Chemical, pharmacological, and in vitro metabolic stability studies on enantiomerically pure RC-33 compounds: Promising neuroprotective agents acting as σ1 receptor agonists

Article abstract of DOI:10.1002/cmdc.201300218

Our recent research efforts identified racemic RC-33 as a potent and metabolically stable σ₁ receptor agonist. Herein we describe the isolation of pure RC-33 enantiomers by chiral chromatography, assignment of their absolute configuration, and

Full text of DOI:10.1002/cmdc.201300218

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DOI: 10.1002/cmdc.201300218
Chemical, Pharmacological, and in vitro Metabolic Stability
Studies on Enantiomerically Pure RC-33 Compounds:
Promising Neuroprotective Agents Acting as s1 Receptor
Agonists
Daniela Rossi,[a] Alice Pedrali,[a] Raffaella Gaggeri,[a] Annamaria Marra,[a] Luca Pignataro,[b]
Erik Laurini,[c] Valentina Dal Col,[c] Maurizio Fermeglia,[c] Sabrina Pricl,*[c, d] Dirk Schepmann,[e]
Bernhard Wꢀnsch,[e] Marco Peviani,[f] Daniela Curti,[f] and Simona Collina*[a]
Our recent research efforts identified racemic RC-33 as
a potent and metabolically stable s1 receptor agonist. Herein
we describe the isolation of pure RC-33 enantiomers by chiral
chromatography, assignment of their absolute configuration,
and in vitro biological studies in order to address the role of
chirality in the biological activity of these compounds and
their metabolic processing. The binding of enantiopure RC-33
to the s1 receptor was also investigated in silico by molecular
dynamics simulations. Both RC-33 enantiomers showed similar
affinities for the s1 receptor and appeared to be almost equally
effective as s1 receptor agonists. However, the R-configured
enantiomer showed higher in vitro hepatic metabolic stability
in the presence of NADPH than the S enantiomer. Overall, the
results presented herein led us to select (R)-RC-33 as the opti-
mal candidate for further in vivo studies in an animal model of
amyotrophic lateral sclerosis.
Introduction
Over the last 10 years our research group has conducted ex-
tensive studies aimed at discovering novel s1 receptor ligands
as potential neuroprotective agents.[1] The s receptor system
includes two subtypes: s1 and s2. The s2 subtype seems to be
involved mainly in death signaling of cancer cells.[2] Published
evidence suggests involvement of the s1 receptor in such con-
ditions as drug addiction, depression, neurodegeneration,
pain-related disorders, and cancer.[2a] Interestingly, a mutation
in the s1 receptor gene was recently found to be associated
with frontotemporal lobar degeneration (FTLD), which is the
most common cause of dementia under age 65,[3] with associ-
ated motor neuron disorders and familial juvenile amyotrophic
lateral sclerosis (ALS).[4] Additionally, the s1 receptor has been
implicated in neurite sprouting and elongation in vitro, sug-
gesting a role for this protein in neuroplasticity.[5] Accordingly,
the identification of new, potent, and highly selective s1 ago-
nists is of significant interest, not only for a better understand-
ing of the role played by s1 receptors in various pathologies,
but also, as a more challenging task, to develop neuroprotec-
tive agents representing an innovative pharmacological ap-
proach for the treatment and prevention of neurodegenerative
diseases. In this context, our recent efforts have been ad-
dressed to the design, synthesis, and biological investigation
of new s1 receptor ligands based on arylalkenylamine and
arylalkylamine scaffolds (Figure 1).[1]
[a] Dr. D. Rossi, Dr. A. Pedrali, Dr. R. Gaggeri, Dr. A. Marra, Prof. S. Collina
Department of Drug Sciences
Medicinal Chemistry and Pharmaceutical Technology Section
University of Pavia, Viale Taramelli 12, 27100 Pavia (Italy)
E-mail: simona.collina@unipv.it
[b] Dr. L. Pignataro
Dipartimento di Chimica, Universitꢀ degli Studi di Milano
Istituto di Scienze e Tecnologie Molecolari (ISTM) del CNR
Via Golgi 19, 20133 Milan (Italy)
Among our compound library, the most promising molecule
is 1-[3-(1,1’-biphen)-4-yl]butylpiperidine hydrochloride (RC-
33·HCl, Figure 1), showing excellent s1 receptor affinity (Ki =
0.70ꢀ0.3 nm) along with high selectivity over the s2 subtype,
m- and k-opioid receptors, and the PCP binding site of NMDA
receptors.[1c] RC-33·HCl also turned out to be a potent s1 recep-
tor agonist in our validated PC12 cell model of neuronal differ-
entiation. Indeed, this molecule is able to potentiate both
nerve growth factor (NGF)-induced neurite outgrowth and
elongation in the absence of toxic effects and at lower doses
than the well-characterized s1 receptor agonist PRE-084.[1c]
Finally, RC-33·HCl was found to be metabolically stable in sev-
eral biological matrices such as mouse and rat blood, and rat,
[c] Dr. E. Laurini, Dr. V. Dal Col, Prof. M. Fermeglia, Prof. S. Pricl
MOSE-DEA, University of Trieste, Via Valerio 10, 34127 Trieste (Italy)
E-mail: sabrina.pricl@di3.units.it
[d] Prof. S. Pricl
National Interuniversity Consortium for Material Science and Technology
(INSTM), Research Unit MOSE-DEA, University of Trieste, Trieste (Italy)
[e] Dr. D. Schepmann, Prof. B. Wꢁnsch
Institute of Pharmaceutical and Medicinal Chemistry
University of Muenster, Correnstrasse 48, 48149 Mꢁnster (Germany)
[f] Dr. M. Peviani, Dr. D. Curti
Department of Biology and Biotechnology “L. Spallanzani”
Laboratory of Cellular and Molecular Neuropharmacology
University of Pavia, Via Ferrata 9, 27100 Pavia (Italy)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300218.
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Figure 1. Structures of arylalkenylamines, arylalkylamines, and the most
promising compound, RC-33.
dog and human plasma. Significant degradation was only ob-
served in rat and human liver S9 fractions in the presence of
NADPH.[1d] According to these results, we identified RC-33·HCl
as an optimal candidate for the hit-to-lead process. Because
the enantiomers of a chiral biologically active molecule may
behave differently under physiological conditions (i.e., they
can exhibit different pharmacological and/or toxicological pro-
files), the investigation of enantiopure forms is a key step in
the drug discovery and development processes.[6] Accordingly,
in the present work we directed our efforts toward investigat-
ing the role of chirality in the biological activity and metabolic
processing of RC-33. To this end, we isolated the pure enantio-
mers of RC-33 by chiral chromatography and carried out the
assignment of their absolute configuration. The binding of
enantiopure RC-33 at the s1 receptor was then investigated
both in silico, by molecular dynamics (MD) simulations using
our recently developed homology model for the s1 receptor,[7]
and in vitro, by competition experiments with radioligand. The
in vitro binding affinities of RC-33 enantiomers for opioid re-
ceptors and the PCP binding site of the NMDA receptor were
also investigated, together with their capacity to promote
NGF-induced neurite outgrowth in PC12 cells. As the last step
in the present hit-to-lead process, both RC-33 enantiomers
underwent toxicity and in vitro metabolic studies.
Figure 2. Analytical separation of RC-33 on a Chiralcel OJ-H column
(1=0.46 cm, l=15 cm, 5 mm). Mobile phase: CH3OH/Et3N 100:0.1; flow rate:
0.5 mLminꢁ1; detection at l=250 nm.
1.58). Hence, these experimental conditions were selected for
the chromatographic scale-up. Briefly, RC-33 (62.5 mg) was pro-
cessed in 50 cycles according to conditions reported in Table 1,
yielding 26.4 mg of the first eluted enantiomer and 27.8 mg of
the second eluted enantiomer, together with 5.2 mg of an in-
termediate fraction as a mixture of the two enantiomers.
Table 1. Semi-preparative resolution of compound RC-33 on a Chiralcel
OJ-H column (1=1 cm, l=25 cm, 5 mm).
CH3OH/Et3N
100:0.1
Flow rate
tR1
[min]
tR2
[min]
Inj. Vol.
[mL]
Conc.
