olivier, I. van Wijngaarden, W. Soudjin, Serotonin Receptors and their
Ligands. Elsevier, The Netherlands, 1997.
5.2.6. a1 Adrenergic binding assay
[7] B. Olivier, W. Soudjin, I. van Wijngaarden, The 5-HT1A receptor and its
ligands: structure and function, Prog. Drug Res. 52 (1999) 103e165.
[8] M.L. Lopez-Rodriguez, D. Ayala, B. Benhamu, M.J. Morcillo, A. Viso, Arylpi-
perazine derivatives acting at 5-HT1A receptors, Curr. Med. Chem. 9 (2002)
443e469.
[9] J.A. Bikker, S. Trump-Kallmeyer, C. Humblet, G-protein coupled receptors:
models, mutagenesis, and drug design, J. Med. Chem. 41 (1998) 2911e2927.
[10] S. Trumpp-Kallmeyer, J. Hoflack, A. Bruinvels, M. Hibert, Modeling of G-
protein-coupled receptors: application to dopamine, adrenaline, serotonin,
acetylcholine, and mammalian opsin receptors, J. Med. Chem. 35 (1992)
3448e3462.
[11] F. Fiorino, E. Magli, E. Perissutti, B. Severino, F. Frecentese, A. Esposito, F. De
Angelis, G.M. Incisivo, P. Massarelli, C. Nencini, E. Di Gennaro, A. Budillon, A. Di
Cinto, V. Santagada, G. Caliendo, Synthesis of 1-naphthylpiperazine deriva-
tives as serotoninergic ligands and their evaluation as antiproliferative agents,
Eur. J. Med. Chem. 46 (6) (2011) 2206e2216.
[12] F.G. Boess, I.L. Martin, Molecular biology of 5-HT receptors, Neuropharma-
cology 33 (3e4) (1994) 275e317.
[13] M. Bancila, D. Verge, O. Rampin, J.R. Backstrom, E. Sanders-Bush,
K.E. McKenna, L. Marson, A. Calas, F. Giuliano, 5-Hydroxytryptamine2C
receptors on spinal neurons controlling penile erection in the rat, Neurosci-
ence 92 (4) (1999) 1523e1537.
[14] Y. Kimura, K. Hatanaka, Y. Naitou, K. Maeno, I. Shimada, A. Koakutsu,
F. Wanibuchi, T. Yamaguchi, Pharmacological profile of gYM348, a novel,
potent and orally active 5-HT2C receptor agonist, Eur. J. Pharmacol. 483 (1)
(2004) 37e43.
[15] G.K. Aghajanian, G.J. Marek, Serotonin model of schizophrenia: emerging role
of glutamate mechanisms, Brain Res. Rev. 31 (2e3) (2000) 302e312.
[16] S. Butini, G. Campiani, S. Franceschini, F. Trotta, V. Kumar, E. Guarino,
G. Borrelli, I. Fiorini, E. Novellino, C. Fattorusso, M. Persico, N. Orteca,
K. Sandager-Nielsen, T.A. Jacobsen, K. Madsen, J. Scheel-Kruger, S. Gemma,
Discovery of bishomo(hetero)arylpiperazines as novel multifunctional ligands
targeting dopamine D3 and serotonin 5-HT1A and 5-HT2A receptors, J. Med.
Chem. 53 (2010) 4803e4807.
The procedure used in the radioligand binding assay has been
reported in detail by Greengrass and Brenner [40]. Brain cortex was
homogenized in 30 vol. (w/v) ice-cold 50 mM TriseHCl buffer, (pH
7.2 at 25 ꢀC) using a Polytron PT10 (setting 5 for 20 s). Homogenates
were centrifuged twice for 10 min at 50,000 g with resuspension of
the pellet in fresh buffer. The final pellet was resuspended in
50 mM ice-cold TriseHCl, (pH 7.4 at 25 ꢀC). Each assay tube con-
tained 50
kin Elmer Life Sciences, Boston, MA, USA) to achieve a final
concentration of 0.4 nM, and 900 L resuspended membranes
mL drug solution, 50 m
L [3H]prazosin (80.5 Ci/mmol, Per-
m
(10 mg fresh tissue). The tubes were incubated for 30 min at 25 ꢀC
and the incubation was terminated by rapid filtration under
vacuum through Whatman GF/B glass fibre filters. The filters were
washed three times with 5 mL ice-cold 50 mM TriseHCl, buffer (pH
7.2 at 25 ꢀC). The radioactivity bound to the filters was measured by
a liquid scintillation counter. Specific [3H]prazosin binding was
defined as the difference between binding in the absence or in the
presence of 10 mM phentolamine.
