Novel enantiopure sigma receptor modulators: Quick (semi-)preparative chiral resolution via hplc and absolute configuration assignment

Article abstract of DOI:10.3390/molecules21091210

The identification of novel pan-sigma receptor (SR) modulators, potentially useful in cancer treatment, represents a new goal of our research. Here, we report on the preparation of novel chiral compounds characterized by a 3-C alkyl chain bridging an arom

Full text of DOI:10.3390/molecules21091210

molecules
Article
Novel Enantiopure Sigma Receptor Modulators:
Quick (Semi-)Preparative Chiral Resolution via
HPLC and Absolute Configuration Assignment
Marta Rui 1,2, Annamaria Marra 1, Vittorio Pace 2, Markus Juza 3, Daniela Rossi 1
and Simona Collina 1,
*
1
Department of Drug Sciences, Medicinal Chemistry and Pharmaceutical Technology Section,
University of Pavia, Viale Taramelli 12, Pavia 27100, Italy; marta.rui01@universitadipavia.it (M.R.);
annamaria.marra@unipv.it (A.M.); daniela.rossi@unipv.it (D.R.)
Department of Pharmaceutical Chemistry, University of Vienna, Althanstrasse 14, Vienna 1090, Austria;
vittorio.pace@univie.ac.at
2
3
Corden Pharma Switzerland LLC, Eichenweg 1, Liestal 4410, Switzerland; markus.juza@cordenpharma.com
Correspondence: simona.collina@unipv.it; Tel.: +39-0382-987-379
*
Academic Editors: Claudio Villani and Derek J. McPhee
Received: 20 July 2016; Accepted: 6 September 2016; Published: 10 September 2016
Abstract: The identification of novel pan-sigma receptor (SR) modulators, potentially useful in
cancer treatment, represents a new goal of our research. Here, we report on the preparation of
novel chiral compounds characterized by a 3-C alkyl chain bridging an aromatic portion to a
4-benzyl-piperidine moiety. All of the studied compounds have been prepared both in racemic and
enantiomerically-pure form, with the final aim to address the role of chirality in the SR interaction.
To isolate and characterize enantiomeric compounds, high-performance liquid chromatography
(HPLC) procedures were set up. A systematic analytical screening, involving several combinations of
chiral stationary and mobile phases, allowed us to optimize the analytical resolution and to set up the
(semi-)preparative chromatographic conditions. Applying the optimized procedure, the enantiomeric
resolution of the studied compounds was successfully achieved, obtaining all of the compounds
with an enantiomeric excess higher than 95%. Lastly, the absolute configuration has been empirically
assigned to enantiopure compounds, combining the electronic circular dichroism (ECD) technique to
the elution order study.
Keywords: sigma receptor (SR) modulators; amylose- and cellulose-derived CSPs; chiral resolution;
enantioselective HPLC; electronic circular dichroism (ECD)
1. Introduction
Sigma receptors (SRs) are involved in different pharmacological and pathological pathways.
They modulate cell survival and excitability and sub-serve many critical functions in the human
body. To date, two subtypes have been identified: Sigma 1 (S1R) and Sigma 2 receptors (S2R). S1Rs
are overexpressed in the central nervous system (CNS), while S2Rs are overexpressed in tumor cells
and tissues in proliferation [
several CNS diseases, such as anxiety, depression, schizophrenia, drug addiction, Parkinson’s and
Alzheimer’s diseases [ ], whereas S2R ligands could have a relevant role in cancer diagnosis and
therapy [ ]. Since some research groups identified an overexpression of S1R in different typologies of
cancer cells and demonstrated a strict correlation between S1R and this pathological manifestation, S1R
has been also proposed as a potential target for treating cancer conditions [ ]. As a result, therapies
1]. Accordingly, S1R modulators could be useful for the treatment of
2
3
4–6
that have S1R and S2R as targets might play a role against a wide spectrum of cancer types. Indeed,
recent literature highlights the anticancer potential of ligands able to bind both receptor subtypes,
Molecules 2016, 21, 1210; doi:10.3390/molecules21091210
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1210
2 of 12
called pan-SR ligands [
7]. The work presented here falls into this field, and it is a part of our ongoing
research focused on the identification of novel SR modulators. Along the years, we prepared and
studied a wide compound library of SR modulators [ ]. The SAR analysis evidenced that a good
affinity towards both SRs is related to the presence of a bulky aminic moiety [ 10]. Particularly, the
4-benzylpiperidine derivative RC-106 (Figure 1) is characterized by a good affinity toward both S1R
and S2R (Ki S1R = 12.0 nM 6.0; Ki S2R = 22.0 nM 1.1). Moreover, the SAR studies of chiral SR
8
9,
±
±
modulators showed that small changes in the ligand structure markedly influence the ability of SRs to
discriminate the enantiomeric form. For example, racemic and enantiomeric RC-33 (Figure 1) interact
in a non-stereoselective manner with SRs, both enantiomers showing a similar affinity toward both
S1R and S2R [11–13]. A different behavior was observed for the analogue RC-34, characterized by the
presence of an electronegative element (the only structural difference with respect to RC-33). In this
case, the (S)-configured enantiomer resulted in being the eutomer, with the following eudismic ratio
(ER, Ki distomer/Ki eutomer):ERS1R = 8.3 and ERS2R = 2.5 [14]. According to these results, the capability
of SRs to discriminate between the enantiomers of a ligand seems to be strictly related to its structural
features [15].
