Improving selectivity preserving affinity: New piperidine-4-carboxamide derivatives as effective sigma-1-ligands

Article abstract of DOI:10.1016/j.ejmech.2014.12.018

We report the design, synthesis and binding evaluation against ₁ and ₂ receptors of a series of new piperidine-4-carboxamide derivatives variously substituted on the amide nitrogen atom. Specifically, we assessed the effects exerted on receptor affinity by substituting the N-benzylcarboxamide group present on a series of compounds previously synthesized in our laboratory with different cyclic or linear moieties. The synthesized compounds 2a-o were tested to estimate their affinity and selectivity toward ₁ and₂ receptors. Very high ₁ affinity (K_i Combining double low line 3.7 nM) and K_i2/K_i1 selectivity ratio (351) were found for the tetrahydroquinoline derivative 2k, featuring a 4-chlorobenzyl moiety linked to the piperidine nitrogen atom.

Full text of DOI:10.1016/j.ejmech.2014.12.018

European Journal of Medicinal Chemistry 90 (2015) 797e808
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Improving selectivity preserving affinity: New piperidine-4-
carboxamide derivatives as effective sigma-1-ligands
,
*
,
,
Daniele Zampieri a 1, Erik Laurini b 1, Luciano Vio a, Maurizio Fermeglia b,
Sabrina Pricl b **, Bernhard Wünsch d, Dirk Schepmann d, Maria Grazia Mamolo a
, c,
a Department of Chemistry & Pharmaceutical Sciences, Piazzale Europa 1, University of Trieste, 34127 Trieste, Italy
b Molecular Simulation Engineering (MOSE) Laboratory, DI3, Piazzale Europa 1, University of Trieste, 34127 Trieste, Italy
c National Interuniversity Consortium for Material Science and Technology (INSTM), Research Unit MOSE-DEA, University of Trieste, Trieste, Italy
d Department of Pharmaceutical and Medicinal Chemistry, Corrensstrasse 48, 48149 Münster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the design, synthesis and binding evaluation against s1 and s2 receptors of a series of new
piperidine-4-carboxamide derivatives variously substituted on the amide nitrogen atom. Specifically, we
Received 24 July 2014
Received in revised form
19 November 2014
Accepted 11 December 2014
Available online 12 December 2014
assessed the effects exerted on
s receptor affinity by substituting the N-benzylcarboxamide group
present on a series of compounds previously synthesized in our laboratory with different cyclic or linear
moieties. The synthesized compounds 2aeo were tested to estimate their affinity and selectivity toward
s1 and s2 receptors. Very high s1 affinity (Ki ¼ 3.7 nM) and Kis2/Kis1 selectivity ratio (351) were found
for the tetrahydroquinoline derivative 2k, featuring a 4-chlorobenzyl moiety linked to the piperidine
nitrogen atom.
Keywords:
s-Receptors
© 2014 Elsevier Masson SAS. All rights reserved.
Piperidine-4-carboxamide derivatives
Radioligand binding assays
1. Introduction
[7] recognized in recent year as a small-ligand operated chaperone
essential for the regulation of the passage of Ca2þ form the endo-
After the initial, erroneous classification as opioid receptor
subtype [1], receptors ( -Rs) have been shown to represent a
non-opioid, non-phencyclidine but haloperidol-sensitive receptor
family [2]. At least two distinct receptor subtypes e designated as
plasmic reticulum (ER) to the mitochondria [12aec]. Recently, its
association with the PGRMC-1 (Progesterone Receptor Membrane
Component 1) protein has been hypothesized, with subsequent
role in signaling and apoptosis [13]. Furthermore, it has been pro-
s
s
s
s1-R and s2-R, respectively e have been identified so far [3e5],
characterized by different tissue distribution and dissimilar binding
profile [6].
posed that both s-R subtypes are involved in cellular apoptotic
response [14,15] and in the release of Ca2þ via an IP3-independent
mechanism [16,17].
The s1-R, originally cloned from guinea pig liver [7] and then
from several other sources including human placenta choriocarci-
noma cells [8], consists of 223 amino acids and shares about 90%
identity and 95% similarity across species [7]. s1 receptors are
involved in the regulation of ion channels and in the modulation of
neurotransmitter systems [9e11]. Much less is known about the s2
receptor subtype. The protein has not been cloned yet, but its
molecular weight has been determined as approximately 21.5 KDa
Many studies described the cytotoxic effects of several s1 an-
tagonists and s2 agonists [18,19]. However, their impact on cell
cycle or mechanisms of cell death is not clearly understood. Further,
several synthetic molecules belonging to different structural classes
were found to bind to the s1 receptor. Among these, (þ)-pentazo-
cine, showing high s1-affinity and selectivity, represents a suitable
tool for structural and functional studies on s1-R and, as such, is
currently used as preferred radioligand [20]. On the other hand,
endogenous compounds such as progesterone, D-erythro-sphin-
gosine, and N,N-dimethyltryptamine have good s1-R affinity/
selectivity and play an important role in modulating s1-Rs [7,21].
From the medicinal chemistry point of view the design and the
development of new, potent and selective s1-R ligands able to
interfere with the biological activity of this receptor are becoming
crucial issues. Under this perspective, new different structures
* Corresponding author.
** Corresponding author. Molecular Simulation Engineering (MOSE) Laboratory,
DI3, Piazzale Europa 1, University of Trieste, 34127 Trieste, Italy.
E-mail addresses: dzampieri@units.it (D. Zampieri), sabrina.pricl@di3.units.it
(S. Pricl).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2014.12.018
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

798
D. Zampieri et al. / European Journal of Medicinal Chemistry 90 (2015) 797e808
endowed with s1-R affinity and selectivity, such as benzoox-
azolones [22a,b], alkyl and arylcarboxamide [22b,23e25], ary-
lalkylamines [26aef], and spirocyclic pyranopyrazoles [27] were
identified and reported by various research groups. In addition,
some arylacetamides derivatives synthesized by Huang Y. and coll.
[28a,b] showed a remarkable affinity towards s1-R and good
selectivity against s2-R subtype. In our previous work we reported
the synthesis of some carboxamide derivatives, in which the amide
group at position 4 of the piperidine is inverted with respect to the
compounds in Huang's series. All our derivatives are endowed with
good s1 affinity but showing only moderate selectivity towards the
s2 receptor [22b]. Within this series, compounds 1a,b (Fig. 1)
showed the most interesting s1 binding profile (Kis1 ¼ 22.5 nM and
12.9 nM, respectively) coupled with modest selectivity against the
s2 subtype (Kis2/Kis1 ¼ 8 and 11, respectively).
Fig. 2. Mapping of 1b on the s1 receptor 3D pharmacophore model. The hypothesis
features are portrayed as meshed spheres, color-coded as follows: red, positive
ionizable (PI); light blue, hydrophobic aromatic (HYAr); pink, generic hydrophobic
(HY), light green, hydrogen bond acceptor (HBA). (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
As such, both derivatives were exploited to validate a three-
dimensional (3D) pharmacophore model [22b] and the only 3D
homology model of s1 receptor available to date [23]. Importantly,
the results of the 3D pharmacophoric modeling offered
a
molecular-based rationale for the remarkable binding profile of 1b.
As we see from Fig. 2, the structure of this carboxamide derivative is
provided with all pharmacophoric requirements for optimal s1
binding: the basic piperidine nitrogen atom matches the positive
ionizable feature, the amide oxygen atom is able to accept a
hydrogen bond from a donor residue on the receptor while the two
benzyl rings fulfill the hydrophobic features of the model.
Accordingly, the predicted Kis1 value of 5.5 nM substantiates
compound 1b as a potent s1 ligand, in agreement with the exper-
imental Kis1 affinity of 12.9 nM [22b]. Despite compound 1a is
missing one hydrophobic pharmacophoric feature because of its
unsubstituted phenyl ring, its mapping onto the s1-R 3D pharma-
cophore model (data not shown) confirmed it as a good s1-R binder
with a predicted affinity of 37 nM, fairly close to the experimental
value (22.5 nM) [22b]. Ultimately, these evidences support the
hypothesis that appropriated substitutions on the phenyl ring of
these derivatives act as optimizing elements for s1-R ligand binding
affinity.
