Studies on the Enantiomers of RC-33 as Neuroprotective Agents: Isolation, Configurational Assignment, and Preliminary Biological Profile

Article abstract of DOI:10.1002/chir.22223

In this study we addressed the role of chirality in the biological activity of RC-33, recently studied by us in its racemic form. An asymmetric synthesis procedure was the first experiment, leading to the desired enantioenriched RC-33 but with an enantiom

Full text of DOI:10.1002/chir.22223

CHIRALITY 25:814–822 (2013)
Studies on the Enantiomers of RC-33 as Neuroprotective Agents:
Isolation, Configurational Assignment, and Preliminary
Biological Profile
DANIELA ROSSI,1 ALICE PEDRALI,1 ANNAMARIA MARRA,1 LUCA PIGNATARO,2 DIRK SCHEPMANN,3 BERNHARD WÜNSCH,3
1
LIAN YE,4 KRISTINA LEUNER,4 MARCO PEVIANI,5 DANIELA CURTI,5 ORNELLA AZZOLINA,1 AND SIMONA COLLINA
*
1Department of Drug Sciences, Medicinal Chemistry and Pharmaceutical Technology section, University of Pavia, Pavia, Italy
2Dipartimento di Chimica, Università degli Studi di Milano, Istituto di Scienze e Tecnologie Molecolari (ISTM) del CNR, Milan, Italy
3Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Münster, Germany
4Molecular & Clinical Pharmacy, FAU Erlangen/Nuremberg, Erlangen, Germany
5Department of Biology and Biotechnology “L. Spallanzani”, Laboratory of Cellular and Molecular Neuropharmacology, University of
Pavia, Pavia, Italy
ABSTRACT
In this study we addressed the role of chirality in the biological activity of
RC-33, recently studied by us in its racemic form. An asymmetric synthesis procedure was
the first experiment, leading to the desired enantioenriched RC-33 but with an enantiomeric
excess (ee) not good enough for supporting the in vitro investigation. An enantioselective
high-performance liquid chromatography (HPLC) procedure was then successfully carried out,
yielding both RC-33 enantiomers in amounts and optical purity suitable for the pharmacological
study. The absolute configuration of pure enantiomers was easily assigned exploiting the asym-
metric synthesis previously devised. As emerged in the preliminary in vitro biological investiga-
tion, (S)- and (R)-RC-33 possess a comparable affinity towards the σ1 receptor and a very a
similar behavior in the calcium influx assay, resulting in an equally effective σ1 receptor agonist.
Overall, the results obtained so far suggest that the interaction with the biological target is
nonstereoselective and leads us to hypothesize that there is a lack of stereoselectivity in the
biological activity of RC-33. Chirality 25:814–822, 2013. © 2013 Wiley Periodicals, Inc.
KEY WORDS: absolute configuration assignment; enantioselective HPLC; asymmetric
hydrogenation; neuroprotective agents; σ1 receptor agonists; chemical correlation
INTRODUCTION
out to be a potent σ1 receptor agonist in our validated PC12 cell
model of neuronal differentiation10 and showed high metabolic
stability in several biological matrices (i.e., mouse and rat blood,
rat, dog, and human plasma).11 Accordingly rac-RC-33 · HCl
was identified as the optimal candidate to be further
investigated. It has to be noted that the enantiomers of a chiral
drug must be considered as two different chemical entities,
potentially possessing different behavior in a physiological envi-
ronment (i.e., they can exhibit differentpharmacological and/or
toxicological profile). Accordingly, the racemate (1:1 mixture of
the two enantiomers) could be defined as a mixture in which the
eutomer (the more active and/or the less toxic enantiomer) is
impure of the distomer (the less active and/or the more toxic
enantiomer). Thus, in the case of a chiral molecule already
investigated as a racemate, the first issue to be addressed is
the study of the role of chirality in the biological activity by pre-
paring the pure enantiomers and evaluating their pharmacolog-
ical activity. Among the different approaches for the preparation
of enantiopure compounds, chiral resolution of racemates,
manipulation of chiral starting material (the so-called chiral pool),
Sigma1 (σ1) receptors are involved in several dysfunctions
of the central nervous system (CNS), such as depression,
anxiety, schizophrenia, acute and chronic neurodegenerative
diseases, in pain control, as well as in the etiology of some
types of cancer.1 Moreover, a mutation in the σ1 receptor
gene was recently found to be associated with frontotemporal
lobar degeneration (FTLD), which is the most common cause
of dementia under the age of 65 years, with associated motor
neuron disorders2 and with a familial juvenile form of
amyotrophic lateral sclerosis.3 Additionally, the σ1 receptor
has been found implicated in neurite sprouting and elonga-
tion in vitro, suggesting
a role for the receptor in
neuroplasticity.4–7 Accordingly, the research field related to
σ1 receptors is highly attractive. This drove, and it is still
driving, researchers of the medicinal chemistry and biology
fields to the discovery of potent and selective σ1 receptor
ligands and to the development of cell models able to
distinguish between agonists and antagonists.
In the last 10 years our research group has conducted
extensive studies aimed at discovering novel σ1 receptor
ligands as potential neuroprotective agents.8–15 In this
context, a drug discovery library based on arylalkenyl- and
arylalkylaminic scaffolds was prepared (Fig. 1).
