Azetidine derivatives as novel γ-aminobutyric acid uptake inhibitors: Synthesis, biological evaluation, and structure-activity relationship

Article abstract of DOI:10.1016/j.ejmech.2010.02.029

In this study azetidine derivatives representing conformationally constrained GABA or β-alanine analogs were evaluated for their potency as GABA-uptake inhibitors. The study comprised derivatives substituted in 2- as well as in 3-position with either an acetic acid moiety or a carboxylic acid function. In addition, azetidine derivatives bearing a tetrazole ring as a bioisosteric substitute for a carboxylic acid group were included. 3-Hydroxy-3-(4-methoxyphenyl)azetidine derivatives were explored as analogs of the known GABA-uptake inhibitor NNC-05-2045 exhibiting an azetidine ring instead of a piperidine ring present in the latter. Both, N-unsubstituted compounds as well as their N-alkylated lipophilic derivatives, were biologically evaluated for their affinity to the GAT-1 and GAT-3 transporters. Azetidin-2-ylacetic acid derivatives provided with a 4,4-diphenylbutenyl or 4,4-bis(3-methyl-2-thienyl)butenyl moiety as lipophilic residue were found to exhibit the highest potency at GAT-1 with IC₅₀ values of 2.83 ± 0.67 μM and 2.01 ± 0.77 μM, respectively. The most potent GAT-3 inhibitor among these compounds appeared to be the β-alanine analog 1-{2-[tris(4-methoxyphenyl)methoxy]ethyl}azetidine-3-carboxylic acid (12d) displaying an IC₅₀ value of 15.3 ± 4.5 μM. Whereas the tetrazole derivatives showed no potency as GABA-uptake inhibitors, the 3-hydroxy-3-(4-methoxyphenyl)azetidine derivatives exhibited moderate affinity to GAT-1 (compound 18b: IC₅₀ = 26.6 ± 3.3 μM) and to GAT-3 (compound 18e: IC₅₀ = 31.0 ± 4.7 μM).

Full text of DOI:10.1016/j.ejmech.2010.02.029

European Journal of Medicinal Chemistry 45 (2010) 2453e2466
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Azetidine derivatives as novel g-aminobutyric acid uptake inhibitors: Synthesis,
biological evaluation, and structureeactivity relationship
1
*
Mark R. Faust , Georg Höfner, Jörg Pabel, Klaus T. Wanner
Department Pharmazie, Zentrum für Pharmaforschung, LMU München, Butenandtstr. 5-13, D-81377 München, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study azetidine derivatives representing conformationally constrained GABA or b-alanine analogs
Received 28 August 2009
Received in revised form
8 February 2010
Accepted 10 February 2010
Available online 14 February 2010
were evaluated for their potency as GABA-uptake inhibitors. The study comprised derivatives substituted
in 2- as well as in 3-position with either an acetic acid moiety or a carboxylic acid function. In addition,
azetidine derivatives bearing a tetrazole ring as a bioisosteric substitute for a carboxylic acid group were
included. 3-Hydroxy-3-(4-methoxyphenyl)azetidine derivatives were explored as analogs of the known
GABA-uptake inhibitor NNC-05-2045 exhibiting an azetidine ring instead of a piperidine ring present in
the latter. Both, N-unsubstituted compounds as well as their N-alkylated lipophilic derivatives, were
biologically evaluated for their affinity to the GAT-1 and GAT-3 transporters. Azetidin-2-ylacetic acid
derivatives provided with a 4,4-diphenylbutenyl or 4,4-bis(3-methyl-2-thienyl)butenyl moiety as lipo-
Dedicated to Prof. H. Wagner with best
wishes on the occasion of his 80th birthday.
Keywords:
GABA-uptake inhibitors
GAT-1
GAT-3
Azetidine
philic residue were found to exhibit the highest potency at GAT-1 with IC50 values of 2.83 ꢀ 0.67
2.01 ꢀ 0.77 M, respectively. The most potent GAT-3 inhibitor among these compounds appeared to be
the -alanine analog 1-{2-[tris(4-methoxyphenyl)methoxy]ethyl}azetidine-3-carboxylic acid (12d) dis-
playing an IC50 value of 15.3 ꢀ 4.5 M. Whereas the tetrazole derivatives showed no potency as GABA-
uptake inhibitors, the 3-hydroxy-3-(4-methoxyphenyl)azetidine derivatives exhibited moderate affinity
to GAT-1 (compound 18b: IC50 ¼ 26.6 ꢀ 3.3 M) and to GAT-3 (compound 18e: IC50 ¼ 31.0 ꢀ 4.7 M).
Ó 2010 Elsevier Masson SAS. All rights reserved.
mM and
m
b
m
m
m
1. Introduction
other neurotransmitters such as dopamine, serotonin, norepi-
nephrine, and glycine [13e16]. Recently, the three-dimensional
structure of a bacterial homologue of Naþ/Clꢁ-dependent neuro-
transmitter transporters, the leucine transporter from Aquifex
aeolicus has been elucidated by X-ray crystallographic analysis [17].
With this analysis a major advance toward the understanding of
structure-function relationship in this important class of trans-
porters has been achieved. Meanwhile, the structure of this leucine
transporter has also been used, for example, as a template for the
construction of three-dimensional models of the GABA transporter
GAT-1 [18,19]. Molecular cloning techniques revealed the existence
of four distinct GABA transporters in humans and other species.
These are termed as GAT-1, GAT-2, GAT-3, and BGT-1 according to
a nomenclature introduced by Borden et al. which will be used in
this paper (for murine GABA transport proteins nomenclature is
different) [20e22]. Our research is focused on selective targeting
GABA transporter subtypes, especially GAT-1 and GAT-3, which
immunocytochemical and in situ hybridization studies have shown
to be the most prevalent GABA transporters in the brain. Subtype
selective compounds are of great interest as they could serve as
valuable tools for the characterization of the physiological function
of the various GABA transporters. Moreover, inhibitors of the
different GABA-uptake proteins can be expected to be of
Alterations in GABAergic function have been found to be
involved in a number of CNS disorders, including epilepsy [1],
Huntington's chorea [2], migraine [3], Parkinson disease [4], and
depression [5e8]. Enhancement of the GABA function can be ach-
ieved through several mechanisms including direct receptor acti-
vation by agonists, allosteric activation by modulators such as
benzodiazepines and barbiturates or inhibition of the enzymatic
GABA degradation by for example vigabatrin [9]. The long-term
utility of both vigabatrin and benzodiazepines as anticonvulsants is
limited by the rapid development of tolerance [10,11]. An efficient,
alternative strategy to palliate GABA deficiency is the inhibition of
GABA transport proteins. These high-affinity carriers regulate
synaptic and extra-synaptic availability of GABA by transporting
the neurotransmitter from the synaptic cleft into presynaptic nerve
terminals and into surrounding astrocytes [12]. GABA-uptake
proteins belong to the large family of Naþ/Clꢁ-dependent trans-
porter proteins, which also include the transporters for several
* Corresponding author. Tel.: þ49 89 2180 77249; fax: þ49 89 2180 77247.
