Synthesis and biological evaluation of 2-(5-methyl-4-phenyl-2- oxopyrrolidin-1-yl)-acetamide stereoisomers as novel positive allosteric modulators of sigma-1 receptor

Article abstract of DOI:10.1016/j.bmc.2013.03.016

Novel positive allosteric modulators of sigma-1 receptor represented by 2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamide enantiomers were synthesised using an asymmetric Michael addition of 2-nitroprop-1-enylbenzene to diethyl malonate. Following the

Full text of DOI:10.1016/j.bmc.2013.03.016

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 2-(5-methyl-4-phenyl-
2-oxopyrrolidin-1-yl)-acetamide stereoisomers as novel
positive allosteric modulators of sigma-1 receptor
⇑
Grigory Veinberg , Maxim Vorona, Liga Zvejniece, Reinis Vilskersts, Edijs Vavers, Edvards Liepinsh,
Helena Kazoka, Sergey Belyakov, Anatoly Mishnev, Jevgenijs Kuznecovs, Sergejs Vikainis, Natalja Orlova,
Anton Lebedev, Yuri Ponomaryov, Maija Dambrova
Latvian Institute of Organic Synthesis, 21 Aizkraukles Str., Riga LV 1006, Latvia
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel positive allosteric modulators of sigma-1 receptor represented by 2-(5-methyl-4-phenyl-2-oxo-
pyrrolidin-1-yl)-acetamide enantiomers were synthesised using an asymmetric Michael addition of 2-
nitroprop-1-enylbenzene to diethyl malonate. Following the chromatographic separation of the methyl
erythro- and threo-4-nitro-3R- and 3S-phenylpentanoate diastereoisomers, target compounds were
obtained by their reductive cyclisation into 5-methyl-4-phenylpyrrolidin-2-one enantiomers and the
attachment of the acetamide group to the heterocyclic nitrogen. Experiments with electrically stimulated
rat vas deference contractions induced by the PRE-084, an agonist of sigma-1 receptor, showed that
(4R,5S)- and (4R,5R)-2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamides with an R-configuration
at the C-4 chiral centre in the 2-pyrrolidone ring were more effective positive allosteric modulators of
sigma-1 receptor than were their optical antipodes.
Received 24 November 2012
Revised 21 February 2013
Accepted 1 March 2013
Available online xxxx
Keywords:
2-(5-Methyl-4-phenyl-2-oxopyrrolidin-1-
yl)-acetamide
Enantiomers
Sigma-1 receptor
Agonist
Ó 2013 Elsevier Ltd. All rights reserved.
Modulation
1. Introduction
R
a) piracetam(R=R1=R2=H);
During the past decade, there has been significant interest in
the investigation of Structure–Activity Relationship (SAR) aimed
at searching for new nootropic pharmaceuticals for the treat-
ment of cognition/memory disorders. Currently, drugs 1a–f con-
taining the pyrrolidin-2-one pharmacophore and representing
the so-called racetam family play a key role in the treatment
of these disorders. For example, piracetam (1a), pramiracetam
(1b), etiracetam (1c), nefiracetam (1d), oxiracetam (1e) and
phenylpiracetam (1f) are drugs of choice for the specific therapy
of cognition/memory disorders.1 However, the discovery of new
effective pharmaceuticals that improve neurotransmission in
the human brain is critical because it affects mental and cogni-
tive abilities.
1
2
O
i
b) pramiracetam(R=R =H, R =(CH2)2N(Pr- )2);
NH R2
N
R1
c) etiracetam(R=R2=H, R1=Et);
d) nefiracetam(R=R2=H, R1=2,6-Me2C6H3)
e) oxiracetam (R=OH, R1=R2=H);
;
O
f) phenylpiracetam (R=Ph, R1=R2=H)
1a-f
Our previous investigations in this field of medicinal chemistry
were aimed at the resolution of racemic phenylpiracetam 1f into
individual stereoisomers and their subsequent pharmaceutical
analysis. The R-phenylpiracetam 4R-1f was found to be a more effec-
tive antidepressant, analgesic, muscle relaxant and psycho-stimu-
lating compound than was the S-antipode 4S-1f.2 These results
were in good agreement with pharmacological data demonstrating
the effectiveness of employing the conformational variation of chiral
centre(s)of racemicmolecules, whereinthe enantiomeric resolution
can be used to afford the optimal biological effect.
The objective of this investigation is the resolution of a known
racemic 4,5-disubstituted piracetam analogue, 2-(5-methyl-4-phe-
nyl-2-oxopyrrolidin-1-yl)-acetamide (2),3 whose nootropic prop-
erties could be improved by regulating the stereochemistry of
the C4 and C5 positions of the pyrrolidin-2-one ring.
⇑
Corresponding author. Tel.: +371 67014941; fax: +371 67550338.
E-mail address: veinberg@osi.lv (G. Veinberg).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.016
Please cite this article in press as: Veinberg, G.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.03.016

2
G. Veinberg et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Ph
Ph
Ph
Ph
Me
Ph
O
O
O
*
*
O
O
N
N
N
N
N
Me
Me
Me
NH2
NH2
NH2
Me
NH2
NH2
O
O
O
O
O
2
(4R,5S)-2a
(4R,5R)-2b
(4S,5R)-2c
(4S,5S)-2d
acetamide (2) or the asymmetric synthesis of its separate enantio-
mers from chiral or nonchiral reagents. Accordingly, we describe
the special methodology for the preparation of these compounds.
The crucial part of the synthesis consists of the asymmetric Mi-
chael addition of 2-nitroprop-1-enylbenzene (3) to diethyl malon-
ate (4) catalysed by the chiral 2,20-cyclopropylidene-bis-oxazoline
compound 5, magnesium triflate and an organic base according to
the methodology developed by Barnes et al.9 As a result, diethyl
(1R)-2-(2-nitro-1-phenylpropyl)-malonate (R-6) was obtained as
a mixture of erythro- and threo-diastereoisomers in 87% yield and
with optical purity 94% in the case of (3aR,30aR,8aS,80aS)-2,20-cyclo-
propylidenebis-[3a,8a]-dihydro-8H-indeno-[1,2-d]-oxazole
((3aR,30aR,8aS,80aS)-5a). The substitution of the catalyst by its opti-
cal antipode (3aS,30aS,8aR,80aR)-5b resulted in the preparation of a
second pair of diastereoisomers S-6 in 83% yield and with optical
purity 95%. The introduction of the methyl group in nitrostyrene
at least 10 times hindered the rate of the condensation compared
with that of 2-nitrovinylbenzene, as discussed in the study.9 The
usage of twofold amount of the chiral catalyst 5 allowed to reduce
the reaction time up to 72 h (Scheme 1).