[mLminꢁ1
]
[mgmLꢁ1
]
3
11.6
13.0
1
1.25
To approach the configurational study of enantiopure RC-33,
an enantioselective synthetic procedure suitable to obtaining
enantioenriched RC-33 with known absolute configuration was
devised (Scheme 1). As highlighted in Scheme 1, the enantiose-
lective hydrogenation of (E)-1 using (R,R)-Ir(ThrePHOX) complex
as catalyst is the key step of the synthetic pathway. The enan-
tioselective hydrogenation of (E)-2 was also performed as a ref-
erence reaction for configurational assignment purposes. Thus,
we started our synthetic approach with the preparation of (E)-
1 and (E)-2, essentially according to the procedures described
by Lemay et al. and Simard-Mercier et al.[9] Compounds (E)-
1 and (E)-2 were then subjected to enantioselective hydroge-
nation in a Parr multireactor (H2, 70 bar, room temperature)
using the (R,R)-Ir(ThrePHOX) complex as catalyst, yielding com-
pounds (+)-4 and (+)-5. The absolute configuration of the
latter compound is known to be S.[10] Both compounds were
obtained in 87% ee, as demonstrated by chiral HPLC analysis
(see the Experimental Section below). The configurational as-
signment of (+)-4 was performed by comparing its circular di-
chroism (CD) spectrum with that of (+)-(S)-5. As expected,
both (+)-4 and (+)-(S)-5 showed a similar positive Cotton
Results and Discussion
RC-33 enantiomer isolation and assignment of configuration
Because chiral HPLC is a rapid and efficient method for directly
obtaining both enantiomers with high optical purity, the reso-
lution of racemic RC-33 by semi-preparative HPLC using chiral
stationary phases (CSPs) was performed to isolate each enan-
tiomer in quantities suitable for in-depth investigations of bio-
logical behavior. Firstly, the racemic compound was synthe-
sized via catalytic reduction of the corresponding arylalkenyla-
mine under a hydrogen atmosphere using Pd0 EnCat 30NP as
catalyst.[1c] A standard screening protocol was then applied to
an analytical Chiralcel OJ-H column, the chiral selector of
which is cellulose tris-(4-methylbenzoate) coated on a silica gel
substrate.[8] As shown in Figure 2, baseline separation of RC-33
enantiomers was achieved by eluting with methanol/diethyl-
amine 100:0.1 (tR1 =9.91 min; tR2 =11.44 min; a=1.25; RS =
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Receptor binding studies
The affinities of the pure RC-
33·HCl enantiomers for s1 and s2
receptors were determined in
competition experiments with
radioligands. The highly s1-selec-
tive radioligand [3H](+)-pentazo-
cine and homogenates of guinea
pig brain cortex were used in
the s1 assay. Nonspecific binding
was recorded in the presence of
non-radiolabeled
(+)-pentazo-
cine in large excess. In the s2
assay, membrane preparations of
rat liver served as the source for
s2 receptors. The nonselective
radioligand [3H]DTG was em-
ployed in the s2 assay because
a s2-selective radioligand is not
yet commercially available. To
mask the s1 receptors, an excess
of non-tritiated (+)-pentazocine
was added to the assay solution,
while a high concentration of
non-tritiated DTG was used to
determine nonspecific binding.
Scheme 1. Synthetic pathway for the preparation of enantiomeric RC-33. Reagents and conditions: a) Bu4NI, DCE,
reflux, 24 h; b) arylboronic acid, Pd2(dba)3, PtBu3 HBF4, Cs2CO3, anhydrous THF, reflux, 4 h; c) H2 (70 bar), anhydrous
CH2Cl2, 258C, 20 h; d) 2m NaOH, EtOH, RT, 2 h; e) piperidine, TBTU, DIPEA, THF, MW (808C, 25 W, 20 min); f) LiAlH4,
anhydrous THF, RT, 3 h.
effect (CE) at ~215 nm (lmax 216, Demax 1.13 and lmax 215,
Table 2. Chiroptical properties of RC-33 enantiomers.
Demax 1.38 for (+)-4 and (+)-(S)-5, respectively). Moreover, com-
pound (+)-4 exhibited an additional positive CE at higher
wavelength (lmax 249, Demax 1.73). This behavior is consistent
with the compound’s UV spectra (Supporting Information).
Based on these considerations, the absolute configuration S
was assigned to (+)-4. Compound (+)-(S)-4 was then fully con-
verted, by alkaline hydrolysis, into the corresponding acid
(+)-(S)-6 (87% ee, determined by chiral HPLC; Experimental
Section) which, in turn, was condensed with piperidine in the
presence of TBTU to yield the tertiary amide (+)-(S)-7 (87% ee,
determined by chiral HPLC: Experimental Section). Compound
(+)-(S)-7 was finally converted into the desired product (+)-(S)-
RC-33 (87% ee, Table 2) by reduction with lithium aluminum
hydride. Importantly, chiral HPLC analysis performed after hy-
drolysis, amidation, and reduction reactions revealed that no
racemization occurs, demonstrating that the S configuration
was correctly assigned to (+)-RC-33 by chemical correlation to
its precursor (+)-(S)-4. As the final step in the configurational
study of enantiopure RC-33 isomers obtained by chiral chro-
matography, their optical rotation values and retention times
from chiral HPLC–UV traces were compared with those of
enantioenriched RC-33 obtained by enantioselective synthesis
(Table 2). In this way, the S configuration was assigned to the
first eluted enantiomer, and the R configuration to the more
retained one. Pure (R)- and (S)-RC-33 were finally converted
into the corresponding hydrochlorides, suitable for biological
investigations.
Compd
[a]D20 (CH3OH)
ee [%][a]
tR [min]
9.88
11.41
9.45 (major)
10.89 (minor)
(+)-RC-33[b]
(ꢁ)-RC-33[b]
+22.1 (c=0.3)
ꢁ22.2 (c=0.3)
+19.2 (c=1)
99.5
99.6
87.0
(+)-(S)-RC-33[c]
[a] Determined by chiral HPLC on Chiralcel OJ-H (Figure 2). [b] Obtained
by semi-preparative chiral HPLC (Table 1). [c] Obtained by enantioselec-
tive synthesis (Scheme 1).
To investigate receptor selectivity, the binding properties of
pure RC-33·HCl enantiomers were also investigated for m-, k-,
d-opioid receptors and the PCP binding site of NMDA recep-
tors using guinea pig brain (m and k), rat brain (d) and pig
brain cortex (PCP binding site of NMDA) as sources of receptor
material. [3H]DAMGO (m), [3H]-U-69,593 (k), [3H]DPDPE (d), and
[3H]MK-801 (NMDA) were used as specific radioligands. Non-
specific binding was determined with unlabeled naloxone (m),
unlabeled U-69,593 (k), unlabeled morphine (d), and unlabeled
(+)-MK-801 (PCP binding site of NMDA). The residual binding
of the radioligand is given at a test compound concentration
of 1 mm (opioid receptor binding assays) or 10 mm (NMDA re-
ceptor binding assays). Binding results are summarized in
Table 3, together with data for RC-33·HCl, reported for compar-
ison purposes.
The enantiomeric arylalkylamines (S)-RC-33·HCl and (R)-RC-
33·HCl presented herein showed interesting binding properties
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Table 3. Affinities of compounds RC-33·HCl toward s1, s2, NMDA, m-, k-
and d-opioid receptors.
Compd[a]
Ki ꢀSEM [nm][b]
Displacement of specific radioligands [%]
s1
s2
m
k
d
NMDA
RC-33
0.9ꢀ0.3 29ꢀ5
0
12
14
36
0
0
0
0
5
25
11
8
(S)-RC-33 1.9ꢀ0.2 34ꢀ8
(R)-RC-33 1.8ꢀ0.1 45ꢀ16
[a] Compounds were tested as hydrochlorides. [b] Values are means ꢀ
SEM of three experiments.
toward the s1 receptor subtype (Ki values <2 nm, Table 3) and
good selectivity over the s2, m-, k- and d-opioid receptors, as
well as the PCP binding site of NMDA receptors. Concerning
the role of chirality in s1 receptor binding, the two enantio-
mers and rac-RC-33·HCl are endowed with similar affinities for
the s1 receptor. Importantly, this observation supports the
notion that the interaction of these compounds with this bio-
logical target is non-stereoselective.
MD simulations
For a molecular-level explanation of the non-stereoselective
binding of RC-33 enantiomers to the s1 receptor, the putative
binding modes of (R)- and (S)-RC-33 on our s1 receptor 3D ho-
mology model were retrieved by exploiting the currently avail-
able preliminary information on sequence–structure relation-
ships and mutagenesis studies.[7,11] To summarize briefly, a pro-
tein isoform lacking residues 119–149 was found devoid of
ligand binding capacity, and the conversion of residues Asp126
and Glu172 into glycine led to a severalfold decrease in ligand
binding at the s1 receptor.[11c] Moreover, our hydrophobicity
analysis[7] and other structure–activity relationship (SAR) studie-
s[11a,b] identified, the transmembrane (TM) regions aside, a third
hydrophobic region matching the steroid binding domain
like II (SBDLII) region and centered on Asp188, a residue specif-
Figure 3. a) Overlay of representative structures for (R)-RC-33 (light-blue ball-
and-stick model) and (S)-RC-33 (light-green ball-and-stick) in complex with
the s1 receptor (orange ribbon), taken from equilibrated MD snapshots.
Some Na+ and Clꢁ ions are also shown as purple and light-green spheres,
respectively. Hydrogen atoms and water molecules are omitted for clarity.
b) Zoomed-in view of the binding modes of (R)- (blue) and (S)-RC-33 (green)
in the s1 binding pocket. In this image, the protein is portrayed in semi-
transparent ribbon, colored according to the respective ligand.