E-mail address: klaus.wanner@cup.uni-muenchen.de (K.T. Wanner).
1
3M ESPE AG, ESPE Platz, D-82229 Seefeld, Germany.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.029

2454
M.R. Faust et al. / European Journal of Medicinal Chemistry 45 (2010) 2453e2466
therapeutic benefit as well, as, for example, GAT-1 is already
a proven target for the therapy of epilepsy.
[38,39]. Replacing the nipecotic acid in (S)-SNAP-5114 (9), we found
compound 11, which compares well to (S)-SNAP-5114 (9) withrespect
A number of conformationally restricted cyclic GABA analogs
such as nipecotic acid (1) and guvacine (2) were found to be inhib-
itors of [3H]-GABA uptake in in vitro experiments (Scheme 1) [23].
to both, its potency at (IC50 ¼ 3.1
mM) and subtype selectivity for GAT-
3 as compared to GAT-1 (22:1) [35,36].
As part of an ongoing project with the aim to explore con-
b
-Alanine has been reported to exhibit moderate affinity to GABA-
uptake transporters with GAT-3 and GAT-2 selectivity (GAT-3:
M) [24]. The use of these amino
formationally restricted GABA and b-alanine analogs of various ring
sizes as potential subtype selective GABA-uptake inhibitors, we
performed the synthesis of a series of azetidine derivatives
provided with a carboxylic acid moiety and studied their in vitro
GABA-uptake activity (see Scheme 3). As parent compounds the
azetidine derivatives 12ae15a have been selected in which the acid
functionality either resides in the 2- or 3-position of the hetero-
cyclic ring system. Variation of the position of the acid functionality
was expected to shed some light on the structureeactivity rela-
tionships for that class of compounds. In potent GABA-uptake
inhibitors as for example in SK&F-89976-A (4), SK&F-100330-A (6),
tiagabine (5), and (S)-SNAP-5114 (9) typically lipophilic N-substit-
IC50 ¼ 110
mM; GAT-2: IC50 ¼ 66
m
acids as pharmacological agents has been hampered by their poor
penetration of the blood brain barrier [23,25]. Thus, considerable
efforts have been made to develop selective and potent GABA
transport inhibitors displaying favorable pharmacokinetic proper-
ties. Ali et al. [26] showed that adding the lipophilic 4,4-diphe-
nylbut-3-en-1-yl moiety to the nitrogen atom of nipecotic acid (1) or
guvacine (2) leading to SK&F-89976-A (4) (IC50 ¼ 0.20
mM) and
SK&F-100330-A (6) (IC50 ¼ 0.2 M), respectively, (Scheme 1)
m
significantly increases the ability of these compounds to enter the
brain and their potency as GABA-uptake inhibitors. To date,
numerous lipophilic derivatives of nipecotic acid (1) and guvacine
(2) have been synthesized and biologically evaluated for their
potency as GABA-uptake inhibitors [27]. Tiagabine (5), which pref-
erentially targets GAT-1, has been in clinical use since 1997 [28e33].
It is an effective and safe drug for the management of epilepsy. But, it
possesses an unfavorable pharmacokinetic profile characterized by
a short half-life [34]. Besides, tiagabine (5) exhibits moderate side
effects, including dizziness, fatigue, and confusion which seem to
limit its usefulness [34]. Thus, the search for GABA-uptake inhibitors
with improved pharmacological properties continues.
uent of
a specific structure are present, which significantly
contribute to the potency, subtype selectivity, and improved
pharmacokinetic properties of these compounds. Accordingly,
derivatives of the amino acids 12ae15a provided with residues bed
should be included in this study as well. In addition, the azetidine
derivatives 16a and 17a, in which the carboxylic acid moiety is
replaced by the bioisosteric tetrazole group should be included in
this study. Furthermore, we were interested in derivatives of 18a,
substituted with both a 4-methoxyphenyl and a hydroxy group in
the 3-position of the azetidine ring, and its derivatives 18be18e as
analogs of NNC-05-2045 (10).
Previously [35,36], we reported the synthesis of the pyrrolidine
derivative 7 and 8, which may be considered as analogs of tiagabine
(5) and SK&F-89976-A (4), respectively, in which the nipecotic acid
subunit is replaced by homoproline (3). Both compounds 7 and 8
show a high potency at and subtype selectivity for the GAT-1
2. Chemistry
2.1. Synthesis of unsubstituted azetidine derivatives
protein (7: IC50 ¼ 0.396
mM, 8: 0.343 mM).
Dhar et al. [37] succeeded in the synthesis of the first inhibitor
withhighpotencyatand subtype selectivity for GAT-3, (S)-SNAP-5114
Azetidine-3-carboxylic acid (12a), azetidine-3-ylacetic acid
(13a), (S)-azetidine-2-carboxylic acid (14a), and 3-[4-methox-
yphenyl]azetidine-3-ol (18a) (see Scheme 3) were synthesized
according to literature procedures [39e42]. For the synthesis of
azetidin-2-ylacetic acid (15a) a method has been patented by Orr
(9) (Scheme 2) with an IC50 value of 5 mM and a selectivity of 78:1 for
GAT-3 versus GAT-1 [37]. Thomsen et al. [32,38] presented NNC-05-
2045 (10), a 4-methoxyphenylpiperidin-4-ol derivative, which was
found to be a potent inhibitor at BGT-1. But, the compound displayed
only moderate subtype selectivity for this transporter (BGT-1:
[43]. It starts from
g-butyrolactone and requires seven steps. We
were able to develop a more convenient synthesis leading to 15a in
only five steps (Scheme 4). The synthetic strategy is based on
a method reported by Shono et al. [44] for the preparation of the
Ki ¼ 1.6 ꢀ 0.4
mM; GAT-1: Ki ¼ 27 ꢀ 2
mM; GAT-3: Ki ¼ 6.1 ꢀ1.3
mM)
COOH
COOH
COOH
N
H
N
H
N
H
nipecotic acid (1)
guvacine (2)
homoproline (3)
COOH
COOH
COOH
COOH
COOH
S
N
N
N
N
N
S
S
S
SK&F-89976-A ( )
tiagabine ( )
SK&F-100330-A ( )
7
8
4
5
6
Scheme 1. Representative GAT-1 selective GABA-uptake inhibitors.

M.R. Faust et al. / European Journal of Medicinal Chemistry 45 (2010) 2453e2466
2455
OMe
HO
COOH
COOH
OMe
N
N
N
O
O
N
MeO
MeO
OMe
OMe
OMe
(S)-SNAP-5114 (9)
NNC-05-2045 (10)
11
Scheme 2. Representative BGT-1 and GAT-3 selective GABA-uptake inhibitors.