In our compound library, the most promising molecule is rac-
1-[3-(1,1’-biphen)-4-yl]butyl-piperidine · HCl (rac-RC-33 · HCl,
Fig. 1), showing excellent σ1 receptor affinity (Ki = 0.70 0.3
nM), combined with high selectivity over various receptors
expressed in the CNS.10 Additionally, rac-RC-33 · HCl turned
Additional Supporting Information may be found in the online version of this
article.
*Correspondence to: Simona Collina, Department of Drug Sciences, Medici-
nal Chemistry and Pharmaceutical Technology section, University of Pavia,
Viale Taramelli 12, 27100 Pavia (Italy). E-mail: simona.collina@unipv.it
Received for publication 24 May 2013; Accepted 19 June 2013
DOI: 10.1002/chir.22223
Published online 7 September 2013 in Wiley Online Library
(wileyonlinelibrary.com).
© 2013 Wiley Periodicals, Inc.

ENANTIOMERS OF RC-33 AS NEUROPROTECTIVE AGENTS
815
Fig. 1. Structures of arylalkenyl- and arylalkylamines and of the most interesting compound rac-RC-33.
70 bar. The reaction was stirred overnight at room temperature (RT)
under pressure of hydrogen, then hydrogen was released, DCM was
evaporated, and the conversion was determined by nuclear magnetic
resonance NMR analysis of the crude. The desired products were puri-
fied by flash chromatography (FC) and then the enantiomeric excess
(ee) was determined by enantioselective HPLC.
asymmetric synthesis, and (semi)-preparative enantioselective
high-performance liquid chromatography (HPLC) using chiral
stationary phases (CSPs) are the most widely used ones. Partic-
ularly, asymmetric synthesis has become the most powerful and
commonly employed method for the preparation of homochiral
compounds, especially due to recent progress in technologies re-
lated to catalytic asymmetric synthesis. Since the development of
the first commercialized catalytic asymmetric synthesis of L-
DOPA,16 enantioselective hydrogenation has played a relevant
role in the preparation of chiral compounds, especially in indus-
trial processes, and it is still one of the most reliable catalytic
methods for the preparation of optically active molecules.17–19
Besides asymmetric synthesis, enantioselective HPLC resolu-
tion on CSPs has also been successfully employed for the isola-
tion of enantiopure compounds, being a viable route for
straightforward and rapid access to both enantiomers with high
optical purity and yields.20–22 The determination of the absolute
configuration of enantiopure compounds is then another un-
avoidable and complementary step to enantiopreparation, for
which single-crystal X-ray diffraction is still considered the exper-
imental technique of choice.23 However, this technique presents
some drawbacks, mainly related to sample characteristics (i.e.,
adequate resonant scattering properties and presence of heavy
atoms), and the need of highly specific equipment.23 Electronic
circular dichroism (ECD) and vibrational circular dichroism
(VCD), as well as chemical correlation with a chiral compound
of known configuration, have also been successfully applied to
the absolute configurational assignment of biologically active
molecules.21,24–27
Particularly, chemical correlation is a reliable empirical
method for configurational assignment. To this end, the
condition that all reactions involved in the stereoselective
process either do not influence the compound configuration
or do so in a predictable way (i.e., SN2 reactions) is strictly
required (or must be strictly fulfilled).
In this paper we focused on the preparation and the
configurational assignment of RC-33 enantiomers in order
to investigate the role of chirality in the biological activity.
To this aim, an integrated strategy combining asymmetric
synthesis, ECD analysis, and chiral HPLC was applied. A
preliminary biological profile of RC-33 pure enantiomers
was also drawn.
(+)-(S/R:93.5/6.5)-(S)-Ethyl 3-(biphenyl-4-yl)butanoate [(+)-(S/
R:93.5/6.5)-3]. Purification by FC (n-hexane/ethyl acetate 97:3)
gave the desired product as a yellow oil. Yield: 739 mg (95%). [α]2D0 = +
29.5 (c 0.5, CHCl3). IR (ATR): ν = 3030, 2971, 2926, 1908, 1731, 1487,
1163, 1033, 765, 697 cm-1. 1H NMR (400 MHz, CDCl3) δ = 7.58 (m, 2H),
7.54 (d, 3J(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, 3J(H,H) = 7.1 Hz, 2H), 3.34
(m, 1H), 2.66 (dd, AB 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 (t, 3J(H,H) = 7.1 Hz, 3H); 13C NMR (100 MHz,
CDCl3): δ = 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. MS (ESI) m/z 268.12 [M]. HPLC (Table 1): tRmajor
8.69 min, tRminor = 11.24 min.
=
(+)-(S/R:93.5/6.5)-Ethyl 3-phenylbutanoate [(+)-(S/R:93.5/6.5)-4].
Purification by FC (n-hexane/ethyl acetate 98:2) gave the desired product as a
colorless oil. Yield: 546 mg (98%). [α]2D0 = + 18.3 (c 0.5, CHCl3). Spectroscopic
properties comply with those reported in the literature.10 HPLC (Table 1):
tRminor =5.36min, tRmajor =5.91min.