Figure 1. Structures of RC-33, RC-34 and RC-106.
In this paper, we report on the preparation of the potential chiral SR modulators 1–6, characterized
by an alkyl or alcoholic spacer bridging an aromatic moiety to the 4-benzyl-piperidine portion (Table 1)
and discuss in detail our efforts to develop easy-to-use chiral chromatographic methods, this approach
being effective for both analytical and preparative purposes [16
configuration assignment studies of enantiomerically-pure , combining electronic circular dichroism
(ECD) and elution order studies. Our final aim is to have enantiopure in a sufficient amount for
–19]. Moreover, we report on absolute
1–6
1–6
the biological investigation, in order to study the intriguing aspect of the influence of chirality in
SR interaction.
Table 1. Molecules under investigation.
Compound
Ar
R
1
2
OH
H
3
4
OH
H
5
6
OH
H
2. Results and Discussion
2.1. Synthesis
As reported in Scheme 1, the synthesis of 4-(4-benzyl-piperidin-1-yl)-butan-2-one, common
intermediate ( ), was accomplished in a good yield via the Michael addition of 4-benzyl piperidine to
A

Molecules 2016, 21, 1210
3 of 12
but-3-en-2-one in absolute ethanol and glacial acetic acid. Briefly, Compounds
1
and
3
were obtained
adopting a nucleophilic addition strategy of in situ-generated organolithium species to ketones.
The appropriate aryl-bromine was lithiated with an excess of tert-butyllithium (t-BuLi) in anhydrous
◦
diethyl ether (Et2O) at
−
78 C, followed by the addition of
A
: the desired tertiary alcohols
1
and
3
were
obtained in practical yields. Subsequently, the so-obtained alcohols were subjected to dehydration
with trifluoroacetic anhydride under copper(I) triflate catalytic conditions [20]. Purification on alumina
column chromatography provided the olefins 2a and 4a, which were hydrogenated (Pd (0)/C 10%
(p/p)) to give the corresponding reduced amines
naphthol derivatives and . However, prior to lithiation, the protection of the aromatic alcohol as
TBS-ether was required to prevent interference during the metalation step. Additional points merit
mention: (1) keeping the temperature at , (2) employing THF as
50 ◦C after the quenching with
the solvent and (3) using the less basic n-BuLi as the lithiating agent resulted in being beneficial to
2 and 4. The same tactic was applied for accessing
5
6
−
A
maximize the yield. Lastly, under identical conditions to those ones reported for the synthesis of
2 and
4, Compound 6 was obtained in a good yield.
Scheme 1. Synthesis of Compounds
acid, abs EtOH, rt; Compounds
to 0 ◦C; ( ) Cu(OTf)2, (CF3O)2O, anhydrous DCM, 0 ◦C to rt; (
) H2O rt; (
abs EtOH, rt; Compounds
) NH4Cl (aq.), rt; (
1
: (
–
a
6
. Experimental conditions:
β
-aminoketone A: (
78 ◦C to rt; (
) ketone
) H2, Pd(0)/C 10% (p/p),
a
) glacial acetic
1
–
4
) t-BuLi, anhydrous Et2O,
−
b
A
,
−
78 ◦C
c
d
e
5–
6
: (
a
) TBDMSCl, DMF; (
) TBAF, CH2Cl2, 0 C; (
b
) n-BuLi, THF,
−
78 ◦C; (
c
) ketone
A,
−
50 ◦C;
) TBAF,
◦
◦
(d
e
f) TFAA, Cu(OTf)2 (2 mol
·
%) CH2Cl2, 0 C to rt; (
g
CH2Cl2; (h) H2 (1 atm) Pd/C (10 mol %) EtOH, rt.

Molecules 2016, 21, 1210
4 of 12
2.2. Analytical Screening and Development of a Scalable Resolution Method
The strategy we adopted for obtaining enantiopure 1–6 is based on our knowledge and extensive
experience in chiral liquid chromatography that led us to have a wide range of chiral analytical and
(semi-)preparative columns in-house. As a first choice, we used amylose- and cellulose-based CSPs
immobilized on silica gel chiral columns, due to their wide solvent compatibility, versatility and
robustness. In details, Chiralpak IC (cellulose tris(3,5-dichlorophenylcarbamate immobilized on silica
gel) and Chiralpak IA (amylose tris(3,5-dimethylphenylcarbamate, immobilized on silica gel) have
been used, eluting with alcohols (MeOH or/and EtOH) or with n-hep in the presence of polar modifiers
(EtOH or IPA), DEA 0.1% and TFA 0.3%.
