Design, synthesis and SAR studies of GABA uptake inhibitors derived from 2-substituted pyrrolidine-2-yl-acetic acids

Article abstract of DOI:10.1016/j.bmc.2015.01.035

In this paper, we disclose the design and synthesis of a series of 2-substituted pyrrolidine-2-yl-acetic acid as core structures and the N-arylalkyl derivatives thereof as potential GABA transport inhibitors. The 2-position in the side chain of pyrrolidin

Full text of DOI:10.1016/j.bmc.2015.01.035

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and SAR studies of GABA uptake inhibitors derived
from 2-substituted pyrrolidine-2-yl-acetic acids
⇑
Tobias Steffan, Thejavathi Renukappa-Gutke, Georg Höfner, Klaus T. Wanner
Department für Pharmazie—Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, D-81377 Munich, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In this paper, we disclose the design and synthesis of a series of 2-substituted pyrrolidine-2-yl-acetic acid
Received 1 December 2014
Revised 20 January 2015
Accepted 21 January 2015
Available online xxxx
as core structures and the N-arylalkyl derivatives thereof as potential GABA transport inhibitors. The
2-position in the side chain of pyrrolidine-2-yl-acetic acid derivatives was substituted with alkyl,
hydroxy and amino groups to modulate the activity and selectivity to mGAT1 and mGAT4 proteins.
SAR studies of the compounds performed for the four mouse GABA transporter proteins (mGAT1–
mGAT4) implied significant potencies and subtype selectivities for 2-hydroxy-2-pyrrolidine-2-yl-acetic
acid derivatives. The racemate rac-(u)-13c exhibited the highest potency (pIC50 5.67) at and selectivity
for mGAT1 in GABA uptake assays. In fact, the potency of rac-(u)-13c at hGAT-1 (pIC50 6.14) was even
higher than its potency at mGAT1. These uptake results for rac-(u)-13c are in line with the binding affin-
Keywords:
CNS
GABA uptake
SAR
i i
ities to the aforesaid proteins mGAT1 (pK 6.99) and hGAT-1 (pK 7.18) determined by MS Binding Assay
2
-Substituted pyrrolidine-2-yl-acetic acid
N-Acylpyrrolidinium ion
based on NO711 as marker quantified by LC–ESI-MS–MS analysis. Interestingly, the 2-hydroxy-2-
pyrrolidine-2-yl-acetic acid rac-(u)-13d containing 2-{[tris(4-methoxyphenyl)]methoxy} ethyl group at
the nitrogen atom of the pyrrolidine ring showed high potency at mGAT4 and a comparatively better
selectivity for this protein (>15 against mGAT3) than the well known mGAT4 uptake inhibitor
(S)-SNAP-5114.
Ó 2015 Elsevier Ltd. All rights reserved.
1
. Introduction
experiments have revealed the existence of four distinct GABA
1
5,16
transporters in humans and other species.
They are named dif-
ferently depending on the species they originate from.1
1,17
GATs
GABA (c-amino butyric acid, 1, Fig. 1) is the most important
1
–4
inhibitory neurotransmitter in the central nervous system.
Attenuation in GABAergic neurotransmission plays an important
role in the etiology of several neurological disorders including epi-
lepsy, Alzheimer’s disease, Huntington’s chorea, migraine, Par-
kinson’s disease, neuropathic pain, and depression. Increase of
GABAergic activity may be achieved for instance through direct
are thus termed as GAT-1, BGT-1, GAT-2, and GAT-3 with a prefix
‘r’ or ‘h’, for example, rGAT-1 or hGAT-1 when cloned from rats or
1
1
human cells, respectively.
Corresponding GABA transport
2
5
6
7
11
proteins cloned from mouse cells are named as mGAT1–4. Alter-
natively, the nomenclature given by the Human Genome Organisa-
8
9
10
1
1,14b
tion (HUGO)
to describe the human GABA transporters as
agonism at the GABA
A
receptors, inhibition of enzymatic break-
GAT1 (slc6a1), BGT1 (slc6a12), GAT2 (slc6a13), and GAT3 (slc6a11)
is also in use, which will be applied for universal description of the
GABA transport proteins irrespective of the species they originate
from in this study. For our biological assays, we have used GABA
transporter proteins originating from both mouse and human cells,
which are respectively termed as mGAT1–4 and hGAT-1.
down of GABA, or by inhibition of the GABA transport proteins
1
1–13
(
GATs).
The GABA transporters belong to a large family of
membrane bound sodium and chloride ion dependent transporter
1
4
proteins. They mediate the cellular transport of GABA from the
synaptic cleft into the presynaptic neurons and the surrounding
glials. Hence, to inhibit the GATs results in a prolonged availability
of the physiologically released GABA in the synaptic cleft and
extrasynaptically that can restore the normal GABA neurotrans-
The GATs differ in their distribution in the brain and the
1
5–19
body.
Of the four GABA transporter subtypes, mGAT1 and
mGAT4 are most prevalent in the brain. mGAT1 is observed in
the cerebral cortex, cerebellum, hippocampus, basal ganglia, brain
stem, spinal cord, retina, and olfactory bulb forming an important
component of the GABAergic system whereas high expression of
mGAT4 in the brain is found in the retina, olfactory bulb, thalamus,
hypothalamus, the spinal cord and the brain stem. Furthermore,
1
3
mission. This makes GATs attractive drug targets in brain disor-
ders associated with decreased GABA activity. Molecular cloning
⇑
Corresponding author. Tel.: +49 (0) 89 2180 77249; fax: +49 (0) 89 2180 77247.
E-mail address: klaus.wanner@cup.uni-muenchen.de (K.T. Wanner).
http://dx.doi.org/10.1016/j.bmc.2015.01.035
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
0
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2
T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
H
2
N
H
HO
H
H
2
N
COOH
H
2
N
COOH
H N
COOH
H N
COOH
2
2
HO
H
1
(S)-2
(S)-3
(RS)-4
COOH
COOH
COOH
COOH
H
H
COOH
COOH
N
N
R
(R)-6
N
N
R
N
N
R
(S)-6
R
R
R
5
(RS)-7
(R)-8
(S)-8
R:
a
b
c
d
e
H
MeO
MeO
MeO
S
O
S
OMe
MeO
OMe
Figure 1. Examples of GABA uptake inhibitors.
mGAT1 is recognized as being responsible for neuronal uptake
whereas mGAT4 predominantly takes care of the transport into
the surrounding glial cells.13 mGAT2 and mGAT3 have
been observed in high concentrations in kidney and liver whereas
their expression in the brain is mainly confined to the
leptomeninges (mGAT2, mGAT3) and some cerebral blood vessels
(S)-3, and (RS)-4, it seemed interesting to study the influence of
alkyl, hydroxy, and amino substituents in 2-position of (RS)-8 on
potency at and selectivity for the transporters mGAT1–4 of these
compounds. Hence, compounds 9–14 designed by addition of OH,
2
NH and alkyl groups in 2-position of the aforementioned pyrroli-
3
0
dine-2-yl-acetic acid 8 were studied for their inhibitory profile at
mGAT1–4. Compounds exhibiting pronounced activity and sub-
type selectivity were further modified by introduction of residues
like the 4,4-diphenylbut-3-enyl and 4,4-bis-(3-methylthiophene-
2-yl)but-3-enyl moiety known to be favourable for enhanced
potency at and selectivity for mGAT1 and by introduction of the
2-{[tris(4-methoxyphenyl)]methoxy}ethyl group being similarly
beneficial for mGAT4 potency and selectivity. All compounds, from
the individual diastereomers of the parent structures of 9, 10, 11,
12, 13a, 14a, and to those of the N-arylalkyl derivatives 13b–d
(only the structures and biological activity of compounds 13b
and 13c have been previously published by us in the context of a
novel MS Binding Assay using MALDI-MS-MS for detection and
1
6,17,20,21
(
mGAT3).
This in turn makes mGAT1 and mGAT4 the pre-
dominant drug targets for enhancing GABA neurotransmission by
inhibiting its reuptake from the synaptic cleft.
In the past decades, intensive studies have given considerable
insight with regard to molecular recognition features for the afore-
2
2
said targets. Initial efforts led to the development of several
GABA mimetics in the form of both acyclic4 (e.g.: (S)-2,4-diamin-
obutyric acid [(S)-DABA] (S)-2, (S)-(À)-4-amino-2-hydroxybutyric
acid (S)-3, (RS)-4-amino-3-hydroxybutyric acid (RS)-4, etc, see
Fig. 1) and cyclic analogues4 {e.g.: guvacine (5a), (R)- and (S)-
nipecotic acid [(R)-6a and (S)-6a], (RS)-pyrrolidine-3-yl-acetic acid
,23
[
(RS)-7a], see Fig. 1} of GABA. The introduction of diphenylbutenyl
3
1
group as a lipophilic residue at the N-atom of the parent amino
acids 5a, (R)-6a, (S)-6a, and (RS)-7a made them blood brain barrier
penetrable and increased their inhibitory activity in vivo.24 Studies
on the structural variations of these lipophilic residues led to the
quantification of MS-marker ) and 14b–c were, after having been
synthesized, tested for their inhibition of mouse GABA transport
Ò
25
discovery of Tiagabine (Gabitril ) (R)-6c, a highly potent and
mGAT1 selective uptake inhibitor which has been in clinical use
since 1997, for the treatment of epilepsy. The first mGAT3/mGAT4
H
rac-(l)-9
rac-(u)-9
Me
Me
COOH
N
H
Me
selective compound (S)-SNAP-5114 (S)-6d,26 containing
a
H
rac-(l)-10
rac-(u)-10
Et
Et
2
-{[tris(4-methoxyphenyl)]methoxy}ethyl group at the N-atom of
COOH
N
H
the amino acid (S)-6a was discovered by Dhar et al. Recently, we
reported on the design of DDPM-1457 (S)-6e,27 as a chemically sta-
ble carba analogue of (S)-6d displaying an inhibitory potency at
mGAT4 which is at least as high as that of (S)-6d, thus representing
one of the two most potent mGAT4 inhibitor known to date. To fur-
ther improve potency and subtype selectivity, the search for new
GABA uptake inhibitors is still ongoing.2
Et
H
COOH
COOH
(RS)-11
(RS)-12
H
N
H
COOH
N
R
H
8–30
(
RS)-8a
N
H
Previously, we have reported a novel class of GAT inhibitors
derived from pyrrolidine-2-yl-acetic acid [(R)-8a–c, (S)-8a–c], see
Figure 1, in their enantiomerically pure form as analogues of
R = a, b, c or d (see
Figure 1)
H
rac-(l)-13a-d
rac-(u)-13a-d
OH
OH
COOH
OH
3
0
GABA. Compounds (S)-8b and (S)-8c exhibited high potencies
at mGAT1 (IC50 = 0.40 M and 0.34 M, respectively) whereas
derivative (R)-8d showed reasonable potency at mGAT4
IC50 = 3.1 M) nearly equalling that of (S)-6d. Extending the scope
N
R
l
l
a
H
rac-(l)-14a-c
rac-(u)-14a-c
NH
2
(
l
COOH
N
R
NH2
of this project, we aimed at designing new parent compounds
based on the amino acid core structure (RS)-8a.30 Additionally,
starting from the structures of the known GAT inhibitors (S)-2,
NH
2
Figure 2. Target structures derived from pyrrolidine-2-yl-acetic acid.
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T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
3
9–45
proteins mGAT1–4, and in addition for hGAT-1 in case of N-arylal-
kyl derivatives 13b–d and 14b–c (see Fig. 2).
1.7–2.0 equiv of the respective ketene silyl acetals 22–25
pre-
3
as
3
9,40
pared as described in literature
catalyst in acetonitrile at 0 °C (Table 1). The yields of the
alkylation products 28–31 amounted to 73–87% (Table 1, entries
in presence of Sc(OTf)
a
-amido-
2
2
. Results and discussion
.1. Chemistry
1
mers
–4) whereby 28 and 29 were obtained as mixtures of diastereo-
4
6
[rac-(l)-28 /rac-(u)-28 = 55:45;
rac-(l)-29/rac-(u)-29 =
5
4:46] (Table 1). Interestingly, the diastereomeric composition of
the -amidoalkylation products 28 and 29 remained mostly
unchanged when mixtures of isomeric silyl ketene acetals (E/Z)-
and (E/Z)-23 , respectively, of different composition were
applied. Due to the difficulties encountered in the separation of
a
Our general strategy for synthesizing 2-substituted pyrrolidine-
-yl-acetic acid derivatives was based on the well known electro-
2
3
9
41
2
2
philic
a
-amidoalkylation reactions as a key step to introduce
-position of the pyrrolidine core struc-
For the application of the aforementioned reaction to
substituents in the
a
3
2–35
the diastereomeric mixtures of 28 [rac-(l)-28/rac-(u)-28] and 29
ture.
[
rac-(l)-29/rac-(u)-29], these were used as such for subsequent
reactions. In the final steps compounds 28–31 were subjected to
hydrolysis (KOH in MeOH/H O) and catalytic hydrogenolysis (H
the preparation of our target compounds, we intended to react
N-acylpyrrolidinium ions 17 generated from a suitable precursor
1
5 by Lewis acid activation with respective trialkylsilyl ketene ace-
2
2
over Pd/C in MeOH), which led to the carboxylic acids 34–37 and
finally to the free amino acids 9–12 in total yields over both steps
from 77–92%, (Scheme 3). The diastereomeric composition of the
outlined amino acids 9 and 10 was identical or only marginally dif-
ferent from that of the starting compounds 28 and 29 amounting
tals 16 (Scheme 1). Hence, using appropriately substituted acetic
acid derivatives in form of their silyl ketene acetals 16 would
enable the introduction of the desired substituents (alkyl, OH and
2
NH ) in the 2-position to the carboxylic group of the pyrrolidine-2-
yl-acetic acid derivatives. The reagent to be employed for the gen-
eration of the N-acyliminium ions was envisaged to consist of a
pyrrolidine ring with an acyl or alkoxycarbonyl group attached to
nitrogen and to exhibit a leaving group such as an alkoxy moiety
4
7
to 55:45 and 53:47 [rac-(l)-9/rac-(u)-9; rac-(l)-10/rac-(u)-10],
respectively. For the biological characterization the mixtures were
used in this form.
The parent compounds rac-(l)-13a48 and rac-(u)-13a48 contain-
or a halogen in the
or carbamate moiety and of the ester function of the
ation product 18 would finally lead to the free amino acids 9–12,
3a and 14a. For selected amino acids exhibiting high potencies
a-position. Subsequent cleavage of the amide
ing an OH group in
a-position of the carboxylic acid group were
a
-amidoalkyl-
prepared as laid out in Table 1 and Scheme 3. For the preparation
of required silyl ketene acetals (E/Z)-26 with a TBDMS protected
1
4
4
OH group, the literature method was slightly modified. Adding
the ester to a solution of LDA in THF with chlorotrimethylsilane
already present (instead of subsequently adding the reagent)
increased the yield of (E/Z)-26 from 34% to 55%. Reaction of silyl
as GABA uptake inhibitors, derivatives 13b–d and 14b–c displaying
N-substituents common for GABA uptake inhibitors should be syn-
thesized (Scheme 1).
4
4
ketene acetals (E/Z)-26
with the a-amidoalkylation reagent
2
.1.1. Preparation of parent amino acids
(
RS)-21 (in MeCN at 0 °C) in the presence of 10 mol % of Sc(OTf)
3
For the implementation of our synthetic strategy, pyrrolidine
derivative (RS)-21 was required as a starting compound. It was
4
9
gave a diastereomeric mixture of rac-(l)-32 and rac-(u)-32 with
a ratio of 45:55 (determined by H NMR) and a total yield of 67%
1
3
6,37
found that in addition to literature methods,
efficiently synthesized starting from the commercially available
-aminobutanal diethyl acetal 19. N-protection of 19 with benzyl
(RS)-21 can be
3
5
49
(Table 1, entry 5). Subsequent deprotection of rac-(l)-32 and
rac-(u)-32 with TBAF in THF at 0 °C yielded the mixture of
4
5
0
3
8
rac-(l)-38 /rac-(u)-38 (95% isolated yield) which, upon repeated
flash chromatography gave the pure diastereomers rac-(l)-3850
and rac-(u)-38 in 36% and 27% yield, respectively. Saponification
chloroformate and subsequent cyclisation of 20, without prior
purification, by means of catalytic amounts of Sc(OTf) (1%) pro-
vided the -amidoalkylation reagent (RS)-21 in 90% yields over
both steps (Scheme 2).
3
a
5
0
of the pure diastereomers rac-(l)-38 and rac-(u)-38 (0.1 M LiOH)
and catalytic hydrogenolysis (H over Pd/C in MeOH) yielded
The preparation of the parent amino acids 9–12, 13a and 14a by
2
4
8
48
finally the free amino acids rac-(l)-13a and rac-(u)-13a (74%
a-amidoalkylation reactions was accomplished as depicted in
and 78%, for last two steps).
Table 1 and in Schemes 3 and 4. The
a-amidoalkylation reactions
The synthesis of
acid derivatives (Table 1 and Scheme 4) was similarly accom-
plished utilizing an
-amidoalkylation reaction as key step.35 Reac-
tion of -amidoalkylation reagent (RS)-21 with a silyl ketene acetal
a-amino substituted pyrrolidine-2-yl-acetic
were carried out according to a method developed by Kobayashi
et al.3 for the reaction of N-benzyloxycarbonyl-2-methoxypiperi-
dine with silyl enol ethers mediated by Lewis acids. Thus, in the
first step the pyrrolidine derivative (RS)-21 was reacted with
5
a
a
Lewis acid
O
O
O
N
Cbz
X
N
Cbz
N
OR3
N
H
OH
N
R
OH
R1
R2
O
SiMe
CbzR1 R2
R1 R2
R1 R2
3
OR3
9-12, 13a, 14a
13b-d, 14b-c
1
5
17
18
16
R = a, b, c, d see Figure 1
Scheme 1. Synthetic strategy.
a
EtO
b
EtO
NHCbz
NH
2
OEt
N
OEt
OEt
Cbz
2038
not isolated)
(RS)-2137
(90% from 19)
19
(
Scheme 2. Synthesis of precursor (RS)-21.37 Reagents and conditions: (a) Cbz-Cl, Na
2 3 2 2 2 3
CO , CH Cl /H O, 0 °C, 3 h; (b) 1 mol % Sc(OTf) , heptane/EtOAc, rt, 3 h.
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T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 1
a
-Amidoalkylation reactions
R1
R2
a
OTMS
OMe
COOMe
N
Cbz
OEt
N
Cbz
R1 R2
(
RS)-21
22-27
Time (h)
28-33
Entry
Silyl ketene acetals
E/Z
Products
Ratioa
Yield (%)
l/u
H
COOMe
OTMS
OMe
N
MeHC
Cbz Me
1
78/22
89/11
3
46
55:45
54:46
73
rac-(l)-28
28
Me
Me
(
E/Z)-2239
rac-(u)-
OTMS
OMe
E/Z)-2341
H
EtHC
COOMe
N
2
1.5
77
Cbz Et
(
rac-(l)-29
rac-(u)-29
Et
Et
OTMS
OMe
H
COOMe
3
4
—
—
1.5
—
—
87
73
N
Cbz
(
2
442
5
7
RS)-30
OTMS
OMe
H
COOMe
4
N
Cbz
2
543
(
RS)-31
OTMS
H
COOMe
N
5
6
TBDMSO
OMe
69/31
19
19
45:55
41:59
67
Cbz OTBDMS
E/Z)-2644
(
rac-(l)-3249
rac-(u)-32
OTBDMS
OTBDMS
Me Me
H
N
Si
OTMS
COOMe
N
2
3
2(l)
2(u)
Si
—
OMe
Cbz
NH2
Me Me
rac-(l)-33
rac-(u)-33
NH
NH
2
2
E)-2745
50
(
a
Determined by 1H NMR spectroscopy; (a) 10 mol % Sc(OTf)
3
, CH
3
CN, 0 °C.
with a stabase-protected amino group (E)-2745,51 in MeCN at 0 °C
in the presence of 10 mol % Sc(OTf) for 19 h gave a diastereomeric
mixture of the pyrrolidine derivative rac-(l)-33 and rac-(u)-33
hydrogenolysis). Therefore, to obtain the pure diastereoisomer
rac-(u)-14aÁ2TFA an alternative synthetic route was followed. In
that case, the N-Cbz protecting groups of rac-(u)-40 were both first
replaced by N-Boc protecting groups. This was accomplished by
3
50
50
with a ratio of 41:59 Table 1, entry 6. As expected the primary
amino function of the silyl ketene acetal had been completely freed
from the stabase protection group during the reaction. This, how-
ever, did not hamper performing the next steps. Thus, chromato-
catalytic hydrogenation (H
give rac-(u)-42Á2TFA, (72%) and subsequent Boc-protection (Boc
2
over Pd/C in MeOH) of rac-(u)-40 to
O,
2
NEt3, MeOH) that yielded rac-(u)-43 (82%). Then, rac-(u)-43 was
transformed into the free acid rac-(u)-44 in high yield (93%) by
hydrolysis with 0.1 M LiOH in dioxane/water. Removal of the
5
0
graphic separation of the mixture yielded 22% of rac-(l)-33 and
0,52
3
2% of rac-(u)-33.5
To facilitate the isolation of the product
resulting from ester hydrolysis, the primary amino groups in the
diastereomers rac-(l)-33 and rac-(u)-33 were first N-Cbz protected.
The respective compounds rac-(l)-40 and rac-(u)-40 were obtained
in a yield of 96% and 87%, respectively. Hydrolysis (0.3 M LiOH in
dioxane/water) of rac-(l)-40 with LiOH (0.3 M) in dioxane/water
yielded 95% of rac-(l)-41 and subsequent, N-deprotection through
2 2
Boc-groups with trifluoroacetic acid in CH Cl afforded finally the
free amino acid rac-(u)-14a as its TFA salt in 99% yield without
any detectable epimerization (Scheme 4).
2.1.2. Preparation of N-substituted derivatives
The ring nitrogen substituted hydroxypyrrolidine-2-yl-acetic
acid derivatives 13b–d and aminopyrrolidine-2-yl-acetic acid
derivatives 14b–c were synthesized as depicted in Scheme 5. For
the preparation of the N-alkylated hydroxypyrrolydin-2-yl-acetic
acid derivatives 13b–d, the diastereomers rac-(l)-38 and rac-(u)-38
were subjected to catalytic hydrogenation over Pd/C in acetic acid
catalytic hydrogenolysis (H
of trifluoroacetic acid yielded the free amino acid rac-(l)-14aÁ2TFA
81% total yield over both steps). This synthetic sequence turned
2
over Pd/C in MeOH) in the presence
(
out to effect epimerization when applied to the diastereomer
rac-(u)-40 (during the removal of the Cbz group by catalytic
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T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
H
H
H
a
COOH
b
COOH
COOMe
N
N
H
N
Cbz Me
rac-(l)-344
rac-(u)-34
Me
Cbz Me
rac-(l)-28
rac-(u)-28
8
Me /
Me
Me /
Me
rac-(l)-946
Me /
Me
46
rac-(u)-9
dr = 55:45
dr = 56:44 90%
dr = 55:45 97%
a
b
H
H
H
COOMe
COOH
N
COOH
N
H
N
Cbz
Cbz Et
Et
Et
rac-(l)-10
rac-(u)-10
Et /
Et
rac-(l)-29
rac-(u)-29
Et /
Et
rac-(l)-35
rac-(u)-35
Et /
Et
dr = 57:43 99%
dr = 54:46
dr = 57:43 78%
a
b
H
H
H
COOMe
COOH
N
COOH
N
N
Cbz
RS)-3057
Cbz
(RS)-36
H
(
91%
(RS)-11
95%
a
b
H
H
H
COOMe
COOH
COOH
N
N
Cbz
N
H
Cbz
(
RS)-31
(RS)-
37
92%
(RS)-12
100%
H
H
H
COOMe
d
COOH
b
COOH
N
N
N
H
Cbz OH
Cbz OH
OH
H
c
rac-(l)-3850 36%
rac-(l)-3959 81%
rac-(l)-13a48
93%
COOMe
N
+
Cbz OTBDMS
rac-(l)-3249
rac-(u)-32
H
H
OTBDMS /
OTBDMS
H
d
COOH
b
COOH
COOMe
N
N
H
N
Cbz OH
OH
Cbz OH
dr =45:55
rac-(u)-3959 81%
rac-(u)-13a48 97%
rac-(u)-38 27%
Scheme 3. Synthesis of 2-alkyl substituted pyrrolidine-2-yl-acetic acids 9–12 and synthesis of 2-hydroxy substituted pyrrolidine-2-yl-acetic acids 13a. Reagents and
Conditions: (a) KOH, MeOH/H O, reflux, 16–96 h; (b) H , Pd/C, MeOH, rt, 1–24 h; (c) TBAF, THF, rt, 1.5 h; (d) LiOH, dioxane/H O, rt, 1–2 h.
2
2
2
to remove the N-Cbz-protecting groups giving rise to acetic acid
salts of the methyl esters rac-(l)-45 and rac-(u)-45, respectively.
Subsequent alkylation of the methyl esters rac-(l)-45 and rac-(u)-
occurred leading to mixtures of diastereomers.30,53 This inversion
could be suppressed by carrying out the N-alkylation reaction of
rac-(l)-50 with 4,4-diphenylbut-3-en-1-yl bromide using KHCO
instead of K CO as a base yielding 55% of rac-(l)-51. Similarly,
3
4
5 with the alkyl bromides (RBr) 4,4-diphenylbut-3-en-1-yl bro-
2
3
2
2
4
5
mide
mide
(R = b), 4,4-bis-(3-methylthien-2-yl)but-3-en-1-yl bro-
(R = c) and 2-[tris(4-methoxyphenyl)methoxy]ethyl
the N-alkylated derivative rac-(u)-51 was obtained in pure form
in 18% yield by changing the base to DIPEA. The N-alkylation of
rac-(l)-50 with 4,4-bis-(3-methylthien-2-yl)but-3-en-1-yl bromide
2
6
bromide (R = d) in MeCN in the presence of K
iodide
2
CO
3
and potassium
3
0,53
provided the respective N-alkyl derivatives 46–48 in
in MeCN in the presence of KI and K
in 49% yield. The alkylated derivative rac-(u)-52 could be obtained
without any epimerization only when instead of KHCO triethyl-
amine was used as a base (in CH Cl ), in which case, however,
2 3
CO as base afforded rac-(l)-52
yields from 19–60%. Hydrolysis of the N-alkyl derivatives 46–48
with LiOH in dioxane/water finally afforded the target compounds
3
1
3b–d in yields of 95–99%.
2
2
For the synthesis of the target compounds 14b–c, the primary
the yield amounted to only 10%. Finally, saponification of the ester
function of rac-51 and rac-52 with LiOH in a mixture of dioxane
and water followed by deprotection of the primary amino group
by treatment with TFA in CH Cl gave the trifluoroacetic acid salts
2 2
of the target compounds 14b–c in 73–97% (Scheme 5).
amino group present in the aminopyrrolidine-2-yl-acetic acid
derivatives rac-(l)-33 and rac-(u)-3352 was protected to enable
the selective alkylation of the secondary amino group of the pyrrol-
idine ring after its deprotection. This was accomplished by reacting
the diastereoisomers rac-(l)-33 and rac-(u)-33 with Boc anhydride
and triethylamine in dioxane at room temperature for 2.5 h to
yield the corresponding Boc protected compounds rac-(l)-49 and
2.2. Biological evaluation
5
2
rac-(u)-49 in good yields (86% and 95%, respectively). Subsequent
hydrogenolytic removal of the Cbz group (H , Pd/C, MeOH, and ace-
The inhibitory potency of the synthesized amino acids and their
subtype selectivity at mGAT1, mGAT2, mGAT3 and mGAT4 trans-
2
3
tic acid) gave the acetic acid salts rac-(l)-50 and rac-(u)-50 in yields
of 99% and 97%, respectively. Unfortunately, when N-alkylation of
rac-(l)-50 and rac-(u)-50 was attempted with 4,4-diphenylbut-3-
port proteins were determined using well established [ H]GABA
5
4
uptake assays
based on mouse GABA transporters stably
5
4
expressed in HEK cells. The measurements were done in tripli-
en-1-yl bromide (R = b) using K
merization at the chiral center in 2-position of those compounds
2
CO
3
as a base with KI in MeCN epi-
cates and the results are given as pIC50 ± SEM (n = 3). For the com-
pounds that were unable to reduce the [ H]GABA uptake to a value
3
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6
T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
H
a
H
COOMe
2
COOMe
N
N
Cbz NH
Cbz NHCbz
rac-(l)-3350
NH
rac-(l)-40
2
2
NHCbz
NHCbz
96%
87%
rac-(u)-3350 NH
rac-(u)-
40
H
b
H
c
H
COOMe
COOH
COOH .2TFA
N
N
N
H
Cbz NHCbz
Cbz NHCbz
NH
2
.
58
rac-(l)-40
rac-(l)-41 95%
rac-(l)-14a 2TFA
86%
H
d
H
e
H
N
COOMe
COOMe
COOMe
.
N
N
2HOAc
Cbz NHCbz
H
NH
2
Boc NHBoc
.
rac-(u)-40
rac-(u)-42 2HOAc 72%
rac-(u)-43
82%
H
H
.
b
COOH
f
COOH 2TFA
N
N
H
Boc NHBoc
NH
2
rac-(u)-44
93%
rac-(u)-14a 2TFA
.
58
99%
Scheme 4. Synthesis of 2-amino substituted pyrrolidine-2-yl-acetic acids 14a. Reagents and conditions: (a) Cbz-Cl, NaHCO
3
, H
2
O/THF, 0 °C to rt, 4 h; (b) LiOH, dioxane/H
2
O,
rt, 1–2 h; (c) H , Pd/C, MeOH, rt, 1 h, TFA; (d) H , Pd/C, MeOH, rt, 2 h, AcOH; (e) (Boc) O, NEt , MeOH, 50 °C, 30 min; (f) TFA, CH
2
2
2
3
2
Cl , rt, 25 min.
2
of <50% at a specific concentration of 1 mM (pIC50 value is taken as
3) or 100 lM (pIC50 value taken as 64), the results are indicated
3
Additionally, rac-(l)-13a exhibited a pronounced selectivity of >71
for mGAT4 against mGAT1 (Table 3) and >12 against mGAT2. In
addition, this ligand also showed a considerable potency at
mGAT3. In contrast, racemic rac-(u)-13a did not exhibit any signif-
icant potency at or selectivity to GAT proteins (Table 2, entry 8).
Similar to what had been observed for the hydroxyl derivatives
6
as the percentage of the remaining [ H]GABA uptake at this con-
centration of the test compound.
2
.2.1. SAR of parent structures
3
The pIC50 values of the unsubstituted pyrrolidine-2-yl-acetic
rac-(l)-13a and rac-(u)-13a, the results of the [ H]GABA uptake
acids (R)-8a and (S)-8a were used for reference. The parent amino
acids (9–12, 13a, 14a) were all tested in their racemic form either
as diastereomeric mixtures (roughly 1:1) or pure diastereomers
and not as pure enantiomers like the reference compounds, the
homoprolines (R)-8a and (S)-8a. This implies that some constitu-
ents of the mixture or the pure enantiomers may have potencies
somewhat higher than the value reflected by the overall test result.
As shown in Table 2, among the parent amino acids substituted
at the 2-position with alkyl groups (9–12, Table 2, entries 3–6), no
reasonable improvement in the potency at and selectivity to GAT
proteins was observed compared to the values obtained for the
unsubstituted enantiomeric pure homoprolines (R)-8a and (S)-8a
assay for 2-amino-pyrrolidine-2-yl-acetic acids, the diastereomer
rac-(l)-14a in comparison to the homoprolines (R)-8a and (S)-8a
showed an improved binding potency at all four mouse GABA
transport proteins with some selectivity for mGAT3 and mGAT4
proteins (see Table 2).
Based on these results, it is to be inferred that the addition of
sterically demanding lipophilic groups to the 2-position of pyrrol-
idine-2-yl-acetic acid core structure is not tolerated by the GAT
proteins. More importantly, it may be concluded that the presence
of hydrophilic groups like a hydroxy or an amino moiety in the 2-
position to the carboxyl group of homoproline with an appropriate
stereochemical orientation appears to be well tolerated or to even
enhance potency and to give rise to promising selectivities for
mGAT3 and mGAT4 proteins.
(Table 2, entries 1 and 2). The potencies of rac-(l)-9/rac-(u)-9 (dr
5
5:45) at mGAT1–mGAT4 exhibiting a methyl group in the side
chain of pyrrolidine-2-yl-acetic acid moiety were roughly the same
as those found for (R)-8a and (S)-8a, except that the activity at
mGAT1 appears to be somewhat lower (compare Table 2, entry 3
to entries 1 and 2). Further increase in the lipophilicity of the par-
ent compounds by adding an ethyl [rac-(l)-10/rac-(u)-10,
dr = 54:46] or two vicinal methyl groups [(RS)-11] or a 1,3-propa-
ndiyl moiety [(RS)-12] resulted in loss of activity and selectivity at
GAT proteins. For the 2-hydroxy substituted pyrrolidine-2-yl-ace-
tic acids rac-(l)-13a and rac-(u)-13a, rac-(l)-13a though still a race-
mic mixture showed better inhibitory potencies at the mGAT2
2.2.2. SAR of the N-substituted derivatives
Unlike the parent compounds, the N-substituted derivatives
13b–c, 14c were also tested for their inhibitory activity at hGAT-1
in addition to the four mouse GABA transporters mGAT1–4. The
3
[ H]GABA uptake assay for hGAT-1 was identical to the one for
mGAT1 except that membrane preparation of HEK293 cells
5
4
expressing hGAT-1 have been employed. The results of the GABA
uptake experiments and subtype selectivities of the N-alkylated
derivatives of 2-hydroxy-pyrrolidine-2-yl-acetic acid 13b–d and
the 2-amino-pyrrolidine-2-yl acetic acid 14b–c are given in Table 3.
The parent structures 13a and 14a (Table 2), Tiagabine (R)-6c, and
(S)-SNAP-5114 (S)-6d (Table 3, entries 1 and 2) were used as
(
pIC50 = 3.77), mGAT3 (pIC50 = 4.44), and mGAT4 (pIC50 = 4.85)
proteins compared to both of the enantiomerically pure homopro-
lines (R)-8a and (S)-8a (compare Table 3, entry 7 to entry 1 and 2).
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T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
a
H
H
COOMe
.
HOAc
COOMe
N
N
H
Cbz OH
OH
HOAc
rac-(l)-3852
rac-(l)-
45 .
OH
OH
89%
OH
OH
rac-(u)-45 .HOAc
96%
rac-(u)-38
H
H
b
c
COOMe
COOH
OH
N
R
N
R
OH
rac-(l)-13b31
rac-(u)-13b31
OH
OH
95%
rac-(l)-46 (R = b)
rac-(u)-46 (R = b)
OH 60%
OH
96%
4
4%
rac-(l)-13c31 OH
rac-(u)-13c31 OH
rac-(l)-47(R = c)
rac-(u)-47 (R = c)
OH 43%
OH 19%
97%
96%
rac-(l)-48 (R = d)
rac-(u)-48 (R = d)
OH 45%
rac-(l)-13d
rac-(u)-13d
OH
OH
9
9%
OH
59%
98%
H
d
H
a
H
COOMe
COOMe
COOMe . HOAc
NHBoc
N
N
N
H
Cbz NH
2
Cbz NHBoc
rac-(l)-33
NH2
rac-(l)-49
NHBoc 86%
NHBoc 95%
rac-(l)-50 . HOAc
NHBoc 99%
NHBoc 97%
rac-(u)-50 HOAc
.
rac-(u)-3352
rac-(u)-4952
NH
2
H
1. c
H
e-f (R=b)
g-h (R=c)
.
COOH 2TFA
COOMe
N
R
N
R
2
. i
NH
NHBoc
2
.
NH
97%
87%
rac-(l)-51 (R = b)
rac-(u)-51(R = b)
NHBoc
NHBoc
55%
18%
rac-(l)-14b 2TFA
2
rac-(u)-14b 2TFA
.
NH
2
rac-(l)-52 (R = c)
rac-(u)-52 (R = c)
NHBoc
NHBoc
49%
10%
rac-(l)-14c 2TFA
.
NH
2
73%
82%
rac-(u)-14c 2TFA
.
NH
2
R:
b
c
d
MeO
MeO
O
S
S
OMe
Scheme 5. Synthesis of N-arylalkyl substituted hydroxy- and aminopyrrolidine-2-yl-acetic acid derivatives 13b–d and 14b–c. Reagents and conditions: (a) H
MeOH, rt, 1 h; (b) R-Br (R = b, c or d), K CO , KI, CH CN, reflux, 4–6 h; (c) LiOH, dioxane/H O, rt, 1–1.5 h followed by addition of Na HPO /NaH PO
, dioxane, rt, 2.5 h; (e) R-Br (R = b), KHCO , KI, CH CN, reflux, 4 h; (f) R-Br (R = b), DIPEA, CH CN, rt, 72 h; (g) R-Br (R = c), K CO , KI, CH CN, reflux, 17 h; (h) R-Br (R = c),
, CH Cl , rt, 72 h; (i) TFA, CH Cl , rt, 25–30 min.
2
, Pd/C, HOAc,
2
3
3
2
2
4
2
4 2
-buffer (pH = 7); (d) (Boc) O,
NEt
NEt
3
3
3
3
3
2
3
3
2
2
2
2
reference compounds. In a competitive MS Binding Assay devel-
By aiming most of all at compounds highly potent at and selec-
tive for mGAT1 and mGAT4 proteins, we first studied the influence
of substituents known to enhance mGAT1 potency and selectivity
like the 4,4-diphenylbut-3-enyl and the 4,4-bis(3-methylthio-
phen-2-yl)but-3-enyl group at the pyrrolidine ring nitrogen of both
the parent structures 13a and 14a. Addition of a 4,4-diphenylbut-
3-enyl residue to the pyrrolidine ring nitrogen of both racemic dia-
stereomers of 2-hydroxypyrrolidine-2-yl-acetic acid derivatives,
rac-(l)-13a and rac-(u)-13a, resulted in a significant increase of
the inhibitory potency at mGAT1 (and similarly hGAT-1) of about
5
5
oped by us, the aforementioned compounds were also character-
ized for their binding affinities to the GABA transport proteins
hGAT-1 and mGAT1. For this competitive assay, NO7115 was used
6
5
5
as unlabelled marker which was quantified by LC–ESI-MS–MS.
Affinity values obtained from the competitive MS Binding Assays
are given as pK ± SEM (calculated from three independent experi-
i
ments). Again, since the compounds have been tested as racemates,
one of the pure enantiomers may possess better inhibitory proper-
ties or binding affinities than the racemic mixture.
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8
T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 2
3
Inhibitory potency of 2-substituted pyrrolidine-2-yl-acetic acid core structures and pyrrolidine-2-yl-acetic acid in [ H]GABA uptake assays given as pIC50 values [mean ± SEM,
n = 3] at mGAT1, mGAT2, mGAT3 and mGAT4 obtained from three independent experiments
Entry
1
Compounds
mGAT1
mGAT2
mGAT3
mGAT4
H
COOH
3.62 ± 0.05
1 mM (81%)
3.47 ± 0.04
2.95 ± 0.07
N
H
(
R)-8a
H
2
3
COOH
3.39 ± 0.10
1 mM (77%)
3.85 ± 0.01
3.51 ± 0.06
3.76 ± 0.04
3.47 ± 0.02
N
H
(
S)-8a
H
COOH
Me
N
H
*
*
1 mM (83%)
1 mM (59%)
rac-(l)-9/rac-(u)-947
5
5:45
H
COOH
*
*
*
1
mM (78%)
1 mM (82%)
1 mM (78%)
1 mM (95%)
4
N
H
Et
rac-(l)-10/rac-(u)-10
4:46
5
H
*
*
*
*
5
6
COOH
1 mM (83%)
1 mM (108%)
1 mM (93%)
1 mM (73%)
1 mM (95%)
N
H
(
RS)-11
H
COOH
*
*
*
*
1 mM (92%)
1 mM (91%)
1 mM (110%)
N
H
(
RS)-12
H
COOH
7
N
H
3.00
3.77 ± 0.02
4.44 ± 0.03
4.85 ± 0.03
OH
4
8
rac-(l)-13a
H
*
*
*
8
9
COOH
1 mM (53%)
1 mM (95%)
3.57 ± 0.04
4.57 ± 0.03
1 mM (61%)
N
H
OH
4
8
rac-(u)-13a
H
COOH .2TFA
3.91 ± 0.05
3.80 ± 0.06
4.67 ± 0.02
N
H
NH
2
5
8
rac-(l)-14a·2TFA
H
COOH. 2TFA
*
*
*
1
0
1 mM (1%)
1 mM (52%)
1 mM (59%)
3.43 ± 0.03
N
H
NH
2
rac-(u)-14a·2TFA58
*
3
3
Remaining [ H]GABA uptake in the presence of 1 mM test substance as compared to control without inhibitor. Compounds that do not reduce at 1 mM the [ H]GABA
uptake to <50%, a pIC50 of 63 is assumed to ease the comparitive analysis of the obtained results.
1
.5 to 2 log units the pIC50 for rac-(l)-13b and rac-(u)-13b at
a substantial increase in activity up to two orders of magnitude
compared to the core structures rac-(l)-13a and rac-(u)-13a (com-
pare Table 3, entries 5 and 6 to Table 2, entries 7 and 8). Whereas of
the parent compounds rac-(l)-13a had better inhibitory potencies
at mGAT3 and mGAT4 as compared to mGAT1 and mGAT2, the
N-arylalkyl derivatives rac-(l)-13c and rac-(u)-13c exhibited selec-
tivity in favour of mGAT1 [rac-(l)-13c: >29 for mGAT1 against
mGAT2, mGAT3 and mGAT4; rac-(u)-13c: >47 for mGAT1 against
mGAT2 and mGAT4, >20 against mGAT3]. With a pIC50 of 5.67,
racemic rac-(u)-13c exhibited the nominally highest potency at
mGAT1 amounting to 4.60 and 5.64, respectively. At the same time,
the inhibitory potency at mGAT2–mGAT4 remained largely
unchanged or became even lower, thus overall leading to an
increased selectivity for mGAT1 (as compared to mGAT2–mGAT4,
Table 3, entries 3 and 4). A strong increase in potency—this time
for both diastereomers, rac-(l)-13a and rac-(u)-13a—was also
effected by introduction of the 4,4-bis(3-methylthiophen-2-
yl)but-3-enyl group. Here the compounds rac-(l)-13c and rac-(u)-
1
3c exhibited pIC50 values of 5.47 and 5.67 at mGAT1 which means
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T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
Table 3
3
Inhibitory potency of N-arylalkyl derivatives in [ H]GABA uptake assays given as pIC50 values [mean ± SEM, n = 3] at hGAT-1, mGAT1, mGAT2, mGAT3 and mGAT4 obtained from
three independent experiments
Entry
Compounds
R
hGAT-1
mGAT1
mGAT2
mGAT3
mGAT4
COOH
N
R
1
00
l
M
100 lM
*
(64%)
100
(73%)
lM
1
6.42 ± 0.01
6.88 ± 0.12
*
S
Tiagabine (R)
R)-6c
(52%)
S
(
*
COOH
MeO
MeO
N
R
O
100 lM
*
(56%)
2
—
4.07 ± 0.05
5.30 ± 0.02
5.81 ± 0.06
(
S)-SNAP-5114
(S)-6d
OMe
H
COOH
N
R
1
00
l
M
100
(97%)
l
M
100 lM
*
OH
4.86 ± 0.05
4.60 ± 0.04
*
*
3
4
(89%)
(59%)
rac-(l)-13b31
H
COOH
100
(60%)
lM
100 lM
*
(55%)
N
R
5.72 ± 0.03
5.64 ± 0.03
4.00
*
OH
rac-(u)-13b31
H
COOH
N
R
OH
1
00
l
M
100
(90%)
l
M
100 lM
*
5
.38 ± 0.05
5.47 ± 0.10
5.67 ± 0.07
*
*
5
6
rac-(l)-13c31
(63%)
(74%)
S
S
H
COOH
N
R
100 lM
100 lM
*
(59%)
OH
6.14 ± 0.02
*
4.37 ± 0.05
(58%)
rac-(u)-13c31
H
COOH
N
R
OH
100
(68%)
l
M
100 lM
*
MeO
MeO
—
—
4.77 ± 0.05
5.24 ± 0.02
*
7
8
(56%)
rac-(l)-13d
O
H
COOH
OMe
N
R
100
(80%)
l
M
100
l
M
100 lM
*
(70%)
*
*
5.18 ± 0.05
OH
(64%)
rac-(u)-13d
(
continued on next page)
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1
0
T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 3 (continued)
Entry Compounds
R
hGAT-1
mGAT1
mGAT2
mGAT3
4.94
mGAT4
H
COOH. 2TFA
NH
N
R
2
100
l
M
9
—
4.31 ± 0.03
*
4.76 ± 0.04
(
66%)
rac-(l)-14b·2TFA
H
.
COOH 2TFA
N
R
1
0
100
l
M
100
(71%)
l
M
100 lM
*
NH
2
—
—
4.46 ± 0.03
*
*
(
61%)
(83%)
rac-(u)-14b·2TFA
H
.
COOH 2TFA
N
R
NH
2
1
1
2
4.85 ± 0.04
4.37 ± 0.01
4.52 ± 0.03
4.50 ± 0.07
rac-(l)-14c·2TFA
S
H
.
S
COOH 2TFA
N
R
1
100
l
M
100 lM
*
(63%)
NH
2
*
4.00
4.00
4.00
(
57%)
rac-(u)-14c·2TFA
The bold values represent the most potent and highly affine compounds.
*
3
3
Remaining [ H]GABA uptake in the presence of 100
lM test substance as compared to control without inhibitor. Compounds that do not reduce at 100
l
M the [ H]GABA
uptake to 650%, a pIC50 of 64 is assumed to ease the comparative analysis of the obtained results.
mGAT1. At hGAT-1, compounds rac-(l)-13b, rac-(u)-13b, rac-(l)-13c
and rac-(u)-13c exhibited pIC50s similar to those found for mGAT1
with one exception. In case of rac-(u)-13c, the pIC50 found for
hGAT1 was almost 0.5 log units higher than that for mGAT1 (see
Table 3, entry 6).
Table 4
Affinities of selected N-arylalkyl derivatives toward HEK hGAT-1 and HEK mGAT1
membrane preparations given as pK
i
values (mean ± SEM, n = 3) obtained from three
independant experiments
*
Entry
Compounds
pK
i
All of the 2-aminopyrrolidine-2-yl-acetic acid derivatives, that
is, rac-(l)-14b, rac-(u)-14b, rac-(l)-14c and rac-(u)-14c showed only
low to mediocre potencies. As far as there was any preference for
one of the four transporters mGAT1–mGAT4, this was very small.
This was also true for rac-(l)-14b. But this compound, rac-(l)-14b
though exhibiting a diphenylbutenyl moiety favouring the inhibi-
tory potency at mGAT1, showed its highest potency at mGAT3
hGAT-1
mGAT1
1
2
3
4
5
6
7
Tiagabine (R)-6c
rac-(l)-13b
rac-(u)-13b
rac-(l)-13c
rac-(u)-13c
rac-(l)-14c
7.32 ± 0.11
5.40 ± 0.01
6.67 ± 0.01
5.96 ± 0.07
7.18 ± 0.03
4.69 ± 0.03
4.72 ± 0.03
7.43 ± 0.06
5.41 ± 0.03
6.84 ± 0.03
5.97 ± 0.05
6.99 ± 0.04
4.76 ± 0.03
4.43 ± 0.02
rac-(u)-14c
(
9
pIC50 = 4.94) followed by mGAT4 (pIC50 = 4.76; see Table 3, entry
).
Finally, we also studied the influence of the 2-{[tris(4-methoxy-
The bold values represent the most potent and highly affine compounds.
K : Affinity constant measured with competitive MS Binding Assay based on
i
unlabelled NO711 as marker.
*
55
phenyl)]methoxy}ethyl group (known to generally increase the
potency at and selectivity for mGAT4)26 on the inhibitory activity
of the core structures of 2-hydroxypyrrolidine-2-yl-acetic acids,
rac-(l)-13a and rac-(u)-13a, at mGAT1–mGAT4. The N-substituted
compound rac-(l)-13d exhibited a pIC50 of 5.24 at mGAT4 with a
selectivity of >17 for mGAT4 against mGAT1 and mGAT2 and also
a slight selectivity against mGAT3 the pIC50 at which transporter
amounted to 4.77 (see Table 3, entry 7). Though the inhibitory
potency of rac-(l)-13d at mGAT4 is quite satisfying, the N-substitu-
tion has given rise to only a small increase of the former (compare
Table 2, entry 7 with Table 3, entry 7). In case of rac-(u)-13a, the
same N-substituent was far more effective causing an increase of
the pIC50 for mGAT4 by more than two log units (compare Table 2,
entry 8 to Table 3 entry 8). In addition rac-(u)-13d exhibited an
improved selectivity of >15 for mGAT4 as compared to mGAT1,
mGAT2 and mGAT3.
i
obtained for hGAT-1 and mGAT1 (given as pK in Table 4) were
all very similar for the individual compounds and in good accord
3
with those from the [ H]GABA uptake assay for the same trans-
i
porter (given as pIC50 in Table 3). The pK values were generally
somewhat higher (ꢀ0.5–1.5 log units, see Table 4) than the corre-
sponding pIC50 values which is a general phenomenon and thought
to be due to the varying NaCl concentrations in the buffer system
used in the uptake and binding assays as outlined previously.55
Interestingly, the pK
Table 4, entry 5) is very close to the pK
((R)-6c, pK = 7.32, Table 4, entry 5), but markedly lower for mGAT1
(mGAT1: Tiagabine (R)-6c, pK = 7.43; rac-(u)-13c, pK = 6.99; see
i
value of rac-(u)-13c for hGAT-1 (pK
i
7.18,
i
value of Tiagabine
i
i
i
Table 4, entries 1 and 5). Similarly, rac-(u)-13c had reached a
pIC50 value in the GABA uptake assay for hGAT-1 of 6.14 (Table 3,
entry 6) and thus close to that for Tiagabine ((R)-6c) in the same
test system (hGAT-1: pIC50 = 6.42), whereas for the mouse GABA
transporter mGAT1 the potency of rac-(u)-13c was again distinctly
Compounds 13b–c and 14c have also been characterized with
regard to their binding affinities to hGAT-1 and mGAT1 in the
respective MS Binding Assays (Table 4). The binding affinities
Please cite this article in press as: Steffan, T.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.01.035