Synthesis of (+)-(S/R:93.5/6.5)-3-(biphenyl-4-yl)butanoic acid
[(+)-(S/R:93.5/6.5)-5]. The compound was prepared according to
the procedure described for the corresponding racemate (see Supporting
Information), starting from (+)-(S/R:93.5/6.5)-3. White solid. Yield:
401 mg (98%), mp: 110-112°C. [α]2D0 = + 33.6 (c 1.0, CH3OH). IR (ATR): ν
= 3033, 2961, 2925, 1705, 1697, 1488, 1409, 1297, 947, 834, 761, 687 cm-1.
1H NMR (400 MHz, [D6]acetone) δ = 10.6 (br. s, 1H), 7.64 (m, 2H), 7.59
(d, 3J(H,H) = 8.3 Hz, 2H), 7.44 (t, 3(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
(d, 3J(H,H) = 7.0 Hz, 3H); 13C NMR (100 MHz, [D6]acetone): δ = 173.4,
146.3, 141.7, 139.8, 129.6, 128.2, 128.0, 127.7, 127.6, 42.6, 36.8, 22.4. MS
(ESI) m/z 239.11 [M-H]-. HPLC (Table 1), analytes were dissolved in a
mixture of n-heptane/2-propanol (90:10, v/v), tRmajor = 8.31 min,
tRminor = 9.85 min.
Synthesis of (+)-(S/R:93.5/6.5)-3-(biphenyl-4-yl)-1-(piperidin-1-yl)
butan-1-one [(+)-(S/R:93.5/6.5)-6]. The compound was prepared
according to the procedure described for the corresponding racemate
(see Supporting Information), starting from (+)-(S/R:93.5/6.5)-5. Yellow
oil. Yield: 176 mg (96%). [α]2D0 = + 11.6 (c 0.5, CH3OH). IR (ATR): ν = 3648,
3545, 3026, 2931, 2852, 2665, 1908, 1631, 1433, 1254, 1214, 1007, 763,
696 cm-1. 1H NMR (400 MHz, CDCl3) δ = 7.57 (m, 2H), 7.53 (d, 3J
(H,H) = 8.2 Hz, 2H), 7.43 (t, 3J(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, 2J(H,H) = 14.7 Hz, 3J
(H,H) = 7.9 Hz, 1H), 1.59-1.47 (m, 6H), 1.38 (d, 3J(H,H) = 7.0 Hz, 3H);
13C NMR (100 MHz, CDCl3): δ = 170.8, 145.5, 141.2, 139.6, 128.9, 127.6,
127.4, 127.3, 127.2, 41.5, 36.9, 26.1, 24.6, 21.8. MS (ESI) m/z 308.22
[M + H]+. HPLC (Table 1), tRmajor = 10.09 min, tRminor = 12.03 min.
MATERIAL AND METHODS
Chemicals, equipments, synthesis, and chiral analysis of all racemic
compounds, materials, and protocols of the biological assays are supplied
in the Supporting Information.
Asymmetric Synthesis of RC-33
Synthesis of (+)-(S/R:93.5/6.5)-3 and (+)-(S/R:93.5/6.5)-4. Cata-
lyst (R,R)-Ir(ThrePHOX)9 (49.9 mg, 0.029 mmol) was weighed in a special
glass vessel. The vessel was purged with nitrogen and a 0.362 M
dichloromethane (DCM) 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 the autoclave and purged three times with hydrogen at
Synthesis of (+)-(S/R:93.5/6.5)-1-(3-(biphenyl-4-yl)butyl)piperi-
dine [(+)-(S/R:93.5/6.5)-RC-33]. The compound was prepared
according to the procedure described for the corresponding racemate
(see Supporting Information), starting from (+)-(S/R:93.5/6.5)-6. Yellow
Chirality DOI 10.1002/chir

816
ROSSI ET AL.
TABLE 1. Summary of the chromatographic behavior of analytes 3, 4, 5, 6 and RC-33
Compound
Chiral column
Chiralcel OJ-H
Eluent (v/v/v/v)
k1
k2
α
RS
rac-3
n-heptane/2-propanola
90:10
2.62
3.68
1.40
2.64
rac-4
Chiralcel OJ-H
Chiralpak IC
Chiralcel OJ-H
Chiralpak IC
n-heptane/2-propanola
90:10
0.24
1.03
3.21
1.36
0.36
1.40
4.01
1.53
1.19
1.37
1.25
1.11
0.81
2.33
1.30
1.10
rac-5
n-heptane/2-propanol/TFAa
96:4:0.1
rac-6
n-heptane/2-propanola
90:10
rac-RC-33
n-heptane/EtOH/TEA/TFAb
90:10:0.1:0.3
Detection: λ = 250 nm.
a
Flow rate: 0.8 ml/min; b1 ml/min.
oil. Yield: 106mg (90%). [α]2D0 = + 19.2 (c 1.0, CH3OH). IR (neat): ν = 3056,
3027, 2927, 2850, 2799, 2761, 1598, 1486, 1450, 1153, 1119, 835, 764 cm-1.