Therefore, the HPLC screening protocol of Table 2 was applied, and only when the results of this
screening were encouraging, but not fully successful, the mobile phase was slightly modified in order to
achieve a compound baseline separation. Since no satisfactory chiral separation for all compounds was
obtained, conventional cellulose-based chiral column Chiralcel OJ-H (cellulose tris(4-methylbenzoate,
coated on silica gel) was also experimented with, again eluting with alcohols (MeOH or/and EtOH)
or n-hep in the presence of IPA as polar modifier and adding DEA 0.1%. Results of the analytical
screening are reported in Tables 3–5 and are expressed as retention (k), selectivity (
α
) and resolution
(Rs) factors.
Table 2. Screening protocol: mobile phase composition.
Mobile Phase Composition (%)
Entry
MeOH
EtOH
n-hep
IPA
1
2
3
4
100
50
-
-
-
-
-
50
10
-
-
90
90
-
-
10
Chiralpak IC provided a baseline separation of Compounds
1
,
2
,
4
and 6, even if the
chromatographic conditions were not suitable for a productive scale-up (Table 3). In detail, no
enantioresolution was observed eluting with alcohols (data not shown), while modest or good values
of
are quite long (tR of the second eluted enantiomer ranging from 30–90 min). Moreover, to solve
Compound , a slight modification of the mobile phase composition of the screening protocol, eluting
α and Rs were obtained eluting with n-hep/EtOH or n-hep/IPA, even if the retention times (tR)
1
with n-hep/IPA (92/8, v/v), has been necessary. Nevertheless, a modest resolution was achieved
(α = 1.12 and Rs = 1.44).
Table 3. Analytical screening on Chiralpak IC with different mobile phases containing 0.1% DEA and
0.3% TFA.
Mobile Phase
Compound
K1
K2
A
Rs
n-hep (%)
EtOH (%)
IPA (%)
90
90
92
10
-
-
10
8
1.52
4.45
1
1
-
-
(R/S)-1
-
7.76
7.83
8.72
9.62
1.12
1.44
90
90
10
-
-
10
1.93
1
1.23
-
(R/S)-2
(R/S)-3
(R/S)-4
(R/S)-5
(R/S)-6
2.36
90
90
10
-
-
10
1.29
1.35
1
1
-
-
90
90
10
-
-
10
1.56
1
1.33
-
6.52
8.68
3.08
90
90
10
-
-
10
3.24
15.79
1
1
-
-
90
90
10
-
-
10
4.07
1
1.11
-
27.14
29.97
1.26
Flow rate: 1 mL/min. Detection: 254 nm (Compounds 1, 2, 5, 6) and 220 nm (Compounds 3, 4).

Molecules 2016, 21, 1210
5 of 12
Chiralpak IA gave rise to better results, being effective in resolving Compounds
Interestingly, the presence of n-hep in the mobile phase was essential for the separation of
gave rise to quite long retention times (tR of the second eluted enantiomer ranging from 15–65 min).
Conversely, and (endowed with an alkyl spacer) have been successfully resolved eluting with
1–
6
(Table 4).
1, 3 and 5 and
2,
4
6
pure MeOH in short times (tR second eluted enantiomer less than 10 min).
Table 4. Analytical screening on Chiralpak IA with different mobile phases containing 0.1% DEA.
Mobile Phase
Compound
(R/S)-1
(R/S)-2
(R/S)-3
(R/S)-4
(R/S)-5
(R/S)-6
K1
K2
α
Rs
n-hep (%)
MeOH (%)
EtOH (%)
IPA (%)
-
90
90
100
-
-
-
10
-
-
-
10
5.48
3.48
1
1
1.11
-
-
5.00
1.00
5.55
1.24
1.69
-
90
90
100
-
-
-
10
-
-
-
10
1.24
1
1.19
1.73
-
2.08
2.62
4.24
2.77
0.57
5.07
3.25
0.81
-
90
90
100
-
-
-
10
-
-
-
10
0.53
3.84
1
1.17
1
-
1.67
-
-
90
90
100
-
-
-
10
-
-
-
10
1.42
1
1
3.06
-
-
1.99
3.21
-
90
90
100
-
-
-
10
-
-
-
10
0.35
1
1.27
1.26
-
13.72
14.68
17.38
18.55
3.04
2.95
-
90
90
100
-
-
-
10
-
-
-
10
0.96
8.90
19.76
1.27
10.45
23.86
1.32
1.18
1.21
2.49
3.22
2.94
Flow rate: 1 mL/min. Detection: 254 nm (Compounds 1, 2, 5, 6) and 220 nm (Compounds 3, 4).
On the basis of these results and keeping in mind that our purpose is to set up an economic and
productive preparative enantiomer separation for , we turned our attention to a further analytical
screening, using the Chiralcel OJ-H column. Baseline separation of Compounds and has been
1–6
1
–
3
5
obtained in short retention times, less than 12 min (referred to the second eluted enantiomer). Results
are reported in Table 5. Interestingly, high enantioselectivity and good resolution have been achieved
eluting only with alcohols, while no separation occurs eluting with n-hep, with the only exception of
Compound 1.
Table 5. Analytical screening on Chiralcel OJ-H with different mobile phases containing 0.1% DEA.