T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
11
lower than that of Tiagabine ((R)-6c) (mGAT1: rac-(u)-13c
pIC50 = 5.67, (R)-6c pIC50 = 6.88). This represents a clear example
that test results obtained for mouse GAT1 do not necessarily corre-
spond closely to those for the human protein.
showed also an acceptable selectivity of >15 for mGAT4 against
mGAT1, mGAT2 and mGAT3.
Overall, the structure–activity relationships so far established
for GAT inhibitors have been distinctly widened by the results pre-
sented in this study. As demonstrated the pyrrolidine-2-yl-acetic
acid moieties with a hydroxy or an amino group in the 2-position
represent novel and promising core structures for the development
of new GAT inhibitors which may show altered physicochemical
properties.
To summarize, the N-substituted 2-hydroxy pyrrolidine-2-yl-
acetic acids [rac-(l)-13b, rac-(u)-13b, rac-(l)-13c and rac-(u)-13c]
are potent and selective inhibitors and binders at mGAT1 and
hGAT-1, the (u)-configured diastereoisomers showing slightly
higher affinity and activity than their (l)-configured counterparts.
Remarkably, N-alkylation with groups known to enhance selectiv-
ity for mGAT1 gave rise to a shift in the selectivity of the parent
compound (rac)-(l)-13a (pIC50 mGAT4/pIC50 mGAT1 >71) in favour
of mGAT4 to a selectivity in favour of mGAT1 for the substituted
compounds (see e.g., rac-(l)-13c, Table 3, entry 5). Above results
also confirm the general ability of the 2-{[tris(4-methoxy-
phenyl)]methoxy}ethyl moiety to enhance inhibitory potency at
mGAT4, the N-substituted compounds rac-(l)-13d and
rac-(u)-13d showing higher pIC50 values for inhibition of mGAT4
than the parent compounds rac-(l)-13a and rac-(u)-13a.
4
4
. Experimental
.1. Chemistry
The melting points were determined on a Büchi melting point
apparatus no. 510 (Dr. Tottoli) and are uncorrected. IR spectra were
recorded with a Perkin Elmer FT-IR spectrometer Paragon 1600.
NMR spectra were obtained with JEOL JNMR-GX 400 (400 MHz),
JNM-ECP 400 (400 MHz) or JNM-ECP 500 (500 MHz) spectrome-
ters, respectively. Tetramethylsilane was used as internal standard
and in exceptional cases the known chemical shift of solvent traces
was used to nominate the spectra. The NMR spectra were recalcu-
lated with NUTS, 2D version 2002 from Acorn NMR. MS spectra
were recorded on a Hewlett Packard 5989 A mass spectrometer
with 59980 B particle beam LC/MS interface. LC–MS/MS-Mass
spectrometer API 2000 from Applied Bio systems was used for
the MS-binding assays. HRMS spectra were obtained with a JEOL
JMS 700 and a JEOL JMS GC-Mate II mass spectrometer. CHN-anal-
yses were determined an HERAEUS Rapid or an ELEMENTAR Vario
EL elemental analysator. Column chromatography (CC) was per-
formed as flash chromatography with silica gel 60 (Merck, 0.040–
3
. Conclusions
In summary, a new series of core structures delineated from
pyrrolidine-2-yl-acetic acid by substituting the 2-position with
lipophilic groups like methyl, ethyl, vicinal dimethyl, 1,3-propa-
ndiyl and with hydrophilic moieties like the hydroxy and the
amino groups has been studied for their potency as GABA uptake
inhibitors. For the synthesis of these compounds a strategy com-
prising electrophilic
a-amidoalkylation reaction based on N-acy-
lpyrrolidinium ions as key steps was followed. Pharmacological
evaluation of the activity of the parent compounds at mouse GABA
uptake proteins mGAT1, mGAT2, mGAT3 and mGAT4 revealed that
lipophilic groups like ethyl, vicinal methyl or the sterically more
demanding 1,3-propandiyl group at the 2-position of the pyrroli-
dine-2-yl-acetic acids are not well tolerated. The amino acids
rac-(l)-13a and rac-(l)-14a resulting from addition of hydrophilic
moieties, that is, hydroxy or amino groups, respectively, exhibited
an improved selectivity profile for mGAT3 and mGAT4 in GABA
uptake assays associated with an increase in potencies for these
proteins in comparison to the unsubstituted core amino acids,
the homoprolines (R)-8a and (S)-8a.
0
0
.063 mm) in the normal phase and silanised silica gel 60 (Merck,
.063–0.200 mm) in the reversed-phase chromatography. Analyti-
cal HPLC comprised of L-6200, L-7100 or L7100 pump, L-4000 or L-
400, UV/Vis. and Diode Array detector L-7450 and L-4000-UV,
Software D-7000 HPLC-System-Manager (Merck-Hitachi), LiChro-
7
Cart with LiChroSpherÒ 100 RP-18 cartridge (5
Ò
l
m, 250 Â 4 mm
with precolumn 4 Â 4 mm) and LiChrosorbÒ Si 60 (5
lm,
2
50 Â 4 mm with precolumn 4 Â 4 mm) (Merck). Preparative HPLC
comprised of L-7150 pump, UV–VIS-detector L-4000, integrator D-
Ò
2
000 (Merck–Hitachi), LiChrosorb Si 60 (7
LiChrosorb RP-18 (7
ried out in vacuum dried glassware under nitrogen atmosphere.
l
m, 250 Â 25 mm) and
The
rac-(l)-13b, rac-(u)-13b, rac-(l)-13c and rac-(u)-13c displaying a
,4-diphenyl-3-butenyl group or the 4,4-bis(3-methyl-
2-hydroxypyrrolidine-2-yl-acetic
acid
derivatives
Ò
l
m, 250 Â 25 mm). All reactions were car-
4
All reagents were used as commercially available. Solvents were
thiophen-2-yl) but-3-enyl group at the ring nitrogen exhibited
high selectivity for mGAT1 in GABA uptake assays in addition to
high potencies for this target in comparison to the core structures
rac-(l)-13a and rac-(u)-13a. The 2-hydroxypyrrolidine-2-yl-acetic
acid derivative rac-(u)-13c was the most potent GAT1 inhibitor
with pIC50 values of 5.67 and 6.14 at mGAT1 and hGAT-1, respec-
tively. In MS Binding Assays, this compound, rac-(u)-13, exhibited
dried prior to use. DME, THF, Et
,4-dioxane, toluene were freshly distilled from sodium metal/
benzophenone ketyl, CH Cl , CDCl , DMF, CH CN from CaH and
the alcohols CH OH, C OH from Mg prior to use. The silyl ketene
2 3
O, NEt , diisopropylamine, DIPEA,
1
2
2
3
3
2
3
2 5
H
3
9
41
42
43
44
45
acetals E/Z-22, E/Z-23, 24, 25, E/Z-26 and E-27 were pre-
pared according to the literature.
3
9,40
pK
the value for the human GABA transporter being close to that
observed for Tiagabine (R)-6c at the hGAT-1 protein (pK 7.32).
i
values of 6.99 and 7.18 for mGAT1 and hGAT-1, respectively,
4
2
.1.1. (2RS)-1-[(Benzyloxy)carbonyl]-2-ethoxypyrrolidine [(RS)-
3
7
1]
A solution of Na
water was added to a solution of 4-aminobutyraldehyde diethyl
acetal (2.64 g, 16.6 mmol) in 50 ml of CH Cl and cooled to 0 °C.
i
2 3
CO (5.81 g, 54.8 mmol, 3.3 equiv) in 50 ml of
In addition, the N-alkylated (u)-configured diastereomers
rac-(u)-13b and rac-(u)-13c exhibited higher potencies at and
selectivities for mGAT1 than their corresponding (l)-configured
counterparts in the GABA uptake assays. The N-substituted 2-ami-
nopyrrolidine-2-yl-acetic acids showed negligible to no selectivity.
By introduction of 2-{[tris(4-methoxyphenyl)methoxy}ethyl
residue known to enhance mGAT4 selectivity to the N-atom of
the pyrrolidine ring of the two racemic diastereomers of
2
2
Benzyl chloroformate (3 g, 17.6 mmol, 2.5 ml, 1.07 equiv) was
added dropwise and the reaction mixture was stirred at rt for
3
h, after which the organic phase was separated and evaporated
3
8
to dryness in vacuum. The residue 20 was cooled to 0 °C and stir-
red with a solution of Sc(OTf) (81 mg, 0.17 mmol, 1 mol %) in
66 ml of heptane/EtOAc (70:30). After 45 min, the reaction was
3
1
2
-hydroxy-pyrrolidine-2-yl-acetic acid gave rac-(l)-13d and
rac-(u)-13d with a reasonably high potency of 5.24 and 5.18
pIC50 in GABA uptake assays), respectively, of which the latter
warmed to rt and stirred for further 105 min followed by removal
of solvent in vacuum. The crude product on purification by CC
(
Please cite this article in press as: Steffan, T.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.01.035