2. Results and discussion
2.1. Molecular modeling
To further rationalize the s1-R affinity of compound 1b we
adopted an in silico approach based on the combination of ligand-
based (3D-pharmacophore modeling) and receptor-based compu-
tational methodologies [22b,23,24] starting from the mapping of
the optimized model of 1b onto our 3D-pharmacophore model
(Fig. 2). To get further insight on the molecular interactions be-
tween compound 1b and its biological target we then docked
[22e25,27] the optimized structure of 1b in the putative binding
pocket of the 3D s1 receptor homology model [22,23] and esti-
mated the corresponding drug/protein free energy of binding
(DGbind) via MM/PBSA (Molecular Mechanics/Poisson-Boltzmann
Surface Area) calculations [29]. As exemplified in Fig. 3, all func-
tional groups identified by pharmacophore mapping establish
stabilizing interactions with the s1 receptor, confirming the idea
that the N-(p-chlorobenzyl)piperidine carboxamide (NpCPC) moi-
ety possesses the three prototypical binding requirements charac-
terizing potent s1 binders [22e24].
On the basis of the results summarized above, with the aim of
improving 1b s1-R selectivity without affecting its high affinity for
this receptor, in the present effort we designed, synthesized and
evaluated the binding constants for the new series of piperidine
carboxamide derivatives 2aeo shown in Scheme 1.
Indeed, the equilibrated MD trajectory of the s1-R/1b complex
As shown in Scheme 1, the N-benzylcarboxamide moiety in 1b
was replaced with various aliphatic or alicyclic moieties (2aei) or
with the tetrahydroquinoline and tetrahydroisoquinoline residues
(2jeo), respectively. Compounds 2aeo resulted from the applica-
tion of a computational procedure combining pharmacophore
modeling and receptor-based ligand design.
reveals the presence of stabilizing
p interactions between the p-
chlorobenzyl ring of 1b and the side chains of Trp121 and Arg119.
Moreover, the basic nitrogen is engaged in a persistent salt bridge
with the COOꢀ group of Asp126 while a stable hydrogen bond
between the donor hydroxyl group of Thr151 and the acceptor
counterpart in the amide moiety of compound 1b is also detected
during the entire course of the MD simulation. Of note, the hy-
drophobic pocket lined by the side chains of the receptor residues
Ile128, Phe133, and Tyr173 with the further stabilizing contribution
of Glu172 perfectly encase the unsubstituted phenyl ring of 1b. As
consequence of this favorable binding mode, MM/PBSA endowes
compounds 1b with a very good affinity towards the s1 receptor, as
testified by the calculated binding free energy value of ꢀ11.12 kcal/
mol (Table 1). As is often the case in protein/ligand binding, the
main favorable contribution to
Waals ( EVDW) and electrostatic (
phase, while polar solvation energies (
nents (ꢀT S) tend to oppose binding (Table 1).
D
Gbind is provided by the van der
EELE) components in the gas
GPB) and entropy compo-
D
D
D
D
The driving force leading to s1-R/1b complex formation was
further investigated by deconvoluting the free energy of binding on
a per-residue basis to generate the receptor/residue interaction
spectrum presented in Fig. 4.
Fig. 1. Structure of lead compounds 1a,b.

D. Zampieri et al. / European Journal of Medicinal Chemistry 90 (2015) 797e808
799
Scheme 1. Structure of compounds 2aeo.
Glu172, and Tyr173. In detail, the salt bridge between the piperidine
eNHþ atom and the side chain of Asp126 is responsible for a
favorable contribution to binding of ꢀ2.57 kcal/mol while the
hydrogen bond involving the hydroxyl group of Thr151 supports
the binding with an enthalpic contribution of ꢀ1.85 kcal/mol.
Moreover, the encasement of the aromatic 4-chlorophenyl ring by
the side chains of the s1 receptor residues Arg119, Tyr120 and
Trp121 contributes ꢀ2.90 kcal/mol of stabilizing van der Waals and
hydrophobic interactions. Finally, the insertion of the benzylcar-
boxamide ring into the binding cavity surrounding the s1-R residue
Ile128, Phe133, Glu172, and Tyr173 provides further, overall favor-
able contribution of ꢀ5.89 kcal/mol.
On the basis of these results we proceeded with the design of
new carboxamide derivatives able to maintain very good s1-R af-
finity. To the purpose, we considered that the N-(p-chlorobenzyl)
piperidine carboxamide scaffold and the corresponding in-
teractions (NpCPCi) were crucial molecular determinants for
effective s1-R binding. Thus, to preserve the aromatic characteris-
tics of the substituent in the structure of 1b, we initially chose to
modify the substituent on the amide nitrogen with a cycloalkyl
group (2b), a bulky or small alkyl moiety (2e and 2h, respectively),
and a tetrahydroquinoline (2k) and tetrahydroisoquinoline ring
(2n), respectively. In principle, all these new residues should be
efficiently encased in the s1 receptor binding pocket and thereby
establish the appropriate interactions with residues Ile128, Phe133,
Glu172, and Tyr173 as observed for compound 1b.
Fig. 3. Equilibrated MD snapshot of the s1 receptor in complex with 1b. The image is a
zoomed view of the receptor binding site. The ligand is portrayed in sticks-and-balls
and colored by element, while the protein residues mainly involved in the interac-
tion with 1b are highlighted as colored sticks and labeled. Salt bridges and H-bonds
interactions are shown as dotted black lines. Some water molecules and ions are
shown as transparent spheres colored by element.
As shown in Fig. 4, all ligand/receptor favorable interactions
discussed above are energetically confirmed and quantified by this
analysis: the major stabilizing contributions are indeed afforded by
the s1-R residues clustering in the region Arg119 e Phe133, besides
those yielded by a few other receptor residues such as Thr151,

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D. Zampieri et al. / European Journal of Medicinal Chemistry 90 (2015) 797e808
Table 1
computational procedure applied to 1b on the new derivatives and
predicted the relevant affinities towards the receptor. These results
are listed in Table 2.
According to our calculations, all new derivatives exhibited s1
receptor affinity values comparable to compound 1b (Table 2) with
the notable exception of the N-isopropyl derivative 2h, for which a
Binding free energy (DGbind) and its components for 1b in
complex with the s1 receptor. All energy values are in kcal/
mol. The experimental and calculated Ki values (nM) are also
reported for comparison.
Components
1b
D
D
D
D
D
D
D
EVDW
EELE
EMM
GPB
GNP
GSOL
Hbind
ꢀ48.33 0.09
ꢀ150.12 0.13
ꢀ198.45 0.16
164.98 0.15
ꢀ5.29 0.01
159.69 0.16
ꢀ38.76 0.23
27.64 0.26
strong decrease of the free energy of binding
were predicted. The best s1 binder of the series was the tetrahy-
droisoquinoline derivative 2n (
DGbind and the Kis1
D
Gbind ¼ ꢀ11.47 kcal/mol, Kis1(calc)
of 3.9 nM) for which the optimized conformation assumed in
complex within the putative binding pocket of s1 receptor is utterly
similar to that of its compound precursor 1b, as illustrated in
Fig. 5A, B (see SI for details on all other compounds).
ꢀT
DSbind
Gbind
D
ꢀ11.12 0.34
Substantially, the tetrahydroisoquinoline substituent allowed
preserving the favorable hydrophobic interactions within the re-
ceptor binding cavity without affecting the optimal binding pose
orientation of the NpCPC portion (Fig. 5A, B).
To quantify the effect of the different substituents on the affinity
toward the s1 receptor, the decomposition of the enthalpic
Kis1(exp)
12.9 0.8
7.1
a
Kis1(calc)
a
The Kis1
values were obtained from the corre-
Gbind values using the relationship
(calc)
sponding
ln(1/Ki).
D
D
Gbind ¼ ꢀRT
component of
DGbind was carried out on the entire series of these
newly designed compounds. To better rationalize these results, we
clustered the contributions of the s1-R residues mainly involved in
ligand binding in two subclasses (Fig. 5C), defined as follows: i) a
contribution afforded by the portion of the molecular structure left
unchanged, previously termed NpCPCi and contributed by residues
Arg119, Trp121, Asp126, and Thr151, and ii) another contribution
brought about by residues Ile128, Phe133, Glu172, and Tyr173
clustered together to represent the global effect of the structure
modification on the enthalpy-driven binding.
