Azetidinic amino acids: Stereocontrolled synthesis and pharmacological characterization as ligands for glutamate receptors and transporters

Article abstract of DOI:10.1039/b509514j

A set of ten azetidinic amino acids, that can be envisioned as C-4 alkyl substituted analogues of trans-2-carboxyazetidine-3-acetic acid (t-CAA) and/or conformationally constrained analogues of (R)- or (S)-glutamic acid (Glu) have been synthesized in a diastereo- and enantiomerically pure form from ss-amino alcohols through a straightforward five step sequence. The key step of this synthesis is an original anionic 4-exo-tet ring closure that forms the azetidine ring upon an intramolecular Michael addition. This reaction was proven to be reversible and to lead to a thermodynamic distribution of two diastereoisomers that were easily separated and converted in two steps into azetidinic amino acids. Azetidines 35-44 were characterized in binding studies on native ionotropic Glu receptors and in functional assays at cloned metabotropic receptors mGluR1, 2 and 4, representing group I, II and III mGlu receptors, respectively. Furthermore, azetidine analogues 35, 36 and 40 were also characterized as potential ligands at the glutamate transporter subtypes EAAT1-3 in the FLIPR Membrane Potential (FMP) assay. The (2R)-azetidines 35, 37, 39, 41 and 43 were inactive in iGlu, mGlu and EAAT assays, whereas a marked change in the pharmacological profile at the iGlu receptors was observed when a methyl group was introduced in the C-4 position, compound 36 versus t-CAA. At EAAT1-3, compound 35 was inactive, whereas azetidines 36 and 40 were both identified as inhibitors and showed selectivity for the EAAT2 subtype. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b509514j

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A R T I C L E
Azetidinic amino acids: stereocontrolled synthesis and
pharmacological characterization as ligands for glutamate
receptors and transporters
Hans Bra¨uner-Osborne,b Lennart Bunch,*†b Nathalie Chopin,a Franc¸ois Couty,*a
Gwilherm Evano,a Anders A. Jensen,b Mie Kusk,b Birgitte Nielsenb and Nicolas Rabassoa
a SIRCOB, UMR CNRS 8086, Universite´ de Versailles, 45, avenue des Etats-Unis, 78035,
Versailles Cedex, France. E-mail: couty@chimie.uvsq.fr
b Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences,
Universitetsparken 2, DK-2100, Copenhagen, Denmark. E-mail: lebu@dfuni.dk
Received 6th July 2005, Accepted 30th August 2005
First published as an Advance Article on the web 29th September 2005
A set of ten azetidinic amino acids, that can be envisioned as C-4 alkyl substituted analogues of trans-2-
carboxyazetidine-3-acetic acid (t-CAA) and/or conformationally constrained analogues of (R)- or (S)-glutamic acid
(Glu) have been synthesized in a diastereo- and enantiomerically pure form from b-amino alcohols through a
straightforward five step sequence. The key step of this synthesis is an original anionic 4-exo-tet ring closure that
forms the azetidine ring upon an intramolecular Michael addition. This reaction was proven to be reversible and to
lead to a thermodynamic distribution of two diastereoisomers that were easily separated and converted in two steps
into azetidinic amino acids. Azetidines 35–44 were characterized in binding studies on native ionotropic Glu
receptors and in functional assays at cloned metabotropic receptors mGluR1, 2 and 4, representing group I, II and
III mGlu receptors, respectively. Furthermore, azetidine analogues 35, 36 and 40 were also characterized as potential
R
ligands at the glutamate transporter subtypes EAAT1–3 in the FLIPRꢀ Membrane Potential (FMP) assay. The
(2R)-azetidines 35, 37, 39, 41 and 43 were inactive in iGlu, mGlu and EAAT assays, whereas a marked change in the
pharmacological profile at the iGlu receptors was observed when a methyl group was introduced in the C-4 position,
compound 36 versus t-CAA. At EAAT1–3, compound 35 was inactive, whereas azetidines 36 and 40 were both
identified as inhibitors and showed selectivity for the EAAT2 subtype.
intramolecular Michael addition to form the four-membered
ring through carbon–carbon bond formation. The azetidinic
amino acids, 35–44, prepared via this novel methodology may
Introduction
Conformationally constrained amino acids (AAs) continue to
be a topic of extensive research for both synthetic and medic-
inal chemists, since such compounds may provide important
information about a ligand’s conformational binding mode
be envisioned as C-4 alkyl substituted analogues of trans-2-
carboxyazetidine-3-acetic acid (t-CAA)9 and/or conformation-
ally constrained analogues of (R)- or (S)-glutamic acid (Glu),
to a receptor or an enzyme, and are basic tools used in the
the latter being the major excitatory neurotransmitter in the
design of novel peptides and peptidomimetics.1 The synthesis
central nervous system (CNS).10 Activation of the glutaminergic
of novel amino acids in which the constraint is brought by
neurotransmitter system in the CNS is involved in important
a heterocyclic ring which holds the nitrogen atom of the
neurophysiological processes such as memory and learning,
amino acid moiety, has received considerable attention,2 as
motor functions, and neural plasticity and development. How-
illustrated by numerous synthetic approaches towards proline,3
ever in the diseased brain, it is believed that neurodegenerative
pipecolic acid,4 and glutamate analogues.5 In contrast, synthesis
diseases such as Alzheimer’s disease, dementia, Huntington’s
of the four-membered heterocyclic amino acid, norproline (2-
disease, amyotrophic lateral sclerosis (ALS), epilepsy and cere-
carboxyazetidine derivative) is underdeveloped,6 which may
bral stroke may be directly related to disordered glutamatergic
be attributed to the lack of efficient synthetic methodologies
neurotransmission originating from dysfunction of either the
for the preparation of functionalized azetidines, especially in
Glu receptors (iGluR or mGluR) and/or the Glu re-uptake
enantiomerically pure forms.7 In this paper, we report a new
system (EAATs).10,11 In the ongoing process to characterize and
synthetic route towards azetidinic amino acids of general
understand the function of the glutaminergic neurotransmitter
structure depicted in Fig. 1, in which the nature of the R
system in the CNS, we have characterized the synthesized
group located at C-4 comes from enantiomerically pure b-
azetidinic amino acids, 35–44 as ligands at the ionotropic Glu
amino alcohols.8 This synthesis relies on an unprecedented
receptors (iGluR), the metabotropic Glu receptors (mGluR) and
the Glu transporters (EAAT1–3),12,13 and present results, includ-
ing a structure–activity study (SAR) in the second part of this
paper.
† To whom correspondence regarding the glutamate research area
should be addressed.
Fig. 1
T h i s j o u r n a l i s
3 9 2 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 9 2 6 – 3 9 3 6
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

View Online
Results and discussion
Chemistry
Azetidinic amino acids 35–44 were prepared through a straight-
forward five step sequence starting from commercially available
(S)-b-amino alcohols 1–5 possessing an alkyl substituent of
various size. The first three steps include: (i) a N-benzylation
through reductive amination of benzaldehyde to give 6–10, (ii)
an alkylation with bromoacetonitrile yielding 12–16 (compound
11 was previously reported),14 (iii) a one-pot two-step Swern
oxidation and Wittig olefination giving the unsaturated amino
esters 17–22.15 The key step of this synthetic sequence is an
original anionic 4-exo-tet ring closure that forms the azetidine
ring upon treatment of these compounds with LiHMDS.16 This
reaction affords a mixture of 2,3-cis and 2,3-trans isomers in a
3 : 7 to 4 : 6 ratio. These diastereoisomers were easily separated by
chromatography and were finally submitted to acidic hydrolysis
followed by hydrogenolysis of the N-benzyl protecting group.
Purification through ion-exchange chromatography yielded the
desired amino acids 35–44 as white solids.
The relative configuration of the substituents in azetidines 25
and 26 was determined on the basis of NOE experiments. In
3
fact, the examination of the values of J coupling constants in
these heterocycles does not give clear cut information about
the relative stereochemistries. In the case of azetidines 23
and 24, the relative configurations were determined by X-ray
crystallography performed on 23.17 The relative configurations
in the other compounds were deduced from (i) the chemical
shift of the proton located at C-2, that constantly appears more
deshielded of nearly 1 ppm in the cis-isomers than in the trans-
isomers, (ii) the diastereomeric distribution of the products
since the cis-isomer is always the minor one, and, (iii) the
biological activity of the amino acid, the cis-isomer, presenting
the unnatural (R) absolute configuration for the amino acid
moiety being not active (vide supra) (Scheme 1).
The diastereoselectivity of this intramolecular Michael addi-
tion deserves comment. First of all, we were able to get some
experimental evidence for thermodynamic control operating in
the cyclization step. Indeed, when diastereoisomerically pure
compounds 25 or 26 were treated under the same conditions
used for ring closure (LiHMDS, −78 to 0 ◦C), an equilibration
occurred that gave in both cases a mixture of 25 and 26 in a
ratio almost identical to the one observed in the initial ring
closure. Furthermore, lithiation of 25 at −78 ◦C immediately
followed by deuteration with CD3OD gave a-deuterated ester at
the methylene position exclusively. These observations suggest
the occurrence of a retro-Michael process that induces a
base-catalyzed equilibration between 25 and 26. Secondly, the
exclusive formation of 3,4-trans isomers in these ring closures
can be explained by considering the different transition states
of this reaction. All of them involving substrate 18 (R = Me)
are depicted in Fig. 2. In the course of the cyclization following
˚
Scheme 1 Reagents and conditions: (a) PhCHO, 4 A MS, CH2Cl2
then NaBH4, EtOH (b) bromoacetonitrile, K2CO3, CH3CN (c) DMSO,
◦
=
(COCl)2, Et3N, CH2Cl2, −78 C, then (C6H5)3P CHCO2Et (d) LiH-
MDS, THF, −78 ◦C to 0 ◦C (e) 6N HCl–CCl4 (f) H2, Pd/C cat., EtOH.
transition state A, a severe A1,3 strain is developed between the
methyl substituent of the azetidine ring and the olefinic proton
of the unsaturated ester, forbidding this pathway. This is not the
case in transition state B, leading to 26. A similar A1,3 strain
occurs in C but not in D, leading to 25, the second isomer being
formed in the reaction. To summarize, cyclization would proceed
through transition states B and D, leading to 25 and 26, which
are in equilibrium.
Attempts to improve the diastereoselectivity of this cyclization
in favor of the 2,3-trans isomer, presenting the (S)-configuration
at C-2 were also done. The stereochemistry of the alkene
Michael acceptor in 18 was first switched to (Z) using Still–
Gennari methodology,18 but the cyclization step occurred for
this substrate with similar yields and diastereoselectivity, which
is in accordance with the reversibility of the mechanism. A more
encouraging result was obtained by replacing the nitrile by a
tert-butyl ester. Thus, compound 46, prepared in a similar way
Fig. 2 Possible transition states for the anionic ring-closure.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 9 2 6 – 3 9 3 6
3 9 2 7

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Scheme 2 Reagents and conditions: (a) NaI, tert-butylbromoacetate, DMF, rt, 76% (b) DMSO, (COCl)2, Et3N, CH2Cl2, −78 ◦C, then
(C6H5)3P CHCO2Et, 77% (c) t-BuOK, THF, −50 ◦C, 50% (de >95%).