1
2
T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
(
(
SiO
RS)-21 as colourless oil.
Bp: 94–96 °C/2.0 mbar {Lit. bp.: 83 °C, 2 mmHg}. IR (KBr):
~ = 2976, 2894, 1708, 1498, 1455, 1407, 1359, 1325, 1287, 1211,
2
, Isohexane/EtOAc/EDMA = 95:5:3) afforded 3.67 g (90%) of
4.1.2.2.
(2RS)-2-{(2RS)-1-[(Benzyloxy)carbonyl]pyrrolidine-2-
yl}butyric acid methyl ester [rac-(l)-29] and (2SR)-2-{(2RS)-1-
37
[(benzyloxy)carbonyl]pyrrolidine-2-yl}butyric
ester [rac-(u)-29]. According to GP1, (RS)-21 (105 mg,
0.42 mmol), [(E/Z)-23] (146 mg, 0.84 mmol), MeCN (4.2 ml) and
Sc(OTf) (22 mg, 44 mol) for 1.5 h. CC (SiO pentane/
acid
methyl
m
À1
1
41
1
179, 1092, 1075, 971, 918, 772, 754, 698, 603, 567 cm
NMR (500 MHz, C Cl , 90 °C): d = 1.14 (m, 3H, CH ), 1.75–1.98
m, 3H, CH –CH ), 2.04–2.17 (m, 1H, CH –CH ), 3.39–3.66 (m,
H, N–CH , O–CH ), 5.18 (dd, J = 22.7/12.8 Hz, 2H, CH –Ar), 5.30
s, 1H, CH–O), 7.31–7.42 (m, 5H, Har) ppm. 13C NMR (500 MHz,
CDCl , 23.3 °C, TMS): d = 15.27 (CH ), 15.36 (CH ), 21.78 (N–CH
CH ), 22.72 (N–CH –CH ), 32.35 (CH–CH ), 32.89 (CH–CH ), 45.73
N–CH ), 45.90 (N–CH ), 63.15 (O–CH –CH ), 63.82 (O–CH –CH ),
6.85 (CH –Ar), 67.08 (CH –Ar), 87.09 (CH), 87.73 (CH), 127.84
CHar), 128.00 (CHar), 128.05 (CHar), 128.47 (CHar), 136.55 (Car),
. H
D
2
4
3
3
l
2
,
2
(
4
(
2
2
2
2
EtOAc = 95:5) afforded 99 mg (77%) of [rac-(l)-29]/[rac-(u)-29] in
a diastereomeric ratio of 54/46 ( H NMR) as colourless oil.
1
2
2
2
IR (KBr):
1103, 990, 920, 769, 752, 698, 605 cm . H NMR (500 MHz, CDCl
24.8 °C, TMS): d = 0.79–0.95 (m, 3H, CH –CH ), 1.30–2.01 (m, 6H,
CH –CH , N–CH–CH -CH
m
~ = 2965, 2878, 1732, 1702, 1498, 1410, 1359, 1200,
À1
1
3
3
3
2
–
3
,
2
2
2
2
2
2
3
(
6
(
2
2
2
3
2
3
2
3
2
2
), 2.70–2.77 (m, 0.46 Â 0.39 Â 1H, CH–
2
2
COO), 2.79–2.87 (m, 0.54 Â 0.42 Â 1H, CH–COO), 2.89–2.97 (m,
0.54 Â 0.58 Â 1H, CH–COO), 2.97–3.03 (m, 0.46 Â 0.61 Â 1H, CH–
1
CH
36.72 (Car), 154.87 (O–CO–N), 155.76 (O–CO–N) ppm. MS (CI,
2 3
COO), 3.22–3.39 (m, 1H, N–CH ), 3.46–3.68 (m, 4H, O–CH , N–
+
+
5
); m/z (%) = 250 (5) [M+H] , 204 (100), 160 (54), 114 (25). Anal.
(249.31): C, 67.45; H, 7.68; N, 5.62. Found: C,
7.03; H, 7.64; N, 5.54.
The spectral data for the compound (RS)-21 were not given in
CH
2
), 4.03–4.11 (m, 0.46 Â 1H, N–CH), 4.15–4.21 (m, 0.54 Â 1H,
calcd for C14
6
H
19NO
3
N–CH), 5.07–5.22 (m, 2H, CH
2
–Ar), 7.28–7.42 (m, 5H, Har) ppm.
Rotamer ratios of the individual diastereomer from [rac-(l)-29]
and [rac-(u)-29] at 24.8 °C were 58:42 und 61:39, respectively.
3
7
13
the literature.
C NMR (500 MHz, CDCl
–CH ), 19.30 (CH
), 23.48 (CH ), 23.82 (CH
), 28.14 (CH ), 46.54 (N–CH
), 49.08 (CH–COO), 49.77 (CH–COO), 50.13 (CH–
COO), 50.52 (CH–COO), 51.49 (O–CH ), 51.52 (O–CH ), 58.56 (N–
CH), 58.89 (N–CH), 59.22 (N–CH), 59.65 (N–CH), 66.60 (CH –Ar),
66.65 (CH –Ar), 66.85 (CH –Ar), 67.02 (CH –Ar), 127.73 (CHar),
3
, 25.8 °C, TMS): d = 12.20 (CH
), 19.62 (CH ), 22.66 (CH ), 23.00
), 24.13 (CH ), 27.00 (CH ), 27.44
), 46.79 (N–CH ), 46.99 (N–CH ),
2 3
–CH ),
1
2.59 (CH
2
3
2
2
2
4
.1.2. General procedure for the
a-amidoalkylation of the
(CH
(CH
2
2
2
2
2
35
precursor (RS)-21 (GP1):
The reported procedure for the
in that the given amount of the precursor (RS)-21 and 1.7–
equiv of the respective silylketene acetal (22–27)39–45 dissolved
in MeCN (c = 1.0 M) was stirred with Sc(OTf) (10 mol %) at 0 °C.
After the given time, the reaction was quenched with saturated
aqueous NaHCO solution. The aqueous layer was extracted several
times with CH Cl , dried over MgSO and evaporated to dryness in
vacuum. The residue was then purified by CC.
2
2
2
2
2
a
-amidoalkylation was modified
47.45 (N–CH
2
37
3
3
2
2
3
2
2
2
127.83 (CHar), 127.90 (CHar), 128.04 (CHar), 128.45 (CHar), 136.76
(Car), 137.01 (Car), 137.12 (Car), 154.93 (N–CO–O), 155.18 (N–CO–
O), 174.08 (COO), 174.35 (COO), 174.73 (COO) ppm. MS (CI, CH
3
+
2
2
4
5
);
+
m/z (%) = 306 (100) [M+H] , 274 (27), 262 (30), 204 (19), 198
22), 170 (20), 160 (24), 128 (7). HRMS (70 eV): calcd for
(
+
4
.1.2.1. (2RS)-2-{(2RS)-1-[(Benzyloxy)carbonyl]pyrrolidine-2-yl}
17
C H23NO
4
[M] , 305.1627; found: 305.1622.
46
propionic acid methyl ester [rac-(l)-28] and (2RS)-2-{(2RS)-1-
(benzyloxy)carbonyl]-pyrrolidine-2-yl}propionic acid methyl
ester [rac-(u)-28]. According to GP1, (RS)-21 (705 mg,
.83 mmol), (E/Z)-223 (776 mg, 4.84 mmol), MeCN (28.3 ml) and
Sc(OTf) (139 mg, 0.28 mmol) for 5.5 h. CC (SiO , isohexane/
[
4.1.2.3.
2-{(2RS)-1-[(Benzyloxy)carbonyl]pyrrolidine-2-yl]-2-
5
7
methylpropionic acid methyl ester [(RS)-30]
.
According
to GP1, (RS)-21 (52 mg, 0.21 mmol), 24 (64 mg, 0.37 mmol),
MeCN (5.6 ml) and Sc(OTf) (11 mg, 21 mol) for 1 h 40 min. CC
(SiO , isohexane/EtOAc = 80:20) afforded 55 mg (87%) of [(RS)-30]
7
42
2
3
2
3
l
EtOAc = 85:15) afforded 603 mg (73%) of [rac-(l)-28]/[rac-(u)-28]
in a diastereomeric ratio of 55:45 ( H NMR) as colourless oil.
2
1
as colourless crystals.
IR (KBr):
262, 1206, 1103, 770, 698, 668 cm
5.6 °C, TMS): d = 1.00–1.18 (m, 3H, CH
–CH –CH
), 2.79–2.89 (m, 0.16 Â 1H, CH–CH
.48 Â 1H, CH–CH ), 3.25–3.41 (m, 1.36H, N–CH
.69 (m, 4H, O–CH N–CH ), 4.08 (ddd, J = 7.9/5.8/4.3 Hz,
.45 Â 1H, N–CH), 4.27 (dt, J = 8.2/4.9 Hz, 0.55 Â 1H, N–CH),
.08–5.20 (m, 2H, CH –Ar), 7.28–7.40 (m, 5H, Har) ppm. Rotamers
m
~ = 2952, 2881, 1734, 1702, 1456, 1412, 1360, 1339,
Mp: 55 °C. IR (KBr): m
~ = 2976, 2950, 1731, 1702, 1455, 1404,
À1
1
À1 1
1
2
CH
0
3
0
5
.
H NMR (500 MHz, CDCl
), 1.73–2.03 (m, 4H, N–
), 3.07–3.18 (m,
, CH–CH ), 3.48–
3
,
1349, 1266, 1193, 1134, 987, 699 cm . H NMR (400 MHz, CDCl
23.4 °C, TMS): d = 1.15 (s, 3H, CH ), 1.18 (s, 3H, CH ), 1.67–1.88 (m,
3H, N–CH –CH , CH –CH), 1.91–2.04 (m, 1H, CH –CH), 3.22–3.30
(m, 1H, N–CH ), 3.54–3.88 (m, 4H, O–CH , N–CH ), 4.31–4.36 (m,
1H, N–CH), 5.11 (br s, 2H, CH –Ar), 7.28–7.39 (m, 5H, Har) ppm.
3
,
3
3
3
2
2
2
3
2
2
2
2
3
2
3
2
3
2
3
,
2
2
1
3
C NMR (400 MHz, CDCl
21.98 (C–CH ), 22.37 (C–CH
CH –CH ), 27.29 (N–CH–CH ), 28.18 (N–CH–CH
47.88 (C–COO), 51.91 (O–CH
66.93 (CH –Ar), 127.84 (CHar), 127.93 (CHar), 128.45 (CHar),
136.92 (Car), 156.27 (O–CO–N), 177.26 (COO) ppm. MS (CI, CH
3
, 21.3 °C, TMS): d = 21.45 (C–CH
), 23.69 (N–CH –CH
), 47.39 (N–CH
3
),
2
3
3
2
2
), 24.34 (N–
13
existed but the ratio could not be determined. C NMR (500 MHz,
CDCl , 25.2 °C, TMS): d = 10.39 (CH–CH ), 10.50 (CH–CH ), 13.99
CH–CH ), 14.12 (CH–CH ), 22.98 (CH ), 23.68 (CH ), 23.80 (CH
4.27 (CH ), 26.84 (CH ), 27.42 (CH ), 28.80 (CH ), 29.72 (CH
0.90 (CH–CH ), 41.39 (CH–CH ), 41.93 (CH–CH
), 46.74 (N–CH ), 47.00 (N–CH ), 47.20 (N–CH
), 51.66 (O–CH ), 58.32 (N–CH), 58.99 (N–CH), 59.63 (N–CH),
–Ar), 66.93 (CH –Ar), 127.74 (CHar),
2
2
2
2
2
),
3
3
3
3
), 62.94 (N–CH), 63.60 (N–CH),
(
3
3
2
2
2
2
),
),
2
+
2
4
2
2
2
2
5
);
m/z (%) = 306 (100) [M+H] , 274 (60), 262 (22), 204 (16), 198
(19), 170 (9), 160 (18). Anal. calcd for C17 (305.38): C,
+
3
3
3
), 42.49 (CH–
), 47.67 (N–
CH
CH
3
2
2
2
H23NO
4
2
3
66.86; H, 7.59; N 4.59. Found: C, 66.85; H, 7.58; N, 4.67.
6
1
C
0.43 (N–CH), 66.64 (CH
2
2
The spectral data for the compound [(RS)-30] were not given in
5
7
27.81 (CHar), 127.93 (CHar), 128.46 (CHar), 128.48 (CHar), 136.78
ar), 136.99 (Car), 137.13 (Car), 154.86 (O–CO–N), 155.23 (O–CO–
the literature.
(
N), 174.66 (COO), 174.91 (COO), 175.16 (COO), 175.21 (COO)
4.1.2.4. 1-{(2RS)-1-[(Benzyloxy)carbonyl]pyrrolidine-2-yl}cyclo-
butane-1-carboxylic acid methyl ester [(RS)-31]. According
to GP1, (RS)-21 (55 mg, 0.22 mmol), 25 (71 mg, 0.38 mmol), MeCN
(2.2 ml) and Sc(OTf) (11 mg, 21 mol) for 4 h. CC (SiO , isohexane/
EtOAc = 80:20) afforded 51 mg (73%) of [(RS)-31] as colourless oil.
IR (KBr):
~ = 2950, 2891, 1727, 1702, 1498, 1447, 1405, 1354,
1280, 1243, 1211, 1154, 1114, 1029, 968, 918, 770, 753, 698,
ppm. MS (CI, CH+
); m/z (%) = 292 (100) [M+H] , 260 (18), 248
27), 204 (14), 184 (20), 160 (17), 156 (21). HRMS (70 eV): calcd
+
5
(
+
for C16
H21NO
4
[M] , 291.1471; found: 291.1470.
3
l
2
1
The analytical data of the rac-(l)-diastereomers ( H NMR) were
in accord with the values reported in the literature for a pure like
m
4
6
enantiomer.
Please cite this article in press as: Steffan, T.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.01.035