1H NMR (400 MHz, CD2Cl2) δ = 7.62 (m, 2H), 7.55 (d, 3J(H,H) = 8.3 Hz,
2H), 7.44 (t, 3J(H,H) = 7.7 Hz, 2H), 7.36-7.32 (m, 1H), 7.30 (d, 3J
(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, 2H), 1.29 (d, 3J(H,H) = 7.0Hz, 3H). 13C
NMR (100 MHz, CD2Cl2): δ = 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. MS (ESI) m/z 294.25 [M+ H]+. HPLC
(Table 1), tRmajor = 9.91 min, tRminor = 11.44 min.
Isolation of RC-33 Enantiomers Via Enantioselective
Chromatography
In order to identify the best conditions to be properly scaled up to (semi)-
preparative scale, an analytical screening of cellulose-based CSPs was firstly
performed using Chiralcel OD-H (0.46cm diameter x 15cm length, 5 μm)
and Chiralcel OJ-H (0.46cm diameter x 15cm length, 5μm) columns and
eluting with a flow rate of 0.4 ml/min and 0.5 ml/min, respectively. Analytes
were detected photometrically at 250 nm. Unless otherwise specified, sam-
ple solutions were prepared dissolving analytes at 0.5 mg/ml in methanol
and filtered through 0.45μm PTFE membranes before analysis. The injec-
tion volume was 10 μL. The mobile phases employed are reported in Table 2.
The enantiomers of RC-33 were then completely resolved by a (semi)-pre-
parative process using a Chiralcel OJ-H column (1 cm diameter × 25 cm
length, 5 μm), eluting with methanol/diethylamine 100:0.1 (v/v) at RT with
a flow rate of 3 ml/min (Table 3). The eluate was properly partitioned
according to the UV profile (detection preformed at 250 nm). Analytical
control of collected fractions was performed on Chiralcel OJ-H eluting with
Electronic Circular Dichroism
The solutions of (+)-(S/R:93.5/6.5)-3 (c 6.25x10-5 M in n-hexane, optical
pathway 1 cm) and of (+)-(S/R:93.5/6.5)-4 (c 2.5x10-5 M in n-hexane, opti-
cal pathway 1 cm) were analyzed in a nitrogen atmosphere. ECD spectra
were scanned at 10 nm/min with a spectral band width of 2 nm and data
resolution of 0.2 nm.
TABLE 2. Screening results for the enantiomer separation of RC-33 on various CSPs
Chiralcel OD-Hb
Chiralcel OJ-Hc
Eluenta
k1
k2
α
RS
k1
k2
α
RS
A
B
C
D
E
F
G
H
I
0.18
—
—
—
—
—
—
—
—
—
—
—
—
—
—
2.08
1.08
0.70
n.t.d
n.t.d
1.13
0.77
0.64
1.97
0.50
1.67
1.07
0.83
2.22
1.16
0.76
1.07
1.07
1.09
—
—
—
d
n.t.
—
—
2.00
—
—
—
—
—
—
—
—
—
n.t.d
0.13
0.22
n.t.d
n.t.d
0.22
0.89
0.79
0.66
0.46
0.37
—
—
0.26
—
—
—
1.20
1.06
—
—
—
—
—
—
—
L
0.59
2.09
1.27
0.91
1.18
1.25
1.19
1.10
M
N
O
1.58
0.96
—
Bold type indicates best result obtained.
aEluent composition: A, n-heptane/EtOH/diethylamine (99:1:0.1, v/v/v); B, n-heptane/EtOH/ diethylamine (98:2:0.1, v/v/v); C, n-heptane/EtOH/diethylamine
(95:5:0.1, v/v/v) D, n-heptane/EtOH/diethylamine (90:10:0.1, v/v/v); E, n-heptane/2-propanol/diethylamine (99:1:0.1, v/v/v); F, n-heptane/2-propanol/
diethylamine (98:2:0.1, v/v/v); G, n-heptane/2-propanol/diethylamine (95:5:0.1, v/v/v); H, n-heptane/2-propanol/diethylamine (90:10:0.1, v/v/v); I, 2-propanol/
diethylamine (100:0.1, v/v); L, acetonitrile/diethylamine (100:0.1, v/v); M, MeOH/diethylamine (100:0.1, v/v); N, MeOH/EtOH/diethylamine (50:50:0.1, v/v/v);
O, EtOH/diethylamine (100:0.1, v/v).
bFlow rate: 0.4 ml/min.
cFlow rate: 0.5 ml/min.
dn.t.: not tested.
Chirality DOI 10.1002/chir

ENANTIOMERS OF RC-33 AS NEUROPROTECTIVE AGENTS
817
TABLE 3. (Semi)-preparative resolution of compound RC-33 on a Chiralcel OJ-H column (1 cm diameter × 25 cm length, 5 μm)
Flow rate
(ml/min)
Injection
volume (ml)
Concentration
(mg/ml)
MeOH/diethylamine (v/v)
100:0.1
tR1
tR2
3
11.6
13.0
1
1.25
methanol/diethylamine 100:0.1 (v/v) (flow rate 0.5 ml/min, UV detector at
250nm). The fractions obtained containing the enantiomers were
evaporated at reduced pressure.
and Pfaltz, showed high activity in the enantioselective hydro-
genation of the β-methylcinnamic ester (E)-2 (conversion
% > 99%, ee of the (R) product = 92%),28 which is structurally
related to (E)-1, we decided to take advantage of this catalyst
for our purposes.