Subsequent chemical manipulations with diastereoisomeric
mixtures R-6 and S-6 were aimed at obtaining suitable derivatives
for resolution by column chromatography. Methyl (3R)- and (3S)-
4-nitro-3-phenylpentanoates R-8 and S-8 were found to be the
most suitable candidates for this purpose. According to this
precondition, compounds R-6 and S-6 were subjected to acidic
hydrolysis and decarboxylation. Intermediate (3R)- and (3S)-4-ni-
tro-3-phenylpentanoic acids R-7 and S-7 were esterified by meth-
anol, and the obtained diastereoisomeric mixtures of esters R-8
and S-8 were separated by column chromatography on silica gel,
affording the erythro- and threo-isomers of 4-nitro-3-phenylpent-
anoate: 3R,4S-8a, 3R,4R-8b, 3S,4R-8c and 3S,4S-8d (Scheme 2).
The resulting methyl 4-nitro-3-phenylpentanoates 8a–d were
converted into target compounds 4R,5S-2a, 4R,5R-2b, 4S,5R-2c,
and 4S,5S-2d using typical reactions for the preparation of 2-(2-
oxopyrrolidin-1-yl)-acetamide analogues:
They attracted our attention due to the unexpected activity dis-
covered for compound (4R,5S)-2a during in vitro CNS pharmaco-
logical target profiling, which was performed in a commercially
available panel of 77 radioligand binding assays (CEREP, France).4
The only target site for its activity (inhibition or enhancement of
radioligand binding for more than 20%) was sigma receptor, where
this compound increased the specific binding of radioligand, thus
suggesting a positive modulatory activity of (4R,5S)-2a on sigma
receptors.
Recent studies consider sigma-1 receptor (sig-1R) as an emerg-
ing new CNS drug target. This receptor plays an important role in
neuronal plasticity, a process implicated in the pathophysiology
of neuropsychiatric diseases, such as Alzheimer’s disease, major
depressive disorders, and schizophrenia.5,6 Sig-1R has been thor-
oughly studied to elucidate possible neuropharmacological appli-
cations, mainly in learning and memory processes, including
depression, anxiety, schizophrenia, analgesia and some effects of
drug abuse.7 As a chaperone protein, sig-1R modulates the intracel-
lular calcium signalling of the endoplasmatic reticulum. Several
studies suggest that sig-1R receptor is involved in memory pro-
cesses and their agonists have demonstrated effectiveness in the
treatment of cognitive impairments in experimental animal
models.8
To date, we have not found any data about the effect of pirace-
tam and its structural analogues on sig-1R. Therefore, our research
is aimed at the synthesis of 2-(5-methyl-4-phenyl-2-oxopyrroli-
din-1-yl)-acetamide (2) four individual enantiomers 4R,5S-2a,
4R,5R-2b, 4S,5R-2c, and 4S,5S-2d for the purpose to compare the
modulatory activity of these compounds with respect to sig-1R
using the isolated vas deferens experimental model.
2. Chemistry
No evidence was found in the literature related to the chiral res-
olution of racemic 2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-
H
H
H
O
O
N
N
Me
Ph
NO2
H
3aR,3'aR,8aS,8'aS-5a
R
COOEt
COOEt
R-6
O
eritro:treo=3:1
NO2
OEt
OEt
H
H
H
H
Ph
+
O
O
Me
O
4
N
N
Me
Ph
NO2
3
3aS,3'aS,8aR,8'aR-5b
S
COOEt
COOEt
S-6
eritro:treo=3:1
Scheme 1. The preparation of diethyl (1R)- and (1S)-2-(2-nitro-1-phenylpropyl)-malonates R-6 and S-6.
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G. Veinberg et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
Me
NO2
COOMe
Me
Ph
Ph
NO2
COOH
Me
Ph
NO2
COOMe
a
b
c
3R,4S-8a (60%)
R-6
42%
Me
NO2
R-8
R-7
COOMe
Ph
3R,4R-8b (14%)
Me
NO2
Me
Ph
NO2
COOH
Me
Ph
NO2
COOMe
a
COOMe
Ph
3S,4R-8c (59%)
b
c
S-6
44%
Me
-8
S
S-7
NO2
COOMe
Ph
3S,4S-8d (13%)
Scheme 2. The preparation of methyl (3R)- and (3S)-4-nitro-3-phenylpentanoates 8a–d. Reagents and conditions: (a) 36% HCl and CH3COOH mixture (1:3), reflux, 18 h; (b)
MeOH, SOCl2 (cat) 20 h, reflux; (c) chromatographic separation on silica gel.
(a) Hydrogenation of each stereoisomer 8a–d in the presence of
Ni Raney catalyst accompanied by the cyclisation of inter-
mediate ethyl 4-amino-3-phenylpentanoates into the appro-
3. Results and discussion
The positive allosteric modulatory activity of the 2-(5-methyl-
4-phenyl-2-oxopyrrolidin-1-yl)-acetamide enantiomers 2a–d with
respect to sig-1R was evaluated using the electrically stimulated
rat vas deferens model.11 The action was determined by comparing
the vas deferens contraction heights induced by selective sig-1R
agonist PRE-084 (2-morpholin-4-ylethyl-1-phenylcyclohexane-1-
carboxylate) in the absence of the tested compound (control) and
priate
5-methyl-4-phenylpyrrolidin-2-ones:
4R,5S-9a,
4R,5R-9b, 4S,5R-9c, and 4S,5S-9d.
(b) The treatment of the individual 5-methyl-4-phenylpyrroli-
din-2-ones 9a–d with sodium hydride and ethyl
bromoacetate.
(c) Carbamoylation of the ethyl 2-(5-methyl-2-oxo-4-phenyl-
pyrrolidin-1-yl)-acetates 4R,5S-10a, 4R,5R-10b, 4S,5R-10c,
and 4S,5S-10d with ammonium hydroxide.3,10
after preincubation with a 10 lM solution of each enantiomer.
The intensity of the electrically stimulated contractions of rat
H2,
Ni Raney
NaH,
BrCH2COOEt
Ph
O
Ph
O
Ph
O
NH4OH
8a-d
O
O
NH
N
N
CH3
CH3
CH3
OEt
NH2
9a-d
10a-d
2a-d
2, 8, 9, 10 a = 4R,5S; b= 4R,5R; c= 4S,5R;d = 4S,5S
The target enantiomers 4R,5S-2a, 4R,5R-2b, 4S,5R-2c, and 4S,5S-
2d were recrystallised from water and according to chiral chroma-
tography data their optical purity was in the range 90–99%. The
angles of optical rotation for these compounds were in agreement
with the stereochemistry of phenyl and methyl groups (Table 1)
and consistent with the X-ray analysis of the two enantiomers
4R,5S-2a and 4R,5R-2b (Figs. 1 and 2). The ORTEP diagram of these
compounds demonstrates that the crystal structure of 4R,5R-2b
features two independent molecules in the asymmetric unit. Both
molecules are connected by a centre of pseudoinversion. The main
bond lengths and angles of 4R,5S-2a and 4R,5R-2b are provided in
Table 2.
vas deferens in the presence of 100 lM of PRE-084 was
122 11%. The tested compounds alone did not alter the height
of contractions of electrically stimulated vas deferens. In contrast,
the preincubation of vas deferens in 10 lM solution of tested enan-
tiomers 10 min before the addition of the PRE-084 increased the
Table 1
Optical rotation angles for 2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-1-yl)-acetamide
(2) stereoisomers
½ ꢀ
a 2D0
Compound
Solvent, concentration
4R,5S-2a
4R,5R-2b
4S,5R-2c
4S,5S-2d
ꢁ96.7°
+22.9°
+94.1°
ꢁ26.0°
c 0.05, MeOH
c 0.05, MeOH
c 0.05, MeOH
c 0.05, MeOH
Figure 1. ORTEP diagram of the crystal structure of 4R,5S-2a.