T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
13
À1
6
(
CH
CH
06 cm . 1H NMR (500 MHz, CDCl
m, 6H, C –CH –CH , N–CH –CH –CH
), 3.27–3.35 (m, 1H, N–CH ), 3.56–3.78 (m, 4H, O–CH
), 4.22–4.46 (m, 0.42 Â 1H, N–CH), 4.35–4.46 (m, 0.58 Â 1H,
–Ar), 7.28–7.40 (m, 5H, Har) ppm.
3
, 24.7 °C, TMS): d = 1.57–2.00
), 2.05–2.42 (m, 4H, C
, N–
C–CH
2.16 (m, 2H, CH
CH
3
), 0.91 (s, 0.55 Â 9H, C–CH
3
), 1.65–1.84 (m, 2H, CH
2
), 1.93–
q
2
2
2
2
2
q
–
2
), 3.30–3.38 (m, 1H, –CH
2
), 3.44–3.57 (m, 1H, N–
),
2
2
3
2
), 3.60 (s, 0.45 Â 3H, O–CH
3
), 3.67 (s, 0.55 Â 3H, O–CH
3
2
4.03–4.07 (m, 0.45 Â 1H, N–CH), 4.14–4.19 (m, 0.55 Â 1H, N–CH),
4.47 (d, J = 4.6 Hz, 0.45 Â 1H, CH–O), 4.64 (d, J = 4.1 Hz,
N–CH), 5.06–5.24 (m, 2H, CH
2
13
Rotamer ratio (24.7 °C) 58:42. C NMR (500 MHz, CDCl
TMS): d = 16.42 (CH ), 23.40 (N–CH –CH ), 24.06 (N–CH
7.70 (CH ), 28.58 (CH ), 29.22 (CH ), 29.48 (CH ), 47.73 (N–
CH ), 48.13 (N–CH ), 53.60 (C–COO), 53.78 (C–COO),
–Ar), 127.82 (CHar),
3
, 25.6 °C,
0.55 Â 1H, CH–O), 5.11–5.18 (m, 2H, CH
2
–Ar), 7.27–7.40 (m, 5H,
1
3
2
2
2
2
–CH ),
2
3
Har) ppm. Rotamers ratio (24 °C) 55:45. C NMR (500 MHz, CDCl ,
2
2
2
2
2
26 °C): d = À5.15 (Si–CH
(C–CH ), 18.53 (C–CH ), 23.16 (CH
26.06 (C–CH ), 27.05 (CH ), 27.51 (CH
(N–CH ), 60.59 (N–CH), 61.24 (N–CH), 66.93 (CH
(CH –Ar), 72.05 (CH–O), 72.19 (CH–O), 127.91 (CHar), 128.15
3
), À4.93 (Si–CH
3
), À4.77 (Si–CH
3
), 18.46
2
2
), 51.99 (CH
3
3
3
2
), 24.09 (CH
2
3
), 25.95 (C–CH ),
6
1
1.69 (N–CH), 62.23 (N–CH), 66.92 (CH
2
3
2
2 2
), 47.41 (N–CH ), 47.78
27.93 (CHar), 128.14 (CHar), 128.20 (CHar), 128.44 (CHar), 136.61
ar), 136.99 (Car), 155.95 (O–CO–N), 156.43 (O–CO–N), 176.72
COO) ppm. MS (CI, CH
2
2
–Ar), 67.51
(
(
C
2
+
+
5
); m/z (%) = 318 [M+H] (100), 286 (50),
(CHar), 128.42 (CHar), 128.68 (CHar), 128.77 (CHar), 128.84 (CHar),
2
74 (23), 225 (8), 210 (20), 204 (14), 182 (11), 160 (16), 105
136.94 (Car), 137.42 (Car), 155.08 (O–CO–N), 155.39 (O–CO–N),
+
+
(
13). MS (EI, 70 eV); m/z (%) = 317 (0) [M] , 204 (17), 182 (5), 160
172.87 (COO), 173.02 (COO) ppm. MS (CI, CH
5
); m/z (%) = 408
+
+
(
28), 91 (100), 65 (9). HRMS (70 eV): calcd for C18
17.1627; found: 317.1617. Anal. calcd for C18 23NO
C, 68.12; H, 7.30; N, 4.41. Found: C, 68.37; H, 6.85; N, 4.43.
H23NO
4
[M] ,
(100) [M+H] , 392 (5), 364 (12), 350 (9), 300 (15), 204 (21), 160
+
3
H
4
(317.39):
(19). HRMS (70 eV): calcd for C21
H33NO
5
Si [M] , 407.2128; found:
407.2140. Anal. calcd for C21 33NO
H
5
Si (407.59): C, 61.88; H, 8.16;
N, 3.44. Found: C, 61.84; H, 8.21; N 3.42.
4
.1.2.5. (2RS)-2-[(tert-Butyldimethylsilanyl)oxy]-2-{(2RS)-1-(be-
nzyloxy)carbonyl]pyrrolidine-2-yl}acetic acid methyl ester
4.1.2.6. (2RS)-2-Amino-2-{(2RS)-1-[(benzyloxy)carbonyl]pyrrol-
idine-2-yl}acetic acid methyl ester [rac-(l)-33] und (2SR)-
4
9
50
[
2
rac-(l)-32]
and (2SR)-2-[(tert-butyldimethyl-silanyl)oxy]-
-{(2RS)-1-(benzyloxy)carbonyl]pyrrolidine-2-yl}-acetic acid
According to GP1, (RS)-21 (1.58 g,
.33 mmol), (E/Z)-26 (4.88 g, 12.7 mmol), MeCN (63 ml) and
Sc(OTf) (0.62 g, 1.26 mmol) for 19 h. CC (SiO isohexane/
amino-2-{(2RS)-1-[(benzyloxy)carbonyl]pyrrolidine-2-yl}acetic
5
0
methyl ester [rac-(u)-32].
6
acid methyl ester [rac-(u)-33]
.
According to GP1, (RS)-21
(830 mg, 3.33 mmol), (E)-27 (2.01 g, 6.6 mmol), MeCN (33 ml)
and Sc(OTf) (163 mg, 0.332 mmol) for 19 h. Purification and sepa-
ration of diastereoisomers through multiple CC (SiO , pentane/
4
4
45
3
2
,
3
4
9
EtOAc = 90:10) afforded 1.72 g (67%) of [rac-(l)-32] /[rac-(u)-32]
in a diastereomeric ratio of 45/55 ( H NMR).
2
1
EtOAc/EDMA = 50:50:2) afforded 217 mg (22%) of rac-(l)-33 and
310 mg (32%) of rac-(u)-33. The analytical data of both the diaste-
reoisomers were in accord with the literature values.50
In order to obtain the analytical data, the experiment was
repeated and the crude product was subjected to repeated CC
(
SiO
2
,
isohexane/EtOAc = 70:30 followed by SiO
2
,
pentane/
EtOAc = 95:5). This afforded 11% of [rac-(l)-38]/[rac-(u)-38], 15%
of [rac-(l)-32] as colourless oil and 12% of [rac-(u)-32] also as col-
4.1.3. (2RS)-2-Hydroxy-2-{(2RS)-1-[(benzyloxy)carbonyl]pyrrol-
idine-2-yl}acetic acid methyl ester [rac-(l)-38] and (2SR)-Hydroxy-
4
9
50
ourless oil.
2-{(2RS)-1-[(benzyloxy)carbonyl]pyrrolidine-2-yl}acetic acid methyl
ester [rac-(u)-38]
4
9
[rac-(l)-32]: IR (KBr):
m
~ = 2954, 2885, 2857, 1756, 1734, 1702,
According to GP1, (RS)-21 (1.86 g, 7.45 mmol), (E/Z)-2643
1
6
0
0
413, 1360, 1257, 1201, 1145, 1114, 1060, 1004, 913, 837, 779,
À1
1
97, 605 cm
.35 Â 3H, Si–CH
.35 Â 3H, Si–CH ), 0.02 (s, 0.65 Â 3H, Si–CH
), 0.87 (s, 0.65 Â 9H, C–CH ), 1.88–1.96 (m, 2H, CH
.07 (m, 2H, CH ), 3.35–3.42 (m, 1H, N–CH ), 3.46–3.58 (m, 1H,
N–CH ), 3.71 (s, 0.65 Â 3H, O–CH ),
), 3.70 (s, 0.35 Â 3H, O–CH
.17–4.24 (m, 1H, N–CH), 4.66 (d, J = 2.7 Hz, 0.35 Â 1H, CH–O),
.93 (d, J = 2.7 Hz, 0.65 Â 1H, CH–O), 5.08–5.24 (m, 2H, CH –Ar),
.28–7.43 (m, 5H, Har) ppm. Rotamer ratio (22.1 °C) 65:35.
, 25.4 °C): d = À5.24 (Si–CH ), À5.20 (Si–
), À4.99 (Si–CH ), 18.46 (C–CH ), 24.19 (CH ),
), 25.71 (CH ), 25.93 (C–CH ), 26.00 (C–CH ), 26.70
), 47.58 (N–CH ), 48.08 (N–CH ), 52.09 (O–CH ), 52.15 (O
), 60.16 (N–CH), 60.72 (N–CH), 67.04 (CH –Ar), 67.14 (CH
.
H NMR (500 MHz, CDCl
), À0.07 (s, 0.65 Â 3H, Si–CH
), 0.85 (s, 0.35 Â 9H,
), 1.93–
3
, 22.1 °C): d = À0.14 (s,
(4.12 g, 14.9 mmol), MeCN (75 ml) and Sc(OTf)
3
(0.37 g,
0.75 mmol) for 19 h. After the usual work up, a solution of tetrabu-
tylammonium fluoride trihydrate (5.92 g, 18.8 mmol) in THF
(75 ml) was added to the product [rac-(l)-32]/[rac-(u)-32] and stir-
red at rt for 1 h. The reaction was quenched by adding 200 ml of
3
3
), À0.06 (s,
3
3
C–CH
2
3
3
2
2
2
CH
2 2
Cl
and washed with NaOH (100 ml, 2 M). The organic phase
and concentrated to dryness. CC (SiO , iso-
2
3
3
4
4
7
was dried over MgSO
4
2
hexane/EtOAc = 60:40) of the crude product afforded 2.07 g (95%)
2
1
3
C
of the diastereomeric mixture [rac-(l)-38]/[rac-(u)-38] with diaste-
1
NMR (500 MHz, CDCl
CH
), À5.14 (Si–CH
4.85 (CH
CH
3
3
reomeric ratio being 39:61 ( H NMR). Separation of the diastereo-
2
meric mixture by repeated CC (SiO , isohexane/EtOAc = 70:30)
3
3
3
3
2
5
0
2
(
2
2
3
3
afforded 784 mg (36%) of [rac-(l)-38] as colourless oil, the analyt-
ical data of which were in accord with the reported values in the
2
2
2
3
5
0
CH
3
2
2
–
literature and 592 mg (27%) of [rac-(u)-38] also a colourless oil.
[rac-(u)-38]: IR (KBr):
~ = 3443, 3032, 2953, 2887, 1741, 1697,
1414, 1358, 1274, 1211, 1108, 1010, 766, 698 cm
(500 MHz, CDCl , 23.4 °C, TMS): d = 1.77–2.16 (m, 4H, N–CH
CH –CH
), 2.89 (br s, 0.23 Â 1H, OH), 3.46–3.64 (m, 1.77H, N–
CH , OH), 3.67 (m, 3H, O–CH ), 4.16–4.30 (m, 2H, N–CH, CH–O),
4.95–5.28 (m, 2H, CH –Ar), 7.27–7.39 (m, 5H, Har) ppm. Rotamers
Ar), 71.75 (CH–O), 73.23 (CH–O), 128.27 (CHar), 128.37 (CHar),
m
À1
1
1
28.44 (CHar), 128.77 (CHar), 128.84 (CHar), 137.07 (Car), 137.24
.
H NMR
(
(
C
ar), 154.77 (O–CO–N), 154.96 (O–CO–N), 172.84 (COO), 173.04
3
2
–
+
COO) ppm. MS (CI, CH
5
); m/z (%) = 408 (100) [M+H]+, 392 (8),
2
2
3
64 (5), 350 (18), 300 (13), 204 (32), 160 (29). HRMS (70 eV): calcd
2
3
+
for C21
H33NO
5
Si [M] , 407.2128; found: 407.2167. Anal. calcd for
2
1
3
C
21
5
H33NO Si (407.59): C, 61.88; H, 8.16; N 3.44. Found: C, 62.16;
ratio (23.4 °C) 77:23. C NMR (400 MHz, CDCl
(N–CH –CH ), 24.14 (N–CH –CH ), 28.86 (N–CH–CH
CH–CH ), 47.61 (N–CH ), 48.06 (N–CH ), 52.42 (O–CH
(O–CH ), 59.29 (N–CH), 60.30 (N–CH), 67.18 (CH –Ar), 72.58
3
, 20.7 °C): d = 23.19
), 29.69 (N–
), 52.73
H, 8.20; N 3.52.
2
2
2
2
2
The spectral data for the compound [rac-(l)-32] were not given
2
2
2
3
4
9
in the literature.
rac-(u)-32]: IR (KBr):
412, 1360, 1339, 1253, 1207, 1114, 1006, 986, 837, 778, 697,
3
2
[
m
~ = 2952, 2930, 2886, 2857, 1752, 1705,
(CH–OH), 73.74 (CH–OH), 127.89 (CHar), 128.09 (CHar), 128.56
1
6
0
0
(CHar), 136.64 (Car), 154.97 (O–CO–N), 156.40 (O–CO–N), 173.68
À1
1
+
04 cm
.45 Â 3H, Si–CH
.55 Â 3H, Si–CH ), 0.08 (s, 0.55 Â 3H, Si–CH
.
H
NMR (500 MHz, CDCl
), À0.05 (s, 0.45 Â 3H, Si–CH
), 0.84 (s, 0.45 Â 9H,
3
,
24 °C): d = À0.10 (s,
(COO), 173.91 (COO) ppm. MS (CI, CH
5
); m/z (%) = 294 (93)
+
[M+H] , 250 (79), 204 (11), 186 (100), 160 (16), 158 (10). HRMS
(DEI+): calcd for C15H19NO [M] , 293.1263; found: 293.1249.
5
3
3
), 0.08 (s,
+
3
3
Please cite this article in press as: Steffan, T.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.01.035

1
4
T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
4
.1.4. General procedure for the preparation of Cbz-protected
given pressure and duration, after which it was filtered several
times and the solvent evaporated to dryness in vacuum.
primary and secondary amines (GP2)
3
The given amount of amine together with NaHCO (2.5 equiv)
was dissolved in THF/H
2
O-mixture (1:1, c = 0.1 M) and cooled to
4
.1.5.1. (2SR)-2-Amino-2-[(2RS)-pyrrolidine-2-yl]acetic acid
According to GP3
method C), rac-(u)-40 (152 mg, 0.52 mmol), Pd/C (16.6 mg), HOAc
68.5 mg, 1.14 mmol, 65.5 l), MeOH (10.4 ml) and the reaction
0
°C. Benzyl chloroformate (1.1 equiv) was then added dropwise
methyl esterÁ2HOAc [rac-(u)-42Á2HOAc].
to the cooled solution. After the described reaction time, the mix-
ture was extracted several times with a suitable organic solvent
after which the organic phase was dried over MgSO
to dryness in vacuum followed by CC.
(
(
l
4
, evaporated
time being 1 h. This afforded 104 mg (72%) of the acetic acid salt
of the free amine rac-(u)-42Á2HOAc as colourless oil.
~ = 3383, 2956, 1739, 1561, 1402, 1014, 651 cmÀ1. 1H
IR (KBr): m
4
.1.4.1. (2RS)-2-[(Benzyloxycarbonyl)amino]-2-{(2RS)-1-[(ben-
NMR (500 MHz, CDCl , 24.1 °C, TMS): d = 1.74–1.83 (m, 1H, N–CH–
3
zyloxy)carbonyl]-pyrrolidine-2-yl}acetic acid methyl ester
CH ), 1.87–2.08 (m, 9H, N–CH–CH , N–CH –CH , CH –COO), 3.16 (t,
2
2
2
2
3
[
rac-(l)-40].
According to GP2, rac-(l)-33 (51 mg, 0.17 mmol),
2 2
J = 7.1 Hz, 2H, N–CH ), 3.56–3.62 (m, 2H, N–CH–CH , CH–COO),
1
3
NaHCO
3
(37 mg, 0.43 mmol), benzyl chloroformate (32 mg,
3.76 (s, 3H, O–CH ), 6.00 (br s, 5H, NH) ppm. C NMR (500 MHz,
3
0
.19 mmol, 33
being 2.5 h. The extraction with CH
was done after acidifying the reaction mixture to pH = 1 with
M HCl. CC (SiO , isohexane/EtOAc = 75:25) of the crude product
afforded 71 mg (96%) of rac-(l)-40 as colourless oil.
IR (KBr):
~ = 3367, 3032, 2953, 2888, 1725, 1702, 1528, 1454,
415, 1358, 1338, 1212, 1100, 1040, 917, 740, 698, 603 cm
NMR (400 MHz, C NO , 140 °C): d = 1.76–2.04 (m, 2H, N–CH
CH ), 2.05–2.16 (m, 2H, N–CH–CH ), 3.37–3.45 (m, 1H, N–CH
.62–3.70 (m, 1H, N–CH ), 3.72 (s, 3H, O–CH
N–CH–CH ), 4.98 (dd, J = 8.8/3.5 Hz, 1H, CH–COO), 5.13–5.30 (m,
–Ar), 6.13 (br s, 1H, NH), 7.23–7.46 (m, 10H, Har) ppm.
C NMR (400 MHz, MeOD, 20.3 °C): d = 24.38 (CH ), 25.03 (CH ),
8.41 (CH ), 28.69 (CH ), 48.17 (N–CH ), 48.56 (N–CH ), 53.11
), 53.18 (O–CH ), 57.21 (CH–COO), 57.33 (CH–COO), 60.04
N–CH–CH ), 60.74 (N–CH–CH ), 67.96 (CH –Ar), 68.28 (CH –Ar),
–Ar), 129.15 (CHar), 129.18 (CHar), 129.31 (CHar),
29.75 (CHar), 129.79 (CHar), 138.11 (Car), 138.48 (Car), 156.69
l
l), THF (0.9 ml), H
2
O (0.9 ml) and the reaction time
CDCl , 26.5 °C, TMS): d = 22.81 (CH –COO), 24.71 (N–CH –CH ),
28.38 (N–CH–CH ), 45.12 (N–CH ), 52.44 (O–CH ), 56.27 (CH–
2 2 3
3
3
2
2
2
Cl
2
(3 Â 10 ml) and work up
COO), 60.79 (N–CH–CH ), 173.56 (CH–COO), 177.16 (CH -COO)
2
3
+
2
2
ppm. MS (ESI); m/z (%) = 159 (100) [M+H] , 142 (46). HRMS (ESI):
calcd for C H N O [M+H] , 159.1134; found: 159.1130.
+
7
15
2 2
m
À1
1
1
. H
4
.1.5.2. (2RS)-2-Hydroxy-2-[(2RS)-pyrrolidine-2-yl]acetic acid
methyl esterÁHOAc [rac-(l)-45ÁHOAc]. According to GP3 with
rac-(l)-38 (40 mg, 0.14 mmol) in MeOH (2.8 ml), Pd/C (8 mg) and
HOAc (9.0 l, 0.15 mmol) at rt for 1 h afforded 26.8 mg (89%) of
rac-(l)-45ÁHOAc as colourless crystals.
Mp: 81 °C. IR (KBr):
~ = 3397, 2973, 2769, 1749, 1566, 1407,
297, 1233, 1133, 1085, 1023, 930, 653 cm
D
6 5
2
2
–
),
2
2
2
3
2
3
), 4.35–4.41 (m, 1H,
l
2
4
H, CH
2
m
1
3
À1 1
2
2
1
CDCl
CH
(
.
H NMR (400 MHz,
, 17.6 °C, TMS): d = 1.83–2.09 (m, 7H, N–CH –CH –CH
), 3.78 (s, 3H, O–CH ), 3.89
td, J = 7.7/2.9 Hz, 1H, N–CH), 4.69 (d, J = 2.9 Hz, 1H, CH–OH)
2
2
2
2
2
3
2
2
2
,
(
O–CH
3
3
3
–COO), 3.16–3.20 (m, 2H, N–CH
2
3
(
6
1
2
2
2
2
8.80 (CH
2
13
ppm. C NMR (500 MHz, MeOD, 25.4 °C, TMS): d = 23.80 (CH
3
–
3
),
3
–
COO), 25.24 (CH
6
COO) ppm. MS (CI, CH
2
), 25.64 (CH
2
), 47.38 (N–CH
2
), 53.01 (O–CH
(
O–CO–N), 157.08 (O–CO–N), 158.78 (O–CO–N), 158.92 (O–CO–
1.99 (N–CH), 70.09 (CH–OH), 173.10 (CH–COO), 179.96 (CH
+
N), 172.50 (COO), 172.77 (COO) ppm. MS (CI, CH
5
); m/z (%) = 427
100) [M+H] , 383 (60), 319 (32), 275 (44), 204 (28), 185 (29),
60 (15). Anal. calcd for C23 (426.47): C, 64.78; H, 6.15;
N, 6.57. Found: C, 64.83; H, 6.23; N, 6.49.
+
+
5
); m/z (%) = 160 (100) [M+H] , 145 (11).
+
(
1
+
HRMS (FAB, NBA): calcd for C
7 3
H14NO [M+H] , 160.0974; found:
26 2 6
H N O
1
60.0980.
4
.1.5.3. (2SR)-2-Hydroxy-2-[(2RS)-pyrrolidine-2-yl]acetic acid
According to GP3
with rac-(u)-38 (101 mg, 0.34 mmol) in MeOH (5 ml), Pd/C
14 mg) and HOAc (22 l, 0.38 mmol) at rt for 1 h to afford
2.3 mg (96%) of rac-(u)-45ÁHOAc as colourless crystals.
Mp: 83 °C. IR (KBr):
~ = 2961, 1741, 1637, 1560, 1406, 1228,
4
.1.4.2. (2SR)-2-[(Benzyloxycarbonyl)amino]-2-{(2RS)-1-[(ben-
zyloxy)carbonyl]-pyrrolidine-2-yl}acetic acid methyl ester
rac-(u)-40]. According to GP2, rac-(u)-33 (151 mg,
.35 mmol), NaHCO (75 mg, 1.04 mmol), benzyl chloroformate
67 mg, 0.39 mmol, 67 l), THF (1.8 ml), H O (1.8 ml) and the reac-
tion time being 2 h. The extraction with CH Cl
(3 Â 10 ml) and
work up was done after acidifying the reaction mixture to pH = 1
with 2 M HCl. CC (SiO , isohexane/EtOAc = 70:30) of the crude
product afforded 191 mg (87%) of rac-(u)-40 as colourless oil.
IR (KBr):
~ = 3420, 3032, 2953, 2888, 1704, 1519, 1454, 1413,
358, 1337, 1212, 1102, 1059, 916, 740, 698, 604 cm
400 MHz, C NO , 140 °C): d = 1.79–1.99 (m, 2H, N–CH
.04–2.12 (m, 2H, N–CH–CH ), 3.39–3.47 (m, 1H, N–CH ), 3.59–
.67 (m, 1H, N–CH ), 3.74 (s, 3H, O–CH ), 4.44–4.50 (m, 1H, N–
methyl esterÁHOAc [rac-(u)-45ÁHOAc].
[
0
(
(
7
l
3
l
2
m
1
À1
2
2
1
126, 1085, 652 cm
–COO), 1.91–2.03 (m, 2H, N–CH
.03–2.11 (m, 1H, N–CH –CH ), 2.13–2.20 (m, 1H, N–CH–CH
.23–3.27 (m, 2H, N–CH ), 3.79 (s, 3H, O–CH
.7/5.2 Hz, 1H, N–CH), 4.35 (d, J = 5.2 Hz, 1H, CH–OH) ppm.
.
H NMR (500 MHz, MeOD, 24.3 °C):
d = 1.90 (CH
3
2
–CH
2
, N–CH–CH
2
),
2
2
3
7
2
2
2
),
2
3
), 3.83 (ddd, J = 8.8/
m
13
C
À1
1
1
(
2
3
.
H NMR
–CH ),
NMR (500 MHz, MeOD, 20.1 °C): d = 23.93 (CH
CH ), 28.47 (CH ), 47.18 (N–CH ), 53.30 (O–CH
1.11 (CH–OH), 173.44 (COOCH ), 179.95 (CH
3
–COO), 25.33
6
D
5
2
2
2
(
7
2
2
2
3
), 62.88 (N–CH),
–COO) ppm. MS
5
); m/z (%) = 160 (100) [M+H] . HRMS (FAB, NBA): calcd
2
2
3
3
+
+
2
3
(
CI, CH
CH), 4.67 (dd, J = 8.1/6.2 Hz, 1H, CH–COO), 5.16–5.27 (m, 4H,
CH –Ar), 5.94–6.06 (m, 1H, NH), 7.23–7.44 (m, 10H, Har) ppm.
MS (CI, CH
+
for C H14NO [M+H] , 160.0974; found: 160.0984.
7 3
2
+
+
5
); m/z (%) = 427 (100) [M+H] , 383 (69), 319 (24), 293
10), 275 (24), 204 (24), 185 (13). Anal. calcd for C23
426.47): C, 64.78; H, 6.15; N, 6.57. Found: C, 64.78; H, 6.25; N,
4
.1.5.4. (2RS)-2-[(tert-Butoxycarbonyl)amino]-2-[(2RS)-pyrroli-
(
(
26 2 6
H N O
dine-2-yl]acetic acid methyl esterÁHOAc [rac-(l)-50ÁHOAc]. Accord-
ing to GP3 with rac-(l)-49 (48 mg, 0.12 mmol), Pd/C (11.4 mg),
6
.54.
HOAc (8
l
l, 0.14 mmol) and MeOH (5 ml) at rt for 1 h afforded
8 mg (99%) of rac-(l)-50ÁHOAc as colourless crystals.
Mp: 98 °C (decomp.). IR (KBr):
~ = 3378, 2978, 1742, 1713, 1393,
367, 1290, 1252, 1168, 1020, 870 cm . H NMR (500 MHz, CDCl
3.7 °C, TMS): d = 1.45 (s, 9H, C–CH ), 1.64–1.73 (m, 1H, N–CH–
), 1.80–1.93 (m, 3H, N–CH–CH , N–CH –CH ), 2.01 (s, 3H,
–COO), 2.95–3.03 (m, 1H, N–CH ), 3.03–3.12 (m, 1H, N–CH ),
3
4
.1.5. General procedure for the hydrogenolytic removal of Cbz-
m
protecting groups (GP3)
À1 1
1
2
CH
CH
3
,
The reactant was treated with 10% m/m of Pd/C in the given
amount of solvent (0.03–0.1 M) and when required acid was
added. The reaction mixture was hydrogenated at rt under the
3
2
2
2
2
3
2
2
Please cite this article in press as: Steffan, T.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.01.035