=
as described for the amino nitrile cyclized upon treatment with
t-BuOK at −50 ◦C in THF to give a unique 2,3-trans isomer 47,
albeit with a modest yield of 50% (Scheme 2).
conformation when agonizing the AMPA and KAIN preferring
iGlu receptors,19 while an extended conformation of Glu is
the agonist conformation at the mGlu receptors.20 First, we
submitted the compounds t-CAA, 36, 38, 40, 42 and 44 to
a stochastic conformational search in the modelling software
program MOE (Table 2). An extended Glu conformation was
identified as the low-energy conformation for the parental
azetidinic system t-CAA and its C-4 analogues 36, 38, 40 and
44, whereas the C-4 azetidine analogue 42 was forced into
a folded Glu conformation, due to its small and bulky C-4
substituent (i-Pr). To fit the low-energy extended conformation
of t-CAA, 36, 38, 40 and 44 to the respective folded Glu
conformation, a systematic rotation of the 3-carboxymethyl
group was carried out. This suggested a very small energy
difference (average +0.49 kcal mol−1) between the low-energy
extended conformation and the respective folded conformations.
Secondly, we wanted to investigate the flexibility of the R group
at C-4. This was done by a systematic rotation of the C–C bonds
of this group, keeping the conformation of the ammonium and
two carboxylate groups fixed in the folded conformation. The
number of structures within +1 kcal mol−1 were counted and
the results are summarized in Table 2. We conclude that the
degree of flexibility increases with increasing length of the R
group (Me to n-Pr), whereas the small and bulky substituent, i-
Pr, is locked into one position in space and the i-Bu is predicted
to be less flexible, as compared with the n-Pr group.
Pharmacology and SAR at iGlu and mGlu receptors
The pharmacological properties of the C-4 substituted azetidine
analogues 35–44 were characterized in binding studies on native
ionotropic Glu receptors and in functional assays at cloned
metabotropic receptors mGluR1, 2 and 4, representing group
I–III mGlu receptors, respectively (Table 1). The (2S)-azetidine
analogues 36, 38, 40, 42 and 44 exhibited low affinity for a-
amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) pre-
ferring iGlu receptors (IC50 >100 lM). Affinities for the kainite
(KAIN) preferring iGlu receptors were in the mid-to-low micro
molar range, decreasing as the C-4 substituent was extended
from a Me group, compound 36, (IC50 = 5 lM) to an n-Pr group,
compound 40, (IC50 = 25 lM). Furthermore, the short but
more bulky substituent i-Pr, compound 42, showed mid-range
affinity (IC50 = 6.8 lM) equivalent to compound 36 (R = Me),
while the longer and more bulky substituent i-Bu, compound
44, resulted in loss of binding affinity (IC50 >100 lM). At the N-
methyl-D-aspartate (NMDA) preferring group of iGlu receptors,
compounds 36, 38 and 40 showed decreasing affinity in the lM
mid-range, while the two compounds with bulky substituents,
42 and 44, both exhibited low affinity. In the functional mGlu
assays, the (2S)-4-Me-azetidine analogue 36 was character-
ized as an agonist, with weak potency (EC50 = 220 lM) at
mGluR2 (group II) and neglectable potency (EC50 >1000 lM) at
mGluR1 and 4.
We next wanted to address the affinity differences observed
for compounds t-CAA and its 4-Me analogue 36 at the AMPA
preferring receptors. The low-energy folded conformations of
the two molecules were superimposed on KAIN in the iGluR2–
S1S2 domain construct (PDB file name: 1FTK, crystallized with
KAIN) (Fig. 3), by fitting the ammonium group and the two
carboxylate groups. It can be seen that the ammonium groups
We then conducted a SAR study to explain the observed
pharmacological profile of the azetidine analogues at the iGlu
and mGlu receptors. It is well-accepted that Glu enters a folded
Table 1 Pharmacological evaluation of (2S)-azetidine analogues 36, 38, 40, 42 and 44 at iGlu receptors. Data for (2R)-azetidine analogues 35, 37,
39, 41 and 43 (IC50 >100 lM in AMPA, KAIN and NMDA assays, and 35 only: EC50 >1000 lM in mGluR1, 2 and 4 assays) omitted for clarity
[3H]AMPA
IC50/lM
[3H]KAIN
IC50/lM
[3H]CGP39653
Ki/lM
mGluR1
EC50/lM
mGluR2
EC50/lM
mGluR4
EC50/lM
R
t-CAA
36
H
Me
Et
n-Pr
i-Pr
i-Bu
13
>100
>100
>100
>100
>100
0.7
5
7.5
25
6.8
49a
2
8.7
22
>100
>100
210
>1000
n.t.
n.t.
n.t.
>600
220
n.t.
n.t.
n.t.
>300
>1000
n.t.
n.t.
n.t.
38
40
42
44
>100
n.t.
n.t.
n.t.
a The ligand [3H]CCP was used and IC50 value reported. n.t. = not tested.
Table 2 Modelling study of (2S)-azetidine analogues 36, 38, 40, 42 and 44. Identification of biologically relevant low-energy conformations
Low-energy conformation
Folded conformation
No. of R group conformations at
R
DG/kcal mol−1a
DDG/kcal mol−1b
DDG + 1/kcal mol−1c
t-CAA
36
H
Me
Et
n-Pr
i-Pr
i-Bu
−121.45
−117.97
−118.93
−118.71
−113.09
−113.00
+1.02
+0.36
+0.40
+0.34
−1.78
+0.34
0
1
2
4
1
2
38
40
42
44
a Located via a stochastic conformational search. b Systematic rotation of the distal carboxymethyl group starting with the molecule in its low-energy
conformation. c Systematic rotation of the R group, starting with the molecule in its folded conformation. See experimental section for details.
3 9 2 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 9 2 6 – 3 9 3 6

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and the amino acid residue Lys409. As these two amino acids
residues are conserved throughout the mGlu receptor subtypes,
we believe that the before mentioned disfavoured van der Waals
interactions are the major reasons for the observed lack of
activity at the mGlu receptors.
Pharmacology and SAR at Glu transporters
The azetidinic amino acid analogues 35, 36 and 40 were
evaluated as ligands for the glutamate transporters EAAT1–3.
While the (2R)-azetidine analogue, compound 35, was inactive,
(2S)-azetidines 36 and 40 were found to be inhibitors at EAAT1–
3, with neglectable potency at subtype EAAT1, medium potency
at subtype EAAT2, equivalent to dihydrokainic acid (DHK),
and low potency at subtype EAAT3, decreasing as the C-4
substituent is extended from a Me group to an n-Pr group
(Table 3). In contrast to the non-selective ligand t-CAA, which
holds the azetidinic parental system, we observe selectivity for
the EAAT2 subtype, upon stereospecific introduction of a (4S)-
4-alkyl substituent. In fact we see increasing selectivity for
subtype EAAT2 over subtype EAAT3 (ratio 4 vs. 8), as the
alkyl substituent is extended to an n-Pr group.
Fig. 3 Superimposition of t-CAA (green) and 36 (purple) with KAIN
(type code) crystallized in iGluR2 (Brookhaven PDB-file name 1FTK),
by fitting the ammonium group and the two carboxylate groups.
and the carboxylate groups of t-CAA and 36 are in favourable
positions to interact with the amino acid residues, essential for
agonist activity (Arg485, Pro478, Thr655 and Glu705). However
for compound 36, an unfavourable steric interaction can also be
observed between the 4-Me group and the amino acid Met708,
which we believe is the main reason for the observed low-
affinities of 36 (and thus 38, 40, 42 and 44) at the AMPA
preferring Glu receptors.
Even though the azetidinic amino acids t-CAA, 36, 38, 40, 42
and 44 readily adopt the extended conformation, required for
agonist activity at the mGlu receptors, no potency was observed
(Table 1). To address this finding, we did a superimposition study
of t-CAA and 36 with Glu in the mGluR1 crystal structure
(PDB file name: 1EWK, crystallized with Glu), by fitting the
ammonium group and the two carboxylate groups (Fig. 4).
The compounds overlaid well with Glu in the crystal structure,
however with two critical steric conflicts between the azetidic
C-4 and amino acid residue Asp318, and the C-4 substituent
It has been suggested by us21 and others22–24 that the inhibitory
binding conformation at EAAT1–3 is similar to the folded Glu
conformation also observed for iGluR agonist activity, while
substrates bind in an extended Glu conformation, however
different from the conformation observed for mGluR agonist
activation. Selective inhibition of the EAAT2 subtype has
previously been observed for the natural product KAIN as well
as its close structural analogue DHK. We therefore performed
a superimposition study of the four low-energy folded confor-
mations found for azetidine analogue 40 with the low-energy
conformation of KAIN, to investigate the degree of overlap of
the two substituents (Fig. 5). However, we see that the n-Pr group
at the best is at the brink of the isopropenyl group in KAIN,
allowing us to suggest that the EAAT2 transporter protein may
be able to accommodate larger substituents than an isopropenyl
group. We believe that the synthesis of other azetidinic amino
acids with longer and more bulky alkyl substituents in the 4
position would be beneficial in the search for potent selective
EAAT2 subtype inhibitors.
Table 3 Pharmacological evaluation of azetidine analogues 35, 36 and
40 at Glu transporters. Data for KAIN and DHK taken from ref. 31;
data for t-CAA taken from ref. 9
R
EAAT1 Ki/lM EAAT2 Ki/lM EAAT3 Ki/lM
KAIN
DHK
t-CAA
—
—
H
>3000
>3000
5
60
31
5
>3000
>3000
5
(2R)-35 Me
(2S)-36 Me
(2S)-40 n-Pr
>1000
>1000
>1000
>1000
32
>1000
126
375
Fig. 4 Superimposition of t-CAA (green) and 36 (purple) with
Glu (type code) crystallized in mGluR1 (Brookhaven PDB-file name
1EWK), by fitting the ammonium group and the two carboxylate groups.