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4
G. Veinberg et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Figure 2. ORTEP diagram of the crystal structure of 4R,5R-2b.
Table 2
contractions in the case of 4R,5S-2a and 4R,5R-2b were observed
to be at least two times higher than those of the control (Table 3).
Principal bond lengths (l) and valence angles (x) for 4R,5S-2a and 4R,5R-2b
compounds
Bond
l (Å)
4. Conclusions
4R,5S-2a
4R,5R-2b
Molecule A
Molecule B
In summary, four individual enantiomers of 2-(5-methyl-4-
phenyl-2-oxopyrrolidin-1-yl)-acetamide
4R,5S-2a,
4R,5R-2b,
N1ꢁC1
N1ꢁC12
C1ꢁC2
C2ꢁC3
C3ꢁC4
C3ꢁC6
C4ꢁN1
C4ꢁC5
C12ꢁC13
C13ꢁO1
C13ꢁN2
Angle
1.345 (2)
1.449 (2)
1.512 (2)
1.524 (2)
1.551 (2)
1.513 (2)
1.476 (2)
1.518 (2)
1.520 (2)
1.235 (2)
1.319 (2)
1.354 (6)
1.437 (6)
1.499 (7)
1.523 (7)
1.528 (7)
1.508 (7)
1.480 (6)
1.502 (8)
1.530 (7)
1.231 (5)
1.318 (7)
1.325 (6)
1.457 (6)
1.521 (7)
1.531 (7)
1.536 (7)
1.525 (7)
1.472 (6)
1.490 (7)
1.514 (7)
1.244 (5)
1.332 (7)
4S,5R-2c, and 4S,5S-2d were obtained using an asymmetric Mi-
chael addition of 2-nitroprop-1-enylbenzene to diethyl malonate
catalysed by the chiral 2,20-cyclopropylidene-bis-oxazoline. This
reaction was followed by the chromatographic separation of the
diastereoisomeric mixtures of methyl erythro- and threo-4-nitro-
3R-phenylpentanoate (R-8) and methyl erythro- and threo-4-ni-
tro-3S-phenylpentanoate (S-8), the reductive cyclisation of each
enantiomer into the appropriate pyrrolidin-2-one and the final
conversion of these compounds into target N-acetamide deriva-
tives. The evaluation of the in vitro biological effect of these com-
pounds using electrically stimulated rat vas deferens contractions
induced by selective sigma-1 receptor agonist PRE-084 demon-
strated that enantiomers 4R,5S-2a and 4R,5R-2b with the R-con-
figuration at the C-4 chiral centre in the 2-pyrrolidone ring are
more effective positive allosteric modulators of sigma-1 receptor
than are their optical antipodes.
x
(°)
4R,5S-2a
4R,5R-2b
Molecule A
Molecule B
C1ꢁN1ꢁC4
C1ꢁN1ꢁC12
C4ꢁN1ꢁC12
N1ꢁC1ꢁC2
C1ꢁC2ꢁC3
113.9 (1)
123.4 (1)
122.1 (1)
107.7 (1)
103.6 (1)
103.0 (1)
100.7 (1)
113.9 (1)
119.0 (1)
118.0 (1)
112.2 (4)
122.8 (4)
125.0 (5)
108.4 (5)
104.5 (5)
102.9 (5)
103.0 (5)
111.2 (5)
120.8 (5)
115.9 (5)
115.0 (4)
121.3 (4)
121.1 (4)
107.1 (5)
102.9 (5)
103.0 (5)
100.0 (5)
112.0 (5)
120.5 (5)
116.3 (5)
C2ꢁC3ꢁC4
C3ꢁC4ꢁN1
N1ꢁC12ꢁC13
C12ꢁC13ꢁO1
C12ꢁC13ꢁN2
5. Experimental
5.1. Chemistry
All chemicals were supplied by Acros and Aldrich. The melting
points were determined using a Boetius PHMK melting point
apparatus and are reported uncorrected. 1H and 13C NMR spectra
were obtained using a Varian 400 MHz Mercury-400 spectrome-
ter and CDCl3 as the solvent. The chemical shifts are reported in
d values (ppm) relative to an internal TMS standard. The abbrevi-
ations are s = singlet, d = doublet, t = triplet, m = multiplet, and
br = broad. High-resolution mass spectra were obtained using a
Micromass Quatro Micro™ API with MeCN as the solvent. Optical
rotation values at 405 nm were measured on a Perkin–Elmer 141
Polarimeter. Elemental analyses (C, H, N) were performed on a
Carlo Erba 1108 analyser and were found to be within 0.4% of
the theoretical values. The purity of the compounds was deter-
mined by TLC using Merck 60F254 silica gel, whereas Merck Kie-
selgel (silica gel 0.063–0.230 mm) was used for column
chromatography.
Table 3
Positive allosteric modulating effect of enantiomers 2a–d in rat vas deferens
contraction experiments
Enantiomers
% Of increase*
4R,5S-2a
4R,5R-2b
4S,5R-2c
4S,5S-2d
222 37**
191 23**
141 40
147 31
*
The response of isolated vas deferens to selective sig-1R agonist PRE-084 was
compared without (control) or in the presence of enantiomers 2a–d at the con-
centration of 10 M. All results are expressed as a mean SEM of six vas deferens.
l
**
P <0.01 paired student t-test.
intensity of observed contractions. As shown in Table 3, this in-
crease in activity was inherent to all enantiomers. However, the
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G. Veinberg et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
5.1.1. Diethyl (1R)-2-(2-nitro-1-phenylpropyl)-malonate (R-6)
To a solution of (3aR,30aR,8aS,80aS)-2,20-cyclopropylidenebis-
a ratio of 4:1. Yield: 42% (579 mg). erythro-Isomer 1H NMR (CDCl3);
d = 1.48 (d, 3H, J = 6.6 Hz, 5-CH3), 2.61–2.91 (m, 2H, 2-CH2), 3.54–
3.70 (m, 1H, 3-H), 4.77–4.87 (m, 1H, 4-H), 7.05–7.48 (m, 5H,
C6H5); threo-isomer 1H NMR (CDCl3); d = 1.27 (d, 3H, J = 6.6 Hz, 5-
CH3), 2.61–2.91 (m, 2H, 2-CH2), 3.54–3.70 (m, 1H, 3-H), 4.66–
4.70 (m, 1H, 4-H), 7.05–7.48 (m, 5H, C6H5); HRMS: calculated for
[C11H13NO4+H]+: 224.0921; found: 224.0917.