T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
15
3
5
.72–3.79 (m, 4H, N–CH–CH
.72 (d, J = 5.5 Hz, 1H, NH), 6.82 (br s, 2H, NH
, 27 °C, CDCl ): d = 22.47 (CH –COO), 25.28 (CH
), 28.23 (C–CH ), 46.25 (N–CH ), 52.50 (O–CH ), 56.17
), 80.38 (C–CH ), 156.03 (N–CO–O),
71.58 (CH–COO), 176.59 (CH –COO) ppm. MS (ESI); m/z
%) = 259 (70) [M+H] , 245 (23), 217 (13), 203 (100), 189 (33),
59 (28), 142 (28). HRMS (FAB, NBA): calcd for C12
2
, O–CH
3
), 4.48–4.54 (m, 1H, CH–COO),
62.41 (N–CH), 180.10 (COO), 181.20 (COO) ppm. MS (CI, CH+
5
); m/
+
13
+
2
) ppm. C NMR
),
z (%) = 158 (100) [M+H] . HRMS (FAB, NBA): calcd for C
[M+H] , 158.1181; found: 158.1199.
8
H16NO
2
+
(
500 MHz, CDCl
3
3
3
2
2
7.15 (CH
2
3
2
3
(CH–COO), 60.06 (N–CH–CH
2
3
4.1.5.8. 2-Methyl-2-[(2RS)-pyrrolidine-2-yl]propionic acid [(RS)-
11]. According to GP3 with carboxylic acid (RS)-36 (171 mg,
0.59 mmol), Pd/C (18 mg), MeOH (5.8 ml) for 4 h afforded 87 mg
1
(
1
3
+
23 2 4
H N O
(
95%) of (RS)-11 as colourless crystals.
Mp: 136 °C (decomp.). IR (KBr):
~ = 3440, 2970, 1618, 1471,
396, 1350, 1292, 1227, 1045, 941, 650, 576 cm
O, 24.7 °C): d = 1.16 (s, 3H, CH ), 1.28 (s, 3H,
–CH–N), 1.94–2.11 (m, 2H, CH –CH
–CH–N), 3.28–3.39 (m, 2H, CH –N),
+
[
M+H] , 259.1658; found: 259.1656.
m
À1
1
1
.
H
4
2
.1.5.5. (2SR)-2-[(tert-Butoxycarbonyl)amino]-2-[(2RS)-pyrrolidine-
-yl]acetic acid methyl esterÁHOAc [rac-(u)-50ÁHOAc]. According
NMR (500 MHz, D
CH ), 1.56–1.65 (m, 1H, CH
N), 2.19–2.26 (m, 1H, CH
2
3
3
2
2
2
2
–
to GP3 with rac-(u)-49 (194 mg, 0.49 mmol), Pd/C (21 mg), HOAc
2
(
(
31
l
l, 0.54 mmol) and MeOH (5 ml) at rt for 1 h afforded 153 mg
13
3
.46 (dd, J = 10.5/7.3 Hz, 1H, CH–N) ppm. C NMR (400 MHz,
97%) of rac-(u)-50ÁHOAc as colourless crystals.
D
(
2
O, 21.3 °C, Acetone): d = 23.26 (CH
CH ), 27.42 (N–CH–CH ), 44.07 (C
CH), 184.04 (COO) ppm. MS (CI, CH
3
), 24.21 (N–CH
2
–CH
), 68.02 (N–
2
), 25.58
Mp: 101 °C. IR (KBr):
m
1
~ = 3397, 2979, 1740, 1702, 1527, 1367,
3
2
q
), 45.81 (N–CH
2
À1
1
255, 1165, 1017 cm
.
H NMR (400 MHz, CDCl
–COO, N–CH
), 3.76 (s, 3H, O–CH ), 4.00 (td,
), 4.61 (dd, J = 9.4/3.5 Hz, 1H, CH–
3
, 20.7 °C, TMS):
+
5
); m/z (%) = 158 (100)
d = 1.46 (s, 9H, C–CH
CH ), 3.13–3.31 (m, 2H, N–CH
J = 8.8/3.5 Hz, 1H, N–CH–CH
COO), 7.12 (d, J = 9.4 Hz, 1H, NH), 9.16 (br s, 2H, NH
NMR (400 MHz, CDCl , 22.1 °C, CDCl ): d = 22.98 (CH
4.40 (CH ), 27.60 (CH ), 28.22 (C–CH ), 45.70 (N–CH ), 52.71
O–CH ), 54.10 (CH–COO), 59.32 (N–CH–CH ), 80.08 (C ), 156.45
O–CO–N), 170.76 (CH–COO), 177.42 (CH –COO) ppm. MS (ESI);
3
), 1.78–2.14 (m, 7H, CH
3
2
–CH
2
–
+
+
[
M+H] . MS(DEI+); m/z (%) = 158 (2) [M+H] , 113 (13), 110 (19),
2
2
3
9
6 (82), 84 (50), 81 (58), 70 (100), 68 (48), 56 (35), 43 (60), 41
2
(
[
8 2
67), 39 (25), 30 (53), 28 (54). HRMS (DEI+): calcd for C H16NO
M+H] , 158.1181; found: 158.1220.
+
13
) ppm.
C
+
2
3
3
3
–COO),
2
(
(
2
2
3
2
4
.1.5.9. 1-[(2RS)-Pyrrolidine-2-yl]cyclobutane-1-carboxylic acid
3
2
q
[(RS)-12]. According to GP3 with carboxylic acid (RS)-37
3
+
(502 mg, 1.65 mmol), Pd/C (51 mg), MeOH (16.5 ml) for 4 h affor-
ded 282 mg (100%) of (RS)-12 as colourless crystals.
m/z (%) = 259 (100) [M+H] , 245 (17), 203 (87), 189 (25), 159
+
(
23 2 4
71), 142 (20). HRMS (FAB, NBA): calcd for C12H N O [M+H] ,
Mp: 198 °C (decomp.). IR (KBr): m
~ = 3430, 2976, 2930, 2879,
196, 1960, 1878, 1560, 1398, 1364, 1351, 1280, 1246, 1156,
112, 1028, 1010, 995, 946, 856, 816, 802, 749, 688, 668, 630,
2
59.1658; found: 259.1684.
.1.5.6. (2RS)-2-[(2RS)-Pyrrolidine-2-yl]propionic acid [rac-(l)-9] and
2
1
4
À1
1
47
600 cm . H NMR (500 MHz, D
N–CH–CH ), 1.61–1.97 (m, 6H, N–CH
H, N–CH–CH , N–CH –CH ), 2.29–2.39 (m, 1H, CH
(m, 2H, N–CH ), 3.74–3.83 (m, 1H, N–CH) ppm.
400 MHz, D O, 21.1 °C, Acetone): d = 14.72 (CH ), 24.15 (CH
6.34 (N–CH–CH ), 27.19 (CH ), 30.08 (N–CH –CH ), 45.88
), 48.61 (C ), 65.26 (N–CH), 182.93 (COO) ppm. MS
2
O, 19.7 °C): d = 1.28–1.42 (m, 1H,
–CH , CH ), 1.99–2.14 (m,
), 3.07–3.20
(2SR)-2-[(2RS)-pyrrolidine-2-yl]propionic acid [rac-(u)-9] . Accord-
46
2
2
2
2
ing to GP3 with carboxylic acids [rac-(l)-34] /[rac-(u)-34] (419 mg,
.51 mmol), Pd/C (43 mg), MeOH (15 ml) for 4 h afforded 210 mg
2
2
2
2
2
1
(
13
97%) of [rac-(l)-9]/[rac-(u)-9]47 as yellow crystals.
2
C
NMR
),
(
2
2
2
2
Mp: 170–176 °C (decomp.). IR (KBr): m
~ = 3425, 2972, 2879,
2
2
2
2
2
1
759, 2567, 2465, 1572, 1458, 1400, 1366, 1287, 1197, 1145,
À1
(N–CH
2
q
100, 1059, 1033, 958, 875, 837, 786, 692, 644, 588, 506 cm
.
+
+
1
3
(CI, CH
DEI+); m/z (%) = 169 (2) [M] , 140 (7), 128 (5), 124 (10), 122 (8),
13 (8), 110 (15), 97 (68), 93 (19), 84 (51), 82 (12), 79 (19), 70
100), 68 (30), 56 (17), 53 (13), 43 (31), 41 (36), 39 (20), 31 (30),
5
); m/z (%) = 170 (100) [M+H] , 152 (6), 126 (6). MS
C NMR (400 MHz, D
CH ), 23.80 (N–CH –CH
), 44.04 (CH–COO), 44.24 (CH–COO), 45.75 (N–CH
), 63.50 (N–CH), 181.56 (COO), 182.37 (COO) ppm. HRMS
2
O, 21.5 °C, Acetone): d = 15.57 (CH
), 28.35 (N–CH–CH ), 28.98 (N–CH–
), 45.85
3
), 16.42
+
(
1
(
(
CH
3
2
2
2
2
2
(N–CH
DEI+): calcd for C
2
+
+
28 (35). HRMS (DEI+): calcd for C
169.1095.
9 2
H15NO [M] , 169.1103; found:
(
7
H
13NO
2
[M] , 143.0946; found: 143.0948.
1
The analytical data ( H NMR and MS) of the diastereomeric
mixture [rac-(l)-9]/[rac-(u)-9] were in accord with the values
reported for the mixture in the literature.47
4
.1.5.10. (2RS)-2-Hydroxy-2-[(2RS)-pyrrolidine-2-yl]acetic acid
4
8
[rac-(l)-13a]
. According to GP3 with carboxylic acid rac-(l)-39
(
6
132 mg, 0.47 mmol), Pd/C (13 mg), MeOH (4.7 ml) for 4 h afforded
3.3 mg (93%) of rac-(l)-13a as yellow crystals.
Mp: 190 °C (decomp.).
4
(
.1.5.7. (2RS)-2-[(2RS)-Pyrrolidine-2-yl]butyric acid [rac-(l)-10] and
2SR)-2-[(2RS)-pyrrolidine-2-yl]butyric acid [rac-(u)-10]. Accord-
ing to GP3 with carboxylic acids rac-(l)-35/rac-(u)-35 (26 mg,
.51 mmol), Pd/C (4.5 mg), EtOAc (5 ml) for 24 h afforded 14 mg
99%) of rac-(l)-10/rac-(u)-10 as colourless crystals.
The analytical data of rac-(l)-13a were in accord with the
1
(
literature values reported for one of the pure like enantiomer.48
Mp: 174–177 °C (decomp.). IR (KBr): m
~ = 3431, 2964, 2877,
595, 1560, 1458, 1394, 1333, 1301, 1244, 1190, 804, 682 cm .
À1
4.1.5.11. (2SR)-2-Hydroxy-2-[(2RS)-pyrrolidine-2-yl]acetic acid
1
4
8
1
[rac-(u)-13a]
.
According to GP3 with carboxylic acid
H NMR (500 MHz, D
2
O, 23.9 °C): d = 0.93 (t, J = 7.4 Hz, 0.43 Â 3H,
), 0.93 (t, J = 7.4 Hz, 0.57 Â 3H, CH ), 1.56–1.74 (m, 3H, CH
, N–CH–CH ), 1.91–2.11 (m, 2H, N–CH –CH ), 2.18 (dtd,
), 2.25 (dtd, J = 13.2/7.4/
), 2.41 (dt, J = 9.6/7.4 Hz, 0.43 Â 1H,
rac-(u)-39 (41.6 mg, 0.15 mmol), Pd/C (4.2 mg), MeOH (1.5 ml)
for 4 h afforded 20.5 mg (97%) of rac-(u)-13a as yellow crystals.
Mp: 224 °C (decomp.). The analytical data of rac-(u)-13a were
in accord with the literature values reported for one of the pure
CH
CH
3
3
2
–
3
2
2
2
J = 13.2/7.1/3.8 Hz, 0.43 Â 1H, N–CH–CH
.6 Hz, 0.57 Â 1H, N–CH–CH
2
3
2
4
8
unlike enantiomer.
CH–COO), 2.47 (dt, J = 9.1/5.5 Hz, 0.57 Â 1H, CH–COO), 3.28–3.37
(
(
m, 2H, N–CH
2
), 3.55 (td, J = 9.6/6.9 Hz, 0.43 Â 1H, N–CH), 3.65
dt, J = 10.2/6.5 Hz, 0.57 Â 1H, N–CH) ppm, diastereomeric ratio
4.1.5.12. (2RS)-2-Amino-2-[(2RS)-pyrrolidine-2-yl]acetic acidÁ2TFA
1
3
58
dr = 57:43. C NMR (500 MHz, 25.6 °C, D
2
O, MeOH): d = 11.05
), 23.68 (CH ), 24.26
), 28.74 (N–CH–CH ), 45.47 (N–CH ),
), 50.59 (CH–COO), 52.72 (CH–COO), 61.71 (N–CH),
[rac-(l)-14aÁ2TFA]
rac-(l)-41 (24 mg, 59
(26 mg, 0.23 mmol, 17
rac-(l)-14aÁ2TFA as a solid.
.
According to GP3 with carboxylic acid
lmol), Pd/C (2.4 mg), MeOH (5.8 ml), TFA
(
(
4
CH
3
), 11.21 (CH
), 28.65 (N–CH–CH
5.54 (N–CH
3
), 23.29 (CH
2
), 23.57 (CH
2
2
CH
2
2
2
2
ll) for 1 h afforded 19 mg (86%) of
2
Please cite this article in press as: Steffan, T.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.01.035

1
6
T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Mp: 185–188 °C (decomp.). IR (KBr):
m
~ = 3431, 1674, 1522,
3
80.05 (C–CH ), 127.81 (CHar), 127.94 (CHar), 128.11 (CHar), 128.45
À1
1
1
D
2
2
437, 1207, 1136, 840, 802, 724, 520 cm
.
H NMR (500 MHz,
O, 23.9 °C, Dioxan): d = 1.88–1.97 (m, 1H, N–CH–CH ), 2.01–
–CH ), 2.15–2.23 (m, 1H, N–CH –CH ), 2.32–
.39 (m, 1H, N–CH–CH ), 3.43 (dd, J = 8.5/6.3 Hz, 2H, N–CH ),
.89 (td, J = 9.6/6.9 Hz, 1H, N–CH–CH ), 3.97 (d, J = 9.6 Hz, 1H,
O, 18.4 °C, Dioxan):
), 46.31 (N–CH ), 54.14
(CHar), 136.51 (Car), 136.78 (Car), 154.56 (O–CO–N), 155.31 (O–
CO–N), 155.55 (O–CO–N), 155.71 (O–CO–N), 171.34 (COO) ppm.
2
2
+
+
.12 (m, 1H, N–CH
2
2
2
2
MS (ESI); m/z (%) = 431 (2) [M +K], 415 (12) [M+Na] , 393 (29)
+
28 2 6
[M+H] , 337 (20), 293 (100), 249 (15). Anal. calcd for C20H N O
2
2
3
2
(392.46): C, 61.21; H, 7.19; N, 7.14. Found: C, 61.13; H, 6.89; N,
7.18.
1
3
CH–COO) ppm.
d = 23.42 (N–CH
C
NMR (400 MHz, D
2
2 2
–CH ), 28.96 (N–CH–CH
2
2
(
CH–COO), 59.03 (N–CH–CH
2
), 171.05 (CH–COO) ppm. MS (ESI);
4.1.6.3. (2SR)-2-[(tert-Butoxycarbonyl)amino]-2-{(2RS)-1-[(ben-
+
+
m/z (%) = 167 (11) [M+Na] , 145 (100) [M+H] . HRMS (FAB, NBA):
calcd for C
zyloxy)carbonyl]pyrrolidine-2-yl}acetic acid methyl ester
+
52
53
H
13
N
2
O
2
[M+H] , 145.0977; found 145.0980.
[rac-(u)-49]
.
According to GP4: rac-(u)-33
(752 mg,
6
The spectral data for the compound rac-(l)-14aÁ2TFA were not
2.57 mmol) taken in dioxane (17 ml), di-tert-butyl dicarbonate
5
8
given in the literature.
(1.17 g, 5.34 mmol) and NEt
the crude product by CC (SiO
3
(0.54 ml, 3.9 mmol). Purification of
, isohexane/EtOAc = 70:30) afforded
2
4
.1.6. General procedure for the preparation of Boc-protected
957 mg (95%) of rac-(u)-49 as colourless oil.
primary and secondary amines (GP4)
The given amount of amine together with di-tert-butyl carbon-
ate (2 equiv per one amino function) was dissolved in dioxane
The analytical data of rac-(u)-49 were in accord with the litera-
ture values reported for one of the pure unlike enantiomer.52
(
2
c = 0.1 M) and stirred with the required amount of NEt
equiv per amino function) for 2.5 h at rt. After which the solvent
was evaporated in vacuum followed by CC of the crude product.
3
(1.5–
4.1.7. General procedure for the removal of Boc-protecting
groups (GP5)
2 2
The given amount of the substance was dissolved in CH Cl and
treated with TFA. After stirring for 30 min at rt, the solvent was
evaporated to obtain the respective trifluoro acetates.
4
.1.6.1. (2SR)-2-[(tert-Butoxycarbonyl)amino]-2-{(2RS)-1-[(tert-
butoxy)carbonyl]pyrrolidine-2-yl}acetic acid methyl ester
[
(
rac-(u)-43].
152 mg, 0.55 mmol), di-tert-butyl carbonate (263 mg, 1.21 mmol),
(0.18 g, 1.8 mmol, 0.25 ml) and dioxane (3 ml). CC (SiO , iso-
hexane/EtOAc = 80:20) of the crude product afforded 161 mg
According to GP4 with rac-(u)-42Á2HOAc
4.1.7.1. 2-(RS)-2-Amino-2-[(2RS)-pyrrolidine-2-yl]acetic acidÁ2TFA
5
8
[rac-(u)-14aÁ2TFA]
.
According to GP5 with carboxylic acid
NEt
3
2
2 2
rac-(u)-44 (11.6 mg, 32.3 lmol), CH Cl (2 ml) and TFA (1.53 g,
13.5 mmol, 1 ml) afforded 12.4 mg (99%) of rac-(u)-14aÁ2TFA as
(
82%) of rac-(u)-43 as colourless oil.
IR (KBr):
~ = 3432, 2977, 1746, 1718, 1699, 1510, 1392, 1367,
253, 1162, 1101, 1055, 1024, 869, 775 cm
Cl , 120 °C): d = 1.49 (s, 9H, C–CH ), 1.53 (s, 9H, C–CH
.82–1.92 (m, 1H, N–CH –CH ), 1.92–2.06 (m, 3H, N–CH –CH
), 3.27–3.35 (m, 1H, N–CH ), 3.44–3.53 (m, 1H,
), 3.78 (s, 3H, O–CH ), 4.19–4.25 (m, 1H, N–CH–CH ), 4.30–
.36 (m, 1H, CH–COO), 5.47–5.57 (m, 1H, NH) ppm. C NMR
400 MHz, C Cl , 120 °C): d = 23.35 (N–CH –CH ), 28.42 (C–
CH ), 28.52 (C–CH ), 28.95 (N–CH–CH ), 46.92 (N–CH ), 52.00
O–CH ), 57.74 (CH–COO), 58.31 (N–CH–CH ), 79.70 (C–CH ),
0.05 (C–CH ), 155.21 (N–CO–O), 155.34 (N–CO–O), 171.62
COO) ppm. MS (CI, CH
100), 229 (14), 203 (62), 185 (25), 170 (15), 114 (45). HRMS
colourless crystals.
m
Mp: 191–193 °C (decomp.). IR (KBr):
1617, 1428, 1208, 1175, 1131, 837, 797, 723 cm
(500 MHz, D O, 24.6 °C, Dioxane): d = 1.72–1.81 (m, 1H, N–CH–
), 2.02–2.13 (m, 1H, N–CH –CH ), 2.13–2.22 (m, 1H, N–CH
), 2.32 (dtd, J = 13.1/7.3/3.7 Hz, 1H, N–CH–CH ), 3.39–3.49 (m,
), 4.04 (ddd, J = 10.1/7.3/4.7 Hz, 1H, N–CH–CH ), 4.40
NMR (400 MHz, O,
), 26.33 (N–CH–CH ),
), 116.93 (q,
–COO), 170.40 (CH–COO) ppm.
m
~ = 3435, 3031, 1684,
À1
1
À1
1
1
C
1
.
H NMR (400 MHz,
),
.
H NMR
2
D
2
4
3
3
2
2
,
CH
CH
2
2
2
2
–
2
2
2
N–CH–CH
N–CH
4
2
2
2
2
2H, N–CH
(d, J = 4.7 Hz, 1H, CH–COO) ppm.
21.8 °C, Dioxane): d = 24.32 (N–CH
2
2
2
3
2
1
3
13
C
D
2
(
D
2 2
4
2
2
2
–CH
2
2
46.90 (N–CH
2
), 52.38 (CH–COO), 58.35 (N–CH–CH
), 162–156 (m, CF
5
MS (CI, CH ); m/z (%) = 145 (100) [M+H] , 128 (8), 115 (2). HRMS
2
3
3
2
2
(
8
3
2
3
J = 291.3 Hz, CF
3
3
+
+
3
+
+
+
(
(
(
5
); m/z (%) = 359 (3) [M+H] , 303 (12), 247
(FAB, NBA): calcd for
145.0966.
6 13 2 2
C H N O [M+H] , 145.0977; found:
+
FAB, NBA): calcd for C17
H
31
N
2
O
6
[M+H] , 359.2182; found:
The spectral data for the compound rac-(u)-14aÁ2TFA were not
58
3
59.2231.
given in the literature.
4
.1.6.2. (2RS)-2-[(tert-Butoxycarbonyl)amino]-2-{(2RS)-1-[(ben-
4.1.8. General procedure for the N-alkylation of the amino acid
core structure (GP6)
The given amount of the reactant, the given base (1–3 equiv), KI
(2.7 equiv) and the required amount of the alkyl halide (1.2 equiv)
zyloxy)carbonyl]pyrrolidine-2-yl}acetic acid methyl ester
rac-(l)-49]. According to GP4: rac-(l)-33 (138 mg,
.47 mmol) taken in dioxane (4.7 ml), di-tert-butyl dicarbonate
202 mg, 0.93 mmol) and NEt (100 l, 0.71 mmol). Purification
of the crude product by CC (SiO , isohexane/EtOAc = 75:25) affor-
ded 158 mg (86%) of rac-(l)-49 as colourless oil.
IR (KBr):
~ = 3425, 2977, 1706, 1499, 1456, 1415, 1364, 1339,
250, 1168, 1102, 1024, 770, 698 cm
NO 120 °C, NO ): d = 1.49–1.53 (m, 9H, C–CH
.78–1.89 (m, 1H, N–CH –CH ), 1.95–2.14 (m, 3H, N–CH –CH
), 3.39–3.49 (m, 1H, N–CH ), 3.62–3.69 (m, 1H, N–
), 3.70–3.74 (m, 3H, O–CH ), 4.34–4.41 (m, 1H, N–CH), 4.94
dd, J = 8.6/3.7 Hz, 1H, CH–COO), 5.21 (d, J = 12.5 Hz, 1H, CH –Ar),
–Ar), 5.94 (br s, 1H, NH), 7.24–7.47
[
0
(
l
was dissolved in MeCN or CH
the solvent was evaporated in vacuum followed by CC of the crude
product or the reaction mixture twice in Et O followed by extrac-
tion in CH Cl
(3 Â 10 ml), the unified organic phase was dried over
MgSO , evaporated to dryness and subjected to CC.
2 2
Cl and heated under reflux. Either
3
2
2
m
2
2
À1
1
1
C
1
.
H NMR (400 MHz,
),
4
6
D
5
2
,
C
6
D
5
2
3
2
,
4.1.8.1. (2RS)-2-Hydroxy-2-{[(2RS)-1-(4,4-diphenylbut-3-en-1-yl)]
pyrrolidine-2-yl}acetic acid methyl ester [rac-(l)-46]. According
to GP6 with rac-(l)-45.AcOH (19 mg, 0.1 mmol) in MeCN (4.1 ml),
CO (45 mg, 0.33 mmol), KI (63 mg, 0.38 mmol) and 4,4-diphe-
nylbut-3-en-1-yl bromide (74 mg, 0.12 mmol) at 80 °C for 16 h.
Purification of the crude product by CC (SiO , isohexane/EtOAc =
75:25) afforded 22.3 mg (60%) of rac-(l)-46 as colourless oil.
IR (KBr):
~ = 3442, 2965, 2802, 1755, 1733, 1494, 1443, 1361,
1221, 1128, 1086, 762, 701, 632 cm
20.9 °C, TMS): d = 1.62–1.76 (m, 4H, N–CH
2
2
2
N–CH–CH
CH
(
2
2
2
3
K
2
3
2
5
.30 (d, J = 12.5 Hz, 1H, CH
2
1
3
(
m, 5H, Har) ppm.
C NMR (500 MHz, CDCl
3
, 20.9 °C, TMS):
, N–
), 55.57
), 60.38
–Ar), 79.76 (C–CH ),
2
d = 23.21 (N–CH
2
–CH
), 46.99 (N–CH
2
), 23.90 (N–CH
), 47.13 (N–CH
CH–COO), 56.22 (CH–COO), 59.30 (N–CH–CH
N–CH–CH ), 66.96 (CH –Ar), 67.27 (CH
2
–CH
2 3
), 28.30 (C–CH
), 52.37 (O–CH
CH–CH
(
(
2
2
2
3
m
À1
1
.
H NMR (500 MHz, CDCl
3
,
2
2
–CH –CH ), 2.14–2.21
2
2
2
2
2
3
Please cite this article in press as: Steffan, T.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.01.035

