New 5-HT1A receptor ligands containing a N′-cyanoisonicotinamidine nucleus: Synthesis and in vitro pharmacological evaluation

Article abstract of DOI:10.1016/j.bmcl.2010.02.106

N′-Cyanoisonicotinamidine derivatives, linked to an arylpiperazine moiety, were prepared to identify highly selective and potent 5-HT_1A ligands as potential pharmacological tools in studies of wide spread psychiatric disorders. The combination of structural elements (heterocyclic nucleus, alkyl chain and 4-substituted piperazine) known to be critical in order to have affinity on 5-HT_1A receptor 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-HT_1A and moderate to no affinity for other relevant receptors (5-HT_2A, 5-HT_2C, D₁, D₂, α₁ and α₂). N′-Cyano-N-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)isonicotinamidine (4o) with K_i = 0.038 nM, was the most active and selective derivative for the 5-HT_1A receptor with respect to other serotoninergic, dopaminergic and adrenergic receptors.

Full text of DOI:10.1016/j.bmcl.2010.02.106

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2978–2982
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New 5-HT1A receptor ligands containing a N0-cyanoisonicotinamidine
nucleus: Synthesis and in vitro pharmacological evaluation
Ferdinando Fiorino a, Beatrice Severino a, Francesca De Angelis a, Elisa Perissutti a, Elisa Magli a,
Francesco Frecentese a, Antonella Esposito 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 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-Cyanoisonicotinamidine derivatives, linked to an arylpiperazine moiety, were prepared to identify
highly selective and potent 5-HT1A ligands as potential pharmacological tools in studies of wide spread
psychiatric disorders. The combination of structural elements (heterocyclic nucleus, alkyl chain and 4-
substituted piperazine) known to be critical in order to have affinity on 5-HT1A receptor 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
Article history:
Received 4 February 2010
Revised 25 February 2010
Accepted 26 February 2010
Available online 3 March 2010
Keywords:
at 5-HT1A and moderate to no affinity for other relevant receptors (5-HT2A, 5-HT2C, D1, D2, a1 and
a2).
N0-Cyanoisonicotinamidine nucleus
Arylpiperazines derivatives
5-HT1a receptor ligands
N0-Cyano-N-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)isonicotinamidine (4o) with Ki = 0.038 nM, was
the most active and selective derivative for the 5-HT1A receptor with respect to other serotoninergic,
dopaminergic and adrenergic receptors.
Ó 2010 Elsevier Ltd. All rights reserved.
The serotoninergic system is implicated in numerous physiolog-
ical and pathophysiological processes.1 Serotonin (5-hydroxytryp-
tamine, 5-HT)2–5 is found throughout the CNS in high levels and
participates in modulating mood. According to the classic seroto-
nin hypothesis, anxiety is usually associated with increased endog-
enous 5-HT, and anxiolytics tend to decrease endogenous 5-HT.6
Discovery of ligands for 5-HT receptors (5-HTRs) is an area of in-
tense research because of the potential for new therapeutic drugs.
Among the 13 different serotonin receptors, belonging to the G
protein coupled receptors superfamily,5 5-HT1A and 5-HT2A recep-
tors, are most frequently considered as important targets for
neurobiological research and drug development. The activation of
these receptors leads to a number of physiological changes that
can be easily quantified.7–9 Agonists and partial agonists have been
proven to be effective in anxiety and depression.10–13
most studied group is that of long-chain arylpiperazines (LCAPs).9
Their general chemical structure contains an alkyl chain (2–4
methylene units) attached to the N-4 atom of the piperazine moi-
ety on a terminal amide or imide fragment. The 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 structure–activity relationship studies (SAR). In particular,
much effort has been devoted to understand the role of the termi-
nal part in the ligand–receptor interaction and, in consequence, a
great number of many different fragments were used.16,17 However,
a limitation of many 5-HT1A receptor ligands for their potential use
as drugs or pharmacological tools is their undesired high affinity
for other receptor subtypes. The dopaminergic D2 receptor and
a1-adrenoceptor are two examples of undesired binding sites to
which several 5-HT1A ligands bind with high affinity.
5-HT1A receptor, as a member of the group of G protein coupled
receptors (GPCRs),14 shows high similarity with other members of
the family. A particular case is represented by the similarity be-
tween 5-HT1AR and
of homology (45%) in their amino acid sequence.
Several structurally different compounds are already known for
their high affinity toward these receptors and, from a chemical
point of view, they can be subdivided into different classes. The
In our laboratories, there has been an ongoing effort to develop
more selective 5-HT1A ligands18–26 in order to have novel pharma-
cological tools that could improve our knowledge of the signal
transduction mechanism and lead to compounds with high affinity
and selectivity. In continuation of our research program, we have
analyzed a new set of derivatives where the piperazine-N-alkyl
moiety has been linked to a novel N0-cyanoisonicotinamidine frag-
ment as terminal part of LCAPs (Scheme 1). To gain insight into its
influence on the serotoninergic activity, this original nucleus was
linked via three methylene spacing units to piperazines substituted
in position 4 with aliphatic and/or aromatic moieties. The choice of
a
1-adrenoreceptor15 that show a high degree
* Corresponding author. Tel./fax: +39 081 678649.
E-mail address: caliendo@unina.it (G. Caliendo).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.106