[3a,8a]-dihydro-8H-indeno-[1,2-d]-oxazole
(5a)
(420 mg,
1.18 mM) in chloroform (amylene stabilised) (5 ml) in a 250 ml
reaction flask, magnesium triflate (378 mg, 1.18 mM) and water
(25 ll) were added at room temperature, and the mixture was al-
lowed to stir under argon for 1 h. Molecular sieves (1.0 g) were
added to the mixture, and the mixture was stirred for an additional
30 min. The obtained suspension was diluted with 45 ml of a chlo-
roform solution containing diethylmalonate (1.67 g, 10.2 mM),
2-nitroprop-1-enylbenzene (1.63 g, 10.0 mM) and a mixture of
5.1.4. (3S)-4-Nitro-3-phenylpentanoic acid (S-7)
The substitution of R-6 with its optical antipode S-6 in Sec-
tion 5.1.3 afforded a diastereoisomeric mixture of erythro- and
threo-isomers of (3S)-4-nitro-3-phenylpentanoic acid in a ratio of
4:1. Yield: 44% (606 mg). erythro-Isomer 1H NMR (CDCl3);
d = 1.48 (d, 3H, J = 6.6 Hz, 5-CH3), 2.61–2.91 (m, 2H, 2-CH2), 3.54–
3.70 (m, 1H, 3-H), 4.77–4.87 (m, 1H, 4-H), 7.05–7.48 (m, 5H,
C6H5); threo-isomer 1H NMR (CDCl3); d = 1.27 (d, 3H, J = 6.6 Hz, 5-
CH3), 2.61–2.91 (m, 2H, 2-CH2), 3.54–3.70 (m, 1H, 3-H), 4.66–
4.70 (m, 1H, 4-H), 7.05–7.48 (m, 5H, C6H5); HRMS: calculated for
[C11H13NO4+H]+: 224.0921; found: 224.0919.
morpholine (46 ll) and tetra-methylguanidine (46 ll). The reac-
tion mixture was stirred at 20–25 °C for 72 h. The degree of conver-
sion and the selectivity were determined by a chiral HPLC analysis
[Chiralpak IC, 4.6 ꢂ 250 mm, 1.0 ml/min, eluent i-PrOH–hexane
(1:9)] every 12 h. After the completion of the reaction, the reaction
mixture was diluted with hexane (50 ml) and stirred for 20 min.
Subsequently, the solid was filtered off and the filtrate was washed
with 5% aqueous HCl (2 ꢂ 50 ml), brine (2 ꢂ 50 ml), dried over
anhydrous Na2SO4. The drying reagent was removed by filtration
and the solution was concentrated under reduced pressure. The
residue was purified by silica chromatography using ethylace-
tate/hexane (1:10) and fractions with Rf 0.28 were collected. Yield:
87% (2.8 g). According to the chiral HPLC, the obtained low-melting
yellow solid was a mixture of erythro- and threo-isomers of diethyl
(1R)-2-(2-nitro-1-phenylpropyl)-malonate in a ratio 3:1. Optical
purity: 94%. erythro-Isomer 1H NMR (CDCl3); d = 0.85 (t, 3H,
J = 7.0 Hz, CH2CH3), 1.15–1.27 (m, 3H, CH2CH3), 1.37 (d, 3H,
J = 6.8 Hz, CH3CHNO2), 3.63–3.93 (m, 3H, CH2CH3, COCHCO),
4.07–4.29 (m, 3H, CH2CH3), 5.07–5.16 (m, 1H, CHNO2), 6.99–7.28
(m, 5H, C6H5); threo-isomer 1H NMR (CDCl3); d = 0.93 (t, 3H,
J = 7.0 Hz, 3CH2CH3), 1.15–1.27 (m, 3H, CH2CH3), 1.29 (d, 1H,
J = 6.8 Hz, CH3CHNO2), 3.63–3.93 (m, 3H, CH2CH3, COCHCO),
4.07–4.29 (m, 3H, CH2CH3, PhCH), 4.29–5.06 (m, 1H, CHNO2),
6.99–7.28 (m, 5H, C6H5); HRMS: calculated for [C16H21NO6+H]+:
324.1444; found: 324.1442. Anal. Calcd for C16H21NO6 (323.35):
C, 59.43; H, 6.55; N, 4.33. Found: C, 59.51; H, 6.46; N, 4.27.
5.1.5. Methyl erythro-(3R,4S)-4-nitro-3-phenylpentanoate
(3R,4S-8a)
A mixture of erythro- and threo-isomers of (3R)-4-nitro-3-phe-
nylpentanoic acid (R-7) (500 mg, 2.42 mM) and thionyl chloride
(61 ll, 1.0 mM) in methanol (20 ml) was refluxed for 6 h. The reac-
tion mixture was cooled and concentrated under reduced pressure.
The residue was purified by silica column chromatography using
ethylacetate/hexane (1:15). The fractions with Rf 0.18 containing
methyl erythro-(3R,4S)-4-nitro-3-phenylpentanoate were collected
and evaporated under reduced pressure. Yield: 345 mg (60%) of
low melting yellow solid. Optical purity 93% according to chiral
HPLC. 1H NMR (CDCl3); d = 1.48 (d, 3H, J = 6.6 Hz, 5-CH3), 2.58–
2.85 (m, 2H, CH2), 3.53 (s, 3H, OCH3), 3.56–3.71 (m, 1H, 3-H),
4.79–4.88 (m, 1H, 4-H), 7.07–7.31 (m, 5H, C6H5); HRMS: calculated
for [C12H15NO4+H]+: 238.1077; found: 238.1074. Anal. Calcd for
C12H15NO4 (237.26): C, 60.75; H, 6.37; N, 5.90. Found: C, 60.53;
H, 6.24; N, 5.82.
5.1.2. Diethyl (1S)-2-(2-nitro-1-phenylpropyl)-malonate (S-6)
The substitution of chiral catalyst 5a with its optical antipode
5b in Section 5.1.1 afforded the diastereoisomeric mixture S-6.