44
Fig. 5 Superimposition of the low-energy conformation of KAIN (type code) and four low-energy conformations of 40 (purple and orange, for
clarity), by fitting the ammonium group and the two carboxylate groups.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 9 2 6 – 3 9 3 6
3 9 2 9

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Introduction of a methyl substituent in the amino acid’s
assay. While 35 was inactive, azetidines 36 and 40 were both
ꢀ
a position has previously been done, maintaining potency at
the EAATs.25 Thus, in rat forebrain synaptosomes, an EAAT
subtype non-specific assay, the amino acids L-trans-2,3-PDC
and its cis-5-methyl derivative (Fig. 7) were shown to inhibit
D-[3H]aspartate uptake by 70%. On the other hand, the C-5
diastereomer trans-5-methyl-L-trans-2,3-PDC (Fig. 7) did not
inhibit D-[3H]aspartate uptake. We decided to overlay low-
energy conformations of cis-5-methyl-L-trans-2,3-PDC with our
azetidine analogue 36 to investigate the orientation of the two
methyl groups in space (Fig. 6). It can be seen that the two
methyl groups are positioned very closely in space pointing in
the same direction. This superimposition study also provides
the information that the C-4 diastereomers of our azetidine
analogues are likely not ligands at the EAATs.
identified as inhibitors and showed selectivity for the EAAT2
subtype. On the basis of the new pharmacological data obtained,
the modelling study provided new insight into the SAR of the
glutamate receptors and transporters.
Experimental
Chemistry
General comments. 1H and 13C spectra (CDCl3 solution
unless otherwise stated) were recorded on a Bruker AC 200
or 300 spectrometer at 200, 300 (1H), 50.3 and 75.5 (13C)
MHz; chemical shifts are reported in ppm from TMS. Optical
rotations were determined with a Perkin Elmer 241 instru-
ment. All reactions were carried out under argon. Column
chromatography was performed on silica gel 230–400 mesh
by using various mixtures of diethyl ether (E), ethyl acetate
(AcOEt), cyclohexane (CyH) and petroleum ether (PE). TLC
was run on Merck Kieselgel 60F254 plates. Melting points are
uncorrected. THF was distilled from sodium–benzophenone
ketyl. Dichloromethane and triethylamine were distilled from
calcium hydride. Benzaldehyde was distilled before use. Other
reagents were used as purchased. Mention of “usual workup”
means: (i) decantation of the organic layer, (ii) extraction of
the aqueous layer with ether, (iii) washing the combined organic
layers with brine and drying of the combined organic phases over
MgSO4, (iv) solvent evaporation under reduced pressure. Com-
positions of stereoisomeric mixtures were determined by NMR
analysis on crude products before any purification. Elementary
analyses were realized by “Le service re´gional de microanalyse
de lꢀUniversite´ de pharmacie de Chaˆtenay-Malabry”. HRMS
was performed on a Finnagan MAT-90 spectrometer at Boston
University and in the laboratory of “Ecole Nationale Supe´rieure
de Paris” (compounds 35 and 36). Mass spectra were recorded
on a GC-MS HP MS 5989B spectrometer at the University of
Versailles.
Fig. 6 Superimposition of low-energy conformations of cis-5-Me-L-
trans-2,3-PDC (type code) and 36 (purple) by fitting the ammonium
group and the two carboxylate groups.
General procedure for the N-benzylation of (S)-amino alcohols
To
a solution of the b-amino alcohol (26 mmol) in
˚
dichloromethane (50 mL) was added 4 A molecular sieves
(5 g) and benzaldehyde (2.60 mL, 26 mmol). After 3 h at rt
without stirring, the suspension was filtered and concentrated
under reduced pressure. The residue was dissolved in EtOH
(50 mL) and sodium borohydride was added portionwise (1.16 g,
31 mmol). After 2 h at rt, the reaction was hydrolyzed by addition
of a saturated aqueous solution of NH4Cl and concentrated
under reduced pressure. Basification of the aqueous residue
with 1N NaOH was followed by the usual workup and gave
crude N-benzyl amino alcohols that were used without further
purification for the next step.
Fig. 7 Known EAAT inhibitors.
In conclusion, for the synthetic part, we have discovered a
very efficient synthetic methodology for the construction of a
functionalized azetidine ring that allows for a straightforward
preparation of an understudied class of cyclic amino acids.
The nature of the thermodynamic driving force overriding
the developing torsional strain in the four-membered ring
produced during the key anionic ring closure remains to be
fully understood but the efficiency of this challenging reaction
contributes to putting into perspective the preconceived strain
in azetidinic heterocycles.
(S)-N-Benzyl-2-aminopropan-1-ol 6. Yield: ◦63%; Rf: 0.12
(E–30% aqueous ammonia, 98 : 2); mp 38–42 C; [a]2D0 +58.9
(c 1.0, CHCl3); dH(250 MHz, CDCl3, Me4Si): 1.34 (d, 3H, J =
6.5, Me), 2.81 (s, 2H, NH and OH), 3.09 (quintd, 1H, J = 4.0, 6.5,
CHCH3), 3.56 (dd, 1H, J = 7.1, 10.8, CHHOH), 3.84 (dd, 1H,
J = 4.0, 10.8, CHHOH), 3.98 (d, 1H, J = 12.9, CHHPh), 4.13
(d, 1H, J = 12.9, CHHPh), 7.51–7.61 (m, 5H, Ph); dC(62.9 MHz,
CDCl3, Me4Si): 17.0 (CH3), 51.1 (CH2), 53.8 (CH), 65.8 (CH2),
127.2, 128.2, 128.5 (CHAr), 140.1 (CqAr).
The ten synthesized novel conformationally restricted Glu
analogues 35–44 were characterized in binding studies on native
ionotropic Glu receptors and in functional assays at cloned
metabotropic receptors mGluR1, 2 and 4, representing group
I–III mGlu receptors, respectively. The (2R)-azetidines 35, 37,
39, 41 and 43 were all inactive, whereas a marked change in the
pharmacological profile at the iGlu receptors was observed when
a methyl group was introduced in the C-4 position, compound 36
versus t-CAA. However, within the series of azetidine analogues
presented here, no receptor-class nor subgroup selectivity could
be demonstrated. Azetidine analogues 35, 36 and 40 were also
characterized as potential ligands at the glutamate transporter
(S)-N-Benzyl-2-aminobutan-1-ol 7. Yield: 88%; Rf: 0.4
◦
(AcOEt); mp 72 C; [a]D20 +28.0 (c 1.2, CHCl3); dH(300 MHz,
CDCl3, Me4Si): 0.85 (t, 3H, J = 7.3, Me), 1.42–1.50 (m, 2H,
CH2CH3), 2.18 (bs, 2H, NH and OH), 2.50–2.58 (m, 1H,
CHCH2), 3.26 (d, 1H, J = 6.3, CHHOH), 3.57 (d, 1H, J =
6.3, CHHOH), 3.67 (d, 1H, J = 12.9, CHHPh), 3.75 (d, 1H,
J = 12.9, CHHPh), 7.16–7.28 (m, 5H, Ph); dC(75 MHz, CDCl3,
Me4Si): 10.4 (CH3), 24.2, 51.0 (CH2), 59.8 (CH), 62.6 (CH2),
127.02, 128.1, 128.5 (CHAr), 140.3 (CqAr); MS (CI, CH4): m/z
180 (MH+), 90.
R
subtypes EAAT1–3 in the FLIPRꢀ Membrane Potential (FMP)
3 9 3 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 9 2 6 – 3 9 3 6

View Online
(S)-N-Benzyl-2-aminopentan-1-ol 8. Yield: 95%; Rf: 0.21
(Et2O); [a]20 +20 (c 1.1, CHCl3); dH(300 MHz, CDCl3, Me4Si):
0.93 (t, 3HD, J = 7.3, Me), 1.25–1.52 (m, 4H, (CH2)2CH3), 2.61–
2.69 (bm, 3H, NH, OH, CHCH2), 3.33 (dd, 1H, J = 6.6 and 11,
CHHOH), 3.63 (d, 1H, J = 3.9 and 11, CHHOH), 3.75 (A part
of an AB syst., 1H, J = 12.7, CHHPh), 3.82 (B part of a AB
syst., 1H, J = 12.9, CHHPh), 7.11–7.23 (m, 5H, Ph); dC(75 MHz,
CDCl3, Me4Si): 14.3 (CH3), 19.3, 33.7, 51.4 (CH2), 58.1 (CH),
63.0 (CH2), 127.2, 128.1, 128.6 (CHAr), 140.7 (CqAr); MS (CI,
CH4): m/z 194 (MH+).
(S)-N-Benzyl valinol 9. Yield: quant.; Rf: 0.4 (AcOEt); [a]20
+16.0 (c 1.4, CHCl3); dH(300 MHz, CDCl3, Me4Si): 0.93 anDd
0.98 (two d, 6H, J = 6.8, Me), 1.84–1.94 (m, 1H, CH(CH3)2),
2.47–2.53 (bm, 3H, NH, OH, CHCHMe2), 3.42 (dd, 1H, J =
3.5 and 11, CHHOH), 3.67 (dd, 1H, J = 6.3 and 11, CHHOH),
3.80 (s, 2H, CH2Ph), 7.18–7.30 (m, 5H, Ph); dC(75 MHz, CDCl3,
Me4Si): 18.6, 19.5 (CH3), 28.9 (CH), 51.5, 60.5 (CH2), 77.2 (CH),
127.0, 128.5, 128.7 (CHAr), 140.4 (CqAr); MS (CI, CH4): m/z
194 (MH+, 18), 193 (100), 162, 91.
CHHOH), 3.69 (dd, 1H, J = 3.7 and 11, CHHOH), 3.76 (d,
1H, J = 13.4, CHHPh), 3.92 (d, 1H, J = 13.3, CHHPh), 7.30–
7.34 (m, 5H, Ph); dC(62.9 MHz, CDCl3, Me4Si): 14.2 (CH3),
20.1, 29.1, 38.2, 53.9, 61.6 (CH2), 64.1 (CH), 117.0 (CN), 127.4,
127.9, 128.9 (CHAr), 137.2 (CqAr).
(S)-N-Benzyl-N-cyanomethyl-valinol 15. Yield: 80%; Rf:
20
D
0.77 (AcOEt); [a] −9 (c 1.6, CHCl3); dH(300 MHz, CDCl3,
Me4Si): 0.99 (t, 3H, J = 6.8, Me), 1.11 (t, 3H, J = 6.8, Me),
1.96–2.08 (m, 1H, CH(CH3)2), 2.35 (bs, 1H, OH), 2.53–2.60 (m,
1H, CHCHMe2), 3.49 (d, 1H, J = 17.5, CHHOH), 3.54 (d,
1H, J = 17.5, CHHOH), 3.72–3.98 (m, 4H, CH2Ph, CH2CN),
7.28–7.39 (m, 5H, Ph); dC(62.9 MHz, CDCl3, Me4Si): 19.8, 21.5
(CH3), 27.6 (CH), 38.4, 55.9, 59.8 (CH2), 69.8 (CH), 117.4 (CN),
127.0, 128.5, 128.9 (CHAr), 137.8 (CqAr); MS (CI, NH3): m/z
206.4, 205.3, 162.3, 90.9; Anal. Calcd. for C14H20N2O: C, 72.38;
H, 8.68; N, 12.06. Found: C, 72.12; H, 8.84; N, 11.95%.