2-substituted azetidines involving the anodic oxidation of a tosy-
lated azetidine to the corresponding 2-acetoxy derivative followed
by the generation of an iminium ion and subsequent trapping
reaction with an appropriate nucleophile. Modifying Shono's
synthetic approach, we used the benzyloxycarbonyl group instead
of the tosyl group to protect the nitrogen atom of the azetidine ring.
The benzyloxycarbonyl group was found to be preferable since it
can be cleaved under milder reaction conditions ensuring that the
azetidine ring remains intact. To access azetidin-2-ylacetic acid
derivatives, we subjected the benzyloxycarbonyl group protected
azetidine 20, which we had synthesized from 19, to anodic oxida-
tion in the presence of sodium acetate to give the N-acyl iminium
ion precursor 21 in 65% yield. Various transformation reactions
were performed to explore the suitability of 21 for the preparation
of 2-substituted azetidine derivatives. In each case 21 was treated
with TiCl4 at ꢁ78 ꢂC (ꢁ70 ꢂC for the reaction with TMSCN and
[1-(4-fluorophenyl)vinyloxy]trimethylsilane) to generate the cor-
responding N-acyl iminium ion which was then reacted with
different nucleophiles. The nucleophilic additions to the interme-
diate N-acyl iminium ion were successfully performed employing
(1-methoxyvinyloxy)trimethylsilane, (1-tert-butoxyvinyloxy)tri-
methylsilane, TMSCN, and [1-(4-fluorophenyl)vinyloxy]trime-
thylsilane resulting in the formation of the addition products 22, 23,
25, and 26 in 55%, 55%, 58%, and 46% yield, respectively (Scheme 4).
Addition product 22 turned out to be well suited for the preparation
of the required azetidin-2-ylacetic acid 15a. Saponification of the
ester functionality with NaOH in MeOH and N-deprotection by
hydrogenation over Pd/C provided it in an overall yield of 28% (from
19). Catalytic hydrogenation of compound 23 provided tert-butyl
ester 27 in 61% yield, required for the synthesis of the N-alkylated
lipophilic azetidine derivatives 15b and 15c (Scheme 3).
Azetidine derivative 16a$HCl exhibiting a tetrazole ring in the
3-position, was synthesized starting from ketone 28 [45,46] (Scheme
5). Reaction of the lithiated tetrazole derivative 29 generated from
1-(4-methoxybenzyl)-1H-tetrazole according to a procedure from
Satoh and Marcopulos [47] with the ketone 28 at ꢁ90 ꢂC provided
N N
OMe
NH
N
N
N
N
COOH
COOH
O
HO
HO
N
COOH
H
COOH
N
R
N
R
N
R
N
R
N
N
R
N
R
R
12
13
14
15
16
17
18
R:
a
b
c
d
e
H
S
O
N
MeO
OMe
Me
Me
S
OMe
Scheme 3. Azetidine derivatives.

2456
M.R. Faust et al. / European Journal of Medicinal Chemistry 45 (2010) 2453e2466
O
COOH
COOH
Me
COOR
c
f
g
a
b
O
N+
Cl
N
N
-
N
N
N
H
H
H
O
h
OBn
24 (85%)
O
d
OBn
O
OBn
O
OBn
20 (93%)
21 (65%)
15a (100%)
19
R = Me: 22 (55%)
R = tBu: 23 (55%)
e
COOR
-OAc
O
N+
F
H
H
CN
N
N
R = tBu: 27 (61%)
O
OBn
26 (46%)
O
OBn
25 (58%)
Scheme 4. Reagents and conditions: (a) benzyl chloroformate, NEt3, CH2Cl2, rt; (b) anodic oxidation, AcOH, NaOAc; (c) (1-methoxyvinyloxy)trimethylsilane or (1-tert-butoxyvinyloxy)
trimethylsilane, TiCl4, CH2Cl2, ꢁ78 ꢂC (2 h), 0 ꢂC (1 h); (d) TMSCN, TiCl4, CH2Cl2, ꢁ70 ꢂC (2 h); (e) [1-(4-fluorophenyl)vinyloxy]trimethylsilane, TiCl4, CH2Cl2, ꢁ70 ꢂC (2 h); (f) 2 N NaOH,
MeOH, 72 h, rt; (g) H2, Pd/C, MeOH, 16 h, rt; (h) Pd/C, H2, MeOH, AcOH, rt, 16 h.
after workup the addition product 30 in 63% yield. Subsequent
removal of the benzhydryl and the 4-methoxybenzyl group by
catalytic hydrogenation with Pearlman's catalyst [48] afforded 3-
(1H-tetrazol-5-yl)azetidin-3-ol hydrochloride (16a$HCl) in 49%
yield. Alternatively, selective removal of the benzhydryl group by
hydrogenation over Pd/C in TFA/acetic acid provided compound 31
in 39% yield. The synthesis of the structurally related 5-(azetidin-3-
yloxy)-1H-tetrazole hydrochloride (17a$HCl) was accomplished as
indicated in Scheme 5. Reaction of the sodium salt of the alcohol 32
[40], generated by treatment with sodium hydride, with iodo
N
N
N
HO
N
H
N
b, c
OMe
N
N
N+
H H
-
O
Cl
N
HO
Ph
N
N
N
a
+
N
N
OMe
N
16a HCl (49%)
Li
d
Ph
28
Ph
Ph
N
30 (63%)
29
N
N
HO
N
OMe
N
H
31 (39%)
OMe
N N
N N
NH
N
N
N
O
N
OH
N
N
O
N
N
b, c
e
N
+
N+
OMe
-
I
Cl
H H
Ph
Ph
34 (39%)
Ph
32
Ph
17a HCl (94%)
33
Scheme 5. Reagents and conditions: (a) THF/TMEDA ¼ 10:1, ꢁ90 ꢂC, 30 min; (b) H2, Pd(OH)2/C, MeOH, rt, 72 h; (c) 2 N HCl; (d) Pd/C, H2, AcOH, TFA, rt, 72 h; (e) NaH, THF, rt, 4 h.

M.R. Faust et al. / European Journal of Medicinal Chemistry 45 (2010) 2453e2466
2457
COOH
trityloxyethyl group, but also for 40b, which underwent decom-
position reactions under these reaction conditions. So all attempts
to obtain the corresponding amino acids 13b and 13d failed. The
cleavage of the tert-butyl esters in compounds 41b and 41c, effected
by means of 20% TFA in dichloromethane at room temperature led
to the amino acids 15b$HCl and 15c. All final products, thus
obtained, (Scheme 7) were then studied for their inhibitory potency
at the GABA transporters GAT-1 and GAT-3.