5.2.7. a2 Adrenergic binding assay
The procedure used in the radioligand binding assay was re-
ported in detail by Perry and U’Prichard [41]. Brain cortex was
homogenized in 30 vol. (w/v) ice-cold 5 mM TriseHCl, 5 mM EDTA
buffer (pH 7.3 at 25 ꢀC) using a polytron PT10 (setting 5 for 20 s).
Homogenates were centrifuged three times for 10 min at 50,000 g
with resuspension of the pellet in fresh buffer. The final pellet was
resuspended in 50 mM ice-cold TriseHCl, 0.5 mM EDTA (pH 7.5 at
[17] G. Caliendo, R. Di Carlo, R. Meli, E. Perissutti, V. Santagada, C. Silipo, A. Vittoria,
Synthesis and trazodone-like pharmacological profile of 1- and 2-[3-[4-(X)-1-
piperazinyl]propyl]benzotriazoles, Eur. J. Med. Chem. 28 (12) (1993)
969e974.
[18] G. Caliendo, R. Di Carlo, G. Greco, R. Meli, E. Novellino, E. Perissutti,
V. Santagada, Synthesis and biological activity of benzotriazole derivatives
structurally related to trazodone, Eur. J. Med. Chem. 30 (1) (1995) 77e84.
[19] G. Caliendo, G. Greco, P. Grieco, E. Novellino, E. Perissutti, V. Santagada,
D. Barbarulo, E. Esposito, A. De Blasi, Structure-affinity relationship studies on
benzotriazole derivatives binding to 5-HT receptor subtypes, Eur. J. Med.
Chem. 31 (3) (1996) 207e213.
25 ꢀC). Each assay tube contained 50
yohimbine (80.5 Ci/mmol, Perkin Elmer Life Sciences, Boston, MA,
USA) to achieve a final concentration of 1 nM, and 900 L resus-
mL drug solution, 50 m
L [3H]
m
pended membranes (10 mg fresh tissue). The tubes were incubated
for 30 min at 25 ꢀC and the incubation was terminated by rapid
filtration under vacuum through Whatman GF/B glass fibre filters.
The filters were washed three times with 5 mL ice-cold 50 mM

F. Fiorino et al. / European Journal of Medicinal Chemistry 47 (2012) 520e529
529
[20] G. Caliendo, F. Fiorino, P. Grieco, E. Perissutti, V. Santagada, S. Albrizio,
L. Spadola, G. Bruni, M.R. Romeo, Synthesis and binding affinities for 5-HT1A,
5-HT2A and 5-HT2C receptors of a series of 1- and 2-(4-arylpiperazinylalkyl)-
4-(benzoyl)-1,2,3-triazole derivatives, Eur. J. Med. Chem. 34 (9) (1999)
719e727.
methionyl-leucyl-phenylalanine (f-Met-Leu-Phe-OR), Can. J. Chem. 63 (11)
(1985) 3089e3101.
[28] M.A. Parker, D.M. Kurrasch, D.E. Nichols, The role of lipophilicity in deter-
mining binding affinity and functional activity for 5-HT2A receptor ligands,
Bioorg. Med. Chem. 16 (2008) 4661e4669.
[21] G. Caliendo, F. Fiorino, P. Grieco, E. Perissutti, V. Santagada, B. Severino,
G. Bruni, M.R. Romeo, Synthesis of new 1,2,3-benzotriazin-4-one-arylpiper-
azine derivatives as 5-HT1A serotonin receptor ligands, Bioorg. Med. Chem. 8
(2000) 533e538.
[29] M.P. Johnson, D.B. Wainscott, V.L. Lucaites, M. Baez, D.L. Nelson, Mutations
of transmembrane IV and V serines indicate that all tryptamines do not
bind to the rat 5-HT2A receptor in the same manner, Brain Res. 49 (1e2)
(1997) 1e6.
[22] (a) G. Caliendo, F. Fiorino, E. Perissutti, B. Severino, S. Gessi, E. Cattabriga,
P.A. Borea, V. Santagada, Synthesis by microwave irradiation and binding
properties of novel 5-HT1A receptor ligands, Eur. J. Med. Chem. 36 (11e12)
(2001) 873e886;
[30] A.P. Monte, D. Marona-Lewicka, M.A. Parker, D.B. Wainscott, D.L. Nelson,
D.E. Nichols, Ddihydrobenzofuran analogues of hallucinogens. 3. Models of 4-
substituted (2,5-dimethoxyphenyl)alkylamine derivatives with rigidified
methoxy groups, J. Med. Chem. 39 (15) (1996) 2953e2961.