Mobile Phase
Compound
K1
K2
α
Rs
n-hep (%)
MeOH (%)
EtOH (%)
IPA (%)
-
-
-
100
50
-
-
50
100
-
-
-
-
2.5
1
-
1.77
1.03
1.56
2.40
1.63
2.27
1.36
1.58
1.46
3.14
3.25
2.35
(R/S)-1
90
-
10
-
-
-
100
50
-
-
50
100
-
-
-
-
3.98
1.87
1.23
4.80
2.25
1.46
1.21
1.20
1.19
1
2.46
1.97
1.83
-
(R/S)-2
90
-
10
9.61

Molecules 2016, 21, 1210
6 of 12
Table 5. Cont.
Mobile Phase
Compound
K1
K2
α
Rs
n-hep (%)
MeOH (%)
EtOH (%)
IPA (%)
-
-
-
100
50
-
-
50
100
-
-
-
-
1.24
0.70
1.80
0.93
1.45
1.33
1
4.05
2.32
-
-
(R/S)-3
(R/S)-4
(R/S)-5
(R/S)-6
2.24
0.46
90
-
10
1
-
-
-
100
50
-
-
50
100
-
-
-
-
1.52
0.74
0.46
0.54
1
1
1
1
-
-
-
-
90
-
10
-
-
-
100
50
-
-
50
100
-
-
-
-
1.10
0.74
0.39
1.61
1.07
0.70
1.46
1.45
1.79
1
2.62
1.99
1.92
-
90
-
10
0.32
-
-
-
100
50
-
-
50
100
-
-
-
-
2.20
1.25
0.73
0.61
1
1
1
1
-
-
-
-
90
-
10
Flow rate: 0.5 mL/min. Detection: 254 nm (Compounds 1, 2, 5, 6) and 220 nm (Compounds 3, 4).
To sum up, we set up methods able to give baseline separation of
time. In detail, the methods foresee the use of Chiralpak IA for solving Compounds
of Chiralcel OJ-H for solving and . In view of these results, we considered the developed
1–
6
in a relatively short analysis
4
and and
6
1
–
3
5
methodologies suitable for the scale-up to the (semi-)preparative scale, and accordingly, no further
attempts were made to extend the screening under HPLC conditions. Figure 2 shows chromatograms
of racemic
1–6, when the highest resolution was reached, that is using Chiralpak IA or Chiralcel OJ-H
columns, eluting with 100% methanol (Compounds 2–6) or with 100% ethanol (Compound 1).
Figure 2. Analytical enantiomer separation of (R/S)-
(4.6 mm 150 mm, dp = 5 m) and of (R/S)- and (R/S)-
dp = 5 µm). (R/S)- elution condition: 100% EtOH, DEA 0.1%, flow rate 0.5 mL/min. (R/S)-
and (R/S)- elution condition: 100% MeOH, DEA 0.1%, flow rate 1.0 mL/min. (R/S)- and (R/S)-
elution condition: 100% MeOH, 0.1% DEA, flow rate 1 mL/min. For all: injection volume 10 µL.
1
, (R/S)-
2
, (R/S)-
3
, (R/S)-
5
on Chiralcel OJ-H
×
µ
4
6
on Chiralpak IA (4.6 mm
×
250 mm,
1
2, (R/S)-
3
6
5
4

Molecules 2016, 21, 1210
7 of 12
2.3. Preparation of Enantiomeric 1–6 through HPLC
During the drug discovery process, (semi-)preparative resolution of enantiomers using HPLC is a
powerful technique for the rapid preparation of enantiomers. Employing this technique, important
prerequisites for an economic and productive preparative enantiomer separation are: (i) retention times
as short as possible; (ii) high solubility of the racemate and the enantiomers in the eluent/injection
solvent; and (iii) the use of a mobile phase consisting of a pure low-cost solvent, facilitating work-up
and re-use of the mobile phase. As previously discussed, using Chiralpak IA or Chiralcel OJ-H columns
and eluting with alcohols added with DEA (0.1%), relatively short retention times (less than 12 min
for the second eluted enantiomer), high enantioselectivity and good resolution could be observed
(Figure 2). Accordingly, these experimental conditions were transferred to a Chiralpak IA and a
Chiralcel OJ-H columns with an ID of 10 mm, on which a maximum of 12.5 mg could be separated in
one run, depending on the solubility of the compound in the mobile phase. Therefore, racemic
were processed in a low number of cycles (Table 6), leading to enantiopure in satisfactory amounts
95% (Table 6), as evidenced by analytical control of the collected fractions
1–6
1–6
and yields, with an ee
(Figure S1).
≥
Table 6. (Semi-)preparative resolution of (R/S)-
(10 mm 250 mm, dp = 5 m) and of (R/S)- and (R/S)-
dp = 5 µm).
1
, (R/S)-
2
6
, (R/S)-
on Chiralpak IA (10 mm
3, (R/S)-5 on Chiralcel OJ-H
×
µ
4
×
250 mm,
Processed
Amount (mg)
No.