T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
17
+
+
(
m, 1H, N–CH
J = 12.1/6.0 Hz, 1H, N–CH
CH –CH, N–CH), 2.99–3.04 (m, 1H, N–CH
OCH ), 4.30 (d, J = 3.3 Hz, 1H, CH–OH), 6.06 (t, J = 7.7 Hz, 1H,
C@CH), 7.15–7.40 (m, 10H, Har) ppm. 13C NMR (500 MHz, CDCl
2
–CH
2
–CH
2
), 2.31–2.36 (m, 2H, C@CH–CH
–CH –CH), 2.83–2.92 (m, 2H, N–CH
–CH –CH ), 3.76 (s, 3H,
2
), 2.45 (dt,
(%) = 428 (32) [M+Na] , 406 (100) [M+H] , 316 (9), 308 (17), 247
+
2
2
2
–
(38), 149 (16). HRMS (FAB, NBA): calcd for C21
406.1511; found: 406.1519.
3 2
H28NO S [M+H] ,
2
2
2
2
3
3
,
4.1.8.4. (2SR)-2-Hydroxy-2-{(2RS)-1-[4,4-bis(3-methylthiophen-
2
5
6
1
0.9 °C, CDCl
3
): d = 23.40 (CH
2
), 24.83 (CH
2
), 28.97 (C@CH–CH
–CH –CH
2
),
),
2-yl)but-3-en-1-yl]pyrrolidine-2-yl}acetic acid methyl ester
2.00 (O–CH
3
), 53.20 (N–CH –CH
2
2
–CH), 53.48 (N–CH
2
2
2
[rac-(u)-47].
(29.5 mg, 0.14 mmol) in MeCN (5.4 ml), K
According to GP6 with rac-(u)-45.HOAc
CO (56 mg, 0.41 mmol),
5.46 (N–CH), 69.18 (CH–OH), 126.71 (C@CH), 127.00 (CHar),
27.03 (CHar), 127.16 (CHar), 128.11 (CHar), 128.19 (CHar), 129.74
2
3
KI (61 mg, 0.37 mmol) and 4,4-bis-(3-methylthien-2-yl)but-3-en-
(
CHar), 139.95 (Car), 142.33 (Car), 143.31 (C@CH), 172.79 (COO)
1-yl bromide (93 mg, 0.28 mmol) at 80 °C for 17 h. Purification of
+
+
ppm. MS (ESI); m/z (%) = 388 (9) [M+Na] , 366 (100) [M+H] , 276
(
3
the crude product by CC (SiO
10 mg (19%) of rac-(u)-47 as yellow oil.
IR (KBr):
~ = 3447, 2949, 1748, 1436, 1267, 1199, 1115, 1013,
67, 718 cm
d = 1.65–1.81 (m, 3H, N–CH
.95–2.09 (m, 7H, Ar–CH , N–CH
N–CH –CH –CH , C@CH–CH ), 2.41–2.51 (m, 1H, N–CH
CH), 2.59 (dt, J = 11.9/8.1 Hz, 1H, N–CH –CH –CH), 3.01–3.10 (m,
H, N–CH –CH –CH N–CH), 3.69 (s, 3H, O–CH ), 3.87 (d,
2
, isohexane/EtOAc = 80:20) afforded
+
3
9), 129 (17). HRMS (FAB, NBA): calcd for C23H28NO [M+H] ,
66.2069; Found 366.2068.
m
À1
1
9
.
H
NMR (400 MHz, CD
N–CH –CH –CH
–CH –CH ), 2.20–2.31 (m, 3H,
–CH
2
Cl
2
,
17.9 °C, TMS):
2
2
2
2
, N–CH
2
–CH –CH ),
2
2
4
.1.8.2. (2SR)-2-Hydroxy-2-{[(2RS)-1-(4,4-diphenylbut-3-en-1-yl)]
1
3
2
2
2
pyrrolidine-2-yl}acetic acid methyl ester [rac-(u)-46]. Accord-
ing to GP6 with rac-(u)-45.HOAc (19 mg, 0.1 mmol) in MeCN
2
2
2
2
2
2
–
(
3.8 ml), K
,4-diphenylbut-3-en-1-yl bromide (35 mg, 0.12 mmol) at 80 °C
for 7 h. Purification of the crude product by CC (SiO , isohexane/
2 3
CO (42 mg, 0.3 mmol), KI (44 mg, 0.26 mmol) and
2
2
2
2
2
2
,
3
4
J = 1.8 Hz, 1H, CH–OH), 6.04 (t, J = 7.3 Hz, 1H, C@CH), 6.78 (d,
J = 5.1 Hz, 1H, Har), 6.88 (d, J = 5.1 Hz, 1H, Har), 7.08 (d, J = 5.1 Hz,
2
EtOAc = 80:20) afforded 15.9 mg (44%) of rac-(u)-46 as colourless
oil.
1
3
1
H, Har), 7.26 (d, J = 5.1 Hz, 1H, Har) ppm. C NMR (500 MHz, CD
2
-
Cl , 23.9 °C, TMS): d = 14.57 (Ar–CH ), 14.91 (Ar–CH ), 24.88 (N–
2
3
3
IR (KBr):
073, 1017, 762, 701 cm
TMS): d = 1.65–1.84 (m, 3H, N–CH
dq, J = 12.5/9.2 Hz, 1H, N–CH–CH
CH , N–CH –CH –CH ), 2.47 (ddd, J = 12.1/6.8/5.3 Hz, 1H, N–CH
CH –CH), 2.60 (dt, J = 12.1/8.1 Hz, 1H, N–CH –CH –CH), 2.94–3.00
m, 1H, N–CH –CH –CH ), 3.09 (ddd, J = 9.2/4.0/2.0 Hz, 1H,
N–CH), 3.73 (s, 3H, O–CH ), 3.92 (d, J = 2.0 Hz, 1H, CH–OH), 6.07
m
~ = 3429, 2924, 1748, 1494, 1443, 1269, 1201, 1116,
À1
1
CH –CH –CH ), 29.94 (N–CH –CH –CH), 30.88 (N–CH –CH –CH ),
2
2
2
2
2
2
2
2
1
.
H NMR (400 MHz, CDCl
–CH –CH , N–CH–CH
), 2.17–2.35 (m, 3H, C@CH–
3
, 18.3 °C,
5
6
1
1
2.11 (O–CH
3
), 54.67 (N–CH
2
–CH
2
–CH
2
), 55.59 (N–CH
2
–CH
2
–CH),
2
2
2
2
), 2.03
6.41 (N–CH), 72.73 (CH–OH), 123.11 (CHar), 124.71 (CHar),
28.75 (C@CH), 129.99 (CHar), 131.56 (CHar), 133.75 (C@CH),
34.13 (Car), 135.77 (Car), 135.90 (Car), 139.95 (Car), 174.84 (COO)
(
2
2
2
2
2
2
–
2
2
2
+
+
ppm. MS (ESI); m/z (%) = 428 (54) [M+Na] , 406 (100) [M+H] ,
16 (7), 308 (19), 247 (38), 149 (16). HRMS (FAB, NBA): calcd for
(
2
2
2
3
3
+
1
3
3 2
C21H28NO S [M+H] , 406.1511; found: 406.1490.
(
(
2
t, J = 7.5 Hz, 1H, C@CH), 7.15–7.41 (m, 10H, Har) ppm. C NMR
500 MHz, CDCl 25.8 °C, TMS): d = 24.37 (N–CH –CH –CH ),
9.02 (C@CH–CH ), 30.36 (N–CH–CH ), 52.03 (CH ), 54.20
N–CH –CH –CH ), 55.85 (N–CH –CH –CH), 66.31 (N–CH), 72.19
3
,
2
2
2
2
2
3
4
.1.8.5. (2RS)-2-Hydroxy-2-[(2RS)-1-{2-[tris(4-methoxyphenyl)
methoxy]ethyl}-pyrrolidine-2-yl]acetic acid methyl ester
rac-(l)-48].
According to GP6 with rac-(l)-45ÁHOAc (29 mg,
.13 mmol) in MeCN (2.7 ml), CO (56 mg, 0.4 mmol), KI
60 mg, 0.36 mmol) and 2-{[tris(4-methoxyphenyl)]methoxy}ethyl
bromide (74 mg, 0.16 mmol) at 80 °C for 16 h. Purification of the
crude product by CC (SiO , isohexane/EtOAc = 60:40) afforded
2.1 mg (45%) of rac-(l)-48 as colourless oil.
IR (KBr):
~ = 3443, 2952, 2836, 1757, 1733, 1607, 1507, 1463,
1441, 1302, 1249, 1175, 1124, 1074, 1034, 827, 583 cm
NMR (400 MHz, CD Cl , 18.4 °C, TMS): d = 1.61–1.73 (m, 4H, N–
(
(
(
2
2
2
2
2
CH–OH), 126.38 (C@CH), 127.06 (CHar), 127.10 (CHar), 127.22
[
CHar), 128.16 (CHar), 128.25 (CHar), 129.80 (CHar), 139.98 (Car),
0
K
2
3
+
1
42.37 (Car), 143.35 (C
q
), 174.19 (COO) ppm. MS (CI, CH
5
); m/z
(
+
(
(
%) = 366 (81) [M+H] , 306 (4), 276 (18), 193 (3), 172 (100). HRMS
+
FAB, NBA): calcd for
23 3
C H28NO [M+H] , 366.2069; found:
2
3
66.2085.
3
m
À1
1
4
2
.1.8.3. (2RS)-2-Hydroxy-2-{(2RS)-1-[4,4-bis(3-methylthiophen-
-yl)but-3-en-1-yl]pyrrolidine-2-yl}acetic acid methyl ester
. H
2
2
[
rac-(l)-47].
According to GP6 with rac-(l)-45.HOAc (23 mg,
2 2 2 2 2 2
CH –CH –CH ), 2.23–2.32 (m, 1H, N–CH –CH –CH ), 2.53 (dt,
0
.1 mmol) in MeCN (4.1 ml), CO (47 mg, 0.34 mmol), KI
K
2
3
2 2
J = 13.0/4.6 Hz, 1H, N–CH –CH –O), 2.89–3.00 (m, 2H, N–CH, N–
(
64 mg, 0.39 mmol) and 4,4-bis-(3-methylthien-2-yl)but-3-en-1-
yl bromide (133 mg, 0.41 mmol) at 80 °C for 17 h. Purification of
the crude product by CC (SiO , isohexane/EtOAc = 80:20) afforded
8 mg (43%) of rac-(l)-47 as yellow oil.
IR (KBr):
~ = 3444, 2950, 2923, 1756, 1733, 1438, 1265, 1211,
126, 1008, 738, 712 cm
2 2 2 2 2 2
CH –CH –O), 3.08–3.14 (m, 2H, N–CH –CH –CH , O–CH ), 3.21
(ddd, J = 9.9/7.9/4.6 Hz, 1H, O–CH ), 3.71 (s, 3H, COOCH ), 3.77 (s,
2
3
2
3
9H, Ar–OCH ), 4.28 (d, J = 3.5 Hz, 1H, CH–OH), 6.80–6.85 (m, 6H,
1
3
1
1
H ), 7.30–7.35 (m, 6H, H ) ppm. C NMR (400 MHz, CD Cl ,
ar
ar
2
2
m
20 °C, TMS): d = 24.04 (N–CH
CH ), 52.06 (COOCH ), 53.64 (N–CH
CH –CH ), 55.59 (Ar–OCH
(CH–OH), 86.19 (O–C ), 113.38 (CHar), 130.07 (CHar), 137.27 (Car),
158.84 (Car), 173.10 (COO) ppm. MS (ESI); m/z (%) = 558 (7)
2
–CH
2
–CH
2
2
), 25.14 (N–CH –CH
2
–
À1
1
.
H NMR (400 MHz, CD
–CH –CH ), 2.00 (s, 3H, Ar–
), 2.15–2.25 (m, 1H, N–CH
–CH –CH), 2.41–2.49 (m, 1H, N–CH
–CH), 2.82–2.93 (m, 2H, N–CH, N–CH –CH
m, 1H, N–CH –CH –CH ), 3.71 (s, 3H, O–CH ), 4.26 (d, J = 3.3 Hz,
H, CH–OH), 6.06 (t, J = 7.3 Hz, 1H, C@CH), 6.78 (d, J = 5.1 Hz, 1H,
2
Cl
2
, 20.9 °C,
2
3
2
2 2
–CH –O), 54.75 (N–CH
–
TMS): d = 1.63–1.70 (m, 4H, N–CH
CH ), 2.05 (s, 3H, Ar–CH
.29–2.37 (m, 2H, N–CH
CH
2
2
2
2
2
3 2
), 62.71 (O–CH ), 66.01 (N–CH), 69.67
3
3
2
–CH
2
–CH
2
),
–
q
2
2
2
2
+
+
2
2
2
–CH), 3.04–3.10
[M+Na] , 536 (5) [M+H] , 333 (100). HRMS (DEI): calcd for
+
(
1
2
2
2
3
31 7
C H38NO [M+H] , 536.2648; found: 536.2623.
H
(
ar), 6.87 (d, J = 5.1 Hz, 1H, Har), 7.07 (d, J = 5.1 Hz, 1H, Har), 7.25
Cl , 21.3 °C,
), 23.58 (N–CH
4.1.8.6. (2SR)-2-Hydroxy-2-[(2RS)-1-{2-[tris(4-methoxyphenyl)
methoxy]ethyl}-pyrrolidine-2-yl]acetic acid methyl ester
d, J = 5.1 Hz, 1H, Har) ppm. 13C NMR (500 MHz, CD
2
2
CD
CH
2
Cl
), 24.85 (N–CH–CH
2.94 (N–CH –CH –CH), 53.51 (N–CH
2
): d = 14.21 (Ar–CH
3
), 14.57 (Ar–CH
–CH
–CH
3
2
–CH
–CH), 51.73 (O–CH
–CH
2
–
),
[rac-(u)-48].
(71 mg, 0.32 mmol) in MeCN (6.4 ml), K
KI (144 mg, 0.87 mmol) and 2-{[tris(4-methoxyphenyl)]
methoxy}ethyl bromide (0.18 g, 0.39 mmol) at 80 °C for 16 h. Puri-
fication of the crude product by CC (SiO , isohexane/EtOAc = 70:30)
afforded 102 mg (59%) of rac-(u)-48 as colourless oil.
According to GP6 with rac-(u)-45ÁHOAc
2
2
), 29.33 (N–CH
2
2
3
2
CO (133 mg, 0.96 mmol),
3
5
6
2
2
2
2
2
), 65.72 (N–CH),
9.31 (CH–OH), 122.78 (CHar), 124.40 (CHar), 128.59 (Car), 129.64
(
1
CHar), 131.20 (CHar), 133.27 (C@CH), 133.78 (Car), 135.31 (Car),
35.58 (Car), 139.52 (C@CH), 172.70 (COO) ppm. MS (ESI); m/z
2
Please cite this article in press as: Steffan, T.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.01.035

1
8
T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
IR (KBr):
302, 1250, 1176, 1117, 1071, 1034, 828, 584 cm
NMR (400 MHz, CD Cl , 20.4 °C, TMS): d = 1.67–1.80 (m, 3H, N–
CH –CH –CH , N–CH–CH ), 1.93–2.06 (m, 1H, N–CH–CH ), 2.29–
.38 (m, 1H, N–CH –CH –CH ), 2.59 (dt, J = 13.2/5.1 Hz, 1H, N–
CH –CH –O), 2.73–2.81 (m, 1H, N–CH –CH –O), 3.02–3.15 (m,
H, N–CH, N–CH –CH –CH , O–CH ), 3.70 (s, 3H, COOCH ), 3.78
s, 9H, O–CH ), 3.87 (d, J = 2.9 Hz, 1H, CH–OH), 6.80–6.85 (m, 6H,
m
~ = 3433, 2952, 2836, 1748, 1608, 1508, 1463, 1441,
(m, 1H, N–CH–CH
2
), 3.68 (s, 0.12 Â 3H, O–CH
3
), 3.70 (s,
À1
1
1
.
H
0.88 Â 3H, O–CH ), 3.95–4.00 (m, 0.12 Â 1H, CH–COO), 4.15 (dd,
3
J = 9.1/1.1 Hz, 0.88 Â 1H, CH–COO), 5.35–5.41 (m, 0.12 Â 1H, NH),
5.61 (d, J = 9.1 Hz, 0.88 Â 1H, NH), 6.05 (t, J = 7.4 Hz, 1H, C@CH),
2
2
2
2
2
2
2
1
3
2
2
2
2
7.17–7.40 (m, 10H, Har) ppm. Rotamer ratio (22.7 °C) 88:12.
C
),
NMR (500 MHz, CDCl
28.31 (C–CH ), 29.16 (N–CH
(O–CH ), 54.22 (N–CH ), 54.87 (N–CH
(N–CH–CH ), 79.53 (C –CH ), 126.85 (CHar), 126.92 (CHar), 127.26
3
, 22 °C, TMS): d = 23.77 (N–CH
–CH –CH), 29.99 (N–CH–CH
), 56.09 (CH–COO), 64.24
2 2
–CH –CH
2
2
2
2
2
4
(
2
2
2
2
3
3
2
2
2
), 52.06
3
3
2
2
H
2
5
ar), 7.30–7.35 (m, 6H, Har) ppm. 13C NMR (500 MHz, CD
2
Cl
), 30.58 (N–CH–CH
2
,
),
2
q
3
5.2 °C, TMS): d = 25.06 (N–CH
2.67 (COOCH ), 55.55 (N–CH –CH
–CH –O), 63.10 (CH
6.22 (O–C ), 113.38 (CHar), 130.14 (CHar), 137.31 (Car), 158.87
ar), 174.71 (COO) ppm. MS (ESI); m/z (%) = 558 (1) [M+Na] , 536
2
–CH
2
–CH
2
2
(CHar), 127.50 (C@CH), 128.09 (CHar), 128.16 (CHar), 129.89 (CHar),
140.19 (CHar), 142.65 (Car), 142.72 (Car), 156.25 (O–CO–N), 172.94
(COO) ppm. MS (ESI); m/z (%) = 487 (4) [M+Na] , 465 (79) [M+H] ,
3
2
2
–CH
2 3
), 55.60 (O–CH ), 56.31
+
+
(
N–CH
2
2
2
–O), 66.67 (N–CH), 73.07 (CH–OH),
8
q
409 (100), 276 (22). HRMS (FAB, NBA): calcd for C28
H
37
N
2
O
4
+
+
(
(
[
C
[M+H] , 465.2753; found: 465.2714.
+
15) [M+H] , 333 (100). HRMS (FAB, NBA): calcd for C31
H38NO
7
+
M+H] , 536.2648; found: 536.2693.
4.1.8.9. (2RS)-2-[(tert-Butoxycarbonyl)amino]-2-{(2RS)-1-[4,4-
bis(3-methylthio-phen-2-yl)but-3-en-1-yl]pyrrolidine-2-yl}ace-
tic acid methyl ester [rac-(l)-52].
rac-(l)-50.HOAc (19 mg, 59 mol) taken in MeCN (2.4 ml), K
28 mg, 0.2 mmol), KI (31 mg, 0.19 mmol) and 4,4-bis-(3-meth-
ylthien-2-yl)but-3-en-1-yl bromide (58 mg, 0.18 mmol) at 80 °C
for 17 h. Purification of the crude product by CC (SiO2, isohex-
ane/EtOAc = 90:10) afforded 14.6 mg (49%) of rac-(l)-52 as colour-
less oil
According to the GP6 with
4
.1.8.7. (2RS)-2-[(tert-Butoxycarbonyl)amino]-2-{[(2RS)-1-(4,4-
diphenylbut-3-en-1-yl)]pyrrolidine-2-yl}acetic acid methyl
ester [rac-(l)-51].
According to GP6 with rac-(l)-50ÁHOAc
11 mg, 36 mol), KHCO (7 mg, 71 mol), KI (13 mg, 79 mol)
taken in CH CN (1.4 ml) with 4,4-diphenylbut-3-en-1-yl bromide
22 mg, 77 mol) and heated to reflux for 4 h. The reaction mixture
was extracted with ether (2 Â 10 ml), CH Cl (3 Â 10 ml) and the
unified organic phase dried over MgSO , concentrated in vacuum
and purified by CC (SiO , isohexane/EtOAc = 95:5) to afford 9 mg
55%) of rac-(l)-51 as colourless oil.
l
2
CO
3
(
(
l
3
3
l
l
(
l
2
2
IR (KBr): m
~ = 3423, 2973, 1716, 1483, 1366, 1163, 1064, 1023,
4
À1 1
7
11 cm
.
H NMR (400 MHz, CDCl
), 1.62–1.76 (m, 4H, N–CH
), 2.10–2.19 (m, 1H, N–CH
–CH –CH, N–CH –CH –CH), 2.77–2.84
), 2.84–2.92 (m, 1H, N–CH –CH –CH), 3.04–
–CH –CH ), 3.70 (s, 3H, O–CH ), 4.26 (br s,
3
, 50 °C, TMS): d = 1.41 (s, 9H,
–CH –CH ), 2.02 (s, 3H, Ar–
–CH –CH ),
2
C–CH
3
2
2
2
(
CH
2
(
3
3
), 2.04 (s, 3H, Ar–CH
3
2
2
2
IR (KBr): m
~ = 3427, 2972, 1716, 1495, 1399, 1164, 1064, 1025,
.24–2.38 (m, 3H, N–CH
2
2
2
2
À1
1
7
9
62, 701 cm
H, C–CH
.
H NMR (500 MHz, CDCl
3
, 23 °C, TMS): d = 1.41 (s,
–CH –CH , N–CH –CH
2
), 2.21–2.34 (m, 3H, N–
m, 1H, N–CH–CH
2
2
2
3
), 1.63–1.75 (m, 4H, N–CH
), 2.06–2.13 (m, 1H, N–CH –CH –CH
–CH), 2.74–2.82 (m, 1H, N–CH–CH
–CH –CH), 3.01–3.07 (m, 1H, N–CH
2
2
2
2
2
–
.10 (m, 1H, N–CH
2
2
2
3
CH
CH
2
2
2
1
H, CH–COO), 5.10 (br s, 1H, NH), 6.03–6.06 (m, 1H, C@CH), 6.74
2
–CH
2
–CH, N–CH
2
–CH
2
2
),
–
(
d, J = 5.1 Hz, 1H, Har), 6.83 (d, J = 5.1 Hz, 1H, Har), 7.03 (d,
2
.82–2.90 (m, 1H, N–CH
2
2
2
13
J = 5.1 Hz, 1H, Har), 7.19 (d, J = 5.1 Hz, 1H, Har) ppm. C NMR
CH –CH
2
2
), 3.69 (s, 3H, O–CH
3
), 4.06–4.21 (m, 0.15 Â 1H, CH–
(
2
500 MHz, CDCl
2.44 (N–CH –CH
CH ), 29.02 (N–CH
CH –CH), 53.10 (N–CH
CH–CH ), 79.77 (C –CH
3
3
, 22.2 °C): d = 14.37 (Ar–CH
–CH ), 25.78 (N–CH –CH
–CH –CH), 52.07 (O–CH
–CH –CH
), 122.68 (CHar), 124.30 (CHar), 128.24
3
), 14.79 (Ar–CH
–CH
), 53.01 (N–CH
3
),
COO), 4.24–4.31 (m, 0.85 Â 1H, CH–COO), 5.00 (br s, 0.15 Â 1H,
NH), 5.19 (s, 0.85 Â 1H, NH), 6.10 (t, J = 6.9 Hz, 1H, C@CH), 7.15–
2
2
2
2
2
2
), 28.29 (C–
–
13
3
2
2
3
2
7
.40 (m, 10H, Har) ppm. Rotamer ratio (23 °C) 85:15. C NMR
2
2
2
2
), 54.36 (CH–COO), 64.09 (N–
(
500 MHz, CDCl
3
,
21.6 °C, TMS): d = 22.49 (N–CH
2
–CH
), 28.94 (N–CH
–CH ), 53.69 (N–CH
), 79.78 (C –CH
2
–CH
2
),
2
–
2
–
3
),
2
q
2
5.95 (N–CH
2
–CH
2
–CH
2
), 28.30 (C
q
–CH
3
2
–CH
(
Car), 129.53 (CHar), 131.10 (CHar), 133.23 (C@CH), 133.52 (Car),
CH), 52.07 (O–CH
CH
3
), 53.24 (N–CH
2
–CH
2
2
1
35.31 (Car), 139.58 (C@CH), 155.97 (O–CO–N), 172.06 (COO)
2
–CH), 54.55 (CH–COO), 64.23 (N–CH–CH
2
q
+
+
ppm. MS (ESI); m/z (%) = 527 (28) [M+Na] , 505 (97) [M+H] , 449
100), 427 (11), 316 (19), 247 (16). HRMS (FAB, NBA): calcd for C26
1
1
26.89 (CHar), 126.96 (CHar), 127.14 (C@CH), 127.20 (CHar),
28.09 (CHar), 128.20 (CHar), 129.81 (CHar), 140.11 (Car), 142.52
(
-
+
37 2 4 2
H N O S [M+H] , 505.2195; found: 505.2238.
(
C
q
), 142.57 (C
q
), 155.94 (N–CO–O), 172.11 (COO) ppm. MS (ESI);
+
+
m/z (%) = 487 (2) [M+Na] , 465 (71) [M+H] , 409 (100), 276 (20).
HRMS (FAB, NBA): calcd for C28H N O [M+H] , 465.2753; found:
37 2 4
4
.1.8.10. (2SR)-2-[(tert-Butoxycarbonyl)amino]-2-{(2RS)-1-[4,4-
bis(3-methylthio-phen-2-yl)but-3-en-1-yl]pyrrolidine-2-yl}ace-
tic acid methyl ester [rac-(u)-52]. According to GP6 with
rac-(u)-50ÁHOAc (17 mg, 54 mol) and 4,4-bis-(3-methylthien-2-
yl)but-3-en-1-yl bromide (36 mg, 0.11 mmol) in CH Cl (2 ml)
with NEt (10 mg, 14 l, 0.1 mmol) stirred at rt for 72 h. Concentra-
tion of the crude product in vacuum followed by CC (SiO , isohex-
+
4
65.2741.
l
4
.1.8.8. (2SR)-2-[(tert-Butoxycarbonyl)amino]-2-{[(RS)-1-(4,4-
2
2
diphenylbut-3-en-1-yl)]pyrrolidine-2-yl}acetic acid methyl
ester [rac-(u)-51].
According to GP6 with rac-(u)-50ÁHOAc
20 mg, 63 mol) and 4,4-diphenylbut-3-en-1-yl bromide (38 mg,
.13 mmol) in MeCN (1.3 ml) and DIPEA (81 mg, 0.63 mmol) at rt
for 72 h. The reaction mixture was concentrated in vacuum and
the crude product on CC (SiO , isohexane/EtOAc = 95:5) afforded
2 mg (18%) of rac-(u)-51 as colourless oil.
IR (KBr):
~ = 3423, 2972, 1750, 1716, 1490, 1365, 1321, 1258,
204, 1164, 760, 702 cm
3
l
2
(
0
l
ane/EtOAc = 95:5) afforded 2.3 mg (10%) of rac-(u)-52 as colourless
oil.
IR (KBr):
m
~
= 3419, 2972, 1749, 1716, 1488, 1365, 1164,
H NMR (400 MHz, CDCl , 20.5 °C, TMS): d = 1.45 (s,
), 1.62–1.74 (m, 3H, N–CH–CH , N–CH –CH –CH ),
1.94–2.06 (m, 7H, Ar–CH , N–CH–CH ), 2.13 (t, J = 8.2 Hz, 1H, N–
CH –CH –CH ), 2.22–2.29 (m, 2H, N–CH –CH –CH), 2.29–2.36 (m,
1H, N–CH –CH –CH), 2.60 (dt, J = 11.0/7.7 Hz, 1H, N–CH –CH
CH), 2.99–3.04 (m, 1H, N–CH –CH –CH ), 3.12–3.17 (m, 1H, N–
CH–CH ), 3.70 (s, 3H, O–CH
À1
1
668 cm
.
3
2
5
1
9H, C–CH
3
2
2
2
2
m
3
2
À1
1
.
H NMR (500 MHz, CDCl
), 1.44 (s, 0.12 Â 9H, C–CH
N–CH –CH –CH ), 1.98 (dq,
), 2.07 (q, J = 8.5 Hz, 1H, N–CH –CH
–CH –CH, N–CH –CH –CH), 2.51–
–CH), 2.82–2.88 (m, 0.88 Â 1H, N–CH
), 2.96–3.03 (m, 0.12 Â 1H, N–CH –CH –CH ), 3.07–3.17
3
, 22.7 °C,
2
2
2
2
2
TMS): d = 1.40 (s, 0.88 Â 9H, C–CH
3
3
),
2
2
2
2
–
1
.58–1.71 (m, 4H, N–CH–CH
2
,
2
2
2
2
2
2
J = 12.4/8.5 Hz, N–CH–CH
2
2
2
–
2
3
), 3.98 (d, J = 7.7 Hz, 0.10 Â 1H, CH–
CH
.58 (m, 1H, N–CH
CH –CH
2
), 2.16–2.36 (m, 3H, N–CH
2
2
2
2
COO), 4.17 (dd, J = 9.3/1.6 Hz, 0.90 Â 1H, CH–COO), 5.27–5.36 (m,
0.10 Â 1H, NH), 5.54 (d, J = 9.3 Hz, 0.90 Â 1H, NH), 6.03 (t,
J = 7.1 Hz, 1H, C@CH), 6.76 (d, J = 4.9 Hz, 1H, Har), 6.85 (d,
2
2
–CH
2
2
–
2
2
2
2
2
Please cite this article in press as: Steffan, T.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.01.035

