Towards the development of 5-HT7 ligands combining serotonin-like and arylpiperazine moieties

Article abstract of DOI:10.1016/j.ejmech.2014.04.034

Many known 5-HT₇ ligands contain either a serotonin-like or an arylpiperazine structure that, in published SAR studies, are generally supposed to bind the same receptor pocket. Conversely, we explored the hypothesis that two such moieties can c

Full text of DOI:10.1016/j.ejmech.2014.04.034

European Journal of Medicinal Chemistry 80 (2014) 8e35
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Towards the development of 5-HT7 ligands combining serotonin-like
and arylpiperazine moieties
Gilberto Spadoni a, Annalida Bedini a, Silvia Bartolucci a, Daniele Pala b, Marco Mor b,
Teresa Riccioni c, Franco Borsini c, Walter Cabri c, Diana Celona c, Mauro Marzi c,
,
c,
*
Giorgio Tarzia a, Silvia Rivara b , Patrizia Minetti
*
a Dipartimento di Scienze Biomolecolari, Università degli Studi di Urbino, Piazza Rinascimento 6, I-61029 Urbino, PU, Italy
b Dipartimento di Farmacia, Università degli Studi di Parma, Parco Area delle Scienze 27/A, I-43124 Parma, Italy
c Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., Via Pontina Km 30,400, I-00040 Pomezia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Many known 5-HT7 ligands contain either a serotonin-like or an arylpiperazine structure that, in pub-
lished SAR studies, are generally supposed to bind the same receptor pocket. Conversely, we explored the
hypothesis that two such moieties can co-exist in the same ligand, binding to different pockets. We thus
designed and synthesized a set of compounds including both a 5-hydroxyindol-3-ylethyl and a 1-
arylpiperazine moieties connected by a short linker. The compounds were tested for their affinity for
human 5-HT7 serotonin receptor. We further prepared a novel series of 5-HT7 ligands, where the 5-
hydroxyindol-3-ylethyl moiety was bioisosterically replaced by a 3-hydroxyanilinoalkyl one. Among
the newly synthesized compounds, potent ligands at the 5-HT7 receptor, behaving as antagonists in
functional tests, were identified, even if they showed limited subtype selectivity. Docking studies within
a model of the 5-HT7 receptor showed that the binding site can actually accommodate both moieties,
with the serotonin-like one in the putative orthosteric site and the arylpiperazine one occupying an
accessory pocket. The present results demonstrate that it is possible to devise and develop new 5-HT7
ligands merging two privileged structures in the same molecule.
Received 18 October 2013
Received in revised form
18 March 2014
Accepted 10 April 2014
Available online 13 April 2014
Keywords:
5-HT7
Medicinal chemistry
Chemical synthesis
Molecular modeling
Serotonin
Structureeactivity relationships
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
[5e7]. Since its discovery, the 5-HT7 receptor has been cloned
from different species, including rat, man, mouse, pig, guinea pig,
Serotonin (5-hydroxytryptamine, 5-HT, Fig. 1) is an important
neurotransmitter that mediates a wide range of physiological
functions by interacting with seven serotoninergic receptor fam-
ilies (5-HT1e7) subdivided into at least 14 subpopulations [1].
Drugs which interact specifically with serotonin receptors are
clinically approved for the treatment of migraine (sumatriptan: 5-
HT1D/1F agonist), psychosis (risperidone: 5-HT2/D2 antagonist),
anxiety disorders (buspirone: 5-HT1A agonist) and emesis
(ondansetron: 5-HT3 antagonist) [2e4]. The most recently
discovered and cloned member of the 5-HT receptor family is 5-
HT7, which belongs to the G protein-coupled receptor (GPCR) su-
perfamily and is positively coupled to adenylyl cyclase. The 5-HT7
receptor was cloned independently by three laboratories in 1993
honeybee, Xenopus laevis, Aedes aegypti and Caenorhabditis elegans
[8]. It is expressed in the central and peripheral nervous system
and also in the periphery. In the latter, the 5-HT7 receptor has
been detected predominantly in smooth muscle cells of the car-
diovascular, gastrointestinal, and reproductive system, where it
was consistently shown to induce smooth muscle relaxation [9]. In
the central nervous system, the 5-HT7 receptor is most abundant
in hippocampus, thalamus, hypothalamus and cerebral cortex. The
presence in the hypothalamus correlates with its involvement in
circadian rhythm, thermoregulation, and endocrine regulation.
Thalamic and cortical 5-HT7 receptors may be of importance for
sleep, mood regulation and epilepsy. The distribution of 5-HT7
receptors in the hippocampus is of relevance for its role in
learning and memory [10]. Finally, its localization in the spinal
cord is in agreement with putative functions in nociception and
locomotion. A consistent body of evidence, using both 5-HT7 re-
ceptor knockout mice and selective ligands, supports the
involvement of this receptor in various pathophysiological
* Corresponding authors.
E-mail addresses: silvia.rivara@unipr.it (S. Rivara), patrizia.minetti@virgilio.it
(P. Minetti).
http://dx.doi.org/10.1016/j.ejmech.2014.04.034
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
9
processes. Therefore, this receptor has become an attractive target
for drug discovery and in the past years intense efforts have led to
the identification of different structural classes of 5-HT7 receptor
agents [for a recent review see Ref. [11]].
Ligands characterized by a 1-arylpiperazine moiety are well
known for their affinity to several members of the family of sero-
toninergic receptors, and recently different research groups have
modified this template in order to identify selective 5-HT7 receptor
ligands [12]. On the other hand, it is known that appropriate mo-
lecular modifications of tryptamine can lead to derivatives that
display an enhanced selectivity for different 5-HT receptor sub-
types [13], even if the features conferring a subtype selective profile
are not fully established.
A number of studies defining 5-HT7 pharmacophore and ho-
mology (using rhodopsin crystal structures as templates) models
have been published [12c,14], and they are characterized by at least
four pharmacophoric features: a positive ionizable atom, a H-
bonding acceptor group, and two hydrophobic regions. In these
models, it is generally assumed that the 1-arylpiperazine and the
indolylethylamine fragments are reciprocal bioisosteres, with their
aromatic portions occupying the same hydrophobic region of the 5-
HT7 receptor.
In some structures of shorter 5-HT7 ligands (aec in Fig. 1), the
basic amine group is bound to a serotonin-like fragment, i.e. a
tryptamine or a 2-aminotetralin one, where the 5-hydroxyl and the
8-hydroxyl groups can be strictly superposed, and conserve a sig-
nificant role for structureeactivity relationships. On the other hand,
in other ligands, such as aminochromanes (d, e), 5-aryl-2-
aminotetralines (f) and mercaptopyridines (g) the similarity with
the tryptamine scaffold of 5-HT is less evident, with the lack of a
hydroxyl group or its bioisostere mimicking the 5-hydroxyl group
of 5-HT. 1-Arylpiperazines (hej), an important class of 5-HT7 li-
gands, resemble more this second chemotype. In fact, the 7’-
methoxy group of h can be replaced by a hydrogen atom with no
loss of receptor affinity [14e]. Moreover, the 1-arylpiperazine
moiety can be connected to a lipophilic tail with good 5-HT7 af-
finity, as in compounds i and j, while no “longer” 5-HT7 ligands
with a tryptamine or 8-hydroxy-2-aminotetralin scaffold have been
described.
These observations led us to hypothesize that a serotonin-like
moiety could be combined with an arylpiperazine one, as they
may occupy two distinct hydrophobic cavities. Although such
distinct binding pockets have been proposed both in ligand-based
and receptor-based models [14], compounds including both moi-
eties have not been reported so far, nor has this hypothesis been
investigated through structureeactivity relationships.
We thus synthesized a first series of putative 5-HT7 ligands,
characterized by the presence of a serotonin-like scaffold, where
the basic nitrogen is enclosed in a piperazine nucleus, linked to
different lipophilic portions through its second nitrogen. As this
lipophilic region showed SARs that resembled those of known 1-
arylpiperazine ligands, we thus explored the possibility to bio-
isosterically replace the tryptamine moiety. In a previous work on
melatonin receptor ligands, we had observed that
a 2-(3-
methoxyanilino)ethyl and a 3-(3-methoxyanilino)propyl fragment
could replace the 5-methoxyindol-3-ylethyl portion of melatonin
[15]. We therefore synthesized a novel series of putative 5-HT7 li-
gands, composed by
a 2-(3-hydroxyanilino)ethyl or a 3-(3-
hydroxyanilino)propyl moiety, potentially mimicking the indole
scaffold of 5-HT, and by an arylpiperazine moiety. Furthermore, we
explored the SARs for this novel class of compounds and analyzed
their possible binding modes at the 5-HT7 receptor by docking
simulations within a homology-based model of the receptor.
Selected compounds, endowed with good receptor affinity, were
further examined for their intrinsic activity and selectivity against
different 5-HT receptor subtypes.
2. Chemistry
A previously reported method [16] was used to prepare the [(4-
substituted-piperazin-1-yl)ethyl]-indole derivatives 5iep (Scheme
1); the procedure involves acylation of the suitable indole 1ae
d with oxalyl chloride, followed by reaction with the appropriate N-
substituted-piperazine to give the (4-substituted-piperazin-1-yl)
ethane-1,2-dione derivatives 3aeh; these latter are then converted
to the corresponding 5-hydroxyindole derivatives 5iep by reduc-
tion with LiAlH4 and subsequent O-deprotection by hydrogenolysis
or by treatment with boron tribromide.
Fig. 1. Representative 5-HT7 ligands [12d,14b,55e58].

10
G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
Scheme 1. Reagents and conditions: a) (COCl)2, THF, RT, 1 h; b) TEA, THF, RT, 1.5 h; c) LiAlH4, THF, reflux, 1 h; d) BBr3, CH2Cl2, RT, 2 h (for 5iek and 5oep) or H2, 10% Pd/C, MeOH, RT,
6 h (for 5len).
The (anilinoethyl)-piperazino derivatives 10hel were prepared
as depicted in Scheme 2. Briefly, the (4-substituted-piperazine)
acetamido derivatives 8deg were synthesized by N-acylation of
anilines (6aec) with bromoacetyl chloride in the presence of
NaHCO3 followed by substitution of the intermediate bromoace-
tamides 7aec with the suitable N-substituted-piperazine. The
target compounds 10hej were then obtained by LiAlH4 reduction of
the amides 8def, followed by O-deprotection of the intermediates
9def by treatment with BBr3 (for 10hei) or by hydrogenolysis
catalyzed by palladium on carbon (for 10j). The nitro-compound 9g,
obtained by BH3 amide reduction of 8g, was hydrogenated over 10%
Pd/C to give the corresponding anilino derivative 10k that could be
converted into the N-methanesulfonamido target compound 10l,
by treatment with methanesulfonyl chloride.
Following a similar stepwise synthetic route (after changing the
N-acylating reagent to 3-bromopropanoyl chloride), the (anilino-
propyl)-piperazino derivatives 14beo were obtained from the
corresponding anilines 6a, 11bei as shown in Scheme 3. In a similar
manner, the higher homologs 18aed, 18feh and the piperidine
analog 18e were synthesized starting from 3-benzyloxyaniline (6b)
Reductive amination of the aniline derivative 17d with acetal-
dehyde or benzaldehyde in the presence of NaBH3CN and subse-
quent O-debenzylation yielded, respectively, the N-ethyl- (18j) or
N-benzyl- (18k) final product (Scheme 4).
Compounds 14reu were prepared from the ethyl ester deriva-
tive 14g following the step sequence shown in Scheme 5. In
particular 14g was reduced by LiAlH4 to the hydroxymethyl de-
rivative 14t, that was oxidized by MnO2 to the carboxaldehyde
derivative 14u. By treatment of 14g with magnesium nitride in
methanol we obtained a mixture of the primary amide 14r and of
the methyl ester derivative 14s, that was separated by
chromatography.
N-substituted-[3-(4-phenylpiperazin-1-yl)propyl]anilines 24e
27 were prepared from the aniline derivative 14h (Scheme 6) that
can be converted to the N-propyl-anilino derivative 24 by N-acyl-
ation with propionic anhydride, followed by BH3 amide reduction
and subsequent O-demethylation with boron tribromide. Alterna-
tively, 14h is converted to the N-benzyl- (25) or to the N-benze-
nesulfonyl- (27) target compound by reaction with benzyl bromide
or benzenesulfonyl chloride, respectively, and subsequent O-
demethylation with BBr3. The N-phenyl derivative 26 was achieved
by palladium-catalyzed amination [Pd(OAc)2/BINAP/KOtBu] of 14h
with iodobenzene and subsequent O-demethylation with boron
tribromide.
or 3-benzyloxy-N-methylaniline (11i) [17] and the suitable
u-
alkanoylchloride (Scheme 4). The phenol-hexaneamido derivative
16i was obtained by O-debenzylation with BCl3 of the corre-
sponding benzyloxy derivative 16f (Scheme 4).
The N-acetamido- (14p) and the N-methanesulfonamido- (14q)
target compounds were obtained by catalytic hydrogenation (10%
Pd/C) of the nitro-compound 14f (Scheme 3) and subsequent
treatment of the crude aniline intermediate with acetic anhydride
or methanesulfonyl chloride, respectively.
Preparation of the 4-substituted piperidine derivatives 30bec
started with the reaction of 12a with the suitable piperidine giving
the corresponding piperidine-propanamido derivatives 28bec,
that were then reduced with BH3, and finally O-deprotected by
treatment with BBr3 (Scheme 7).

G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
11
Scheme 2. Reagents and conditions: a) 2-bromoacetyl chloride, NaHCO3, EtOAc, H2O, RT, 20 min; b) TEA, DMF, RT, 1e16 h; c) LiAlH4, THF, reflux, 2 h (for 9def) or BH3, THF, RT, 16 h
(for 9g); d) BBr3, CH2Cl2, RT, 2 h (for 10hei) or H2, 10% Pd/C, MeOH, 60 ꢀC, 3 h (for 10j); e) H2, 10% Pd/C, EtOH, EtOAc, RT, 5 h; f) methanesulfonyl chloride, TEA, THF, RT, 1 h.
Phenolalkyl-piperazino derivative 32aec were synthesized by
condensation of the appropriate carboxylic acid with N-phenyl
piperazine in the presence of 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC), followed by amide reduction with 1 M B2H6 in
THF and O-deprotection (for 32c) (Scheme 8).
biphenyl and p-biphenyl derivatives 5l and 5m, abolished affinity
for the receptor. On the other hand, an -naphthyl group (5i) had a
favorable effect and led to a sixfold increase of binding affinity
compared to the phenyl derivative 5n. When small alkyl groups
a
were inserted at position 2 of the indole ring in the a-naphthyl
Compound 35 was prepared from 3-(benzyloxy)phenol
following the three-step sequence (O-alkylation, amide reduction,
O-debenzylation) illustrated in Scheme 9.
The tetralin derivative 37 was synthesized by reductive amina-
tion of 8-methoxy-2-tetralone with N-phenyl-piperazine and sub-
sequent O-demethylation with BBr3 (Scheme 10).
derivative, a methyl group (5k) was tolerated and an ethyl one (5j)
doubled 5-HT7 binding affinity. This behavior differs from what
observed for other tryptamine derivatives. In fact, insertion of a
methyl or an ethyl group in position 2 of N,N-dimethyl-5-
methoxytryptamine led to a decrease of binding affinity, with an
increase from tenfold to fiftyfold in Ki values [14b,18]. Replacement
of the aromatic substituent on the piperazine nitrogen with an alkyl
one, such as a n-butyl chain (5o) or a cyclopentyl ring (5p), led to
derivatives devoid of any significant 5-HT7 binding affinity. This
observation is not in line with what reported for a series of 6-
bromo-1-(homo-piperazinyl)ethylindoles, in which the presence
of alkyl substituents on the homo-piperazine nitrogen afforded
compounds with binding affinities in the nanomolar range [19].
3. Results and discussion
Binding affinities at the human 5-HT7 receptor for the newly
synthesized compounds that combine
a serotonin-like (5-
hydroxyindol-3-ylethylamine) with an arylpiperazine moiety are
reported in Table 1. Compared with 5-HT, the phenylpiperazinyl
derivative 5n showed moderate binding affinity (Ki ¼ 63 nM),
suggesting that the presence of the phenyl-piperazine moiety did
not prevent accommodation of the 5-hydroxyindole portion within
the 5-HT7 binding site. Bulkier aromatic substituents, in the m-
Thus, both a phenyl- and an a-naphthyl-piperazine moiety could be
combined with a serotonin-like moiety with limited loss of re-
ceptor affinity. This preliminary result was confirmed preparing
compound 37, where the 5-hydroxyindol-3-ylethyl substructure

12
G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
Scheme 3. Reagents and conditions: a) 3-bromopropanoyl chloride, NaHCO3, EtOAc, H2O, RT, 20 min; b) TEA, DMF, RT, 1e16 h; c) BH3, THF, RT, 16 h; d) H2, 10% Pd/C, MeOH, 60 ꢀC, 3 h;
e) BBr3, CH2Cl2, RT, 2 h; f) H2, 10% Pd/C, EtOH, EtOAc, RT, 5 h; g) Ac2O, TEA, THF, RT, 1 h; h) methanesulfonyl chloride, TEA, THF, RT, 1 h.
was replaced by a 8-hydroxytetralin one, which had been shown to
be a bioisostere on several 5-HT receptors.
compound 5i the protonated piperazine nitrogen interacts with
Asp1623.32 through a salt bridge and the ligand molecule spans the
helix bundle from TM5 to TM7 (Fig. 2a). The indole ring of 5i is
located in a cavity delimited by TM3, TM5 and TM6 and is engaged
To investigate the binding mode of our indole derivatives, we
built a homology model of the 5-HT7 receptor based on the three-
dimensional coordinates of the 5-HT1B serotonin receptor co-
crystallized with the agonist dihydroergotamine. Crystal struc-
tures of different GPCRs have shown that agonist and antagonist
compounds can be accommodated within binding sites having very
similar location and size, with only subtle differences [20]. There-
fore, given the level of accuracy of GPCR homology models, we
considered the 5-HT1B crystal structure as a suitable template and
we used the 5-HT7 model for docking studies on our compounds,
for which a functional characterization was not systematically
carried out. As already observed by Kolaczkowsky et al. [14e] for a
rhodopsin-based 5-HT7 receptor model, the putative binding re-
gion is characterized by the presence of Asp162 (3.32 according to
BallesteroseWeinstein’s nomenclature [21]) on transmembrane
(TM) helix 3 which is supposed to be the counterpart for basic
groups of ligands and which divides the binding site into two
portions, one located within the helix bundle and delimited by
TM3, 5 and 6 and one which lays towards the extracellular side of
TM7. Accordingly, in the best induced-fit docking solution of
in several hydrophobic contacts with Val1633.33
,
Ala2475.46
,
Phe3436.51 and Phe3446.52; the
a-naphthyl-piperazine moiety is
accommodated towards the extracellular portion of the helix
bundle, close to TM7, and undertakes a number of favorable in-
teractions with Phe1583.28, Ile1593.29, Leu232 (extracellular loop 2)
and Leu3707.39. Interestingly, the region occupied by the
a-naphthyl
substituent of compound 5i is partially superposed to that occupied
by the cyclic tripeptide of dihydroergotamine in the crystallized 5-
HT1B receptor (Fig. 2a). A 100 ns long molecular dynamics (MD)
simulation of the 5-HT7e5i complex was conducted in an explicit
solvated lipid bilayer. The stability of the receptor structure was
confirmed by the time evolution of the root mean squared devia-
tion (RMSD) calculated for TM alpha carbons, which showed a
plateau after 30 ns of simulation (Fig. S1). Compound 5i retained its
initial conformation during the whole MD simulation as well as the
key ionic interaction with Asp1623.32, and subtle rearrangements
involving the indole and the
a-naphthyl rings were detected
(Fig. 2b). In particular, an outward shift of the extracellular half of

G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
13
Scheme 4. Reagents and conditions: a)
u-bromoalkanoyl chloride, NaHCO3, EtOAc, H2O, RT, 20 min; b) TEA, DMF, RT, 1e16 h; c) BCl3, CH2Cl2, RT, 1 h; d) LiAlH4, THF, reflux, 2 h; e)
BCl3, CH2Cl2, RT, 2 h (for 18eeh) or H2, 10% Pd/C, MeOH, 60 ꢀC, 3 h (for 18aec); f) CH3CHO (for 17j) or benzaldehyde (for 17k), MeOH, AcOH, NaBH3CN, RT, 16 h.
TM5 was observed at the beginning of the MD simulation, allowing
a slight rotation of the indole ring of 5i and the consequent for-
mation of a hydrogen bond between its 5-hydroxyl group and
Ser2435.42, which was maintained throughout the MD simulation.
Although no mutagenesis data are available for this amino acid,
replacement of the corresponding serine of the 5-HT1A receptor
with an alanine decreased 5-HT binding affinity [22]. Moreover, the
serine residue in position 5.42 of the b1 receptor is hydrogen
bonded to the hydroxyl groups of isoprenaline [23], suggesting that
the amino acid in this position may interact with polar groups of
other aminergic neurotransmitters. The a-naphthyl substituent of
5i slightly rearranged during MD simulation. In particular, it moved
slightly away from Phe1583.28 on TM3 and was accommodated in a
crevice located at the tips of TM6 and TM7, delimited by Phe3436.51
,
Leu3466.54 and Leu3707.39. The shape and the location of this pocket
could also explain the loss of binding affinity brought by the
introduction of meta- or para-biphenyl substituents (compounds 5l
and 5m). Indeed, these bulkier groups could not be accommodated,
due to unfavorable steric clashes. This hydrophobic region,
extending beyond the anionic site of Asp1623.32, can thus represent
the binding pocket for the arylpiperazine moieties of our hybrid
ligands.
The binding model depicted in Fig. 2b is also consistent with the
possibility to replace the serotonin-like portion with bioisosteric
substructures. In fact, compound 37, having a 8-hydroxytetralin
scaffold, showed binding affinity similar to that of the serotonin-
like derivative 5n (Ki ¼ 81.4 nM and Ki ¼ 63.1 nM, respectively).
To further test this hypothesis, we replaced the indole scaffold with
an aniline one and we prepared a small series of anilino-alkyl-
piperazine derivatives, exemplified by compound 10h (Table 2). In
Scheme 5. Reagents and conditions: a) Mg3N2, MeOH, 80 ꢀC, 24 h; b) LiAlH4, THF, RT,
1 h; c) MnO2, CH2Cl2, RT, 18 h.

14
G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
Scheme 6. Reagents and conditions: a) (EtCO)2O, TEA, THF, RT, 24 h; b) BH3, THF, RT, 16 h; c) benzyl bromide, TEA, toluene, 80 ꢀC, 16 h; d) Pd(OAc)2, (ꢁ)-BINAP, tert-BuO-Kþ
iodobenzene, toluene, reflux, 24 h; e) benzenesulfonyl chloride, TEA, THF, RT, 48 h; f) BBr3, CH2Cl2, RT, 2 h.
,
fact, successful bioisosteric replacement of the 5-methoxyindol-3-
ylethyl fragment with a meta-methoxyanilinoethyl or a meta-
methoxyanilinopropyl one had already been described for mela-
tonin (N-acetyl-5-methoxytryptamine) derivatives [15], and the
anilino scaffold had resulted more versatile and synthetically
phenylpiperazine derivative 14o had Ki ¼ 20.0 nM, compared to
Ki ¼ 161.4 nM for the shorter analog 10h. As already observed in the
indole series, replacement of the phenyl substituent with an
naphthyl one significantly increased binding affinity (14m,
-naphthyl group (14n) was hardly tolerated.
a
-
Ki ¼ 2.9 nM), while a
b
feasible. Fig.
3
depicts N,N-dimethyl-5-hydroxytryptamine (b,
When docked into the 5-HT7 receptor binding site, compound 14m
showed an accommodation and a pattern of interactions similar to
those described for the indole derivative 5i. The meta-hydroxyl
group is hydrogen-bonded to Ser2435.42, the protonated piperazine
nitrogen interacts with Asp1623.32 and the naphthyl substituent
Fig. 1) in the bioactive conformation proposed by Vermeulen et al.
[14b], superposed to the tetralin derivative 8-OH-DPAT (c, Fig. 1)
and to 3-{[2-(dimethylamino)ethyl](methyl)amino}phenol. The
close superposition of this last compound highlights the possibility
for a meta-hydroxyanilino-ethylamine fragment to bioisosterically
replace the other two scaffolds.
occupies the region defined by Phe3436.51 Leu3466.54 and
,
Leu3707.39 (Fig. 2c). Within the anilinopropyl series, a slight in-
crease in binding affinity was registered when the aniline nitrogen
was demethylated (14l) or replaced by a methylene group, with
Ki ¼ 3.1 nM for compound 32c. The meta-hydroxyl group was
fundamental to achieve high potency. In fact, its replacement with
other substituents reduced binding affinity, as observed for a
methyl group (14b), for halogens, such as fluoro (14e), chloro (14d)
and bromo (14c), and for acetylamino (14p) and methyl-
sulfonylamino (14q) groups. A similar decrease of binding affinity
was obtained, on the anilino-NH scaffold, with hydroxymethyl
(14t), methoxycarbonyl (14s) and carboxamido (14r) groups, with
only the formyl substituent (14u) maintaining the same binding
affinity as the meta-hydroxyl derivative 14l. Four different sub-
stituents were introduced on the aniline nitrogen to look for
additional interactions with the binding site. Our receptor model
suggested that the 5-HT7 receptor binding site has room enough to
accommodate substituents at nitrogen slightly bulkier than the
methyl group. Indeed, a n-propyl chain (24) did not affect binding
affinity, while the phenyl (26) and benzyl (25) groups reduced Ki to
about 90 nM. Interestingly, the phenylsulfonyl derivative 27 was
the best one, with higher binding affinity than the reference 14o.
Docking of 27 into the receptor model gave two families of solu-
tions. One of them was consistent with our hypothesis (Fig. 2d),
while the other one showed inverted orientation, i.e. with the
Compound 10h carries a hydroxyl group in meta position, to-
pologically corresponding to position 5 of the indole ring of 5-HT,
and a spacer with two methylene units connects the aniline ni-
trogen to the arylpiperazine portion. Compound 10h showed a
limited loss of binding affinity (Table 2), being about three times
less potent than the corresponding indole derivative 5n. An
investigation on the importance of the hydroxyl group was carried
out inserting in meta position substituents with different size and
ability to form hydrogen bonds. While amino (10k) and nitro (9g)
substituents led to a decrease of binding affinity, a methyl-
sulfonylamino group had a positive effect, leading to a Ki value of
57.0 nM for compound 10l. Replacement of the phenyl ring on the
piperazine nitrogen with a benzyl group markedly reduced binding
affinity (10i), which stresses the role of the arylpiperazine moiety.
Removal of the methyl substituent from the aniline nitrogen
slightly reduced binding affinity (10j), while replacement of N-
methyl with an oxygen atom (35) almost abolished binding affinity.
On the other hand, replacement with a methylene, as in the phe-
nylpropyl derivative 32a, led to
a
recovery in potency
(Ki ¼ 40.0 nM). SARs related to the lengthening of the alkyl spacer
were also investigated, progressively increasing the number of
methylene groups from two to six. Addition of one methylene
group had
a positive effect on binding affinity, since the

G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
15
Scheme 7. Reagents and conditions: a) TEA, DMF, RT, 16 h; b) BH3, THF, RT, 16 h; c) BBr3, CH2Cl2, RT, 2 h.
Scheme 8. Reagents and conditions: a) N-phenylpiperazine, EDC, HOBT, Et3N, CH2Cl2, 2 h at 0 ꢀC and 16 h at RT; b) B2H6, THF, RT, 4 h; c) BBr3,CH2Cl2, RT, 2 h.
phenyl-piperazine moiety in the serotonin-like binding cavity and
vice versa. These alternative solutions can be the result of model
errors, due to its homology-based nature, or simply a consequence
of the choice to select a model adapted, by MD refinement, to a
smaller ligand (14m), lacking the N-phenylsulfonyl group.
Attempts to probe the size of the binding site portion accom-
modating the arylpiperazine moiety with bulkier substituents were
not successful, as derivatives carrying 4,40-diphenylpiperidinyl
(30b) and 4-(N,N-diphenylamino)piperidinyl (30c) groups mark-
edly reduced binding affinity. Lengthening of the alkyl spacer to
four (18a), five (18b) and six (18c) methylene units did not
significantly affect binding affinity, ending up with the most potent
compound 18c having Ki ¼ 12.0 nM.
Selected anilino-alkyl-piperazines with two to six methylene
spacers were tested in functional assays, to evaluate their agonist or
antagonist behavior. Compound 18a produced no increase of cAMP
level when tested up to the concentration of 10ꢂ5 M. On the other
hand it decreased the accumulation of cAMP stimulated by 5-HT,
with a KB ¼ 57 nM. In the same test, compounds 10l, 14o, 18b and
18c at the concentration of 1 mM produced a reduction of cAMP
accumulation induced by 5-HT to 48%, 49%, 69% and 56%, respec-
tively (Table 2), suggesting antagonist behavior.
Scheme 9. Reagents and conditions: a) K2CO3, DMF, 40 ꢀC, 16 h; b) B2H6, THF, RT, 4 h; c) BCl3, CH2Cl2, RT, 1 h.