Figure 4, in both cases the entire RC-33 molecule is nicely
lined by the side chains of several other s1 residues, and the
relevant hydrophobic interactions which originate from these
intermolecular contacts all contribute to stabilize receptor–
ligand binding.
ically
photolabeled
with
[
125I]3-iodo-4-azidococaine
([125I]IACoc).[12] Having localized this protein region as a possible
zone for ligand binding, the two enantiomers of RC-33 were
then docked into the putative binding site of our 3D receptor
model, and their affinity toward the receptor was scored by
MM/PBSA analysis.[13]
The similar binding mode and the equivalent type and
number of interactions predicted for (R)- and (S)-RC-33 in com-
plex with the s1 receptor constitute the molecular rationale,
according to which our MM/PBSA[13] estimated binding affini-
ties (DGbind) for these two enantiomers for the protein are
nearly identical, that is, DGbind =ꢁ11.42ꢀ0.29 kcalmolꢁ1 for
(R)-RC-33 and DGbind =ꢁ11.15ꢀ0.31 kcalmolꢁ1 for (S)-RC-33. In
accordance with the components of the binding free energy in
Figure 5a, for both s1–RC-33 enantiomer complexes, van der
Waals (DEVDW) and electrostatic (DEELE) interactions in the gas
phase provide the major favorable contributions to ligand
binding. Nonpolar solvation energies (DGNP), resulting from the
burial of RC-33 solvent-accessible surface area, also afford posi-
tive contributions to the binding affinity. Conversely, polar sol-
vation energies (DGPB) and entropy components (ꢁTDS)
oppose binding by making unfavorable contributions to
In a typical structure of the MD-simulated s1–ligand com-
plexes, both (R)- and (S)-RC-33 are oriented horizontally inside
the receptor binding pocket and adopt similar binding poses,
as illustrated in Figure 3. For both enantiomers, the ꢁNH+
moiety of the ligand piperidine ring is anchored around the
negatively charged side chain of Asp126 from the s1 protein,
interacting with each other through a permanent salt bridge.
As tracked by MD simulations, the average distance for the salt
bridge through the proton at the cationic moiety of RC-33 and
the COOꢁ group of s1 Asp126 is 2.9ꢀ0.1 ꢂ for the R enantio-
mer and 2.7ꢀ0.1 ꢂ for the S enantiomer (Figure 4). The two ar-
omatic rings of RC-33 are packed parallel to Tyr120 and per-
pendicularly with respect to Tyr173, resulting in strongly stabi-
lizing p–p interactions. Lastly, as can be appreciated from
DGbind
.
To analyze in detail the similarities and differences in the
binding modes of the two RC-33 enantiomers with the s1 re-
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Figure 5. a) Energy components for the binding of (R)- and (S)-RC-33 to the
s1 receptor. DEVDW, van der Waals energy; DEELE, electrostatic energy; DGNP,
nonpolar solvation energy; DGPB, polar solvation energy;
DGNP,T =DEVDW +DGNP; DGP,T =DEELE +DGPB; ꢁTDS, total entropy contribu-
tion; DGbind =DGNP,T +DGP,TꢁTDS. b) Per-residue free-energy contribution to
the binding of (R)- and (S)-RC-33 to the s1 receptor.
Figure 4. Equilibrated MD snapshots of a) (R)-RC-33 and b) (S)-RC-33 in com-
plex with the s1 receptor; the images are zoomed-in views of the receptor
binding site. The protein is depicted in semi-transparent light-blue and
light-green ribbon, respectively, while both enantiomers are shown as ball-
and-stick models colored according to atom type (C, gray; N, blue; H atoms
omitted). Protein residues mainly involved in interaction with the ligands are
highlighted as colored sticks and are labeled. Salt bridge interactions are
shown as dashed black lines. Water molecules and counterions are omitted
for clarity.
NGF-induced neurite outgrowth in PC12 cells and
cytotoxicity
Pure enantiomers of RC-33·HCl were tested in our validated
PC12 cell model of neuronal differentiation to analyze their
effect on NGF-induced neurite outgrowth. In previous experi-
ments on PC12 cells rac-RC-33·HCl displayed an agonist profile,
consistently and significantly potentiating NGF-induced neurite
outgrowth at concentrations as low as 0.25 mm.[1c] Therefore,
we chose this concentration to test (S)- and (R)-RC-33·HCl on
PC12 cells. rac-RC-33·HCl and its enantiomers appear to be
almost equally effective. Indeed, both enantiomers were able
to increase the percentage of cells with neurite outgrowth
with respect to NGF alone (152.8ꢀ7.6%, p<0.01 and 143.1ꢀ
3.8% p<0.01 for (S)- and (R)-RC-33·HCl, respectively), showing
an effect very similar to that of rac-RC-33·HCl (157.7ꢀ11.0%,
p<0.001) (Figure 6). Additionally, their effect was totally
blocked by co-administration of the selective s1 antagonist NE-
100. Taken together, these results confirm that (S)- and (R)-RC-
33·HCl similarly modulate NGF-induced neurite outgrowth, spe-
cifically acting as s1 receptor agonists.
ceptor, the binding free energy was further decomposed into
ligand–residue pairs to create the receptor–residue interaction
spectrum shown in Figure 5b. As can be observed, the interac-
tion spectra of the two complexes are similar to each other,
with the major contributions stemming from a few groups
centered around residues Asp126, Tyr120, and Glu172 in both
cases, as expected. Furthermore, for interacting residues that
are not shared by the two enantiomers of RC-33 upon binding
to the s1 receptor (i.e., Thr127, Trp169, and Met170 for the R
enantiomer, and Arg119, Trp121, and Phe133 in the case of the
S enantiomer), a sort of compensatory effect is present, ulti-
mately resulting in the non-stereoselective binding of the s1
receptor to RC-33.
Finally, an MTT-based cytotoxicity assay, performed after
treating HaCaT cells with (S)- or (R)-RC-33·HCl and with rac-RC-
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showed high metabolic stability in all the investigated matri-
ces, with the only exception of liver S9 fractions in the pres-
ence of NADPH. Therefore, in the present work we addressed
our attention at evaluating the hepatic metabolism of (R)- and
(S)-RC-33·HCl in rat and human liver S9 fractions both in the
presence and in the absence of NADPH. To this aim, our previ-
ously validated and reproducible method using an ultrafast
liquid chromatography system interfaced with a photodiode
array detector (UPLC/UV/PAD, Acquity, Waters) was applied.[1d]
Briefly, UPLC/UV/PAD analyses were carried out with a BEH
Shield RP18 column, eluting with a mixture of water and meth-
anol, containing 0.1% formic acid, in gradient at a flow rate of
0.5 mLminꢁ1. Calibration curves in the range of 1–200 mm
(eight points of calibration) were determined, adding known
concentrations of each enantiomer of RC-33·HCl to each con-
sidered biological matrix, previously deproteinized. In all cases
calibration curves with a quadratic correlation coefficient (R2)
of 0.9999 were obtained. Quantification was performed by
comparing the chromatographic peak areas for test solutions
with those of external standard. The matrices could be directly
analyzed by UPLC without clean-up steps.
Figure 6. Potentiating effect of s1 receptor ligands RC-33·HCl and enantio-
mers (at 0.25 mm) on neurite outgrowth induced by NGF (2.5 ngmLꢁ1). NE-
100 (3 mm) co-administration totally blocked the potentiating effect. Histo-
grams represent the mean ꢀSEM of at least three different experiments.
Data are expressed as percentage of control (CTR; NGF alone). ANOVA:
p<0.0005; Tukey’s post-hoc test: ***p<0.001; **p<0.01 vs. NGF alone;
#
888p<0.001 vs. (ꢁ)-(R)-RC-33·HCl; p<0.05 vs. (+)-(S)-RC-33·HCl.
Similarly to the racemate, both RC-33·HCl enantiomers un-
derwent relatively weak non-oxidative metabolism, with degra-
dation of ~30% within 4 h at 378C, both in rat and human
(Figure 8). In contrast, they were subjected to a relevant oxida-
tive metabolism in both rat and human liver S9 fractions
within the same frame time at 378C. Notably, degradation of
~50% in both rat and human was evidenced for (R)-RC-33·HCl,
while degradation of ~70% in both the biological matrices
Figure 7. Effect of s1 receptor ligands RC-33·HCl and enantiomers on HaCaT
cell viability. Values are the mean ꢀSEM of at least three different experi-
ments. ANOVA: p<0.0001; Tukey’s post-hoc test: *p<0.0001 CTR vs. (R)-RC-
33·HCl, (S)-RC-33·HCl, and rac-RC-33·HCl.
33·HCl for 48 h (Figure 7) or 72 h (data not shown) confirmed
that, similarly to the racemic mixture, both enantiomers are
nontoxic at the concentration used in our NGF-induced neurite
outgrowth experiments.
In vitro metabolic stability studies
To investigate the role of chirality on metabolic processes, the
hepatic metabolic stability of RC-33·HCl enantiomers was
tested in vitro using rat and human liver S9 fraction. We previ-
ously described rac-RC-33·HCl in vitro metabolic stability in
several biological matrices, such as mouse and rat blood, rat,
dog and human plasma, as well as rat and human liver S9 frac-
tions, containing microsomes (metabolic enzymes of phase I)
and cytosol (metabolic enzymes of phase II).[1d] rac-RC-33·HCl
Figure 8. Degradation time courses of a) (S)-RC-33·HCl and b) (R)-RC-33·HCl
in rat and human liver S9 fractions in the absence of NADPH at 378C. Data
are expressed as the mean ꢀSD of two independent experiments.