Yield: 83% (2.67 g). According to the chiral HPLC, the obtained
low-melting yellow solid was a mixture of erythro- and threo-iso-
mers of diethyl 2-(2-nitro-1(S)-phenylpropyl)-malonate in a ratio
of 3:1. Optical purity: 95%. erythro-Isomer 1H NMR (CDCl3);
d = 0.85 (t, 3H, J = 7.0 Hz, CH2CH3), 1.15–1.27 (m, 3H, CH2CH3),
1.37 (d, 3H, J = 6.8 Hz, CH3CHNO2), 3.63–3.93 (m, 3H, CH2CH3,
COCHCO), 4.07–4.29 (m, 3H, CH2CH3, PhCH), 5.07–5.16 (m, 1H,
CHNO2), 6.99–7.28 (m, 5H, C6H5); threo-Isomer 1H NMR (CDCl3);
d = 0.93 (t, 1H, J = 7.0 Hz, CH2CH3), 1.15–1.27 (m, 3H, CH2CH3),
1.29 (d, 1H, J = 6.8 Hz, CH3CHNO2), 3.63–3.93 (m, 3H, CH2CH3,
COCHCO), 4.07–4.29 (m, 3H, CH2CH3, PhCH), 4.29–5.06 (m, 1H,
CHNO2), 6.99–7.28 (m, 5H, C6H5); HRMS: calculated for
[C16H21NO6+H]+: 324.1444; found: 324.1443. Anal. Calcd for
5.1.6. Methyl threo-(3R,4R)-4-nitro-3-phenylpentanoate (3R,4R-
8b)
The fractions with Rf 0.26 containing methyl threo-(3R,4R)-4-ni-
tro-3-phenylpentanoate obtained during the chromatographic sep-
aration in Section 5.1.5 were collected and evaporated under
reduced pressure. Yield: 80 mg (14%) of low melting yellow solid
threo-(3R,4R)-4-nitro-3-phenylpentanoate. Optical purity 82%
according to chiral HPLC. 1H NMR (CDCl3); d = 1.27 (d, 3H,
J = 6.6 Hz, 5-CH3), 2.58–2.85 (m, 2H, 2-CH2), 3.46 (s, 3H, OCH3),
3.56–3.71 (m, 1H, 3-H), 4.68–4.77 (m, 1H, 4-H), 7.07–7.31 (m,
5H, C6H5); HRMS: calculated for [C12H15NO4+H]+: 238.1077; found:
238.1075. Anal. Calcd for C12H15NO4 (237.26): C, 60.75; H, 6.37; N,
5.90. Found: C, 60.39; H, 6.28; N, 5.75.
5.1.7. Methyl erythro-(3S,4R)-4-nitro-3-phenylpentanoate
(3S,4R-8c)
C
16H21NO6 (323.35): C, 59.43; H, 6.55; N, 4.33. Found: C, 59.49;
A mixture of erythro- and threo-isomers of (3S)-4-nitro-3-phe-
nylpentanoic acid (S-7) (500 mg, 2.42 mM) and thionyl chloride
H, 6.54; N, 4.27.
(61 ll, 1.0 mM) in methanol (20 ml) was refluxed for 6 h. The reac-
5.1.3. (3R)-4-Nitro-3-phenylpentanoic acid (R-7)
tion mixture was cooled and concentrated under reduced pressure.
The residue was purified by silica column chromatography using
ethylacetate/hexane (1:15). The fractions with Rf 0.18 containing
methyl erythro-(3S,4R)-4-nitro-3-phenylpentanoate were collected
and evaporated under reduced pressure. Yield: 340 mg (59%) of
low melting yellow solid. Optical purity 93% according to chiral
HPLC. 1H NMR (CDCl3); d = 1.48 (d, 3H, J = 6.6 Hz, 5-CH3), 2.58–
2.85 (m, 2H, CH2), 3.53 (s, 3H, OCH3), 3.56–3.71 (m, 1H, 3-H),
4.79–4.88 (m, 1H, 4-H), 7.07–7.31 (m, 5H, C6H5); HRMS: calculated
Diethyl (1R)-2-(2-nitro-1-phenylpropyl)-malonate (2.00 g,
6.18 mM) was refluxed in a mixture of acetic and 36% hydrochloric
acids at a ratio of 1:3 (30 ml) for 18 h. After the completion of the
reaction, the reaction mixture was cooled and concentrated under
reduced pressure. The residue was purified by silica column chro-
matography using ethylacetate/hexane (1:5), and fractions with Rf
0.22 were collected. The obtained yellow solid was a mixture of er-
ythro- and threo-isomers of (3R)-4-nitro-3-phenylpentanoic acid in
Please cite this article in press as: Veinberg, G.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.03.016

6
G. Veinberg et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
for [C12H15NO4+H]+: 238.1077; found: 238.1074. Anal. Calcd for
12H15NO4 (237.26): C, 60.75; H, 6.37; N, 5.90. Found: C, 60.63;
H, 6.29; N 5.78.
7.33 (m, 5H, C6H5); 13C NMR (CDCl3); d = 20.38, 39.36, 49.64,
57.71, 127.22 (C-2, C-6 aromatic), 127.37, 128.82 (C-3, C-5 aro-
matic), 140.85, 176.59; HRMS: calculated for [C11H13NO+Na]+:
198.0895; found: 198.0890.
C
5.1.8. Methyl threo-(3S,4S)-4-nitro-3-phenylpentanoate (3S,4S-
8d)
5.1.13. Ethyl erythro-(4R,5S)-2-(5-methyl-4-phenylpyrrolidin-1-
yl)-acetate (4R,5S-10a)
The factions with Rf 0.26 containing methyl threo-(3S,4S)-4-ni-
tro-3-phenylpentanoate obtained during the chromatographic sep-
aration in Section 5.1.7 were collected and evaporated under
reduced pressure. Yield: 78 mg (13%) of low melting yellow solid.
Optical purity 94% according to chiral HPLC. 1H NMR (CDCl3);
d = 1.27 (d, 3H, J = 6.6 Hz, 5-CH3), 2.58–2.85 (m, 2H, 2-CH2), 3.46
(s, 3 H, OCH3), 3.56–3.71 (m, 1H, 3-H), 4.68–4.77 (m, 1H, 4-H);
7.07–7.31 (m, 5H, C6H5); HRMS: calculated for [C12H15NO4+H]+:
238.1077; found: 238.1073. Anal. Calcd for C12H15NO4 (237.26):
C, 60.75; H, 6.37; N, 5.90. Found: C, 60.60; H, 6.31; N, 5.77.
A solution of erythro-(4R,5S)-5-methyl-4-phenylpyrrolidin-2-
one (4R,5S-9a) (351 mg, 2.00 mM) in toluene (30 ml) was added
to a suspension of sodium hydride (56 mg, 2.35 mM) in toluene
(30 ml). The stirred mixture was heated at 80–90 °C for 30 min
and then cooled to room temperature. Ethyl bromoacetate
(368 mg, 2.20 mM) was added to the reaction mixture, which
was heated to 110–120 °C for 6 h and concentrated under reduced
pressure. The residue was dissolved in toluene (30 ml). The ob-
tained solution was washed with 5% aqueous HCl (2 ꢂ 50 ml),
brine (2 ꢂ 50 ml), dried over anhydrous Na2SO4. The drying re-
agent was removed by filtration, and the solution was concen-
trated under reduced pressure. The residue was purified by silica
column chromatography using CH2Cl2/MeOH (20:1). The fractions
with Rf 0.48 were collected and evaporated under reduced pres-
sure, affording ethyl (4R,5S)-2-(5-methyl-4-phenylpyrrolidin-1-
yl)-acetate (367 mg, 70%) as a colourless oil. 1H NMR (CDCl3);
d = 0.72 (d, 3H, J = 6.6 Hz, 5-CH3), 1.23 (t, 3H, J = 7.0 Hz, CH2CH3),
2.60–2.91 (d, 2H, J = 8.5 Hz, 3-CH2), 3.65–3.74 (m, 1H, 4-H), 3.66
(d, 2H, J = 17.7 Hz, NCH2COO), 4.01–4.10 (m, 1H, 5-H), 4.10–4.20
(m, 2H, CH2CH3), 4.38 (d, 1H, J = 17.7 Hz, NCH2COO), 7.09–7.31
(m, 5H, C6H5); 13C NMR (CDCl3); d = 14.14, 14.62, 35.21, 42.07,
42.29, 57.62, 61.34, 127.14 (C-2, C-6 aromatic), 128.03, 128.51
(C-3, C-5 aromatic), 138.88, 168.89, 174.69; HRMS: calculated for
[C15H29NO3+H]+: 262.1443; found: 262.1433.