16
G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
affinity, and insertion of an a-naphthyl ring on the piperazine had a
moderate negative effect (18f), opposite to what observed for
shorter derivatives. Recovery of binding affinity could be attained
by the anilide derivative 16i having Ki ¼ 8.1 nM. The 5-HT7 receptor
was poorly tolerant to substituents on the piperidine nitrogen of
compounds with a six-methylene linker. Indeed, insertion of a
trifluoromethyl group in meta position on the phenyl ring (18g)
reduced binding affinity, and a bulkier 7-chloro-quinolin-4-yl
fragment replacing the phenyl ring led to compound 18h devoid
of any binding affinity. Substituents on the aniline nitrogen were
tolerated also in this longer series, even if an ethyl chain (18j) and a
benzyl group (18k) slightly decreased binding affinity compared to
the methyl derivative 18c.
Four compounds from both the indole (5n, 5i) and the aniline
series (14o, 16i) were also tested on other 5-HT receptors (Table S1)
[26]. These compounds interacted with different receptor subtypes
with high affinity, in particular with the 5-HT1A receptor, which is
known to bind different classes of 5-HT7 ligands This behavior
highlights the necessity to optimize the structure of these com-
pounds to gain receptor subtype selectivity while maintaining their
good 5-HT7 binding affinity.
Scheme 10. Reagents and conditions: a) PTSA, toluene, reflux, 24 h; b) H2, EtOH/THF,
PtO2, RT, 24 h; c) BBr3,CH2Cl2, RT, 2 h.
The antagonists 18a and 18c, having a four-methylene and six-
methylene spacers, respectively, were docked into the 5-HT7 re-
ceptor binding site. Both were accommodated similarly to the
shorter 14m, with the same polar interactions formed by the
piperazine nitrogen and hydroxyl substituent (Fig. 4). The
hydroxylanilino moiety is accommodated within the serotonin-like
binding pocket where agonists are supposed to bind, while the
phenyl ring on the piperazine lies in the region of the binding site
delimited by TM2, 3 and 7. This binding pose could be consistent
with their antagonist behavior. Indeed, for the 5-HT7 receptor [24]
and, in general, for biogenic amine receptors [25] it has been pro-
posed that antagonist and agonist compounds occupy the same
binding region, with antagonists undertaking interactions with an
additional portion of the receptor.
4. Conclusions
Our data and models are consistent with the existence of two
separate binding pockets in the 5-HT7 receptor. The first one can
interact with the serotonin-like 5-hydroxyindole substructure or its
bioisosteres in the putative orthosteric site, and the second one can
accommodate an arylpiperazine moiety. For this reason the two
substructures can be combined to yield new ligands endowed with
good receptor affinity and antagonist potency, while subtype
selectivity still needs further investigations.
Other structural modifications were evaluated in the presence of
the six-methylene spacer. Replacement of the phenyl-piperazine
with a 4-phenyl-piperidine (18e) remarkably decreased binding
5. Experimental section
5.1. Chemistry
Table 1
Melting points were determined on a Buchi B-540 capillary
melting point apparatus and are uncorrected. 1H NMR spectra and
13C NMR were recorded on a Bruker AVANCE 200 spectrometer (1H:
200 MHz; 13C: 50 MHz) using CDCl3 as solvent unless stated
Binding affinity of the newly synthesized indole and tetralin derivatives for human
5-HT7 receptors.
otherwise. Chemical shifts (d scale) are reported in parts per million
(ppm) relative to the central peak of the solvent. Coupling constants
(J values) are given in Hz. EI-MS spectra (70 eV) were taken on a
Fisons Trio 1000 instrument. Only molecular ions (Mþ) and base
peaks are given. ESI-MS spectra were taken on a Waters Micromass
Zq instrument. Only molecular ions (M þ H)þ are given. Infrared
spectra were obtained on a Nicolet Avatar 360 FT-IR spectrometer;
absorbances are reported in
n
(cmꢂ1). Elemental analyses for C, H
Compd.
R1
R2
Ki (nM)a (95% confidence interval)
or % displacement at fixed conc.
and N were performed on a Carlo Erba analyzer, and the results are
within 0.4% of the calculated values. Column chromatography pu-
rifications were performed under “flash” conditions using Merck
230e400 mesh silica gel. Analytical thin-layer chromatography
(TLC) was carried out on Merck silica gel 60 F254 plates. The starting
indoles 1aed, anilines 6aec and 11beh, 3-(3-hydroxyphenyl)pro-
5-HT
5n
5l
1.4 (0.8e2.2)
63.1 (35ꢂ.85e111.1)
88%@10ꢂ6
Ph
H
H
m-Biphenyl
42%@10ꢂ5
5m
p-Biphenyl
H
32%@10
pionic
acid,
3-(benzyloxy)phenol,
8-methoxy-3,4-
10%@10ꢂ7
dihydronaphthalen-2(1H)-one, 4-phenylpiperidine and all the 4-
substituted-piperazines were commercially available.
5i
5k
5j
5o
5p
37b
a
a
a
-Naphthyl
-Naphthyl
-Naphthyl
H
11.6 (6.8e19.7)
12.8 (8.7e18.8)
6.6 (3.5e12.6)
ꢂ9%@10ꢂ7
Me
Et
H
n-Butyl
c-Pentyl
5.1.1. General procedure for 1-(5-substituted-indol-3-yl)-2-(4-
substituted-piperazin-1-yl)ethane-1,2-dione derivatives 3aeh
A solution of oxalyl chloride (0.14 mL, 1.6 mmol) in dry THF
(2.5 mL) was added dropwise to an ice-cooled stirred solution of
the suitable indole 1aec (1.3 mmol) in dry THF (6.5 mL), under N2.
Upon completion of the addition, the reaction mixture was allowed
H
1%@10ꢂ7
81.4 (44.3e149.4)
a
Ki values were calculated from IC50 values, obtained from competition curves by
the method of Cheng and Prusoff [33], and are the mean of four determinations
performed in duplicate.
b
Tested as racemate.

G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
17
Fig. 2. a) Induced-fit docking of compound 5i (orange) within the 5-HT7 receptor model (light gray). Dihydroergotamine is represented as transparent cyan carbons. b) MD
simulation of the 5-HT7e5i complex: superposition of the starting (transparent sticks) and the final energy-minimized structures (opaque sticks and cartoons). c) Best docking pose
of 14m (green carbons) within the 5-HT7 binding site. 5i (transparent orange carbons) is depicted for comparison. d) Best docking pose of 27 (pink carbons) within the 5-HT7
binding site. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
to warm to room temperature and stirred at this temperature for
1 h. The solvent and excess oxalyl chloride were removed under
reduced pressure and the residue dissolved in dry THF. After
cooling to 0 ꢀC, a solution of TEA (0.42 mL) and of the suitable N-
substituted-piperazine (1.95 mmol) in dry THF (6.5 mL) was added,
and the resulting mixture was stirred at room temperature for 1.5 h.
The solvent was removed by distillation in vacuo, the residue par-
titioned between EtOAc and water, and the aqueous phase extrac-
ted with EtOAc. The combined organic extracts were washed with
brine, dried (Na2SO4) and concentrated under reduced pressure, to
give a crude residue that was purified by flash-chromatography on
silica gel: cyclohexane/EtOAc 1:1 as eluant (EtOAc/MeOH, 95:5 for
3g and 3h).
piperazine), 3.13 (m, 2H, piperazine), 3.61 (m, 2H, piperazine), 3.79
(s, 3H, OCH3), 3.94 (m, 2H, piperazine), 6.84 (dd, 1H, J ¼ 2.0 and 8.0,
Ar-H), 7.14 (m, 1H, Ar-H), 7.33 (d, 1H, J ¼ 8.0, Ar-H), 7.39e7.65 (m,
5H, Ar-H), 7.90 (m, 1H, Ar-H), 8.19 (m, 1H, Ar-H), 12.01 (brs, 1H, NH);
ESI-MS (m/z): 428 (M þ H)þ.
5.1.1.4. 1-[5-(Benzyloxy)-1H-indol-3-yl]-2-[4-(biphenyl-3-yl)piper-
azin-1-yl]ethane-1,2-dione (3d). White solid (0.45 g, 67%); mp:
221e222 ꢀC (EtOAc); 1H NMR (CDCl3):
d
¼ 3.25 (m, 2H, piperazine),
3.36 (m, 2H, piperazine), 3.75 (m, 2H, piperazine), 3.96 (m, 2H,
piperazine), 5.17 (s, 2H, CH2Ph), 6.95 (m, 1H, Ar-H), 7.06 (dd, 1H,
J ¼ 2.5 and 8.5, Ar-H), 7.13e7.17 (m, 2H, Ar-H), 7.31e7.60 (m,12H, Ar-
H), 7.98 (m, 2H, Ar-H), 8.80 (brs, 1H, NH); MS (EI, 70 eV): m/z 515
(Mþ), 175 (100).
5.1.1.1. 1-(5-Methoxy-1H-indol-3-yl)-2-[4-(naphthalen-1-yl)piper-
azin-1-yl]ethane-1,2-dione (3a). White amorphous solid (0.33 g,
62%); 1H NMR (acetone-d6):
d
¼ 3.15 (m, 4H, piperazine), 3.80 (m,
5.1.1.5. 1-[5-(Benzyloxy)-1H-indol-3-yl]-2-[4-(biphenyl-4-yl)piper-
azin-1-yl]ethane-1,2-dione (3e). Yellowish solid (0.50 g, 75%); mp:
2H, piperazine), 3.87 (s, 3H, CH3), 4.01 (m, 2H, piperazine), 6.93 (dd,
1H, J ¼ 2.0 and 8.0, Ar-H), 7.18 (m, 1H, Ar-H), 7.40e7.65 (m, 5H, Ar-
H), 7.81 (d, 1H, J ¼ 2.0, Ar-H), 7.89 (m, 1H, Ar-H), 8.16 (m, 1H, Ar-H),
8.29 (m, 1H, Ar-H), 11.23 (brs, 1H, NH); ESI-MS (m/z): 414 (M þ H)þ.
216e217 ꢀC (EtOAc); 1H NMR (DMSO-d6):
d
¼ 3.16 (m, 2H, pipera-
zine), 3.32 (m, 2H, piperazine), 3.49 (m, 2H, piperazine), 3.77 (m,
2H, piperazine), 5.14 (s, 2H, CH2Ph), 6.98 (dd, 1H, J ¼ 2.5 and 8.5, Ar-
H), 7.03 (d, 1H, J ¼ 8.5, Ar-H), 7.23e7.70 (m, 15H, Ar-H), 7.74 (d, 1H,
J ¼ 2.5, Ar-H), 12.23 (brs, 1H, NH); ESI-MS (m/z): 516 (M þ H)þ.
5.1.1.2. 1-(2-Ethyl-5-methoxy-1H-indol-3-yl)-2-[4-(naphthalen-1-yl)
piperazin-1-yl]ethane-1,2-dione (3b). Yellowish amorphous solid
(0.30 g, 52%); 1H NMR (CDCl3):
d
¼ 1.41 (t, 3H, J ¼ 7.5, CH2CH3), 3.18
5.1.1.6. 1-[5-(Benzyloxy)-1H-indol-3-yl]-2-(4-phenylpiperazin-1-yl)
ethane-1,2-dione (3f). White solid (0.43 g, 75%); mp: 136e137 ꢀC
(q, 2H, J ¼ 7.5, CH2CH3), 3.22 (m, 4H, piperazine), 3.41 (m, 2H,
piperazine), 3.75 (m, 2H, piperazine), 3.90 (s, 3H, OCH3), 6.90 (dd,
1H, J ¼ 2.0 and 8.0, Ar-H), 7.08 (d, 1H J ¼ 8.0, Ar-H), 7.26 (m, 1H, Ar-
H), 7.38e7.63 (m, 5H, Ar-H), 7.85 (m, 1H, Ar-H), 8.20 (m, 1H, Ar-H),
8.75 (brs, 1H, NH); ESI-MS (m/z): 442 (M þ H)þ.
(EtOAc); 1H NMR (CDCl3):
d
¼ 3.16 (m, 2H, piperazine), 3.27 (m, 2H,
piperazine), 3.68 (m, 2H, piperazine), 3.91 (m, 2H, piperazine), 5.14
(s, 2H, CH2Ph), 6.91e7.02 (m, 4H, Ar-H), 7.23e7.51 (m, 8H, Ar-H),
7.78 (d, 1H, J ¼ 3.0, Ar-H), 7.95 (d, 1H, J ¼ 2.0, Ar-H), 9.73 (brs, 1H,
NH); 13C NMR (CDCl3):
d
¼ 185.5, 166.3, 156.1, 150.8, 137.1, 135.7,
5.1.1.3. 1-(5-Methoxy-2-methyl-1H-indol-3-yl)-2-[4-(naphthalen-1-
131.5, 129.3, 128.6, 127.9, 127.7, 126.1, 120.8, 116.9, 115.4, 114.5, 112.8,
104.6, 70.6, 50.0, 49.6, 46.1,41.5; MS (EI, 70 eV): m/z 439 (Mþ), 330
(100).
yl)piperazin-1-yl]ethane-1,2-dione (3c). Yellowish amorphous solid
(0.24 g, 43%); 1H NMR (DMSO-d6):
d
¼ 2.62 (s, 3H, CH3), 3.00 (m, 2H,

18
G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
Table 2
Binding affinity of the newly synthesized anilino-alkyl-piperazine derivatives for human 5-HT7 receptors (Ki (nM) or % displacement at fixed concentration) and functional
evaluation at the cAMP test.
Compd.
R1
X
n
R2
Ki (nM)a (95% confidence interval)
or % displacement at fixed conc.
% Serotonin response on cAMP accumulation at 1 mM
10h
10k
OH
NH2
N-Me
N-Me
2
2
Ph
Ph
161.4 (7ꢂ85.3e332.9)
85%@10
40%@10ꢂ6
9g
10l
10i
NO2
NHSO2Me
OH
N-Me
N-Me
N-Me
2
2
2
Ph
Ph
Bn
332.0 (96.3e1177.0)
57.0 (38ꢂ.95e83.2)
82%@10
48%
42%@10ꢂ6
10j
35
OH
OH
NH
O
2
2
Ph
Ph
252.1 (1ꢂ454.4e555.5)
21%@10ꢂ6
42%@10
32a
14o
14m
14n
32c
14l
14b
14e
14d
14c
14p
14q
14t
14s
14r
14u
24
OH
OH
OH
OH
OH
OH
Me
F
CH2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Ph
Ph
40.0 (22.9e69.9)
20.0 (12.0e33.3)
2.9 (1.0e8.3)
434.2 (117.6e1602.0)
3.1 (1.3e7.6)
N-Me
N-Me
N-Me
CH2
49%
a
b
-Naphthyl
-Naphthyl
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
NH
10.1 (5.2e19.6)
84.8 (26.1e275.2)
111.7 (55.2e226.3)
179.2 (72.9e440.5)
161.2 (49.5e525.3)
161.1 (73.8e351.5)
39.6 (14.9e104.7)
108.8 (8.9e1325.0)
77.9 (40.8e148.6)
63.3 (32.7e122.4)
13.2 (7.2e24.1)
20.8 (11.8e36.4)
90.1 (28.3e287.0)
92.5 (47.0e181.8)
12.1 (6.2e23.8)
N-Me
N-Me
N-Me
N-Me
N-Me
N-Me
NH
NH
NH
NH
Cl
Br
NHCOMe
NHSO2Me
CH2OH
COOMe
CONH2
CHO
OH
OH
OH
OH
N-nPr
N-Ph
N-Bn
N-SO2Ph
26
25
27
30b
242.1 (150.8e388.6)
30c
18a
310.0 (170.1e564.9)
30.0 (14.0e59.3)
OH
N-Me
4
Ph
39%
IC50 ¼ 540 nM
KB ¼ 57 nM
69%
18b
18c
OH
OH
N-Me
N-Me
5
6
Ph
Ph
16.7 (7.7e35.9)
12.0 (8.3e17.2)
56%
18e
18f
16i
18g
226.0 (141.9e360.0)
39.4 (18.2e85.6)
8.1 (4.6e14.2)
OH
OH
N-Me
N-Me
6
6
a-Naphthyl
m-CF3-Ph
95.6 (45.5e201.3)

G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
19
Table 2 (continued )
Compd.
R1
X
n
R2
Ki (nM)a (95% confidence interval)
or % displacement at fixed conc.
% Serotonin response on cAMP accumulation at 1 mM
18h
OH
N-Me
6
7-Cl-quinolin-4-yl
74%@10ꢂ5
28%@10ꢂ6
18j
18k
OH
OH
N-Et
N-Bn
6
6
Ph
Ph
191.0 (51.8e703.7)
137.5 (90.0e210.3)
a
Ki values were calculated from IC50 values, obtained from competition curves by the method of Cheng and Prusoff [33], and are the mean of four determinations performed
in duplicate.
5.1.1.7. 1-(4-Butylpiperazin-1-yl)-2-(5-methoxy-1H-indol-3-yl)
ethane-1,2-dione (3g). Yellowish solid (0.36 g, 82%); 1H NMR
(CDCl3):
CH2CH2CH2CH3), 2.43 (m, 6H, piperazine and CH2CH2CH2CH3), 3.52
(m, 2H, piperazine), 3.76 (m, 2H, piperazine), 3.88 (s, 3H, OCH3),
6.89 (dd, 1H, J ¼ 2.0 and 8.0, Ar-H), 7.24 (d, 1H, J ¼ 8.0, Ar-H), 7.71
(brs, 1H, Ar-H), 7.79 (d, 1H, J ¼ 2.0, Ar-H), 9.87 (brs, 1H, NH); ESI-MS
(m/z): 344 (M þ H)þ.
addition was completed, the reaction mixture was refluxed for 1 h.
Aftercoolingto0 ꢀC, waterwas addeddropwise todestroy theexcess
hydride and the resulting mixture was filtered on Celite. The filtrate
was concentrated in vacuo, and partitioned between water and
EtOAc. The combined organic layers were washed with brine, dried
(Na2SO4) and concentrated under reduced pressure to afford a crude
residue which was purified by flash chromatography on silica gel
(cyclohexane/EtOAc 1:1 or EtOAc/MeOH 9:1 for 4geh as eluant).
d
¼ 0.92 (t, 3H, J ¼ 7.0, CH2CH2CH2CH3), 1.39 (m, 4H,
5.1.2.1. 5-Methoxy-3-{2-[4-(naphthalen-1-yl)piperazin-1-yl]ethyl}-
5.1.1.8. 1-(4-Cyclopentylpiperazin-1-yl)-2-(5-methoxy-1H-indol-3-
1H-indole (4a). Amorphous solid (0.23 g, 68%); 1H NMR (CDCl3):
yl)ethane-1,2-dione (3h). Yellowish solid (0.28 g, 62%); 1H NMR
d
¼ 2.06 (m, 2H, CH2Ar), 2.82e3.09 (m, 6H, piperazine and CH2-
(CDCl3):
d
¼ 1.41e1.86 (m, 8H, c-pentyl), 2.55 (m, 5H, piperazine
piperazine), 3.24 (m, 4H, piperazine), 3.90 (s, 3H, OCH3), 6.88 (dd,
1H, J ¼ 2.0 and 8.0, Ar-H), 7.07e7.30 (m, 4H, Ar-H), 7.39e7.60 (m,
4H, Ar-H), 7.84 (m, 1H, Ar-H), 7.92 (brs, 1H, NH), 8.23 (m, 1H, Ar-H);
ESI-MS (m/z): 386 (M þ H)þ.
and CH), 3.55 (m, 2H, piperazine), 3.79 (m, 2H, piperazine), 3.90 (s,
3H, OCH3), 6.92 (dd, 1H, J ¼ 2.0 and 10.0, Ar-H), 7.27 (d, 1H, J ¼ 10,
Ar-H), 7.77 (brs, 1H, Ar-H), 7.80 (d, 1H, J ¼ 2.0, Ar-H), 9.65 (brs, 1H,
NH); MS (EI, 70 eV) m/z: 355 (Mþ), 174 (100).
5.1.2.2. 2-Ethyl-5-methoxy-3-{2-[4-(naphthalen-1-yl)piperazin-1-yl]
5.1.2. General procedure for 3-[2-(4-substituted-piperazin-1-yl)
ethyl]-1H-indoles 4aeh
LiAlH4 (0.2 g, 5.4 mmol) was added portionwise to an ice-cooled
suspension of the suitable ethane-1,2-dione derivative 3aeh
(0.9 mmol) in dry THF (9 mL) under a N2 atmosphere. Once the
ethyl}-1H-indole (4b). Amorphous solid (0.32 g, 85%); 1H NMR
(CDCl3):
d
¼ 1.32 (t, 3H, J ¼ 7.5, CH2CH3), 2.86 (m, 10H, CH2CH3,
piperazine and ArCH2CH2N), 3.26 (m, 4H, piperazine), 3.90 (s, 3H
OCH3), 6.81 (dd, 1H, J ¼ 2.0 and 8.0, Ar-H), 7.06 (d, 1H, J ¼ 2.0, Ar-H),
7.19 (m, 2H, Ar-H), 7.50 (m, 4H, Ar-H), 7.73 (brs, 1H, NH), 7.85 (m,1H,
Ar-H), 8.24 (m, 1H, Ar-H); ESI-MS (m/z): 414 (M þ H)þ.
5.1.2.3. 5-Methoxy-2-methyl-3-{2-[4-(naphthalen-1-yl)piperazin-1-
yl]ethyl}-1H-indole (4c). Amorphous solid (0.26 g, 74%); 1H NMR
(CDCl3):
d
¼ 2.40 (s, 3H, CH3), 2.70 (m, 2H, CH2Ar), 2.93 (m, 6H,
piperazine and CH2-piperazine), 3.23 (m, 4H, piperazine), 3.88 (s,
Fig. 3. Superposition of N,N-dimethyl-5-hydroxytryptamine (b in Fig. 1, green car-
bons), 8-OH-DPAT (c in Fig. 1, orange carbons) and 3-{[2-(dimethylamino)ethyl](-
methyl)amino}phenol (yellow carbons). (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Superposition of the best docking poses of 18a (green carbons) and 18c (yellow
carbons) within the 5-HT7 binding site. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)