COOMe
a
N
+ Cl
H
N
H
H
12a
35 (97%)
COOH
COOMe
b
3. Biological results
N
+ Cl
N
H
H
As standard assay for the characterization of GABA-uptake
inhibitors, we had used a procedure based on bovine brain material
[54]. Because of emerging cases of bovine spongiform encephalop-
athy (BSE) in German cattle during the course of this study, we were,
however, forced to establish a new test system. We decided for
porcine brain material as a new protein source, as this was regarded
as non-hazardous. The reliability of the newly developed assay
system based on porcine brain was evaluated using a series of
reference compounds. Results were found to be in accordance with
both, the previously used assay based on bovine brain material as
well as the alternative assay systems published by other research
groups. The results of the biological evaluation of the azetidine
carboxylic acid and azetidinylacetic acid core structure and the
corresponding N-alkylated derivatives as GABA-uptake inhibitors
are summarized inTable 1. The values of some reference compounds
can be found in Table 2. Azetidine-3-carboxylic acid (12a) displayed
moderate GABA-uptake inhibition at GAT-3 with a selectivity of 34:1
for this transporteras compared to GAT-1 which is in the range of the
O
O
36
37 (58%)
Scheme 6. Reagents and conditions: (a) SOCl2, MeOH, ꢁ60 ꢂC (30 min), rt (16 h);
(b) AcCl, MeOH, rt, 16 h.
derivative 33 led to compound 34 in 39% yield. This was then
transformed into the desired compound 17a$HCl in 94% yield by
concurrent removal of the benzhydryl and the 4-methoxybenzyl
group, again accomplished by hydrogenation over Pearlman's
catalyst.
2.2. Synthesis of N-alkylated azetidine derivatives
For the synthesis of the N-alkylated azetidine derivatives
provided with a carboxylic acid moiety, in addition, the carboxylic
acid esters 35 and 37 (Scheme 6) of the amino acids 12a [49] and
13a, (Scheme 3) had to be prepared. Azetidin-3-carboxylic acid 12a
was converted to the corresponding ester 35 [50] employing SOCl2
in methanol (Scheme 6). For the synthesis of methyl azetidin-3-
ylethanoate hydrochloride 37 [51], a method published by Nudel-
man et al. [52] was followed. This method allows a one-step
conversion of N-Boc protected amino acids to the corresponding N-
unprotected amino acid esters by treating the starting material
with acetyl chloride in methanol. When applied to Boc protected
azetidin-3-ylacetic acid 36 [40] (Scheme 6), the desired ester
compound 37 was obtained in a 58% yield.
The N-alkylation of the amino acid esters 27, 35, and 37, the
tetrazole derivative 31 and the 4-hydroxy-4-phenyl derivative 18a
[42] (Scheme 7) was accomplished in analogy to a procedure
published by Dhar et al. [37], which is based on alkyl bromides and
an in situ Finkelstein reaction to generate alkyl iodides as alkylation
agents (Scheme 7). In all cases the desired alkylation products were
obtained, but yields varied considerably and were, in general, not
higher than 60% (see Scheme 7).
GAT-3 selectivity observed for the structurally related
b-alanine
(GAT-3/GAT-1 81:1 [55]). Despite the affinity of 12a to the GAT
proteins, its N-alkylated derivatives 12bec with lipophilic residues
were found to be more or less inactive at 100
estingly, derivative 12d substituted with a trityloxyethyl group,
showed almost no activity at GAT-1 (100 M: 76%), but an IC50 value
of 15.3 M for GAT-3 inhibition. This observation resembles the
mM (Table 1). Inter-
m
m
affinity and selectivity of the GAT-3 selective inhibitor (S)-SNAP-
5114 (9) and its homoproline analog 11 (Table 2). Azetidin-3-ylacetic
acid 13a displayed IC50 values of 43.3 mM for GAT-1 and 39.8 mM for
GAT-3. A preliminary screening of product 13b, that from the
saponification had been obtained in impure form only, showed no
activity at GAT-1 and GAT-3. Therefore, we waived further efforts in
synthesizing the N-substituted derivatives of 13a. The naturally
occurring azetidin-2-carboxylic acid (14a) showed only negligible
affinity to both, the GAT-1 as well as the GAT-3 transport protein.
Thus, no N-alkylated derivatives of 14a were prepared for biological
evaluation. Among the azetidine derivatives bearing a carboxylic
acid moiety, the lowest IC50 values were observed for the azetidin-2-
ylacetic acid derivative 15c, which is structurally related to tiagabine
(5) and the homoproline derivative 8 (Table 2). The parent structure
15a only showed low affinity for GAT-1 and GAT-3, but the
2.3. Deprotection of the azetidine derivatives
N-alkylated 15c displayed an IC50 value of 2.01
mM (tiagabine (5):
To gain access to the free amino acids, the amino acid esters
39bed and 41bec obtained from the alkylation reaction were
subjected to a saponification reaction with 2 N NaOH at room
temperature (see Scheme 7). In general, the resulting amino acids
were isolated as their hydrochloride salts, except for 12d provided
with a trityloxyethyl moiety, which was extracted as free amino
acid as the trityloxyethyl residue is known to be sensitive to acidic
conditions and heat. A rearrangement to lactones occurred in case
of compounds 40b and 40d under the conditions of the saponifi-
cation reaction obviating the isolation of the desired compounds
[53]. Using acidic conditions (10% HCl at 50 ꢂC) for the hydrolysis of
the ester group was not successful either, not only for compound
40d for which it was expected because of the presence of the
0.159 M, 8: 0.343 M) with a selectivity for GAT-1 as compared to
GAT-3 of 48:1. For the diphenylbutenyl derivative 15b$HCl struc-
turally related to SK&F-89976-A (4) and the homoproline derivative
m
m
7 a potencyat GAT-1 was found (15b$HCl: IC50 ¼ 2.83
7: 0.396 M; Table 2) similar to that of 15c, but its selectivity for GAT-
1 as compared to GAT-3 was distinctly lower (11:1).
mM, 4: 1.18 mM,
m
The IC50 values of both, of the tetrazole and tetrazoloxy
substituted azetidine compounds as well as of the 4-hydroxy-4-
(4-methoxyphenyl)azetidine derivatives are reported in Table 3.
The tetrazole derivatives 16a$HCl and 17a$HCl showed even at high
concentration (1000 mM and 100 mM, respectively) no or only
negligible inhibition of GAT-1 and GAT-3. 4-Hydroxy-4-
(4-methoxyphenyl)azetidine (18a), which was synthesized as an

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M.R. Faust et al. / European Journal of Medicinal Chemistry 45 (2010) 2453e2466
R:
b
c
d
e
S
O
N
MeO
OMe
Me
Me
S
OMe
COOMe
COOMe
COOH
a
b
N+
Cl
+
R
Br
N
R
N
R
H2
38b
38c
38d
39b (21%)
39c (51%)
39d (20%)
12b HCl (92%)
12c HCl (95%)
12d (94%)
35
COOMe
Cl
COOMe
COOH
a
N+
H2
N
R
+
R
Br
N
R
38b
38d
40b (27%)
40d (16%)
13b
13d
37
COOtBu
COOtBu
COOH
a
c
N+
H2
N
R
N
R
+
R
Br
AcO-
27
38b
38c
38d
41b (35%)
41c (16%)
41d (25%)
15b HCl (100%)
15c (69%)
OMe
OMe
HO
HO
a
+
R
Br
N
R
N
H
38b
38c
38d
18b (58%)
18c (52%)
18d (25%)
18e (53%)
18a
N
N
N
N
N
N
HO
HO
N
N
a
OMe
OMe
+
R
Br
N
N
R
H
31
35e
42e (35%)
Scheme 7. Reagents and conditions: (a) K2CO3, NaI; (b) 2 N NaOH, H2O, MeOH, rt, 48 h; (c) TFA, CH2Cl2, rt, 2 h. Where the term “HCl” has been added to the compound number, the
compound has been isolated as hydrochloride salt though only the neutral form is listed.