F. Fiorino et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2978–2982
NCN
2979
NCN
CN
NH(CH2)3Br
OCH(CH3)2
i
ii
N
N
N
(3)
iii
(1)
(2)
NCN
N
N
H
N
N
X
(4a-q)
Scheme 1. Reagents and conditions: (i) CH3ONa, H2NCN, iPrOH; (ii) Br(CH2)3NH2ꢀHBr, CH3ONa, anhydrous MeOH; (iii) 4-X-substituted-piperazine, K2CO3, NaI, CH3CN, 70 °C, 4 h.
the alkyl chain length (three units), was performed on the basis of
our previous investigations, where, as a general trend, compounds
with piperazinylpropyl chain linked to different nuclei showed
good and preferential affinity for the 5-HT1AR, with respect to com-
pounds in which the spacer is one atom shorter. Instead the choice
of aliphatic substituents on the N-4 of the piperazine moiety, that
could appear in contrast with the statement that protonable nitro-
gen and aromatic system are necessary for binding of aminergic li-
gand,32,35 was done in order to obtain a complete structure–affinity
and structure–selectivity relationship study. All the new com-
pounds were tested for their affinity for 5-HT1A, 5-HT2A and
5-HT2C receptors. Moreover, the multireceptor profiles of promis-
ing derivatives were also evaluated in terms of binding affinities
rat brain cortex37; (c) serotonin 5-HT2C receptor, [3H]mesulergine,
rat brain cortex37; (d) dopamine D1 receptor, [3H]SCH-23390, rat
striatum38; (e) dopamine D2 receptor, [3H]spiperone, rat stria-
tum39; (f) a1 adrenergic receptor, [3H]prazosin, rat brain cortex40
;
(g) a2 adrenergic receptor, [3H]yohimbine, rat brain cortex.41
Non-specific binding was determined as described in the Sup-
plementary data, and specific binding as the difference between to-
tal 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
‘
EASYFIT’ program28 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 inhibition
constants (Ki) by the method of Cheng and Prusoff,29 from the fol-
lowing equation: Ki = IC50/(1 + S/KD) where S represents the con-
centration of the hot ligand used and KD its receptor dissociation
constant (KD values, obtained by Scatchard analysis,30 were calcu-
lated for each labeled ligand).
Some of the reported derivatives were potent 5-HT1A receptor li-
gands, in fact, they showed nanomolar or even subnanomolar 5-
HT1A receptor affinities (Table 1). Besides the outstanding 5-HT1A
receptor affinity of compound 4o (Ki = 0.038 nM), other interesting
Ki values were those of compounds 4d (1.47 nM), 4l (6.17 nM) and
4f (21.3 nM) while compounds 4h, 4i, 4m and 4n where less active
with Ki values of 101, 166, 141 and 172 nM, respectively. Other
derivatives showed Ki values of above 104 nM or no affinity.
The derivatives 4a–q differ in the substituents on the N-4 of the
piperazine moiety that represents a critical feature in determining
5-HT1A receptor affinity and selectivity. These results confirm
those obtained with previously described compounds25,26 in fact,
the introduction of non aromatic or larger substituents on the
piperazine nitrogen, even in this series of derivatives, causes a dra-
matic decrease of the receptor affinity. These results were also in
accordance with recently reported structure affinity relationships
of new model of arylpiperazines.31
for dopaminergic (D1 and D2) and adrenergic (a1 and a2) receptors.
The synthetic strategy employed for the preparation of the target
compounds (Table 1) is summarized in Scheme 1. The general proce-
dure is as follows: isopropyl-N-cyano-4-pyridinecarboximidate (2)
was prepared starting from 4-cyanopyridine (1) by base-promoted
reaction with isopropanol followed by treatment with cyanamide
in aqueous phosphate buffer (NaH2PO4ꢀ2H2O/Na2HPO4 = 4:1). Com-
pound (2) was converted to N-(3-bromopropyl)-N0-cyanoisonicoti-
namidine (3) by reaction with 3-bromopropylamineꢀ ꢀ ꢀHBr and
NaOMe in 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 4a–q. Purification of each final product was obtained
by chromatography on silica gel column and further by crystalliza-
tion from the appropriate solvent. All new compounds gave satisfac-
tory elemental analyses 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 the 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 the final compounds 4a–q, confirmed that all final compounds ex-
ist predominantly as the cyanoimino form (I). Moreover, about the
configurational determination of geometrical isomers (E/Z) on the
amidine bond C@N, the N-cyanoamidine derivatives 4a–q 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 the literature.27
The compounds dissolved in ethanol or in 5% DMSO were tested
for in vitro affinity for 5-HT1A, 5-HT2A and 5-HT2C receptors by radi-
oligand binding assays. The compounds showing the highest affin-
ity towards serotonin receptors have been selected and evaluated
for their affinity for dopaminergic (D1 and D2) and adrenergic (a1
Concerning the influence of the N-4 substituent of the pipera-
zine moiety, the pyridin-2-yl group, (4o), and the 2-methoxy-
phenyl group (4d), conferred the highest affinity and selectivity
for the 5-HT1A receptor. The presence of a 3-chlorophenyl group
and 2-ethoxyphenyl group on the N-4 of the piperazine moiety,
led also to compounds which exhibited high affinity for 5-HT1A
receptor (4l Ki = 6.17 nM; 4f Ki = 21.3 nM). Non aromatic moiety
as well as pyrimidinic and piperonyl groups seem especially unfa-
vorable determining a high decrease in binding affinity compared
to the other N-4 substituents at the piperazine ring.
and
a
2) receptors. All The following specific radioligands and tissue
The 5-HT2A and 5-HT2C receptor affinities of the tested com-
pounds were always lower than those observed for 5-HT1A recep-
tors except for compounds 4m and 4f that show higher affinities
sources were used: (a) serotonin 5-HT1A receptor, [3H]-8-OH-DPAT,
rat brain cortex36; (b) serotonin 5-HT2A receptor, [3H]ketanserin,