20
G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
3H, OCH3), 6.78 (dd,1H, J ¼ 2.0 and 8.0, Ar-H), 7.03 (d,1H, J ¼ 2.0, Ar-
H), 7.15 (m, 2H, Ar-H), 7.38e7.59 (m, 4H, Ar-H), 7.69 (brs, 1H, NH),
7.83 (m, 1H, Ar-H), 8.23 (m, 1H, Ar-H); ESI-MS (m/z): 400 (M þ H)þ.
122.9, 114.5, 112.7, 112.6, 111.5, 111.2, 102.5, 59.2, 53.6, 53.1, 22.9; MS
(EI, 70 eV) m/z: 371 (Mþ), 146 (100); Anal. calcd for C24H25N3O: C
77.60, H 6.78, N 11.31, found C 77.81, H 6.90, N 11.03.
5.1.2.4. 5-(Benzyloxy)-3-{2-[4-(biphenyl-3-yl)piperazin-1-yl]ethyl}-
5.1.3.2. 2-Ethyl-3-{2-[4-(naphthalen-1-yl)piperazin-1-yl]ethyl}-1H-
1H-indole (4d). White solid (0.33 g, 75%); mp: 99e100 ꢀC (EtOAc/n-
indol-5-ol (5j). White solid (0.28 g, 70%); mp: 200e201 ꢀC (CH2Cl2/
hexane); 1H NMR (CDCl3):
3.00 (m, 2H, CH2-piperazine), 3.35 (m, 4H, piperazine), 5.14 (s, 2H,
CH2Ph), 6.95e7.62 (m, 18H, Ar-H), 7.93 (brs, 1H, NH); MS (EI, 70 eV):
m/z 487 (Mþ), 251 (100).
d
¼ 2.78 (m, 6H, piperazine and CH2Ar),
petroleum ether); 1H NMR (CDCl3):
d
¼ 1.29 (t, 3H, J ¼ 7.5, CH3CH2),
2.76 (q, 2H, J ¼ 7.5, CH3CH2), 2.77 (m, 2H, CH2Ar) 2.90 (m, 6H,
piperazine and CH2CH2Ar), 3.25 (m, 4H, piperazine), 6.73 (dd, 1H,
J ¼ 2.0 and 8.0, Ar-H), 6.89 (d, 1H, J ¼ 2.0, Ar-H), 7.14 (m, 2H, Ar-H),
7.37e7.59 (m, 4H, Ar-H), 7.69 (brs, 1H, NH), 7.85 (m, 1H, Ar-H), 8.21
5.1.2.5. 5-(Benzyloxy)-3-{2-[4-(biphenyl-4-yl)piperazin-1-yl]ethyl}-
1H-indole (4e). White solid (0.28 g, 64%); mp: 92e93 ꢀC (EtOAc/n-
(m, 1H, Ar-H); 13C NMR (CDCl3):
d
¼ 149.7, 149.5, 138.3, 134.7, 130.3,
129.3,128.9,128.4,125.9,125.8,125.3,123.5,123.5,114.8,111.1,111.0,
107.9, 103.1, 59.4, 53.7, 52.6, 21.6, 19.5, 14.4; IR (nujol):
hexane); 1H NMR (acetone-d6):
d
¼ 2.70 (m, 6H, piperazine and
CH2Ar), 2.97 (m, 2H, CH2-piperazine), 3.32 (m, 4H, piperazine), 5.19
(s, 2H, CH2Ph), 6.89 (dd, 1H, J ¼ 2.0 and 8.0, Ar-H), 7.09 (m, 2H, Ar-
H), 7.24 (m, 2H, Ar-H), 7.29e7.48 (m, 7H, Ar-H), 7.53e7.68 (m, 6H,
Ar-H), 9.92 (brs, 1H, NH); ESI-MS (m/z): 488 (M þ H)þ.
n
¼ 3308 cmꢂ1; ESI-MS (m/z): 400 (M þ H)þ; Anal. calcd for
C
26H29N3O: C 78.16, H 7.32, N 10.52, found C 78.01, H 7.21, N 10.29.
5.1.3.3. 2-Methyl-3-{2-[4-(naphthalen-1-yl)piperazin-1-yl]ethyl}-
1H-indol-5-ol (5k). White solid (0.30 g, 79%); mp: 192e193 ꢀC
5.1.2.6. 5-(Benzyloxy)-3-[2-(4-phenylpiperazin-1-yl)ethyl]-1H-
indole (4f). White solid (0.26 g, 71%); mp: 133e134 ꢀC (EtOAc/n-
(CH2Cl2/petroleum ether); 1H NMR (CDCl3):
d
¼ 2.39 (s, 3H, CH3),
2.70 (m, 2H, CH2Ar), 2.90 (m, 6H, piperazine and CH2CH2Ar), 3.23
(m, 4H, piperazine), 6.68 (dd, 1H, J ¼ 2.0 and 8.0, Ar-H), 6.91 (d, 1H,
J ¼ 2.0, Ar-H), 7.13 (m, 2H, Ar-H), 7.37e7.58 (m, 4H, Ar-H), 7.64 (brs,
1H, NH), 7.83 (m, 1H, Ar-H), 8.21 (m, 1H, Ar-H); 13C NMR (DMSO-
hexane); 1H NMR (CDCl3):
d
¼ 2.77 (m, 6H, piperazine and CH2Ar),
2.99 (m, 2H, CH2-piperazine), 3.29 (m, 4H, piperazine), 5.14 (s, 2H,
CH2Ph), 6.85e7.00 (m, 4H, Ar-H), 7.05 (d, 1H, J ¼ 2.0, Ar-H), 7.17 (d,
1H, J ¼ 2.0, Ar-H), 7.25e7.53 (m, 8H, Ar-H), 7.92 (brs, 1H, NH); 13C
d6):
d
¼ 150.6, 149.9, 134.8, 132.6, 130.1, 129.5, 128.7, 128.6, 126.5,
NMR (CDCl3):
d
¼ 153.1, 151.3, 137.7, 131.6, 129.1, 128.5, 127.8, 127.6,
126.3, 125.8, 123.8, 123.4, 115.0, 111.1, 110.2, 107.9, 102.2, 59.4, 53.7,
122.4, 119.7, 116.1, 114.1, 112.9, 111.8, 102.5, 71.0, 59.2, 53.3, 49.2,
53.2, 22.1, 11.8; IR (nujol):
n
¼ 3238 cmꢂ1; ESI-MS (m/z): 386
23.0; MS (EI, 70 eV): m/z 411 (Mþ), 175 (100).
(M þ H)þ; Anal. calcd for C25H27N3O: C 77.89, H 7.06, N 10.90, found
C 77.92, H 7.16, N 10.85.
5.1.2.7. 3-[2-(4-Butylpiperazin-1-yl)ethyl]-5-methoxy-1H-indole
(4g). Oil (0.18 g, 63%); 1H NMR (CDCl3):
d
¼ 0.93 (t, 3H, J ¼ 7.0,
5.1.3.4. 3-[2-(4-Butylpiperazin-1-yl)ethyl]-1H-indol-5-ol
(5o).
¼ 0.90 (t,
CH2CH2CH2CH3), 1.20e1.60 (m, 4H, CH2CH2CH2CH3), 2.39e3.00 (m,
14H, piperazine, CH2Ar and 2 ꢃ CH2N), 3.87 (s, 3H, OCH3), 6.85 (dd,
1H, J ¼ 2.0 and 8.0, Ar-H), 7.02 (d, 1H, J ¼ 2.0, Ar-H), 7.03 (d, 1H,
J ¼ 2.0, Ar-H), 7.23 (d, 1H, J ¼ 8.0, Ar-H), 8.04 (brs, 1H, NH); ESI-MS
(m/z): 316 (M þ H)þ.
Amorphous solid (0.12 g, 42%); 1H NMR (acetone-d6):
d
3H, J ¼ 7.0, CH2CH2CH2CH3), 1.16e1.54 (m, 4H, CH2CH2CH2CH3),
2.28e2.88 (m, 14H, piperazine, CH2CH2CH2CH3 and CH2CH2Ar),
6.68 (dd,1H, J ¼ 2.0 and 8.0, Ar-H), 6.96 (d, 1H, J ¼ 2.0, Ar-H), 7.09 (d,
1H, J ¼ 2.0, Ar-H), 7.18 (d, 1H, J ¼ 8.0, Ar-H), 7.61 (brs, 1H, NH), 9.70
(brs, 1H, OH); 13C NMR (CDCl3):
d
¼ 150.0, 131.2, 128.0, 122.6, 112.8,
5.1.2.8. 3-[2-(4-Cyclopentylpiperazin-1-yl)ethyl]-5-methoxy-1H-
112.7, 111.9, 103.7, 58.9, 58.4, 52.6, 29.7, 28.6, 22.4, 20.7, 14.0; IR
indole (4h). Amorphous solid (0.21 g, 71%); 1H NMR (CDCl3):
(nujol):
n
¼ 3452, 3142 cmꢂ1; ESI-MS (m/z): 302 (M þ H)þ; Anal.
d
¼ 1.26e2.00 (m, 8H, c-pentyl), 2.45e3.00 (m, 13H, piperazine,
calcd for C18H27N3O: C 71.72, H 9.03, N 13.94, found C 71.63, H 9.21,
N 14.02.
CH2Ar, CH2N and CH), 3.86 (s, 3H, OCH3), 6.86 (dd, 1H, J ¼ 2.5 and
8.0, Ar-H), 7.02 (d, 1H, J ¼ 2.5, Ar-H), 7.05 (d, 1H, J ¼ 2.5, Ar-H), 7.23
(d, 1H, J ¼ 8.0, Ar-H), 7.93 (brs, 1H, NH); MS (EI, 70 eV): m/z 327
(Mþ), 167 (100).
5.1.3.5. 3-[2-(4-Cyclopentylpiperazin-1-yl)ethyl]-1H-indol-5-ol (5p).
Amorphous solid (0.15 g, 47%); 1H NMR (acetone-d6):
d
¼ 1.32e1.89
(m, 8H, c-pentyl), 2.35e3.31 (m, 13H, piperazine, CH2CH2Ar and
CH), 6.68 (dd, 1H, J ¼ 2.0 and 8.0, Ar-H), 6.95 (d, 1H, J ¼ 2.0, Ar-H),
7.09 (d, 1H, J ¼ 2.0, Ar-H), 7.18 (d, 1H, J ¼ 8.0, Ar-H), 7.63 (brs, 1H,
5.1.3. O-Demethylation with BBr3. General procedure for 5-
hydroxyindole derivatives 5iek and 5oep
A solution of BBr3 (1 M in CH2Cl2, 3 mL, 3 mmol) diluted with dry
CH2Cl2 (6 mL) was added dropwise to a solution of the suitable
methoxy derivative 4aec or 4geh (1 mmol) in dry CH2Cl2 (4 mL) at
0 ꢀC, and the resulting mixture was stirred at room temperature for
2 h. The reaction mixture was neutralized with a 2 N aqueous so-
lution of Na2CO3 and extracted with EtOAc. The organic phases
were combined, dried (Na2SO4) and concentrated under reduced
pressure, to give a crude product that was purified by flash chro-
matography (silica gel; EtOAc for 5iek or EtOAc/MeOH 9:1 for 5oe
p as eluant) and crystallization.
NH), 9.70 (brs, 1H, OH); 13C NMR (CDCl3):
d
¼ 149.9, 131.2, 128.0,
122.6, 113.0, 112.8, 111.9, 103.7, 67.6, 59.0, 52.7, 51.8, 30.2, 24.1, 22.4;
IR (nujol):
n
¼ 3165, cmꢂ1; ESI-MS (m/z): 314 (M þ H)þ; Anal. calcd
for C19H27N3O: C 72.81, H 8.68, N 13.41, found C 72.85, H 8.74, N
13.58.
5.1.4. O-Debenzylation by hydrogenolysis. General procedure for 5-
hydroxyindole derivatives 5len
A solution of the benzyloxy derivative 4def (0.73 mmol) was
dissolved in anhydrous MeOH (12 mL) and then hydrogenated
(4 atm) at room temperature in the presence of 10% Pd/C (0.053 g)
for 6 h. The reaction mixture was filtered on Celite, the filtrate was
evaporated under reduced pressure to yield a crude residue that
was purified by crystallization.
5.1.3.1. 3-{2-[4-(Naphthalen-1-yl)piperazin-1-yl]ethyl}-1H-indol-5-
ol (5i). Pink Solid (0.16 g, 44%); 1H NMR (CDCl3):
d
¼ 2.96 (m, 8H,
piperazine and CH2CH2Ar), 3.24 (m, 4H, piperazine), 6.80 (dd, 1H,
J ¼ 2.0 and 8.0, Ar-H), 6.99 (d, 1H, J ¼ 2.0, Ar-H), 7.06 (d, 1H, J ¼ 2.0,
Ar-H), 7.10e7.27 (m, 2H, Ar-H), 7.48 (m, 4H, Ar-H and NH), 7.85 (m,
5.1.4.1. 3-{2-[4-(Biphenyl-3-yl)piperazin-1-yl]ethyl}-1H-indol-5-ol
2H, Ar-H), 8.21 (m, 1H, Ar-H); 13C NMR (acetone-d6):
d
¼ 150.5,
(5l). White solid (148 mg, 51%); mp: 194e195 ꢀC dec. (EtOAc/pe-
149.9, 134.9, 128.9, 128.5, 128.3, 125.9, 125.7, 125.1, 123.6, 123.1,
troleum ether); 1H NMR (DMSO-d6):
d
¼ 2.64 (m, 6H, piperazine

G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
21
and CH2Ar), 2.80 (m, 2H, CH2CH2Ar), 3.25 (m, 4H, piperazine), 6.60
(dd, 1H, J ¼ 2.0 and 8.0, Ar-H), 6.85 (d, 1H, J ¼ 2.0, Ar-H), 6.97e7.49
(m, 9H, Ar-H), 7.65 (m, 2H, Ar-H), 8.55 (brs, 1H, NH), 10.48 (brs, 1H,
5.1.5.5. 3-Bromo-N-methyl-N-m-tolylpropanamide
(12b). The
product was prepared starting from 3-methyl-N-methylaniline and
3-bromopropanoyl chloride (Scheme 3). Oil; ESI-MS (m/z): 256e
258 (M þ H)þ.
OH); 13C NMR (DMSO-d6):
d
¼ 152.1, 150.6, 141.6, 141.4, 131.3, 129.9,
129.2, 128.4, 127.7, 127.3, 123.4, 117.8, 115.0, 114.1, 112.2, 112.1, 111.6,
102.7, 59.3, 53.3, 48.8, 23.1; IR (nujol):
n
¼ 3198 cmꢂ1; MS (EI, 70 eV)
5.1.5.6. 3-Bromo-N-(3-bromophenyl)-N-methylpropanamide (12c).
The product was prepared starting from 3-bromo-N-methylaniline
and 3-bromopropanoyl chloride (Scheme 3). Oil; ESI-MS (m/z):
320e322e324 (M þ H)þ.
m/z: 397 (Mþ), 251 (100); Anal. calcd for C26H27N3O: C 78.56, H
6.85, N 10.57, found C 78.77, H 6.97, N 10.75.
5.1.4.2. 3-{2-[4-(Biphenyl-4-yl)piperazin-1-yl]ethyl}-1H-indol-5-ol
(5m). Gray solid (122 mg, 42%); mp: 216e217 ꢀC dec. (MeOH); 1H
5.1.5.7. 3-Bromo-N-(3-chlorophenyl)-N-methylpropanamide (12d).
The product was prepared starting from 3-chloro-N-methylaniline
and 3-bromopropanoyl chloride (Scheme 3). Oil; ESI-MS (m/z):
276e278e280 (M þ H)þ.
NMR (acetone-d6):
d
¼ 2.67 (m, 6H, piperazine and CH2Ar), 2.88 (m,
2H, CH2CH2Ar), 3.27 (m, 4H, piperazine), 6.68 (dd, 1H, J ¼ 2.5 and
8.0, Ar-H), 6.97 (d, 1H, J ¼ 2.5, Ar-H), 6.97e7.61 (m, 12H, Ar-H and
NH), 9.73 (brs, 1H, OH); 13C NMR (CDCl3):
d
¼ 150.8, 150.6, 140.5,
131.3,130.8,129.2, 128.4,127.6, 126.7, 126.3,123.5,116.0, 112.1,112.0,
5.1.5.8. 3-Bromo-N-(3-fluorophenyl)-N-methylpropanamide (12e).
The product was prepared starting from 3-fluoro-N-methylaniline
and 3-bromopropanoyl chloride (Scheme 3). Oil; ESI-MS (m/z):
260e262 (M þ H)þ.
111.7, 102.7, 59.0, 53.0, 48.3, 26.8; IR (nujol):
n
¼ 3162 cmꢂ1; ESI-MS
(m/z): 398 (M þ H)þ; Anal. calcd for C26H27N3O: C 78.56, H 6.85, N
10.57, found C 7 8.69, H 6.90, N 10.43.
5.1.4.3. 3-[2-(4-Phenylpiperazin-1-yl)ethyl]-1H-indol-5-ol
White solid (145 mg, 62%); mp: 202e203 ꢀC (MeOH/Et2O); 1H NMR
(acetone-d6):
(5n).
5.1.5.9. 3-Bromo-N-methyl-N-(3-nitrophenyl)propanamide
(12f).
The product was prepared starting from 3-nitro-N-methylaniline
and 3-bromopropanoyl chloride (Scheme 3). Oil; ESI-MS (m/z):
300e302 (M þ H)þ.
d
¼ 2.69 (m, 6H, piperazine and CH2Ar), 2.90 (m, 2H,
CH2CH2Ar), 3.22 (m, 4H, piperazine), 6.70 (ddd, 1H, J ¼ 0.5, 2.0 and
9.0, Ar-H), 6.78 (m, 1H, Ar-H) 6.94e6.99 (m, 3H, Ar-H), 7.13 (d, 1H,
J ¼ 2.0, Ar-H), 7.17e7.26 (m, 3H, Ar-H), 7.53 (brs, 1H, NH), 9.67 (brs,
5.1.5.10. Ethyl 3-(3-bromo-N-methylpropanamido)benzoate (12g).
The product was prepared starting from ethyl 3-aminobenzoate
and 3-bromopropanoyl chloride (Scheme 3). Oil; ESI-MS (m/z):
287e289 (M þ H)þ.
1H, OH); 13C NMR (DMSO-d6):
d
¼ 151.6, 150.6, 131.3, 129.3, 128.4,
123.4, 119.1, 115.7, 112.1, 112.1, 111.6, 102.7, 59.3, 53.2, 48.7, 23.1; IR
(nujol):
n
¼ 3205 cmꢂ1; MS (EI, 70 eV): m/z 321 (Mþ), 175 (100);
Anal. calcd for C20H23N3O: C 74.74, H 7.21, N 13.07, found C 74.96, H
7.19, N 13.32.
5.1.5.11. 3-Bromo-N-(3-methoxyphenyl)propanamide
(12h).
The product was prepared starting from 3-methoxyaniline and 3-
bromopropanoyl chloride (Scheme 3). Oil; ESI-MS (m/z): 258e260
(M þ H)þ.
5.1.5. N-Acylation of anilines with
u
-bromoalkanoyl chlorides.
General procedure for
u-bromoalkanamido derivatives 7aec, 12aei
and 15aed
The suitable
u-bromoalkanoyl chloride (0.7 mmol) was added
5.1.5.12. N-[3-(Benzyloxy)phenyl]-3-bromo-N-methylpropanamide
(12i). The product was prepared starting from 3-benzyloxy-N-
methylaniline [17] and 3-bromopropanoyl chloride (Scheme 3). Oil;
MS (EI, 70 eV) m/z: 347e349 (Mþ), 91 (100).
dropwise to a stirred ice-cooled solution of the suitable aniline
(0.33 mmol) and NaHCO3 (0.10 g, 1.22 mmol) in EtOAc (0.4 mL) and
water (0.4 mL), and the resulting mixture was stirred at room
temperature for 20 min. Water was added and the aqueous phase
was extracted three times with EtOAc. The combined organic
phases were washed with brine, dried (Na2SO4) and concentrated
under reduced pressure to afford a crude residue that was used for
the next step, without further purification.
5.1.5.13. N-[3-(Benzyloxy)phenyl]-4-bromo-N-methylbutanamide
(15a). The product was prepared starting from 3-benzyloxy-N-
methylaniline [17] and 4-bromobutanoyl chloride (Scheme 4). Oil;
MS (EI, 70 eV) m/z: 361e363 (Mþ), 91 (100).
5.1.5.1. 2-Bromo-N-(3-methoxyphenyl)-N-methylacetamide
(7a).
5.1.5.14. N-[3-(Benzyloxy)phenyl]-5-bromo-N-methylpentanamide
(15b). The product was prepared starting from 3-benzyloxy-N-
methylaniline [17] and 5-bromopentanoyl chloride (Scheme 4). Oil;
The product was prepared starting from 3-methoxy-N-methylani-
line and 2-bromoethanoyl chloride (Scheme 2). Oil; MS (EI, 70 eV)
m/z: 257e259 (Mþ), 136 (100) [27].
1H NMR (CDCl3):
d
¼ 1.67e1.87 (m, 4H, CH2CH2CH2CH2), 2.09 (t, 2H,
J ¼ 7.0, CH2CO), 3.25 (s, 3H, CH3), 3.31 (t, 2H, J ¼ 6.5, CH2Br), 5.09 (s,
2H, CH2Ph), 6.76e6.79 (m, 2H, Ar-H), 7.00 (dd, 1H, J ¼ 2.0 and 8.0,
5.1.5.2. N-[3-(Benzyloxy)phenyl]-2-bromo-N-methylacetamide (7b)
[28]. The product was prepared starting from 3-benzyloxyaniline
and 2-bromoethanoyl chloride (Scheme 2). Oil; ESI-MS (m/z):
334e336 (M þ H)þ.
Ar-H), 7.27e7.46 (m, 6H, Ar-H); 13C NMR (CDCl3):
d
¼ 172.6, 159.6,
145.0, 136.3, 130.6, 128.7, 128.2, 127.5, 119.6, 114.3, 114.0, 70.2, 37.3,
33.3, 33.0, 32.3, 24.1; MS (EI, 70 eV) m/z: 375e377 (Mþ), 91 (100).
5.1.5.3. 2-Bromo-N-methyl-N-(3-nitrophenyl)acetamide (7c) [27].
The product was prepared starting from 3-nitro-N-methylaniline
and 2-bromoethanoyl chloride (Scheme 2). Oil; ESI-MS (m/z): 272e
274 (M þ H)þ.
5.1.5.15. N-[3-(Benzyloxy)phenyl]-6-bromo-N-methylhexanamide
(15c). The product was prepared starting from 3-benzyloxy-N-
methylaniline [17] and 3-bromohexanoyl chloride (Scheme 4). Oil;
MS (EI, 70 eV) m/z: 389e391 (Mþ), 91 (100).
5.1.5.4. 3-Bromo-N-(3-methoxyphenyl)-N-methylpropanamide
(12a). The product was prepared starting from 3-methoxy-N-
methylaniline and 3-bromopropanoyl chloride (Scheme 3). Oil; ESI-
MS (m/z): 272e274 (M þ H)þ.
5.1.5.16. N-[3-(Benzyloxy)phenyl]-6-bromohexanamide
(15d).
The product was prepared starting from 3-benzyloxyaniline and 3-
bromohexanoyl chloride (Scheme 4). Oil; ESI-MS (m/z): 375e377
(M þ H)þ.