From Fig. 5C we can observe that the replacement of the N-
benzyl of compound 1b with a more rigid aromatic portion (com-
pounds 2k and 2n) did not affect both classes of binding in-
teractions while a slight decrease in binding stabilization is
detected for the non-aromatic derivatives 2b and 2e which, in turn,
reflects in a moderate reduction of the corresponding overall re-
Fig. 4. Per residue binding free energy decomposition for the s1 receptor in complex
with 1b. Only s1-R amino acids from position 100 to 200 are shown, as for all the
remaining protein residues the contribution to ligand binding is irrelevant.
ceptor affinities (
D
Gbind ¼ ꢀ10.15 kcal/mol and Kis1(calc) ¼ 36 nM
for 2b and
DGbind ¼ ꢀ9.78 kcal/mol and Kis1(calc) ¼ 68 nM for 2e,
respectively, Table 2). Conversely, the N-isopropyl substitution in
2h led to a strong decrement in favorable binding enthalpy: in fact,
the small aliphatic substituent cannot originate the required
network of hydrophobic interactions with the side chains of the
To validate this hypothesis, before engaging in the synthesis of
this new series of s1-R ligands we carried out the same
Table 2
Binding free energies
D
Gbind (kcal/mol) and predicted Kis1(calc) values (nM) for 1b, 2b, 2e, 2h, 2k and 2n in complex with the s1 receptor. Errors are given in parenthesis as
Gbind values (
Gbind ¼ ꢀRT ln(1/Kis1(calc))), are also reported.
standard errors of the mean. The calculated Kis1(calc) (nM) values, as estimated from the corresponding
D
D
Compound
R1
R2
DH (kcal/mol)
ꢀT
D
S (kcal/mol) Gbind (kcal/mol) is1(calc)[nM]
D
K
2b
Cl
ꢀ37.47 (0.20)
27.32 (0.28)
ꢀ10.15 (0.34)
36
2e
Cl
Cl
ꢀ37.03 (0.19)
27.25 (0.29)
ꢀ9.78 (0.35)
68
2h
ꢀ33.94 (0.23)
ꢀ38.01 (0.21)
26.03 (0.30)
27.11 (0.28)
ꢀ7.91 (0.38)
1600
10.3
2k
Cl
ꢀ10.90 (0.35)
2n
1b
Cl
ꢀ38.54 (0.22)
ꢀ38.76 (0.23)
27.07 (0.27)
27.64 (0.26)
ꢀ11.47 (0.34)
ꢀ11.12 (0.34)
3.9
7.1
e
e

D. Zampieri et al. / European Journal of Medicinal Chemistry 90 (2015) 797e808
801
Fig. 5. (A, B) Comparison between the optimized MD binding conformations within the s1 receptor putative binding site between compounds 1b (light see green) and 2n
(firebrick). In both panels, the ligands are portrayed as sticks-and-balls, while the protein residues mainly involved in the interactions with the derivatives are depicted in sticks,
labeled and colored accordingly. (C) Comparison of per-residue binding enthalpy decomposition for compounds 1b, 2b, 2e, 2h, 2k, and 2n in complex with the s1 receptor. Critical
receptor residues are clustered according to the specific underlying interactions as explained in the legend (see also main text for more details). (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
residues belonging to the second cluster in Fig. 5C. As a conse-
quence, the ligand assumes an unproductive pose, which exerts a
negative influence also on the NpCPCi portion of the receptor
binding site (see Figs. S1C and S2C).
TFA to afford intermediates 4aee. The final step was the N-alkyl-
ation of the piperidine nitrogen atom with different benzyl chlo-
rides to produce compounds 2aeo.
The encouraging results predicted by our in silico approach
discussed above thus prompted us to synthesize the new potential
s1-R ligands 2b, 2e, 2h, 2k and 2n and test their affinity for the s1
receptor in biological assays. Also, based on the previous experi-
mental and in silico results achieved with compound 1a
4. Receptor binding studies
The s1 and s2 receptor affinity of the test compounds was
determined in competition experiments by radiometric assays.
Compounds 2aeo were then tested at s1 and s2 receptors of animal
origin prepared from guinea pig brain and rat liver, respectively.
The principles of these receptor binding studies are reported in the
pharmacology section below; s1-R assays were performed with
[3H]-(þ)-pentazocine as radioligand while [3H]-DTG was used as
radioligand in the s2 receptor assays.
The collected affinity results for the new derivatives 2aeo are
reported in Table 4. As we can see, all compounds are provided with
high s1-R/s2-R selectivity and some of those exhibited a s1-R af-
finity very similar to reference compound 1b, in agreement with
the in silico predictions. In particular, it was confirmed that the
introduction of another chlorine atom in the ortho position dras-
tically reduces the s1-R affinity of all 2,4-dichlorosubstituted de-
rivatives (2c, 2f, 2i, 2l, and 2o) while it seems not to affect the
affinity for the s2 protein. In fact, the Kis1 values of these molecules
are at least one order of magnitude higher compared to those of the
corresponding unsubstituted and 4-chloro substituted derivatives.
Pleasingly, the introduction of a cyclohexyl moiety (2aeb) or a
bulky alkyl chain (2dee) on the amide nitrogen atom led to a
substantial preservation of the s1-R affinity (7.7e20 nM) paralleled
by a strong increment in the selectivity toward the s2-R subtype.
Indeed, among the derivative subset 2aee, compound 2b exhibited
the best s1 receptor affinity (Ki(s1) ¼ 7.7 nM) and highest selec-
tivity (Kis2/Kis1 ¼ 234). On the other hand as predicted by
(D
Gbind ¼ ꢀ10.24 kcal/mol, Kis1(calc) ¼ 31 nM, and Kis1 ¼ 22.5 nM,
[24]), we chose to complete the series of the new carboxamide
molecules by synthesizing the corresponding unsubstituted N-
benzyl-piperidine derivatives 2a, 2d, 2g, 2j, and 2m. Moreover, we
further added to the series the 2,4-dichloro derivatives substituted
on the same aromatic ring (2c, 2f, 2i, 2l, and 2o) since our previous
works [22a,b] revealed that this modification decreases the s1-R
affinity of the relevant compounds without affecting their s2 re-
ceptor binding capability. In this way, we could also have a defin-
itive confirmation of the alleged hypothesis according to which the
effect on s1-R ligand selectivity in the present series of compounds
is borne exclusively by the structural modifications suggested by
the computer-based drug-design approach.
3. Chemistry
The new piperidine-4-carboxamide derivatives 2aeo (Table 3)
have been prepared (Scheme 2) starting from the commercially
available 4-piperidinecarboxylic acid, which was first protected on
nitrogen atom with common Boc-anhydride and subsequently
treated with SOCl2 to afford the corresponding acyl chloride. The
various amides 3aee were obtained in situ using the corresponding
amines in presence of Et3N and DMAP and then deprotected with

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D. Zampieri et al. / European Journal of Medicinal Chemistry 90 (2015) 797e808
Table 3
s1-R ligands 2aeo by testing them against other receptor systems
Characterization of derivatives 2aeo.
such as NMDA (Table 4). As we can see from the affinity values
reported in Table 4, none of the compounds exhibited considerable
NMDA affinity, highlighting once again their intrinsic preference
towards s1 receptors.
5. Conclusions
Cpd R1
R2
H
Yield (%) M.p. (ꢁC) CHN
Compound 1b, endowed with high s1 receptor affinity
(Kis1 ¼ 12.9 nM) has been chosen as lead compound for compu-
tational design and subsequent synthesis of a new series of de-
rivatives 2aeo, with the aim of improving the original low
selectivity with respect to the s2 receptor subtype. In the design
process we maintained the N-p-chlorobenzylpiperidine-4-
carboxamide scaffold of 1b, whereas the benzyl moiety linked to
the amide nitrogen atom has been replaced with cycloalkyl (2b)
and alkyl (2e and 2h) groups or with residues containing an aro-
matic ring as the 3,4-dihydroquinolin-1(2H)-yl (2k) or 3,4-
dihydroisoquinolin-2(1H)-yl moieties (2n). Once synthesized and
characterized the new series of potential s1-R binders, we evalu-
2a
26.3
131-3
165-7
183-5
Oil
C19H28N2O
N
H
2b
2c
2d
4-Cl
33.1
C19H27ClN2O
C19H26Cl2N2O
C20H32N2O
N
H
2,4(Cl)2 24.4
N
H
H
95.4
48.4
2e
2f
4-Cl
84-6
C20H31ClN2O
C20H30Cl2N2O
C16H24N2O
ated their biological affinity against
s receptors and NMDA receptor
(PCP site).