2980
F. Fiorino et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2978–2982
Table 1
Affinities of compounds 4a–q for 5-HT1A, 5-HT2A and 5-HT2C receptors
CN
N
N
H
N
N
N
X
Compd
Receptor affinity Ki SD (nM)
X
5-HT1A [3H]8OH-DPAT
5-HT2A [3H]Ketanserin
5-HT2C [3H]Mesulergine
No affinity
4a
4b
No affinity
No affinity
No affinity
>104
No affinity
OCH3
4c
>104
No affinity
>104
No affinity
OCH2CH3
H3CO
4d
1.47 0.05
No affinity
OCH3
4e
4f
>104
>104
No affinity
11.5 3.0
H3CH2CO
21.3 0.8
1900 100
NC
4g
4h
4i
No affinity
101 38
>104
>104
>104
>104
No affinity
No affinity
No affinity
No affinity
H3C
CH3
Cl
166
8
Cl
4l
6.17 0.14
CF3
4m
4n
141
9
32.1 0.5
No affinity
No affinity
N
172 24
No affinity
N
N
4o
4p
0.038 0.001
>104
No affinity
>104
No affinity
No affinity
O
O
O
4q
>104
>104
No affinity
O
For purpose of comparison, 8-OH-DPAT, Ketanserine and Mesulergine binds 5-HT1A, 5-HT2A and 5-HT2C receptors with values of 0.80, 0.85 and 1.90 nM, respectively, under
these assay conditions.
CN
CN
N
HN
N
H
N
N
N
N
N
N
N
X
X
I
II
Figure 1. Tautomeric equilibrium about sp2carbon of the cyanoamidine derivatives.