(S)-N-Benzyl-N-cyanomethyl-leucinol 16. Yield: 82%; Rf:
0.33 (E–CyH, 1 : 1); [a]2D0 +51 (c 1.5, CHCl3); dH(200 MHz,
CDCl3, Me4Si): 0.97 (d, 6H, J = 6.1, 2Me), 1.21–1.34 (m,
CH(Me)2), 1.58–1.74 (m, 2H, CHCH2CH), 2.52 (bs, 1H, OH),
2.99–3.09 (m, 1H, CHN), 3.47–3.71 (m, 4H, CH2OH, CH2CN),
3.78 (d, 1H, J = 13.6, CHHPh), 3.78 (d, 1H, J = 13.6, CHHPh),
7.20–7.40 (m, 5H, Ph); dC(62.9 MHz, CDCl3, Me4Si): 22.3, 23.3
(CH3), 25.4 (CH), 35.8, 38.2, 53.9 (CH2), 61.2 (CH), 62.4 (CH2),
116.9 (CN), 127.9, 128.9, 129.0 (CHAr), 137.2 (CqAr); MS (IC,
NH3): m/z 220.3, 162.7, 91.9.
(◦S)-N-Benzyl leucinol 10. Yield: 97%; Rf: 0.45 (AcOEt); mp
70 C; [a]2D0 +31.0 (c 1.0, CHCl3); dH(200 MHz, CDCl3, Me4Si):
0.89 and 0.92 (two d, 6H, J = 6.8, Me), 1.19–1.50 (m, 1H,
CH(CH3)2), 1.58–1.68 (m, 2H, CH2CH(Me)2), 2.13 (bs, 2H,
NH and OH), 2.73–2.80 (m, 1H, CHN), 3.29 (d, 1H, J = 11,
CHHOH), 3.42 (d, 1H, J = 11, CHHOH), 3.81 (d, J = 2.4, 2H,
CH2Ph), 7.27–7.36 (m, 5H, Ph); dC(75 MHz, CDCl3, Me4Si):
22.7, 23.1 (CH3), 25.0 (CH), 40.9, 50.8, 56.1 (CH2), 63.0 (CH),
127.0, 128.0, 128.3 (CHAr), 139.5 (CqAr); MS (CI, NH3): m/z
221.2, 220.3, 206.4, 162.7, 91.9; Anal. calcd. for C13H21NO: C,
75.32; H, 10.21; N, 6.76. Found: C, 75.28; H, 10.32; N, 6.65%.
General procedure for the “one-pot” Swern reaction and Wittig
olefination
To a solution of oxalyl chloride (0.16 mL, 1.8 mmol) in
dichloromethane (10 mL) was added dropwise at −50 ◦C DMSO
(0.15 mL, 1.7 mmol). After five minutes, the reaction mixture
was cooled to −78 ◦C and the amino alcohol (1.2 mmol) in
dichloromethane (5 mL) was added dropwise. After 30 min at
−78 ◦C, triethylamine (0.63 mL, 3.7 mmol) was added dropwise,
followed after 5 minutes by a solution of carboethoxymethylen-
etriphenylphosphorane (0.8 g, 2.3 mmol) in dichloromethane
(5 mL). The reaction mixture was then gradually allowed to
reach room temperature and was hydrolysed by addition of
water. Usual workup (dichloromethane) gave a residue that was
purified by flash chromatography.
General procedure for alkylation with bromoacetonitrile
To a solution of N-benzyl aminoalcohol (9.66 mmol) in
acetonitrile (80 mL) was added potassium carbonate (2.06 g,
14.9 mmol) and bromoacetonitrile (1.90 mL, 27 mmol). The
resulting mixture was refluxed for 2.5 h and, after cooling to rt,
concentrated to dryness under reduced pressure. After addition
of water and ether, the usual workup was followed by flash
chromatography (AcOEt–CyH, 2 : 8) of the crude residue and
gave the following compounds:
(S)-N-Benzyl-N-cyanomethyl-2-aminopropan-1-ol 12. Yield:
68%; Rf: 0.34 (E–CyH, 8 : 2); [a]2D5 +75.2 (c 1.5, CHCl3); IR (film):
3441, 2965, 2930, 2879, 2228, 1496, 1445 cm−1; dH(250 MHz,
CDCl3, Me4Si): 0.98 (d, 3H, J = 6.8, Me), 2.39 (bs, 1H, OH),
2.81–2.96 (m, 1H, CHMe), 3.22 (d, 2H, J = 5.9, CH2CN), 3.31
(d, 2H, J = 6.2, CH2OH), 3.48 (d, 1H, J = 13.3, CHHPh), 3.66
(d, 1H, J = 13.3, CHHPh), 7.05–7.15 (m, 5H, Ph); dC(62.9 MHz,
CDCl3, Me4Si): 11.8 (CH3), 38.5, 53.1 (CH2), 59.6 (CH), 63.5
(CH2), 116.8 (CN), 128.0, 128.9, 129.0 (CHAr), 136.9 (CqAr);
Anal. Calcd. for C12H16N2O: C, 70.56; H, 7.89; N, 13.71. Found:
C, 69.78; H, 7.90; N, 13.32%.
(2E,4S)-4-(Benzyl-cyanomethyl-amino)-4-phenyl-but-2-enoic
acid ethyl ester 17. Yield: 83%; Rf: 0.72 (E–CyH, 6 : 4); [a]D25
−40 (c 0.9, CHCl3); dH(250 MHz, CDCl3, Me4Si): 1.19 (t, 3H,
J = 7.3, CH3), 3.27 (d, 1H, J = 17.5, CHHCN), 3.28 (d, 1H,
J = 17.5, CHHCN), 3.60 (AB syst., 2H, J = 13.5, CH2Ph),
4.09 (q, 2H, J = 7.3, OCH2), 4.23 (d, 1H, J = 9.3, NCHPh),
6.11 (d, 1H, J = 15.5, CHCOOEt), 6.96 (dd, 1H, J = 9.8,
=
15.8, CH CHCO2Et), 7.18–7.41 (m, 10H, Ph); dC(62.9 MHz,
CDCl3, Me4Si): 14.3 (CH3), 39.2, 55.6, 60.8 (CH2), 69.6 (CH),
114.1 (CN), 123.4 (CH), 128.0, 128.1, 128.8, 128.9, 128.9, 129.5
(CHAr), 136.9, 139.0 (Cq Ar), 147.2 (CH), 166.0 (CO); Anal.
Calcd. for C21H22N2O2: C, 75.42; H, 6.63; N, 8.38. Found: C,
74.93; H, 6.73; N, 8.02%.
(S)-N-Benzyl-N-cyanomethyl-2-aminobutan-1-ol 13. Yield:
85%; Rf: 0.85 (AcOEt–PE, 1 : 1); [a]20 +35.0 (c 1.1, CHCl3);
dH(300 MHz, CDCl3, Me4Si): 0.99 (t, D3H, J = 7.2, Me), 1.36–
1.92 (m, 2H, CH2CH3), 2.70 (bs, 1H, OH), 2.76–2.87 (m, 1H,
CHCH2), 3.46–3.70 (m, 4H, CH2CN and CH2OH), 3.78 (d, 1H,
J = 13.4, CHHPh), 3.91 (d, 1H, J = 13.3, CHHPh), 7.15–7.45
(m, 5H, Ph); dC(62.9 MHz, CDCl3, Me4Si): 11.5 (CH3), 20.2,
38.2, 53.9 (CH2), 61.2 (CH), 65.9 (CH2), 117.1 (CN), 127.9,
128.5, 128.9 (CHAr), 137.2 (CqAr); MS (CI, NH3): m/z 192.6,
162.8, 92.5; Anal. Calcd. for C13H18N2O: C, 71.53; H, 8.31; N,
12.83. Found: C, 71.60; H, 8.39; N, 12.72%.
(2E,4S )-4-(Benzyl-cyanomethyl-amino)-pent-2-enoic acid
ethyl ester 18. Yield: 73%; Rf: 0.53 (E–CyH, 1 : 1); [a]2D5 −37.9
(c 0.9, CHCl3); dH(250 MHz, CDCl3, Me4Si): 1.04 (t, 3H, J =
7.3, CH3), 1.08 (d, 3H, J = 6.8, CH3), 3.15 (AB syst., 2H, J =
17.6, CH2CN), 3.18–3.32 (m, 1H, CHCH3), 3.48 (AB syst., 2H,
J = 17.6, CH2Ph), 3.95 (q, 2H, J = 7.1, CH2CH3), 5.78 (dd,
1H, J = 1.0, 15.8, CHCOOEt), 6.66 (dd, 1H, J = 7.4, 15.7,
=
(S)-N-Benzyl-N-cyanomethyl-2-aminopentan-1-ol 14. Yield:
74%; Rf: 0.89 (AcOEt–PE, 1 : 1); [a]20 +28.0 (c 1.1, CHCl3);
dH(300 MHz, CDCl3, Me4Si): 0.96 (t, D3H, J = 7.2, Me), 1.27–
1.41 (m, 4H, (CH2)2CH3), 2.58 (bs, 1H, OH), 2.91–3.01 (m, 1H,
CHN), 3.46 (s, 2H, CH2CN), 3.59 (dd, 1H, J = 6.6 and 11,
CH CHCO2Et), 7.00–7.09 (m, 5H, Ph); dC(62.9 MHz, CDCl3,
Me4Si): 14.3, 17.5 (CH3), 38.5, 55.2, 60.7 (CH2), 115.7 (CN),
123.0 (CH), 127.9, 128.8, 128.9 (CHAr), 137.0 (Cq Ar), 149.3
(CH), 166.2 (CO); Anal. Calcd. for C16H20N2O2: C, 70.56; H,
7.40; N, 10.29. Found: C, 70.64; H, 7.55; N, 10.18%.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 9 2 6 – 3 9 3 6
3 9 3 1

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(2E,4S)-4-(Benzyl-cyanomethyl-amino)-hex-2-enoic acid ethyl
ester 19. Yield: 84%; Rf: 0.66 (AcOEt–PE, 1 : 1); [a]25 −37
(c 1.1, CHCl3); dH(300 MHz, CDCl3, Me4Si): 0.95 (t, 3H, JD= 7.3,
CH3), 1.31 (t, 3H, J = 7.1, CH3), 1.57–1.58 (m, 2H, CH2CH3),
3.21–3.29 (m, 1H, CHN), 3.37 (d, 1H, J = 17.9, CHHCN), 3.45
(d, 1H, J = 17.9, CHHCN), 3.68 (d, 1H, J = 13.5, CHHPh),
3.80 (d, 1H, J = 13.5, CHHPh), 4.22 (q, 2H, J = 7.0, CH2O),
6.03 (d, 1H, J = 15.7, CHCOOEt), 6.90 (dd, 1H, J = 8.6, 15.7,
(3R,4S,5R)-(1-Benzyl-2-cyano-4-phenyl-azetidin-3-yl)-acetic
acid ethyl ester 23. R = Ph: overall yield: 58%. Cis–trans ratio,
38 : 62.