Cycles
Isolated
Amount (mg)
Yield
(%)
[α]2D0
(MeOH)
Compound
(R/S)-1
(R/S)-2
(R/S)-3
(R/S)-4
(R/S)-5
(R/S)-6
CSP
Enantiomer
ee (%)
(+)-1
(−)-1
(+)-2
(−)-2
(+)-3
(−)-3
(+)-4
(−)-4
(+)-5
(−)-5
(+)-6
(−)-6
14.1
14.3
96.0
97.0
94.0
95.3
+40.5
−42.3
+6.1
−6.3
+10.5
−9.2
+8.2
−8.3
+24.2
−24.8
+11.8
−12.0
Chiralcel
OJ-H
30
30
50
16
22
25
3
3
4
2
4
3
13.8
12.5
95.0
95.0
92.0
83.3
Chiralcel
OJ-H
22.9
23.0
99.9
98.0
91.6
92.0
Chiralcel
OJ-H
6.1
6.3
99.9
99.9
76.3
78.8
Chiralpak
IA
9.1
8.9
99.9
99.9
82.7
80.9
Chiralcel
OJ-H
10.5
9.8
99.9
99.9
84.0
78.4
Chiralpak
IA
Flow rate: 2.5 mL/min. Detection: 254 nm (Compounds
volume: 1.0 mL.
1, 2, 5, 6) and 220 nm (Compounds 3, 4). Injection
Figure 3. Representative chromatogram of (semi-)preparative resolution: compound (R/S)-1, Chiralcel
OJ-H (10 mm 250 mm, dp = 5 m). Elution condition: 100% EtOH, DEA 0.1%, flow rate 2.5 mL/min.
Injection volume 1.0 mL. 1a: (+)-1, 1b: (−)-1.
×
µ

Molecules 2016, 21, 1210
8 of 12
A representative example of (semi-)preparative resolution is reported in Figure 3. Actually, 30 mg
of racemic were processed in three runs (45 min in total) at a flow rate of 2.5 mL/min at room
1
temperature. According to the chromatographic profile (Figure 3), a small intermediate fraction was
collected. Final analysis of 1 enantiomers is shown in Figure 3.
2.4. Absolute Configurational Assignment
As the last step of this work, the absolute configuration at the stereogenic center of enantiomeric
1
–
6
was established by combining electronic circular dichroism (ECD) and chiral HPLC analysis. The
electronic circular dichroism (ECD) spectra have been compared with the ECD curves of structural
analogues RC-33 and RC-34, whose configuration was already assigned by us [13 14]. In detail, the
ECD spectra of enantiomeric arylamino alcohols and were compared with that of (S)-RC-34
(Figure 1) and the ECD spectra of enantiomeric 2, 4 and 6, with that of (S)-RC-33.
Briefly, comparable Cotton effects (CEs) for (+)- , (+)- , (+)- and (+)-(S)-RC-34 compounds are
,
1,
3
5
1
3
5
evident in two ranges of wavelengths. Between 195 and 207 nm, there are negative CEs, and between
216 and 223 nm there are positive CE, attributable to 1La and 1Lb electronic transitions of benzene and
naphthalene (Figure 4). The sign of the CEs of 1La is consistently opposite that at the longer wavelength
(1Lb). Some evident differences in the profile of the curves are in agreement with the UV spectra and
could depend on the substitution pattern of the aromatic moiety. Basing on these considerations, the
(S) absolute configuration of (+)-(S)-RC-34 may be proposed also for (+)-
1, (+)-3 and (+)-5, because the
sequence around the stereogenic center is the same for all four compounds.
Further support to this configurational assignment was derived from the study of the elution order
of chiral HPLC analysis, taking into account that the absolute configuration of structurally-related
compounds usually follows the same chiral recognition mechanism in the chromatographic process on
a given chiral stationary phase, using the same mobile phase. Accordingly, (S)-RC-34, (+)-
1, (+)-3 and
(+)- were analyzed under the same chromatographic conditions (Chiralcel OJ-H, 50% MeOH and 50%
5
EtOH, 0.1% DEA) and resulted in all cases in the first eluted enantiomers. The elution order results
confirmed that the (S) absolute configuration may be attributed also to (+)-1, (+)-3 and (+)-5.
Empirical absolute configuration assignment of
ECD spectra of have been compared with that of the structurally-related compound (S)-RC-33,
as a reference of known stereochemistry. The enantiomers (+)- , (+)- , (+)- and (S)-RC-33 displayed
2, 4, 6 was effected applying a similar approach.
2, 4, 6
2
4
6
a similar negative CE in the wavelength range between 198 and 230 nm and a CE between 214 and
255 nm, associated with 1La and 1Lb electronic transitions of the aromatic chromophores, respectively.
1
Again, the sign of the CEs of La is consistently opposite that at the longer wavelength (1Lb), and
the differences in the profile of the curves could depend on the different aromatic nucleus. Based
on these considerations, the (S) absolute configuration of (+)-(S)-RC-33 may be proposed also for
(+)-
2
, (+)-
4
and (+)-
6
(Figure 5), having the same substituents around the stereogenic center. Again,
were analyzed under the same chromatographic conditions (Chiralcel OJ-H,
(S)-RC-33 and (+)-
2
50% MeOH and 50% EtOH, 0.1% DEA) and resulted in both cases in the first eluted enantiomers.