M.R. Faust et al. / European Journal of Medicinal Chemistry 45 (2010) 2453e2466
2459
Table 1
Uptake inhibition [IC50
(m
M)] at GAT-1 and GAT-3 exhibited by azetidine carboxylic acid derivatives.
COOH
COOH
COOH
COOH
N
R
N
R
N
R
N
R
IC50
[m
M]
IC50
[
mM]
IC50
[m
M]
IC50
[
mM]
R
GAT-1
GAT-3
GAT-1
GAT-3
GAT-1
GAT-3
GAT-1
GAT-3
H
1550 ꢀ 150a
45.5 ꢀ 3.5b
43.3 ꢀ 34.8a 39.8 ꢀ 17.5b 1950 ꢀ 300a 706 ꢀ 26b
111 ꢀ 71a
312 ꢀ 120b
12a
12a
13a
13a
14a
14a
15a
15a
(CH2)2
101%a (n ¼ 1)
12b$HCl
81.6%b (n ¼ 1)
12b$HCl
2.83 ꢀ 0.67a
15b$HCl
31.4 ꢀ 11.5f
15b$HCl
e*
e*
e
e
S
(CH2)2
95.6%a (n ¼ 1)
12c$HCl
96.6%b (n ¼ 1)
12c$HCl
2.01 ꢀ 0.77e
96.6 ꢀ 30.9f
e
e
e
e
15c
15c
S
OMe
76.10%a (n ¼ 1)
15.3 ꢀ 4.5b
MeO
O(CH2)2
e
e
e
e
e
e
12d
12d
OMe
If not stated otherwise the results are given as means of IC50-values ꢀ SEM of three independent experiments each performed in triplicate, n ¼ 1 denotes value from a single
experiment, percentages represent specific binding remaining in presence of 100
m
M inhibitor. The abbreviations aef specify the used biological material: abfcP2B, bbbsP2C,
f
ccfcP2B, dcbsP2C, epfcP2B, pbsP2C (see Experimental section).
Where “HCl” has been added to the compound number the compound has been tested as hydrochloride of the drawn neutral structure.
Preliminary screening of the impure saponification product 13b showed no activity.
*
N-unsubstituted analog of NNC-05-2045 (10) with reduced ring
size was devoid of any significant activity at GAT-1 as well as GAT-3.
However, N-alkylated derivatives of 18a exhibited reasonable
potencies as GABA-uptake inhibitors. The highest affinity was
observed for the diphenylbutenyl derivative 18b showing IC50
GAT-3 with an IC50 value of 15.3 mM and a selectivity greater than
7:1 (GAT-3/GAT-1). The tetrazole substituted azetidine derivatives
16a$HCl and 17a$HCl appeared to be inactive as GABA-uptake
inhibitors when tested at a concentration of 100 mM or 1000 mM,
respectively. Compound 18e as an analog of NNC-05-2045 (10) of
values of 26.6
Compound 18e, which is a direct analog of 10 with a reduced ring
size, was found to display an IC50 value of 31.0 M at GAT-3 (10:
IC50 ¼ 3.34 M) with a moderate preference for this transporter.
m
M and 48.5
m
M at GAT-1 and GAT-3, respectively.
a smaller ring size, showed only a moderate potency at and subtype
selectivity for GAT-3 (IC50 ¼ 31.0
mM).
m
m
5. Experimental section
4. Conclusion
5.1. Methods and materials
Azetidine derivatives were synthesized as structural analogs of
the known GABA-uptake inhibitors tiagabine (5), SK&F-89976-A
(4), (S)-SNAP-5114 (9), and NNC-05-2045 (10). The activity of the
azetidine derivatives was found to be strongly dependent on both
the position of the carboxylic acid moiety and on the nature of the
lipophilic groups. Among the investigated compounds, the highest
affinity at GAT-1 was observed for the azetidin-2-ylacetic acid
analog 15c of tiagabine (5) displaying an IC50 value of 2.01 mM and
a selectivity of 48:1 for GAT-1 over GAT-3. The azetidine-3-
carboxylic acid derivative 12d was the most potent inhibitor of
Anhydrous reactions were carried out in vacuum dried glass-
ware under nitrogen atmosphere. THF, Et2O, NEt3, DME, diiso-
propylamine, 1,4-dioxane, and toluene were freshly distilled from
sodium metal/benzophenone ketyl, CH2Cl2, DMF, and acetonitrile
from CaH2 and CH3OH from Mg prior to use. Other common
solvents for recrystallization and column chromatography were
always distilled before use. Purchased chemical reagents were
used without further purification. TLC plates were made from
silica gel 60 F254 on aluminum sheets (Merck). Compounds were
stained with 5% (NH4)6Mo7O24$4H2O, 0.2% Ce(SO4)2$4H2O, and 5%

2460
M.R. Faust et al. / European Journal of Medicinal Chemistry 45 (2010) 2453e2466
Table 2
Uptake inhibition [IC50 (mM)] at GAT-1 and GAT-3 for some reference compounds.
COOH
COOH
COOH
COOH
COOH
N
R
N
R
N
R
N
R
N
R
IC50
[m
M]
IC50
[m
M]
IC50
[m
M]
IC50
[
mM]
IC50
[m
M]
R
GAT-1
GAT-3
GAT-1
GAT-3
GAT-1
GAT-3
GAT-1
GAT-3
GAT-1
GAT-3
H
4.77 ꢀ 1.17b 17.3 ꢀ 2.36b 2.15 ꢀ 0.11b
18.5 ꢀ 5.6b 32.6 ꢀ 6.5b 218 ꢀ 54b
214 ꢀ 16b
(R)-3
602 ꢀ 59b
482 ꢀ 39b
(S)-3
74.2 ꢀ 6.2b
(rac)-1
(rac)-1
(R)-1
(R)-1
(S)-1
(S)-1
(R)-3
(S)-3
(CH2)2
1.18 ꢀ 0.24b 290 ꢀ 151b
0.396 ꢀ 0.026b 64.8 ꢀ 12.1b
e
e
e
e
e
e
4
4
7
7
S
(CH2)2
b
b
0.159 ꢀ 0.029b 483 ꢀ 83b
0.343 ꢀ 0.044
26.6 ꢀ 4.3
e
e
e
e
e
e
5
5
8
8
S
OMe
83.3 ꢀ 15.7b 1.08 ꢀ 0.24b 67.8 ꢀ 19.0b 3.10 ꢀ 0.45b
MeO
O(CH2)2
e
e
e
e
e
e
9
9
11
11
OMe
If not stated otherwise the results are given as means of IC50-values ꢀ SEM of three independent experiments each performed in triplicate, percentages represent specific
binding remaining in presence of 100 M inhibitor. The abbreviation b specifies the used biological material: bbsP2C (see experimental section).
m
conc. H2SO4. If nothing else is stated, Merck silica gel (mesh
cloudy (pH 5e6) and extracted with CH2Cl2 (3ꢃ). The organic layers
230e400) was used as stationary phase for flash chromatography.
were dried (MgSO4) and concentrated in vacuo.