Yield: 22%; Rf: 0.72 (E–CyH, 60 : 40); [a]2D5 +91.6 (c 0.4,
CHCl3); dH(250 MHz, CDCl3, Me4Si): 1.08 (t, 3H, J = 7.5,
CH3), 2.61 (dd, 1H, J = 4.6, 12.9, CHHCO2Et), 2.73–2.98 (m,
2H, CHHCO2Et and CHCH2), 3.70 (AB syst., 2H, J = 13.3,
CH2Ph), 3.97 (brquad, 3H, J = 7.1, OCH2 and CHPh), 4.34 (d,
1H, J = 7.1, CHCN), 7.18–7.38 (m, 10H, Ar); dC(62.9 MHz,
CDCl3, Me4Si): 14.2 (CH3), 34.2 (CH2), 40.6, 55.3 (CH), 56.0,
61.0 (CH2), 72.7 (CH), 115.7 (CN), 127.1, 127.7, 128.6, 128.8,
129.2 (CHAr), 136.5, 139.5 (CqAr), 170.7 (CO); Anal. Calcd.
for C21H22N2O2: C, 75.42; H, 6.63; N, 8.38. Found: C, 74.93; H,
6.73; N, 8.02%.
=
CH CHCO2Et), 7.27–7.34 (m, 5H, Ph); dC(62.9 MHz, CDCl3,
Me4Si): 10.1, 14.2 (CH3), 24.6, 38.5, 54.9, 60.6 (CH2), 64.6 (CH),
115.7 (CN), 124.5 (CH), 127.8, 128.7, 128.8, 129.1 (CHAr), 137.1
(Cq Ar), 146.3 (CH), 170.0 (CO); MS (IC, NH3): m/z: 286.6,
256.9, 91.9; Anal. Calcd. for C17H22N2O2: C, 71.30; H, 7.74; N,
9.78. Found: C, 71.29; H, 7.82; N, 9.76%.
(3S,4R,5R)-(1-Benzyl-2-cyano-4-phenyl-azetidin-3-yl)-acetic
acid ethyl ester 24. Yield: 36%; mp: 68–70 ◦C; Rf: 0.5 (E–CyH,
1 : 1); [a]2D5 −68.7 (c 1.5, CHCl3); dH(250 MHz, CDCl3, Me4Si):
1.10 (t, 3H, J = 6.8, CH3), 2.48 (ABX syst., 1H, CHHCO2Et),
2.50 (ABX syst., 1H, CHHCO2Et), 2.83 (dquad, 1H, J = 5.9
and 6.0, CHCH2), 3.49 (d, 1H, J = 7.7, CHPh), 3.52 (d, J =
12.9, CHHPh), 3.75 (d, 1H, J = 7.1, CHCN), 3.83 (d, J = 12.9,
CHHPh), 4.00 (dquad, 2H, J = 1.5 and 7.4, CH2O), 7.18–7.38
(m, 10H, Ar); dC(62.9 MHz, CDCl3, Me4Si): 14.2 (CH3), 36.4
(CH2), 43.6, 53.1 (CH), 59.9, 61.2 (CH2), 71.3 (CH), 118.9
(CN), 127.1, 128.0, 128.5, 128.6, 128.7, 129.6 (CHAr), 135.0,
139.8 (CqAr), 170.3 (CO); Anal. Calcd. for C21H22N2O2: C,
75.42; H, 6.63; N, 8.38. Found: C, 75.27; H, 6.72; N, 8.12%.
(2E,4S)-4-(Benzyl-cyanomethyl-amino)-hept-2-enoic acid
ethyl ester 20. Yield: 92%; Rf: 0.7 (AcOEt–PE, 1 : 1); [a]D25
−37 (c 1.1, CHCl3); dH(300 MHz, CDCl3, Me4Si): 0.95 (t, 3H,
J = 7.1, CH3), 1.30 (t, 3H, J = 7.1, CH3), 1.31–1.90 (m, 4H,
(CH2)2CH3), 3.32–3.38 (m, 1H, CHN), 3.43 (s, 2H, CH2CN),
3.68 (d, 1H, J = 13.5, CHHPh), 3.84 (d, 1H, J = 13.5, CHHPh),
4.24 (q, 2H, J = 7.0, CH2O), 6.03 (d, 1H, J = 14.2, CHCOOEt),
=
6.90 (dd, 1H, J = 8.6, 14.7, CH CHCO2Et), 7.25–7.35 (m, 5H,
Ph); dC(62.9 MHz, CDCl3, Me4Si): 14.0, 14.2 (CH3), 19.0, 33.8,
38.5, 54.9, 60.6 (CH2), 63.0 (CH), 115.8 (CN), 124.3 (CH), 127.8,
128.7, 128.8 (CHAr), 137.1 (Cq Ar), 146.5 (CH), 165.9 (CO).
(2E,4S)-4-(Benzyl-cyanomethyl-amino)-5-methyl-hex-2-enoic
acid ethyl ester 21. Yield: 94%; Rf: 0.53 (AcOEt–PE, 2 : 8);
[a]2D5 −32 (c 3.3, CHCl3); dH(300 MHz, CDCl3, Me4Si): 0.91 (d,
3H, J = 6.8, CH3), 1.01 (d, 3H, J = 6.8, CH3), 1.35 (t, 3H,
J = 7.1, CH3), 2.03–2.10 (m, 1H, CHMe2), 3.48 (dd, 1H, J =
7.1 and 9.8, CHN), 3.35 (d, 1H, J = 17.6, CHHCN), 3.43 (d,
1H, J = 17.6, CHHCN), 3.43 (d, 1H, J = 13.5, CHHPh), 3.43
(d, 1H, J = 13.5, CHHPh), 4.22 (q, 2H, J = 7.1, OCH2CH3),
6.00 (d, 1H, J = 15.8, CHCOOEt), 6.85 (dd, 1H, J = 10 and
(3R,4S,5S)-(1-Benzyl-2-cyano-4-methyl-azetidin-3-yl)-acetic
acid ethyl ester 25. R = Me: overall yield: 74%. Cis–trans
ratio, 37 : 63.
Yield: 27%; Rf: 0.59 (E–CyH, 1 : 1); [a]2D5 +101.5 (c 0.2, CHCl3);
IR (neat): 2966, 2925, 2848, 2223, 1726 cm−1; dH(250 MHz,
CDCl3, Me4Si): 1.05 (d, 3H, J = 6.2, CH3), 1.18 (t, 3H, J =
7.1, CH3), 2.54–2.70 (m, 3H, CHCH2CO2Et), 3.10–3.23 (m,
1H, CHCH3), 3.62 (d, 1H, J = 13.3, CHHPh), 3.75 (d, 1H,
J = 13.3, CHHPh), 4.06 (q, 2H, J = 7.1, CH2O), 4.30 (d, 1H,
J = 5.9, CHCN), 7.18–7.30 (m, 5H, Ar); dC(62.9 MHz, CDCl3,
Me4Si): 14.3, 19.8 (CH3), 34.5 (CH2), 37.8, 55.9 (CH), 56.5, 61.0
(CH2), 66.5 (CH), 116.1 (CN), 127.6, 128.6, 129.2 (CHAr), 136.9
(CqAr), 171.1 (CO); Anal. Calcd. for C16H20N2O2: C, 70.56; H,
7.40; N, 10.29. Found: C, 70.63; H, 7.58; N, 10.18%.
=
15.7, CH CHCO2Et), 7.22–7.35 (m, 5H, Ph); dC(62.9 MHz,
CDCl3, Me4Si): 14.2, 17.8, 20.1 (CH3), 28.7 (CH), 38.6, 54.9,
60.4 (CH2), 69.2 (CH), 115.6 (CN), 125.9 (CH), 127.8, 128.7,
130.2 (CHAr), 137.1 (Cq Ar), 144.2 (CH), 165.5 (CO); MS (CI,
NH3): m/z: 301.2, 300.3, 257.3, 108.0, 90.9; Anal. Calcd. for
C18H24N2O2: C, 71.97; H, 8.05; N, 9.33. Found: C, 71.98; H,
7.98; N, 9.27%.
(3S,4S,5S)-(1-Benzyl-2-cyano-4-methyl-azetidin-3-yl)-acetic
acid ethyl ester 26. Yield: 47%; Rf: 0.47 (E–CyH, 1 : 1); [a]D25
−15.8 (c 0.5, CHCl3); IR (neat): 2980, 2925, 2863, 2238, 1726,
1491, 1450 cm−1; dH(250 MHz, CDCl3, Me4Si): 1.02 (d, 3H, J =
5.9, CH3), 1.19 (t, 3H, J = 7.15, CH3), 2.42 (d, 1H, J = 3.1,
CHHCO2Et), 2.45 (d, 1H, J = 0.9, CHHCO2Et), 2.52–2.67
(m, 1H, CHCH2), 2.90 (qd, 1H, J = 6 and 7.5, 1H, CHCH3),
3.35 (d, 1H, J = 7.4, CHCN), 3.64 (AB syst., 2H, J = 12.9,
CH2Ph), 4.08 (q, 2H, J = 7.1, CH2O), 7.19–7.27 (m, 5H, Ar);
dC(62.9 MHz, CDCl3, Me4Si): 14.3, 20.6 (CH3), 36.7 (CH2),
41.0, 54.3 (CH), 60.8, 61.2 (CH2), 65.8 (CH), 119.0 (CN), 128.0,
128.6, 129.4 (CHAr), 135.7 (CqAr), 170.6 (CO); Anal. Calcd.
for C16H20N2O2: C, 70.56; H, 7.40; N, 10.29. Found: C, 70.54;
H, 7.51; N, 10.24%.
(2E,4S)-4-(Benzyl-cyanomethyl-amino)-6-methyl-hept-2-
enoic acid ethyl ester 22. Yield: 77%; Rf: 0.43 (E–CyH, 85 :
15); [a]2D5 −16 (c 1.1, CHCl3); dH(300 MHz, CDCl3, Me4Si): 0.83
(d, 6H, J = 6.8, 2CH3), 1.31 (t, 3H, J = 7.2, CH3), 1.40–2.69 (m,
3H, CH2CHMe2), 3.35–3.48 (m, 3H, CHN, CH2CN), 3.66 (d,
1H, J = 13.4, CHHPh), 3.85 (d, 1H, J = 13.4, CHHPh), 4.23
(q, 2H, J = 7.1, OCH2CH3), 6.02 (d, 1H, J = 16, CHCOOEt),
=
6.89 (dd, 1H, J = 8 and 16, CH CHCO2Et), 7.21–7.35 (m,
5H, Ph); dC(62.9 MHz, CDCl3, Me4Si): 14.2, 22.1, 23.1 (CH3),
24.6 (CH), 38.3, 40.5, 54.7, 60.6 (CH2), 61.2 (CH), 116.6 (CN),
124.3 (CH), 127.8, 128.7, 130.2 (CHAr), 137.1 (Cq Ar), 146.2
(CH), 165.5 (CO); MS (CI, NH3): m/z: 315.2, 314.3, 256.8,
92.4; Anal. Calcd. for C19H26N2O2: C, 72.58; H, 8.33; N, 8.91.