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Figure 9. Degradation time courses of (S)-RC-33·HCl (*) and (R)-RC-33·HCl (*) in a) rat and b) human liver S9 fractions in presence of NADPH at 378C. Data
are expressed as the mean ꢀSD of two independent experiments. Enantiomers of RC-33·HCl showed significant degradation in liver S9 fractions in the pres-
ence of NADPH, following first-order kinetics, as confirmed by the respective semi-logarithmic plots for c) rat and d) human.
was observed for (S)-RC-33·HCl (Figure 9a,b). Moreover, as
clearly shown in the semi-logarithmic plots shown in Fig-
ure 9c,d, the oxidative degradation of both enantiomers fol-
lows first-order kinetics, with a half-life (t1/2) of 112 min in rat
and 130 min in human for (S)-RC-33·HCl, and of 225 min in rat
and 246 min in human for (R)-RC-33·HCl. Overall, results of the
in vitro hepatic metabolic stability studies clearly show that
RC-33·HCl enantiomers are similarly metabolized by non-oxida-
tive processes in both rat and human; on the other hand, their
degradation in the presence of NADPH was unequivocally
proven to be enantioselective with a preference for the S
enantiomer in both species.
binding site of our 3D receptor model, and their affinity
toward the receptor was scored by MM/PBSA analysis. The re-
sults of our modeling investigations confirm that both enantio-
mers of RC-33 can be accommodated within the s1 binding
site and establish the same network of stabilizing interactions
with the target, supporting the non-stereoselective binding of
RC-33 to the s1 receptor. Lastly, the hepatic metabolic stability
of RC-33 enantiomers was also tested in vitro using rat and
human liver S9 fractions. (S)- and (R)-RC-33 were similarly me-
tabolized by non-oxidative processes in both rat and human.
Interestingly, compared with the corresponding enantiomer,
(S)-RC-33·HCl was preferentially metabolized in the presence of
NADPH in both rat and human, thus proving that the oxidative
metabolic processes possess the same enantiomeric preference
in both species.
Conclusions
In the present work we addressed the role of chirality in the
biological activity of RC-33, recently studied by us in its race-
mic form. RC-33 enantiomers were isolated by enantioselective
semi-preparative HPLC, and their absolute configuration was
assigned by applying an integrated CD analysis/chemical corre-
lation strategy. Their in vitro binding affinities toward s, opioid,
and PCP binding site of the NMDA receptors were investigat-
ed, and their agonist/antagonist profiles at s1 receptor were
derived. In synthesis, (R)-RC-33 and (S)-RC-33 showed nearly
the same affinity for the s1 receptor and were equally effective
as s1 receptor agonists, without toxic effects at the concentra-
tion shown to be effective. To provide a rational in silico ex-
planation of the experimental evidence that the interaction be-
tween RC-33 enantiomers and the s1 receptor is non-stereose-
lective, the two enantiomers were docked into the putative
In summary, based on the overall results of the biological in-
vestigations, (R)-RC-33 emerged as the eutomer. Because s1 re-
ceptor agonists such as PRE-084 were recently shown to be
promising candidates for a therapeutic strategy for ALS,[14] (R)-
RC-33 represents the optimal candidate for the in vivo investi-
gation. Its pharmacokinetic profile and results of both short-
and long-term investigations as drug candidate in an animal
model of ALS will be reported in due course.
Experimental Section
Chemistry
Reagents and instrumentation: Reagents and solvents for synthe-
sis were obtained from Aldrich (Italy). Unless otherwise specified,
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commercially available reagents were used as received from the
supplier. The catalyst (R,R)-Ir(ThrePHOX) was purchased from Strem
Chemicals, Inc. (Bichheim, France) and stored under nitrogen in
a Schlenk flask. Solvents were purified according to the guidelines
in Purification of Laboratory Chemicals.[15] Microwave dielectric heat-
ing was performed in a Discover LabMate instrument (CEM Corpo-
ration) specifically designed for organic synthesis and following an
appropriate microwave program. Enantioselective hydrogenation
reactions were carried out using a Parr multireactor, allowing up to
six reactions in parallel under hydrogen pressure. All solvents were
evaporated under reduced pressure using a Heidolph Laborota
4000 instrument. Melting points were measured on an SMP3 Stuart
Scientific apparatus and are uncorrected. Analytical thin-layer chro-
matography (TLC) was carried out on silica gel pre-coated glass-
backed plates (Fluka Kieselgel 60 F254, Merck) and visualized by UV
light, acidic ammonium molybdate(IV), or potassium permanga-
nate. Flash chromatography (FC) was performed with silica gel 60
(particle size 230–400 mesh) purchased from Nova Chimica (Cinisel-
lo Balsamo, Italy). IR spectra were recorded on a Jasco (Cremella,
LC, Italy) FTIR-4100 spectrophotometer with ATR module; only no-
table absorptions are given. 1H NMR spectra were recorded on
a Bruker Avance 400 spectrometer operating at 400.13 MHz; chem-
ical shifts (d) are reported in ppm with the solvent reference rela-
tive to tetramethylsilane (TMS) employed as the internal standard
(CDCl3, d=7.26 ppm; CD2Cl2, d=5.32 ppm; [D6]acetone, d=
2.05 ppm). The following abbreviations are used to describe spin
multiplicity: s=singlet, d=doublet, t=triplet, q=quartet, m=
multiplet, br=broad signal, dd=double-doublet, td=triple-dou-
blet. Coupling constant values are reported in Hz. 13C NMR spectra
tection at l=250 nm. The fractions obtained containing the enan-
tiomers were evaporated at reduced pressure.
(+)-(S)-1-(3-(Biphenyl-4-yl)butyl)piperidine [(+)-(S)-RC-33]: Yellow
oil: 99.5% ee determined by analytical chiral HPLC: tR =9.91 min;
[a]D20 = +22.1 (c=0.3 in CH3OH); 1H NMR (400 MHz, CD2Cl2): d=
3
3
7.62 (m, 2H), 7.55 (d, J(H,H)=8.3 Hz, 2H), 7.44 (t, J(H,H)=7.7 Hz,
3
2H), 7.36–7.32 (m, 1H), 7.30 (d, J(H,H)=8.2 Hz, 2H), 2.82 (m, 1H),
2.32 (m, 4H), 2.26–2.14 (m, 2H), 1.79 (m, 2H), 1.55 (m, 4H), 1.43 (m,
3
2H), 1.29 ppm (d, J(H,H)=7.0 Hz, 3H); 13C NMR (100 MHz, CD2Cl2):
d=147.6, 141.6, 139.2, 129.3, 128.0, 127.6, 127.5, 58.0, 55.2, 38.3,
36.1, 26.8, 25.2, 22.8 ppm; IR (ATR): n˜ =3056, 3027, 2927, 2850,
2799, 2761, 1598, 1486, 1450, 1153, 1119, 835, 764 cmꢁ1; HRMS-ESI:
m/z [M+H]+ calcd for C21H28N: 294.2222, found 294.2215. By treat-
ment of (+)-(S)-RC-33 with 37% HCl in CH3OH under stirring
(30 min) followed by solvent evaporation, pure (+)-(S)-RC-33·HCl
20
was obtained as a white solid: mp: 165–1668C (dec.); [a]D
+15.8 (c=0.2 in CH3OH).
=
(ꢁ)-(R)-1-(3-(Biphenyl-4-yl)butyl)piperidine [(ꢁ)-(R)-RC-33]: Yellow
oil: 99.6% ee determined by analytical chiral HPLC: tR =11.44 min;
[a]D20 =ꢁ22.2 (c=0.3 in CH3OH). Spectroscopic properties comply
with those reported for (S)-RC-33. HRMS-ESI: m/z [M+H]+ calcd for
C21H28N: 294.2222, found 294.2218. By treatment of (ꢁ)-(R)-RC-33
with 37% HCl in CH3OH under stirring (30 min) followed by solvent
evaporation, pure (ꢁ)-(R)-RC-33·HCl was obtained as a white solid:
mp: 165–1668C (dec.); [a]D20 =ꢁ15.9 (c=0.2 in CH3OH).
Typical procedure for the synthesis of (E)-1 and (E)-2: A solution
of ethyl 2-butynoate (2.2 mL, 19 mmol), in 1,2-dichloroethane (DCE,
150 mL) and tetrabutylammonium iodide (21 g, 57 mmol) was
heated at reflux for 24 h. The reaction mixture was then cooled, di-
luted with CH2Cl2, washed with NaHSO3 (20% aqueous solution),
saturated NaHCO3, and brine. The organic phase, dried over
Na2SO4 and evaporated, gave crude (E)-3, that was finally purified
by FC, eluting with n-hexane, then n-hexane/EtOAc 9:1, yielding
pure (E)-3 as a colorless oil (4.36 g, 84%), the spectroscopic proper-
ties of which comply with reported values.[9a] (E)-3 (2.1 g, 7.6 mmol)
was then added to a solution of the appropriate boronic acid
(3.7 g for phenyl boronic acid and 6.0 g for biphenyl boronic acid,
30.4 mmol), Cs2CO3 (7.4 g, 22.8 mmol), Pd2(dba)3 (696 mg,
0.76 mmol), P(tBu)3·HBF4 (882 mg, 3.04 mmol) in freshly distillated
THF (92 mL) under nitrogen atmosphere. The reaction mixture was
heated at reflux for 4 h, then cooled, diluted with Et2O, washed
with brine, dried over Na2SO4, and evaporated to dryness, yielding
crude (E)-1 and (E)-2. Pure products were finally obtained by FC.
were recorded on
a 400 MHz spectrometer operating at
100.56 MHz, with complete proton decoupling. Carbon chemical
shifts (d) are reported in ppm relative to TMS with the respective
solvent resonance as the internal standard (CDCl3, d=77.23 ppm;
CD2Cl2, d=54.00 ppm; [D6]acetone, d=29.84 ppm). High-resolution
MS spectra were recorded with a Fourier transform ion cyclotron
resonance (FT-ICR) instrument equipped with an ESI source. MS
analyses were performed on
a Finnigan LCQ Fleet system
equipped with an ESI source, controlled by Xcalibur software 1.4
(Thermo Finnigan, San Jose, CA, USA). ESI mass spectra were gen-
erated in both positive and negative ion modes as necessary. High-
performance liquid chromatography (HPLC) runs were conducted
on a Jasco (Cremella, LC, Italy) HPLC system equipped with a Jasco
AS-2055 plus autosampler, a PU-2089 plus quaternary gradient
pump, and an MD-2010 plus multi-wavelength detector. Experi-
mental data were acquired and processed by Jasco Borwin PDA
and Borwin Chromatograph Software. Solvents used for chiral
chromatography were HPLC grade and supplied by Carlo Erba
(Milan, Italy). All HPLC analyses were performed at room tempera-
ture. UPLC analyses were performed with a UPLC system (Acquity,
Waters) interfaced with a UV detector. Optical rotation values were
measured on a Jasco (Cremella, LC, Italy) photoelectric polarimeter
DIP 1000 with a 1 dm cell at the sodium D line (l=589 nm);
sample concentration values (c) are given in 10ꢁ2 gmLꢁ1. Circular
dichroism (CD) spectra were recorded on a Jasco J-710 instrument.