5.1.9. erythro-(4R,5S)-5-Methyl-4-phenylpyrrolidin-2-one
(4R,5S-9a)
The hydrogenation was performed using a stirring suspension
of methyl erythro-(3R,4S)-4-nitro-3-phenylpentanoate (3R,4S-8a)
(600 mg, 2.52 mM) in ethanol (40 ml) and 1 ml of 50% Ni Raney
slurry in water at 50 °C and 50 atm for 18 h. After the completion
of the reaction, the reaction mixture was cooled, and the catalyst
was filtered off and washed with 30 ml of ethanol. The filtrate
was concentrated under reduced pressure. The purification of the
residue by column chromatography on silica gel using CH2Cl2/
EtOH (20:1) and collecting fractions with Rf 0.40 afforded ery-
thro-(4R,5S)-5-methyl-4-phenylpyrrolidin-2-one as a white solid.
Yield: 80% (353 mg). 1H NMR (CDCl3); d = 0.75 (d, 3H, J = 6.5 Hz,
5-CH3), 2.55–2.69 (m, 2H, 3-CH2), 3.64–3.72 (m, 1H, 4-H), 3.96–
4.04 (m, 1H, 5-H), 6.78 (br s, 1H, NH), 7.07–7.33 (m, 5H, C6H5);
13C NMR (CDCl3); d = 17.50, 35.22, 44.08, 53.72, 126.51, 127.12,
127.92, 128.50, 128.80, 138.81, 177.84; HRMS: calculated for
[C11H13NO+Na]+: 198.0895; found: 198.0900.
5.1.14. Ethyl threo-(4R,5R)-2-(5-methyl-4-phenylpyrrolidin-1-
yl)-acetate (4R,5R-10b)
The substitution of 4R,5S-9a with 4R,5R-9b in Section 5.1.13
afforded
(4R,5R)-2-(5-methyl-4-phenylpyrrolidin-1-yl)-acetate.
5.1.10. threo-(4R,5R)-5-Methyl-4-phenylpyrrolidin-2-one
(4R,5R-9b)
Yield: 72% (377 mg). 1H NMR (CDCl3 d = 1.16 (d, 3H, J = 6.3 Hz, 5-
CH3), 1.23 (t, 3H, J = 7.0 Hz, CH2CH3), 2.53–2.63 (m, 1H, CH2),
2.76–2.86 (m, 1H, CH2), 2.92–3.01 (m, 1H, 4-H), 3.71 (d, 1H,
J = 17.7 Hz, NCH2COO), 3.74–3.83 (m, 1H, 5-H), 4.10–4.20 (m, 3H,
CH2CH3), 4.38 (d, 1H, J = 17.8 Hz, NCH2COO), 7.18–7.33 (m, 5H,
C6H5); 13C NMR (CDCl3), d = 14.14, 17.97, 38.89, 41.88, 47.22,
61.22, 61.35, 127.25 (C-2, C-6 aromatic), 127.54, 128.33 (C-3, C-5
aromatic), 141.07, 168.75, 174.23; HRMS: calculated for
[C15H29NO3+H]+: 262.1443; found: 262.1423.
The substitution of 3R,4S-8a with 3R,4R-8b in Section 5.1.9
afforded (4R,5R)-5-methyl-4-phenylpyrrolidin-2-one. Yield: 85%
(375 mg). 1H NMR (CDCl3); d = 1.20 (d, 3H, J = 6.5 Hz, 5-CH3),
2.48–2.57 (m, 1H, 3-CH2), 2.65–2.74 (m, 1H, 3-CH2), 2.98–3.07
(m, 1H, 4-H) 3.65–3.75 (m, 1H, 5-H), 6.76 (br s, 1H, NH), 7.07–
7.33 (m, 5H, C6H5); 13C NMR (CDCl3); d = 20.39, 39.33, 49.66,
57.68, 127.23 (C-2, C-6 aromatic), 127.37, 128.82 (C-3, C-5 aro-
matic), 140.84, 176.48;. HRMS: calculated for [C11H13NO+Na]+:
198.0895; found: 198.0888.
5.1.15. Ethyl erythro-(4S,5R)-2-(5-methyl-4-phenylpyrrolidin-1-
yl)-acetate (4S,5R-10c)
5.1.11. erythro-(4S,5R)-5-Methyl-4-phenylpyrrolidin-2-one
(4S,5R-9c)
The substitution of 4R,5S-9a with 4S,5R-9c in Section 5.1.13
afforded
(4S,5R)-2-(5-methyl-4-phenylpyrrolidin-1-yl)-acetate.
The substitution of 3R,4S-8a with 3S,4R-8c in Section 5.1.9
afforded (4S,5R)-5-methyl-4-phenylpyrrolidin-2-one. Yield: 84%
(371 mg). 1H NMR (CDCl3); d = 0.75 (d, 3H, J = 6.5 Hz, 5-CH3),
2.55–2.69 (m, 2H, 3-CH2), 3.64–3.72 (m, 1H, 4-H), 3.96–4.04 (m,
1H, 5-H), 6.78 (br s, 1H, NH), 7.07–7.33 (m, 5H, C6H5). 13C NMR
(CDCl3); d = 17.53, 35.21, 44.07, 53.72, 126.50, 127.13, 127.91,
128.51, 128.80, 138.83, 177.74; HRMS: calculated for [C11H13NO+
Na]+: 198.0895; found: 198.0901.
Yield: 70% (367 mg). 1H NMR (CDCl3); d = 0.72 (d, 3H, J = 6.6 Hz,
5-CH3), 1.23 (t, 3H, J = 7.0 Hz, CH2CH3), 2.60–2.91 (d, 2H,
J = 8.5 Hz, 3-CH2), 3.65–3.74 (m, 1H, 4-H), 3.66 (d, 2H, J = 17.7 Hz,
NCH2COO), 4.01–4.10 (m, 1H, 5-H), 4.10–4.20 (m, 2H, CH2CH3),
4.38 (d, 1H, J = 17.7 Hz, NCH2COO), 7.09–7.31 (m, 5H, C6H5); 13C
NMR (CDCl3), d = 14.13, 14.65, 35.21, 42.07, 42.29, 57.62, 61.34,
127.14 (C-2, C-6 aromatic), 128.03, 128.51 (C-3, C-5 aromatic),
138.88, 168.89, 174.68; HRMS: calculated for [C15H29NO3+H]+:
262.1443; found: 262.1448.