22
G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
5.1.6. General procedure for
alkanamides 8deg, 13bek and 16aeh
The suitable N-substituted-piperazine (0.6 mmol) and TEA
(0.83 mL) were added to a solution of the appropriate crude
u
-(4-substituted-piperazin-1-yl)
4H, piperazine), 2.76 (apt, 2H, J ¼ 7.0, COCH2CH2N), 3.16 (m, 4H,
piperazine), 3.28 (s, 3H, NCH3), 6.90 (m, 3H, Ar-H), 7.10e7.45 (m,
6H, Ar-H); ESI-MS (m/z): 358 (M þ H)þ.
u-
bromoalkanamide (from 0.5 mmol of substituted-aniline) in DMF
(0.14 mL) and the resulting mixture was stirred at room tempera-
ture for 1e16 h, monitoring the progress of the reaction by TLC on
silica gel. The reaction mixture was poured into water, and
extracted with EtOAc. After drying over Na2SO4, the combined
organic phases were concentrated in vacuo and the resulting crude
product was purified by column flash chromatography on silica gel
(EtOAc or cyclohexane/EtOAc, 1:1 for 8f as eluant) and/or
crystallization.
5.1.6.8. N-(3-Fluorophenyl)-N-methyl-3-(4-phenylpiperazin-1-yl)
propanamide (13e). The product was prepared starting from 12e
and N-phenylpiperazine (Scheme 3). Amorphous solid (128 mg,
75%); 1H NMR (CDCl3):
d
¼ 2.36 (apt, 2H, J ¼ 7.0, CH2CO), 2.53 (m,
4H, piperazine), 2.76 (apt, 2H, J ¼ 7.0, COCH2CH2N), 3.16 (m, 4H,
piperazine), 3.29 (s, 3H, NCH3), 6.82e7.47 (m, 9H, Ar-H); ESI-MS (m/
z): 342 (M þ H)þ.
5.1.6.9. N-Methyl-N-(3-nitrophenyl)-3-(4-phenylpiperazin-1-yl)
propanamide (13f). The product was prepared starting from 12f
and N-phenylpiperazine (Scheme 3). Yellow amorphous solid
5.1.6.1. N-(3-Methoxyphenyl)-N-methyl-2-(4-phenylpiperazin-1-yl)
acetamide (8d). The product was prepared starting from 7a and N-
phenylpiperazine (Scheme 2). Oil (154 mg, 91%); 1H NMR (CDCl3):
(147 mg, 80%); 1H NMR (CDCl3):
d
¼ 2.38 (apt, 2H, J ¼ 7.0, CH2CO),
2.58 (m, 4H, piperazine), 2.81 (apt, 2H, J ¼ 7.0, COCH2CH2N), 3.18
(m, 4H, piperazine), 3.36 (s, 3H, NCH3), 6.88 (m, 3H, Ar-H), 7.25 (m,
2H, Ar-H), 7.64 (m, 2H, Ar-H), 8.13 (m, 1H, Ar-H), 8.23 (m, 1H, Ar-H);
ESI-MS (m/z): 369 (M þ H)þ.
d
¼ 2.67 (m, 4H, piperazine), 3.05 (s, 2H, CH2CO), 3.21 (m, 4H,
piperazine), 3.29 (s, 3H, NCH3), 3.85 (s, 3H, OCH3), 6.86 (m, 6H, Ar-
H), 7.30 (m, 3H, Ar-H); MS (EI, 70 eV): m/z 339 (Mþ), 175 (100).
5.1.6.2. 2-(4-Benzylpiperazin-1-yl)-N-(3-methoxyphenyl)-N-methyl-
acetamide (8e). The product was prepared starting from 7a and N-
benzylpiperazine (Scheme 2). Oil (116 mg, 66%); 1H NMR (CDCl3):
5.1.6.10. Ethyl 3-[3-(4-phenylpiperazin-1-yl)propanamido]benzoate
(13g). The product was prepared starting from 12g and N-phe-
nylpiperazine (Scheme 3). White solid (86 mg, 45%); mp 127e
d
¼ 2.51 (m, 8H, piperazine), 2.96 (s, 2H, CH2CO), 3.26 (s, 3H, NCH3),
128 ꢀC (EtOAc) 1H NMR (CDCl3):
d
¼ 1.30 (t, 3H, J ¼ 7.0, CH2CH3),
3.52 (s, 2H, CH2Ph), 3.83 (s, 3H, OCH3), 6.81 (m, 3H, Ar-H), 7.32 (m,
2.60 (m, 2H, CH2CO), 2.79 (m, 6H, piperazine and COCH2CH2N), 3.31
(m, 4H, piperazine), 4.31 (q, 2H, J ¼ 7.0, CH2CH3), 6.93 (m, 3H, Ar-H),
7.25e7.41 (m, 3H, Ar-H), 7.74 (m, 1H, Ar-H), 7.86 (m, 1H, Ar-H), 8.04
(m, 1H, Ar-H), 11.15 (brs, 1H, NH); ESI-MS (m/z): 382 (M þ H)þ.
6H, Ar-H); MS (EI, 70 eV): m/z 353 (Mþ), 91 (100).
5.1.6.3. N-[3-(Benzyloxy)phenyl]-2-(4-phenylpiperazin-1-yl)acet-
amide (8f). The product was prepared starting from 7b and N-
phenylpiperazine (Scheme 2). White solid (160 mg, 80%); mp 119e
5.1.6.11. N-(3-Methoxyphenyl)-3-(4-phenylpiperazin-1-yl)prop-
anamide (13h). The product was prepared starting from 12h and N-
phenylpiperazine (Scheme 3). Amorphous solid (151 mg, 89%); 1H
120 ꢀC (EtOAc/petroleum ether); 1H NMR (CDCl3):
d
¼ 2.82 (m, 4H,
piperazine), 3.24 (s, 2H, CH2CO), 3.29 (m, 4H, piperazine), 5.09 (s,
2H, CH2Ph), 6.76 (dd, 1H, J ¼ 2.0 and 8.0, Ar-H), 6.98 (m, 4H, Ar-H),
7.25e7.48 (m, 9H, Ar-H), 9.19 (brs, 1H, NH); ESI-MS (m/z): 402
(M þ H)þ.
NMR (CDCl3):
d
¼ 2.59 (m, 2H, CH2CO), 2.80 (m, 6H, piperazine and
COCH2CH2N), 3.33 (m, 4H, piperazine), 3.80 (s, 3H, CH3), 6.64 (dd,
1H, J ¼ 2.0 and 8.0, Ar-H), 6.95 (m, 4H, Ar-H), 7.16e7.43 (m, 4H, Ar-
H), 10.95 (brs, 1H, NH); ESI-MS (m/z): 340 (M þ H)þ.
5.1.6.4. N-Methyl-N-(3-nitrophenyl)-2-(4-phenylpiperazin-1-yl)
acetamide (8g). The product was prepared starting from 7c and N-
phenylpiperazine (Scheme 2). Yellow oil (136 mg, 77%); 1H NMR
5.1.6.12. N-[3-(Benzyloxy)phenyl]-N-methyl-3-(4-phenylpiperazin-
1-yl)propanamide (13i). The product was prepared starting from
12i and N-phenylpiperazine (Scheme 3). Oil (131 mg, 61%); 1H NMR
(CDCl3):
d
¼ 2.71 (m, 4H, piperazine), 3.14 (s, 2H, CH2CO), 3.20 (m,
4H, piperazine), 3.31 (s, 3H, NCH3), 6.87 (m, 3H, Ar-H), 7.28 (m, 2H,
Ar-H), 7.62 (m, 2H, Ar-H), 8.14 (m, 1H, Ar-H), 8.24 (m, 1H, Ar-H); ESI-
MS (m/z): 355 (M þ H)þ.
(CDCl3):
d
¼ 2.35 (apt, 2H, J ¼ 7.0, CH2CO), 2.52 (m, 4H, piperazine),
2.76 (apt, 2H, J ¼ 7.0, COCH2CH2N), 3.16 (m, 4H, piperazine), 3.27 (s,
3H, NCH3), 5.09 (s, 2H, CH2Ph), 6.81e6.99 (m, 6H, Ar-H), 7.22e7.43
(m, 8H, Ar-H); MS (EI, 70 eV): m/z 429 (Mþ), 91 (100).
5.1.6.5. N-Methyl-3-(4-phenylpiperazin-1-yl)-N-m-tolylpropana-
mide (13b). The product was prepared starting from 12b and N-
phenylpiperazine (Scheme 3). Amorphous solid (106 mg, 63%); 1H
5.1.6.13. N-(3-Methoxyphenyl)-N-methyl-3-[4-(naphthalen-1-yl)
piperazin-1-yl]propanamide (13j). The product was prepared
starting from 12a and 1-(naphthalen-1-yl)piperazine (Scheme 3).
NMR (CDCl3):
d
¼ 2.33 (apt, 2H, J ¼ 7.0, CH2CO), 2.39 (s, 3H, Ar-CH3),
2.51 (m, 4H, piperazine), 2.76 (apt, 2H, J ¼ 7.0, COCH2CH2N), 3.15 (m,
4H, piperazine), 3.27 (s, 3H, NCH3), 6.82e7.35 (m, 9H, Ar-H); ESI-MS
(m/z): 338 (M þ H)þ.
Amorphous solid (103 mg, 51%); 1H NMR (CDCl3):
d
¼ 2.29 (apt, 2H,
J ¼ 7.0, CH2CO), 2.61 (m, 4H, piperazine), 2.70 (apt, 2H, J ¼ 7.0,
COCH2CH2N), 3.10 (m, 4H, piperazine), 3.24 (s, 3H, NCH3), 3.80 (s,
3H, OCH3), 6.82 (m, 3H, Ar-H), 7.09 (m, 1H, Ar-H), 7.30e7.61 (m, 5H,
Ar-H), 7.85 (m, 1H, Ar-H), 8.21 (m, 1H, Ar-H); ESI-MS (m/z): 404
(M þ H)þ.
5.1.6.6. N-(3-Bromophenyl)-N-methyl-3-(4-phenylpiperazin-1-yl)
propanamide (13c). The product was prepared starting from 12c
and N-phenylpiperazine (Scheme 3). Amorphous solid (140 mg,
70%); 1H NMR (CDCl3):
d
¼ 2.34 (apt, 2H, J ¼ 7.0, CH2CO), 2.52 (m,
4H, piperazine), 2.75 (apt, 2H, J ¼ 7.0, COCH2CH2N), 3.16 (m, 4H,
piperazine), 3.29 (s, 3H, NCH3), 6.93e7.38 (m, 9H, Ar-H); ESI-MS (m/
z): 402e404 (M þ H)þ.
5.1.6.14. N-(3-Methoxyphenyl)-N-methyl-3-[4-(naphthalen-2-yl)
piperazin-1-yl]propanamide (13k). The product was prepared
starting from 12a and 1-(naphthalen-2-yl)piperazine (Scheme 3).
Oil (95 mg, 47%); 1H NMR (CDCl3):
d
¼ 2.34 (apt, 2H, J ¼ 7.0, CH2CO),
5.1.6.7. N-(3-Chlorophenyl)-N-methyl-3-(4-phenylpiperazin-1-yl)
propanamide (13d). The product was prepared starting from 12d
and N-phenylpiperazine (Scheme 3). Amorphous solid (144 mg,
2.58 (m, 4H, piperazine), 2.79 (apt, 2H, J ¼ 7.0, COCH2CH2N), 3.27
(m, 4H, piperazine), 3.28 (s, 3H, NCH3), 3.85 (s, 3H, OCH3), 6.75e
6.93 (m, 3H, Ar-H), 7.08 (m, 1H, Ar-H), 7.22e7.44 (m, 4H, Ar-H), 7.70
(m, 3H, Ar-H); ESI-MS (m/z): 404 (M þ H)þ.
81%); 1H NMR (CDCl3):
d
¼ 2.34 (apt, 2H, J ¼ 7.0, CH2CO), 2.53 (m,

G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
23
5.1.6.15. N-[3-(Benzyloxy)phenyl]-N-methyl-4-(4-phenylpiperazin-1-
yl)butanamide (16a). The product was prepared starting from 15a
and N-phenylpiperazine (Scheme 4). Oil (51 mg, 23%); 1H NMR
piperazine), 3.19 (s, 3H, CH3), 3.31 (m, 4H, piperazine), 5.01 (s, 2H,
CH2Ph), 6.76 (m, 2H, Ar-H), 6.86e7.08 (m, 4H, Ar-H), 7.19e7.40 (m,
7H, Ar-H). ESI-MS: m/z 540 (M þ H)þ.
(CDCl3):
d
¼ 1.81 (m, 2H, CH2CH2CH2), 2.14 (apt, 2H, J ¼ 7.0, CH2CO),
2.32 (apt, 2H, J ¼ 7.0, COCH2CH2CH2N), 2.55 (m, 4H, piperazine),
3.16 (m, 4H, piperazine), 3.26 (s, 3H, NCH3), 5.08 (s, 2H, CH2Ph),
6.79e6.99 (m, 6H, Ar-H), 7.26e7.43 (m, 8H, Ar-H); MS (EI, 70 eV): m/
z 443 (Mþ), 91 (100).
5.1.6.22. N-[3-(Benzyloxy)phenyl]-6-[4-(7-chloroquinolin-4-yl)
piperazin-1-yl]-N-methylhexanamide (16h). The product was pre-
pared starting from 15c and 7-chloro-4-(piperazin-1-yl)quinoline
(Scheme 4). Oil (81 mg, 29%); 1H NMR (CDCl3):
d
¼ 1.27 (m, 2H,
CH2CH2CH2CH2CH2), 1.58 (m, 2H, CH2CH2CH2), 1.97 (m, 2H,
CH2CH2CH2), 2.14 (m, 2H, CH2CO), 3.01e3.68 (m, 6H), 4.18 (m, 2H),
4.42 (m, 2H), 3.18 (s, 3H, CH3), 5.01 (s, 2H, CH2Ph), 6.75 (m, 2H. Ar-
H), 6.92 (m, 1H, Ar-H), 7.16 (m, 1H, Ar-H), 7.18e7.41 (m, 6H, Ar-H),
7.48 (m, 1H, Ar-H), 7.88 (m, 1H, Ar-H), 8.22 (m, 1H, Ar-H), 8.32 (m,
1H Ar-H). ESI-MS: m/z 557 (M þ H)þ.
5.1.6.16. N-[3-(Benzyloxy)phenyl]-N-methyl-5-(4-phenylpiperazin-1-
yl)pentanamide (16b). The product was prepared starting from 15b
and N-phenylpiperazine (Scheme 4). Oil (94 mg, 41%); 1H NMR
(CDCl3):
d
¼ 1.54 (m, 4H, CH2CH2CH2CH2), 2.13 (apt, 2H, J ¼ 7.0,
CH2CO), 2.34 (t, 2H, J ¼ 7.5, CH2-piperazine), 2.57 (m, 4H, pipera-
zine), 3.19 (m, 4H, piperazine), 3.25 (s, 3H, CH3), 5.08 (s, 2H, CH2Ph),
6.78e6.99 (m, 6H, Ar-H), 7.23e7.46 (m, 8H, Ar-H); 13C NMR (CDCl3):
5.1.7. Reduction of the alkanamido derivatives with LiAlH4. General
procedure for compounds 9def and 17aeh
d
¼ 172.9, 159.6, 151.3, 145.3, 136.4, 130.4, 129.1, 128.7, 128.2, 127.5,
119.8, 119.6, 116.0, 114.2, 114.0, 70.2, 58.3, 53.2, 49.0, 37.2, 33.8, 26.4,
LiAlH4 (0.59 g, 15.5 mmol) was added portionwise to an ice-
cooled solution of the suitable alkanamido derivative 8def, 16ae
h (2.5 mmol) in dry THF (30 mL) under a N2 atmosphere. Once the
addition was completed, the reaction mixture was refluxed for 2 h.
After cooling to 0 ꢀC, water was added dropwise to destroy the
excess hydride and the resulting mixture was filtered on Celite. The
filtrate was concentrated in vacuo and partitioned between water
and EtOAc. The combined organic layers were washed with brine,
dried (Na2SO4) and concentrated under reduced pressure to afford
a crude residue which was purified by flash chromatography on
silica gel.
23.5; MS (EI, 70 eV): m/z 457 (Mþ), 91 (100).
5.1.6.17. N-[3-(Benzyloxy)phenyl]-N-methyl-6-(4-phenylpiperazin-1-
yl)hexanamide (16c). The product was prepared starting from 15c
and N-phenylpiperazine (Scheme 4). Oil (94 mg, 40%); 1H NMR
(CDCl3):
d
¼ 1.27 (m, 2H, CH2CH2CH2CH2CH2), 1.57 (m, 4H,
CH2CH2CH2CH2CH2), 2.09 (apt, 2H, J ¼ 7.5, CH2CO), 2.42 (apt, 2H,
J ¼ 7.5, CH2N), 2.66 (m, 4H, piperazine), 3.24 (m, 4H, piperazine),
3.25 (s, 3H, CH3), 5.09 (s, 2H, CH2Ph), 6.77e6.99 (m, 6H, Ar-H), 7.24e
7.45 (m, 8H, Ar-H); MS (EI, 70 eV): m/z 471 (Mþ), 91 (100).
5.1.6.18. N-[3-(Benzyloxy)phenyl]-6-(4-phenylpiperazin-1-yl)hex-
anamide (16d). The product was prepared starting from 15d and N-
phenylpiperazine (Scheme 4). Oil (103 mg, 45%); 1H NMR (CDCl3):
5.1.7.1. 3-Methoxy-N-methyl-N-[2-(4-phenylpiperazin-1-yl)ethyl]an-
iline (9d). The product was prepared starting from 8d (Scheme 2).
Purification by flash chromatography on silica gel (EtOAc as eluant).
d
¼
1.28 (m, 2H, CH2CH2CH2CH2CH2), 1.61 (m, 4H,
Oil (292 mg, 36%); 1H NMR (CDCl3):
d
¼ 2.63 (apt, 2H, J ¼ 7.5,
CH2CH2CH2CH2CH2), 2.13 (apt, 2H, J ¼ 7.5, CH2CO), 2.43 (apt, 2H,
J ¼ 7.5, CH2-piperazine), 2.71 (m, 4H, piperazine), 3.21 (m, 4H,
piperazine), 5.08 (s, 2H, CH2-Ph), 6.73e6.98 (m, 5H, Ar-H), 7.21e
7.55 (m, 9H, Ar-H), 9.03 (brs, 1H, NH); ESI-MS: m/z 458 (M þ H)þ.
CH2CH2NCH3), 2.70 (m, 4H, piperazine), 2.98 (s, 3H, NCH3), 3.23 (m,
4H, piperazine), 3.54 (apt, 2H, J ¼ 7.5, CH2NCH3), 3.82 (s, 3H, OCH3),
6.35 (m, 3H, Ar-H), 6.91 (m, 3H, Ar-H), 7.23 (m, 3H, Ar-H); 13C NMR
(CDCl3):
d
¼ 160.9, 151.3, 150.6, 130.0, 129.1, 119.7, 116.0, 105.3, 101.0,
98.7, 55.1, 54.9, 53.7, 50.5, 49.1, 38.7; MS (EI, 70 eV): m/z 325 (Mþ),
5.1.6.19. N-[3-(Benzyloxy)phenyl]-N-methyl-6-(4-phenylpiperidin-1-
yl)hexanamide (16e). The product was prepared starting from 15e
and 4-phenylpiperidine (Scheme 4). Oil (94 mg, 40%); 1H NMR
150 (100).
5.1.7.2. N-[2-(4-Benzylpiperazin-1-yl)ethyl]-3-methoxy-N-methyl-
aniline (9e). The product was prepared starting from 8e (Scheme
2). Purification by flash chromatography on silica gel (EtOAc/
cyclohexane 8:2 as eluant). Oil (246 mg, 29%); 1H NMR (CDCl3):
(CDCl3):
d
¼ 1.19 (m, 2H, CH2CH2CH2CH2CH2), 1.58 (m, 4H,
CH2CH2CH2CH2CH2), 1.81 (m, 2H, piperidine), 2.01e2.25 (m. 6H,
CH2N, CH2CO and piperidine), 2.45 (m, 1H, CH), 2.60 (m, 4H,
piperidine), 3.20 (s, 3H, CH3), 5.01 (s, 2H, CH2Ph), 6.71 (m, 2H, Ar-H),
6.92 (m, 1H, Ar-H), 7.16e7.42 (m, 11H, Ar-H); ESI-MS: m/z 471
(M þ H)þ.
d
¼ 2.53 (m, 10H, piperazine and CH2CH2NCH3), 2.93 (s, 3H, NCH3),
3.46 (m, 2H, CH2NCH3), 3.51 (s, 2H, CH2Ph), 3.78 (s, 3H, OCH3), 6.29
(m, 3H, Ar-H), 7.08e7.33 (m, 6H, Ar-H); MS (EI, 70 eV): m/z 339
(Mþ), 91 (100).
5.1.6.20. N-[3-(Benzyloxy)phenyl]-N-methyl-6-[4-(naphthalen-1-yl)
piperazin-1-yl]hexanamide (16f). The product was prepared start-
ing from 15c and 1-(naphthalen-1-yl)piperazine (Scheme 4). Oil
5.1.7.3. 3-(Benzyloxy)-N-[2-(4-phenylpiperazin-1-yl)ethyl]aniline
(9f). The product was prepared starting from 8f (Scheme 2). Puri-
fication by flash chromatography on silica gel (EtOAc/cyclohexane
(99 mg, 38%). 1H NMR (CDCl3):
d
¼
1.32 (m, 2H,
CH2CH2CH2CH2CH2), 1.61 (m, 2H, CH2CH2CH2), 1.78 (m, 2H,
CH2CH2CH2), 2.13 (apt, 2H, J ¼ 7.0, CH2CO), 2.79 (m, 2H, CH2-
piperazine), 2.91e3.51 (m, 8H, piperazine), 3.22 (s, 3H, CH3), 5.09 (s,
2H, CH2Ph), 6.80 (m, 2H, Ar-H), 6.99 (m, 1H, Ar-H), 6.38 (m, 1H, Ar-
H), 7.28e7.55 (m, 9H, Ar-H), 7.61 (m, 1H, Ar-H), 7.84 (m, 1H, Ar-H),
8.13 (m, 1H, Ar-H); ESI-MS: m/z 522 (M þ H)þ.
4:6 as eluant). Oil (822 mg, 85%); 1H NMR (CDCl3):
d
¼ 2.69 (m, 6H,
piperazine and CH2CH2NH), 3.22 (m, 6H, piperazine and CH2NH),
4.38 (brs, 1H, NH), 5.06 (s, 2H, CH2Ph), 6.35 (m, 3H, Ar-H), 6.93 (m,
3H, Ar-H), 7.07e7.45 (m, 8H, Ar-H); MS (EI, 70 eV): m/z 387 (Mþ), 91
(100).
5.1.7.4. 3-(Benzyloxy)-N-methyl-N-[4-(4-phenylpiperazin-1-yl)
butyl]aniline (17a). The product was prepared starting from 16a
(Scheme 4). Purification by flash chromatography on silica gel
(EtOAc/cyclohexane 7:3 as eluant). Amorphous solid (257 mg, 24%);
5.1.6.21. N-[3-(Benzyloxy)phenyl]-N-methyl-6-{4-[3-(tri-
fluoromethyl)phenyl]piperazin-1-yl}hexanamide (16g). The product
was prepared starting from 15c and 1-[3-(trifluoromethyl)phenyl]
piperazine (Scheme 4). Oil (124 mg, 46%); 1H NMR (CDCl3):
(m, 2H, CH2CH2CH2CH2CH2), 1.56 (m, 4H, CH2CH2CH2CH2CH2), 2.02
d
¼ 1.19
1H NMR (CDCl3):
CH2-piperazine), 2.65 (m, 4H, piperazine), 2.93 (s, 3H, CH3), 3.26 (m,
4H, piperazine), 3.34 (m, 2H, CH2NCH3), 5.06 (s, 2H, CH2Ar), 6.35
d
¼ 1.63 (m, 4H, CH2CH2CH2CH2), 2.46 (m, 2H,
(apt, 2H, J ¼ 7.0, CH2CO), 2.55 (m, 2H, CH2-piperazine), 2.68 (m, 4H,