(E)-Ethyl 3-(biphenyl-4-yl)but-2-enoate [(E)-1]: Purification by FC
(n-hexane, n-hexane/Et2O 99:1, then n-hexane/Et2O 95:5) gave the
desired product as a white solid (1.64 g, 81%): mp: 82–848C. Spec-
troscopic properties agree with published data.[16]
(E)-Ethyl 3-phenylbut-2-enoate [(E)-2]: Purification by FC (n-
hexane, then n-hexane/Et2O 99:1) gave the desired product as
a colorless oil (1.23 g, 85%). Spectroscopic properties comply with
those reported previously.[9b]
General procedure for the enantioselective hydrogenation reac-
tion: Catalyst (R,R)-Ir(ThrePHOX) (49.9 mg, 0.029 mmol) was weigh-
ed in a special glass vessel. The vessel was purged with nitrogen,
and a 0.362m CH2Cl2 solution (8 mL) of the appropriate substrate
(772 mg for (E)-1 and 552 mg for (E)-2, 2.9 mmol) was added. The
vessel was placed into an autoclave and purged three times with
hydrogen at 70 bar. The reaction was stirred overnight at RT under
pressure of hydrogen; the hydrogen was then released, CH2Cl2 was
evaporated, and the conversion was determined by NMR analysis
Chiral chromatography: The enantiomers of RC-33 were com-
pletely resolved by a semi-preparative process using a Daicel Chir-
alcel OJ-H column (1=1 cm, l=25 cm, 5 mm), eluting with
CH3OH/Et3N 100:0.1 at RT at a flow rate of 3 mLminꢁ1 (Table 1). The
eluate was properly partitioned according to the UV profile. Analyt-
ical control of collected fractions was performed on a Daicel Chiral-
cel OJ-H column (1=0.46 cm, l=15 cm, 5 mm) eluting with
CH3OH/Et3N 100:0.1 at RT at a flow rate of 0.5 mLminꢁ1 and UV de-
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of the crude. The desired products were purified by FC, and enan-
tiomeric excess was determined by chiral HPLC.
141.2, 139.6, 128.9, 127.6, 127.4, 127.3, 127.2, 41.5, 36.9, 26.1, 24.6,
21.8 ppm; IR (neat): MS (ESI): m/z 308.22 [M+H]+.
(+)-(S)-Ethyl 3-(biphenyl-4-yl)butanoate [(+)-(S)-4]: Purification by
FC (n-hexane/EtOAc 97:3) gave the desired product as a yellow oil
(739 mg, 95%): 87% ee determined by analytical chiral HPLC
[Daicel Chiralcel OJ-H (1=0.46 cm, l=15 cm, 5 mm), n-heptane/ 2-
(+)-(S)-1-(3-(Biphenyl-4-yl)butyl)piperidine [(+)-(S)-RC-33]: A so-
lution of (+)-(S)-7 (61 mg, 0.2 mmol) in anhydrous THF (5 mL) was
added dropwise to
a stirred suspension of LiAlH4 (23 mg,
0.6 mmol) in anhydrous THF (6 mL). The reaction mixture was
stirred at RT for 3 h, cooled to 08C, slowly quenched with H2O,
stirred for 15 min at RT and finally filtered on a pad of Celite (wash-
ing with EtOAc). The filtrate was evaporated, the residue dissolved
in EtOAc (30 mL) and extracted with H2O (3ꢃ20 mL). The organic
phase was dried over anhydrous Na2SO4 and evaporated, yielding
the desired compound as a yellow oil (53 mg, 90%): 87% ee deter-
mined by analytical chiral HPLC [Daicel Chiralcel OJ-H (1=
0.46 cm, l=15 cm, 5 mm), CH3OH/Et3N 100:0.1, flow rate=
propanol 90:10, flow rate=0.8 mLminꢁ1
, l=250 nm]: tRmajor =
8.69 min,
t
Rminor =11.24 min; [a]D20 = +29.5 (c=0.5 in CHCl3);
3
1H NMR (400 MHz, CDCl3): d=7.58 (m, 2H), 7.54 (d, J(H,H)=8.3 Hz,
2H), 7.43 (t, 3J(H,H)=7.6 Hz, 2H), 7.34 (m, 1H), 7.31 (d, 3J(H,H)=
8.3 Hz, 2H), 4.10 (q, J(H,H)=7.1 Hz, 2H), 3.34 (m, 1H), 2.66 (dd, AB
3
system, 2J(H,H)=15 Hz, 3J(H,H)=7.1 Hz, 1H), 2.58 (dd, AB system,
2J(H,H)=15 Hz, 3J(H,H)=8.1 Hz, 1H), 1.35 (d, 3J(H,H)=7.0 Hz, 3H),
1.20 ppm (t, 3J(H,H)=7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): d=
172.6, 145.1, 141.2, 139.5, 128.9, 127.4, 127.3, 127.2, 60.5, 43.2, 36.4,
22.0, 14.4 ppm; MS (ESI): m/z: 269.12 [M+H]+.
0.5 mLminꢁ1
,
l=250 nm]:
t
Rmajor =9.91 min,
tRminor =11.44 min;
[a]D20 = +19.2 (c=1.0 in CH3OH); MS (ESI): m/z 294.25 [M+H]+.
Spectroscopic properties comply with those reported above for
(+)-(S)-RC-33 obtained by semi-preparative HPLC.
(+)-(S)-Ethyl 3-phenylbutanoate [(+)-(S)-5]: Purification by FC (n-
hexane/EtOAc 98:2) gave the desired product as colorless oil
(546 mg, 98%): 87% ee determined by analytical chiral HPLC
[Daicel Chiralcel OJ-H (1=0.46 cm, l=15 cm, 5 mm), n-heptane/ 2-
Circular dichroism: Solutions of (+)-4 (c=6.25ꢃ10ꢁ5 m in n-
hexane, optical pathway 1 cm) and of (+)-(S)-5 (c=2.5ꢃ10ꢁ5 m in
n-hexane, optical pathway 1 cm) were analyzed in a nitrogen at-
mosphere. CD spectra were scanned at 10 nmminꢁ1 with a spectral
band width of 2 nm and data resolution of 0.2 nm. Experimental
data are reported in the Supporting Information.
propanol 90:10, flow rate=0.8 mLminꢁ1
, l=250 nm]: tRminor =
5.36 min, tRmajor =5.91 min; [a]D20 = +18.3 (c=0.5 in CHCl3). Spec-
troscopic properties comply with those reported earlier.[10]
(+)-(S)-3-(Biphenyl-4-yl)butanoic acid [(+)-(S)-6]: To a solution of
(+)-(S)-4 (456 mg, 1.7 mmol) in EtOH (23 mL), 2.0m NaOH (23 mL)
was added. The mixture was stirred at RT for 2 h, concentrated
under vacuum, adjusted to pH 2 with HCl (1.0m) and extracted
with CH2Cl2 (3ꢃ40 mL). The combined organic phases were dried
over anhydrous Na2SO4 and evaporated to dryness, yielding the de-
sired product as a white solid (401 mg, 98%): 87% ee determined
by analytical chiral HPLC [Daicel Chiralpak IC (1=0.46 cm, l=
15 cm, 5 mm), n-heptane/2-propanol/TFA 96:4:0.1, flow rate=
0.8 mLminꢁ1, l=250 nm]: tRmajor =8.31 min, tRminor =9.85 min; mp:
110–1128C; [a]D20 = +33.6 (c=1.0 in CH3OH); 1H NMR (400 MHz,
[D6] acetone): d=10.6 (brs, 1H), 7.64 (m, 2H), 7.59 (d, 3J(H,H)=
8.3 Hz, 2H), 7.44 (t, 3J(H,H)=7.6 Hz, 2H), 7.39 (d, 3J(H,H)=8.2 Hz,
2H), 7.34 (m, 1H), 3.29 (m, 1H), 2.70–2.58 (m, 2H), 1.33 ppm (d,
3J(H,H)=7.0 Hz, 3H); 13C NMR (100 MHz, [D6] acetone): d=173.4,
146.3, 141.7, 139.8, 129.6, 128.2, 128.0, 127.7, 127.6, 42.6, 36.8,
22.4 ppm; MS (ESI): m/z 239.11 [MꢁH]ꢁ.