2,4(Cl)2 25.7
89-91
143-5
From the view point of the s1-R ligand molecular structure re-
quirements, the main results of our combined in silico/in vitro ef-
forts can be summarized as follows:
2g
H
34.9
43.3
(i) with respect to the lead compound 1b, hydrophobic moieties
as bulky alkyl, cycloalkyl or residues containing an aromatic ring
linked to the amide nitrogen atom preserved good s1-R affinity and,
at the same time, improved selectivity towards the s2 receptor; (ii)
small alkyl groups (e.g., isopropyl) are not able to generate the
necessary hydrophobic interaction within the receptor binding
pocket for an optimal binding, so that the corresponding de-
rivatives were almost devoid of s1-R affinity; and (iii) the 2,4-
dichloro substitution on the N-benzylpiperidine moiety strongly
reduced the s1-R affinity.
The data presented in this work constitute an important starting
point for the design and synthesis of new s1 receptor binders able
to establish optimized, stabilizing interactions with both clusters of
receptor residues mainly involved in protein/ligand complex
formation.
2h
2i
4-Cl
167-9
140-2
Oil
C16H23ClN2O
C16H22Cl2N2O
C22H26N2O
2,4(Cl)2 39.8
2j
H
62.6
91.1
2k
2l
4Cl
Oil
C22H25ClN2O
C22H24Cl2N2O
Lastly, due to their specificity the best compounds of this new
carboxamide series could be exploited in further, specific biological
tests on the s1 receptor activity with the purpose of obtaining more
information about the pharmacological pathways of this very
interesting target.
2,4(Cl)2 91.9
104-7
2m
2n
2o
H
43.2
63.6
82-5
Oil
C22H26N2O
6. Experimental
4-Cl
C22H25ClN2O
C22H24Cl2N2O
6.1. Computational details
2,4(Cl)2 94.6
Oil
The optimized structure of selected compounds 2b, 2c, 2h, 2k
and 2n was docked into the s1-R putative binding pockets by
applying a consolidated procedure [23e26,29]. All docking exper-
iments were performed with Autodock 4.3/Autodock Tools 1.4.6 [31]
on a win64 platform. The resulting docked conformations were
clustered and visualized; then, for each compound, only the mo-
lecular conformation satisfying the combined criteria of having the
lowest (i.e., more favorable) Autodock energy and belonging to a
highly populated cluster was selected to carry for further modeling.
The ligand/s1-R complex obtained from the docking procedure
was further refined in Amber 12 [31] using the quenched molecular
dynamics (QMD) method as previously described [23e26,29]. Ac-
cording to QMD, the best energy configuration of each complex
resulting from this step was subsequently solvated by a cubic box of
TIP3P [33] water molecules extending at least 10 Å in each direction
modeling head of synthesis, compounds 2gei, in which the car-
boxamide nitrogen atom has been linked to a small isopropyl
group, are almost devoid of receptor affinity. However, the best
result was achieved with the introduction of an aromatic scaffold
on the nitrogen atom: 3,4-dihydroquinoline-1(2H)-yl derivatives
2jek and 3,4-dihydroisoquinoline-2(1H)-yl derivatives 2men are,
in effect, the most active compounds of the entire series, with Kis1
values of 4.6, 3.7, 8.8, and 8.0 nM respectively. Importantly, the
selectivity of these derivatives is very high, with compound 2j
endowed with the best Kis2/Kis1 ratio (>435).
As a final step we checked the selectivity spectrum of the new

D. Zampieri et al. / European Journal of Medicinal Chemistry 90 (2015) 797e808
803
Scheme 2. Reagents and conditions: a) Boc2O, K2CO3, THF/H2O, 4 h rt; b) SOCl2/Py; c) ReNHeR0, Et3N, DMAP, CH2Cl2, 14 h rt; d) TFA 24 h, rt; e) Acetone, K2CO3, 4 h, reflux.
from the solute. The system was neutralized and the solution ionic
strength was adjusted to the physiological value of 0.15 M by
adding the required amounts of Naþ and Clꢀ ions. Each solvated
system was relaxed by 500 steps of steepest descent followed by
500 other conjugate-gradient minimization steps and then gradu-
ally heated to a target temperature of 300 K in intervals of 50 ps of
NVT MD, using a Verlet integration time step of 1.0 fs. The Langevin
thermostat was used to control temperature, with a collision fre-
quency of 2.0 psꢀ1. The protein was restrained with a force constant
of 2.0 kcal/(mol Å), and all simulations were carried out with pe-
riodic boundary conditions. Subsequently, the density of the system
was equilibrated via MD runs in the isothermalꢀisobaric (NPT)
ensemble, with isotropic position scaling and a pressure relaxation
time of 1.0 ps, for 50 ps with a time step of 1 fs. All restraints on the
protein atoms were then removed, and each system was further
equilibrated using NPT MD runs at 300 K, with a pressure relaxation
time of 2.0 ps. Three equilibration steps were performed, each 2 ns
long and with a time step of 2.0 fs. To check the system stability, the
fluctuations of the rmsd of the simulated position of the backbone
atoms of the s1 receptor with respect to those of the initial protein
were monitored. All chemicophysical parameters and rmsd values
showed very low fluctuations at the end of the equilibration pro-
cess, indicating that the systems reached a true equilibrium
condition.
the s1 receptor was estimated by resorting to the MM/PBSA
approach implemented in Amber 12. According to this well-
validated methodology [23e26,29e34], the free energy was
calculated for each molecular species (complex, receptor, and
ligand), and the binding free energy was computed as the
difference:
DGbind ¼ Gcomplex ꢀ ðGreceptor þ GligandÞ ¼ DEMM þ DGsol ꢀ TDS
in which
DEMM represents the molecular mechanics energy,
D
Gsol
includes the solvation free energy and TDS is the conformational
entropy upon ligand binding.
The per residue binding free energy decomposition was per-
formed exploiting the MD trajectory of each given compound/s1-R
complex, with the aim of identifying the key residues involved in
the ligandereceptor interaction. This analysis was carried out using
the MM/GBSA approach [35], and was based on the same snapshots
used in the binding free energy calculation.
All simulations were carried out using the Pmemd modules of
Amber 12, running on the EURORA-CPU/GPU calculation cluster of
the CINECA supercomputer facility (Bologna, Italy). The entire MD
simulation and data analysis procedure was optimized by inte-
grating Amber 12 in modeFRONTIER, a multidisciplinary and mul-
tiobjective optimization and design environment [36].
The equilibration phase was followed by a data production run
consisting of 40 ns of MD simulations in the canonical (NVT)
ensemble. Only the last 20 ns of each equilibrated MD trajectory
were considered for statistical data collections. A total of 1000
trajectory snapshots were analyzed the each ligand/receptor
complex.
6.2. Chemistry, general methods
Melting points were determined with a Buchi 510 capillary
apparatus, and are uncorrected. Infrared spectra in nujol mulls
were recorded on a Perkin Elmer Spectrum RXI. Proton nuclear
magnetic resonance (1H NMR) spectra were determined on a
The binding free energy,
DGbind, between the two ligands and

804
D. Zampieri et al. / European Journal of Medicinal Chemistry 90 (2015) 797e808
disappearing on deuteration). MS: m/z 327 [MHþ].