Unfortunately, the OJ-H column is not able to solve Compounds and . To confirm that a set of
4
6
structurally-related compounds is characterized by the same absolute configuration, they must be
analyzed under the same chromatographic conditions. Indeed, chiral recognition mechanisms on chiral
stationary phases may be sensitive to even minor structural or conditional changes. Therefore, we took
into consideration the chromatographic profiles of (S)-RC-33, (+)-
(90/10 n-hep/IPA, 0.1% DEA), a condition that ensures the baseline separation of all of the studied
compound enantiomers. (S)-RC-33, (+)- , (+)- and (+)- showed the same behaviors, confirming that
2, (+)-4 and (+)-6 on Chiralpak IC
2
4
6
all of the first eluted enantiomers are characterized by the same absolute configuration, that is the (S)
configuration. These results are in agreement with the data collected through ECD technique, and the
(S) absolute configuration may be attributed also to (+)-1, (+)-3 and (+)-5.

Molecules 2016, 21, 1210
9 of 12
(A)
λmin
(B)
Compound
∆εmin
λ
max
∆ε
max
(S)-RC-34
(+)-1
206.5
208.5
195.5
207.5
−2.78
−3.58
−5.19
−4.82
253.0
222.5
216.5
223.5
1.09
7.59
1.57
6.31
(+)-3
(+)-5
Figure 4. ECD curves of (
A
) (+)-(S)-RC-34 and (
B) (+)-
1
, (+)-
3
, (+)-
5
and their Cotton effect (CE) in the
wavelength range 190–300 nm.
A
B
Compound
λmin
∆εmin
λ
max
∆ε
max
(S)-RC-33
(+)-2
229.8
214.0
198.5
198.0
−0.61
−9.04
−2.26
−4.34
255.0
232.5
223.0
214.0
0.47
3.56
3.85
2.18
(+)-4
(+)-6
Figure 5. ECD curves of (A) (+)-(S)-RC-33, B) (+)-2, (+)-4, (+)-6 and their Cotton effect (CE) in the
wavelength range of 190–300 nm.
3. Experimental Section
3.1. HPLC-UV Chiral Resolution
In order to identify the best conditions to be properly scaled up to the (semi-)preparative scale,
an analytical screening of cellulose- and amylose-based CSPs was firstly performed using Chiralcel
OJ-H (Chiral technologies Europe, Illkirch-Cedex, France, Europe, 4.6 mm diameter
×
150 mm
length, dp 5 µm), Chiralpack IC (Chiral technologies Europe, Illkirch-Cedex, France, Europe, 4.6 mm
diameter × 250 mm length, dp 5 µm) and Chiralpack IA (Chiral technologies Europe, Illkirch-Cedex,

Molecules 2016, 21, 1210
10 of 12
France, Europe, 4.6 mm diameter
of 0.5 mL/min, 1 mL/min and 1 mL/min, respectively. Analytes were detected photometrically
at 220 and 254 nm. Unless otherwise specified, sample solutions were prepared dissolving analytes
×
250 mm length, dp 5
µ
m) columns and eluting with a flow rate
at 1 mg/mL in ethanol and filtered through 0.45-µm PTFE membranes before analysis. The injection
volume was 10 L. The mobile phases consisted of alcohols (MeOH or/and EtOH) or mixtures of
µ
n-hep and polar modifiers (EtOH or IPA). In all cases, 0.1% of DEA was added to the mobile phase. In
the case of the Chiralpak IC columns, the analyses were carried out also in the presence of 0.3% of TFA.
The retention factor (k) was calculated using the equation k = (tR
time and t0 the dead time (t0 was considered to be equal to the peak of the solvent front and was taken
from each particular run). The enantioselectivity ( ) and the resolution factor (Rs) were calculated as
follows: = k2/k1 and Rs = 2 (tR2 tR1)/(w1 + w2), where tR2 and tR1 are the retention times of the
−
t0)/t0, where tR is the retention
α
α
−
second and the first eluted enantiomers and w1 and w2 are the corresponding base peak widths. The
best conditions found by the screening protocol were applied to a (semi-)preparative scale-up. The
enantiomers of
Chiralcel OJ-H column (10 mm diameter
) or MeOH (for Compounds and
1
,
2
,
3
and
5
were then completely resolved by a (semi-)preparative process using a
250 mm length, 5 m), eluting with EtOH (for Compound
) at rt with a flow rate of 2.5 mL/min. Compounds and
×
µ
1
2
,
3
5
4
6
were resolved on Chiralpak IA (10 mm diameter × 250 mm length, 5 µm) using MeOH at a flow rate
of 2.5 mL/min as the eluent.
The eluate was fractioned according to the UV profile (detection at 220 and 254 nm). The fractions
obtained containing the enantiomers were evaporated at reduced pressure. Analytical control of the
collected fractions was performed using the analytical columns.
3.2. Electronic Circular Dichroism
The solutions of
in n-hexane), (+)-3 (c: 3.09
6.29
10−6 M in n-hexane), (+)-6 (c: 1.31
(c: 2.5
10−5 M in n-hexane; optical pathway 1 cm) and (+)-(S)-RC-33 (c: 9.12
optical pathway 1 cm) were analyzed in a nitrogen atmosphere. ECD spectra were scanned at
50 nm/min with a spectral band width of 2 nm and a data resolution of 0.5 nm (Figures 4 and 5).