Biological investigations
In vitro binding assays
Materials: Guinea pig brains, rat liver, and rat brains for the s1, s2,
m-, k-, and d-opioid receptor binding assays were commercially
available (Harlan–Winkelmann, Borchen, Germany). Pig brains for
binding assays to the PCP binding site of the NMDA receptor were
a kind donation from a local slaughterhouse (Coesfeld, Germany).
Homogenizer: Elvehjem Potter (B. Braun Biotech International, Mel-
sungen, Germany) and Soniprep 150, MSE, London, UK). Centrifug-
es: Cooling centrifuge model Rotina 35R (Hettich, Tuttlingen, Ger-
many) and High-speed cooling centrifuge model Sorvall RC-5C plus
(Thermo Fisher Scientific, Langenselbold, Germany). Multiplates:
standard 96-well multiplates (Diagonal, Muenster, Germany).
Shaker: self-made device with adjustable temperature and tum-
bling speed (scientific workshop of the institute). Vortexer: Vortex
Genie 2 (Thermo Fisher Scientific, Langenselbold, Germany). Har-
vester: MicroBeta FilterMate-96 Harvester. Filter: Printed Filtermat
Type A and B. Scintillator: Meltilex (Type A or B) solid-state scintilla-
tor. Scintillation analyzer: MicroBeta Trilux (all PerkinElmer LAS,
Rodgau-Jꢀgesheim, Germany). Chemicals and reagents were pur-
chased from various commercial sources and were of analytical
grade.
(+)-(S)-3-(Biphenyl-4-yl)-1-(piperidin-1-yl)butan-1-one [(+)-(S)-7]:
In a microwave vial, TBTU (321 mg, 1 mmol) was added to a solu-
tion of (+)-(S)-6 (120 mg, 0.5 mmol) in THF (16 mL). The mixture
was stirred at RT for 5 min, and then a solution of N,N-diisopropyl-
ethylamine (DIPEA, 0.2 mL, 1 mmol) and piperidine (0.1 mL,
1 mmol) in THF (2 mL) was added. The reaction mixture was irradi-
ated by microwave at 25 W, 808C for 20 min, and then the solvent
was evaporated under vacuum. The residue was then dissolved
with CH2Cl2 (30 mL), and the organic phase was washed with 0.5m
HCl (3ꢃ15 mL), dried over anhydrous Na2SO4, and evaporated,
yielding the desired product as a yellow oil (147 mg, 96%): 87% ee
determined by analytical chiral HPLC (Daicel Chiralcel OJ-H (1=
0.46 cm, l=15 cm, 5 mm), n-heptane/2-propanol 90:10, flow rate=
Preparation of membrane homogenates from guinea pig brain: Five
guinea pig brains were homogenized with the potter (500–
800 rpm, 10 up-and-down strokes) in six volumes of cold 0.32m su-
crose. The suspension was centrifuged at 1200 g for 10 min at 48C.
The supernatant was separated and centrifuged at 23500 g for
20 min at 48C. The pellet was resuspended in 5–6 volumes of
buffer (50 mm Tris, pH 7.4) and centrifuged again at 23500 g
(20 min, 48C). This procedure was repeated twice. The final pellet
was resuspended in 5–6 volumes of buffer and frozen (ꢁ808C) in
0.8 mLminꢁ1
[a]D20 = +11.6 (c=0.5 in CH3OH); 1H NMR (400 MHz, CDCl3): d=
, l=250 nm): tRmajor =10.09 min, tRminor =12.03 min;
3
3
7.57 (m, 2H), 7.53 (d, J(H,H)=8.2 Hz, 2H), 7.43 (t, J(H,H)=7.6 Hz,
2H), 7.35–7.31 (m, 3H), 3.59–3.29 (m, 5H), 2.67 (dd, AB system,
2J(H,H)=14.7 Hz, 3J(H,H)=6.5 Hz, 1H), 2.56 (dd, AB system,
3
2J(H,H)=14.7 Hz, J(H,H)=7.9 Hz, 1H), 1.59–1.47 (m, 6H), 1.38 ppm
1.5 mL portions containing ~1.5 (mg protein)mLꢁ1
.
3
(d, J(H,H)=7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): d=170.8, 145.5,
Preparation of membrane homogenates from rat liver: Two rat livers
(Sprague–Dawley rats) were cut into small pieces and homogen-
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ized with the potter (500–800 rpm, 10 up-and-down strokes) in six
volumes of cold 0.32m sucrose. The suspension was centrifuged at
1200 g for 10 min at 48C. The supernatant was separated and cen-
trifuged at 31000 g for 20 min at 48C. The pellet was resuspended
in 5–6 volumes of buffer (50 mm Tris, pH 8.0) and incubated at RT
for 30 min. After incubation, the suspension was centrifuged again
at 31000 g for 20 min at 48C. The final pellet was resuspended in
5–6 volumes of buffer and stored at ꢁ808C in 1.5 mL portions con-
well was washed five times with 300 mL of water. Subsequently, the
filtermats were dried at 958C. The solid scintillator was melted on
the dried filtermats at 958C for 5 min. After solidifying of the scin-
tillator at RT, the trapped radioactivity in the filtermats was mea-
sured with the scintillation analyzer. Each position on the filtermat
corresponding to one well of the multiplate was measured for
5 min with the [3H]-counting protocol. The overall counting effi-
ciency was 20%. The IC50 values were calculated with GraphPad
Prism 3.0 (GraphPad Software, San Diego, CA, USA) by nonlinear
regression analysis. The IC50 values were subsequently transformed
into Ki values using the equation of Cheng and Prusoff.[19] The Ki
values are given as mean value ꢀSEM from three independent
experiments.
taining ~2 (mg protein)mLꢁ1
.
Preparation of membrane homogenates from rat brain: Five rat
brains (Sprague–Dawley rats) were homogenized with the potter
(500–800 rpm, 10 up-and-down strokes) in six volumes of cold
0.32m sucrose. The suspension was centrifuged at 1200 g for
10 min at 48C. The supernatant was separated and centrifuged at
23500 g for 20 min at 48C. The pellet was resuspended in 5–6 vol-
umes of buffer (50 mm Tris, pH 7.4) and centrifuged again at
23500 g (20 min, 48C). This procedure was repeated twice. The
final pellet was resuspended in 5–6 volumes of buffer and stored
s1 receptor binding assay: The assay was performed with the radio-
ligand [3H](+)-pentazocine (22.0 Cimmolꢁ1
; PerkinElmer). The
thawed membrane preparation of guinea pig brain cortex (~
100 mg protein) was incubated with various concentrations of test
compounds, 2 nm [3H](+)-pentazocine, and Tris buffer (50 mm,
pH 7.4) at 378C. The nonspecific binding was determined with
10 mm unlabeled (+)-pentazocine. The Kd value of (+)-pentazocine
is 2.9 nm.
at ꢁ808C in 1.5 mL portions containing ~1.5 (mg protein)mLꢁ1
.
Preparation of membrane homogenates from pig brain cortex: Fresh
pig brain cortex was homogenized with the potter (500–800 rpm,
10 up-and-down strokes) in six volumes of cold 0.32m sucrose. The
suspension was centrifuged at 1200 g for 10 min at 48C. The super-
natant was separated and centrifuged at 31000 g for 20 min at
48C. The pellet was resuspended in 5–6 volumes of Tris/EDTA
buffer (5 mm/1 mm, pH 7.5) and centrifuged again at 31000 g
(20 min, 48C). The final pellet was resuspended in 5–6 volumes of
buffer and frozen (ꢁ808C) in 1.5 mL portions containing ~0.8 (mg
s2 receptor binding assay: The assays were performed with the
radioligand [3H]DTG (specific activity 50 Cimmolꢁ1; ARC, St. Louis,
MO, USA). The thawed membrane preparation of rat liver (~100 mg
protein) was incubated with various concentrations of the test
compound, 3 nm [3H]DTG, and buffer containing (+)-pentazocine
(500 nm (+)-pentazocine in 50 mm Tris, pH 8.0) at RT. The nonspe-
cific binding was determined with 10 mm unlabeled DTG. The Kd
value of [3H]DTG is 17.9 nm.
protein)mLꢁ1
.
Protein determination: The protein concentration was determined
by the method of Bradford[17] modified by Stoscheck.[18] The Brad-
ford solution was prepared by dissolving 5 mg of Coomassie Bril-
liant Blue G 250 in 2.5 mL EtOH (95% v/v). Deionized H2O (10 mL)
and phosphoric acid (85% w/v, 5 mL) were added to this solution,
and the mixture was stirred and filled to a total volume of 50 mL
with deionized water. Calibration was carried out using bovine
serum albumin as a standard in nine concentrations (0.1, 0.2, 0.4,
0.6, 0.8, 1.0, 1.5, 2.0, and 4.0 mgmLꢁ1). In a 96-well standard multi-
plate, 10 mL of the calibration solution or 10 mL of the membrane
receptor preparation were mixed with 190 mL of the Bradford solu-
tion. After 5 min, the UV absorption of the protein–dye complex at
l=595 nm was measured with a plate reader (Tecan Genios,
Tecan, Crailsheim, Germany).
k opioid receptor binding assay: The assay was performed with the
radioligand [3H]U-69,593 (55 Cimmolꢁ1, Amersham, Little Chalfont,
UK). The thawed guinea pig brain membrane preparation (~100 mg
protein) was incubated with various concentrations of test com-
pounds, 1 nm [3H]U-69,593, and Tris/MgCl2 buffer (50 mm, 8 mm
MgCl2, pH 7.4) at 378C. The nonspecific binding was determined
with 10 mm unlabeled U-69,593. The Kd value of U-69,593 is
0.69 nm.
m opioid receptor binding assay: The assay was performed with the
radioligand [3H]DAMGO (51 Cimmolꢁ1, PerkinElmer). The thawed
guinea pig brain membrane preparation (~100 mg protein) was in-
cubated with various concentrations of test compounds, 3 nm
[3H]DAMGO, and Tris/MgCl2 buffer (50 mm, 8 mm MgCl2, pH 7.4) at
378C. The nonspecific binding was determined with 10 mm unla-
beled Naloxon. The Kd value of DAMGO is 0.57 nm.