24
G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
(m, 3H, Ar-H), 6.92 (m, 3H, Ar-H), 7.10e7.48 (m, 8H, Ar-H); MS (EI,
70 eV): m/z 429 (Mþ), 91 (100).
2H, Ar-H), 6.83e7.09 (m, 4H, Ar-H), 7.15e7.42 (m, 7H, Ar-H). ESI-MS:
m/z 526 (M þ H)þ.
5.1.7.5. 3-(Benzyloxy)-N-methyl-N-[5-(4-phenylpiperazin-1-yl)pen-
tyl]aniline (17b). The product was prepared starting from 16b
(Scheme 4). Purification by flash chromatography on silica gel
(EtOAc/cyclohexane 7:3 as eluant). Oil (764 mg, 69%); 1H NMR
5.1.7.11. 3-(Benzyloxy)-N-{6-[4-(7-chloroquinolin-4-yl)piperazin-1-
yl]hexyl}-N-methylaniline (17h). The product was prepared starting
from 16h (Scheme 4). Purification by flash chromatography on
silica gel (EtOAc as eluant). Oil (528 mg, 39%); 1H NMR (CD3OD):
(CDCl3):
d
¼ 1.36 (m, 2H, CH2CH2CH2CH2CH2), 1.61 (m, 4H,
d
¼ 1.42 (m, 4H, CH2CH2CH2CH2CH2CH2), 1.58 (m, 2H, CH2CH2CH2),
CH2CH2CH2CH2CH2), 2.44 (apt, 2H, J ¼ 8.0, CH2-piperazine), 2.65
(m, 4H, piperazine), 2.92 (s, 3H, CH3), 3.25 (m, 4H, piperazine), 3.31
(t, 2H, J ¼ 7.5, CH2NCH3), 5.07 (s, 2H, CH2Ar), 6.35 (m, 3H, Ar-H), 6.91
1.81 (m, 2H, CH2CH2CH2), 3.20 (s, 3H, CH3), 3.23 (m, 2H, CH2NCH3),
3.49 (m, 4H, piperazine), 3.79 (m, 2H), 3.92 (m, 2H), 4.38 (m, 2H),
5.18 (s, 2H, CH2Ph), 7.10 (m, 3H, Ar-H), 7.30e7.51 (m, 7H, Ar-H), 7.78
(m,1H, Ar-H), 8.05 (m,1H, Ar-H), 8.24 (m,1H, Ar-H), 8.78 (m, 1H, Ar-
H). ESI-MS: m/z 543 (M þ H)þ.
(m, 3H, Ar-H), 7.11e7.49 (m, 8H, Ar-H); 13C NMR (CDCl3):
d
¼ 160.1,
151.2, 150.7, 137.4, 129.8, 129.1, 128.6, 127.9, 127.6, 119.8, 116.1, 105.5,
101.4, 99.6, 69.9, 58.6, 53.2, 52.7, 49.0, 38.4, 26.7, 26.6, 25.1; MS (EI,
70 eV): m/z 443 (Mþ), 91 (100).
5.1.8. Reduction of the alkanamido derivatives with BH3. General
procedure for compounds 9g, 14bek
5.1.7.6. 3-(Benzyloxy)-N-methyl-N-[6-(4-phenylpiperazin-1-yl)hex-
yl]aniline (17c). The product was prepared starting from 16c
(Scheme 4). Purification by flash chromatography on silica gel
(EtOAc/cyclohexane 7:3 as eluant). Oil (320 mg, 28%); 1H NMR
A 1 M solution of BH3 in THF (3 equiv) was added dropwise to an
ice-cooled solution of the suitable alkanamido derivative 8g, 13bek
(1 mmol) in dry THF (7 mL) under a N2 atmosphere. Once the
addition was completed, the reaction mixture was stirred at room
temperature for 16 h; 6 N HCl (0.7 mL) [a 1.25 N solution of HCl in
EtOH for 14g] was added and the resulting mixture was heated to
reflux for 1 h. After cooling to room temperature the reaction
mixture was made alkaline with 1 N NaOH (2.7 mL) and then
extracted with EtOAc. The combined organic layers were washed
with brine, dried (Na2SO4) and concentrated to afford a crude
residue which was purified by flash chromatography on silica gel
and/or crystallization.
(CDCl3):
d
¼ 1.35 (m, 4H, CH2CH2CH2CH2CH2CH2), 1.58 (m, 4H,
CH2CH2CH2CH2CH2CH2), 2.45 (m, 2H, CH2-piperazine), 2.66 (m, 4H,
piperazine), 2.92 (s, 3H, CH3), 3.29 (m, 6H, piperazine and
CH2NCH3), 5.06 (s, 2H, CH2Ph), 6.34 (m, 3H, Ar-H), 6.86 (m, 3H, Ar-
H), 7.10e7.48 (m, 8H, Ar-H); MS (EI, 70 eV): m/z 457 (Mþ), 91 (100).
5.1.7.7. 3-(Benzyloxy)-N-[6-(4-phenylpiperazin-1-yl)hexyl]aniline
(17d). The product was prepared starting from 16d (Scheme 4).
Purification by flash chromatography on silica gel (EtOAc as eluant).
Oil (443 mg, 40%); 1H NMR (CDCl3):
d
¼
1.38 (m, 4H,
5.1.8.1. N-Methyl-3-nitro-N-[2-(4-phenylpiperazin-1-yl)ethyl]anili-
ne$HCl (9g). The product was prepared starting from 8g (Scheme
2). Purification by flash chromatography on silica gel (EtOAc/
cyclohexane 1:1 as eluant); the product was then dissolved in a
solution of HCl in MeOH and the solvent was removed by distilla-
tion under vacuum obtaining the final product as white solid
CH2CH2CH2CH2CH2CH2), 1.59 (m, 4H, CH2CH2CH2CH2CH2CH2), 2.50
(m, 2H, CH2-piperazine), 2.71 (m, 4H, piperazine), 3.11 (m, 2H,
CH2NH), 3.30 (m, 4H, piperazine), 3.65 (brs, 1H, NH), 5.05 (s, 2H,
CH2Ph), 6.21 (m, 3H, Ar-H), 6.83e7.61 (m, 11H, Ar-H); ESI-MS: m/z
444 (M þ H)þ.
(207 mg, 55%); 1H NMR (DMSO-d6):
d
¼ 3.01 (s, 3H, CH3), 3.20 (m,
5.1.7.8. 3-(Benzyloxy)-N-methyl-N-[6-(4-phenylpiperidin-1-yl)hexyl]
aniline (17e). The product was prepared starting from 16e (Scheme
4). Purification by flash chromatography on silica gel (EtOAc/
cyclohexane 8:2 as eluant). Oil (513 mg, 45%); 1H NMR (CDCl3):
4H, piperazine), 3.31 (m, 2H, CH2-piperazine), 3.61 (m, 2H, piper-
azine), 3.82 (m, 2H, piperazine), 3.96 (m, 2H, CH2NCH3) 6.86 (m, 1H,
Ar-H), 7.02 (m, 2H, Ar-H), 7.25e7.34 (m, 3H, Ar-H), 7.43e7.52 (m,
3H, Ar-H), 11.50 (brs, 1H, NHþ); ESI-MS (m/z): 341 (M þ H)þ. Anal.
calcd for C19H25ClN4O2$0.15 H2O: C 60.12, H 6.72, N 14.76, found C
60.28, H 6.93, N 14.55.
d
¼
1.22 (m, 4H, CH2CH2CH2CH2CH2CH2), 1.57 (m, 4H,
CH2CH2CH2CH2CH2CH2), 1.83 (m, 2H, piperidine), 1.88e2.57 (m, 9H,
piperidine, and CH2N), 2.95 (s, 3H, CH3), 3.24 (m, 2H, CH2NCH3),
5.02 (s, 2H, CH2Ph), 6.70 (m, 2H, Ar-H), 6.95 (m, 1H, Ar-H), 7.13e7.45
(m, 11H, Ar-H); ESI-MS: m/z 457 (M þ H)þ.
5.1.8.2. N,3-Dimethyl-N-[3-(4-phenylpiperazin-1-yl)propyl]aniline
(14b). The product was prepared starting from 13b (Scheme 3).
Purification by flash chromatography on silica gel (EtOAc/cyclo-
5.1.7.9. 3-(Benzyloxy)-N-methyl-N-{6-[4-(naphthalen-1-yl)piper-
azin-1-yl]hexyl}aniline (17f). The product was prepared starting
from 16f (Scheme 4). Purification by flash chromatography on silica
gel (EtOAc/cyclohexane 7:3 as eluant). Oil (532 mg, 42%); 1H NMR
hexane 7:3 as eluant). Oil (145 mg, 45%); 1H NMR (CDCl3):
d
¼ 1.82
(m, 2H, CH2CH2CH2), 2.34 (s, 3H, Ar-CH3), 2.46 (t, 2H, J ¼ 7.5, CH2-
piperazine), 2.63 (m, 4H, piperazine), 2.95 (s, 3H, NCH3), 3.25 (m,
4H, piperazine), 3.41 (t, 2H, J ¼ 7.5, CH2NCH3), 6.57 (m, 3H, Ar-H),
(CDCl3):
d
¼ 1.36 (m, 4H, CH2CH2CH2CH2CH2CH2), 1.60 (m, 4H,
6.90 (m, 3H, Ar-H), 7.22 (m, 1H, Ar-H), 7.23e7.34 (m, 2H, Ar-H); 13
C
CH2CH2CH2CH2CH2CH2), 2.65 (m, 2H, CH2-piperazine), 2.71 (m, 4H,
piperazine), 2.90 (s, 3H, CH3), 3.29 (m, 6H, piperazine and
CH2NCH3), 5.08 (s, 2H, CH2Ph), 6.78 (m, 2H, Ar-H), 6.99 (m, 1H, Ar-
H), 7.13 (m, 1H, Ar-H), 7.25e7.59 (m, 9H, Ar-H), 7.63 (m, 1H, Ar-H),
7.82 (m, 1H, Ar-H), 8.15 (m, 1H, Ar-H); ESI-MS: m/z 508 (M þ H)þ.
NMR (CDCl3):
d
¼ 151.3, 149.5, 138.8, 129.1, 129.0, 119.7, 118.6, 117.0,
116.0, 113.0, 109.5, 55.8, 53.3, 50.7, 49.2, 38.2, 24.3, 22.0; ESI-MS (m/
z): 324 (M þ H)þ. Anal. calcd for C21H29N3: C 77.97, H 9.04, N 12.99,
found C 77.90, H 9.18, N 13.07.
5.1.8.3. 3-Bromo-N-methyl-N-[3-(4-phenylpiperazin-1-yl)propyl]an-
iline (14c). The product was prepared starting from 13c (Scheme 3).
Purification by flash chromatography on silica gel (EtOAc/cyclo-
5.1.7.10. 3-(Benzyloxy)-N-methyl-N-(6-{4-[3-(trifluoromethyl)
phenyl]piperazin-1-yl}hexyl)aniline (17g). The product was pre-
pared starting from 16g (Scheme 4). Purification by flash chroma-
tography on silica gel (EtOAc/cyclohexane 7:3 as eluant). Oil
hexane 3:7 as eluant). Oil (314 mg, 81%); 1H NMR (CDCl3):
d
¼ 1.80
(m, 2H, CH2CH2CH2), 2.43 (t, 2H, J ¼ 7.0, CH2-piperazine), 2.62 (m,
4H, piperazine), 2.94 (s, 3H, CH3), 3.25 (m, 4H, piperazine), 3.41 (t,
2H, J ¼ 7.0, CH2NCH3), 6.65 (dd, 1H, J ¼ 2.0 and 8.0, Ar-H), 6.78e7.00
(m, 5H, Ar-H), 7.07 (dd, 1H, J1 ¼ J2 ¼ 8.0, Ar-H), 7.27 (d, 1H, J ¼ 7.0,
(670 mg, 51%); 1H NMR (CDCl3):
d[1.37 (m, 4H,
CH2CH2CH2CH2CH2CH2), 1.59 (m, 4H, CH2CH2CH2CH2CH2CH2), 2.43
(m, 2H, CH2-piperazine), 2.69 (m, 4H, piperazine), 2.90 (s, 3H, CH3),
3.31 (m, 6H, piperazine and CH2NCH3), 5.02 (s, 2H, CH2Ph), 6.76 (m,
Ar-H), 7.31 (d, 1H, J ¼ 8.0, Ar-H); 13C NMR (CDCl3):
d
¼ 151.3, 150.6,

G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
25
130.3, 129.1, 123.5, 119.7, 118.6, 116.1, 114.8, 110.5, 55.4, 53.3, 50.3,
piperazine), 2.62 (m, 4H, piperazine), 2.93 (s, 3H, CH3), 3.23 (m, 4H,
piperazine), 3.39 (apt, 2H, J ¼ 7.0, CH2NCH3), 5.06 (s, 2H, CH2Ph),
6.37 (m, 3H, Ar-H), 6.90 (m, 3H, Ar-H), 7.11e7.49 (m, 8H, Ar-H); MS
(EI, 70 eV): m/z 415 (Mþ), 91 (100).
49.2, 38.1, 24.1. ESI-MS (m/z): 388e390 (M þ H)þ. Anal. calcd for
C
20H26BrN3: C 61.86, H 6.75, N 10.82, found C 61.69, H 6.69, N 12.63.
5.1.8.4. 3-Chloro-N-methyl-N-[3-(4-phenylpiperazin-1-yl)propyl]an-
iline (14d). The product was prepared starting from 13d (Scheme
3). Purification by flash chromatography on silica gel (EtOAc/
cyclohexane 3:7 as eluant). Oil (250 mg, 73%); 1H NMR (CDCl3):
5.1.8.10. 3-Methoxy-N-methyl-N-{3-[4-(naphthalen-1-yl)piperazin-
1-yl]propyl}aniline (14j). The product was prepared starting from
13j (Scheme 3). Purification by flash chromatography on silica gel
(EtOAc/cyclohexane 1:1 as eluant). Oil (323 mg, 83%);1H NMR
d
¼ 1.82 (m, 2H, CH2CH2CH2), 2.45 (t, 2H, J ¼ 7.0, CH2-piperazine),
2.64 (m, 4H, piperazine), 2.96 (s, 3H, CH3), 3.28 (m, 4H, piperazine),
3.44 (t, 2H, J ¼ 7.0, CH2NCH3), 6.70 (m, 3H, Ar-H), 6.94 (m, 3H, Ar-H),
7.16 (t, 1H, Ar-H), 7.28e7.37 (m, 2H, Ar-H); 13C NMR (CDCl3):
(CDCl3):
d
¼ 1.86 (m, 2H, CH2CH2CH2), 2.52 (apt, 2H, J ¼ 7.0, CH2-
piperazine), 2.77 (m, 4H, piperazine), 2.97 (s, 3H, NCH3), 3.19 (m,
4H, piperazine), 3.44 (apt, 2H, J ¼ 7.0, CH2NCH3), 3.82 (s, 3H, OCH3),
6.28 (m, 2H, Ar-H), 6.41 (m, 1H, Ar-H), 7.15 (m, 2H, Ar-H), 7.38e7.59
(m, 4H, Ar-H), 7.84 (m, 1H, Ar-H), 8.22 (m, 1H, Ar-H); ESI-MS (m/z):
390 (M þ H)þ.
d
¼ 151.4, 150.5, 135.1, 130.1, 129.2, 119.7, 116.1, 115.7, 111.9, 110.1,
55.5, 53.3, 50.3, 49.2, 38.2, 24.2. ESI-MS (m/z): 344 (M þ H)þ. Anal.
calcd for C20H26ClN3: C 69.85, H 7.62, N 12.22, found C 70.01, H 7.78,
N 12.35.
5.1.8.11. 3-Methoxy-N-methyl-N-{3-[4-(naphthalen-2-yl)piperazin-
1-yl]propyl}aniline (14k). The product was prepared starting from
13k (Scheme 3). Purification by flash chromatography on silica gel
(EtOAc/cyclohexane 1:1 as eluant). Oil (222 mg, 57%); 1H NMR
5.1.8.5. 3-Fluoro-N-methyl-N-[3-(4-phenylpiperazin-1-yl)propyl]an-
iline (14e). The product was prepared starting from 13e (Scheme 3).
Purification by flash chromatography on silica gel (EtOAc/cyclo-
hexane 3:7 as eluant). White solid (245 mg, 75%); mp: 64e65 ꢀC
(Et2O/petroleum ether); 1H NMR (CDCl3):
d
¼ 1.79 (m, 2H,
(CDCl3):
d
¼ 1.84 (m, 2H, CH2CH2CH2), 2.46 (apt, 2H, J ¼ 7.0, CH2-
piperazine), 2.67 (m, 4H, piperazine), 2.96 (s, 3H, NCH3), 3.34 (m,
4H, piperazine), 3.42 (apt, 2H, J ¼ 7.0, CH2NCH3), 3.82 (s, 3H, OCH3),
6.29 (m, 2H, Ar-H), 6.40 (m, 1H, Ar-H), 7.16 (m, 2H, Ar-H), 7.25e7.45
(m, 3H, Ar-H), 7.73 (m, 3H, Ar-H); ESI-MS (m/z): 390 (M þ H)þ.
CH2CH2CH2), 2.42 (t, 2H, J ¼ 7.0, CH2-piperazine), 2.61 (m, 4H,
piperazine), 2.94 (s, 3H, CH3), 3.23 (m, 4H, piperazine), 3.40 (t, 2H,
J ¼ 7.0, CH2NCH3), 6.31e6.53 (m, 3H, Ar-H), 6.84e7.32 (m, 6H, Ar-
H); 13C NMR (CDCl3):
d
¼ 164.2 (d, J ¼ 239.5), 151.3, 151.1 (d,
J ¼ 10.5), 130.1 (d, J ¼ 10.5), 129.1, 119.7, 116.0, 107.5 (d, J ¼ 2), 102.3
(d, J ¼ 21.5), 99.0 (d, J ¼ 26), 55.5, 53.3, 50.4, 49.2, 38.3, 24.2; ESI-MS
(m/z): 328 (M þ H)þ. Anal. calcd for C20H26FN3: C 73.36, H 8.00, N
12.83, found C 73.21, H 7.91, N 12.85.
5.1.9. O-Demethylation of methoxyanilino-derivatives with BBr3.
General procedure for compounds 10hei, 14len
A solution of BBr3 (1 M in CH2Cl2, 3 mL, 3 mmol) diluted with dry
CH2Cl2 (6 mL) was added dropwise to a solution of the suitable
methoxy derivative 9dee,14h,14jek (1 mmol) in dry CH2Cl2 (4 mL)
at 0 ꢀC, and the resulting mixture was stirred at room temperature
for 2 h. The reaction mixture was neutralized with a 2 N aqueous
solution of Na2CO3 and extracted with EtOAc. The organic phases
were combined, dried (Na2SO4) and concentrated under reduced
pressure, to give a crude product that was purified by flash chro-
matography on silica gel and/or crystallization.
5.1.8.6. N-Methyl-3-nitro-N-[3-(4-phenylpiperazin-1-yl)propyl]ani-
line (14f). The product was prepared starting from 13f (Scheme 3).
Purification by flash chromatography on silica gel (EtOAc as eluant).
Yellow oil (241 mg, 68%); 1H NMR (CDCl3):
d
¼ 1.86 (m, 2H,
CH2CH2CH2), 2.47 (t, 2H, J ¼ 7.5, CH2-piperazine), 2.65 (m, 4H,
piperazine), 3.03 (s, 3H, CH3), 3.27 (m, 4H, piperazine), 3.51 (t, 2H,
J ¼ 7.5, CH2NCH3), 6.95 (m, 4H, Ar-H), 7.31 (m, 3H, Ar-H), 7.51 (m,
2H, Ar-H); ESI-MS (m/z): 355 (M þ H)þ.
5.1.9.1. 3-{Methyl[2-(4-phenylpiperazin-1-yl)ethyl]amino}phenol
(10h). The product was prepared starting from 9d. Purification by
flash chromatography on silica gel (cyclohexane/EtOAc 1:1) and
subsequent crystallization. White solid (106 mg, 34%); mp: 155e
5.1.8.7. Ethyl
3-[3-(4-phenylpiperazin-1-yl)propylamino]benzoate
(14g). The product was prepared starting from 13g (Scheme 3).
Purification by flash chromatography on silica gel (EtOAc/cyclo-
hexane 1:1 as eluant). Amorphous solid (253 mg, 69%); 1H NMR
156 ꢀC (Et2O); 1H NMR (CDCl3):
d
¼ 2.63 (t, 2H, J ¼ 8.0,
(CDCl3):
d
¼ 1.37 (t, 3H, J ¼ 7.0, CH2CH3), 1.87 (m, 2H, CH2CH2CH2),
CH2CH2NCH3), 2.66 (m, 4H, piperazine), 2.95 (s, 3H, NCH3), 3.24 (m,
4H, piperazine), 3.53 (t, 2H, J ¼ 8.0, CH2NCH3), 5.51 (brs, 1H, OH),
6.24 (m, 3H, Ar-H), 6.90 (m, 3H, Ar-H), 7.08 (t,1H, Ar-H), 7.28 (m, 2H,
2.58 (t, 2H, J ¼ 7.0, CH2-piperazine), 2.66 (m, 4H, piperazine), 3.26
(m, 6H, piperazine and CH2NH), 4.35 (q, 2H, J ¼ 7.0, CH2CH3), 4.89
(brs, 1H, NH), 6.75e6.99 (m, 4H, Ar-H), 7.17e7.43 (m, 5H, Ar-H); ESI-
MS (m/z): 368 (M þ H)þ.
Ar-H); 13C NMR (CDCl3):
116.1, 104.7, 103.4, 99.2, 54.7, 53.6, 50.2, 49.0, 38.7; MS (EI, 70 eV):
m/z 311 (Mþ), 136 (100); Anal. calcd for C19H25N3O: C 73.28, H 8.09,
N 13.49, found C 73.52, H 8.33, N 13.20.
d
¼ 157.0, 151.1, 150.6, 130.2, 129.1, 119.9,
5.1.8.8. 3-Methoxy-N-[3-(4-phenylpiperazin-1-yl)propyl]aniline
(14h). The product was prepared starting from 13h (Scheme 3).
Purification by flash chromatography on silica gel (EtOAc as eluant).
Amorphous solid (292 mg, 90%); 1H NMR (CDCl3):
d
¼ 1.86 (m, 2H,
5.1.9.2. 3-{[2-(4-Benzylpiperazin-1-yl)ethyl]methylamino}phenol
(10i). The product was prepared starting from 9e. Purification by
flash chromatography on silica gel (cyclohexane/EtOAc 2:8) and
subsequent crystallization. White solid (159 mg, 49%); mp: 99e
CH2CH2CH2), 2.57 (t, 2H, J ¼ 7.0, CH2-piperazine), 2.66 (m, 4H,
piperazine), 3.24 (m, 6H, piperazine and CH2NH), 3.79 (s, 3H, CH3),
4.78 (brs, 1H, NH), 6.16e6.30 (m, 3H, Ar-H), 6.92 (m, 3H, Ar-H), 7.09
(dd, 1H, J1 ¼ J2 ¼ 8.0, Ar-H), 7.30 (m, 2H, Ar-H); ESI-MS (m/z): 326
(M þ H)þ.
100 ꢀC (Et2O/petroleum ether); 1H NMR (CDCl3):
piperazine and CH2CH2NCH3), 2.92 (s, 3H, NCH3), 3.47 (apt, 2H,
J ¼ 7.5, CH2NCH3), 3.54 (s, 2H, CH2-Ar), 6.15 (m, 2H, Ar-H), 6.27 (m,
1H, Ar-H), 7.06 (m, 1H, Ar-H), 7.22e7.34 (m, 5H, Ar-H); 13C NMR
d
¼ 2.56 (m, 10H,
5.1.8.9. 3-(Benzyloxy)-N-methyl-N-[3-(4-phenylpiperazin-1-yl)pro-
pyl]aniline (14i). The product was prepared starting from 13i
(Scheme 4). Purification by flash chromatography on silica gel
(EtOAc/cyclohexane, 7:3 as eluant). Oil (286 mg, 69%); 1H NMR
(CDCl3):
d
¼ 157.3,150.5,137.5,130.1, 129.4,128.3,127.2,104.3,103.7,
99.5, 63.0, 54.5, 53.3, 52.7, 50.0, 38.6; ESI-MS (m/z): 326 (M þ H)þ;
Anal. calcd for C20H27N3O: C 73.81, H 8.36, N 12.91, found C 73.98, H
8.44, N 12.95.
(CDCl3):
d
¼ 1.81 (m, 2H, CH2CH2CH2), 2.43 (apt, 2H, J ¼ 7.0, CH2-