1
–
6
enantiomers: (+)-
1
(c: 2.68
×
10−5 M, in n-hexane), (+)-
2
(c: 1.90 × 10−5
M
×
10−5 M in n-hexane), (+)-4 (c: 2.21
×
10−5 M in n-hexane), (+)-5 (c:
×
×
10−5 M in n-hexane; optical pathway 1 cm), (+)-(S)-RC-34
×
×
10−5 M in n-hexane;
4. Conclusions
In this paper, we presented the synthesis of the novel potential SR modulators 1–6 and their
enantioseparation via HPLC chiral resolutions on cellulose- and amylose-based CSPs. A systematic
screening protocol for enantioselective HPLC was established leading to fast and easy-to-use chiral
HPLC separations suitable for a (semi-)preparative scale-up. The separation of the enantiomers was
optimized by varying the chromatographic parameters. Baseline separations were obtained for all of
the studied compounds under optimized chromatographic conditions. The recovery of the enantiomers
after chromatography was in the range of 76%–95%, for the individual enantiomers. (Semi-)preparative
enantioselective chromatography for compounds of interest proves to be a straightforward, productive
and robust methodology for the quick access to the desired amounts of pure enantiomers. It remains
one of the most versatile and cost effective tools for the fast isolation of desired enantiomers from a
racemic mixture. The absolute configuration at the chiral center of enantiomeric 1–6 was empirically
assigned by combining electronic circular dichroism (ECD) and chiral HPLC analysis.
The bioactivity of each enantiomer is now under investigation in order to deeply understand the
role of chirality in the SRs-ligand interaction.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
9/1210/s1.
Acknowledgments: The authors gratefully acknowledge Michela Culanti Indiano for the experimental support.

Molecules 2016, 21, 1210
11 of 12
Author Contributions: S.C. conceived of the work and contributed to reviewing the whole manuscript. She was
also responsible for the correctness of all of the studies. M.R. and A.M. performed the research, analyzed the data
and contributed to the writing of the manuscript. V.P provided guidance on the synthesis of the racemates. D.R.
and M.J. provided guidance on the design of the screening and the up-scaling of the enantiomer separations.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
The following abbreviations are used in this manuscript:
MDPI
DOAJ
DEA
ECD
EtOH
IPA
Multidisciplinary Digital Publishing Institute
Directory of Open Access Journals
Diethylamine
Electronic circular dichroism
Ethanol
Isopropanol
MeOH
n-hep
TFA
Methanol
n-Heptane
Trifluoroacetic acid
Ultraviolet
UV
References
1.
2.
3.
4.
Aydar, E.; Palmer, C.P.; Djamgoz, M.B. Sigma receptors and cancer: Possible involvement of ion channels.
Cancer Res. 2004, 64, 5029–5035. [CrossRef] [PubMed]
Collina, S.; Gaggeri, R.; Marra, A.; Bassi, A.; Negrinotti, S.; Negri, F.; Rossi, D. Sigma receptor modulators: A
patent review. Exp. Opin. Ther. Pat. 2013, 23, 597–613. [CrossRef] [PubMed]
Huang, Y.S.; Lu, H.L.; Zhang, L.J.; Wu, Z. Sigma-2 receptor ligands and their perspectives in cancer diagnosis
and therapy. Med. Res. Rev. 2014, 34, 532–566. [CrossRef] [PubMed]
Wang, B.; Rouzier, R.; Albarracin, C.T.; Sahin, A.; Wagner, P.; Yang, Y.; Smith, T.L.; Meric Bernstam, F.;
Marcelo, A.C.; Hortobagyi, G.N.; Pusztai, L. Expression of sigma 1 receptor in human breast cancer.
Breast Cancer Res. Treat. 2004, 87, 205–214. [CrossRef] [PubMed]
5.
6.
7.
8.
Aydar, E.; Onganer, P.; Perrett, R.; Djamgoz, M.B.; Palmer, C.P. The expression and functional characterization
of sigma (sigma) 1 receptors in breast cancer cell lines. Cancer Lett. 2006, 242, 245–257. [CrossRef] [PubMed]
Crottes, D.; Guizouarn, H.; Martin, P.; Borgese, F.; Soriani, O. The sigma-1 receptor: A regulator of cancer cell
electrical plasticity? Front. Physiol. 2013, 4, 175. [CrossRef] [PubMed]
Megalizzi, V.; le Mercier, M.; Decaestecker, C. Sigma receptors and their ligands in cancer biology: Overview
and new perspectives for cancer therapy. Med. Res. Rev. 2012, 32, 410–427. [CrossRef] [PubMed]
Collina, S.; Loddo, G.; Urbano, M.; Linati, L.; Callegari, A.; Ortuso, F.; Alcaro, S.; Laggner, C.;
Langer, T.; Prezzavento, O.; et al. Design, synthesis, and SAR analysis of novel selective sigma1 ligands.
Bioorg. Med. Chem. 2007, 15, 771–783. [CrossRef] [PubMed]
9.