(+)-(S)-1-(3-(Biphenyl-4-yl)butyl)piperidine (+)-(S)-RC-33. Yellow
oil. [α]2D0 = +22.1 (c 0.3, CH3OH). IR (ATR): ν = 3056, 3027, 2927, 2850,
2799, 2761, 1598, 1486, 1450, 1153, 1119, 835, 764 cm-1. 1H NMR (CDCl3)
δ 1.31 (d, J = 7.0 Hz, 3H), 1.39-1.49 (m, 2H), 1.55-1.64 (m, 4H), 1.79-1.92
(m, 2H), 2.21 (ddd, J = 5.8, 9.9, 12.1 Hz, 1H), 2.29-2.46 (br, 4H), 2.31
(ddd, J = 6.2, 9.8, 12.1 Hz, 1H), 2.78 (sextet, J = 7.2 Hz, 1H), 7.28
(d, J = 8.1 Hz, 2H), 7.32-7.38 (m, 1H), 7.42-7.48 (m, 2H), 7.54
(d, J = 8.2 Hz, 2H), 7.59-7.64 (m, 2H). 13C NMR (100 MHz, CD2Cl2): δ
=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. HPLC: tR = 9.91 min, ee 99.5%. HRMS (ESI) m/z [M
+H]+ calcd. for C21H28N: 294.2222, found 294.2215. By treatment of (+)-
(S)-RC-33 with 37% HCl in methanol under stirring condition, followed
by solvent evaporation, pure (+)-(S)-RC-33 · HCl was obtained as a white
solid. mp: 165-166°C dec.; [α]20D = +15.8 (c 0.2, CH3OH)
Thus, we started our synthetic approach (Scheme 2) with the
preparation of (E)-1 and (E)-2. As shown in Scheme 2A, target
compounds were obtained by the Suzuki-type reaction of (E)-
ethyl-3-cloro-2-iodobut-2-enoate with the appropriate boronic
acid, essentially according to the procedure described by
Simard-Mercier et al.29 The Suzuki substrate was readily
prepared as a single isomer by treating ethyl 2-butynoate with
Bu4NI in refluxing 1,2-dichloroethane (DCE).30 Both (E)-1
and (E)-2 were first hydrogenated with 10% Pd/C under hydro-
gen (Method I, Scheme 2A),8 yielding rac-3 and rac-4, that, in
turn, were used to develop chiral HPLC methods suitable for
evaluating the enantioselectivity of the hydrogenation reaction
catalyzed by the Ir(ThrePHOX) complex. For both analytes,
baseline separation was achieved on Chiralcel OJ-H, eluting
with n-heptane/2-propanol (Table 1).
(–)-(R)-1-(3-(Biphenyl-4-yl)butyl)piperidine (–)-(R)-RC-33. Yellow
oil. [α]2D0 = -22.2 (c 0.3, CH3OH). The IR and NMR spectra are identical
to that of (+)-(S)-RC-33. HPLC: tR = 11.44 min, ee 99.6%. HRMS (ESI)
m/z [M+H]+ calcd. for C21H28N: 294.2222, found 294.2218. By treatment
of (À)-(R)-RC-33 with 37% HCl in methanol under stirring condition,
followed by solvent evaporation, pure (À)-(R)-RC-33 · HCl was obtained
as a white solid. mp: 165-166°C dec.; [α]2D0 = À15.9 (c 0.2, CH3OH).
Compounds (E)-1 and (E)-2 were then subjected to
enantioselective
hydrogenation
using
the
(R,R)-Ir
(ThrePHOX) complex as catalyst (Method II, Scheme 2A).
Briefly, reactions were carried out in a Parr multireactor
using 2.9 mmol of each substrate in DCM, under 70 bar of hy-
drogen pressure at RT. Both (E)-1 and (E)-2 were fully
converted into the corresponding hydrogenated derivatives
(+)-3 and (+)-4, as evidenced by 1H-NMR analysis of the
crude products. Regarding the reaction stereoselectivity,
(+)-3 and (+)-4 were both obtained with an ee of 87%, as
proved by enantioselective HPLC analysis employing our
chiral HPLC methods (Table 1). As mentioned above, the
enantioselective hydrogenation of (E)-2, yielding (+)-4, was
performed as a reference reaction for configurational assign-
ment purposes. Briefly, Appella et al. reported a positive
RESULTS AND DISCUSSION
Asymmetric Synthesis
To have RC-33 enantiomers available in sufficient amounts
for exhaustive investigation of the role of chirality in the bio-
logical activity, an asymmetric synthesis procedure was the
first experiment. As shown in Scheme 1, the retrosynthetic
analysis led us to identify the asymmetric hydrogenation of
(E)-1 as the key step to achieve, after simple modifications,
the desired enantiomerically enriched RC-33. Since the Ir
(ThrePHOX) complex (Scheme 1), developed by Menges
Scheme 1. Retrosynthetic analysis of the (S)-enantiomerically enriched RC-33.