Found: C, 72.69; H, 8.48; N, 8.80%.
(3R,4S,5S)-(1-Benzyl-2-cyano-4-ethyl-azetidin-3-yl)-acetic
acid ethyl ester 27. R = Et: overall yield: 73%. Cis–trans ratio,
42 : 58.
General procedure for azetidine formation
To a solution of amino ester (1.19 mmol) in THF (5 mL) was
added, at −78 ◦C, a solution of LiHMDS (1 M solution in THF,
1.42 mL, 1.42 mmol). The mixture was gradually warmed to
−10 ◦C over a period of 2 h and the progress of the reaction was
monitored by TLC. After completion, hydrolysis by a saturated
aqueous solution of ammonium chloride was followed by the
usual workup (AcOEt). Flash chromatography of the residue
on silica gel (eluant: AcOEt–PE, 5 : 95 then 1 : 9) gave 2,3-cis-
isomers, followed by 2,3-trans-isomers.
Yield: 31%; Rf: 0.69 (AcOEt–PE, 2 : 8); [a]25 +83 (c 0.6,
CHCl3); dH(300 MHz, CDCl3, Me4Si): 0.90 (t, 3H,DJ = 7.5, CH3),
1.27 (t, 3H, J = 7.5, CH3), 1.50–1.56 (m, 2H, CH3CH2), 2.64–
2.84 (m, 3H, CHCH2CO2Et), 3.16 (q, 1H, J = 6.8, CHN), 3.74
(d, 1H, J = 13.2, CHHPh), 3.87 (d, 1H, J = 13.2, CHHPh), 4.18
(q, 2H, J = 7.1, CH2O), 4.38 (d, 1H, J = 7.4, CHCN), 7.27–7.31
(m, 5H, Ar); dC(62.9 MHz, CDCl3, Me4Si): 9.3, 14.1 (CH3), 27.0,
35.0 (CH2), 35.3, 55.6 (CH), 56.8, 60.9 (CH2), 71.6 (CH), 117.0
=
(CN), 127.4, 128.4, 128.9 (CHAr), 138.1 (CqAr), 171.4 (C O);
3 9 3 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 9 2 6 – 3 9 3 6

View Online
MS (CI, NH3): m/z 286.6, 91.9; Anal. Calcd. for C17H22N2O2:
C, 71.30; H, 7.74; N, 9.78. Found: C, 71.23; H, 7.79; N, 9.74%.
NH3): m/z 286.6, 91.9; Anal. Calcd. for C18H24N2O2: C, 71.97;
H, 8.05; N, 9.33. Found: C, 71.87; H, 8.15; N, 9.25%.
(3R,4S,5S)-(1-Benzyl-2-cyano-4-isobutyl-azetidin-3-yl)-acetic
acid ethyl ester 33. R = i-Bu: overall yield: 86%. Cis–trans
ratio, 27 : 63.
(3S,4R,5S)-(1-Benzyl-2-cyano-4-ethyl-azetidin-3-yl)-acetic
acid ethyl ester 28. Yield: 42%; Rf: 0.59 (AcOEt–PE, 2 : 8);
[a]2D5 −9 (c 0.8, CHCl3); dH(300 MHz, CDCl3, Me4Si): 0.86 (t,
3H, J = 7.5, CH3), 1.29 (t, 3H, J = 7.5, CH3), 1.45–1.55 (m,
2H, CH3CH2), 2.44–261 (m, 2H, CHCH2CO2Et), 2.71–2.77
(m, 1H, CHCH2CO2Et), 2.87–2.93 (m, 1H, CHN), 3.45 (d, 1H,
J = 7.5, CHCN), 3.66 (d, 1H, J = 12.9, CHHPh), 3.81 (d, 1H,
J = 12.9, CHHPh), 4.16 (q, 2H, J = 7.3, CH2O), 7.25–7.31 (m,
5H, Ar); dC(62.9 MHz, CDCl3, Me4Si): 9.2, 14.2 (CH3), 27.8,
37.4 (CH2), 38.5, 54.0 (CH), 61.0, 61.4 (CH2), 71.4 (CH), 119.1
(CN), 127.8, 128.5, 129.2 (CHAr), 136.1 (CqAr), 170.5 (CO);
MS (CI, NH3): m/z 286.7, 91.9; Anal. Calcd. for C17H22N2O2:
C, 71.30; H, 7.74; N, 9.78. Found: C, 71.31; H, 7.83; N, 9.75%.
Yield: 32%; Rf: 0.77 (AcOEt–PE, 2 : 8); [a]25 +85 (c 1.2,
CHCl3); dH(300 MHz, CDCl3, Me4Si): 0.88 andD0.91 (2d, 6H,
J = 5.1, CH3), 1.25 (t, 3H, J = 7.0, CH3), 1.41–1.62 (m, 3H,
(CH3)2CHCH2), 2.61–2.83 (m, 3H, CHCH2CO2Et), 3.24 (q,
1H, J = 6.4, CHN), 3.56 (d, 1H, J = 12.6, CHHPh), 3.80 (d,
1H, J = 12.6, CHHPh), 4.13 (q, 2H, J = 7.1, CH2O), 4.35 (d,
1H, J = 6.1, CHCN), 7.28–7.35 (m, 5H, Ar); dC(62.9 MHz,
CDCl3, Me4Si): 14.1, 22.8, 23.2 (CH3), 24.8 (CH), 34.8 (CH2),
36.7 (CH), 44.5, 55.6 (CH2), 56.8 (CH), 60.8 (CH2), 68.5 (CH),
116.0 (CN), 127.4, 128.4, 128.9 (CHAr), 136.7 (CqAr), 170.8
(CO); MS (CI, NH3): m/z 315.3, 314.4, 92.4; Anal. Calcd. for
C19H26N2O2: C, 72.58; H, 8.33; N, 8.91. Found: C, 72.39; H, 8.44;
N, 8.86%.
(3R,4S,5S)-(1-Benzyl-2-cyano-4-propyl-azetidin-3-yl)-acetic
acid ethyl ester 29. R = Pr: overall yield: 72%. Cis–trans ratio,
37 : 63.
(3S,4R,5S)-(1-Benzyl-2-cyano-4-isobutyl-azetidin-3-yl)-acetic
acid ethyl ester 34. Yield: 54%; Rf: 0.53 (AcOEt–PE, 2 : 8); [a]25
+5 (c 1.5, CHCl3); dH(300 MHz, CDCl3, Me4Si): 0.85 and 0.8D8
(2d, 6H, J = 6.0, CH3), 1.28 (t, 3H, J = 7.1, CH3), 1.38–170 (m,
3H, (CH3)2CHCH2), 2.40–2.77 (m, 3H, CHCH2CO2Et), 3.02
(q, 1H, J = 6.6, CHN), 3.44 (d, 1H, J = 7.5, CHCN), 3.53 (d,
1H, J = 13.0, CHHPh), 3.69 (d, 1H, J = 13.0, CHHPh), 4.16
(q, 2H, J = 7.1, CH2O), 7.25–7.45 (m, 5H, Ar); dC(62.9 MHz,
CDCl3, Me4Si): 14.1, 22.8, 23.1 (CH3), 24.5 (CH), 37.2 (CH2),
39.8 (CH), 45.1, 54.0 (CH2), 61.0, 61.4, 67.9 (CH), 119.0 (CN),
127.9, 128.5, 129.2 (CHAr), 135.7 (CqAr), 170.4 (CO); MS (CI,
NH3): m/z 315.3, 314.4, 92.3; Anal. Calcd. for C19H26N2O2: C,
72.58; H, 8.33; N, 8.91. Found: C, 72.73; H, 8.49; N, 8.78%.
Yield: 27%; Rf: 0.72 (AcOEt–PE, 2 : 8); [a]25 +75 (c 0.8,
CHCl3); dH(300 MHz, CDCl3, Me4Si): 0.92 (t, 3H,DJ = 7.5, CH3),
1.32 (t, 3H, J = 7.5, CH3), 1.40–1.58 (m, 4H, CH3 (CH2)2), 2.62–
2.86 (m, 3H, CHCH2CO2Et), 3.10 (q, 1H, J = 6.8, CHN), 3.65
(d, 1H, J = 13.2, CHHPh), 3.76 (d, 1H, J = 13.2, CHHPh),
4.18 (q, 2H, J = 7.1, CH2O), 4.28 (d, 1H, J = 6.3, CHCN),
7.27–7.32 (m, 5H, Ar); dC(62.9 MHz, CDCl3, Me4Si): 14.1, 14.3
(CH3), 18.5, 34.5 (CH2), 35.9, (CH), 36.7 (CH2), 55.6 (CH), 56.8,
60.6 (CH2), 70.1 (CH), 116.0 (CN), 127.5, 128.4, 128.9 (CHAr),
=
136.8 (CqAr), 170.9 (C O); MS (CI, NH3): m/z 300.6, 91.9.
(3S,4R,5S)-(1-Benzyl-2-cyano-4-propyl-azetidin-3-yl)-acetic
acid ethyl ester 30. Yield: 45%; Rf: 0.62 (AcOEt–PE, 2 : 8);
[a]2D5 −18 (c 0.7, CHCl3); dH(300 MHz, CDCl3, Me4Si): 0.88 (t,
3H, J = 7.5, CH3), 1.29 (t, 3H, J = 7.5, CH3), 1.32–1.56 (m,
4H, CH3 (CH2)2), 2.48–2.65 (m, 2H, CHCH2CO2Et), 2.75–2.79
(m, 1H, CHCH2CO2Et), 2.85–2.99 (m, 1H, CHN), 3.45 (d, 1H,
J = 7.5, CHCN), 3.66 (d, 1H, J = 12.9, CHHPh), 3.85 (d, 1H,
J = 12.9, CHHPh), 4.15 (q, 2H, J = 7.3, CH2O), 7.25–7.31 (m,
5H, Ar); dC(62.9 MHz, CDCl3, Me4Si): 14.1 (2CH3), 18.3, 37.3,
37.5 (CH2), 39.1, 54.0 (CH), 61.0, 61.4 (CH2), 69.5 (CH), 114.0
(CN), 127.4, 128.5, 128.9 (CHAr), 135.8 (CqAr), 170.5 (CO).
General procedure for the hydrolysis–debenzylation steps
An emulsion of 2-cyano azetidine (0.75 mmol) in a 1 : 1 mixture
of CCl4 and 6N aqueous HCl (20 mL) was heated for 48 h
with vigorous stirring at 80 ◦C. The mixture was cooled to rt
and concentrated to dryness under reduced pressure. The crude
residue was washed with small portions of diethyl ether to give
the crude chlorhydrate that was used as such for the next step.