Rossi, D.; Urbano, M.; Pedrali, A.; Serra, M.; Zampieri, D.; Mamolo, M.G.; Laggner, C.; Zanette, C.;
Florio, C.; Shepmann, D.; et al. Design, synthesis and SAR analysis of novel selective sigma1 ligands (Part 2).
Bioorg. Med. Chem. 2010, 18, 1204–1212. [CrossRef] [PubMed]
10. Rossi, D.; Pedrali, A.; Urbano, M.; Gaggeri, R.; Serra, M.; Fernandez, L.; Fernandez, M.; Caballero, J.;
Rosinsvalle, S.; Prezzavento, O.; et al. Identification of a potent and selective 1 receptor agonist potentiating
σ
NGF-induced neurite outgrowth in PC12 cells. Bioorg. Med. Chem. 2011, 19, 6210–6224. [CrossRef] [PubMed]
11. Rossi, D.; Marra, A.; Picconi, P.; Serra, M.; Catenacci, L.; Sorrenti, M.; Laurini, E.; Fermeglia, M.; Pricl, S.;
Brambilla, S.; et al. Identification of RC-33 as a potent and selective σ1 receptor agonist potentiating
NGF-induced neurite outgrowth in PC12 cells. Part 2: G-scale synthesis, physicochemical characterization
and in vitro metabolic stability. Bioorg. Med. Chem. 2013, 21, 2577–2586. [CrossRef] [PubMed]
12. Rossi, D.; Pedrali, A.; Gaggeri, R.; Marra, A.; Pignataro, L.; Laurini, E.; DalCol, V.; Fermeglia, M.; Pricl, S.;
Schepmann, D.; et al. Chemical, pharmacological, and in vitro metabolic stability studies on enantiomerically
pure RC-33 compounds: promising neuroprotective agents acting as σ1 receptor agonists. Chem. Med. Chem.
2013, 8, 1514–1527. [CrossRef] [PubMed]

Molecules 2016, 21, 1210
12 of 12
13. Rossi, D.; Pedrali, A.; Marra, A.; Pignataro, L.; Schepmann, D.; Wünsch, B.; Ye, L.; Leuner, K.; Peviani, M.;
Curti, D.; Azzolina, O.; Collina, S. Studies on the Enantiomers of as Neuroprotective Agents: Isolation,
Configurational Assignment, and Preliminary Biological Profile. Chirality 2013, 25, 814–822. [CrossRef]
[PubMed]
14. Rossi, D.; Marra, A.; Rui, M.; Laurini, E.; Fermeglia, M.; Pricl, S.; Schepmann, D.; Wuensch, B.; Peviani, M.;
Curti, D.; et al. A step forward in the sigma enigma: A role for chirality in the sigma1 receptor-ligand
interaction? MedChemComm 2014, 6, 138–146. [CrossRef]
15. Walker, J.M.; Bowen, W.D.; Walker, F.O.; Matsumoto, R.R.; De Costa, B.; Rice, K.C. Sigma receptors: Biology
and function. Pharmacol. Rev. 1990, 42, 355–402. [PubMed]
16. Collina, S.; Loddo, G.; Urbano, M.; Rossi, D.; Mamolo, M.G.; Zampieri, D.; Alcaro, S.; Gallelli, A.; Azzolina, O.
Enantioselective chromatography and absolute configuration of N,N-dimethyl-3-(naphthalen-2-yl)-butan-
1-amines: Potential Sigma1 ligands. Chirality. 2006, 18, 245–253. [CrossRef] [PubMed]
17. Rossi, D.; Nasti, R.; Marra, A.; Meneghini, S.; Mazzeo, G.; Longhi, G.; Memo, M.; Cosimelli, B.; Greco, G.;
Novellino, E.; et al. Enantiomeric 4-Acylamino-6-alkyloxy-2 Alkylthiopyrimidines As Potential A3
Adenosine Receptor Antagonists: HPLC Chiral Resolution and Absolute Configuration Assignment by a
Full Set of Chiroptical Spectroscopy. Chirality 2016. [CrossRef] [PubMed]
18. Gaggeri, R.; Rossi, D.; Collina, S.; Mannucci, B.; Baierl, M.; Juza, M. Quick development of an analytical
enantioselective high performance liquid chromatography separation and preparative scale-up for the
flavonoid Naringenin. J. Chromatogr. A 2011, 1218, 5414–5422. [CrossRef] [PubMed]
19. Rossi, D.; Marra, A.; Rui, M.; Brambilla, S.; Juza, M.; Collina, S. “Fit-for-purpose” development of analytical
and (semi)preparative enantioselective high performance liquid and supercritical fluid chromatography for
the access to a novel σ1 receptor agonist. J. Pharm. Biomed. Anal. 2016, 118, 363–369. [CrossRef] [PubMed]
20. Pace, V.; Martínez, F.; Fernández, M.; Sinisterra, J.V.; Alcántara, A.R. Highly Efficient Synthesis of New
α
-Arylamino-α
0-chloropropan-2-ones via Oxidative Hydrolysis of Vinyl Chlorides Promoted by Calcium
Hypochlorite. Adv. Synth. Catal. 2009, 351, 3199–3206. [CrossRef]
Sample Availability: Samples of all compounds are available from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).