Optical rotations: Polarimeter 241 MC at
l
589 cmꢁ1. Melting
points: mp (uncorrected) were determined with a Büchi 510
Melting Point apparatus. Elementary analysis: Elementar-
analysator Rapid (Heraeus). IR spectroscopy: FT-IR Spectrometer
1600 and Paragon 1000 (Perkin Elmer), oily samples as film, solid
samples as pellets for measurements. Mass spectroscopy: Mass
Spectrometer 5989 A with 59980 B particle beam LC/MS interface
(Hewlett Packard). NMR spectroscopy: NMR spectra were recor-
ded on JNMR-GX (Jeol, 400 MHz and 500 MHz) with TMS as
internal standard and integrated with the program of NMR-soft-
ware Nuts (2D Version 5.097, Acorn NMR, 1995). If nothing else is
stated, measurements were performed at 400 MHz at room
temperature.
5.1.3. General procedure 3 (GP3)
A solution of the respective alkyl bromide was added to a solution
of the respective amine. K2CO3 and NaI were added and the reaction
mixture was stirred for the time given under nitrogen and under
exclusion of light. The solvent was removed in vacuo. The crude
product was extracted with EtOAc (30 ml). The organic layer was
washed withwater (15 ml), dried (NaSO4), and concentratedinvacuo.
5.1.4. General procedure 4 (GP4)
Acetyl chloride (2.5 equiv) was added to the respective amino
acid in MeOH at 0 ꢂC. The reaction mixture was allowed to warm to
rt and was stirred for 24 h. The solvents and excess of acetyl chlo-
ride were removed in vacuo.
5.1.1. General procedure 1 (GP1)
2 N NaOH was added to the respective ester compound dissolved
in alcohol. The reaction mixture was stirred for 48 h. Following acid-
ificationtopH1e2usingHCl(0.01 N)andextractionwithCH2Cl2 (3ꢃ),
the organic layers were dried (MgSO4) and concentrated in vacuo.
5.1.5. 1-(4,4-Diphenylbut-3-en-1-yl)azetidine-3-carboxylic acid
hydrochloride (12b$HCl)
Hydrolysis according to GP1 starting from 39b (71 mg,
0.22 mmol) in 1 ml MeOH and 2 N NaOH (0.275 ml, 0.55 mmol).
Yield: 69 mg (92%); colorless solid, mp 64 ꢂC. 1H NMR (CDCl3):
5.1.2. General procedure 2 (GP2)
d
¼ 2.39 ppm (q, 2 H, J ¼ 7.4 Hz), 3.04 (t, 2 H, J ¼ 7.4 Hz), 3.18e3.23
2 N NaOH was added to the respective ester compound dis-
solved in alcohol. The reaction mixture was stirred for 48 h. The
mixture was acidified using HCl (0.01 N) until the solution turned
(m, 2 H), 3.25 (m, 1 H), 3.44e3.49 (m, 2 H), 6.05 (t, 1 H, J ¼ 7.6 Hz),
¼ 2952 cmꢁ1, 2924, 2849, 2566,
~
7.14e7.38 (m, 10 H). IR (KBr):
n
1718, 1495, 1443, 1379, 1074, 761, 699. MS (CI): m/z (%): 308

M.R. Faust et al. / European Journal of Medicinal Chemistry 45 (2010) 2453e2466
2461
Table 3
Uptake inhibition [IC50
(m
M)] at GAT-1 and GAT-3 exhibited by azetidine derivatives.
N
N
OMe
OMe
N
N
N
NH
N
HO
N
HO
HO
O
H
N
R
N
R
N
R
N
R
IC50
[m
M]
IC50
[m
M]
IC50
[
m
M]
IC50 [mM]
R
GAT-1
GAT-3
GAT-1
GAT-3
GAT-1
GAT-3
GAT-1
GAT-3
H
95.6%c (n ¼ 1)
16a$HCl
106%d (n ¼ 1)
112%e (n ¼ 1)
17a$HCl
95.1%f (n ¼ 1)
102%a (n ¼ 1) 90.7%b (n ¼ 1)
e
e
16a$HCl
17a$HCl
18a
18a
(CH2)2
26.6 ꢀ 3.3a
48.5 ꢀ 12.8b
e
e
e
e
e
e
18b
18b
S
(CH2)2
39.5 ꢀ 3.2a
63.0 ꢀ 28.5b
e
e
e
e
e
e
18c
18c
S
OMe
77.5 ꢀ 4.3a
60.0 ꢀ 3.0b
MeO
O(CH2)2
e
e
e
e
e
e
18d
18d
OMe
61.3%e (n ¼ 1) 31.0 ꢀ 4.7f
51.2e (n ¼ 1) 3.34f (n ¼ 1)
N
(CH2)3
e
e
e
e
18e
18e
10
10
If not stated otherwise the results are given as means of IC50-values ꢀ SEM of three independent experiments each performed in triplicate, n ¼ 1 denotes value from a single
experiment, percentages represent specific binding remaining in presence of 100 mM inhibitor (1 mM in the case of 17a$HCl). The abbreviations aef specify the used biological
material (see Experimental section).
Where “HCl” has been added to the compound number the compound has been tested as hydrochloride of the drawn neutral structure.
[Mþ þ 1 ꢁ HCl] (20), 264 (100). C20H22ClNO2 (343.8): calcd C 69.86,
1083, 1005, 932, 834, 714. MS (CI): m/z (%): 348 [Mþ þ 1 ꢁ HCl] (27),
H 6.45, N 4.07; found C 69.65, H 6.59, N 4.14.
114(100). C18H22ClNO2S2 (383.9): calcd C 56.31, H 5.78, N 3.65;
found C 55.97, H 6.10, N 3.38.
5.1.6. 1-[4,4-Bis(3-methylthien-2-yl)but-3-en-1-yl]azetidine-3-
carboxylic acid hydrochloride (12c$HCl)
Hydrolysis according to GP1 starting from 39c (108 mg,
0.299 mmol) in 2 ml MeOH and 2 N NaOH (0.371 ml, 0.75 mmol).
Yield: 109 mg (95%); yellowish solid, mp 148 ꢂC. 1H NMR (CDCl3):
5.1.7. 1-{2-[Tris(4-methoxyphenyl)methoxy]ethyl}azetidine-3-
carboxylic acid hydrochloride (12d)
Hydrolysis according to GP2 starting from 39d (53 mg,
0.11 mmol) in 0.5 ml MeOH and 2 N NaOH (0.138 ml, 0.27 mmol).