T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
19
J = 4.9 Hz, 1H, Har), 7.05 (d, J = 4.9 Hz, 1H, Har), 7.22 (d, J = 4.9 Hz,
4.1.9.2. (2RS)-2-{(2RS)-1-[(Benzyloxy)carbonyl]pyrrolidine-2-yl}
butyric acid [rac-(l)-35] und (2SR)-2-{(2RS)-1-[(benzyloxy)car-
13
1
H, Har) ppm. Rotamer ratio (20.5 °C) 90:10. C NMR (400 MHz,
CDCl , 22.3 °C, CDCl ): d = 14.37 (CH –Ar), 14.79 (CH –Ar), 23.73
N–CH –CH –CH ), 28.35 (C–CH ), 29.22 (C@CH–CH
N–CH–CH ), 52.03 (O–CH ), 54.11 (N–CH –CH –CH
N–CH –CH
–CH ), 122.64 (CHar), 124.29 (CHar), 128.23 (C@CH), 129.51
CHar), 131.15 (CHar), 133.28 (C@CH), 133.51 (Car), 135.34 (Car),
39.55 (Car), 156.27 (O–CO–N), 172.84 (COO) ppm. MS (ESI); m/z
3
3
3
3
bonyl]pyrrolidine-2-yl}butyric acid [rac-(u)-35].
to GP7, method A with rac-(l)-29/rac-(u)-28 (89 mg, 0.29 mmol),
KOH (2.6 ml, 0.5 M in MeOH), H O (0.3 ml) and the reaction time
was 16 h at 92 °C. Purification through CC (SiO , pentane/EtOAc/
According
(
(
(
(
(
2
2
2
3
2
), 29.80
), 54.41
2
3
2
2
2
2
2
2
–CH), 56.06 (CH–COO), 64.42 (N–CH–CH
2
), 79.54
2
C
q
3
HOAc = 90:10:1) afforded 66 mg (78%) of the diastereomeric mix-
ture rac-(l)-35/rac-(u)-35 as colourless oil.
1
IR (KBr):
1259, 1197, 1114, 1029, 917, 876, 770, 698 cm
m
~ = 2967, 2879, 1731, 1698, 1498, 1455, 1416, 1359,
+
À1
1
(
%) = 505 (100) [M+H] , 449 (60). HRMS (FAB, NBA): calcd for
.
H
+
C
26
H
37
N
2
O
4
S
2
[M+H] , 505.2195; found: 505.2187.
NMR (400 MHz, CDCl , 20.2 °C, TMS): d = 0.80–1.03 (m, 3H, CH ),
3
3
1
.30–2.01 (m, 6H, N–CH
2
–CH
2
–CH
2
, CH
2
–CH
3
), 2.63–3.06 (m, 1H,
4
.1.9. General procedure for the hydrolysis of methyl esters
2 2
CH–COO), 3.29–3.41 (m, 1H, N–CH ), 3.47–3.68 (m, 1H, N–CH ),
(
GP7)
Method A: The ester was refluxed with the given amount of
methanolic KOH (0.5 M) and H O, after which the MeOH was
removed by distillation. The aqueous residue left behind was
extracted thrice with CH Cl and the extracts were discarded. After
acidifying the aqueous phase with HCl (2 M) to pH = 1, it was again
extracted thrice with CH Cl , then the unified organic phase was
dried over MgSO and evaporated to dryness. The crude product
was purified by CC.
4.08–4.15 (m, 0.43 Â 1H, N–CH), 4.21–4.28 (m, 0.57 Â 1H, N–CH),
5.06–5.20 (m, 2H, CH –Ar), 7.27–7.41 (m, 5H, H ) ppm, diastereo-
2
ar
1
3
2
meric ratio dr = 57:43. C NMR (400 MHz, CDCl , 20.8 °C, TMS):
3
d = 12.20 (CH ), 12.65 (CH ), 18.86 (CH ), 19.31 (CH ), 23.09
3
3
2
2
2
2
2 2 2 2 2
(CH ), 23.63 (CH ), 23.83 (CH ), 24.13 (CH ), 27.48 (CH ), 27.72
(CH ), 28.00 (CH ), 28.45 (CH ), 46.81 (N–CH ), 47.13 (N–CH ),
2
2
2
2
2
2
2
47.62 (N–CH ), 49.57 (CH–COO), 50.28 (CH–COO), 58.50 (N–CH),
2
4
58.87 (N–CH), 59.17 (N–CH), 59.51 (N–CH), 66.87 (CH –Ar),
2
127.85 (CH ), 127.97 (CH ), 128.48 (CH ), 136.73 (C ), 155.08
ar
ar
ar
ar
Method B: To the solution of the methyl ester in dioxane was
added the given amount of aq LiOH (1.2 M) at rt. After stirring
for the given time at rt, the aqueous phase was extracted with
(O–CO–N), 155.47 (O–CO–N), 179.13 (COO), 179.57 (COO) ppm.
+
+
MS (CI, CH ); m/z (%) = 292 (61) [M+H] , 274 (18), 248 (100), 204
5
+
(13), 184 (20), 160 (14). MS (EI, 70 eV); m/z (%) = 291 (0) [M] ,
CH
2
Cl
2
and the organic phase discarded. The aq phase was then
Cl
and
204 (13), 160 (24), 91 (100), 65 (9). Anal. calcd for C16H21NO4
(291.1471): C, 65.96; H, 7.27; N, 4.81. Found: C, 65.79; H, 7.28; N
4.68.
acidified with 2 M HCl to pH = 1 and extracted thrice with CH
Finally, the unified organic phase was dried over MgSO
evaporated to dryness in vacuum.
2
2
.
4
Method C: Analogous to GP1, the methyl ester dissolved in diox-
ane was treated with aq LiOH. After stirring for 1.5 to 2 h at rt, the
4.1.9.3.
2-{(2RS)-1-[(Benzyloxy)carbonyl]pyrrolidine-2-yl}-2-
According to GP7, method
methylpropionic acid [(RS)-36].
Na
and extracted twice with CH
with 2 M HCl to pH = 6 and extracted twice with CH
organic phase was dried over MgSO and evaporated to dryness
in vacuum. In exceptional cases, the crude product was purified
by CC or HPLC.
2
HPO
4
/NaH
2
PO
4
-buffer pH = 7 (10 ml) was added to the reaction
Cl . The aqueous phase was acidified
Cl . The
A, with (RS)-30 (44 mg, 0.15 mmol), KOH (20 ml, 0.5 M in MeOH),
2
2
H O (4 ml) and the reaction time at reflux was 96 h. This afforded
2
2
2
42 mg (91%) of (RS)-36 as colourless crystals.
Mp: 100 °C. IR (KBr): m
1137, 697, 668 cm
~ = 3436, 2976, 1701, 1458, 1420, 1362,
. H NMR (500 MHz, CDCl , 24.5 °C, TMS):
3
4
À1
1
d = 1.16 (s, 3H, CH ), 1.19 (s, 3H, CH ), 1.73–1.83 (m, 2H, N–CH–
3
3
2 2 2 2 2
CH , N–CH –CH ), 1.83–1.91 (m, 1H, N–CH –CH ), 1.98–2.07 (m,
4
.1.9.1. (2RS)-2-{(2RS)-1-[(Benzyloxy)carbonyl]pyrrolidine-2-yl}
1H, N–CH–CH ), 3.28 (dt, J = 11.2/7.1 Hz, 1H, N–CH ), 3.72–3.81
2
2
46
propionic acid [rac-(l)-34] und (2SR)-2-{(2RS)-1-[(benzyloxy)
carbonyl]pyrrolidine-2-yl}-propionic acid [rac-(u)-34]. According
to GP7, method
2
(m, 1H, N–CH ), 4.37 (dd, J = 8.2/3.9 Hz, 1H, N–CH), 5.07–5.14 (m,
1
3
2H, CH –Ar), 7.28–7.38 (m, 5H, H ) ppm. C NMR (500 MHz,
2
ar
A
with [rac-(l)-28]46/[rac-(u)-28] (567 mg,
3 3 3 2
CDCl , 25.8 °C, TMS): d = 21.50 (CH ), 22.66 (CH ), 24.13 (N–CH –
1
2
.95 mmol), KOH (130 ml, 0.5 M in MeOH), H O (26 ml) heated for
2 2 2
CH ), 27.82 (N–CH–CH ), 47.39 (C–COO), 48.08 (N–CH ), 63.25
3
0 h at 92 °C. This afforded 486 mg (90%) of the diastereomeric
(N–CH), 67.02 (CH –Ar), 127.98 (br s, 2 CH ), 128.47 (CH ),
2
ar
ar
+
mixture [rac-(l)-34]/[rac-(u)-34] as colourless crystals.
Mp: 118 °C. IR (KBr):
~ = 3090, 2974, 2880, 1724, 1668, 1585,
136.72 (C ), 156.35 (N–CO–O), 182.61 (COO) ppm. MS (CI, CH );
ar
5
+
m
m/z (%) = 292 (28) [M+H] , 274 (19), 248 (50), 195 (38), 184 (30),
1
1
6
1
1
CH
1
(
(
500, 1458, 1429, 1361, 1344, 1316, 1286, 1267, 1205, 1166,
137, 1109, 1029, 1006, 988, 960, 909, 802, 768, 748, 699, 630,
158 (100), 145 (51), 127 (59). Anal. calcd for C16H21NO (291.35):
C, 65.96; H, 7.27; N, 4.81. Found: C, 65.68; H, 7.39; N, 4.58.
4
À1
1
02, 476 cm
.08 (m, 0.56 Â 3H, CH
.75–1.94 (m, 3H, CH –CH
–CH–N), 2.74–3.24 (m, 0.56 Â 1H, CH–COO), 3.31–3.43 (m,
.44H, N–CH , CH–COO), 3.51–3.69 (m, 1H, N–CH ), 4.10–4.14
m, 0.44 Â 1H, CH–N), 4.29–4.34 (m, 0.56 Â 1H, CH–N), 5.07–5.19
m, 2H, CH –Ar), 7.28–7.38 (m, 5H, Har) ppm, diastereomeric ratio
.
H NMR (500 MHz, CDCl
), 1.11–1.19 (m, 0.44 Â 3H, CH
–N, CH –CH–N), 1.96–2.05 (m, 1H,
3
, 19.9 °C, TMS): d = 1.01–
3
3
),
4.1.9.4. 1-{(2RS)-1-[(Benzyloxy)carbonyl]pyrrolidine-2-yl}cyclo-
2
2
2
butane-1-carboxylic acid [(RS)-37].
According to GP7.
2
Method A with (RS)-31 (753 mg, 2.37 mmol), KOH (132 ml, 0.5 M
2
2
in MeOH), H O (26 ml) and the reaction time was 24 h at 92 °C.
2
This afforded 664 mg (92%) of (RS)-37 as colourless crystals.
Mp: 87 °C. IR (KBr):
1194, 1116, 771, 735, 698, 606 cm
25.3 °C, TMS): d = 1.67–2.04 (m, 6H, N–CH –CH , N–CH–CH ,
m
~ = 3422, 2952, 1700, 1419, 1354, 1281,
2
+
+
À1 1
dr = 56:44 (l:u). MS (CI, CH
5
); m/z (%) = 278 (52) [M+H] , 260 (10),
. H NMR (500 MHz, CDCl3,
2
1
2
34 (100), 204 (9), 173 (5), 170 (24), 160 (12), 155 (6), 144 (46),
2
2
2
+
42 (18), 127 (18), 105 (9). MS (EI, 70 eV); m/z (%) = 277 (1) [M] ,
2 2 2
CH ), 2.16–2.30 (m, 3H, CH ), 2.34–2.42 (m, 1H, CH ), 3.36–3.43
09 (8), 204 (10), 160 (16), 142 (5), 111 (6), 105 (12), 97 (15), 94
2 2
(m, 1H, N–CH ), 3.65–3.73 (m, 1H, N–CH ), 4.34–4.38 (m, 1H, N–
13
(
(
71), 91 (100), 85 (10), 83 (13), 81 (10), 79 (7), 77 (7), 73 (5), 71
17), 70 (18), 69 (22), 67 (8), 65 (15), 57 (29), 55 (26). HRMS (70 eV):
CH), 5.12–5.19 (m, 2H, CH –Ar), 7.28–7.39 (m, 5H, H ) ppm.
C
2
ar
3 2
NMR (400 MHz, CDCl , 21.2 °C, TMS): d = 16.41 (N–CH–CH ),
23.92 (N–CH –CH ), 28.37 (CH ), 29.29 (CH ), 48.07 (N–CH ),
2 2 2 2 2
53.56 (C ), 61.72 (N–CH), 67.10 (CH –Ar), 127.99 (CH ), 128.47
q 2 ar
+
calcd for C15
H19NO
4
[M] , 277.1314; Found 277.1314.
1
The analytical data of the rac-(l)-diastereomers ( H NMR) were
in accord with the values reported in the literature for a pure like
enantiomer.
ar ar
(CH ), 136.65 (C ), 156.40 (O–CO–N), 181.84 (COOH) ppm. MS (CI,
CH ); m/z (%) = 304 (84) [M+H] , 286 (43), 260 (100), 210 (5), 204
5
4
6
+
+
Please cite this article in press as: Steffan, T.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.01.035

2
0
T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
(
18), 196 (37), 170 (25), 168 (11), 160 (18), 123 (10).
dryness in vacuum to afford 67.6 mg (95%) of rac-(l)-41 as colour-
less crystals.
HRMS (70 eV): calcd for
C
17
H21NO
4
[M]+, 303.1471; found:
3
03.1469.
Mp: 87 °C (decomp.). IR (KBr):
586, 1522, 1498, 1455, 1419, 1359, 1287, 1241, 1212, 1119, 1061,
m
~ = 3322, 3064, 3033, 2958, 1720,
1
1
À1
1
040, 1028, 982, 914, 824, 769, 738, 697, 603 cm
NO , 140 °C): d = 1.78–1.89 (m, 1H, N–CH
), 1.97–2.08 (m, 1H, N–CH –CH ), 2.13–2.22 (m, 2H,
N–CH–CH ), 3.44–3.53 (m, 1H, N–CH ), 3.64–3.73 (m, 1H,
N–CH ), 4.41–4.47 (m, 1H, N–CH), 5.08 (dd, J = 8.6/2.9 Hz, 1H,
CH–COO), 5.14–5.30 (m, 4H, CH –Ar), 6.40 (br s, 1H, NH), 7.23–
.
H
4
.1.9.5. (2RS)-2-Hydroxy-2-{(2RS)-1-[(benzyloxy)carbonyl]pyr-
NMR (400 MHz, C
CH
6
D
5
2
2
–
59
rolidine-2-yl}acetic acid [rac-(l)-39]
.
According to GP7,
2
2
2
method B with rac-(l)-38 (283 mg, 0.96 mmol), LiOH (71 mg,
mmol, 2.4 ml, 1.2 M in H O), dioxane (21.7 ml) and the reaction
time was 1.5 h. Unlike the GP 6, the extraction at pH 1 was first
done with CH Cl
(3 Â 50 ml) and later with EtOAc (2 Â 50 ml).
CC (SiO , pentane/EtOAc/HOAc = 20:80:2) of the crude product
afforded 217 mg (81%) of rac-(l)-39 as colourless oil.
TLC: R = 0.26 (SiO , Pentane/EtOAc/HOAc = 20:80:2). IR (KBr):
2
2
3
2
2
2
2
2
+
7
.47 (m, 10H, Har) ppm. MS (ESI); m/z (%) = 435 (64) [M+Na] ,
13 (86) [M+H] , 369 (100). Anal. calcd for C22H N O6 (412.45):
24 2
2
+
4
C, 64.07; H, 5.87; N, 6.79. Found: C, 63.82; H, 6.07; N, 6.60.
f
2
m
~ = 3419, 2957, 1682, 1498, 1426, 1360, 1203, 1124, 1078, 1048,
4
.1.9.8. (2SR)-2-[(tert-Butoxycarbonyl)amino]-2-{(2RS)-1-[(tert-
À1
1
1
CDCl
2
029, 911, 770, 736, 698, 669, 606, 552 cm
, 19.6 °C, TMS): d = 1.96–1.81 (m, 1H, N–CH
.06 (m, 2H, N–CH –CH , N–CH–CH ), 2.08–2.19 (m, 1H, N–CH–
CH ), 3.36–3.44 (m, 1H, N–CH ), 3.50–3.59 (m, 1H, N–CH ), 4.16–
.29 (m, 1H, N–CH), 4.57 (d, J = 1.8 Hz, 0.79 Â 1H, CH–O), 4.68 (br
s, 0.21 Â 1H, CH–O), 5.06–5.22 (m, 2H, CH –Ar), 7.27–7.40 (m,
.
H NMR (400 MHz,
butoxy)carbonyl]pyrrolidine-2-yl}acetic acid [rac-(u)-44]. Accord-
ing to GP7, method B with rac-(u)-43 (56 mg, 0.15 mmol), LiOH
3
2
–CH ), 1.85–
2
2
2
2
(
2
29 mg, 1.2 mmol, 1.0 ml, 1.2 M in H O), dioxane (3 ml) and the
2
2
2
reaction time was 1.5 h. This afforded 49.8 mg (93%) of rac-(u)-44
as colourless oil.
4
2
IR (KBr):
054, 864, 774 cm
m
~ = 3441, 2979, 1718, 1508, 1395, 1367, 1255, 1165,
1
3
5
H, Har) ppm. Rotamers ratio (19.6 °C) 79:21. C NMR (400 MHz,
CDCl , 20.4 °C, TMS): d = 24.18 (N–CH –CH ), 27.18 (N–CH–CH ),
7.60 (N–CH ), 61.09 (N–CH), 67.60 (CH –Ar), 72.32 (CH–COO),
27.85 (CHar), 128.19 (CHar), 128.55 (CHar), 136.10 (Car), 156.79
À1
1
1
.
H
NMR (400 MHz,
), 1.54 (s, 9H, CH ), 1.83–2.05 (m, 3H, N–CH
), 3.31–3.40 (m,
), 4.25–4.34 (m, 2H, N–CH–
2 2 4
C D Cl , 120 °C):
3
2
2
2
d = 1.50 (s, 9H, CH
CH
3
3
2
–
4
1
2
2
2
, N–CH–CH
2
), 2.06–2.18 (m, 1H, N–CH–CH
), 3.47–3.56 (m, 1H, N–CH
2
2
1
H, N–CH
2
+
(
[
(
O–CO–N), 175.07 (COO) ppm. MS (CI, CH
5
); m/z (%) = 280 (100)
M+H] , 236 (84), 204 (13), 172 (92), 160 (13), 146 (25), 144
10), 123 (13), 108 (39). Anal. calcd for C14 (279.30): C,
13
CH
C
2
, CH–COO), 5.92 (br s, 1H, NH) ppm. C NMR (400 MHz,
Cl 120 °C): d = 23.29 (N–CH –CH ), 28.39 (CH ), 28.47
), 29.47 (N–CH–CH ), 47.14 (N–CH ), 57.83 (N–CH), 57.85
N–CH), 80.52 (C ), 80.67 (C ), 155.80 (O–CO–N), 156.32 (O–CO–
+
2
D
2
4
,
2
2
3
H17NO
5
(
(
CH
3
2
2
6
1.42; H, 6.53; N, 4.78. Found: C, 61.18; H, 6.61; N, 4.76.
1
q
q
The H NMR data was in accord with the literature values given
+
N), 171.88 (COO) ppm. MS (ESI); m/z (%) = 367 (100) [M+Na] ,
for the diastereomeric mixture rac-(l)-39/rac-(u)-39.5
9
+
+
3
3
45 (12) [M+H] . HRMS (FAB, NBA): calcd for C16
45.2026; found: 345.1985.
H
29
N
2
O
6
[M+H] ,
4
.1.9.6. (2SR)-2-Hydroxy-2-{(2RS)-1-[(benzyloxy)carbonyl]pyr-
59
rolidine-2-yl}-acetic acid [rac-(u)-39]
.
According to GP7,
4.1.9.9. (2RS)-2-Hydroxy-2-{[(2RS)(2RS)-1-(4,4-diphenylbut-3-
en-1-yl)]pyrrolidine-2-yl}acetic acid [rac-(l)-13b]. Accord-
ing to GP7, method C: rac-(l)-46 (22 mg, 60 mol) in dioxane
(6 ml) and aq LiOH (57 mg, 2.4 mmol) at rt for 2 h. This afforded
20 mg (95%) of rac-(l)-13b as colourless crystals. Mp: 75 °C.
method B with rac-(u)-38 (280 mg, 0.96 mmol), LiOH (71 mg,
mmol, 2.4 ml, 1.2 M in H O), dioxane (21.5 ml) and the reaction
time was 1.5 h. Unlike the GP6, the extraction at pH 1 was first
done with CH Cl
(3 Â 50 ml) and later with EtOAc (2 Â 50 ml).
CC (SiO , acetone/pentane/EtOAc/HOAc = 25:50:25:1) of the crude
product afforded 269 mg (81%) of [rac-(u)-39] as colourless oil.
IR (KBr):
~ = 3441, 2960, 1680, 1426, 1359, 1197, 1121, 983,
13, 737, 698, 669, 603, 546 cm
0.8 °C, TMS): d = 1.76–1.89 (m, 1H, N–CH
H, N–CH –CH , N–CH–CH
), 2.09 (s, 0.58 Â 1H, OH), 2.17 (s,
.42 Â 1H, OH), 2.18–2.31 (m, 1H, N–CH–CH ), 3.40–3.48 (m, 1H,
), 3.50–3.60 (m, 1H, N–CH ), 4.10–4.16 (m, 1H, CH–OH),
.16–4.23 (m, 1H, N–CH), 5.13 (br s, 2H, CH –Ar), 7.29–7.40 (m,
, 21.4 °C, TMS): d = 23.96
), 60.43 (N–CH), 67.71 (CH –Ar),
3.69 (CH–COO), 127.93 (CHar), 128.18 (CHar), 128.53 (CHar),
3
2
l
2
2
IR (KBr):
1364, 1122, 1074, 1029, 763, 701, 632 cm
CD OD, 20.9 °C): d = 1.78–2.08 (m, 4H, N–CH
J = 7.9/7.5 Hz, 2H, C@CH–CH ), 2.97–3.08 (m, 1H, N–CH
CH ), 3.10–3.22 (m, 1H, N–CH –CH
CH –CH –CH , N–CH –CH –CH), 3.65–3.75 (m, 1H, N–CH–CH
m
~ = 3406, 3054, 3023, 2957, 2925, 1619, 1494, 1444,
2
À1
1
.
H NMR (400 MHz,
m
3
2
–CH –CH ), 2.60 (dt,
2
2
À1
1
9
2
2
0
.
H NMR (400 MHz, CDCl
3
,
2
2
–CH
2
–
),
–CH), 3.44–3.59 (m, 2H, N–
2
–CH ), 1.93–2.10 (m,
2
2
2
2
2
2
2
2
2
2
2
2
2
4.22 (d, J = 3.3 Hz, 1H, CH–OH), 6.11 (t, J = 7.5 Hz, 1H, C@CH),
2
1
3
N–CH
2
2
7.19–7.48 (m, 10H,
21.9 °C): d = 22.77 (N–CH
(C@CH–CH ), 54.42 (N–CH
H
ar
)
ppm.
–CH –CH
), 55.04 (N–CH
2
C
NMR (400 MHz, CD
), 24.72 (N–CH–CH ), 27.30
), 68.83 (CH–OH),
3
OD,
4
5
2
2
2
2
2
1
3
H, Har) ppm. C NMR (500 MHz, CDCl
), 29.02 (CH ), 47.65 (N–CH
3
2
2
(
CH
2
2
2
2
71.65 (N–CH), 123.33 (C@CH), 128.38 (CHar), 128.63 (CHar),
128.76 (CHar), 129.26 (CHar), 129.73 (CHar), 130.71 (CHar), 140.71
7
1
+
36.05 (Car), 157.13 (O–CO–N), 174.88 (COO) ppm. MS (CI, CH
m/z (%) = 280 (4) [M+H] , 262 (6), 236 (6), 218 (6), 215 (17), 181
5
);
(C
q q q
), 143.11 (C ), 147.21 (C ), 176.09 (COO) ppm. MS (ESI); m/z
+
+
+
(%) = 374 (100) [M+Na] , 352 (75) [M+H] , 276 (9), 129 (17).
HRMS (FAB, NBA): calcd for C22
352.1901.
+
(
(
C
6), 172 (100), 169 (9), 155 (8), 146 (10), 141 (10), 130 (31), 128
3
H26NO [M+H] , 352.1913; found:
71), 126 (9), 123 (15), 108 (41). HRMS (FAB, NBA): calcd for
+
14
H
18NO
5
[M+H] , 280.1185; Found: 280.1140.
1
The H NMR data was in accord with the literature for the dia-
4.1.9.10. (2SR)-2-Hydroxy-2-{[(2RS)-1-(4,4-diphenylbut-3-en-1-
yl)]pyrrolidine-2-yl}acetic acid [rac-(u)-13b]. According to
GP7, method C: rac-(u)-46 (17 mg, 47 mol) in dioxane (6 ml)
and aq LiOH (57 mg, 2.4 mmol) at rt for 2 h. Recrystallisation of
stereomeric mixture rac-(l)-39/rac-(u)-39.59
l
4
.1.9.7. (2RS)-2-[(Benzyloxycarbonyl)amino]-2-{(2RS)-1-[(ben-
zyloxy)carbonyl]-pyrrolidine-2-yl}acetic acid [rac-(l)-41]. Accord-
ing to GP7, method B: rac-(l)-40 (74 mg, 1.73 mmol) together
the crude product in hexane/CH
rac-(u)-13b as colourless crystals.
2
Cl
2
afforded 15.8 mg (96%) of
~ = 3427, 2924, 1624, 764, 702 cm . 1
À1
with LiOH (57 mg, 2.4 mmol, 2.0 ml, 1.2 M in H
6 ml) was stirred at rt for 2 h. The reaction mixture was acidified
to pH = 2 with 2 M HCl and then extracted with CH Cl
(3 Â 10 ml).
The organic phase was pooled, dried over MgSO and evaporated to
2
O) and dioxane
Mp: 44 °C. IR (KBr):
NMR (400 MHz, CD Cl
CH–CH ), 1.83–1.91 (m, 2H, N–CH
N–CH–CH ), 2.52–2.58 (m, 3H, N–CH
m
H
(
2
2
, 17.7 °C, TMS): d = 1.73–1.81 (m, 1H, N–
–CH –CH ), 2.04–2.13 (m, 1H,
–CH –CH , C@CH–CH ),
2
2
2
2
2
2
4
2
2
2
2
2
Please cite this article in press as: Steffan, T.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.01.035