F. Fiorino et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2978–2982
2981
Table 2
antipsychotics combine a concomitant activity at serotoninergic
receptors (5-HT1A and 5-HT2A) with D2 receptor occupancy, to pro-
vide drug therapies for resistant schizophrenic patients, with
prompter therapeutic benefits and the improvement of cognitive
symptoms.34
Affinities of compounds 4d, 4f, 4l, and 4o for D1, D2, a1 and a2 receptors
Compd
Receptor affinity Ki SD (nM)
D1
D2
a1
a2
[3H]SCH-23390
[3H]spiperone
[3H]prazosin
[3H]yohimbine
Finally the bad affinity/selectivity profile of the derivatives
characterized with the presence of an aliphatic group (4a–4c) on
the N-4 position has clarified the role of N-4 position of the piper-
azine moiety on the 5-HT1A receptor affinity and selectivity. Zlat-
ovic´ et al.35 have recently reported that some arylpiperazines can
interact directly with the hydrophobic part of the 5-HT1A receptor
binding site. In particular the hydrophobic part of the binding site
in the 5-HT1A receptor, formed by Trp-358, Phe-361 and Tyr-390, is
significant for the stabilization of the ligand–receptor complex.
Therefore, these aspects can be useful to explain the low affinity
that these new piperazine derivatives have shown; in these com-
pounds, in fact, a non aromatic moiety is less favorable to the
hydrophobic interaction with the receptor, determining the forma-
tion of weaker complexes with the receptor.
In conclusion, in this Letter, we have described the synthesis of
a new series of arylpiperazines as 5-HT1A ligands (4a–q), contain-
ing a novel heterocyclic fragment. Some of the described com-
pounds showed high in vitro affinity and selectivity towards 5-
HT1A receptors. Compound 4o was the most potent (Ki = 0.038 nM)
and selective derivative for the 5-HT1A receptor with respect to the
other serotonin, dopaminergic and adrenergic receptors, besides
appear interesting also the affinity/selectivity profile of compound
4d (Ki = 1.47 nM) supporting on the N-4 of the piperazine moiety
the 2-methoxyphenyl group. The binding data presented in this
study identified the N0-cyanoisonicotinamidine nucleus as an opti-
mal structural element to enhance 5-HT1A receptor binding,
although piperazinylpropyl chain and the nature of the substituent
on the N-4 piperazine ring play an important role in determining
affinity and selectivity.
4d
4f
4l
>104
>104
>104
>104
>104
>104
190 58
912 11
>104
2570 258
516 64
>104
>104
1630 60
4o
>104
>104
towards 5-HT2AR (Ki = 32.1 nM) and 5-HT2C receptor (Ki = 11.5 nM),
respectively. N0-Cyano-N-(3-(4-(pyridin-2-yl)piperazin-1-yl)pro-
pyl)isonicotinamidine (4o) (Ki = 0.037 nM), showed not only the
highest affinity but also the highest selectivity with respect to
other serotoninergic receptors studied.
Additionally, the affinity of the most active compounds (4d, 4f, 4l
and 4o) on several other receptors (a1 and a2 adrenergic and D1 and
D2 dopaminergic receptors) was 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 4l,
which exhibited Ki value of 1630 nM on D1 receptor. Regarding a1
and a2 adrenergic receptors, only compound 4f showed quite mod-
erate affinity (190 nM and 516 nM, respectively), while compounds
4d and 4o showed Ki values higher than 104 nM; these data are very
interesting considering the high degree of homology existing be-
tween these two receptors and demonstrates that these compounds
possess a very good binding profile, preferring 5-HT1ARs over all
other evaluated receptors.
These results emphasize the high selectivity afforded by the dis-
cussed N0-cyanoisonicotinamidine scaffold and confirm that the
propyl alkyl chain together with the aromatic ring substitution
plays a critical role in determining 5-HT1A receptor affinity and
selectivity.
As already observed in a previously described series of norborn-
ene derivatives,24 the exceptional high affinity of 4o, supporting a
pyridin-2-yl moiety, could be explained in terms of solvent acces-
sibility and hydrophobic interaction with the receptor that are
decisive compared to all other compounds, whereas the lower
affinity of 4n appear to agree data already reported in the litera-
ture,32,33 where as general trend the pyrimidin-2yl derivatives re-
sult poorer ligands with respect to 2-methoxyphenyl and
pyridin-2-yl moiety. In fact as already described33 this aspect could
be due to a unfavorable conformation of the pyrimidinylpiper-
azine, where the pyrimidin-2yl ring is parallel to that of the piper-
azine due to delocalization of sp2/sp3 nitrogen into the aromatic
system. Instead the high affinity of 4d is most probably explained
by an additional hydrogen bond between the methoxy substituent
in ortho position and the side chain of Asn-386.
Moreover, results obtained with compound 4f (Ki value of
21.3 nM) supporting an o-ethoxyphenylpiperazine moiety, com-
pared with o-methoxyphenylpiperazine derivative 4d (Ki value of
1.47 nM), could be discussed in order to better explain the influ-
ence of different substituents in ortho position on the phenylpiper-
azine moiety. In fact, the simple change from –OCH3 to –OCH2CH3
produces a reduction of affinity and a loss of selectivity. This result
could be, probably, attributed to unfavorable steric interaction of
the ethoxy group (4f) with the surrounding receptor residues and
loss of the hydrogen bond to Asn-386, that molecular modeling
studies24 suggested as important for o-methoxy analogues. In fact
compound 4f presented a mixed 5-HT1A/5-HT2C affinity with Ki val-
ues of 21.3/11.5 nM, respectively. Additionally 4m, characterized
by a very hydrophobic trifluoromethyl group, exhibits a mixed
5-HT1A/5-HT2A affinity with Ki values of 141/32.1 nM, respectively.
This data is of particular interest and outlines a potential atypical
antipsychotic profile for this derivative. In fact second-generation
Acknowledgments
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.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.02.106.
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