A suspension of the above hydrochloride (0.484 mmol)
in ethanol (15 mL) was stirred until complete dissolution.
Palladium on charcoal (10% wt., 60 mg) was then added and
the suspension was vigorously stirred under an atmosphere
of hydrogen (balloon) for 4 h. After filtration on Celite, the
filtrate was evaporated to dryness. The residue was dissolved
in the minor amount of water and this solution was deposited
on an ion exchange resin (Dowex 50 × 8, H+ form, 3 g) in a
chromatography column. The resin was washed with water until
neutrality and the amino acid was then eluted with an aqueous
solution of ammonia (1%). The elution of the TLCs was done
with EtOH–40% aqueous NH3–H2O = 9 : 3 : 1. Positive fractions
were then lyophilized to give amino acids as solids.
(3R,4S,5S)-(1-Benzyl-2-cyano-4-isopropyl-azetidin-3-yl)-
acetic acid ethyl ester 31. R = i-Pr: overall yield: 87%.
Cis–trans ratio, 30 : 70.
Yield: 26%; Rf: 0.67 (AcOEt–PE, 2 : 8); [a]25 +81 (c 1.1,
CHCl3); dH(300 MHz, CDCl3, Me4Si): 0.73 andD0.74 (2d, 6H,
J = 6.8, CH3), 1.05 (t, 3H, J = 7.1, CH3), 1.53–1.61 (m, 1H,
(CH3)2CH), 2.41–2.66 (m, 2H, CHCH2CO2Et), 2.57–2.58 (m,
1H, CHCH2CO2Et), 2.81–2.84 (m, 1H, CHN), 3.54 (d, 1H, J =
13.5, CHHPh), 3.72 (d, 1H, J = 13.5, CHHPh), 3.93 (q, 2H,
J = 7.1, CH2O), 4.13 (d, 1H, J = 7.4, CHCN), 7.06–7.13 (m,
5H, Ar); dC(62.9 MHz, CDCl3, Me4Si): 14.1, 17.7, 19.0 (CH3),
31.8, 32.7 (CH), 35.5 (CH2), 55.3 (CH), 57.2, 60.9 (CH2), 75.4
(CH), 116.0 (CN), 127.4, 128.4, 128.9 (CHAr), 136.9 (CqAr),
(2R,3S,4S)-3-Carboxymethyl-4-methyl-azetidine-2-carboxylic
acid 35. Yield: 80%; Rf = 0.29 (EtOH–40%NH4OH–H2O, 9 :
3 : 1); [a]25 +97 (c 0.2, H2O); dH(200 MHz, D2O, Me4Si): 1.56 (d,
3H, J =D6.6, Me), 2.26 (dd, 1H, J = 11.4, 15.4, CHHCOOH),
2.50 (dd, 1H, J = 4.8, 15.4, CHHCOOH), 3.06 (dddd, 1H,
J = 4.8, 7.5, 10.1, 11.9, CHCH2), 4.19 (quint, 1H, J = 7.0,
CHMe), 4.70 (d, 1H, J = 10.1, NCHCOOH); dC(75.5 MHz,
D2O, Me4Si): 21.3 (CH3), 43.0 (CH2), 45.3, 62.4, 63.0 (CH),
176.3, 181.5 (CO); Anal. Calcd. for C7H11NO4, 0.75 NH3, 1.50
H2O: C, 39.48; H, 7.69; N, 11.51. Found: C, 39.33; H, 8.02;
N, 11.51%; MS (CI, CH4): m/z 174 (MH+, 19), 173 (34), 156
(100), 143 (10), 130 (12), 128 (19); HRMS (CI, CH4): MH+
(C7H12NO4) Calcd. 174.0766, Found 174.0769.
=
170.9 (C O); MS (CI, NH3): m/z 286.6, 91.9; Anal. Calcd. for
C18H24N2O2: C, 71.97; H, 8.05; N, 9.33. Found: C, 71.84; H, 8.14;
N, 9.24%.
(3S,4R,5S)-(1-Benzyl-2-cyano-4-isopropyl-azetidin-3-yl)-
acetic acid ethyl ester 32. Yield: 61%; Rf: 0.52 (AcOEt–PE,
2 : 8); [a]2D5 +1.5 (c 1.5, CHCl3); dH(300 MHz, CDCl3, Me4Si):
0.74 and 0.75 (2d, 6H, J = 6.9, CH3), 1.12 (t, 3H, J =
7.1, CH3), 1.53–1.64 (m, 1H, (CH3)2CH), 2.25–2.62 (m, 4H,
CHCH2CO2Et and CHN), 3.27 (d, 1H, J = 7.1, CHCN), 3.42
(d, 1H, J = 13.0, CHHPh), 3.77 (d, 1H, J = 13.0, CHHPh), 4.00
(q, 2H, J = 7.1, CH2O), 7.12–7.25 (m, 5H, Ar); dC(62.9 MHz,
CDCl3, Me4Si): 14.1, 17.6, 18.7 (CH3), 32.8, 36.2 (CH), 38.0
(CH2), 53.8 (CH), 61.0, 62.2 (CH2), 75.1 (CH), 119.2 (CN),
127.8, 128.5, 129.1 (CHAr), 136.0 (CqAr), 170.5 (CO); MS (CI,
(2S,3S,4S)-3-Carboxymethyl-4-methyl-azetidine-2-carboxylic
acid 36. Yield: 66%; Rf = 0.25 (EtOH–40%NH4OH–H2O,
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 9 2 6 – 3 9 3 6
3 9 3 3

View Online
9 : 3 : 1); [a]25 +9.5 (c 0.2, H2O); dH(200 MHz, D2O, Me4Si): 1.51
(d, 3H, J =D7.0, Me), 2.38–2.91 (m, 3H, CH–CH2), 4.21 (quint,
1H, J = 8.8, CHMe), 4.33 (d, 1H, J = 8.6, NCHCOOH);
dC(75.5 MHz, D2O, Me4Si): 21.3 (CH3), 43.0 (CH2), 45.3 (CH),
62.4 (CH), 63.0 (CH), 176.3, 181.5 (CO); Anal. Calcd. for
C7H11NO4, 0.75 NH3, 1 H2O: C, 41.22; H, 7.54; N, 12.02.
Found: C, 41.40; H, 8.13; N, 12.03%; MS (CI, CH4): m/z 174
(MH+, 22), 173 (55), 156 (100), 130 (26), 128 (35); HRMS (CI,
CH4): MH+ (C7H12NO4) Calcd. 174.0766, Found 174.0764.
(2R,3S,4S )-3-Carboxymethyl-4-isobutyl-azetidine-2-car-
boxylic acid 43. Yield: 64%; Rf = 0.79 (EtOH–40%NH4OH–
H2O, 9 : 3 : 1); mp: 185 ◦C; [a]D25 +46 (c 0.3, H2O); dH(300 MHz,
D2O, Me4Si): 0.57 (d, 6H, J = 6.5, 2Me), 1.23–1.65 (m, 3H,
CH2CHMe2), 1.96–2.23 (m, 2H, CH2COOH), 2.75–2.84 (m,
1H, CHCH2COOH), 3.80–3.98 (m, 1H, CHN), 4.37 (d, 1H,
J = 8.9, NCHCOOH); dC(75.5 MHz, D2O, Me4Si): 21.0, 21.8
(CH3), 24.9 (CH), 37.1 (CH2), 37.3 (CH), 41.5 (CH2), 59.3,
63.6 (CH), 171.4, 178.0 (CO); MS (CI, NH3): m/z 237.1,
213.0, 199.0, 176.1, 153.0, 142.1, 126.1, 112.1; EIHRMS: M+
(C10H17NO4) Calcd. 215.116, Found 215.116.
(2R,3S,4S)-3-Carboxymethyl-4-ethyl-azetidine-2-carboxylic
acid 37. Yield: 71%; Rf = 0.85 (EtOH–40%NH4OH–H2O, 9 : 3 :
1); [a]2D5 +140 (c 0.2, H2O); dH(300 MHz, D2O, Me4Si): 0.83 (t,
3H, J = 7.5, Me), 1.84 (quint, 2H, J = 7.5, CH2CH3), 2.14–2.45
(m, 2H, CH2COOH), 2.97–3.08 (m, 1H, CHCH2COOH), 3.92
(q, 1H, J = 7.3, CHN), 4.60 (d, 1H, J = 10.2, NCHCOOH);
dC(75.5 MHz, D2O, Me4Si): 8.6 (CH3), 25.7, 37.3 (CH2),
37.7, 59.1, 66.6 (CH), 171.5, 178.7 (CO); MS (CI, NH3): m/z
176.1, 165.0, 158.0, 140.0, 115.0, 112.0, 96.0; EIHRMS: M+
(C8H13NO4) Calcd. 187.084, Found 187.087.
(2S,3S,4S )-3-Carboxymethyl-4-isobutyl-azetidine-2-car-
boxylic acid 44. Yield: 77%; Rf = 0.88 (EtOH–40%NH4OH–
◦
H2O, 9 : 3 : 1); mp: 192 C; [a]2D5 0 (c 0.3, H2O); dH(300 MHz,
D2O, Me4Si): 0.81 (d, 6H, J = 6.5, 2Me), 1.59–1.76 (m, 3H,
CH2CHMe2), 2.43–2.63 (m, 2H, CH2COOH), 2.73–2.77 (m,
1H, CHCH2COOH), 4.10–4.14 (m, 1H, CHN), 4.31 (d, 1H,
J = 8.4, NCHCOOH); dC(75.5 MHz, D2O, Me4Si): 21.1, 21.8
(CH3), 23.9 (CH), 39.8 (CH2), 41.4 (CH), 41.7 (CH2), 60.5,
61.8 (CH), 173.4, 178.3 (CO); MS (CI, NH3): m/z 244.2, 216.2,
157.0, 139.0, 93.0, 81.0, 78.0; EIHRMS: M+ (C10H17NO4)
Calcd. 215.116, Found 215.116.
(2S,3S,4S)-3-Carboxymethyl-4-ethyl-azetidine-2-carboxylic
acid 38. Yield: 58%; Rf = 0.88 (EtOH–40%NH4OH–H2O, 9 :
3 : 1); [a]2D5 0 (c 0.3, H2O); dH(300 MHz, D2O, Me4Si): 0.83 (t,
3H, J = 7.5, Me), 1.78 (quint, 2H, J = 7.5, CH2CH3), 2.35–2.51
(m, 2H, CH2COOH), 2.72–2.79 (m, 1H, CHCH2COOH), 3.96
(q, 1H, J = 7.5, CHN), 4.28 (d, 1H, J = 8.3, NCHCOOH);
dC(75.5 MHz, D2O, Me4Si): 8.5 (CH3), 25.9, 40.5 (CH), 40.6
(CH2), 60.3, 64.8 (CH), 173.5, 178.9 (CO); MS (CI, NH3): m/z
199.1, 186.1, 168.1, 140.1, 112.1, 96.0, 78.0, 63.0; EIHRMS: M+
(C8H13NO4) Calcd. 187.084, Found 187.087.