T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
21
2
5
CH
.82 (dt, J = 12.6/6.0 Hz, 1H, N–CH
.5 Hz, 1H, N–CH –CH –CH ), 3.33 (dt, J = 11.0/5.5 Hz, 1H, N–
–CH –CH), 3.40 (ddd, J = 8.8/7.1/5.5 Hz, 1H, N–CH), 4.11 (d,
J = 5.5 Hz, 1H, CH–OH), 6.04 (t, J = 7.1 Hz, 1H, C@CH), 7.18–7.46
2
–CH
2
–CH), 3.26 (dt, J = 11.0/
crude product in heptane/CH
13d as colourless crystals.
Mp: 62 °C (decomp.). IR (KBr): m
~ = 3431, 2926, 2853, 1608, 1508,
1463, 1302, 1250, 1176, 1034, 827, 583 cm
CH Cl , 19.9 °C, CD Cl ): d = 1.88–2.21 (m, 4H, N–CH –CH –CH ),
2 2 2 2 2 2 2
2 2 2 2 2
2.81–3.00 (m, 2H, N–CH –CH –CH , N–CH –CH –O), 3.23–3.48
2 2 2 2 2 2
(m, 5H, N–CH, N–CH –CH –CH , N–CH –CH –O, O–CH ), 3.74–
2
Cl
2
afforded 23 mg (99%) of rac-(l)-
2
2
2
2
2
À1
1
. H NMR (400 MHz,
1
3
(
m, 10H, Har) ppm. C NMR (500 MHz, CD
d = 23.33 (N–CH –CH –CH ), 25.78 (N–CH–CH
CH –CH), 53.72 (N–CH ), 53.88 (N–CH ), 66.51 (CH–OH), 67.52
N–CH), 122.95 (C@CH), 127.78 (CHar), 127.97 (CHar), 128.01
CHar), 128.59 (CHar), 128.94 (CHar), 129.98 (CHar), 139.69 (C ),
42.17 (C ), 146.40 (C ), 175.56 (COO) ppm. MS (ESI); m/z
%) = 374 (9) [M+Na] , 352 (100) [M+H] . HRMS (FAB, NBA): calcd
2
Cl
2
, 20.3 °C, TMS):
2
2
2
2
), 27.43 (N–CH
2
–
2
2
2
(
(
1
3 ar
3.79 (m, 10H, O–CH , CH–OH), 6.82–6.87 (m, 6H, H ), 7.31–7.36
1
3
q
ar 2 2
(m, 6H, H ) ppm. C NMR (500 MHz, CD Cl , 25.8 °C, TMS):
q
q
2 2 2 2 2
d = 23.03 (N–CH –CH –CH ), 30.11 (N–CH–CH ), 52.24 (N–CH –
CH –CH ), 55.66 (N–CH –CH –O, O–CH ), 59.43 (O–CH ), 67.48
2 2 2 2 3 2
(N–CH), 68.09 (CH–O), 87.41 (C ), 113.68 (CH ), 130.07 (CH ),
q ar ar
+
+
(
+
for C22
H26NO
3
[M+H] , 352.1913; Found 352.1876.
1
36.33 (Car), 159.11 (Car), 175.36 (COO) ppm. MS (ESI); m/z
+
+
4
.1.9.11.
(2RS)-2-Hydroxy-2-{(2RS)-1-[4,4-bis(3-methylthio-
(
%) = 544 (11) [M+Na] , 522 (2) [M+H] , 333 (100). HRMS (FAB,
+
phen-2-yl)but-3-en-1-yl]pyrrolidine-2-yl}acetic acid [rac-(l)-
NBA): calcd for C30
7
H36NO [M+H] , 522.2492; Found 522.2509.
1
3c].
mol) in dioxane (6 ml) and aq LiOH (57 mg, 2.4 mmol) at rt
O/MeCN =
5:25) afforded 11.8 mg (97%) of rac-(l)-13c as yellow oil.
IR (KBr):
~ = 3397, 2923, 2854, 1618, 1457, 1380, 1124, 1010,
32, 712 cm
According to GP7, method C: rac-(l)-47 (13 mg,
3
1 l
4
.1.9.14. (2SR)-2-Hydroxy-[(2RS)-1-{2-[tris(4-methoxyphenyl)
for 2 h. Purification of the crude product by CC (RP2, H
7
2
methoxy]-ethyl} pyrrolidine-2-yl]acetic acid [rac-(u)-13d]. Accord-
ing to GP7, method C: rac-(u)-48 (101 mg, 0.19 mmol) in dioxane
(6 ml) and aq LiOH (77 mg, 3.2 mmol) at rt for 2.5 h. This afforded
97 mg (98%) of rac-(u)-13d as colourless crystals.
m
À1
1
9
.
H
NMR (500 MHz, CD
–CH –CH ), 2.00 (s, 3H, CH
), 2.50–2.56 (m, 2H, C@CH–CH ), 2.77–2..85 (m, 1H, N–
–CH ), 2.85–2.94 (m, 1H, N–CH –CH –CH), 3.09–3.16 (m,
H, N–CH –CH –CH), 3.24 (br s, 1H, N–CH), 3.38–3.46 (m, 1H, N–
CH –CH –CH ), 3.61–3.68 (m, 1H, CH–OH), 6.00 (t, J = 7.4 Hz, 1H,
C@CH), 6.79 (d, J = 5.5 Hz, 1H, Har), 6.91 (d, J = 5.5 Hz, 1H, Har),
2
Cl
2
,
25.9 °C, TMS):
d = 2.01–2.21 (m, 4H, N–CH
H, CH
CH –CH
2
2
2
3
), 2.04 (s,
Mp: 84 °C (decomp.). IR (KBr): ~
302, 1250, 1176, 1092, 1032, 828, 584 cm
CD Cl , 20.1 °C, TMS): d = 1.75–1.83 (m, 1H, N–CH–CH
.93 (m, 2H, N–CH –CH –CH ), 2.08 (dq, J = 13.2/8.5 Hz, 1H, N–
CH–CH ), 2.62–2.69 (m, 1H, N–CH –CH –CH ), 2.75–2.83 (m, 1H,
N–CH –CH –O), 3.29–3.46 (m, 5H, N–CH, N–CH –CH –CH , N–
CH –CH –O, O–CH ), 3.78 (s, 9H, O–CH ), 4.11 (d, J = 5.5 Hz, 1H,
CH–OH), 6.83–6.87 (m, 6H, Har), 7.32–7.36 (m, 6H, Har) ppm. 13
NMR (500 MHz, CD Cl , 23.9 °C, CD Cl ): d = 23.07 (N–CH –CH
CH ), 25.41 (N–CH–CH ), 53.56 (N–CH ), 53.69 (N–CH ), 55.26
O–CH ), 59.13 (CH –O), 66.33 (N–CH), 67.23 (CH–OH), 86.89 (O–
), 113.26 (CHar), 129.70 (CHar), 135.88 (Car), 158.72 (Car),
m
= 3428, 2926, 1608, 1508, 1462,
À1
1
3
3
2
1
.
H NMR (400 MHz,
), 1.84–
2
2
2
2
2
2
2
2
1
2
2
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
3
7
.11 (d, J = 5.5 Hz, 1H, Har), 7.29 (d, J = 5.5 Hz, 1H, Har) ppm.
NMR (500 MHz, CD Cl , 22.4 °C, TMS): d = 14.60 (Ar–CH ), 15.08
), 23.04 (N–CH –CH ), 26.25 (N–CH–CH ), 26.96 (C@CH–
), 52.67 (N–CH –CH –CH, N–CH –CH –CH ), 68.19 (N–
C
2
2
2
3
2
2
3
C
(
Ar–CH
3
2
2
2
2
2
2
2
2
2
–
CH
2
2
2
2
2
2
2
2
2
2
CH, CH–O), 123.80 (Car), 125.22 (Car), 128.51 (C@CH), 130.41
(
3
2
(
C
ar), 131.66 (C@CH), 131.82 (Car), 134.44 (Car), 134.96 (Car),
C
1
3
5
q
+
1
36.55 (Car), 138.90 (Car), 175.34 (COO) ppm. MS (ESI); m/z
75.20 (COO) ppm. MS (FAB); m/z (%) = 522 (1) [M+H] , 391 (21),
+
+
+
(
%) = 414 (5) [M+Na] , 392 (100) [M+H] , 247 (15). HRMS (FAB,
33 (100). HRMS (FAB, NBA): calcd for
22.2492; found: 522.2518.
30 7
C H36NO [M+H] ,
+
3 2
NBA): calcd for C20H26NO S [M+H] , 392.1354; found: 392.1403.
4
.1.9.12. (2SR)-2-Hydroxy-2-{(2SR)-1-[4,4-bis(3-methylthiophen-
4
.1.9.15. (2RS)-2-Amino-2-{[(2RS)-1-(4,4-diphenylbut-3-en-1-yl)]
2-yl)but-3-en-1-yl]pyrrolidine-2-yl} acetic acid [rac-(u)-13c]. Accord-
pyrrolidine-2-yl}-acetic acidÁ2TFA [rac-(l)-14bÁ2TFA]. Accord-
ing to GP7, method C with rac-(l)-51 (35.6 mg, 77 mol) in dioxane
6 ml) and aq LiOH (77 mg, 3.2 mmol, 1.6 M) at rt for 2 h. Followed
by the procedure according to GP5 in CH Cl (2 ml) and TFA (6.1 g,
ing to GP7, method C: rac-(u)-47 (22 mg, 60
l
mol) in dioxane
l
(
6 ml) and aq LiOH (57 mg, 2.4 mmol) at rt for 2 h. This afforded
(
1
1.2 mg (96%) of rac-(u)-13c as yellow oil.
IR (KBr):
NMR (500 MHz, CD
H, N–CH –CH –CH
s, 3H, CH ), 2.04–2.21 (m, 4H, CH
J = 7.7 Hz, 2H, C@CH–CH ), 2.67 (dt, J = 11.0/8.2 Hz, 1H, N–CH
CH –CH ), 2.86 (dt, J = 12.6/7.1 Hz, 1H, N–CH –CH –CH), 3.30 (dt,
J = 12.6/8.2 Hz, 1H, N–CH –CH –CH), 3.38–3.47 (m, 2H, N–CH, N–
CH –CH –CH ), 4.11 (d, J = 5.5 Hz, 1H, CH–OH), 6.02 (t, J = 7.7 Hz,
H, C@CH), 6.80 (d, J = 5.5 Hz, 1H, Har), 6.91 (d, J = 5.5 Hz, 1H, Har),
2
2
~ = 3421, 2922, 1618, 1106, 1007, 933, 714 cm . 1
À1
m
H
4 ml, 54 mmol) afforded 25.9 g (97%) rac-(l)-14bÁ2TFA as yellow
2
Cl
), 1.87–1.94 (m, 2H, N–CH
, N–CH –CH
2
, 21.4 °C, TMS): d = 1.77 (dq, J = 13.7/7.1 Hz,
–CH –CH ), 2.00
–CH ), 2.56 (q,
oil.
À1
1
(
2
2
2
2
2
2
~
m = 3424, 2926, 1676, 1493, 1202, 1130, 763, 701 cm
IR (KBr):
H NMR (500 MHz, D
CH ), 2.00–2.12 (m, 2H, N–CH
CH–CH ), 2.57–2.63 (m, 2H, N–CH
N–CH –CH –CH ), 3.15–3.25 (m, 1H, N–CH
m, 2H, N–CH –CH –CH, N–CH –CH –CH ), 3.76–3.83 (m, 1H, N–
CH–CH ), 3.87 (d, J = 8.8 Hz, 1H, CH–COO), 6.16 (t, J = 7.4 Hz, 1H,
C@CH), 7.27–7.54 (m, 10H, Har) ppm. C NMR (400 MHz, D
.
1
3
3
2
2
2
O, 23.7 °C): d = 1.90–2.00 (m, 1H, N–CH–
–CH –CH ), 2.22–2.32 (m, 1H, N–
–CH –CH), 2.97–3.06 (m, 1H,
–CH –CH), 3.36–3.51
2
2
2
–
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
(
2
2
2
2
2
1
7
2
1
3
1
3
.12 (d, J = 5.5 Hz, 1H, Har), 7.30 (d, J = 5.5 Hz, 1H, Har) ppm.
Cl , 21.5 °C, TMS): d = 14.59 (Ar–CH ), 15.07
), 23.46 (N–CH –CH –CH ), 25.91 (N–CH –CH
7.72 (C@CH–CH ), 53.62 (N–CH –CH –CH), 54.13 (N–CH
C
2
O,
),
), 67.85 (N–CH–
2
), 123.28 (C@CH), 127.91 (CHar), 128.47 (CHar), 128.58 (CHar),
NMR (500 MHz, CD
Ar–CH
2
2
3
1
9.1 °C, MeOH): d = 22.62 (CH
2 2 2
), 23.01 (CH ), 26.40 (C@CH–CH
(
2
3
2
2
2
2
2
–CH
–CH
2
),
–
5
2.66 (CH–COO), 53.53 (N–CH
2
), 56.20 (N–CH
2
2
2
2
2
2
CH
2
CH ), 66.80 (CH–OH), 67.36 (N–CH), 123.86 (CHar), 125.32 (CHar),
1
29.23 (CHar), 129.38 (CHar), 130.34 (CHar), 139.77 (Car), 142.29
1
1
28.48 (C@CH), 130.37 (CHar), 131.84 (CHar), 131.87 (C@CH),
34.45 (Car), 135.01 (Car), 136.55 (Car), 138.83 (Car), 175.54 (COO)
(
C
ar), 146.20 (C@CH), 163.89 (COO) ppm. MS (ESI); m/z (%) = 373
+
+
(
20) [M+Na] , 351 (100) [M+H] , 276 (29). HRMS (FAB, NBA): calcd
+
+
+
ppm. MS (ESI); m/z (%) = 414 (56) [M+Na] , 392 (100) [M+H] , 316
27 2 2
for C22H N O [M+H] , 351.1994; found: 351.2033.
(
[
4), 294 (5), 247 (17). HRMS (FAB, NBA): calcd for C20
M+H] , 392.1354; found: 392.1392.
H26NO
3
S
2
+
4.1.9.16. (2SR)-2-Amino-2-[(2RS)-1-(4,4-diphenylbut-3-en-1-yl)
Accord-
pyrrolidine-2-yl] acetic acidÁ2TFA [rac-(u)-14bÁ2TFA].
4
.1.9.13. (2RS)-2-Hydroxy-[(2RS)-1-{2-[tris(4-methoxyphenyl)
methoxy]-ethyl}-pyrrolidine-2-yl]acetic acid [rac-(l)-13d]. Accord-
ing to GP7, method C: rac-(l)-48 (23 mg, 44 mol) in dioxane (6 ml)
and aq LiOH (77 mg, 3.2 mmol) at rt for 2 h. Recrystallisation of the
ing to GP7, method C with rac-(u)-51 (80 mg, 0.17 mmol) in diox-
ane (6 ml) and aq LiOH (77 mg, 3.2 mmol, 1.6 M) at rt for 2 h.
Followed by the procedure according to GP5 in CH Cl2 (2 ml) and
TFA (6.1 g, 4 ml, 54 mmol). Here the reaction mixture was
l
2
Please cite this article in press as: Steffan, T.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.01.035

2
2
T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
extracted in CH
phase was dried over MgSO
2.5 mg (87%) of rac-(u)-14bÁ2TFA as yellow oil.
2
Cl
2
(2 Â 10 ml) at pH = 7 and pH = 6, the organic
Har), 6.89 (d, J = 5.3 Hz, 1H, Har), 7.24 (d, J = 5.3 Hz, 1H, Har), 7.41
1
3
4
and concentrated in vacuum to afford
(d, J = 5.3 Hz, 1H, Har) ppm. C NMR (500 MHz, D
24.8 °C, MeOD): d = 14.60 (CH ), 14.95 (CH ), 23.68 (N–CH
CH ), 27.80 (N–CH–CH ), 28.00 (N–CH –CH –CH), 53.91 (CH–
COO), 54.29 (N–CH –CH –CH), 54.79 (N–CH –CH –CH ), 67.95
(N–CH–CH ), 124.60 (CHar), 126.15 (CHar), 129.96 (C@CH), 131.03
2
O/MeOD = 1:1,
5
3
3
2 2
–CH –
À1
À1 1
IR (KBr):
NMR (400 MHz, D
.83–1.94 (m, 1H, N–CH
m
~ = 3442 cm , 2924, 1683, 1635, 1205, 1130 cm . H
2
2
2
2
2
O, 20.8 °C): d = 1.64–1.75 (m, 1H, N–CH–CH
–CH –CH ), 1.94–2.06 (m, 1H, N–CH
), 2.20–2.31 (m, 1H, N–CH–CH
–CH), 2.95 (dt, J = 11.2/8.1 Hz, 1H, N–CH
–CH –CH, N–CH –CH –CH
–CH), 3.69 (m, 1H, N–CH–CH
J = 3.7 Hz, 1H, CH–COO), 6.11 (t, J = 7.7 Hz, 1H, C@CH), 7.26–7.51
2
),
2
2
2
2
2
1
2
2
2
2
–
2
CH
CH
2
–CH
–CH
2
2
), 2.61–2.71 (m, 2H, N–
–CH –CH ),
), 3.54–3.64
), 4.10 (d,
(CHar), 132.10 (C@CH), 132.51 (CHar), 135.67 (Car), 137.30 (Car),
139.39 (Car), 135.29 (Car), 175.68 (COO) ppm. MS (ESI); m/z
2
2
2
2
2
+
+
3
.16–3.27 (m, 2H, N–CH
2
2
2
2
2
(%) = 413 (26) [M+Na] , 391 (100) [M+H] , 361 (19), 149 (25).
+
(
m, 1H, N–CH –CH
2
2
2
HRMS (ESI): calcd for C20
391.1509.
27 2 2 2
H N O S [M+H] , 391.1514; found:
1
3
(
m, 10H, Har) ppm. C NMR (400 MHz, D
d = 22.99 (N–CH –CH –CH ), 26.38 (C@CH–CH
CH ), 52.34 (CH–COO), 53.97 (N–CH ), 54.33 (N–CH
CH–CH ), 123.66 (C@CH), 128.02 (CHar), 128.50 (CHar), 128.54
CHar), 129.17 (CHar), 129.33 (CHar), 130.35 (CHar), 139.68 (Car),
2
O, 20.4 °C, dioxane):
), 26.48 (N–CH–
), 68.25 (N–
4.2. Biological evaluation
4.2.1. GABA uptake assays
2
2
2
2
2
2
2
2
[3H] GABA uptake assays with mGAT1, mGAT2, mGAT3, mGAT4
and hGAT-1 expressing HEK293 cells were performed as described
(
1
42.41 (Car), 146.65 (C@CH), 176.00 (COO) ppm. MS (ESI); m/z
+
54
(
%) = 351 (100) [M+H] , 276 (29). HRMS (FAB, NBA): calcd for
earlier. Stably hGAT-1 expressing HEK293 cells were obtained
+
C
22
H
27
N
2
O
2
[M+H] , 351.2073; found: 351.2045.
starting from the cDNA encoding hGAT-1 (kindly provided by Prof.
N. Nelson) which was subcloned from pBluescript into the mamma-
lian expression vector pcDNA3.1(+) (Invitrogen, Karlsruhe, Ger-
many) using EcoR I and Xho I. Transfections with the linearised
plasmid (Sca I) were performed as described earlier for mGAT1–4.54
4
2
1
3
1
.1.9.17. (2RS)-2-Amino-2-{(2RS)-1-[4,4-bis(3-methylthiophen-
-yl)but-3-en-1-yl]-pyrrolidine-2-yl}acetic acidÁ2 TFA [rac-(l)-
4cÁ2TFA]. According to GP7, method C with rac-(l)-52 (20 mg,
9 lmol) taken in dioxane (6 ml) and aq LiOH (57 mg, 2.4 mmol,
.2 M) at rt for 2 h. Followed by procedure according to GP5, in
Cl (2 ml) and TFA (6.1 g, 54 mmol, 4 ml) and on purification
of the crude product by CC (RP2, H O/MeCN/TFA = 60:40:1)
afforded 19.5 mg (73%) of rac-(l)-14cÁ2TFA as yellow oil.
IR (KBr):
~ = 3427, 2925, 1685, 1206, 1129, 834, 803, 721 cm
H NMR (500 MHz, D O, 21.8 °C): d = 1.97–2.17 (m, 9H, CH , N–
CH–CH , N–CH –CH ), 2.30–2.38 (m, 1H, N–CH–CH ), 2.58 (q,
J = 7.1 Hz, 2H, N–CH –CH –CH), 3.13–3.21 (m, 1H, N–CH ), 3.21–
.28 (m, 1H, N–CH –CH –CH), 3.39–3.48 (m, 1H, N–CH
CH), 3.57–3.65 (m, 1H, N–CH ), 3.84–3.90 (m, 1H, N–CH–CH
4
.2.2. MS-binding assays
CH
2
2
MS-binding assays with mGAT1 obtained from a stable HEK293
2
56
cell line and NO 711 as a marker in competitive binding experi-
55
ments were performed as described previously. The hGAT-1
À1
m
.
membrane preparation for the MS-binding assay was obtained in
1
2
3
55
the same way as described for mGAT1.
2
2
2
2
2
2
2
Acknowledgements
3
2
2
2
–CH
2
–
),
2
2
We would like to thank Prof. N. Nelson (University Tel Aviv) for
kindly providing us the cDNA for hGAT-1. We also like to thank
Katharina Heimberger for the editorial assistance and proof
reading.
3
.92–3.95 (m, 1H, CH–COO), 6.08 (t, J = 7.1 Hz, 1H, C@CH), 6.88
(
d, J = 5.2 Hz, 1H, Har), 7.00 (d, J = 5.2 Hz, 1H, Har), 7.26 (d,
13
J = 5.2 Hz, 1H, Har), 7.42 (d, J = 5.2 Hz, 1H, Har) ppm. C NMR
(
2
500 MHz, D
2.70 (N–CH
CH –CH –CH), 52.48 (CH–COO), 53.91 (N–CH
7.87 (N–CH–CH ), 117.05 (q, J = 291.7 Hz, CF
26.07 (CH), 128.56 (CH), 130.79 (CH), 131.67 (Car), 132.34 (CH),
34.61 (Car), 135.54 (Car), 137.14 (Car), 138.95 (C@CH), 163.52 (q,
2
O, 21.4 °C, MeOH): d = 14.40 (CH
–CH –CH ), 26.62 (N–CH –CH –CH
), 56.01 (N–CH
3
), 14.89 (CH
3
),
2
2
2
2
2
2
), 27.50 (N–
),
References and notes
2
2
2
2
6
1
1
2
3
), 124.58 (CH),
1
2
3
.
.
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3
–COO), 170.62 (CH–COO) ppm. MS (ESI); m/z
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+
+
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2
3
47 (25). HRMS (FAB, NBA): [M+H] required for C20
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H
27
N
2
O
2
S ,
2
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According to GP7, method C with rac-(u)-52
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1
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l
2
.4 mmol, 1.2 M) and stirred at rt for 2 h. Followed by procedure
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according to GP5, the residue taken in CH
(
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1
1
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08, 734 cm
d = 1.63–1.73 (m, 1H, N–CH–CH
m
~ = 3433, 2924, 2854, 1629, 1459, 1202, 1175, 1130,
À1
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.
H
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–CH), 2.99 (dt, J = 11.2/7.9 Hz, 1H, N–CH –CH –CH ),
–CH –CH), 3.37–3.45 (m, 1H,
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CH–COO), 6.10 (t, J = 7.5 Hz, 1H, C@CH), 6.87 (d, J = 5.3 Hz, 1H,
D
2
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–CH
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Please cite this article in press as: Steffan, T.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.01.035

T. Steffan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
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Please cite this article in press as: Steffan, T.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.01.035