Table 4
Affinities towards s1-R, s2-R and NMDA (PCP site) of the synthesized compounds
2aeo and haloperidol, DTG, and MK.801 as reference compounds. In case of NMDA
the inhibition of the radioligand [3H]-MK-801 at a concentration of 1
respective test compound is given.
mM of the
6.2.3. tert-Butyl 4-(isopropylcarbamoyl)piperidine-1-carboxylate
3c
Oil Yield (%): 67. 1H NMR (CDCl3/TMS)
d: 1.11 (d, 6H, 2(CH3)
Cpd
Ki SEM (nM)a
s2-R/s1-R selectivity
Kis2/Kis1
NMDA (PCP)
% Inhibition
isopr., J ¼ 6.59 Hz); 1.40e2.00 (m, 4H, CH2, pip.); 1.43 (s, 9H, 3(CH3),
Boc); 2.04e2.55 (m, 1H, CH, pip.); 2.76 (m, 2H, CH2, pip.); 4.06 (m,
3H, CH isopr., CH2 pip.) 5.41 (d,1H, NH disappearing on deuteration,
J ¼ 7.32 Hz). MS: m/z 271 [MHþ].
s1
s2
2a
2b
2c
2d
2e
2f
15.0
7.7 1.4
52.0 16
19.0
20.0
125
1
>2000
1800
>2000
>2000
622
>133
234
>38
>105
31.1
9.6
10
11
20
29
36
17
1
1
6.2.4. tert-Butyl 4-(1,2,3,4-tetrahydroquinoline-1-carbonyl)
1200
2g
2h
2i
2j
2k
2l
2m
2n
1070
637
>2000
4.6 1.2
3.7 0.1
138
8.8 0.2
8.0 2.9
>2000
>2000
>2000
>2000
1300
>2000
1200
627
>2
>3
33
42
24
19
34
22
42
37
piperidine-1-carboxylate 3d
Oil Yield (%): 88. 1H NMR (CDCl3/TMS)
d
0
: 1.38 (s, 9H, 3(CH3),
n.d.b
>435
351
>14
136
78
Boc); 1.50e1.80 (m, 4H, 2(CH2), pip. and H3,3 tetrahydroq.); 1.90 (t,
2H, CH2, pip., J ¼ 6.59 Hz); 2.44e2.70 (t and m, 4H, 2(CH2), pip. and
0
H4,4 tetrahydroq., J ¼ 6.59 Hz); 2.93 (m, 1H, CH, pip.); 3.72 (m, 2H,
0
CH2, H2,2 tetrahydroq., J ¼ 6.59 Hz) 4.04 (d, 2H, CH2, pip.); 7.20 (m,
4H, arom.). MS: m/z 345 [MHþ].
2o
61.0
7
662
11
13
1b
12.9 0.8
6.6 0.9
146
78 2.0
54
n.d.
11
12
0.8
e
e
Haloperidol
DTG
MK-801
n.d.b
n.d.b
3.4 0.8
6.2.5. tert-Butyl 4-(1,2,3,4-tetrahydroisoquinoline-2-carbonyl)
piperidine-1-carboxylate 3e
71
8
8
n.d.
Oil Yield (%): 92. 1H NMR (CDCl3/TMS)
Boc); 1.65 (m, 4H, CH2, pip.); 2.60e2.80 (m, 5H, CH and 2(CH2), pip.
d
: 1.38 (s, 9H, 3(CH3),
a
Triplicates were performed only for high affinity compounds (<100 nM).
Not determined.
b
0
0
and H4,4 tetrahydroisoq.); 3.67 (m, 2H, CH2, H3,3 tetrahydroisoq.,
0
J ¼ 5.86 Hz); 4.10 (d, 2H, CH2, pip.); 6.64 (d, 2H, H1,1 tetrahydroisoq.,
J ¼ 10.98 Hz); 7.10 (m, 4H, arom.). MS: m/z 345 [MHþ].
Varian Gemini 200 spectrometer, chemical shifts are reported as
(ppm) in CDCl3 solution (0.05% v/v TMS). Reaction courses and
d
product mixtures were routinely monitored by thin-layer chro-
matography (TLC) on silica gel precoated F254 Merck plates gener-
ally with CHCl3/EtOH (9:1) as eluent phase. ESI-MS spectra were
obtained on a PE-API I spectrometer by infusion of a solution of the
sample in MeOH. Elemental analyses (C, H, N) were performed on a
Carlo Erba analyzer and were within 0.3 of the theoretical value.
All the commercially available reactants and solvents were
purchase from SigmaeAldrich, Fluka Chemicals and Merk.
6.2.6. General procedure for the deprotection of various
carboxamide into corresponding derivatives 4aee
Trifluoroacetic acid (5 mL) was added to 0.50 g (1.61 mmol) of
the compound 4a and the reaction was stirred, under N2 atmo-
sphere, overnight. The excess of trifluoroacetic acid was eliminated
under reduced pression and the residue was taken by water and
washed with diethyl ether. The aqueous layer was alcalinizated to
pH 12 (NaOH 10%) and extracted with CH2Cl2. The organic phase
was then washed with distilled water, dried over anhydrous Na2SO4
and filtered. A light-yellow solid of N-cyclohexylpiperidine-4-
carboxamide 4a was obtained (0.21 g; yield (%): 62); mp
163e167 ꢁC.
6.2.1. General procedure for the preparation of various 1-(tert-
butoxycarbonyl)piperidine-4-carboxamide derivatives 3aee
To a mixture of N-Boc-4-piperidinecarboxylic acid (3.00 g,
13.1 mmol), pyridine (2.59 g, 32,7 mmol), CH2Cl2 (40 mL) and SOCl2
(1.87 g, 15.7 mmol) were added, under N2 atmosphere at room
temperature, while stirring. After 30 min, a solution of cyclohex-
ylamine (1.43 g, 14.4 mmol), Et3N (4.24 g, 41.9 mmol), and a cata-
lytic amount of DMAP in CH2Cl2 (20 mL) was added dropwise. The
reaction was monitored by TLC. After 12 h, the organic phase was
washed with 1 N HCl (2 ꢂ 20 mL) and distilled water (2 ꢂ 20 mL),
dried with Na2SO4, and concentrated in vacuum to give 3.25 g (80%)
of tert-butyl 4-(cyclohexylcarbamoyl)piperidine-1-carboxylate 3a
as a light-brown solid; mp 129e133 ꢁC.
1H NMR (CDCl3-TMS) ppm (
d): 1.02e2.00 (m 14H, cyclohex. and
pip., NH pip.); 2.10 (m, 1H, CH, pip.); 2.54 (t, 2H, CH2, pip.); 3.10 (d,
2H, CH2, pip.); 3.68 (m,1H, CH cyclohex.); 4.20 (broad sign.,1H, (CO)
NH, disappearing on deuteration). MS: m/z 211 [MHþ].
In the same way the following compounds 4bee were obtained.
6.2.7. N-(heptan-2-yl)piperidine-4-carboxamide 4b
Oil Yield (%): 46. 1H NMR (CDCl3-TMS) ppm (
d): 0.80 (t, 3H, CH3,
hept., J ¼ 6.59 Hz); 1.03 (d, 3H, CH3, hept., J ¼ 6.59 Hz); 1.13e1.84
(m, 12H, 6(CH2) hept. and pip.); 2.02e2.26 (m, 1H, CH pip.); 2.18
(broad sign., 1H, NH, pip., disapp. on deuteration); 2.56 (t, 2H, CH2,
pip., J ¼ 12.08 Hz); 3.08 (d, 2H, CH2, pip., J ¼ 12.08 Hz); 3.91 (m, 1H,
CH, hept.); 5.30 (d, 1H, (CO)NH disappearing on deuteration). MS:
m/z 227 [MHþ].
1H NMR (CDCl3-TMS) ppm (
d): 1.02e2.00 (m, 14H, cyclohex. and
pip.); 1.38 (s, 9H, 3(CH3), Boc); 2.10 (m, 1H, CH, pip., J ¼ 8.05 Hz);
2.66 (t, 2H, CH2, pip.); 3.68 (m, 1H, CH cyclohex.); 4.06 (m, 2H, CH2,
pip.); 4.23 (broad sign., 1H, (CO)NH, disappearing on deuteration).
MS: m/z 311 [MHþ].
In an analogous way the following compounds 3bee were
obtained.
6.2.8. N-isopropylpiperidine-4-carboxamide 4c
Light-yellow solid; M.p.: 125e127 ꢁC; Yield (%): 60. 1H NMR
6.2.2. tert-Butyl 4-(heptan-2-ylcarbamoyl)piperidine-1-
(CDCl3/TMS) ppm (
d
): 1.07 (d, 6H, 2(CH3), isopr., J ¼ 6.59 Hz);
carboxylate 3b
1.13e2.20 (m, 4H, CH2, pip.); 2.01 (d broad, 1H, NH, disappearing on
deuteration); 2.13 (m, 1H, CH, pip.); 2.57 (t, 2H, CH2, pip.,
J ¼ 12.45 Hz); 3.08 (d, 2H, CH2, pip., J ¼ 12.45 Hz); 4.02 (m, 1H, CH
isopr.) 5.24 (broad sign., 1H, NH disappearing on deuteration,
J ¼ 7.32 Hz). MS: m/z 171 [MHþ].