5.1.12. threo-(4S,5S)-5-Methyl-4-phenylpyrrolidin-2-one (4S,5S-
9d)
5.1.16. Ethyl threo-(4S,5S)-2-(5-methyl-4-phenylpyrrolidin-1-
yl)-acetate (4S,5S-10d)
The substitution of 3R,4S-8a with 3S,4S-8d in Section 5.1.9
afforded (4S,5S)-5-methyl-4-phenylpyrrolidin-2-one. Yield: 84%
(371 mg). 1H NMR (CDCl3); d = 1.20 (d, 3H, J = 6.5 Hz, 5-CH3),
2.48–2.57 (m, 1H, 3-CH2), 2.65–2.74 (m, 1H, 3-CH2), 2.98–3.07
(m, 1H, 4-H) 3.65–3.75 (m, 1H, 5-H), 6.76 (br s, 1H, NH), 7.07–
The substitution of 4R,5S-9a with 4S,5S-9d in Section 5.1.13
afforded
(4S,5S)-2-(5-methyl-4-phenylpyrrolidin-1-yl)-acetate.
Yield: 72% (377 mg). 1H NMR (CDCl3); d = 1.16 (d, 3H, J = 6.3 Hz,
5-CH3), 1.23 (t, 3H, J = 7.0 Hz, CH2CH3), 2.53–2.63 (m, 1H, CH2),
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G. Veinberg et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
2.76–2.86 (m, 1H, CH2), 2.92–3.01 (m, 1H, 4-H), 3.71 (d, 1H,
5.1.20. threo-(4S,5S)-2-(5-Methyl-2-oxo-4-phenyl-pyrrolidin-1-
yl)-acetamide (4S,5S-2d)
J = 17.7 Hz, NCH2COO), 3.74–3.83 (m, 1H, 5-H), 4.10–4.20 (m, 3H,
CH2CH3), 4.38 (d, 1H, J = 17.8 Hz, NCH2COO), 7.18–7.33 (m, 5H,
C6H5); 13C NMR (CDCl3), d = 14.14, 17.97, 38.89, 41.87, 47.21,
61.22, 61.35, 127.25 (C-2, C-6 aromatic), 127.53, 128.33 (C-3, C-5
aromatic), 141.06, 168.75, 174.23; HRMS: calculated for
[C15H29NO3+H]+: 262.1443; found: 262.1456.
The substitution of 4R,5S-10a with 4S,5S-10d in Section 5.1.17
afforded (4S,5S)-2-(5-methyl-4-phenylpyrrolidin-1-yl)-acetamide.
Yield: 72% (224 mg). Mp 117–118 °C. ½a D20
ꢁ26.0 (c 0.05, MeOH).
ꢀ
Optical purity 93.8% according to chiral HPLC.
1H NMR (CDCl3); d = 1.23 (d,3H, J = 6.2 Hz, 5-CH3), 2.52–2.62 (m,
1H, 3-CH2), 2.77–2.86 (m, 1H, 3-CH2), 3.95–3.05 (m, 1H, 4-H),
3.67–3.85 (m, 1H, 5-H), 3.85 (d, 1H, J = 16 Hz, NCH2COO), 3.97 (d,
1H, J = 16 Hz, NCH2COO), 5.54 and 6.25 (br s, br s, 2H, NH2),
7.16–7.33 (m, 5H, C6H5). 13C NMR (CDCl3); d = 18.33, 38.55,
45.10, 47.15, 62.51, 127.33 (C-2, C-6 aromatic), 127.43, 128.95
(C-3, C-5 aromatic), 140.65, 170.80, 174.71; HRMS: calculated for
[C13H16N2O2+Na]+: 255.1109; found: 255.1107. Anal. Calcd for
5.1.17. erythro-(4R,5S)-2-(5-Methyl-2-oxo-4-phenyl-pyrrolidin-
1-yl)-acetamide (4R,5S-2a)
A solution of ethyl (4R,5S)-2-(5-methyl-4-phenylpyrrolidin-1-
yl)-acetate (4R,5S-10a) (350 mg, 1.34 mM) in methanol (30 ml)
was treated with 25% aqueous ammonium (10 ml) for 12 h.
The reaction mixture was concentrated under reduced pressure,
and the residue was purified by column chromatography using
CH2Cl2/EtOH (20:1). The fractions with Rf 0.32 were collected
and evaporated under reduced pressure, affording erythro-
(4R,5S)-2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-1-yl)-acetamide
(249 mg, 80%) as a white solid recrystallised from water. Mp
C13H16N2O2 (232.28): C, 67.22; H, 6.94; N, 12.06. Found: C, 67.24;
H, 6.96; N, 12.09.
5.2. Determination of optical purity by chiral HPLC
Chiral HPLC measurements were performed on a Waters Alli-
ance (Waters Corporation, Milford, USA) LC system equipped with
a 2695 separation module, quaternary pump, degasser, autosam-
pler and column thermostat. Waters 2489 double-absorbed UV
detector at 210 nm was used for the analysis. The output signal
was monitored and processed using Waters Empower 2 software.
The separation of R-6 and S-6 was performed on polysaccharide-
based immobilised columns Chiralpak IA (250 ꢂ 4.6 mm I.D., with
169–171 °C. ½a 2D0
ꢁ96.7° (c 0.05, MeOH). Optical purity 99.3%
ꢀ
according to chiral HPLC. 1H NMR (CDCl3); d = 0.77 (d, 3H,
J = 6.6 Hz, 5-CH3), 2.62–2.81 (m, 2H, 3-CH2), 3.66–3.75 (m, 1H,
4-H), 3.75 (d, 1H, J = 16 Hz, NCH2COO), 3.98–4.08 (m, 1H, 5-H),
4.04 (d, 1H, J = 16 Hz, NCH2COO), 5.48 and 6.29 (br s, br s, 2H,
NH2), 7.07–7.32 (m, 5H, C6H5); 13C NMR (CDCl3); d = 14.72,
34.69, 42.40, 45.47, 59.03, 127.33 (C-2, C-6 aromatic), 127.87,
128.65 (C-3, C-5 aromatic), 138.19, 170.92, 175.10; HRMS: calcu-
lated for [C13H16N2O2+Na]+: 255.1109; found: 255.1113. Anal.
Calcd for C13H16N2O2 (232.28): C, 67.22; H, 6.94; N, 12.06.
Found: C, 67.31; H, 6.99; N, 12.10.
a particle size 5 lm) using the i-PrOH/n-hexane (1:9) mobile
phase. The separation of 3R,4S-8, 3R,4R-8, 3S,4S-8, and 3S,4R-8
was performed on coated Lux Cellulose-1 (150 ꢂ 4.6 mm I.D., with
a particle size 5 lm) columns using EtOH/n-hexane (1:99) as the
mobile phase. The separation of 4R,5S-2, 4R,5R-2, 4S,5S-2, and
4S,5R-2 was performed on coated Lux Amylose-2 (150 ꢂ 4.6 mm
5.1.18. threo-(4R,5R)-2-(5-Methyl-2-oxo-4-phenyl-pyrrolidin-1-
yl)-acetamide (4R,5R-2b)
I.D., with a particle size 5 lm) columns using EtOH/n-hexane
(15:85) as the mobile phase. The chromatographic runs were per-
formed at a flow rate of 1.0 ml/min and a column temperature of
25 °C. The injection volume was 10 ll and the analytical sample
concentration was 0.5 mg/ml. The enantiomeric elution order
was established by analysing racemic mixture and individual
enantiomer samples.