(b) G. Caliendo, F. Fiorino, E. Perissutti, B. Severino, D. Scolaro, S. Gessi,
E. Cattabriga, P.A. Borea, V. Santagada, A convenient synthesis by microwave
heating and pharmacological evaluation of novel benzoyltriazole and
saccharine derivatives as 5-HT(1A) receptor ligands, Eur. J. Pharm. Sci. 16
(2002) 15e28.
[31] J.C. Shih, K. Chen, T.K. Gallaher, Structure and function of serotonin 5-HT2
receptors, NIDA Res. Monogr. 146 (1994) 284e296.
[32] M.E. Silva, R. Heim, A. Strasser, S. Elz, S. Dove, Theoretical studies on the
interaction of partila agonists with the 5-HT2A receptor, J. Comput. Aided Mol.
Des. 25 (2011) 51e66.
[23] F. Fiorino, E. Perissutti, B. Severino, V. Santagada, D. Cirillo, S. Terracciano,
P. Massarelli, G. Bruni, E. Collavoli, C. Renner, G. Caliendo, New 5-
hydroxytryptamine(1A) receptor ligands containing a norbornene nucleus:
synthesis and in vitro pharmacological evaluation, J. Med. Chem. 48 (2005)
5495e5503.
[24] F. Fiorino, B. Severino, F. De Angelis, E. Perissutti, F. Frecentese, P. Massarelli,
G. Bruni, E. Collavoli, V. Santagada, G. Caliendo, Synthesis and in-vitro phar-
macological evaluation of new 5-HT1A receptor ligands containing a benzo-
triazinone nucleus, Arch. Pharm. (Weinheim) 341 (2008) 20e27.
[25] F. Fiorino, B. Severino, F. De Angelis, E. Perissutti, E. Magli, F. Frecentese,
A. Esposito, P. Massarelli, C. Nencini, B. Viti, V. Santagada, G. Caliendo,
Synthesis and in vitro pharmacological evaluation of a new series of 5-HT1A
[33] EasyFit 1.4. Matteo Vaccari and Mario Negri Institute, Milan, Italy, 1989e1991.
[34] Y.C. Cheng, W.H. Prusoff, Relationship between the inhibition constant (K1)
and the concentration of inhibitor which causes 50 percent inhibition (I50) of
an enzymatic reaction, Biochem. Pharmacol. 22 (1973) 3099e3108.
[35] G. Scatchard, The attraction of proteins for small molecules and ions, Ann. N.Y.
Acad. Sci. 51 (1949) 660e672.
[36] J.R. Schlegel, S.J. Peroutka, Nucleotide interactions with 5-HT1A binding sites
directly labeled by [3H]-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]-8-OH-
DPAT), Biochem. Pharmacol 35 (1986) 1943e1949.
[37] J.L. Herndon, A. Ismaiel, S.P. Ingher, M. Teitler, R.A. Glennon, Ketanserin
analogues: structure-affinity relationships for 5-HT2 and 5-HT1C serotonin
receptor binding, J. Med. Chem. 35 (1992) 4903e4910.
5-HT2A and 5-HT2C receptor ligands containing
Pharmazie 64 (9) (2009) 555e564.
a
norbornene nucleus,
[38] W. Billard, V. Ruperto, G. Crosby, L.C. Iorio Barnett, Characterization of the
binding of 3H-SCH 23390, a selective D-1 receptor antagonist ligand, in rat
striatum, Life Sci. 35 (18) (1984) 1885e1893.
[39] I. Creese, R. Schneider, S.H. Snyder, 3H-spiroperidol labels dopamine receptors
in pituitary and brain, Eur. J. Pharmacol. 46 (4) (1977) 377e381.
[40] P. Greengrass, R. Bremner, Binding characteristics of 3H-prazosin to rat brain
alpha-adrenergic receptors, Eur. J. Pharmacol. 55 (3) (1979) 323e326.
[41] B.D. Perry, D.C. U’Prichard, [3H]rauwolscine (alpha-yohimbine): a specific
antagonist radioligand for brain alpha 2-adrenergic receptors, Eur. J. Phar-
macol. 76 (4) (1981) 461e464.
[26] F. Fiorino, B. Severino, F. De Angelis, E. Perissutti, E. Magli, F. Frecentese,
A. Esposito, P. Massarelli, C. Nencini, V. Santagada, G. Caliendo, New 5-HT1A
receptor ligands containing a N0-cyanoisonicotinamidine nucleus: synthesis
and in vitro pharmacological evaluation, Bioorg. Med. Chem. Lett. 20 (9)
(2010) 2978e2982.
[27] G. Sauve, V.S. Rao, G. Lajoie, B. Belleau, Backbone-modified oligopeptidic
bioregulators. The synthesis and configuration of thioamide, amidoxime,
cyanoamidine, and amidrazone analogs of the chemotactic peptide N-formyl-