Oil Yield (%): 72. 1H NMR (CDCl3/TMS)
d: 0.85 (t, 3H, CH3, hept.,
J ¼ 6.59 Hz); 1.09 (d, 3H, CH3, hept., J ¼ 6.50 Hz); 1.18e2.20 (m, 12H,
hept. and pip.); 1.44 (s, 9H, 3(CH3), Boc); 2.10e2.50 (m, 1H, CH,
pip.); 2.80 (m, 2H, CH2, pip.); 4.00 (m, 4H, CH hept., CH2 pip. and NH

D. Zampieri et al. / European Journal of Medicinal Chemistry 90 (2015) 797e808
805
6.2.9. (3,4-Dihydroquinolin-1(2H)-yl) (piperidin-4-yl)methanone
N, 8.85%; found: C, 75.80; H, 9.90; N, 8.60%.
4d
Oil Yield (%): 82. 1H NMR (CDCl3/TMS) ppm (
d
): 1.40e1.80 (m,
6.2.15. 1-(4-Chlorobenzyl)-N-(heptan-2-yl)piperidine-4-
0
5H, 2(CH2), pip. and H3,3 tetrahydroq., NH); 1.90 (t, 2H, CH2, pip.,
carboxamide 2e
0
J ¼ 6.59 Hz); 2.44 (dt, 2H, CH2, pip.); 2.64 (t, 2H, CH2, H4,4 tetra-
White solid, melting point: 84e86 ꢁC. Yield (%): 48. I.R. cmꢀ1
hydroq., J ¼ 6.59 Hz); 2.80e2.91 (m, 3H, CH and CH2, pip.); 3.72 (m,
(nujol): 1635, 3251. 1H NMR (CDCl3/TMS) ppm (
d): 0.80 (t, 3H, CH3,
0
2H, CH2, H2,2 tetrahydroq., J ¼ 6.59 Hz) 7.11 (m, 4H, arom.). MS: m/z
hept., J ¼ 6.10 Hz); 1.10 (d, 3H, CH3, hept., J ¼ 6.71 Hz); 1.10e2.10 (m,
15H, 7(CH2), CH hept. and pip.); 2.84 (d, 2H, CH2, pip.); 3.39 (s, 2H,
eCH2eAr); 3.92 (m, 1H, CH, hept., J ¼ 6.71 and 7.93 Hz); 5.20 (d
broad, 1H, (CO)NH disappearing on deuteration, J ¼ 7.93 Hz); 7.20
(m, 4H, arom.). MS: m/z 351 [MHþ], 353 [MHþþ2]. Anal. calcd. for
245 [MHþ].
6.2.10. (3,4-Dihydroisoquinolin-2(1H)-yl) (piperidin-4-yl)
methanone 4e
Oil Yield (%): 70. 1H NMR (CDCl3/TMS) ppm (
d
): 1.68 (m, 4H,
C20H31ClN2O (MW 350.93): C, 68.45; H, 8.90; N, 7.98%; found: C,
2(CH2), pip.); 2.42 (broad sign., 1H, NH disapp. on deuteration);
68.50; H, 8.90; N, 8.20%.
0
2.55e2.90 (m, 5H, CH and 2ꢂ CH2, pip. and H4,4 tetrahydroisoq.);
0
3.12 (d, 2H, CH2, pip.); 3.67 (m, 2H, CH2, H3,3 tetrahydroisoq.,
6.2.16. 1-(2,4-Dichlorobenzyl)-N-(heptan-2-yl)piperidine-4-
carboxamide 2f
J ¼ 10.98 Hz); 7.12 (m, 4H, arom.). MS: m/z 245 [MHþ].
White solid, melting point: 89e91 ꢁC. Yield (%): 26. I.R. cmꢀ1
6.2.11. Synthesis of the final piperidine-4-carboxamide derivatives
2aeo
(nujol): 1635, 3284. 1H NMR (CDCl3/TMS) ppm (
d): 0.80 (t, 3H, CH3,
hept., J ¼ 6.10 Hz); 1.10 (d, 3H, CH3, hept., J ¼ 6.71 Hz); 1.10e2.10 (m,
15H, 7 (CH2), CH, hept. and pip.); 2.87 (d, 2H, CH2, pip.); 3.49 (s, 2H,
eCH2eAr); 3.93 (m, 1H, CH, hept., J ¼ 6.71 and 7.93 Hz); 5.17 (d
broad, 1H, (CO)NH disappearing on deuteration, J ¼ 7.93 Hz); 7.15
(dd, 1H, H5, arom., J ¼ 8.54 Hz); 7.28 (d, 1H, H4, arom.); 7.15 (d, 1H,
H2, arom., J ¼ 8.54 Hz). MS: m/z 385 [MHþ], 387 [MHþþ2]. Anal.
calcd. for C20H31Cl2N2O (MW 385.37): C, 62.33; H, 7.85; N, 7.27%;
found: C, 62.10; H, 7.60; N, 7.00%.
Compound 4a (0.16 g, 0.76 mmol) and K2CO3 (0.13 g, 0.91 mmol)
was dissolved in 50 mL of acetone and 0.10 g (0.76 mmol) of benzyl
chloride was added to the solution. The reaction was allowed to
stirring under reflux for 4 h (monitored by TLC) then solvent was
eliminated under vacuum and the residue was washed with water
then with diethyl ether to afford a white solid of 1-benzyl-N-
cyclohexylpiperidine-4-carboxamide 2a.
Melting point: 131e133 ꢁC. Yield (%): 26. I.R. cmꢀ1 (nujol): 1632,
3221. 1H NMR (CDCl3/TMS) ppm (
d): 0.90e2.00 (m, 17H, cyclohex.
6.2.17. 1-Benzyl-N-isopropylpiperidine-4-carboxamide 2g
and pip.); 2.88 (d, 2H, CH2, pip.); 3.43 (s, 2H, eCH2ePh); 3.70 (m,
1H, CH, cyclohex.); 5.26 (broad sign., 1H, NH, disapp. on deutera-
tion); 7.25 (m, 5H, arom.). MS: m/z 301 [MHþ]. Anal. calcd. for
Light-yellow solid, melting point: 143e145 ꢁC. Yield (%): 35. I.R.
cmꢀ1 (nujol): 1632, 3244. 1H NMR (CDCl3/TMS) ppm (
d): 1.15 (d, 6H,
2(CH3), isopr., J ¼ 6.59 Hz); 1.70e2.10 (m, 7H, 3(CH2), CH, pip.); 2.90
(d, 2H, CH2, pip.); 3.51 (s, 2H, eCH2ePh); 4.08 (m, 1H, CH, isopr.,
J ¼ 6.59 Hz); 5.30 (s broad, 1H, (CO)NH disappearing on deutera-
tion); 7.30e7.35 (m, 5H, arom.). MS: m/z 261 [MHþ]. Anal. calcd. for
C
19H28N2O (MW 300.44): C, 75.96; H, 9.39; N, 9.32%; found: C,
73.80; H, 9.20; N, 9.10%.
In a similar way, starting from compounds 4bee and benzyl, 4-
chlorobenzyl and 2,4-dichlorobenzyl chloride respectively, de-
rivatives 2beo were obtained.
C16H24N2O (MW 260.37): C, 73.81; H, 9.29; N, 10.76%; found: C,
73.80; H, 9.10; N, 10.60%.
6.2.12. 1-(4-Clorobenzyl)-N-cyclohexylpiperidine-4-carboxamide
6.2.18. 1-(4-Chlorobenzyl)-N-isopropylpiperidine-4-carboxamide
2b
2h
White solid, melting point: 165e167 ꢁC. Yield (%): 33. I.R. cmꢀ1
Light-yellow solid, melting point: 167e169 ꢁC. Yield (%): 43. I.R.