General protocol for binding assays: The test compound solutions
were prepared by dissolving ~10 mmol (usually 2–4 mg) of test
compound in 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 presoaked in 0.5% aqueous polyethylenimine solution for 2 h
at RT before use. All binding experiments were carried out in dupli-
cate in 96-well multiplates. The concentrations given are the final
concentrations in the assay. Generally, the assays were performed
by addition of 50 mL of the respective assay buffer, 50 mL test com-
d opioid receptor binding assay: The assay was performed with the
radioligand [3H]DPDPE (69 Cimmolꢁ1, Amersham). The thawed rat
membrane preparation (~75 mg protein) was incubated with vari-
ous concentrations of test compounds, 3 nm [3H]DPDPE, and Tris/
MgCl2/PMSF buffer (50 mm, 8 mm MgCl2, 400 mm PMSF, pH 7.4) at
378C. The nonspecific binding was determined with 10 mm unla-
beled morphine. The Kd value of DPDPE is 0.65 nm.
PCP binding site of the NMDA receptor binding assay: The assay was
pound solution at various concentrations (10ꢁ5, 10ꢁ6, 10ꢁ7, 10ꢁ8
,
performed with the radioligand [3H](+)-MK-801 (22.0 Cimmolꢁ1
;
10ꢁ9 and 10ꢁ10 m), 50 mL of corresponding radioligand solution, and
50 mL of the respective receptor preparation into each well of the
multiplate (total volume 200 mL). The receptor preparation was
always added last. During the incubation, the multiplates were
shaken at a speed of 500–600 rpm at the specified temperature.
Unless otherwise noted, the assays were terminated after 120 min
by rapid filtration using the harvester. During the filtration each
PerkinElmer). The thawed membrane preparation of pig brain
cortex (~100 mg protein) was incubated with various concentra-
tions of test compounds, 2 nm [3H](+)-MK-801, and Tris/EDTA
buffer (5 mm/1 mm, pH 7.5) at RT. The nonspecific binding was de-
termined with 10 mm unlabeled (+)-MK-801. The Kd value of
(+)-MK-801 is 1.26 nm.
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tralized with the addition of 21Na+ and 15Clꢁ counterions; fur-
thermore, the solution ionic strength was adjusted to the physio-
logical value of 0.15m by adding the required amounts of Na+ and
Clꢁ ions. Each solvated system was relaxed by 500 steps of steep-
est descent followed by 500 other conjugate-gradient minimization
steps and then gradually heated to a temperature of 300 K in inter-
vals 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 frequency of 2.0 psꢁ1. The SHAKE method[31] was
used to constrain all of the covalently bound hydrogen atoms,
while long-range nonbonded van der Waals interactions were trun-
cated by using dual cutoffs of 6 and 12 ꢂ. The particle mesh Ewald
(PME) method[32] was applied to treat long-range electrostatic inter-
actions. The protein was restrained with a force constant of 2.0 kcal
molꢁ1 ꢂꢁ1, and all simulations were carried out with periodic boun-
dary conditions. The density of each system was subsequently
equilibrated via MD runs in the isothermal–isobaric (NPT) ensem-
ble, 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 relaxa-
tion 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 stabil-
ity, the fluctuations of the RMSD of the simulated position of the
backbone atoms of the s1 receptor with respect to those of the ini-
tial protein were monitored. All physicochemical parameters and
RMSD values showed very low fluctuations at the end of the equili-
bration process, indicating that the systems reached a true equilib-
rium condition. Each equilibration phase was followed by a data
production run consisting of 4 ns of MD simulations in the canoni-
cal (NVT) ensemble. Only the last 2 ns of each equilibrated MD tra-
jectory were considered for statistical data collections. A total of
100 trajectory snapshots were analyzed for each ligand–receptor
complex. The binding free energy, DGbind, between each ligand and
the s1 receptor was estimated by resorting to the MM/PBSA
approach.[13] According to this well-validated methodolo-
gy,[7,11a,b,20,26,33] the free energy was calculated for each molecular
species (complex, receptor, and ligand), and the binding free
energy was computed as the difference:
Molecular simulations
The model structures of (R)- and (S)-RC-33 were sketched and geo-
metrically optimized using Discovery Studio (DS, version 2.5, Accel-
rys, San Diego, CA, USA). A conformational search was then carried
out using a well-validated, ad hoc developed combined molecular
mechanics/molecular dynamics simulated annealing (MDSA) proto-
col[7,11a,b,20] using Amber 11.[21] Accordingly, the relaxed structures
were subjected to five repeated temperature cycles (from 300 to
1000 K and back) using constant-volume/constant-temperature
(NVT) MD conditions. At the end of each annealing cycle, the struc-
tures were again energy minimized to converge below
10ꢁ4 kcalmolꢁ1 ꢂ, and only the structures corresponding to the
minimum energy were used for further modeling. The atomic par-
tial charges for the geometrically optimized compounds were ob-
tained using the RESP procedure,[22] and the electrostatic potentials
were produced by single-point quantum mechanical calculations
at the Hartree–Fock level with a 6-31G* basis set, using the Merz–
Singh–Kollman van der Waals parameters.[23] Eventual ff03[24] miss-
ing force field parameters for the RC-33 enantiomers were generat-
ed using the general Amber force field (GAFF)[25] of Amber 11. The
optimized structures of (R)- and (S)-RC-33 were then docked into
the s1 putative binding pockets by applying a consolidated proce-
dure;[7,11a,b,26] accordingly, it is described herein only briefly. All
docking experiments were performed with AutoDock 4.3/Auto-
Dock Tools 1.4.6[27] on a win64 platform. DS was employed to
define the size of the binding site, using an opening site of 10 ꢂ
and a grid size of 0.7 ꢂ. The dimensions of the AutoDock grid box,
based on the cavity identified by DS, was large enough to cover all
possible rotations of each ligand. van der Waals interactions and
hydrogen bonding (OꢁH, NꢁH, and SꢁH) were modeled with the
Amber 12-6 and 12-10 Lennard–Jones parameters, respectively,
while the distance-dependent relative permittivity of Mehler and
Solmajer[28] was applied in the generation of the electrostatic grid
maps. A total of 300 Monte Carlo/simulated annealing (MC/SA)
runs were performed, with 100 constant-temperature cycles for si-
mulated annealing. The GB/SA implicit water model[29] was used in
these calculations to mimic the solvated environment. The angles
of the side chains and the rotation of the angles f and y were set
free during the calculations, while all other parameters of the MC/
SA algorithm were kept as default. The structures of the two com-
pounds were subjected to cluster analysis with a 1 ꢂ tolerance for
an all-atom root-mean-square (RMS) deviation from a lower-energy
structure representing each cluster family. The resulting docked
conformations were clustered and visualized; then, for each com-
pound, only the molecular 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. Both ligand–receptor complex ob-
tained from the docking procedure was further refined in
Amber 11 using the quenched molecular dynamics (QMD) meth-
od.[7,11a,b,20,26] According to QMD, 1 ns MD simulations at 300 K
were employed to sample the conformational space of each
ligand–receptor complex in the GB/SA continuum solvation envi-
ronment.[29] The integration step was equal to 1 fs. After each pico-
second, each system was cooled to 0 K, and the structure was ex-
tensively minimized and stored. To prevent global conformational
changes of the protein, the backbone atoms of the protein binding
DGbind ¼ GcomplexꢁðGreceptor þ GligandÞ ¼ DEMM þ DGsolꢁTDS
ð1Þ
The molecular mechanics energy DEMM was calculated as the sum
of the van der Waals and electrostatic interactions:
DEMM ¼ DEVDW þ DEELE
ð2Þ
The solvation free energy term DGsol was composed of the polar
and nonpolar contributions:
DGsol ¼ DGPB þ DGNP
ð3Þ
DGPB was estimated using DelPhi,[34] which solves the Poisson–
Boltzmann equations numerically and calculates the electrostatic
energy according to the electrostatic potential. Dielectric constants
of 1 and 80 were used for solute and solvent, respectively. A grid
spacing of 0.5 per ꢂ, extending 20% beyond the dimensions of
the solute, was employed in these calculations. The nonpolar sol-
vation contribution was determined using the following relation-
ship:[35]
site were constrained by a harmonic force constant of 100 kcalꢂꢁ1
,
whereas the amino acid side chains and ligands were allowed to
move without constraint. The best energy configuration of each
complex resulting from the previous step was subsequently solvat-
ed by a cubic box of TIP3P[30] water molecules extending at least
10 ꢂ in each direction from the solute. The system was then neu-
DGNP ¼ g ꢂ SA þ b
ð4Þ
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in which g=0.00542 kcalmolꢁ1 ꢂꢁ2, b=0.92 kcalmolꢁ1, and SA is