Reaction time: 72 h. Yield: 50 mg (94%); colorless solid, mp 92 ꢂC. 1H
d
¼ 1.93 ppm (s, 3 H), 1.99 (s, 3 H), 2.40 (q, 2 H, J ¼ 7.4 Hz), 3.08 (t, 2
H, J ¼ 7.4 Hz), 3.38 (qui, 1 H, J ¼ 8.2 Hz), 3.73e3.94 (m, 2 H),
4.05e4.28 (m, 2 H), 5.94 (t, 1 H, J ¼ 7.4 Hz), 6.73 (d, 1 H, J ¼ 4.9 Hz),
6.83 (d, 1 H, J ¼ 4.9 Hz), 7.05 (d, 1 H, J ¼ 4.9 Hz), 7.21 (d, 1 H,
NMR (CDCl3):
d
¼ 2.96e3.01 ppm (m, 2 H), 3.37e3.42 (m, 2 H),
3.28e3.38 (m, 1 H), 3.34e3.40 (m, 2 H), 3.55e3.61 (m, 2 H), 3.76 (s, 9
H), 6.78e6.83 (m, 6 H), 7.25e7.30 (m, 6 H). IR (KBr):
2953, 2835, 1607, 1508, 1463, 1249, 1175, 1033, 827, 583. MS (CI): m/z
(%): 333 (100), 227 (100), 145 (11), 101 (7). C28H31NO6 (477.6): calcd C
70.42, H 6.54, N 2.93; found C 70.21, H 6.69, N 2.99.
n
¼ 3429 cmꢁ1
,
~
J ¼ 4.9 Hz). 13C NMR APT (CDCl3):
d
¼ 14.3 ppm, 14.8, 25.2, 34.5,
54.6, 56.3, 123.2, 124.8, 127.9, 129.8, 131.0, 131.4, 133.8, 134.3, 135.8,
~
n
138.4, 176.0. IR (KBr):
¼ 3423 cmꢁ1, 2921, 2853, 1601, 1383, 1221,

2462
M.R. Faust et al. / European Journal of Medicinal Chemistry 45 (2010) 2453e2466
5.1.8. (RS)-Azetidin-2-ylacetic acid (15a) [44]
d
¼ 4.30 ppm (dd, 2 H, J ¼ 12.8, 4.7 Hz), 4.58 (dd, 2 H, J ¼ 12.8, 6.7 Hz),
20% Pd/C (8 mg) was added to a solution of 24(37 mg, 0.15 mmol)
in 1 ml MeOH. The mixture was subjected to hydrogen under
ambient pressure at rt for 16 h and then filtrated. The solvent was
evaporated. Purification by ion exchange chromatography (Dowex
50 W x 8, Fluka, eluent e saturated aqueous NH3) and evaporation in
vacuo afforded 15a. Yield: 17 mg (w100%); colorless crystals. 1H
5.50(tt,1 H, J ¼ 6.7/4.7 Hz).13CNMR (MeOH-d4):
d
¼ 52.0 ppm (CH2),
¼ 3035 cmꢁ1, 2919, 2812, 2628, 2488,
~
70.3 (CH), 162.0. IR (KBr):
n
1718,1621,1357,1146,1066, 733. MS (CI): m/z (%): 142 [Mþ þ 1 ꢁ HCl]
(100). HRMS (ESIþ) for C4H8N5O: calcd 142.0729; found 142.0713.
5.1.13. Benzyl azetidine-1-carboxylate (20)
NMR (D2O):
d
¼ 2.18e2.30 ppm (m, 1 H), 2.39e2.51 (m, 1 H),
Azetidine hydrochloride (19) (5.25 g, 56.1 mmol) was suspended
in 100 ml CH2Cl2 and cooled to 0 ꢂC. NEt3 (23.4 ml,168.3 mmol) was
added followed by the addition of benzyl chloroformate (15.9 ml,
112.2 mmol). The reaction mixture was stirred for 72 h, acidified
using HCl (2 N) and extracted with CH2Cl2 (4 ꢃ100 ml). The
combined organic layers were dried (MgSO4) and concentrated in
vacuo. Purification by CC (n-heptane/EtOAc ¼ 80:20) afforded
compound 20. Yield: 9.97 g (93%), colorless oil. TLC: Rf ¼ 0.22 (n-
2.58e2.72 (m, 2 H), 3.78 (ddd,1 H, J ¼ 15.6,10.1, 5.5), 3.89e3.99 (m,1
¼ 3424 cmꢁ1, 2971, 1573, 1400,
~
n
H), 4.56e4.65 (m, 1 H). IR (Film):
1264, 1075. MS (CI): m/z (%): 116 [Mþ þ 1] (58), 100 (100), 98 (46).
5.1.9. (RS)-1-(4,4-Diphenylbut-3-en-1-yl)azetidin-2-ylacetic acid
hydrochloride (15b$HCl)
TFA (1.2 ml) was added to a solution of 41b (66 mg, 0.17 mmol) in
3 ml CH2Cl2 at 0 ꢂC. The reaction mixture was stirred for 2 h at rt and
acidified with HCl (0.6 ml, 2 N) followed by evaporation of the
solvent. The crude product was washed with Et2O (3 ꢃ 5 ml). Yield:
61 mg (w100%); colorless solid, mp 61 ꢂC. 1H NMR (CDCl3):
heptane/EtOAc ¼ 80:20). 1H NMR (CDCl3):
¼ 2.23 ppm (quint, 2 H,
d
J ¼ 7.6 Hz), 4.03 (t, 4 H, J ¼ Hz), 5.09 (s, 2 H), 7.27e7.39 (m, 5 H). IR
~
(KBr):
n
¼ 2958 cmꢁ1, 2888, 1709, 1415, 1354, 1128, 1174, 982, 751,
698. MS (CI): m/z (%): 192 [Mþ þ 1] (31), 91(100). C11H13NO2 (191.2):
d
¼ 2.31e2.56 ppm (m, 4 H), 2.73e2.83 (m, 1 H), 2.84e2.97 (m, 1 H),
calcd C 69.09, H 6.85, N 7.32; found C 69.14, H 6.96, N 7.32.
3.29e3.41 (m,1 H), 3.42e3.54 (m,1 H), 3.64e3.74 (m,1 H), 4.15e4.25
(m,1 H), 4.39e4.52 (m,1 H), 6.02 (t,1 H, J ¼ 7.3 Hz), 7.11e7.16 (m, 2 H),
7.17e7.25 (m, 4 H), 7.31e7.49 (m, 4 H), 11.6 (s br, 1 H). 13C NMR
5.1.14. (RS)-Benzyl 2-(acetyloxy)azetidine-1-carboxylate (21)
NaOAc (11.55 g) was dissolved in 200 ml of glacial acetic acid.