(nujol): 1627, 3239. 1H NMR (CDCl3/TMS) ppm (
d): 0.90e2.00 (m,
cmꢀ1 (nujol): 1629, 3249. 1H NMR (CDCl3/TMS) ppm (
d): 1.14 (d, 6H,
17H, cyclohex. and pip.); 2.81 (d, 2H, CH2, pip.); 3.40 (s, 2H,
eCH2eAr); 3.70 (m, 1H, CH, cyclohex.); 5.22 (broad sign., 1H, NH,
disapp. on deuteration); 7.20 (m, 4H, arom.). MS: m/z 335 [MHþ],
337 [MHþþ2]. Anal. calcd. for C19H27ClN2O (MW 334.88): C, 75.96;
H, 9.39; N, 9.32%; found: C, 73.80; H, 9.20; N, 9.10%.
2(CH3) isopr., J ¼ 6.59 Hz); 1.60e2.10 (m, 7H, 3(CH2), CH, pip.); 2.90
(d, 2H, CH2, pip.); 3.46 (s, 2H, eCH2ePh); 4.09 (m, 1H, CH, isopr.,
J ¼ 6.59 Hz); 5.30 (s broad, 1H, (CO)NH disappearing on deutera-
tion); 7.28 (m, 4H, arom.). MS: m/z 295 [MHþ], 297 [MHþþ2]. Anal.
calcd. for C16H23ClN2O (MW 294.82): C, 65.18; H, 7.86; N, 9.50%;
found: C, 64.90; H, 7.70; N, 9.50%.
6.2.13. 1-(2,4-Diclorobenzyl)-N-cyclohexylpiperidine-4-
carboxamide 2c
6.2.19. 1-(2,4-Diclorobenzyl)-N-isopropylpiperidine-4-carboxamide
White solid, melting point: 183e185 ꢁC. Yield (%): 25. I.R. cmꢀ1
2i
(nujol): 1639, 3280. 1H NMR (CDCl3/TMS) ppm (
d
): 0.90e2.10 (m,
Light-yellow solid, melting point: 140e142 ꢁC. Yield (%): 40. I.R.
17H, cyclohex. and pip.); 2.84 (d, 2H, CH2, pip.); 3.50 (s, 2H,
eCH2eAr); 3.70 (m, 1H, CH, cyclohex.); 5.22 (broad sign., 1H, NH,
disapp. on deuteration); 7.12e7.42 (m, 3H, arom.). MS: m/z 369
[MHþ], 371 [MHþþ2]. Anal. calcd. for C19H26Cl2N2O (MW 369.33):
C, 61.79; H, 7.10; N, 7.58%; found: C, 61.60; H, 7.00; N, 7.30%.
cmꢀ1 (nujol): 1638, 3286. 1H NMR (CDCl3/TMS) ppm (
d): 1.09 (d, 6H,
2(CH3) isopr., J ¼ 5.86 Hz); 1.50e2.10 (m, 7H, 3(CH2), CH, pip.); 2.90
(d, 2H, CH2, pip.); 3.50 (s, 2H, eCH2ePh); 4.10 (m, 1H, CH, isopr.,
J ¼ 5.86 Hz); 5.20 (s broad, 1H, (CO)NH disappearing on deutera-
tion); 7.20e7.40 (m, 3H, arom.). MS: m/z 329 [MHþ], 331 [MHþþ2].
Anal. calcd. for C16H22Cl2N2O (MW 329.26): C, 58.36; H, 6.73; N,
8.51%; found: C, 58.94; H, 6.70; N, 8.70%.
6.2.14. 1-Benzyl-N-(heptan-2-yl)piperidine- 4-carboxamide 2d
Oil Yield (%): 95. I.R. cmꢀ1 (nujol): 1634, 3269. 1H NMR (CDCl3/
TMS) ppm (
d
): 0.80 (t, 3H, CH3, hept., J ¼ 5.86 Hz); 1.10 (d, 3H, CH3,
6.2.20. (1-Benzylpiperidin-4-yl) (3,4-dihydroquinolin-1(2H)-yl)
hept., J ¼ 6.59 Hz); 1.10e2.00 (m, 15H, 7(CH2), CH hept. and pip.);
2.90 (d, 2H, CH2, pip.); 3.46 (s, 2H, eCH2ePh); 3.90 (m, 1H, CH,
hept., J ¼ 6.59 and 7.32 Hz); 5.20 (d broad, 1H, (CO)NH disappearing
on deuteration, J ¼ 7.93 Hz); 7.10e7.30 (m, 5H, arom.). MS: m/z 317
[MHþ]. Anal. calcd. for C20H32N2O (MW 316.48): C, 75.90; H, 10.19;
methanone 2j
Oil Yield (%): 62. I.R. cmꢀ1 (nujol): 1660. 1H NMR (CDCl3/TMS)
0
ppm (
d
): 1.10e1.90 (m, 9H, H3,3 dihydroq. and 3(CH2), CH pip.); 2.63
0
(t, 2H, H4,4 dihydroq., J ¼ 6.71 Hz); 2.82 (d, 2H, CH2, pip.); 3.39 (s,
0
2H, eCH2ePh); 3.70 (t, H2,2 dihydroq., J ¼ 6.71 Hz.); 7.10e7.40 (m,

806
D. Zampieri et al. / European Journal of Medicinal Chemistry 90 (2015) 797e808
9H, arom.). MS: m/z 335 [MHþ]. Anal. calcd. for C22H26N2O (MW
334.45): C, 79.00; H, 7.84; N, 8.38%; found: C, 78.80; H, 7.70; N,
8.10%.
kind donation of the local slaughterhouse (Coesfeld, Germany).
Homogenizers: Elvehjem Potter (B. Braun Biotech International,
Melsungen, Germany) and (Soniprep 150, MSE, London, UK). Cen-
trifuges: Cooling centrifuge model Rotina 35R (Hettich, Tuttlingen,
Germany) and High-speed cooling centrifuge model Sorvall RC-5C
plus (Thermo Fisher Scientific, Langenselbold, Germany). Multi-
plates: standard 96-well multiplates (Diagonal, Muenster, Ger-
many). Shaker: self-made device with adjustable temperature and
tumbling speed (scientific workshop of the institute). Harvester:
MicroBeta FilterMate-96 Harvester. Filter: Printed Filtermat Typ A
and B. Scintillator: Meltilex (Typ A or B) solid state scintillator.
Scintillation analyzer: MicroBeta Trilux (all Perkin Elmer LAS,
Rodgau-Jügesheim, Germany).
6.2.21. (1-(4-Chlorobenzyl)piperidin-4-yl) (3,4-dihydroquinolin-
1(2H)-yl)methanone 2k
Oil Yield (%): 91. I.R. cmꢀ1 (nujol): 1631. 1H NMR (CDCl3/TMS)
0
ppm (
d
): 1.10e1.90 (m, 9H, H3,3 dihydroq. and 3(CH2), CH pip.); 2.60
0
(t, 2H, H4,4 dihydroq., J ¼ 6.71 Hz); 2.80 (d, 2H, CH2, pip.); 3.40 (s,
0
2H, eCH2eAr); 3.67 (t, H2,2 dihydroq., J ¼ 6.71 Hz.); 7.00e7.20 (m,
8H, arom.). MS: m/z 369 [MHþ], 371 [MHþþ2]. Anal. calcd. for
C
22H25ClN2O (MW 368.90): C, 71.63; H, 6.83; N, 7.59%; found: C,
71.70; H, 6.90; N, 7.70%.
6.2.22. (1-(2,4-Dichlorobenzyl)piperidin-4-yl) (3,4-
dihydroquinolin-1(2H)-yl)methanone 2l
Brown solid, melting point: 104e107 ꢁC. Yield (%): 92. I.R. cmꢀ1
0
7.2. Preparation of membrane homogenates from guinea pig brain
5
guinea pig brains were homogenized with the potter
(nujol): 1643. 1H NMR (CDCl3/TMS) ppm (
d): 1.50e2.00 (m, 6H, H3,3
(500e800 rpm, 10 up-and-down strokes) in 6 volumes of cold
0.32 M sucrose. The suspension was centrifuged at 1200ꢂ g for
10 min at 4 ꢁC. The supernatant was separated and centrifuged at
23,500ꢂ g for 20 min at 4 ꢁC. The pellet was resuspended in 5e6
volumes of buffer (50 mM TRIS, pH 7.4) and centrifuged again at
23,500ꢂ g (20 min, 4 ꢁC). This procedure was repeated twice. The
final pellet was resuspended in 5e6 volumes of buffer and stored
at ꢀ80 ꢁC in 1.5 mL portions containing about 1.5 mg protein/mL.