Chirality DOI 10.1002/chir

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ROSSI ET AL.
Scheme 2. (A) Synthesis of esters 3 and 4 in both racemic and enantioenriched forms. (B) Racemic and asymmetric synthesis of RC-33.
optical rotatory power in chloroform for the (S)-configured
compound 4 ([α]2D5 = + 19, c 1.1, CHCl3, ee = 90%).31 Since com-
pound 4, as obtained by us via enantioselective hydrogenation
of (E)-2 using the (R,R)-configured catalyst, showed a positive
optical rotatory power in chloroform ([α]D = + 18.3, c 0.5, CHCl3,
ee= 87%), the (S) configuration was assigned to (+)-4. This
configuration assignment is consistent with Menges and
Pfaltz’s report, in which the authors described the preparation
of (R)-4 via (S,S)-Ir(ThrePHOX) complex catalyzed hydrogena-
tion of (E)-2.28 The (S) configuration was also proposed for (+)-
3 as obtained by us via enantioselective hydrogenation of (E)-1
using the same (R,R)-configured catalyst.
alkaline hydrolysis rac-3 was fully converted into the corre-
sponding acid rac-5 (98% yield). This intermediate was then
condensed with piperidine in the presence of TBTU to yield
the tertiary amide rac-6 (96% yield), which, in turn, was finally
converted into the desired product rac-RC-33 by means of re-
duction with LiAlH4 (87% yield). Both intermediates rac-5 and
rac-6, together with the final product rac-RC-33, were used
to develop chiral HPLC methods suitable for verifying the
stereochemical behavior of each reaction step. Baseline sepa-
rations were achieved for all the analytes, as summarized in
Table 1. In detail, rac-5 and rac-RC-33 were fully resolved
on a Chiralpak IC column, eluting with a mixture of n-heptane
and 2-propanol or ethanol as polar modifier. With regard to
the chromatographic behavior of rac-6, baseline separation
was obtained on a Chiralcel OJ-H column, using a mixture
of n-heptane-2-propanol as mobile phase.
The synthetic pathway was then applied for the asymmetric
synthesis of RC-33 starting from (+)-(S)-3 (Scheme 2B), op-
erating under the same experimental conditions described
for the preparation of rac-RC-33. As summarized in
Scheme 1, the (S) absolute configuration could be assigned
to enantiomerically enriched RC-33 via chemical correlation
between enantioenriched RC-33 and its enantioenriched pre-
cursor 3, whose absolute configuration is (S), as discussed
above. The chemical correlation is reliable considering that
the conditions of all reactions involved in the stereoselective
process do not influence the compound configuration.
Indeed, enantioselective HPLC analysis performed after each
Empirical proof for this assignment came from ECD exper-
iments, using (+)-(S)-4 as reference of known stereochemis-
try. As shown in Figure 2A, both (+)-3 and (+)-(S)-4 showed
positive ECD curves in the range of wavelength between
210 and 300 nm. They displayed a similar positive Cotton
effect (CE) at about 215 nm (λmax 216, Δεmax 1.13 and λmax
215, Δεmax 1.38 for (+)-3 and (+)-(S)-4, respectively); more-
over, compound (+)-3 exhibited an additional positive CE at
higher wavelength (λmax 249, Δε
1.73). This behavior
max
was consistent with the compound’s UV spectra: as reported
in Figure 2, compound 4 showed only a maximum absorption
at 207 nm (Fig. 2B), while compound 3 exhibited two
maximums, at 207 nm and 247 nm respectively (Fig. 2C).
Hence, the proposed synthetic route was applied to the
preparation of rac-RC-33 starting from rac-3 (see Supporting
Information for experimental details). Briefly, by means of
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ENANTIOMERS OF RC-33 AS NEUROPROTECTIVE AGENTS
819
Fig. 3. Loading behavior of the Chiralcel OJ-H column (0.46 cm diameter x
15 cm length, 5 μm) for the separation of rac-RC-33. Mobile phase: methanol/
diethylamine (100/0.1, v/v); flow rate: 0.5 ml/min; detection at 250 nm;
injected amounts 40 μg (bottom trace), 80 μg, 160 μg, 200 μg, 240 μg, 320
μg (top trace).
are cellulose tris-(3,5-dimethylphenyl carbamate) and cellu-
lose tris-(4-methylbenzoate), respectively, coated on a silica
gel substrate. Elution conditions adopted included mixtures
of n-heptane and polar modifiers (ethanol or 2-propanol),
alcohols (methanol, ethanol, and 2-propanol), and acetoni-
trile; in all cases 0.1% of diethylamine was added to the
mobile phase. Results of the screening protocol are reported
in Table 2 expressed in terms of retention factor (k1 or k2),
separation factor (α), and resolution factor (RS). Generally,
no enantioresolution was observed on Chiralcel OD-H; only
a marginal enantioselectivity was evidenced eluting with n-hep-
tane/ethanol /diethylamine (90:10:0.1, v/v/v). On the contrary,
the results on Chiralcel OJ-H turned out to be quite promising.