26
G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
5.1.9.3. 3-[3-(4-Phenylpiperazin-1-yl)propylamino]phenol
(14l).
(5 mL), and the resulting mixture was stirred at room temperature
for 1 h. After removing the solvent by distillation at reduced
pressure, the residue was taken up in EtOAc, and washed with 1 N
NaOH and with brine. After drying over Na2SO4, the organic layer
was concentrated under reduced pressure and the resulting crude
product was purified by column flash chromatography on silica gel
(EtOAc/MeOH, 95:5 as eluant) to give a crude oily amine that was
dissolved in a solution of HCl in EtOH and stirred for 1 h. After
removing the solvent by distillation under vacuum, the resulting
solid was purified by crystallization in EtOH/Et2O White solid
The product was prepared starting from 14h. Purification by flash
chromatography on silica gel (cyclohexane/EtOAc 3:7) and subse-
quent crystallization. White solid (208 mg, 67%); mp: 140e141 ꢀC
(EtOAc); 1H NMR (CDCl3):
d
¼ 1.87 (m, 2H, CH2CH2CH2), 2.56 (t, 2H,
J ¼ 7.0, CH2-piperazine), 2.66 (m, 4H, piperazine), 3.17 (t, 2H J ¼ 6.5,
CH2NH), 3.23 (m, 4H, piperazine), 6.05 (m, 1H, Ar-H), 6.12e6.21 (m,
2H, Ar-H), 6.85e7.05 (m, 4H, Ar-H), 7.25e7.33 (m, 2H, Ar-H); IR
(nujol):
n
¼ 3390, 3340 cmꢂ1
;
13C NMR (CDCl3):
d
¼ 157.1, 151.1,
150.2, 130.2, 129.1, 119.9, 116.1, 105.7, 104.5, 99.7, 57.1, 53.2, 49.1,
43.4, 25.7. ESI-MS (m/z): 312 (M þ H)þ; Anal. calcd for C19H25N3O: C
73.28, H 8.09, N 13.49, found C 73.09, H 7.78, N 13.55.
(216 mg, 81%). 1H NMR (DMSO-d6):
d
¼ 2.91 (s, 3H, CH2NCH3), 2.97
(s, 3H, SO2CH3), 3.10 (m, 4H, piperazine), 3.25 (apt, 2H, J ¼ 7.0, CH2-
piperazine), 3.58 (m, 2H, piperazine), 3.80 (m, 2H, piperazine), 3.84
(apt, 2H, J ¼ 7.0, CH2NCH3), 6.57e6.69 (m, 3H, Ar-H), 6.87 (t, 1H, Ar-
H), 7.01 (m, 2H, Ar-H), 7.14 (t, 1H, Ar-H), 7.24e7.29 (m, 2H, Ar-H),
9.51 (s, 1H, NH), 11.60 (brs, 1H, NHþ). ESI-MS (m/z): 389 (M þ H)þ.
Anal. calcd for C20H29ClN4O2S$0.5 H2O: C 55.35, H 6.97, N 12.91,
found C 55.49, H 7.13, N 12.79.
5.1.9.4. 3-(Methyl{3-[4-(naphthalen-1-yl)piperazin-1-yl]propyl}
amino)phenol (14m). The product was prepared starting from 14j.
Purification by flash chromatography on silica gel (EtOAc as eluant)
and subsequent crystallization. Beige solid (255 mg, 68%); mp:
124e125 ꢀC (Et2O/petroleum ether); 1H NMR (CDCl3):
d
¼ 1.85 (m,
2H, CH2CH2CH2), 2.56 (apt, 2H, J ¼ 7.0, CH2-piperazine), 2.83 (m, 4H,
piperazine), 2.89 (s, 3H, CH3), 3.21 (m, 4H, piperazine), 3.38 (apt,
2H, J ¼ 7.0, CH2NCH3), 6.18 (m, 2H, Ar-H), 6.31 (m, 1H, Ar-H), 7.09
(m, 2H, Ar-H), 7.37e7.59 (m, 4H, Ar-H), 7.85 (m, 1H, Ar-H), 8.21 (m,
5.1.12. O-Debenzylation of benzyloxyanilino-derivatives by
hydrogenolysis. General procedure for compounds 10j, 14o, 18aec
A solution of the suitable benzyloxy-anilino derivative 9f, 14i,
17aec (1 mmol) in dry MeOH (18 mL) was hydrogenated over 10%
Pd/C (0.075 g) at 4 atm of H2 for 3 h at 60 ꢀC. The catalyst was
filtered on Celite, the filtrate was concentrated under reduced
pressure to give a crude residue that was purified by flash chro-
matography on silica gel and/or crystallization.
1H, Ar-H); 13C NMR (CDCl3):
d
¼ 157.2, 150.9, 149.4, 134.7, 130.1,
128.8, 128.4, 125.9, 125.8, 125.3, 123.6, 123.5, 114.8, 104.7, 103.4,
99.5, 56.0, 53.8, 52.6, 50.6, 38.3, 24.2. ESI-MS (m/z): 376 (M þ H)þ;
Anal. calcd for C24H29N3O: C 76.76, H 7.78, N 11.19, found C 76.99, H
7.83, N 11.32.
5.1.9.5. 3-(Methyl{3-[4-(naphthalen-2-yl)piperazin-1-yl]propyl}
amino)phenol (14n). The product was prepared starting from 14k.
Purification by flash chromatography on silica gel (gradient cyclo-
hexane/EtOAc 1:1 to EtOAc as eluant) and subsequent crystalliza-
tion. White solid (198 mg, 53%); mp: 161e162 ꢀC (acetone); 1H NMR
5.1.12.1. 3-[2-(4-Phenylpiperazin-1-yl)ethylamino]phenol
(10j).
The product was prepared starting from 9f. Purification by flash
chromatography on silica gel (EtOAc/cyclohexane, 6:4 as eluant)
and subsequent crystallization. Beige solid (119 mg, 40%); mp:
159e160 ꢀC (EtOAc); 1H NMR (CDCl3):
d
¼ 2.69 (m, 6H, piperazine
(CDCl3):
d
¼ 1.84 (m, 2H, CH2CH2CH2), 2.47 (apt, 2H, J ¼ 7.5, CH2-
and CH2CH2NH), 3.21 (m, 6H, piperazine and CH2NH), 4.35 (brs, 1H,
NH), 6.10 (dd, 1H, J1 ¼ J2 ¼ 2.0, Ar-H), 6.21 (m, 2H, Ar-H), 6.84e6.98
(m, 3H, Ar-H), 7.03 (dd, 1H, J1 ¼ J2 ¼ 8.0, Ar-H), 7.29 (m, 2H, Ar-H);
piperazine), 2.69 m (4H, piperazine), 2.92 (s, 3H, CH3), 3.37 (m, 6H,
piperazine and CH2NCH3), 6.18 (m, 2H, Ar-H), 6.33 (m, 1H, Ar-H),
7.04e7.14 (m, 2H, Ar-H), 7.25e7.45 (m, 3H, Ar-H), 7.72 (m, 3H, Ar-H);
13C NMR (CDCl3):
d
¼ 156.9, 151.2, 149.9, 147.9, 130.2, 129.1, 119.9,
MS (EI, 70 eV): m/z 325 (Mþ), 91 (100); 13C NMR (CDCl3):
d
¼ 156.8,
116.2, 106.0, 99.8, 56.6, 52.9, 49.1, 40.2; IR (nujol):
n
¼ 3423 cmꢂ1
;
150.9, 149.0, 134.5, 130.0, 128.7, 128.5, 126.7, 126.2, 125.2, 123.4,
119.4, 110.3, 104.9, 103.1, 99.2, 55.8, 53.2, 50.5, 49.4, 38.3, 24.2. ESI-
MS (m/z): 376 (M þ H)þ; Anal. calcd for C24H29N3O: C 76.76, H 7.78,
N 11.19, found C 76.59, H 7.71, N 11.27.
MS (EI, 70 eV): m/z 297 (Mþ),122 (100). Anal. calcd for C18H23N3O: C
72.70, H 7.80, N 14.13, found C 72.39, H 8.03, N 14.00.
5.1.12.2. 3-{Methyl[3-(1-phenylpiperazin-4-yl)propyl]amino}phenol
(14o). The product was prepared starting from 14i (Scheme 4).
Purification by flash chromatography on silica gel (EtOAc as eluant)
and subsequent crystallization. Gray solid (172 mg, 53%); mp: 177e
5.1.10. N1-Methyl-N1-[2-(4-phenylpiperazin-1-yl)ethyl]benzene-
1,3-diamine$HCl (10k)
A solution of the nitro compound 9g (1.0 mmol) in EtOH (10 mL)
and EtOAc (10 mL) was hydrogenated over 10% Pd/C (0.075 g) at
1 atm of H2 for 5 h at room temperature. The catalyst was filtered on
Celite, the filtrate was concentrated under reduced pressure to give
a crude oily amine that was dissolved in a solution of HCl in EtOH
and stirred for 1 h. After removing the solvent by distillation under
vacuum, the resulting solid was purified by crystallization in EtOH/
178 ꢀC (EtOAc); 1H NMR (CDCl3):
d
¼ 1.82 (m, 2H, CH2CH2CH2), 2.44
(apt, 2H, J ¼ 7.0, CH2-piperazine), 2.63 (m, 4H, piperazine), 2.90 (s,
3H, CH3), 3.24 (m, 4H, piperazine), 3.37 (t, 2H, J ¼ 7.0, CH2NCH3),
6.16 (m, 2H, Ar-H), 6.31 (m, 1H, Ar-H), 6.83e7.32 (m, 6H, Ar-H); 13
C
NMR (CDCl3):
d
¼ 156.7, 151.3, 150.9, 130.0, 129.1, 119.7, 116.0, 105.0,
103.0, 99.1, 55.7, 53.3, 50.5, 49.1, 38.3, 24.2; ESI-MS (m/z): 326
(M þ H)þ; Anal. calcd for C20H27N3O: C 73.81, H 8.36, N 12.91, found
C 74.10, H 8.65, N 12.89.
Et2O. White solid (280 mg, 81%). 1H NMR (DMSO-d6):
d
¼ 2.98 (s,
3H, CH3), 3.20 (m, 4H, piperazine), 3.30 (apt, 2H, J ¼ 7.0, CH2-
piperazine), 3.63 (m, 2H, piperazine), 3.82e3.95 (m, 4H, piperazine
and CH2NCH3), 6.66 (m, 1H, Ar-H), 6.82 (m, 1H, Ar-H), 6.87 (m, 1H,
Ar-H), 6.93 (m, 1H, Ar-H), 7.10 (m, 2H, Ar-H), 7.27 (m, 3H, Ar-H),
10.25 (brs, 2H, NH2), 11.50 (brs, 1H, NHþ); ESI-MS (m/z): 311
(M þ H)þ. Anal. calcd for C19H27ClN4$0.3 EtOH: C 65.26, H 8.05, N
15.53, found C 65.12, H 8.24, N 15.31.
5.1.12.3. 3-{Methyl[4-(1-phenylpiperazin-4-yl)butyl]amino}phenol
(18a). The product was prepared starting from 17a (Scheme 4).
Purification by flash chromatography on silica gel (EtOAc, as eluant)
and subsequent crystallization. White solid (217 mg, 64%); mp:
169e170 ꢀC (EtOAc); 1H NMR (CDCl3):
d
¼
1.59 (m, 4H,
CH2CH2CH2CH2), 2.45 (t, 2H, J ¼ 7.0, CH2-piperazine), 2.65 (m, 4H,
piperazine), 2.90 (s, 3H, CH3), 3.26 (m, 6H, piperazine and
CH2NCH3), 6.13 (m, 2H, Ar-H), 6.32 (m, 1H, Ar-H), 6.83e7.32 (m, 6H,
5.1.11. N-(3-{Methyl[2-(4-phenylpiperazin-1-yl)ethyl]amino}
phenyl)methanesulfonamide$HCl (10l)
Methanesulfonyl chloride (0.05 mL, 0.65 mmol) was added to a
solution of 10k (0.25 g, 0.63 mmol) and TEA (0.18 mL) in dry THF
Ar-H); 13C NMR (CDCl3):
116.1, 104.8, 103.1, 99.3, 58.4, 53.2, 52.6, 49.0, 38.4, 24.7, 24.2; ESI-
d
¼ 156.9, 151.2, 150.8, 130.0, 129.1, 119.8,

G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
27
MS (m/z): 340 (M þ H)þ; Anal. calcd for C21H29N3O: C 74.30, H 8.61,
C24H35ClN2O$0.5 H2O: C 71.05, H 8.77, N 6.90, found C 70.88, H 8.61,
N 6.69.
N 12.38, found C 74.15, H 8.44, N 12.27.
5.1.12.4. 3-{Methyl[5-(1-phenylpiperazin-4-yl)pentyl]amino}phenol
(18b). The product was prepared starting from 17b (Scheme 4).
Purification by flash chromatography on silica gel (EtOAc, as eluant)
and subsequent crystallization. White solid (141 mg, 40%); mp:
5.1.13.3. 3-(Methyl{6-[4-(naphthalen-1-yl)piperazin-1-yl]hexyl}
amino)phenol$HCl (18f). The product was prepared starting from
17f (Scheme 4). White solid (385 mg, 85%); 1H NMR (CD3OD):
d
¼
1.60 (m, 4H, CH2CH2CH2CH2CH2CH2), 1.72 (m, 2H,
144e145 ꢀC (EtOAc); 1H NMR (CDCl3):
d
¼
1.33 (m, 2H,
CH2CH2CH2CH2CH2CH2), 2.00 (m, 2H, CH2CH2CH2CH2CH2CH2), 3.36
(m, 4H, piperazine), 3.40 (s, 3H, CH3) 3.62e3.95 (m, 8H, piperazine,
CH2N and CH2NCH3), 7.18 (dd,1H, Ar-H), 7.23 (m, 2H, Ar-H), 7.38 (m,
1H, Ar-H), 7.52e7.75 (m, 4H, Ar-H), 7.81 (m, 1H, Ar-H), 8.06 (m, 1H,
Ar-H), 8.40 (m, 1H, Ar-H); ESI-MS (m/z): 418 (M þ H)þ. Anal. calcd
for C27H36ClN3O$0.2 MeOH: C 70.95, H 8.06, N 9.13, found C 71.21, H
8.28, N 9.33.
CH2CH2CH2CH2CH2), 1.61 (m, 4H, CH2CH2CH2CH2CH2), 2.45 (apt,
2H, J ¼ 8.0, CH2-piperazine), 2.69 (m, 4H, piperazine), 2.88 (s, 3H,
CH3), 3.25 (m, 6H, CH2NCH3 and piperazine), 6.14 (m, 2H, Ar-H),
6.25 (m, 1H, Ar-H), 6.84e7.10 (m, 4H, Ar-H), 7.24e7.32 (m, 2H, Ar-
H); 13C NMR (CDCl3):
104.5, 103.3, 99.4, 58.7, 53.3, 52.5, 48.9, 38.4, 26.4, 26.3, 25.0; ESI-
MS (m/z): 354 (M þ H)þ; Anal. calcd for C22H31N3O: C 74.75, H 8.84,
N 11.89, found C 74.55, H 8.71, N 11.96.
d
¼ 157.2, 151.1, 150.8, 130.0, 129.1, 119.9, 116.2,
5.1.13.4. 3-[Methyl(6-{4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}
hexyl)amino]phenol$HCl (18g). The product was prepared starting
from 17g (Scheme 4). White solid (368 mg, 78%); 1H NMR (CD3OD):
5.1.12.5. 3-{Methyl[6-(4-phenylpiperazin-1-yl)hexyl]amino}phenol
(18c). The product was prepared starting from 17c (Scheme 4).
Purification by flash chromatography on silica gel (EtOAc as eluant)
and subsequent crystallization. White solid (183 mg, 50%); mp:
d
¼
1.42 (m, 4H, CH2CH2CH2CH2CH2CH2), 1.57 (m, 2H,
CH2CH2CH2CH2CH2CH2), 1.81 (m, 2H, CH2CH2CH2CH2CH2CH2), 3.19
(m, 6H, piperazine and CH2N), 3.22 (s, 3H, CH3), 3.62 (m, 4H,
piperazine), 3.91 (m, 2H, CH2NCH3), 6.94 (m, 1H, Ar-H), 7.08 (m, 2H,
Ar-H), 7.21 (m, 1H, Ar-H), 7.32 (m, 2H, Ar-H), 7.42 (m, 2H, Ar-H). ESI-
MS (m/z): 436 (M þ H)þ. Anal. calcd for C24H33ClF3N3O$0.3 MeOH: C
60.60, H 7.16, N 8.72, found C 60.65, H 7.01, N 8.63.
125e126 ꢀC (EtOAc/petroleum ether); 1H NMR (CDCl3):
d
¼ 1.35 (m,
4H, CH2CH2CH2CH2CH2CH2), 1.57 (m, 4H, CH2CH2CH2CH2CH2CH2),
2.41 (apt, 2H, J ¼ 7.5, CH2-piperazine), 2.62 (m, 4H, piperazine), 2.89
(s, 3H, CH3), 3.25 (m, 4H, piperazine), 3.28 (apt, 2H J ¼ 7.0,
CH2NCH3), 6.13 (m, 2H, Ar-H), 6.26 (m, 1H, Ar-H), 6.82e7.10 (m, 4H,
Ar-H), 7.22e7.30 (m, 2H, Ar-H); 13C NMR (CDCl3):
d
¼ 157.1, 151.2,
5.1.13.5. 3-({6-[4-(7-Chloroquinolin-4-yl)piperazin-1-yl]hexyl}meth-
ylamino)phenol (18h)$HCl. The product was prepared starting from
17h (Scheme 4). White solid (381 mg, 78%); 1H NMR (CD3OD):
150.8, 130.0, 129.1, 119.8, 116.1, 104.6, 103.2, 99.4, 58.7, 53.2, 52.6,
48.8, 38.4, 27.3, 26.8, 26.4, 26.3; ESI-MS (m/z): 368 (M þ H)þ; Anal.
calcd for C23H33N3O: C 75.16, H 9.05, N 11.43, found C 74.91, H 8.86,
N 11.56.
d
¼
1.41 (m, 4H, CH2CH2CH2CH2CH2CH2), 1.58 (m, 2H,
CH2CH2CH2CH2CH2CH2), 1.82 (m, 2H, CH2CH2CH2CH2CH2CH2), 3.22
(s, 3H, CH3), 3.25 (m, 2H), 3.51 (m, 4H, piperazine), 3.79 (m, 2H),
3.92 (m, 2H), 4.38 (m, 2H), 6.98 (m, 1H, Ar-H), 7.08 (m, 2H, Ar-H),
7.42 (m, 2H, Ar-H), 7.80 (m, 1H, Ar-H), 8.08 (m, 1H, Ar-H), 8.22 (d,
1H, Ar-H), 8.78 (d, 1H, Ar-H). ESI-MS (m/z): 453.5 (M þ H)þ. Anal.
calcd for C26H34Cl2N4O$0.1 H2O$0.15 MeOH: C 63.31, H 7.07, N 11.29,
found C 63.60, H 7.24, N 11.19.
5.1.13. O-Debenzylation of benzyloxyanilino-derivatives with BCl3.
General procedure for compounds 16i, 18eeh, 18jek
A solution of BCl3 (1 M in CH2Cl2, 3 mL, 3 mmol) was added
dropwise to a solution of the suitable benzyloxy-anilino derivative
16f, 17eeh, or 17jek (1 mmol) in dry CH2Cl2 (6 mL) at 0 ꢀC, and the
resulting mixture was stirred at room temperature for 1 h. The
reaction mixture was neutralized with 2 N NaOH, the organic layer
was washed with brine, dried (Na2SO4) and concentrated under
reduced pressure, to give a crude product that was purified by flash
chromatography on silica gel (CH2Cl2/MeOH, 8: 2 as eluant). The
product was dissolved in a solution of HCl in MeOH and the solvent
was removed by distillation under vacuum to give the desired final
product as hydrochloride.
5.1.13.6. 3-{Ethyl[6-(4-phenylpiperazin-1-yl)hexyl]amino}phenol
(18j)$HCl. The product was prepared starting from 17j (Scheme 4).
White solid (342 mg, 82%); 1H NMR (CD3OD):
d
¼ 1.19 (t, 3H,
CH2CH3), 1.40 (m, 4H, CH2CH2CH2CH2CH2CH2), 1.50 (m, 2H,
CH2CH2CH2CH2CH2CH2), 1.76 (m, 2H, CH2CH2CH2CH2CH2CH2), 3.19
(m, 6H), 3.60 (m, 6H), 3.85 (m, 2H), 6.90e7.10 (m, 6H, Ar-H), 7.30
(m, 2H, Ar-H), 7.43 (m, 1H, Ar-H); ESI-MS (m/z): 382 (M þ H)þ. Anal.
calcd for C24H36ClN3O$0.3 H2O: C 68.08, H 8.71, N 9.92, found C
67.91, H 8.52, N 9.78.
5.1.13.1. N-(3-Hydroxyphenyl)-N-methyl-6-[4-(naphthalen-1-yl)
piperazin-1-yl]hexanamide$HCl (16i). The product was prepared
starting from 16f (Scheme 4). White solid (398 mg, 85%); 1H NMR
5.1.13.7. 3-{Benzyl[6-(4-phenylpiperazin-1-yl)hexyl]amino}phenol
(CD3OD):
d
¼ 1.35 (m, 2H, CH2CH2CH2CH2CH2), 1.70 (m, 4H,
(18k)$HCl. The product was prepared starting from 17k (Scheme
CH2CH2CH2CH2CH2), 2.20 (apt, 2H, CH2CO), 3.20 (m, 4H, pipera-
zine), 3.22 (s, 3H, CH3), 3.50 (m, 4H, piperazine), 3.70 (m, 2H, CH2-
piperazine), 6.67 (m, 2H, Ar-H), 6.82 (m, 1H, Ar-H), 7.21e7.58 (m,
5H, Ar-H), 7.62 (m, 1H, Ar-H), 7.90 (m, 1H, Ar-H), 8.22 (m, 1H, Ar-H).
ESI-MS (m/z): 432 (M þ H)þ. Anal. calcd for C27H34ClN2O2$0.15 H2O
C 69.29, H 7.32, N 8.98, found C 69.49, H 7.51, N 8.69.
4). White solid (379 mg, 79%); 1H NMR (CD3OD):
d
¼ 1.42 (m, 6H,
CH2CH2CH2CH2CH2CH2), 1.75 (m, 2H, CH2CH2CH2CH2CH2CH2), 3.20
(m, 6H, piperazine and CH2N), 3.78 (m, 6H, piperazine and CH2N),
4.77 (s, 2H, CH2Ph), 6.81e7.08 (m, 6H, Ar-H), 7.10e7.23 (m, 8H, Ar-
H); ESI-MS (m/z): 444 (M þ H)þ. Anal. calcd for C29H38ClN3O: C
72.55, H 7.98, N 8.75, found C 72.81, H 8.15, N 8.90.
5.1.13.2. 3-{Methyl[6-(4-phenylpiperidin-1-yl)hexyl]amino}phe-
5.1.14. 3-(Benzyloxy)-N-ethyl-N-[6-(4-phenylpiperazin-1-yl)hexyl]
aniline (17j)
nol$HCl (18e). The product was prepared starting from 17e
(Scheme 4). White solid (322 mg, 80%); 1H NMR (CD3OD):
d
¼ 1.58
A solution of acetaldehyde (132 mg, 3.0 mmol) in MeOH (16 mL)
was added dropwise to a stirred cooled (0 ꢀC) solution of 17d
(1.33 g, 3.0 mmol), AcOH (0.47 mL) and sodium cyanoborohydride
(0.35 g, 5.55 mmol) and the resulting mixture was stirred at room
temperature for 16 h. MeOH was removed by distillation in vacuo,
then a 1N NaOH aqueous solution (5 mL) was added, and the
(m,
4H,
CH2CH2CH2CH2CH2CH2),
1.70
(m,
2H,
CH2CH2CH2CH2CH2CH2), 1.90 (m, 2H, CH2CH2CH2CH2CH2CH2),
2.10e2.30 (m, 4H, piperidine), 3.10 (m, 1H, CH), 3.26 (m, 4H, CH2N),
3.38 (s, 3H, CH3), 3.83 (m, 4H, CH2N), 7.19 (m 3H, Ar-H), 7.44e7.58
(m, 6H, Ar-H). ESI-MS (m/z): 367 (M þ H)þ. Anal. calcd for

28
G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
aqueous phase was extracted with CH2Cl2; after drying over
Na2SO4, the combined organic layers were concentrated under
reduced pressure to give a crude residue which was purified by
flash chromatography on silica gel (EtOAc/cyclohexane 8:2 as
CH2NCH3), 6.47e6.58 (m, 3H, Ar-H), 6.83e6.97 (m, 3H, Ar-H), 7.13e
7.32 (m, 3H, Ar-H); 13C NMR (CDCl3):
¼ 151.2, 150.3, 138.0, 130.2,
d
129.1, 119.8, 116.1, 109.1, 108.0, 104.4, 55.7, 53.2, 50.5, 49.1, 38.9, 38.4,
24.1; IR (nujol):
n
¼ 3248, 1148 cmꢂ1; ESI-MS (m/z): 403 (M þ H)þ;
eluant). Oil (848 mg, 60%). 1H NMR (CDCl3):
d
¼ 1.15 (t, 3H, J ¼ 7.0,
Anal. calcd for C21H30N4O2S: C 62.66, H 7.51, N 13.92, found C 62.54,
H 7.38, N 13.78.
CH2CH3), 1.38 (m, 4H, CH2CH2CH2CH2CH2CH2), 1.56 (m, 2H,
CH2CH2CH2CH2CH2CH2), 1.83 (m, 2H, CH2CH2CH2CH2CH2CH2),
2.62e3.24 (m, 8H, piperazine and 2 ꢃ CH2N), 3.30 (q, 2H, J ¼ 7.0,
CH2CH3), 3.56 (m, 4H, piperazine), 5.02 (s, 2H, CH2Ph), 6.22 (m, 3H,
Ar-H), 6.82e7.52 (m, 11H, Ar-H). ESI-MS: m/z 472 (M þ H)þ.
5.1.19. 3-[3-(4-Phenylpiperazin-1-yl)propylamino]benzamide (14r)
To a stirred ice-cooled solution of the ester 14g (0.12 g,
0.33 mmol) in MeOH (3 mL) in a 5 mL microwave vial, was added
magnesium nitride (0.165 g, 1.64 mmol). The vial was sealed
immediately and allowed to warm to room temperature in a water
bath. After approximately 1 h, the reaction was heated at 80 ꢀC for
24 h. After cooling to room temperature the reaction mixture was
diluted with dichloromethane (12 mL) and water (10 mL), the
aqueous phase was neutralized with 1 N HCl and then extracted
(2 ꢃ 10 mL) with dichloromethane. After drying over Na2SO4, the
combined organic layers were concentrated under reduced pres-
sure and the resulting crude product was purified by column flash
chromatography on silica gel (EtOAc/MeOH, 9:1 as eluant) and
subsequent crystallization. White solid (14 mg, 13%); mp: 147e
5.1.15. N-Benzyl-3-(benzyloxy)-N-[6-(4-phenylpiperazin-1-yl)
hexyl]aniline (17k)
The product was prepared according to the above described
procedure for 17j, using benzaldehyde instead of acetaldehyde.
Purification by flash chromatography on silica gel (EtOAc/cyclo-
hexane 7:3 as eluant). Oil (1.04 g, 65%) 1H NMR (CDCl3):
d
¼ 1.31 (m,
4H, CH2CH2CH2CH2CH2CH2), 1.58 (m, 2H, CH2CH2CH2CH2CH2CH2),
1.92 (m, 2H, CH2CH2CH2CH2CH2CH2), 2.91 (m, 4H, piperazine), 3.38
(m, 2H, CH2-piperazine), 3.60 (m, 6H, piperazine and CH2NAr), 4.78
(d, 2H, J ¼ 9.0, NCH2Ph), 4.98 (s, 2H, OCH2Ph), 6.30 (m, 2H, Ar-H),
6.90e7.41 (m, 17H, Ar-H). ESI-MS: m/z 534 (M þ H)þ.
148 ꢀC (EtOAc); 1H NMR (CDCl3):
d
¼ 1.87 (m, 2H, CH2CH2CH2), 2.57
(t, 2H, J ¼ 6.5, CH2-piperazine), 2.65 (m, 4H, piperazine), 3.23e3.28
(m, 6H, piperazine and CH2NH), 4.95 (brs, 1H, NH), 5.71 (brs, 1H,
NH), 6.07 (brs, 1H, NH), 6.75 (dd, 1H, J ¼ 2.0 and 8.0, Ar-H), 6.85e
5.1.16. N1-Methyl-N1-[3-(4-phenylpiperazin-1-yl)propyl]benzene-
1,3-diamine
A solution of the nitro compound 14f (0.38 g, 1.07 mmol) in
EtOH (10 mL) and EtOAc (10 mL) was hydrogenated over 10% Pd/C
(0.075 g) at 1 atm of H2 for 5 h at room temperature. The catalyst
was filtered on celite, the filtrate was concentrated under reduced
pressure to give a crude oily amine that was used for the next step,
without further purification (342 mg, near quantitative yield). ESI-
MS (m/z): 325 (M þ H)þ.
7.34 (m, 8H, Ar-H); 13C NMR (CDCl3):
d
¼ 170.0, 151.2, 149.0, 134.5,
129.3, 129.1, 119.8, 116.4, 116.0, 115.1, 111.1, 57.3, 53.3, 49.3, 43.4,
25.5; IR (nujol):
n
¼ 3321, 3151, 1661 cmꢂ1; ESI-MS (m/z): 339
(M þ H)þ; Anal. calcd for C20H26N4O: C 70.98, H 7.74, N 16.55, found
C 71.11, H 7.86, N 16.37.
From chromatographic purification we also isolated the methyl
ester derivative 14s (87 mg, 75%) as
transesterification.
a
result of 14g
5.1.17. N-(3-{Methyl[3-(4-phenylpiperazin-1-yl)propyl]amino}
pheny)acetamide (14p)
5.1.20. Methyl 3-[3-(4-phenylpiperazin-1-yl)propylamino]benzoate
(14s)
Ac2O (0.045 mL, 0.48 mmol) was added to a solution of N1-
methyl-N1-[3-(4-phenylpiperazin-1-yl)propyl]benzene-1,3-
diamine (0.15 g, 0.46 mmol) and TEA (0.07 mL) in dry THF (4 mL),
and the resulting mixture was stirred at room temperature for 1 h.
After removing the solvent by distillation at reduced pressure, the
residue was taken up in EtOAc, and washed with 1 N NaOH and
with brine. After drying over Na2SO4, the organic layer was
concentrated under reduced pressure and the resulting crude
product was purified by column flash chromatography on silica gel
(EtOAc/MeOH, 95:5 as eluant). Amorphous solid (145 mg, 86%); 1H
White solid; mp: 102e103 ꢀC (EtOAc/petroleum ether); 1H NMR
(CDCl3):
d
¼ 1.88 (m, 2H, CH2CH2CH2), 2.59 (t, 2H, J ¼ 6.5, CH2-
piperazine), 2.67 (m, 4H, piperazine), 3.24e3.29 (m, 6H, piperazine
and CH2NH), 3.90 (s, 3H, CH3), 4.93 (brs, 1H, NH), 6.79 (ddd, 1H,
J ¼ 1.0, 2.5, 8.0, Ar-H), 6.89 (m, 1H, Ar-H), 6.95 (m, 1H, Ar-H), 6.99
(m, 1H, Ar-H), 7.18e7.38 (m, 5H, Ar-H); 13C NMR (CDCl3):
d
¼ 167.6,
151.2, 148.7, 131.0, 129.2, 129.1, 119.8, 118.2, 117.5, 116.1, 112.8, 57.3,
53.3, 52.0, 49.2, 43.4, 25.5; IR (nujol):
n
¼ 3384, 1707 cmꢂ1; ESI-MS
(m/z): 354 (M þ H)þ; Anal. calcd for C21H27N3O2: C 71.36, H 7.70, N
NMR (CDCl3):
d
¼ 1.80 (m, 2H, CH2CH2CH2), 2.15 (s, 3H, COCH3),
11.89, found C 71.02, H 7.58, N 12.05.
2.42 (apt, 2H, J ¼ 7.0, CH2-piperazine), 2.59 (m, 4H, piperazine), 2.93
(s, 3H, NCH3), 3.21 (m, 4H, piperazine), 3.39 (t, 2H, J ¼ 7.0,
CH2NCH3), 6.49 (dd, 1H, J ¼ 2.0 and 8.0, Ar-H), 6.66 (dd, 1H, J ¼ 1.5
and 8.0, Ar-H), 6.81e6.97 (m, 3H, Ar-H), 7.04e7.08 (m, 2H, Ar-H),
7.13 (dd, 1H, J1 ¼ J2 ¼ 8.0, Ar-H), 7.21e7.32 (m, 1H, Ar-H); 13C NMR
5.1.21. {3-[3-(4-Phenylpiperazin-1-yl)propylamino]phenyl}
methanol (14t)
LiAlH4 (0.085 g, 2.24 mmol) was added portionwise to an ice-
cooled stirred solution of 14g (1.13 mmol) in dry THF (10 mL) un-
der nitrogen atmosphere, and the resulting mixture was stirred for
1 h at room temperature. Water was added dropwise to destroy the
excess of hydride, the resulting mixture was filtered on Celite, and
the filtrate was concentrated in vacuo and partitioned between
EtOAc and water. The combined organic phases were washed with
brine, dried (Na2SO4) and concentrated to afford a crude residue
which was purified by crystallization. White solid (334 mg, 91%);
(CDCl3):
d
¼ 168.2,151.3,150.0,138.9,129.5,129.1,119.6,116.0,108.3,
107.5, 103.7, 55.7, 53.3, 50.6, 49.1, 38.3, 24.8, 24.2; IR (nujol):
n
¼ 3311, 1657 cmꢂ1; ESI-MS (m/z): 367 (M þ H)þ; Anal. calcd for
C
22H30N4O: C 72.10, H 8.25, N 15.29, found C 72.28, H 8.19, N 15.41.
5.1.18. N-(3-{Methyl[3-(4-phenylpiperazin-1-yl)propyl]amino}
phenyl)methanesulfonamide (14q)
The product was prepared according the above described pro-
cedure for 14p, using methanesulfonyl chloride (0.05 mL,
0.65 mmol) instead of Ac2O. Purification by column flash chroma-
tography on silica gel (EtOAc as eluant). Oil (157 mg, 85%); 1H NMR
mp: 142e143 ꢀC (EtOAc); 1H NMR (CDCl3):
d
¼ 1.87 (m, 2H,
CH2CH2CH2), 2.57 (t, 2H, J ¼ 6.5, CH2-piperazine) 2.65 (m, 4H,
piperazine), 3.23e3.28 (m, 6H, piperazine and CH2NH), 4.62 (s, 2H,
CH2OH), 4.74 (brs, 1H, NH), 6.55 (dd, 1H, J ¼ 2.0 and 8.0, Ar-H), 6.61
(m, 2H, Ar-H), 6.92 (m, 3H, Ar-H), 7.17 (dd, 1H, J1 ¼ J2 ¼ 8.0, Ar-H),
(CDCl3):
d
¼ 1.79 (m, 2H, CH2CH2CH2), 2.42 (apt, 2H, J ¼ 7.0, CH2-
piperazine), 2.60 (m, 4H, piperazine), 2.94 (s, 3H, NCH3), 2.99 (s, 3H,
7.31 (m, 2H, Ar-H); 13C NMR (CDCl3):
129.1, 119.8, 116.0, 115.6, 112.1, 111.0, 65.5, 57.2, 53.3, 49.2, 43.4, 25.8;
d
¼ 151.2, 149.0, 142.3, 129.4,
NHSO2CH3), 3.21 (m, 4H, piperazine), 3.39 (apt, 2H, J ¼ 7.0,