The substitution of 4R,5S-10a with 4R,5R-10b in Section 5.1.17
afforded
(4R,5R)-2-(5-methyl-4-phenylpyrrolidin-1-yl)-acetam-
ide. Yield: 72% (224 mg). Mp 117–118 °C. ½a D20
ꢀ
+22.9 (c 0.05,
MeOH). Optical purity 90.6% according to chiral HPLC. 1H NMR
(CDCl3); d = 1.23 (d, 3H, J = 6.2 Hz, 5-CH3), 2.52–2.62 (m, 1H, 3-
CH2), 2.77–2.86 (m, 1H, 3-CH2), 3.95–3.05 (m, 1H, 4-H), 3.67–
3.85 (m, 1H, 5-H), 3.85 (d, 1H, J = 16 Hz, NCH2COO), 3.97 (d,
1H, J = 16 Hz, NCH2COO), 5.54 and 6.25 (br s, br s, 2H, NH2),
7.16–7.33 (m, 5H, C6H5). 13C NMR (CDCl3); d = 18.32, 38.54,
45.10, 47.15, 62.51, 127.33 (C-2, C-6 aromatic), 127.43, 128.95
(C-3, C-5 aromatic), 140.65, 170.80, 174.72; HRMS: calculated
for [C13H16N2O2+Na]+: 255.1109; found: 255.1115. Anal. Calcd
for C13H16N2O2 (232.28): C, 67.22; H, 6.94; N, 12.06. Found: C,
67.25; H, 6.98; N, 12.08.
5.3. X-ray crystallographic analysis
Diffraction data for the erythro-(4R,5S)-2-(5-methyl-2-oxo-4-
phenyl-pyrrolidin-1-yl)-acetamide (4R,5S-2a) and threo-(4R,5R)-
2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-1-yl)-acetamide (4R,5R-
2b) were collected on a Bruker-Nonius KappaCCD diffractometer
using monochromic graphite Mo K
a radiation (k = 0.71073 Å).
The crystal structures were solved by a direct method and refined
by a full-matrix least squares method.12,13 Non-hydrogen atoms
were refined using an anisotropic approximation, whereas H-
atoms were refined by the riding model. Crystal data for 4R,5S-2:
orthorhombic, a = 6.3800(2), b = 9.9220(3), c = 20.1510(6) Å,
V = 1275.61(7) Å3, Z = 4, and the space group is P212121. A total of
2905 reflection intensities were collected up to 2hmax = 55°; for
5.1.19. erythro-(4S,5R)-2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-
1-yl)-acetamide (4S,5R-2c)
The substitution of 4R,5S-10a with 4S,5R-10c in Section 5.1.17
afforded
(4S,5R)-2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-1-yl)-
acetamide. Yield: 72% (224 mg). Mp 169–171 °C. ½a D20
ꢀ
+96.7° (c
0.05, MeOH). Optical purity 99.0% according to chiral HPLC.
1H NMR (CDCl3); d = 0.77 (d, 3H, J = 6.6 Hz, 5-CH3), 2.62–2.81
(m, 2H, 3-CH2), 3.66–3.75 (m, 1H, 4-H), 3.75 (d, 1H, J = 16 Hz,
NCH2COO), 3.98–4.08 (m, 1H, 5-H), 4.04 (d, 1H, J = 16 Hz,
NCH2COO), 5.48 and 6.29 (br s, br s, 2H, NH2), 7.07–7.32 (m, 5H,
C6H5). 13C NMR (CDCl3); d = 14.72, 34.69, 42.40, 45.47, 59.03,
127.33 (C-2, C-6 aromatic), 127.87, 128.65 (C-3, C-5 aromatic),
138.19, 170.91, 175.10; HRMS: calculated for [C13H16N2O2+Na]+:
255.1109; found: 255.1116. Anal. Calcd for C13H16N2O2 (232.28):
C, 67.22; H, 6.94; N, 12.06. Found: C, 67.30; H, 6.95; N, 12.11.
the structural refinement, 2367 reflections with I >2r(I) were used.
The final R-factor was 0.041. Crystal data for 4R,5R-2: monoclinic;
a = 11.2168(5), b = 9.0714(4), c = 13.3621(7) Å, b = 109.849(2)°;
V = 1278.9(1) Å3, Z = 4, and the space group is P21. A total of 3571
reflection intensities were collected up to 2hmax = 58°; for the
structure refinement, 1930 independent reflections with I >3r(I)
were used. The final R-factor was 0.054. Crystallographic data for
the compounds were deposited into the Cambridge Crystallo-
graphic Data Centre as a Supplementary Publication Number CCDC
905537 for 4R,5S-2a and CCDC 904950 for 4R,5R-2b, respectively.
Please cite this article in press as: Veinberg, G.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.03.016

8
G. Veinberg et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Copies of the data could be obtained, free of charge, by application
to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.03.016.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
5.4. The experimental model of vas deferens isolation and
stimulation in vitro
Wistar rats were sacrificed by decapitation. Both vas deferens
were excised, immersed in ice-cold Krebs–Henseleit (K–H) buffer
solution (content in mmol/l: NaCl 118.0, KCl 4.75, CaCl2 2.52,
MgCl2 1.64, NaHCO3 24.88, K2HPO4 1.18, glucose 10.0, EDTA
0.05) and cleaned from the surrounding tissues. The proximal por-
tions of the ductus deferens (ꢃ15 mm) were mounted in 50 ml or-
gan baths and incubated in K–H buffer solution bubbled with 95%
CO2 and 5% O2 at 32 °C. The passive tension was fixed at 1.0 g and
every 15 min, the buffer solution in the organ bath was changed.
After a 60 min adaptation period, the isolated vas deferens were
stimulated with an electrical current at the frequency of 0.1 Hz,
pulse duration of 1 ms and at a voltage of 50 V. When the electrical
current induced stable contraction amplitude, cumulative doses
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(from 1 to 100
added. After reaching the plateau contraction amplitude at the
highest studied agonist concentration (100 M), the electrical
lM) of selective sig-1R agonist PRE-084 were
l
stimulation was turned off and the isolated vas deferens was
washed several times with the K–H buffer solution. After 30 min,
electrical stimulation was resumed using the same parameters.
When the electrical current induced stable contraction amplitude,
the tested compounds were added to the isolated vas deferens at a
concentration of 10 lM. After 10 min of electrical stimulation, a
cumulative dose of PRE-084 was added. The response to the sig-
1R agonist before and after the addition of tested compound was
calculated as a percentage increase relative to the baseline contrac-
tion amplitude. All results are expressed as a mean SEM. The data
were evaluated using an analysis of variance (ANOVA). Whenever
ANOVA was significant, additional multiple comparisons were per-
formed using a Newman–Keuls post-hoc test. P-values of less than
0.05 were considered to be significant.
Acknowledgment
This work was supported by European Regional Development
Fund Project: ERAF 2DP/2.1.1.1.0/10/APIA/VIAA/059.
Please cite this article in press as: Veinberg, G.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.03.016