the molecular surface area estimated by means of the MSMS soft-
ware.[36] The conformational entropy (translation, rotation, and vi-
bration) upon ligand binding [ꢁTDS in Eq. (1)] was estimated using
normal-mode analysis with the Nmode module of Amber 11.[37]
Prior to normal-mode calculations, each MD snapshot of each re-
ceptor–ligand complex was energy minimized using a distance-de-
pendent dielectric constant e=4rij until the RMS of the elements
of the gradient vector was <10ꢁ4 kcalmolꢁ1 ꢂ. To minimize the ef-
fects due to different conformations adopted by individual snap-
shots, and due to the high computational demand of this ap-
proach, we averaged the estimation of entropy over MD 40 snap-
shots for each molecular complex that were evenly extracted from
the last 2 ns of each corresponding MD trajectory. The per-residue
binding free energy decomposition was performed exploiting the
MD trajectory of each given compound–receptor complex, with
the aim of identifying the key residues involved in the ligand–
receptor interaction. This analysis was carried out using the MM/
GBSA approach, and was based on the same snapshots used in
the binding free energy calculation. All simulations were carried
out using the Sander and Pmemd modules of Amber 11, running
in parallel on 256 processors of the PLX calculation cluster of the
CINECA supercomputer facility (Bologna, Italy). The entire MD sim-
ulation and data analysis procedure was optimized by integrating
Amber 11 in modeFRONTIER, a multidisciplinary and multiobjective
optimization and design environment.[38]
Cytotoxicity test: In vitro spontaneously transformed keratinocytes
from histologically normal human skin (HaCaT) were purchased
from CLS (Cell Lines Service, D69214 Eppelhein, Germany) and
grown in DMEM/high glucose; 2 mm glutamine; Pen/Strep 1%;
FBS 10%. Cells were dissociated using an appropriate volume of
pre-warmed TrypLE Select cell dissociation reagent (Sigma–Aldrich)
to the flask (i.e., 1 mL in a T25 cm2 flask). Complete growth
medium was then added, and the cells were pelletted at 250 g for
5 min. HaCaT cells were resuspended in medium with 10% FBS
and plated 5000 cells per well in 96-well plates. After 24 h, cells
were treated with rac-RC-33·HCl, (R)-RC-33, or (S)-RC-33·HCl in the
concentration range of 1ꢃ10ꢁ8 to 1ꢃ10ꢁ5 m and incubated for 48
or 72 h at 378C under 5% CO2 in media with 5% FBS (compounds
were tested in triplicate, in n=4 independent experiments). An
MTT-based cytotoxicity test (CellTiter 96 AQueous One Solution
Cell Proliferation Assay, Promega) was performed, and the optical
density was read in a microplate reader (BioTek Instruments, Inc.).
Statistical analysis: Data are expressed as the mean ꢀ standard
error of the mean (SEM). Statistical analysis was performed by two-
way analysis of variance (ANOVA) and post hoc Tukey’s test. Values
of p<0.05 were considered statistically significant.
In vitro metabolic study
General procedure: Metabolic stability studies were conducted on
liver S9 fraction from rat and human. To make a comparison be-
tween oxidative and non-oxidative metabolism of the compound,
two studies were performed in parallel, with and without the addi-
tion of NADPH cofactor. Briefly, 200 mL of 20 mgmLꢁ1 commercial
liver S9 were firstly diluted to 0.5 mgmLꢁ1 with 0.1m PBS at
pH 7.4; 980 mL of the diluted S9 were then placed in Eppendorf
vials in a thermomixer at 378C and gentle shaking was applied
(300 rpm). NADPH (10 mL, 50 mm in water) was added to the S9 to
obtain a test solution with NADPH, while water alone (10 mL) was
added to the S9 to obtain an NADPH-independent test solution. Fi-
nally, (ꢁ)-(R)-RC-33·HCl or (+)-(S)-RC-33·HCl (10 mL of a 1 mm solu-
tion in DMSO) were added to the test solutions and the kinetics
measurements started. Aliquots (50 mL) from the reaction mixtures
were sampled in duplicate at 5, 10, 15, 30, 60, 120, and 240 min to
determine reaction kinetics. The reaction was quenched by adding
acetonitrile (150 mL) and DMSO (10 mL) to precipitate the proteins.
Samples were vortex mixed and centrifuged at 4000 rpm for
10 min at 48C. The supernatant was directly analyzed by UPLC/UV/
PAD without clean-up steps to estimate the concentration of test
compound at each time point. The extrapolated concentration is
useful to calculate the half-life (t1/2) of enantiomeric RC-33·HCl in
each matrix.
NGF neurite outgrowth in PC12 cells and cytotoxicity
Cell culture: PC12 cells were cultured at 378C, under 5% CO2 in
RPMI 1640 medium supplemented with 5% heat-inactivated fetal
bovine serum (FBS), 10% heat-inactivated horse serum (HS), 1%
Glutamax, 1% Zell (Biochrom). The medium was changed two or
three times a week. When NGF with or without the test com-
pounds had to be added cells were detached from the culture
dishes, centrifuged at 150 g for 5 min and plated at 8000 cellsmLꢁ1
onto glass coverslips coated with poly-d-lysine in 12-well tissue
culture plates; 24 h after plating, the medium was replaced with
RPMI 1640 medium containing 0.5% HS, 1% Glutamax, 1% Zell
and with NGF (2.5 ngmLꢁ1) with or without drugs. Stock solutions
(10 mm) of compounds RC-33·HCl, (S)-RC-33·HCl, and (R)-RC-33·HCl
were dissolved with apyrogenic H2O to 1 mm solution and added
to the cell medium to reach the selected final concentrations
(0.25 mm). In some experiments, the well-characterized s1 receptor
antagonist NE-100 (10 mm stock solution in apyrogenic H2O) was
co-administered with RC-33·HCl, (S)-RC-33·HCl, or (R)-RC-33·HCl at
a final concentration of 3 mm.
Quantification of neurite outgrowth: Five days after incubation with
NGF (2.5 ngmLꢁ1) with or without drugs, PC12 cells, grown on
glass coverslips, were fixed at RT for 15 min in phosphate-buffered
saline (PBS) containing 4% (w/v) paraformaldehyde. Morphometric
analysis was performed on digitized images of fixed cells taken
under phase-contrast illumination with an inverted microscope
(Optika) linked to a digital camera. Images of at least six fields per
coverslip were taken at 20ꢃ magnification in order to count an
average of 300 cells. At least three independent experiments were
performed for each condition. Neurite outgrowth was scored by
measuring the percentage of differentiated cells bearing at least
one neurite longer than the cell body diameter. Cell counting and
neurite length measurements were performed in a blind manner
by two independent observers using NeuronJ plugin[39] of ImageJ
public domain software.
Analytical method: UPLC analyses were carried out on a BEH Shield
RP18, 2.1ꢃ50 mm, 1.7 mm chromatographic column with gradient
mode, using mixtures of mobile phase A [H2O containing 0.1%
formic acid] and mobile phase B [CH3OH containing 0.1% formic
acid], at a flow rate of 0.5 mLminꢁ1. Each analysis lasted 1.7 min
with the following gradient: linear decrease from 90 to 0% mobile
phase A from 0 to 1 min; 100% of mobile phase B was maintained
for 0.5 min before a quick ramp (in 0.1 min) to 90% mobile phase
A. The analysis was continued under initial condition for another
0.1 min in isocratic mode. The injection volume for each sample
was 5 mL and chromatograms were recorded at l=254 nm. The
raw data (average peak area at each time point) were interpolated
using Quanlynx v 4.1 software to extrapolate the concentration of
test compound at each time point.
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Abbreviations
[3H]DAMGO: [3H][d-Ala2,Me-Phe4,Gly-ol5]enkephalin; DCE: 1,2-di-
chloroethane; [3H]DPDPE: [3H](1S,6S,12S)-6-[[(2S)-2-amino-3-[4-hy-
droxyphenyl]propanoyl]amino]-2,2,5,5-tetramethyl-7,10,13-trioxo-
12-(phenylmethyl)-3,4-dithia-8,11,14-triazacyclotetradecane-1-car-
boxylic acid; DTG: [3H]di-o-tolylguanidine; [3H]MK-801: [3H](5R,10S)-
(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-
imine; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; NE-100: 4-methoxy-3-(2-phenylethoxy)-N,N-dipropylben-
zeneethanamine hydrochloride; PRE-084: 2-(4-morpholinethyl)1-
phenylcyclohexanecarboxylate hydrochloride; TBTU: O-(benzotria-
zol-1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate; [3H]U-
69,593: [3H](5R,7R,8b-(ꢁ)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro-
(4-5)dec-8-yl]benzeneacetamide).
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D.R., S.C., M.P., and D.C. gratefully acknowledge financial support
from ARISLA (Grant SaNet-ALS). E.L., V.D.C., M.F., and S.P. grate-
fully acknowledge financial support from ESTECO s.r.l. (Grant
DDOS). Access to CINECA supercomputing facility was granted
through the sponsored Italian Super Computing Resource Alloca-
tion (ISCRA), project MONALISA (to S.P.).
Keywords: biological activity · enantioselectivity · in vitro
metabolism · neuroprotective agents · s1 agonists.
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Received: May 16, 2013
Published online on July 5, 2013
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