G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
29
IR (nujol):
n
¼ 3332, 3121 cmꢂ1; ESI-MS (m/z): 326 (M þ H)þ; Anal.
16 h. After cooling to room temperature, water was added and the
aqueous phase was extracted (3ꢃ) with EtOAc. The combined
organic layers were washed with brine, dried (Na2SO4) and
concentrated under reduced pressure to give a crude residue that
was purified by flash chromatography on silica gel (EtOAc/cyclo-
calcd for C20H27N3O: C 73.81, H 8.36, N 12.91, found C 73.59, H 8.18,
N 12.77.
5.1.22. 3-[3-(4-Phenylpiperazin-1-yl)propylamino]benzaldehyde
(14u)
hexane, 3:7 as eluant). Oil (207 mg, 82%); 1H NMR (CDCl3):
d
¼ 1.90
Activated manganese dioxide (1.28 g) was added to a solution of
the alcohol derivative 14t (0.35 g, 1.08 mmol) in dry dichloro-
methane (15 mL), and the resulting mixture was stirred for 18 h at
room temperature. The mixture was filtered, the filter cake was
washed with hot acetone and the combined filtrates were
concentrated under reduced pressure to yield a crude residue that
was purified by flash chromatography (EtOAc as eluant) and crys-
tallization. Yellow solid (261 mg, 75%); mp: 87e88 ꢀC (EtOAc); 1H
(m, 2H, CH2CH2CH2), 2.45 (t, 2H, J ¼ 7.0, CH2-piperazine), 2.61 (m,
4H, piperazine), 3.23 (m, 4H, piperazine), 3.49 (t, 2H, J ¼ 7.0,
CH2NCH2Ph), 3.75 (s, 3H, OCH3), 4.57 (s, 2H, CH2Ph), 6.27 (m, 2H,
Ar-H), 6.38 (dd, 1H, J ¼ 2.0 and 8.0, Ar-H), 6.92 (m, 3H Ar-H), 7.11
(dd, 1H, J1 ¼ J2 ¼ 8.0, Ar-H), 7.22e7.35 (m, 7H, Ar-H); ESI-MS (m/z):
416 (M þ H)þ.
5.1.26. 3-Methoxy-N-phenyl-N-[3-(4-phenylpiperazin-1-yl)propyl]
aniline (22)
NMR (CDCl3):
d
¼ 1.88 (m, 2H, CH2CH2CH2), 2.58 (t, 2H, J ¼ 6.5, CH2-
piperazine) 2.66 (m, 4H, piperazine), 3.23e3.32 (m, 6H, piperazine
and CH2NH), 5.13 (brs, 1H, NH), 6.84e7.35 (m, 9H, Ar-H), 9.93 (s, 1H,
A Schlenk flask was charged with palladium acetate (0.01 g,
0.044 mmol), (ꢁ)-BINAP (0.031 g, 0.05 mmol), potassium tert-
butoxide (0.147 g, 1.3 mmol) and toluene (2 mL) under a nitrogen
atmosphere. To this mixture a solution of 14h (0.3 g, 0.92 mmol)
and iodobenzene (0.1 mL, 0.92 mmol) in dry toluene (2 mL) was
added dropwise via syringe. After the addition was completed, the
mixture was heated at 110 ꢀC for 24 h. After cooling to room tem-
perature EtOAc was added, the mixture was filtered and the filtrate
was washed with brine. After drying over Na2SO4, the organic layer
was concentrated under reduced pressure and the resulting crude
product was purified by column flash chromatography on silica gel
(cyclohexane/EtOAc 7:3 as eluant). Oil (162 mg, 44%); 1H NMR
CHO); 13C NMR (CDCl3):
d
¼ 193.1, 151.2, 149.2, 137.5, 129.7, 129.2,
120.0, 119.9, 119.5, 116.1, 110.8, 57.3, 53.3, 49.3, 43.4, 25.4; IR (nujol):
n
¼ 3346, 1677 cmꢂ1; ESI-MS (m/z): 324 (M þ H)þ; Anal. calcd for
C
20H25N3O: C 74.27, H 7.79, N 12.99, found C 74.45, H 7.92, N 12.63.
5.1.23. N-(3-Methoxyphenyl)-N-[3-(4-phenylpiperazin-1-yl)
propyl]propionamide (19)
TEA (0.186 mL, 1.34 mmol) and propionic anhydride (0.171 mL,
1.34 mmol) were added to a solution of 14h (0.29 g, 0.89 mmol) in
dry THF (12 mL) and the resulting mixture was stirred at room
temperature for 24 h. The solvent was removed by distillation in
vacuo, water was added and the aqueous phase was extracted (3ꢃ)
with EtOAc. The combined organic layers were washed with brine,
dried (Na2SO4) and concentrated under reduced pressure to give a
crude residue that was purified by flash chromatography on silica
(CDCl3):
d
¼ 1.91 (m, 2H, CH2CH2CH2), 2.47 (t, 2H, J ¼ 7.0, CH2-
piperazine), 2.61 (m, 4H, piperazine), 3.23 (m, 4H, piperazine), 3.76
(s, 3H, OCH3), 3.80 (t, 2H, J ¼ 7.0, CH2NPh), 6.54 (m, 3H, Ar-H), 6.84e
7.32 (m, 11H, Ar-H); MS (EI, 70 eV): m/z 401 (Mþ), 226 (100).
gel (EtOA as eluant). Oil (308 mg, 91%); 1H NMR (CDCl3):
d
¼ 1.06 (t,
5.1.27. N-(3-Methoxyphenyl)-N-[3-(4-phenylpiperazin-1-yl)
propyl]benzenesulfonamide (23)
3H, J ¼ 7.0, CH2CH3), 1.82 (m, 2H, CH2CH2CH2), 2.09 (m, 2H, CH2-
piperazine), 2.41 (q, 2H, J ¼ 7.0, CH2CH3), 2.57 (m, 4H, piperazine),
3.17 (m, 4H, piperazine), 3.77 (t, 2H, J ¼ 7.5, CH2NAr), 3.82 (s, 3H,
OCH3), 6.71e6.93 (m, 6H, Ar-H), 7.21e7.32 (m, 3H, Ar-H); ESI-MS
(m/z): 382 (M þ H)þ.
TEA (0.23 mL, 1.8 mmol) and benzenesulfonyl chloride (0.18 mL,
1.41 mmol) were added to a solution of 14h (0.25 g, 0.77 mmol) in
dry THF (7.5 mL), and the resulting mixture was stirred at room
temperature under nitrogen for 48 h. After removing the solvent by
distillation in vacuo, the residue was partitioned between water and
EtOAc. The combined organic layers were washed with brine, dried
(Na2SO4) and concentrated under reduced pressure to afford a
crude residue which was purified by flash chromatography on silica
gel (EtOAc/cyclohexane, 7:3 as eluant). White solid (250 mg, 70%);
5.1.24. 3-Methoxy-N-[3-(4-phenylpiperazin-1-yl)propyl]-N-
propylaniline (20)
A 1 M solution of BH3 in THF (2.43 mL) was added dropwise to
an ice-cooled solution of 19 (0.31 g, 0.81 mmol) in dry THF (6 mL)
under a N2 atmosphere. Once the addition was completed, the re-
action mixture was stirred at room temperature for 16 h; 6 N HCl
(0.7 mL) was cautiously added and the resulting mixture was
heated to reflux for 1 h. After cooling to room temperature the
reaction mixture was made alkaline with 1 N NaOH (2.7 mL) and
then extracted with EtOAc. The combined organic layers were
washed with brine, dried (Na2SO4) and concentrated to afford a
crude residue which was purified by flash chromatography on silica
gel (cyclohexane/EtOAc, 7:3 as eluant). Oil (193 mg, 65%); 1H NMR
1H NMR (CDCl3):
d
¼ 1.60 (m, 2H, CH2CH2CH2), 2.48 (t, 2H, J ¼ 7.0,
CH2-piperazine), 2.56 (m, 4H, piperazine), 3.18 (m, 4H, piperazine),
3.62 (t, 2H, J ¼ 7.0, CH2NSO2), 3.76 (s, 3H, OCH3), 6.62 (m, 2H, Ar-H),
6.88 (m, 4H, Ar-H), 7.24 (m, 3H, Ar-H), 7.54 (m, 5H, Ar-H); ESI-MS
(m/z): 466 (M þ H)þ.
5.1.28. 3-{[3-(4-Phenylpiperazin-1-yl)propyl]propylamino}phenol
(24)
The product was prepared starting from 20 using the above
general O-demethylation procedure with boron tribromide. Puri-
fication by flash chromatography on silica gel (cyclohexane/EtOAc
1:1 as eluant) and subsequent crystallization. White solid (339 mg,
96%); mp: 132e133 ꢀC (Et2O/petroleum ether); 1H NMR (CDCl3):
(CDCl3):
d
¼ 0.92 (t, 3H, J ¼ 7.0, CH2CH2CH3), 1.63 (m, 2H,
CH2CH2CH3), 1.80 (m, 2H, CH2CH2CH2), 2.43 (t, 2H, J ¼ 7.0, CH2-
piperazine), 2.62 (m, 4H, piperazine), 3.23 (m, 6H, piperazine and
CH2N), 3.35 (t, 2H, J ¼ 7.0, CH2NAr), 3.80 (s, 3H, OCH3), 6.22 (m, 2H,
Ar-H), 6.33 (dd, 1H, J ¼ 2.0 and 8.5, Ar-H), 6.83e6.98 (m, 3H, Ar-H),
7.12 (dd,1H, J1 ¼ J2 ¼ 8.5, Ar-H), 7.23e7.32 (m, 2H, Ar-H); ESI-MS (m/
z): 368 (M þ H)þ.
d
¼ 0.91 (t, 3H, J ¼ 7.5, CH2CH2CH3), 1.67 (q, 2H, J ¼ 7.5, CH2CH2CH3),
1.84 (m, 2H, CH2CH2CH2), 2.45 (t, 2H, J ¼ 7.0, CH2-piperazine), 2.66
(m, 4H, piperazine), 3.16e3.36 (m, 8H, piperazine, 2 ꢃ CH2NAr),
5.86 (brs, 1H, OH), 6.10 (m, 2H, Ar-H), 6.25 (dd, 1H, J ¼ 2.0 and 8.0,
Ar-H), 6.84e7.07 (m, 4H, Ar-H), 7.26e7.33 (m, 2H, Ar-H); 13C NMR
5.1.25. N-Benzyl-3-methoxy-N-[3-(4-phenylpiperazin-1-yl)propyl]
aniline (21)
TEA (0.5 mL, 3.6 mmol) and benzyl bromide (0.37 mL,
3.11 mmol) were added to a solution of 14h (0.20 g, 0.61 mmol) in
toluene (4 mL), and the resulting mixture was heated at 80 ꢀC for
(CDCl3):
d
¼ 157.1, 151.2, 149.7, 130.0, 129.1, 119.9, 116.1, 104.5, 102.6,
99.0, 55.9, 53.3, 52.8, 49.0, 48.9, 24.6, 20.3, 11.5; ESI-MS (m/z): 354
(M þ H)þ; Anal. calcd for C22H31N3O: C 74.75, H 8.84, N 11.89, found
C 74.98, H 8.59, N 11.66.

30
G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
5.1.29. 3-{Benzyl[3-(4-phenylpiperazin-1-yl)propyl]amino}phenol
(25)
5.1.33. 3-[4-(Diphenylamino)piperidin-1-yl]-N-(3-
methoxyphenyl)-N-methylpropanamide (28c)
The product was prepared starting from 21 using the above
general O-demethylation procedure with boron tribromide. Puri-
fication by flash chromatography on silica gel (cyclohexane/EtOAc
3:7 as eluant) and subsequent crystallization. White solid (228 mg,
This compound was prepared using the above described pro-
cedure for 28b, using diphenylpiperidin-4-ylamine [30] instead of
4,4-diphenylpiperidine Purification by flash chromatography on
silica gel (EtOAc as eluant). Oil (113 mg, 51%); 1H NMR (CDCl3):
57%); mp: 166e167 ꢀC (EtOAc); 1H NMR (CDCl3):
d
¼ 1.89 (m, 2H,
CH2CH2CH2), 2.44 (t, 2H, J ¼ 7.0, CH2-piperazine), 2.54 (m, 4H,
piperazine), 3.16 (m, 4H, piperazine), 3.45 (t, 2H, 7.0,
d
¼ 1.39 (m, 2H, piperidine), 1.99 (m, 4H, piperidine), 2.27 (t, 2H,
J ¼ 7.5, CH2CO), 2.66 (t, 2H, J ¼ 7.5, COCH2CH2N), 2.82 (m, 2H,
piperidine), 3.23 (s, 3H, NCH3), 3.80 (s, 3H, OCH3), 3.81 (m, 1H, CH),
6.67e7.01 (m, 9H, Ar-H), 7.19e7.33 (m, 5H, Ar-H); ESI-MS (m/z): 444
(M þ H)þ.
J
¼
CH2NCH2Ph), 4.44 (s, 2H, CH2Ph), 6.08 (m, 1H, Ar-H), 6.16 (dd, 1H,
J ¼ 2.0 and 8.0, Ar-H), 6.31 (dd, 1H, J ¼ 2.0 and 8.0, Ar-H), 6.83e6.94
(m, 3H, Ar-H), 7.06 (dd, 1H, J1 ¼ J2 ¼ 8.0, Ar-H) 7.13e7.35 (m, 7H, Ar-
H); 13C NMR (CDCl3):
126.7, 126.4, 119.9, 116.1, 104.4, 103.6, 99.5, 55.6, 53.5, 53.1, 48.8,
48.5, 24.7; ESI-MS (m/z): 402 (M þ H)þ; Anal. calcd for C26H31N3O:
C 77.77, H 7.78, N 10.46, found C 77.39, H 7.52, N 10.30.
d
¼ 157.3,151.1,150.3,138.5,130.2,129.1,128.6,
5.1.34. N-[3-(4,4-Diphenylpiperidin-1-yl)propyl]-3-methoxy-N-
methylaniline (29b)
A 1 M solution of BH3 in THF (3 mL) was added dropwise to an
ice-cooled solution of 28b (0.43 g, 1 mmol) in dry THF (7 mL) under
a N2 atmosphere. Once the addition was completed, the reaction
mixture was stirred at room temperature for 16 h; 6 N HCl (0.9 mL)
was cautiously added and the resulting mixture was heated to
reflux for 1 h. After cooling to room temperature the reaction
mixture was made alkaline with 1 N NaOH (3 mL) and then
extracted with EtOAc. The combined organic layers were washed
with brine, dried (Na2SO4) and concentrated to afford a crude
residue which was purified by flash chromatography on silica gel
5.1.30. 3-{Phenyl[3-(4-phenylpiperazin-1-yl)propyl]amino}phenol
(26)
The product was prepared starting from 22 using the above
general O-demethylation procedure with boron tribromide. Puri-
fication by flash chromatography on silica gel (cyclohexane/EtOAc
1:1 as eluant) and subsequent crystallization. White solid (248 mg,
64%); mp: 172e173 ꢀC (Et2O); 1H NMR (CDCl3):
d
¼ 1.87 (m, 2H,
CH2CH2CH2), 2.44 (t, 2H, J ¼ 7.0, CH2-piperazine), 2.57 (m, 4H,
piperazine), 3.19 (m, 4H, piperazine), 3.74 (t, 2H, J ¼ 7.0, CH2NPh),
6.33 (m, 2H, Ar-H), 6.51 (m, 1H, Ar-H), 6.83e7.13 (m, 7H, Ar-H),
(EtOAc as eluant). Oil (270 mg, 65%); 1H NMR (CDCl3):
d
¼ 1.78 (m,
2H, CH2CH2CH2), 2.34 (t, 2H, J ¼ 7.5, CH2-piperidine), 2.54 (m, 8H,
piperidine), 2.79 (s, 3H, NCH3), 3.28 (t, 2H, J ¼ 7.5, CH2NCH3), 3.83
(s, 3H, OCH3), 6.16 (m, 3H, Ar-H), 7.00e7.32 (m, 11H, Ar-H); ESI-MS
(m/z): 415 (M þ H)þ.
7.21e7.32 (m, 4H, Ar-H); 13C NMR (CDCl3):
d
¼ 156.8, 151.1, 149.5,
147.6, 130.1, 129.4, 129.1, 123.4, 122.7, 119.9, 116.1, 110.8, 107.1, 105.9,
55.6, 53.1, 50.0, 48.9, 24.8; ESI-MS (m/z): 387 (M þ H)þ; Anal. calcd
for C25H29N3O: C 77.48, H 7.54, N 10.84, found C 77.13, H 7.30, N
10.96.
5.1.35. 1-{3-[(3-Methoxyphenyl)methylamino]propyl}-N,N-
diphenylpiperidin-4-amine (29c)
The product was prepared using the above described procedure
starting from 28c. Purification by flash chromatography on silica gel
(EtOAc/cyclohexane 1:1 as eluant). Oil (202 mg, 47%); 1H NMR
5.1.31. N-(3-Hydroxyphenyl)-N-[3-(4-phenylpiperazin-1-yl)propyl]
benzenesulfonamide (27)
The product was prepared starting from 23 using the above
general O-demethylation procedure with boron tribromide. Puri-
fication by flash chromatography on silica gel (cyclohexane/EtOAc
3:7 as eluant). Amorphous solid (333 mg, 74%); 1H NMR (CDCl3):
(CDCl3):
d
¼ 1.50 (m, 2H, piperidine), 1.72 (m, 2H, CH2CH2CH2), 2.03
(m, 4H, piperidine), 2.34 (t, 2H, J ¼ 7.0, CH2-piperidine), 2.88 (s, 3H,
NCH3), 2.97 (m, 2H, piperidine), 3.30 (t, 2H, J ¼ 7.0, CH2NCH3), 3.77
(s, 3H, OCH3), 3.85 (m, 1H, CH), 6.29 (m, 3H, Ar-H), 6.84 (m, 4H, Ar-
H), 7.05 (m, 3H, Ar-H), 7.22e7.32 (m, 4H, Ar-H); ESI-MS (m/z): 430
(M þ H)þ.
d
¼ 1.69 (m, 2H, CH2CH2CH2), 2.55 (t, 2H, J ¼ 7.0, CH2-piperazine),
2.68 (m, 4H, piperazine), 3.19 (m, 4H, piperazine), 3.55 (t, 2H, J ¼ 7.0,
CH2NSO2), 6.42 (m, 2H, Ar-H), 6.55 (dd, 1H, J ¼ 2.0 and 8.0, Ar-H),
6.83e6.93 (m, 3H, Ar-H), 7.06 (dd, 1H, J1 ¼ J2 ¼ 8.0, Ar-H), 7.22e7.30
5.1.36. 3-{3-[(4,4-Diphenylpiperidin-1-yl)propyl]methylamino}
phenol (30b)
(m, 2H, Ar-H), 7.36e7.58 (m, 5H, Ar-H); 13C NMR (CDCl3):
d
¼ 156.9,
151.0, 139.3, 137.7, 132.7, 129.8, 129.1, 128.8, 127.6, 120.0, 119.1, 116.1,
The product was prepared starting from 29b using the above
general O-demethylation procedure with boron tribromide. Puri-
fication by flash chromatography on silica gel (EtOAc as eluant).
115.5, 55.5, 53.1, 48.6, 48.4, 24.9; IR (nujol):
n
¼ 1164 cmꢂ1; ESI-MS
(m/z): 452 (M þ H)þ; Anal. calcd for C25H29N3O3S: C 66.49, H 6.47, N
9.31, found C 66.20, H 6.55, N 9.66.
Amorphous solid (116 mg, 29%); 1H NMR (CDCl3):
d
¼ 1.76 (m, 2H,
CH2CH2CH2), 2.31 (t, 2H, J ¼ 7.5, CH2-piperidine), 2.52 (m, 8H,
piperidine), 2.82 (s, 3H, NCH3), 3.29 (t, 2H, J ¼ 7.0, CH2NCH3), 6.13
(m, 2H, Ar-H), 6.25 (ddd, 1H, J ¼ 1.5, 3.0 and 8.0, Ar-H), 7.04 (dd, 1H,
J1¼J2 ¼ 8.0, Ar-H), 7.11e7.18 (m, 2H, Ar-H), 7.21e7.30 (m, 8H, Ar-H);
5.1.32. 3-(4,4-Diphenylpiperidin-1-yl)-N-(3-methoxyphenyl)-N-
methylpropanamide (28b)
4,4-diphenylpiperidine [29] (0.6 mmol) and TEA (0.83 mL) were
added to a solution of 3-bromo-N-(3-methoxyphenyl)-N-methyl-
propanamide (12a) (0.5 mmol) in DMF (0.14 mL) and the resulting
mixture was stirred at room temperature for 16 h, monitoring the
progress of the reaction by TLC on silica gel. The reaction mixture
was poured into water, and extracted with EtOAc. After drying over
Na2SO4, the combined organic phases were concentrated in vacuo
and the resulting crude product was purified by column flash
chromatography on silica gel (EtOAc as eluant). Amorphous solid
13C NMR (CDCl3):
d
¼ 157.6, 150.8, 130.0, 128.4, 127.1, 125.8, 104.3,
103.7, 99.5, 56.1, 50.6, 50.5, 44.6, 38.1, 35.6, 29.7, 24.2; ESI-MS (m/z):
401 (M þ H)þ; Anal. calcd for C27H32N2O: C 80.96, H 8.05, N 6.99,
found C 81.24, H 8.22, N 6.68.
5.1.37. 3-({3-[4-(Diphenylamino)piperidin-1-yl]propyl}
methylamino)phenol (30c)
The product was prepared starting from 29c using the above
general O-demethylation procedure with boron tribromide. Puri-
fication by flash chromatography on silica gel (CH2Cl2/MeOH, 95:5).
(103 mg, 48%); 1H NMR (CDCl3):
d
¼ 2.28 (t, 2H, J ¼ 7.5, CH2CO), 2.41
(m, 8H, piperidine), 2.60 (t, 2H J ¼ 7.5, COCH2CH2N), 3.24 (s, 3H,
NCH3), 3.82 (s, 3H, OCH3), 6.68e6.76 (m, 2H, Ar-H), 6.87 (ddd, 1H,
J ¼ 1.0, 3.0 and 8.0, Ar-H), 7.09e7.34 (m, 11H, Ar-H); ESI-MS (m/z):
429 (M þ H)þ.
Amorphous solid (166 mg, 40%); 1H NMR (CDCl3):
d
¼ 1.45e1.81 (m,
4H, piperidine and CH2CH2CH2), 1.94e2.16 (m, 4H, piperidine), 2.36
(t, 2H, J ¼ 7.0, CH2-piperidine), 2.86 (s, 3H, NCH3), 3.00 (m, 2H,