0
dihydroq. and 2(CH2) pip.); 2.60 (t, 2H, H3,3 dihydroq., J ¼ 6.71 Hz);
0
2.50e2.80 (m, 5H, H4,4 dihydroq. and 2(CH2), CH pip.); 3.41 (s, 2H,
0
eCH2eAr); 3.68 (t, H2,2 dihydroq., J ¼ 6.71 Hz.); 7.00e7.40 (m, 7H,
arom.). MS: m/z 403 [MHþ], 405 [MHþþ2]. Anal. calcd. for
C
22H24Cl2N2O (MW 403.34): C, 65.51; H, 6.00; N, 6.95%; found: C,
65.50; H, 6.10; N, 6.90%.
6.2.23. (1-Benzylpiperidin-4-yl) (3,4-dihydroisoquinolin-2(1H)-yl)
methanone 2m
7.3. Preparation of membrane homogenates from rat liver
Brown solid, melting point: 82e85 ꢁC. Yield (%): 43. I.R. cmꢀ1
(nujol): 1631. 1H NMR (CDCl3/TMS) ppm (
3(CH2) pip.); 2.46 (m, 1H, CH pip.); 2.80 (m, 4H, H4,4 dihydroq. and
d
): 1.50e2.00 (m, 6H,
Two rat livers were cut into small pieces and homogenized with
the potter (500e800 rpm, 10 up-and-down strokes) in 6 volumes of
cold 0.32 M sucrose. The suspension was centrifuged at 1200ꢂ g for
10 min at 4 ꢁC. The supernatant was separated and centrifuged at
31,000ꢂ g for 20 min at 4 ꢁC. The pellet was resuspended in 5e6
volumes of buffer (50 mM TRIS, pH 8.0) and incubated at room
temperature for 30 min. After the incubation, the suspension was
centrifuged again at 31,000ꢂ g for 20 min at 4 ꢁC. The final pellet
was resuspended in 5e6 volumes of buffer and stored at ꢀ80 ꢁC in
1.5 mL portions containing about 2 mg protein/mL.
0
0
CH2 pip.); 3.45 (s, 2H, eCH2ePh); 3.60e3.80 (dt, 2H, H3,3 dihydroq.,
0
J ¼ 5.49 and 6.10 Hz.); 4.60 (d, 2H, H1,1 dihydroq., J ¼ 16.5 Hz);
7.10e7.40 (m, 9H, arom.). MS: m/z 335 [MHþ]. Anal. calcd. for
C
22H26N2O (MW 334.45): C, 79.00; H, 7.84; N, 8.38%; found: C,
78.70; H, 7.90; N, 8.30%.
6.2.24. (1-(4-Chlorobenzyl)piperidin-4-yl) (3,4-dihydroisoquinolin-
2(1H)-yl)methanone 2n
Oil Yield (%): 64. I.R. cmꢀ1 (nujol): 1624. 1H NMR (CDCl3/TMS)
ppm (
d
): 1.50e2.00 (m, 6H, 3(CH2) pip.); 2.45 (m, 1H, CH pip.); 2.80
7.4. Preparation of membrane homogenates from pig brain cortex
0
(m, 4H, H4,4 dihydroq. and CH2 pip.); 3.37 (s, 2H, eCH2eAr);
0
3.57e3.80 (dt, 2H, H3,3 dihydroq., J ¼ 5.49 and 6.10 Hz.); 4.60 (d, 2H,
Fresh pig brain cortex was homogenized with the potter
(500e800 rpm, 10 up-and-down strokes) in 6 volumes of cold
0.32 M sucrose. The suspension was centrifuged at 1200ꢂ g for
10 min at 4 ꢁC. The supernatant was separated and centrifuged at
31,000ꢂ g for 20 min at 4 ꢁC. The pellet was resuspended in 5e6
volumes of TRIS/EDTA buffer (5 mM/1 mM, pH 7.5) and centrifuged
again at 31,000ꢂ g (20 min, 4 ꢁC). The final pellet was resuspended
in 5 6 volumes of buffer and stored at ꢀ80 ꢁC in 1.5 mL portions
containing about 0.8 mg protein/mL.
0
H1,1 dihydroq., J ¼ 15.9 Hz); 7.00e7.20 (m, 8H, arom.). MS: m/z 369
[MHþ], 371 [MHþþ2]. Anal. calcd. for C22H25ClN2O (MW 368.90): C,
71.63; H, 6.83; N, 7.59%; found: C, 71.50; H, 6.80; N, 7.30%.
6.2.25. (1-(2,4-Dichlorobenzyl)piperidin-4-yl) (3,4-
dihydroisoquinolin-2(1H)-yl)methanone 2o
Oil Yield (%): 95. I.R. cmꢀ1 (nujol): 1640. 1H NMR (CDCl3/TMS)
ppm (
d
): 1.54e2.16 (m, 6H, 3(CH2) pip.); 2.50 (m, 1H, CH pip.); 2.80
0
(m, 4H, H4,4 dihydroq. and CH2 pip.); 3.49 (s, 2H, eCH2eAr);
0
3.60e3.80 (dt, 2H, H3,3 dihydroq., J ¼ 5.49 and 6.10 Hz.); 4.60 (d,
7.5. Protein determination
0
2H, H1,1 dihydroq., J ¼ 14.0 Hz); 7.00e7.40 (m, 7H, arom.). MS: m/z
403 [MHþ], 405 [MHþþ2]. Anal. calcd. for C22H24Cl2N2O (MW
403.34): C, 65.51; H, 6.00; N, 6.95%; found: C, 65.70; H, 6.20; N,
7.10%.
The protein concentration was determined by the method of
Bradford [37], modified by Stoscheck [38]. The Bradford solution
was prepared by dissolving 5 mg of Coomassie Brilliant Blue G 250
in 2.5 mL of EtOH (95%, v/v). 10 mL deionized H2O and 5 mL
phosphoric acid (85%, m/v) were added to this solution, the mixture
was stirred and filled to a total volume of 50.0 mL with deionized
water. The calibration was carried out using bovine serum albumin
as a standard in 9 concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0
7. Pharmacology
7.1. Materials
The guinea pig brains and rat liver for the s1 and s2 receptor
binding assays were commercially available (Harlan-Winkelmann,
Borchen, Germany). The pig brains for the performance of the
binding assay to the PCP-binding site of the NMDA receptor were a
and 4.0 mg/mL). In a 96-well standard multiplate, 10
calibration solution or 10 L of the membrane receptor preparation
were mixed with 190 L of the Bradford solution, respectively. After
5 min, the UV absorption of the protein-dye complex at
¼ 595 nm
mL of the
m
m
l

D. Zampieri et al. / European Journal of Medicinal Chemistry 90 (2015) 797e808
807
was measured with a platereader (Tecan Genios, Tecan, Crailsheim,
Germany).
and TRIS/EDTA buffer (5 mM/1 mM, pH 7.5) at room temperature.
The non-specific binding was determined with 10 M unlabeled
m
(þ) MK 801. The Kd-value of (þ)-MK-801 is 2.26 nM [42].
7.6. General procedures for the binding assays
Acknowledgment
The test compound solutions were prepared by dissolving
approximately 10
mmol (usually 2e4 mg) of test compound in
The financial support of FRA 2012 (Research Fund University of
DMSO so that a 10 mM stock solution was obtained. To obtain the
required test solutions for the assay, the DMSO stock solution was
diluted with the respective assay buffer. The filtermats were pre-
soaked in 0.5% aqueous polyethylenimine solution for 2 h at room
temperature before use. All binding experiments were carried out
in duplicates in the 96-well multiplates. The concentrations given
are the final concentration in the assay. Generally, the assays were
Trieste-Italy) is gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.12.018.
performed by addition of 50
test compound solution in various concentrations (10ꢀ5, 10ꢀ6, 10ꢀ7
10ꢀ8, 10ꢀ9 and 10ꢀ10 mol/L), 50
L of corresponding radioligand
solution and 50 L of the respective receptor preparation into each
well of the multiplate (total volume 200 L). The receptor prepa-
mL of the respective assay buffer, 50 mL
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