Among alcohol-based mobile phases, good results in terms of α
and RS were evidenced eluting with both methanol/
diethylamine (100:0.1, v/v) and methanol/ethanol/diethylamine
(50:50:0.1, v/v/v): in both cases baseline separation was
achieved. In contrast, no enantioresolution was observed with
2-propanol/diethylamine (100:0.1, v/v); however, in practical ex-
perience, 2-propanol is rarely used as eluent in (semi)-prepara-
tive enantioselective HPLC because of its high viscosity and
difficulty of evaporation from collected fractions.32 Moreover,
the use of acetonitrile did not significantly improve the
enantioresolution. Finally, marginal or null enantioselectivity
was evidenced eluting with n-heptane/alcohol-based mobile
phases. Overall, the analytical results of Table 2 definitely indi-
cated that the best enantiodiscrimination for RC-33 (tR1 =9.91
min; tR2 = 11.44 min; α = 1.25; RS = 1.58) was achieved eluting
with methanol/diethylamine (100:0.1, v/v). Interestingly, these
experimental conditions meet the most important prerequisites
for an economic and productive (semi)-preparative enantiomeric
separation, such as shortest retention times, high solubility of ra-
cemate and enantiomers in the eluent/injection solvent, and the
use of a mobile phase consisting of a pure low-cost solvent, which
ultimately facilitates workup and reuse of the mobile phase.
Accordingly, these experimental conditions were selected for
the next (semi)-preparative scale-up.
Fig. 2. (A) CD curves of (+)-3 and (+)-(S)-4; (B) UV spectra of (+)-(S)-4;
(C) UV spectra of. (+)-3.
step of the synthetic pathway revealed that both intermedi-
ates (+)-(S)-5 and (+)-(S)-6, as well as the target product
(+)-(S)-RC-33, were obtained with an ee of 87%, demonstrat-
ing that no racemization occurs. Unfortunately, (+)-(S)-
RC-33 was obtained with an ee not good enough for
supporting its in vitro pharmacological evaluation. Since a
chemical purity equal or higher than 95% is usually required
for obtaining unambiguous biological results in the pharma-
cological evaluation of achiral molecules, in our opinion an
ee at least of 90% is necessary for proceeding with the pharma-
cological evaluation of enantiopure compounds. Thus, the
proposed asymmetric synthesis does not represent the most
adequate approach for preparing both RC-33 enantiomers
with an optical purity suitable for investigating their
pharmacological behavior.
RC-33 Enantiomers Separation Via Enantioselective
Chromatography
The resolution of rac-RC-33 via enantioselective HPLC
on a (semi)-preparative scale was carried out. A standard
screening protocol for cellulose and amylose derived
CSPs22 was applied to Chiralcel OD-H (0.46 cm diameter
x 15 cm length, 5 μm) and Chiralcel OJ-H (0.46 cm diame-
ter x 15 cm length, 5 μm) columns, whose chiral selectors
In order to determine the best conditions for a (semi)-prepar-
ative scale-up, the loading behavior of the Chiralcel OJ-H col-
umn (0.46 cm diameter x 15cm length, 5 μm) was determined
by injecting increasing amounts of rac-RC-33 (Fig. 3). A
Chirality DOI 10.1002/chir

820
ROSSI ET AL.
Fig. 4. (Semi)-preparative enantiomer separation of RC-33 on Chiralcel OJ-H column (1 cm × 25 cm length, 5 μm); eluent: methanol/diethylamine100:0.1 (v/v),
flow: 3.9 ml/min, detection at 250 nm, injected amount 1.25 mg, cut points given by dashes (—).
TABLE 4. Chiroptical properties of RC-33 enantiomers obtained by (semi)-preparative enantioselective HPLC
Compound
[α]2D0 (MeOH)
ee%
tR (min)
(+)-(S)-RC-33
(À)-(R)-RC-33
+22.1 (c 0.3)
- 22.2 (c 0.3)
99.5
99.6
9.88
11.41
maximum of 200 μg could be separated in a single injection
within 15 min, which allowed predicting a scale-up to the
mg scale.
nonselective radioligand [3H]-DTG was employed in the
σ2 assay because a σ2 selective radioligand is not commer-
cially available. To mask the σ1 receptors, an excess of
nontritiated (+)-pentazocine was added to the assay
solution. A high concentration of nontritiated DTG was
used to determine the nonspecific binding.
The enantiomerically pure arylalkylamines (S)-RC-33 · HCl
and (R)-RC-33 · HCl herein presented showed interesting
binding properties for σ1 receptor (Ki values <2 nM, Table 5)
and a good selectivity over the σ2 receptor. With regard to the
role of chirality in the σ1 receptors binding, the two enantio-
mers and RC-33 appear to posses almost the same affinity
towards the σ1 receptor, thus suggesting that the interaction
with the biological target is nonstereoselective.
To deeply investigate the role of chirality in the agonistic/
antagonistic profile of RC-33 · HCl pure enantiomers, their
effect on KCl-induced depolarization of PC12 cells via L-type
voltage-gated Ca2+ channels (VOCCs) were also investigated.