(4S)-[Benzyl-(2-hydroxy-1-methylethyl)-amino]-acetic acid
tert-butyl ester 45. To a solution of amino alcohol 6 (2.5 g,
15.2 mmol) in DMF (65 mL) were added sodium iodide (4.5 g,
30.5 mmol), potassium hydrogencarbonate (3.05 g, 30.5 mmol)
and tert-butylbromoacetate (5.9 g, 30.5 mmol). After stirring
at rt for 12 h, the mixture was treated with water (100 mL).
Extraction with DCM and the usual workup gave amino alcohol
45 after purification (E–CyH = 1 : 1) by flash chromatography
(3.22 g, 76%).
(2R,3S,4S)-3-Carboxymethyl-4-propyl-azetidine-2-carboxylic
acid 39. Yield: 73%; Rf = 0.85 (EtOH–40%NH4OH–H2O, 9 :
3 : 1); [a]2D5 +105 (c 0.2, H2O); dH(300 MHz, D2O, Me4Si): 0.89
(t, 3H, J = 7.5, Me), 1.22–1.50 (m, 2H, CH2CH3), 1.75–1.85
(m, 2H, CH2CH2CH3), 2.21 (dd, 1H, J = 8, 15, CHHCOOH),
2.51 (dd, 1H, J = 4, 15, CHHCOOH), 3.01–3.18 (m, 1H,
CHCH2COOH), 4.12 (q, 1H, J = 7.3, CHN), 4.75 (d, 1H, J =
10.2, NCHCOOH); EIHRMS: M+ (C9H15NO4) Calcd. 201.100,
Found 201.099.
Rf = 0.38(E–CyH = 6 : 4); [a]2D5 +34.0(c 1.3, CHCl3); IR (film):
3868, 3462, 2976, 2930, 1726, 1455, 1363 cm−1; dH(250 MHz,
CDCl3, Me4Si): 1.01 (d, 3H, J = 6.8, CH3), 1.45 (s, 9H, t-Bu),
2.99–3.13 (m, 1H, CHMe), 3.13 (d, 1H, J = 17.0, CHHPh), 3.29
(d, 1H, J = 17.3, CHHPh), 3.34–3.55 (m, 2H, CH2OH), 3.67
(d, 1H, J = 13.6, NCHH), 3.90 (d, 1H, J = 13.6, NCHH), 3.99
(broadd, 1H, J = 7.1, OH), 7.25–7.45 (m, 5H, Ph); dC(62.9 MHz,
CDCl3, Me4Si): 10.3, 28.2 (CH3), 51.4, 55.1 (CH2), 57.2 (CH),
63.6 (CH2) 81.3 (Cq), 127.3, 128.4, 128.9 (CHAr), 138.9 (CqAr),
172.0 (CO); Anal. Calcd. for C16H25NO3: C, 68.79; H, 9.02; N,
5.01. Found: C, 68.54; H, 9.16; N, 4.91%.
(2S,3S,4S)-3-Carboxymethyl-4-propyl-azetidine-2-carboxylic
acid 40. Yield: 68%; Rf = 0.82 (EtOH–40%NH4OH–H2O, 9 :
3 : 1); [a]25 +5 (c 0.2, H2O); dH(300 MHz, D2O, Me4Si): 0.88 (t,
3H, J = D7.5, Me), 1.20–1.52 (m, 2H, CH2CH3), 1.75–1.95 (m,
2H, CH2CH2CH3), 2.60–3.01 (m, 3H, CHCH2COOH), 4.18
(q, 1H, J = 7.3, CHN), 4.55 (d, 1H, J = 10.5, NCHCOOH);
EIHRMS: M+ (C9H15NO4) Calcd. 201.100, Found 201.099.
(2E,4S)-4-(Benzyl-tert-butoxycarbonylmethyl-amino)-pent-2-
enoic acid ethyl ester 46. Following the general procedure for
the synthesis of 17–22, and starting from 45, compound 46 was
obtained as an oil.
(2R,3S,4S)-3-Carboxymethyl-4-isopropyl-azetidine-2-car-
boxylic acid 41. Yield: 49%; Rf = 0.87 (EtOH–40%NH4OH–
H2O, 9 : 3 : 1); mp: 152 ◦C; [a]25 +32 (c 0.3, H2O); dH(300 MHz,
D2O, Me4Si): 0.83 (d, 3H, JD = 6.5, Me), 0.89 (d, 3H, J =
6.5, Me), 1.99–2.11 (m, 1H, CH(Me)2), 2.21–2.48 (m, 2H,
CH2COOH), 3.08–3.19 (m, 1H, CHCH2COOH), 3.71 (t,
1H, J = 7.5, CHN), 4.55 (d, 1H, J = 10.2, NCHCOOH);
dC(75.5 MHz, D2O, Me4Si): 16.3, 17.2 (CH3), 30.8, 35.9 (CH),
37.8 (CH2), 56.4, 70.3 (CH), 171.4, 178.0 (CO); MS (CI,
NH3): m/z 199.1, 183.1, 156.1, 140.0, 122.1, 112.0, 91.0, 78.0;
EIHRMS: M+ (C9H15NO4) Calcd. 201.100, Found 201.096.
Yield: 77%; Rf = 0.58 (E–CyH = 6 : 4); [a]2D5 −31.8 (c 0.9,
CHCl3); IR (film): 2976, 2925, 1726, 1644, 1454 cm−1;
dH(250 MHz, CDCl3, Me4Si): 1.11 (d, 3H, J = 6.8, Me), 1.19 (t,
3H, J = 7.1, Me), 1.34 (s, 9H, t-Bu), 3.12 (AB syst., 2H, J =
17.0, CH2Ph), 3.54 (dquint, 1H, J = 1.3, 6.8, CHMe), 3.70 (s,
2H, NCH2CO), 4.11 (quad, 2H, J = 7.3, CH2CH3), 5.85 (dd, 1H,
=
J = 1.2, 15.7, CHCO2Et), 6.84 (dd, 1H, J = 6.5, 15.7, CH CH),
7.08–7.31 (m, 5H, Ph); dC(62.9 MHz, CDCl3, Me4Si): 14.3, 16.3,
28.2 (Me), 51.8, 55.2 (CH2), 56.3 (CH), 60.4 (CH2), 80.9 (Cq),
121.9 (CH), 128.1, 128.4, 128.7 (CHAr), 139.4 (CqAr), 150.4
(CH), 166.5, 171.2 (CO); Anal. Calcd. for C20H29NO4: C, 68.14;
H, 8.41; N, 4.03. Found: C, 69.21; H, 8.59; N, 4.00%.
(2S,3S,4S)-3-Carboxymethyl-4-isopropyl-azetidine-2-car-
boxylic acid 42. Yield: 43%; Rf = 0.81 (EtOH–40%NH4OH–
◦
H2O, 9 : 3 : 1); mp: 113 C; [a]2D5 0 (c 0.3, H2O); dH(300 MHz,
(2S,3S,4S )-1-Benzyl-3-ethoxycarbonylmethyl-4-methyl-
azetidine-2-carboxylic acid tert-butyl ester 47. To a solution
of 45 (0.35 mmol, 121 mg) in THF (6 mL) was added, at
−78 ◦C, a 1 M solution of potassium tert-butylate in THF
(0.35 mL, 0.35 mmol). The mixture was warmed to −50 ◦C
within one hour and was hydrolyzed by addition of a saturated
aqueous solution of NH4Cl. Usual workup was followed by
purification by preparative TLC (E–CyH, 6 : 4), which gave
47 as an oil (60 mg, 50%). Rf = 0.52 (E–CyH = 60 : 40);
D2O, Me4Si): 0.56 (d, 3H, J = 6.5, Me), 0.62 (d, 3H, J =
6.5, Me), 1.65–1.77 (m, 1H, CH(Me)2), 2.14–2.31 (m, 2H,
CH2COOH), 2.51–2.63 (m, 1H, CHCH2COOH), 3.51 (t,
1H, J = 7.0, CHN), 4.05 (d, 1H, J = 10.2, NCHCOOH);
dC(75.5 MHz, D2O, Me4Si): 16.0, 17.0 (CH3), 30.8, 39.6 (CH),
40.8 (CH2), 59.5, 68.5 (CH), 173.3, 178.7 (CO); MS (CI,
NH3): m/z 202.1, 79.0, 64.0; EIHRMS: M+ (C9H15NO4) Calcd.
201.100, Found 201.096.
3 9 3 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 9 2 6 – 3 9 3 6

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dH(200 MHz, CDCl3, Me4Si): 1.03 (d, 3H, J = 6.2, Me), 1.25
(t, 3H, J = 7.0, Me), 1.38 (s, 9H, t-Bu), 2.35–2.55 (m, 3H,
CHCH2CO2Et), 2.84 (quint, 1H, J = 7.0, CHMe), 3.17 (d, 1H,
J = 7.5, NCHCO), 3.62 (d, 1H, J = 12.7, CHHPh), 3.79 (d,
1H, J = 12.7, CHHPh), 4.10 (quad, 2H, J = 7.0, CH2CH3),
7.18–7.36 (m, 10H, Ph); dC(75.5 MHz, CDCl3, Me4Si): 14.3,
20.6, 28.0 (CH3), 37.6 (CH2), 39.5 (CH), 60.5, 61.4 (CH2), 64.7,
68.1 (CH), 80.8 (Cq), 127.1, 128.1, 129.6 (CHAr), 137.4 (CqAr),
171.2, 171.4 (CO).
Acknowledgements
We would like to thank CNRS (ATIP jeune e´quipe to FC), the
Danish Medical Research Council, the Lundbeck Foundation
and the Novo Nordisk Foundation for funding.
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R
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occurred on the most sensitive intermediate a-amino aldehyde
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WELK 0–1 column of 250 mm length and of 4 mm diameter. The
flow rate was 1 mL mn−1 and the pressure 39 kg cm−2. (Heptane–
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for 17 was 23.85 min and 22.00 min for ent-17.
16 To the best of our knowledge, intramolecular Michael additions
leading to four-membered rings were not previously reported. Few
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have been reported. For a recent example leading to a pyrrolidine
ring, see: S. Yokoshima, H. Tokuyama and T. Fukuyama, Angew.
Chem., 2000, 112, 4239–4241.
17 Compound 232 crystallised in the space group P212121 (orthorhom-
˚
˚
bic crystal system) with a = 5.558(2) A, b = 17.627(5) A, c = 19.730(4)
A, and dcalcd. = 1.15 g cm−3. The intensity data were measured
˚
on an Enraf-Nonius CAD4 diffractometer (Mo radiation). There
were 1988 independent reflections of which 1118 were considered
observed. Final discrepancy indices were R = 0.0757 and wR =
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