G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
31
piperidine), 3.29 (t, 2H, J ¼ 7.0, CH2NCH3), 3.87 (m, 1H, CH), 6.12 (m,
2H, Ar-H), 6.25 (ddd, 1H, J ¼ 1.5, 3.0 and 8.0, Ar-H), 6.80e6.86 (m,
4H, Ar-H), 6.95e7.07 (m, 3H, Ar-H), 7.23e7.31 (m, 4H, Ar-H); 13C
on silica gel (cyclohexane/EtOAc, 7:3 as eluant). Oil (130 mg, 67%);
1H NMR (CDCl3):
J ¼ 7.0, CH2Ar), 2.63 (m, 6H, piperazine and CH2N), 3.22 (m, 4H,
piperazine), 3.81 (s, 3H, OCH3), 6.73e6.98 (m, 6H, Ar-H), 7.17e7.32
(m, 3H, Ar-H); ESI-MS (m/z): 325 (M þ H)þ.
d
¼ 1.68 (m, 4H, CH2CH2CH2CH2), 2.42 (t, 2H,
NMR (CDCl3):
d
¼ 157.1, 150.8, 146.0, 130.0, 129.3, 122.7, 121.8, 104.7,
103.5, 99.5, 55.7, 55.0, 53.4, 50.7, 38.2, 30.2, 24.2; ESI-MS (m/z): 416
(M þ H)þ; Anal. calcd for C27H33N3O: C 78.03, H 8.00, N 10.11, found
C 78.36, H 8.35, N 10.01.
5.1.42. 3-[4-(4-Phenylpiperazin-1-yl)butyl]phenol (32c)
The product was prepared starting from 32b using the above
described general O-demethylation procedure with boron tri-
bromide. Purification by flash chromatography on silica gel
(cyclohexane/EtOAc 1:1 as eluant) and subsequent crystallization.
White solid (226 mg, 73%); mp: 143e144 ꢀC (Et2O/petroleum
5.1.38. 3-(3-Hydroxyphenyl)-1-(4-phenylpiperazin-1-yl)propan-1-
one (31a)
Phenyl piperazine (500 mg, 3.08 mmol), 3-(3-hydroxyphenyl)
propionic acid (570 mg, 3.4 mmol) and Et3N (1.3 mL, 9.24 mmol)
were added to an ice-cooled solution of 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide [EDC] (885 mg, 4.62 mmol)
and N-hydroxybenzotriazole [HOBT] (582 mg, 4.3 mmol) in CH2Cl2
(16.6 mL). The resulting mixture was stirred for 2 h at 0 ꢀC and at
room temperature overnight. The organic solution was washed
with water, dried (Na2SO4), and concentrated under vacuum,
obtaining a crude residue that was used for the next step without
ether); 1H NMR (CDCl3):
d
¼ 1.63 (m, 4H, CH2CH2CH2CH2), 2.48 (t,
2H, J ¼ 7.0, CH2Ar), 2.58 (t, 2H, J ¼ 7.0, CH2N), 2.69 (m, 4H, piper-
azine), 3.26 (m, 4H, piperazine), 6.59 (m, 2H, Ar-H), 6.70 (d, 1H,
J ¼ 7.5, Ar-H), 6.82e6.98 (m, 3H, Ar-H), 7.12 (dd, 1H, J1 ¼ J2 ¼ 7.5, Ar-
H), 7.24e7.32 (m, 2H, Ar-H); 13C NMR (CDCl3):
d
¼ 156.2,151.1,144.0,
129.4, 129.1, 120.2, 119.9, 116.2, 115.5, 113.0, 58.6, 53.2, 48.8, 35.6,
29.2, 26.0; ESI-MS (m/z): 311 (M þ H)þ; Anal. calcd for C20H26N2O: C
77.38, H 8.44, N 9.02, found C 77.02, H 8.12, N 9.33.
further purification. Oil (827 mg, 86%). 1H NMR (CDCl3):
d
¼ 2.41 (t,
2H, J ¼ 7.5, CH2CO), 2.67 (t, 2H, J ¼ 7.5, CH2Ar), 3.20 (m, 4H,
piperazine), 3.61 (m, 2H, piperazine), 3.85 (m, 2H, piperazine),
6.68e6.99 (m, 6H, Ar-H), 7.17e7.35 (m, 3H, Ar-H); ESI-MS (m/z): 333
(M þ Na)þ.
5.1.43. 2-[3-(Benzyloxy)phenoxy]-1-(4-phenylpiperazin-1-yl)
ethanone (33)
2-Bromo-1-(4-phenylpiperazin-1-yl)ethanone [32] (1.5 g,
6.28 mmol) was added to a solution of 3-benzyloxyphenol (1.9 g,
9.42 mmol) and K2CO3 (1.35 g, 9.42 mmol) in DMF (5 mL), and the
resulting mixture was stirred at 40 ꢀC overnight. After removing the
solvent by distillation under vacuum, EtOAc was added and the
organic solution was washed with 1 M NaOH and then with brine.
After drying over Na2SO4, the organic layer was concentrated under
reduced pressure and the resulting crude product was purified by
column flash chromatography on silica gel (EtOAc/hexane, 1:1 as
5.1.39. 4-(3-Methoxyphenyl)-1-(4-phenylpiperazin-1-yl)butan-1-
one (31b)
This product was obtained according to the above described
procedure for 31a starting from 4-(3-methoxyphenyl)butanoic acid
[31] (0.7 g, 3.6 mmol) instead of 3-(3-hydroxyphenyl)propionic
acid. Purification by flash chromatography on silica gel (cyclo-
hexane/EtOAc, 6:4 as eluant). Oil (770 mg, 74%); 1H NMR (CDCl3):
d
¼ 2.03 (m, 2H, CH2CH2CH2), 2.37 (apt, 2H, J ¼ 7.5, CH2CO), 2.69
eluant). White solid (0.88 g, 35%). 1H NMR (CDCl3):
d
¼ 3.10 (m, 4H,
(apt, 2H, J ¼ 7.5, CH2Ar), 3.15 (m, 4H, piperazine), 3.57 (m, 2H,
piperazine), 3.79 (m, 2H, piperazine), 3.80 (s, 3H, OCH3), 6.72e6.85
(m, 3H, Ar-H), 6.86e6.95 (m, 3H, Ar-H), 7.18e7.34 (m, 3H, Ar-H);
ESI-MS (m/z): 339 (M þ H)þ.
piperazine), 3.71 (m, 4H, piperazine), 4.62 (s, 2H, OCH2CO), 4.98 (s,
2H, CH2Ph), 6.52 (m, 3H, Ar-H), 6.85 (m, 3H, Ar-H), 7.05e7.40 (m,
8H, Ar-H). ESI-MS (m/z): 425 (M þ Na)þ.
5.1.44. 1-{2-[3-(Benzyloxy)phenoxy]ethyl}-4-phenylpiperazine$HCl
(34)
This compound was obtained by reduction of 33 (0.63 g,
1.56 mmol) with 1 M B2H6 in THF according to the above described
procedure for compound 32a. The crude product was dissolved in a
saturated solution of HCl in MeOH and then crystallized by MeOH/
5.1.40. 3-[3-(4-Phenylpiperazin-1-yl)propyl]phenol$HCl (32a)
A 1 M solution of B2H6 in THF (20 mL, 16 mmol) was added
dropwise to an ice-cooled solution of 31a (827 mg, 2.5 mmol) in
THF (18 mL) and the reaction mixture underwent stirring for 4 h at
room temperature. 6 M HCl (7 mL) was added to the solution at 0 ꢀC
and the resulting mixture was evaporated under vacuum. The solid
was dissolved in EtOAc and washed with an 7% aqueous solution of
NaHCO3. The organic layer was washed with water, dried (Na2SO4),
and evaporated under vacuum obtaining a crude product (1.02 g,
3.6 mmol). The oily product was dissolved in a solution of EtOH
(10 mL) and conc. HCl (300 mL, 3.6 mmol) and the mixture was
stirred for 1 h. After removing the solvent by distillation under
vacuum, the resulting solid was purified by crystallization in EtOH.
Et2O. White solid (0.58 g, 87%); 1H NMR (DMSO-d6):
d
¼ 3.02e3.37
(m, 4H, piperazine), 3.57 (m, 4H, piperazine), 3.83 (m, 2H, CH2-
piperazine), 4.40 (m, 2H, OCH2), 5.10 (s, 2H, CH2Ph), 6.64 (m, 3H, Ar-
H), 6.85 (m, 1H, Ar-H), 7.01 (m, 2H, Ar-H), 7.20e7.50 (m, 8H, Ar-H),
10.81 (brs, 1H). ESI-MS (m/z): 389 (M þ H)þ.
5.1.45. 3-[2-(4-Phenylpiperazin-1-yl)ethoxy]phenol$HCl (35)
This compound was obtained starting from compound 34 and
using the above described general O-debenzylation procedure with
White solid (196 mg, 59%); 1H NMR (DMSO-d6):
d
¼ 2.01 (m, 2H,
CH2CH2CH2), 2.57 (m, 2H, CH2-piperazine), 3.01 (m, 6H, piperazine
and CH2Ar), 3.58 (m, 2H, piperazine), 3.78 (m, 2H, piperazine), 6.62
(m, 2H, Ar-H), 6.82 (ddd, 1H, J ¼ 1.5, 3.0 and 8.0, Ar-H), 7.00 (m, 3H,
Ar-H), 7.15 (dd, 1H, J1 ¼ J2 ¼ 8.0, Ar-H), 7.30 (m, 2H, Ar-H); 9.38 (s,
1H, OH), 11.00 (brs, 1H, NHþ); ESI-MS (m/z): 297 (M þ H)þ. Anal.
calcd for C19H24N2O: C 76.99, H 8.16, N 9.45, found C 76.79, H 8.01, N
9.39.
BCl3. White solid (237 mg, 71%); 1H NMR (DMSO-d6):
d¼3.15 (m,
4H, piperazine), 3.60 (m, 4H, piperazine), 3.81 (m, 2H, CH2-piper-
azine), 4.37 (m, 2H, OCH2), 6.42 (m, 3H, Ar-H), 6.85 (m, 1H, Ar-H),
6.98 (m, 2H, Ar-H), 7.08 (m, 1H, Ar-H), 7.25 (m, 2H, Ar-H); ESI-MS
(m/z): 299 (M þ H)þ. Anal. calcd for C18H23ClN2O2$0.5 MeOH: C
63.33, H 7.18, N 7.98, found C 63.51, H 7.40, N 7.83.
5.1.46. 1-(8-Methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)-4-
phenylpiperazine (36)
N-Phenylpiperazine (1.4 g, 8.7 mmol) and p-toluenesulfonic acid
(0.053 g, 0.29 mmol) were added to a solution of 8-methoxy-3,4-
dihydronaphthalen-2(1H)-one (0.5 g, 2.85 mmol) in dry toluene
(25 mL). This solution was heated to reflux in a Dean Stark
5.1.41. 1-[4-(3-Methoxyphenyl)butyl]-4-phenylpiperazine (32b)
This compound was obtained by reduction of 4-(3-
methoxyphenyl)-1-(4-phenylpiperazin-1-yl)butan-1-one (0.2 g,
0.6 mmol) with 1 M B2H6 in THF according to the above described
procedure for compound 32a. Purification by flash chromatography

32
G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
apparatus for 24 h, removing the water of reaction. The solvent was
removed by distillation under vacuum, the resulting crude residue
was dissolved in EtOH/THF (20 mL þ 20 mL) and then hydroge-
nated (4 atm) at room temperature in the presence of PtO2 (0.05 g)
for 24 h. The reaction mixture was filtered on celite, the filtrate was
evaporated under reduced pressure to yield a crude residue that
was purified by flash-chromatography on silica gel (cyclohexane/
EtOAc 8:2 as eluant). White solid (688 mg, 75%); mp: 119e120 ꢀC
vacuum manifold). Filter plates were then washed several times
with ice-cold buffer (50 mM TriseHCl, pH 7.4) and filter-bound
radioactivity measured using a MicroBeta counter (PerkinElmer)
after addition of 30
cocktail (PerkinElmer). Compounds were first tested at fixed (10e1
and/or 0.1 mol/L) concentrations and those active at these con-
mL/well of OptiPhase SuperMix scintillation
m
centrations (50% inhibition vs control) were repeated at scalar
concentrations (ranging from 10ꢂ5 to 10ꢂ10 mol/L).
(Et2O/petroleum ether); 1H NMR (CDCl3):
d
¼ 1.65 (dddd, 1H,
J ¼ 5.0, 6.0, 11.0, 16.5, H3a), 2.17 (m, 1H, H2), 2.53 (dd, 1H, J ¼ 11.0,
16.5, H4a), 2.73 (m, 1H, H3b), 2.90 (m, 6H, NCH2CH2N, H1a and H1b),
3.10 (ddd, 1H, J ¼ 1.5, 5.0, 16.5, H4b), 3.28 (m, 4H, NCH2CH2N), 3.84
(s, 3H, CH3), 6.69 (d, 2H, J ¼ 8.0, Ar-H), 6.74 (d,1H, J ¼ 8.0, Ar-H), 6.88
(m, 1H, Ar-H), 6.97 (m, 2H, Ar-H), 7.11 (dd, 1H, J1 ¼ J2 ¼ 8.0, Ar-H),
5.2.3. Data evaluation
Data were analyzed using nonlinear regression with GraphPad
PRISM commercial software. Test compound concentrations
causing a half maximal inhibition of control values (IC50 calculated
by GraphPad Prism software) were calculated. Inhibitory binding
constant (Ki) values were calculated from IC50 values, according to
the Cheng and Prusoff equation [33], Ki ¼ IC50/(1 þ [C]/Kd), where
[C] is the concentration of the radioligand and Kd its dissociation
constant.
7.25e7.33 (m, 2H, Ar-H); 13C NMR (CDCl3):
d
¼ 157.6, 151.4, 137.7,
129.1, 126.1, 124.6, 120.7, 119.6, 116.1, 106.7, 60.4, 55.2, 49.6, 49.2,
29.7, 25.9, 25.8; MS (EI, 70 eV): m/z 322 (Mþ, 100).
5.1.47. 7-(4-Phenylpiperazin-1-yl)-5,6,7,8-tetrahydronaphthalen-
1-ol (37)
5.2.4. Human 5-HT7 functional assays
The product was prepared according to the above described
general O-demethylation procedure with BBr3 starting from 36.
Purification by flash-chromatography on silica gel (cyclohexane/
EtOAc 1:1 as eluant) and crystallization. White solid (194 mg, 63%);
Agonist and antagonist behavior were evaluated by Cerep [26]
in cAMP functional assays performed in CHO cells, according to
the method of Adham et al. [34]. Agonist behavior was determined
by evaluating the concentration-dependent elevation of intracel-
lular cAMP levels produced by test compounds. Serotonin showed
EC50 ¼ 29 nM. Antagonist behavior of test compounds was deter-
mined evaluating the effect on cAMP accumulation stimulated by
300 nM 5-HT.
IC50 values (concentration causing a half-maximal inhibition of
the control specific agonist response) were determined by non-
linear regression analysis of the concentrationeresponse curves
generated with mean replicate values using Hill equation curve
fitting (Y ¼ D þ [(A ꢂ D)/(1 þ (C/IC50)nH)], where Y ¼ specific
response, D ¼ minimum specific response, A ¼ maximum specific
response, C ¼ compound concentration, and nH ¼ slope factor).
This analysis was performed using a software developed at Cerep
(Hill software). The apparent dissociation constants (KB) were
calculated using the modified Cheng and Prusoff equation
(KB ¼ IC50/(1 þ (A/EC50A)), where A ¼ concentration of reference
agonist in the assay, and EC50A ¼ EC50 value of the reference
agonist). The reference compound mesulergine had KB ¼ 16 nM.
mp: 211 ꢀC dec. (EtOAc); 1H NMR (DMSO-d6):
d
¼ 1.50 (dddd, 1H,
J ¼ 4.5, 6.0, 11.0, 16.5, H6a), 2.02 (m, 1H, H7), 2.39 (dd, 1H, J ¼ 11.0,
16.0, H5a), 2.60e2.85 (m, 7H, NCH2CH2N þ H8a þ H8b þ H6b), 2.86
(dd, 1H, J ¼ 4.5, 16.0, H5b), 3.15 (m, 4H, NCH2CH2N), 6.52 (d, 1H,
J ¼ 7.5, Ar-H), 6.58 (d, 1H, J ¼ 8.0), 6.77 (dd, 1H, J1 ¼ J2 ¼ 7.0, Ar-H),
6.87 (dd, 1H, J ¼ 7.5, 8.0, Ar-H), 6.93 (m, 2H, Ar-H), 7.21 (m, 2H, Ar-
H), 9.17 (brs, 1H, NH); 13C NMR (CD3OD):
d
¼ 154.9, 151.3, 137.2,
128.6, 125.8, 121.9, 119.7, 119.1,116.0, 111.1, 60.8, 49.1, 47.8, 29.0, 25.6,
25.4; MS (EI, 70 eV): m/z 308 (Mþ), 120 (100); Anal. calcd for
C
20H24N2O: C 77.89, H 7.84; N 9.08 found C 77.95, H 7.99, N 8.97.
5.2. Pharmacology
5.2.1. HEK293 cell culture and membrane preparation
HEK293 cells, stably expressing the human serotonin 5-HT7
receptor gene, were grown in Falcon flasks in DMEM (Cambrex,
Verviers, Belgium) supplemented with 10% FBS (Cambrex), and
2 m
g/mL puromincine (SigmaeAldrich, St Louis, MO) at 37 ꢀC in a 5%
CO2 atmosphere. For radioligand binding experiments, the cells, at
80% of confluence, were collected in PBS/EDTA (Lonza, Basel,
Switzerland, cat.# BE02-017F) and centrifuged at 1000 ꢃ g for
10 min. The pellet obtained was resuspended in the same buffer
used in binding experiments and homogenized with Ultra Turrax.
The homogenate was centrifuged at 48,000 ꢃ g for 90 min. The final
pellet was stored at ꢂ80 ꢀC until the day of experiment. The protein
concentration of membrane suspension was determined using the
Bradford method (Pierce, Rockford, IL, USA) with bovine albumin as
standard.
5.2.5. Binding assays on other serotonin receptors
Affinity of compounds 5n, 5i, 14o and 16i for 5-HT1A, 5-HT1B, 5-
HT1D, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT4e, 5-HT5a and 5-HT6
receptors was evaluated by Cerep [26]. % Displacement of reference
ligands at the concentration of 1 mM of 5n, 5i, 14o and 16i are re-
ported in Supplementary Material Table S1.
5.3. Molecular modeling
The X-ray structure of the 5-HT1B serotonin receptor in complex
with the agonist dihydroergotamine (PDB code: 4IAQ) [35] was
used as the template for 5-HT7 receptor modeling. The thermo-
stabilized apocytochrome b562 RIL segment and all non-protein
atoms were removed and hydrogen atoms were added using the
Protein Preparation Wizard [36] implemented in Maestro 9.2 [37].
The amino acid sequence of the human 5-HT7 receptor was
retrieved from the Universal Protein Resource (UniProt ID: P34969)
[38,39]. Initial alignment was carried out with Prime 3.0 [40] and
subsequently optimized considering conserved sequence motifs of
class A GPCRs [21,41] (Fig. S2). Since the intracellular loop (ICL) 3 of
the 5-HT1B receptor was replaced with the thermostabilized
apocytochrome b562 RIL, the ICL3 sequence of the 5-HT7 receptor
was excluded from homology modeling and Ala270, at the N-
5.2.2. Competition binding assay
Competition binding experiments were performed incubating
membranes (5e10 mg of protein/sample) with a single concentra-
tion of [3H]5-HT (5 nmol/L) (Biotrend, Cologne, Germany), in the
presence of test compounds, in 96-well filter plates (MultiScreen
system, cat # MAFBN0B10, Millipore, Billerica, MA, USA) for 30 min
at 25 ꢀC in a total volume of 200
containing 10
acid. Non-specific binding was determined in the presence of
100 M cold 5-HT (SigmaeAldrich). At the end of incubation, bound
and free radioligand were separated by filtering the 96-well filter
plates using Millipore filtration apparatus (MultiscreenHTS
mL/well of TriseHCl 50 mM, pH 7.4,
mmol/L pargyline, 4 mmol/L CaCl2 and 0.05% ascorbic
m
a

G. Spadoni et al. / European Journal of Medicinal Chemistry 80 (2014) 8e35
33
terminus of ICL3, was directly linked to Ser317 belonging to the C-
terminus of ICL3. This should not influence docking results, since
the putative binding pocket of the 5-HT7 receptor is located in the
extracellular portion of the helix bundle. The all-hydrogen 5-HT7
model structure obtained with Prime 3.0 was subjected to a
restrained minimization using Impact 5.7 [42] and the OPLS2005
contained 87 lipid molecules, 12 chlorine atoms and 8284 water
molecules. The system was initially relaxed using a customized
version of a membrane relaxation protocol implemented in the
Desmond package and subsequently simulated for 100 ns in the
NPT ensemble. Temperature and pressure were set to 310 K and
1 atm, respectively, applying the Langevin coupling scheme [50],
and the M-SHAKE algorithm [51] was used to constrain all bond
lengths to hydrogen atoms. VdW and short-range electrostatic in-
ꢀ
force field [43] to an RMSD value of 0.3 A calculated on the protein
heavy atoms. The stereochemical quality of the minimized struc-
ture was evaluated using Procheck [44]. The Ramachandran plot of
the final refined structure showed only Val230 in extracellular loop
(ECL) 3 and Ala270 at the N terminus of IL3 in disallowed regions
(Fig. S3).
ꢀ
teractions were cut off at 9 A and long-range electrostatic in-
teractions were computed using the smooth Particle Mesh Ewald
method [52]. A multistep RESPA scheme [53] was used to integrate
the equations of motion, applying a timestep of 2 fs for the bonded
and the short-range nonbonded interactions and of 6 fs for the
long-range nonbonded interactions. The OPLS2005 force field was
used for both the relaxation and the production phase of the MD
simulation.
Compound 5i was built with Maestro 9.2 and subjected to an
energy minimization procedure using the OPLS2005 force field to a
ꢀꢂ1
convergence threshold of 0.05 kJ molꢂ1
A . Induced-Fit Docking
(IFD) [45,46] was then applied to account for receptor flexibility
during docking calculations. During the initial softened-potential
docking, a Van der Waals (VdW) radii scaling of 0.7 and 0.5 were
applied on protein and ligand non-polar atoms, respectively.
Moreover, Asp1422.65, Arg3677.36 and Leu3707.39 were temporarily
mutated to alanines, to favor initial ligand placement. Asp1623.32
was used to define the location of the binding site and the di-
The last snapshot of the MD simulation was submitted to energy
minimization with Desmond 2.2, applying the OPLS2005 force field
ꢀꢂ1
to a convergence threshold of 0.5 kcal molꢂ1
A . The energy-
minimized 5-HT7 receptore5i complex was then extracted from
the solvated lipid bilayer and used for docking studies of com-
pounds 14m, 27, 18a and 18c. Docking grids were generated with
Glide 5.7 [54] and were centered on the 5i molecule, setting
ꢀ
mensions of bounding and enclosing boxes were set to 10 A and
ꢀ
ꢀ
ꢀ
30 A, respectively. Initial docking runs were performed in standard
bounding and enclosing boxes to 10 A and 30 A on each side,
respectively. To retain the key ionic interaction with Asp1623.32, a
hydrogen-bond constraint was applied between the protonated
piperazine nitrogen of ligands and Asp1623.32 during docking runs.
Twenty docking poses were collected for each ligand and the top-
ranked pose according to the Emodel value was merged into the
5-HT7 model structure. The resulting complexes were subjected to
an energy minimization process using the OPLS2005 force field and
precision (SP) mode, applying a hydrogen-bond constraint between
the protonated piperazine nitrogen of 5i and Asp1623.32. A core
constraint was also applied on compound 5i, to induce the heavy
atoms of the indole ring to superpose to the corresponding atoms of
dihydroergotamine (co-crystallized with the 5-HT1B receptor), with
ꢀ
a tolerance of 1 A. Twenty ligand poses were retained for subse-
quent refinement. Each ligandereceptor complex obtained at the
end of the softened-potential docking run was then submitted to
side chain and backbone refinements with Prime 3.0. During this
phase, the previously removed side chains were re-introduced and
the GB/SA water solvation treatment to a convergence threshold of
ꢀꢂ1
0.5 kJ molꢂ1
A . The energy-minimized complexes are depicted in
Fig. 2c, d and 4.
ꢀ
all residues located within 5.0 A from the ligand molecule were
refined by a side chain conformational search, followed by energy
minimization of the residues and the ligand molecule. To prevent
Glide from searching for alternative accommodations of the ligand
during the final docking stage of the IFD workflow, each ligand
molecule obtained at the end of the Prime stage was not re-docked
into its induced-fit receptor structure, but it was only optimized in
the field of the receptor and subsequently scored using default
Glide settings (VdW radii scaling of 1.0 and 0.8 for receptor and
ligand non-polar atoms, respectively). The resulting ligandeprotein
complexes were ranked according to their IFD score and the 5-HT7
receptore5i complex showing the best IFD score was selected. To
prevent ECL3 from interpenetrating into the lipid bilayer, a loop
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.034.
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