It has been reported that the σ1 receptor interacts with volt-
age-gated Ca2+ channels VOCCs, mainly L-type channel, and
inhibit Ca2+ influx via these channels.33 Particularly, it is well
known that σ agonists inhibit VOCCs mediated Ca2+-influx
while σ antagonists show no activity in this assay. Thus, PC12
cells were preincubated with rac-RC-33 · HCl, (S)-RC-
33 · HCl, and (R)-RC-33 · HCl or the unselective σ agonist
opipramol and afterwards stimulated with KCl, which leads to
a depolarization of PC12 cells via L-type VOCCs. The results
reported in Figure 6 clearly evidenced that rac-RC-33 · HCl
and their enantiomers similarly reduced the VOCCs mediated
Ca2+-influx, supporting the idea that the interaction with the bi-
ological target is nonstereoselective and indicating that they act
as σ1 receptor agonists, as already demonstrated for the race-
mate.10 Moreover, the effect was evidenced at a concentration
Actually, in 50 cycles 62.5 mg of rac-RC-33 were processed
according to the conditions reported in Table 3 and Figure 4
(cutpoints were determined after fraction analysis of test
runs), yielding 26.4 mg of the first eluted enantiomer and
24.8 mg of the second eluted one, along with 5.2 mg of an
intermediate fraction as a mixture of the two enantiomers.
Interestingly, both RC-33 enantiomers were obtained with
an ee 99.5% (Table 4), as evidenced by analytical control
of the collected fractions. As the last step in this work, the
(S) absolute configuration was assigned to the first eluted
enantiomer and, consequently, the (R) configuration to the
more retained one, by comparing the sign of the optical
rotation and the HPLC-UV trace of (+)-(S)-RC-33, as
obtained by us via asymmetric synthesis, with those of the
pure enantiomers obtained by enantioselective chromatogra-
phy (Fig. 5). As previously discussed, these ee values are
certainly good enough for supporting unambiguous in vitro
tests to elucidate the stereoselectivity in the biological
activity of RC-33. Accordingly, (S)- and (R)-RC-33 were
converted into the corresponding hydrochlorides, suitable
for the biological investigation.
Biology
The σ1 and σ2 receptor affinities of RC-33 · HCl pure en-
antiomers were determined in competition experiments
with radioligands. The highly σ1 selective radioligand
[3H]-(+)-pentazocine and homogenates of guinea pig brain
cortex were used in the σ1 assay. The nonspecific binding
was recorded with a large excess of nonradiolabeled (+)-
pentazocine. In the σ2 assay membrane preparations of
rat liver served as the source for σ2 receptors. The
Chirality DOI 10.1002/chir

ENANTIOMERS OF RC-33 AS NEUROPROTECTIVE AGENTS
821
Fig. 6. Behavior of rac-RC-33, (R)- and (S)-RC-33 in the calcium influx as-
say. *P < 0.05 vs. KCl, **P < 0.01 vs. KCl.
10- fold lower than that of opipramol (10 μM for racemic and
enantiopure RC-33, 100 μM for opipramol), thus suggesting
that rac-RC-33 · HCl and their enantiomers are more potent
than the reference compound.
CONCLUSIONS
In this paper we described the isolation of RC-33 pure en-
antiomers and the assignment of their absolute configuration
in order to investigate the role of chirality in the biological ac-
tivity of RC-33, recently identified by us as highly promising
neuroprotective agents acting as σ1 receptor agonists. In our
strategy, an asymmetric synthesis procedure was the first
experiment, leading to the desired enantioenriched RC-33
with an ee not good enough for supporting the in vitro
investigation. An enantioselective HPLC procedure using a
Chiralcel OJ-H column was then successfully applied to the
enantioseparation, furnishing both RC-33 enantiomers in
amounts and optical purity suitable for the pharmacological
study. Importantly, the absolute configuration of pure
enantiomers was easily assigned exploiting the asymmetric
synthesis previously devised. As emerged by the preliminary
in vitro biological investigation, (S)- and (R)-RC-33 possess a
comparable affinity towards the σ1 receptor and a very similar
behavior in the calcium influx assay, resulting in an equally
effective σ1 receptor agonist. Overall, the results obtained so
far suggest that the interaction with the biological target is
nonstereoselective and led us to hypothesize the lack of
stereoselectivity in the biological activity of RC-33. With
the final aim to unambiguously address this issue, and also
to identify the optimal candidate for the in vivo investigation,
our current efforts are directed towards an in-depth investiga-
tion of the biological behavior of RC-33 pure enantiomers.
The results will be reported in due course.
Fig. 5. UV chromatograms (recorded at 250 nm) of (+)-(S)-RC-33 obtained
via asymmetric synthesis and RC-33 enantiomers obtained via
enantioselective HPLC. Experimental conditions: Chiralcel OJ-H (0.46 cm di-
ameter x 15 cm length, 5 μm) column; mobile phase: methanol/diethylamine
(100:0.1, v/v); flow rate: 0.5 ml/min.
ACKNOWLEDGMENTS
TABLE 5. Affinities of compounds RC-33 · HCl towards σ1 and
σ2 receptors
DR, SC, MP and DC gratefully acknowledge the financial sup-
port from ARISLA (Grant SaNet-ALS).
Ki SEM (nM)
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Chirality DOI 10.1002/chir