Compound 20 (4.90 g, 25.6 mmol) was added. The reaction mixture
was subjected to anodic oxidation for 40 h at a constant current of
0.6 A under exclusion of air (surface of Pt-electrode: 2 ꢃ 50 cm2)
while keeping the reaction at a constant temperature of 20 ꢂC using
water cooling. The reaction mixture was poured into water (100 ml)
and extracted with CH2Cl2 (5 ꢃ100 ml). The combined organic
layers were washed with water (50 ml), dried (MgSO4), and
concentrated in vacuo. Purification by CC (n-heptane/
EtOAc ¼ 80:20) afforded compound 21. Yield: 4.15 g (65%); colorless
oil. TLC: Rf ¼ 0.16 (n-heptane/EtOAc ¼ 80:20). 1H NMR (CDCl3):
(CDCl3):
d
¼ 22.1 ppm, 24.8, 37.2, 50.7, 54.9, 65.8, 121.8, 127.2, 127.5,
128.2, 128.6, 129.5, 139.0, 141.4, 145.6, 171.3. IR (KBr):
n
¼ 3425 cmꢁ1, 3023, 1725, 1671, 1494, 1444, 1199, 1136, 765, 702.
~
MS (CI): m/z (%): 322 [Mþ þ 1 ꢁ HCl] (6), 304 (54), 224 (100), 215
(76). HRMS (ESIꢁ) for C21H22NO2: calcd 320.1651; found 320.1663.
5.1.10. (RS)-1-[4,4-Bis(3-methyl-2-thienyl)but-3-en-1-yl]azetidin-
2-ylacetic acid (15c)
TFAwasaddeddropwisetoasolutionof41c (26 mg,0.06 mmol)in
2 mlCH2Cl2 at0 ꢂC until a concentration of 20% (v/v) TFAwasreached.
The reaction mixture was stirred for 2 h at rt followed by evaporation
of the solvent. Purification by CC (CH2Cl2/EtOH ¼ 50:50) afforded 15c.
Yield: 16 mg (69%), colorless solid. TLC: Rf ¼ 0.08 (CH2Cl2/
d
¼ 2.07 ppm (s, 3 H), 2.16e2.26 (m, 1 H), 2.61e2.71 (m, 1 H), 3.83
(ddd, 1 H, J ¼ 8.5/8.0/5.7 Hz), 3.97 (ddd, 1 H, J ¼ 8.9/8.0/5.5 Hz), 5.10
(d, 1 H, J ¼ 12.4 Hz), 5.17 (d, 1 H, J ¼ 12.4 Hz), 6.36 (dd, 1 H, J ¼ 6.4/
EtOH ¼ 50:50). 1H NMR (CDCl3):
d
¼ 1.93 ppm (s, 3 H), 1.99 (s, 3 H),
3.6 Hz), 7.29e7.40 (m, 5 H). IR (KBr):
n
¼
¼ 2970 cmꢁ1 2899,1750,
~
~
n
2.36e2.56 (m, 4 H), 2.80e2.89 (m,1 H), 2.98e3.10 (m,1 H), 3.16e3.25
(dd, 1 H, J ¼ 18.5, 9.2 Hz), 3.51e3.69 (m, 2 H), 4.23e4.34 (m, 1 H),
4.44e4.55(m,1 H), 5.92(t,1H, J ¼ 7.5 Hz), 6.75(d,1 H, J ¼ 5.0 Hz), 6.86
(d, 1 H, J ¼ 5.0 Hz), 7.07 (d, 1 H, J ¼ 5.0 Hz), 7.23 (d, 1 H, J ¼ 5.0 Hz). 13C
1717, 1411, 1351, 1232, 1039, 753, 699. MS (CI): m/z (%): 250 [Mþ þ 1]
(1), 190 (100), 91 (73). C13H15NO4 (249.3): calcd C 62.64, H 6.06, N
5.62; found C 62.64, H 5.97, N 5.58.
NMR APT (CDCl3):
d
¼ 14.2 ppm,14.8, 21.8, 24.9, 51.3, 55.0, 65.7,123.5,
5.1.15. (RS)-Benzyl (2-methoxycarbonylmethyl)azetidine-1-
carboxylate (22)
124.8, 126.4, 130.2, 131.6, 132.1, 133.4, 134.7, 136.3. IR (KBr):
~
n
¼ 3429 cmꢁ1, 2924, 2857, 2615, 1728, 1668, 1418, 1199, 834, 798,
TiCl4 (205 ml, 2.05 mmol, 1 M in CH2Cl2) was added dropwise to
721. MS (CI): m/z (%): 362 [Mþ þ 1] (100), 344 (26), 218 (26),184 (27),
21 (511 mg, 2.05 mmol) in 5 ml CH2Cl2 at ꢁ78 ꢂC and the reaction
mixture was stirred for 10 min. (1-Methoxy-1-vinyloxy)trime-
thylsilane (750 mg, 5.12 mmol) was added dropwise. The reaction
mixture was stirred for 2 h at ꢁ78 ꢂC and for 1 h at 0 ꢂC. The
resulting solution was transferred via syringe to a vigorously stirred
saturated NaHCO3 solution at 0 ꢂC. The reaction mixture was
extracted with CH2Cl2 (3 ꢃ10 ml), dried (MgSO4), and concentrated
in vacuo. Purification by CC (n-heptane/EtOAc ¼ 80:20) afforded
compound 22. Yield: 298 mg (55%); colorless oil. TLC: Rf ¼ 0.12
128 (68). HRMS (CI) for C19H23NO2S2: calcd 361.1170; found 361.1195.
5.1.11. 3-(1H-Tetrazol-5-yl)azetidin-3-ol hydrochloride (16a$HCl)
Compound 30 (1.83 g, 4.28 mmol) was dissolved in 50 ml of
MeOH. Pd(OH)2/C (732 mg, 40%) was added. The mixture was
stirred for 72 h at rt under hydrogen atmosphere. Following
filtration and washing with water, the combined organic and water
phase was concentrated. The residue was acidified using HCl (2 N).
Following extraction with Et2O, the water phase was concentrated
to yield compound 16a$HCl. Yield: 375 mg (49%); colorless solid,
(n-heptane/EtOAc ¼ 80:20). 1H NMR (CDCl3):
d
¼ 1.99e2.10 ppm
(m, 1 H), 2.39e2.50 (m, 1 H), 2.69 (dd, 1 H, J ¼ 15.9, 8.6 Hz),
mp 174 ꢂC. 1H NMR (D2O):
d
¼ 4.41 ppm (d, 2 H, J ¼ 12.5 Hz), 4.63 (d,
2.94e3.06 (m, 1 H), 3.64 (s, 3 H), 3.86e4.00 (m, 2 H), 4.56e4.65 (m,
1 H), 5.09 (s, 2 H), 7.29e7.39 (m, 5 H). IR (KBr):
2893, 1731, 1705, 1413, 1352, 1253, 1135, 753, 698. MS (CI): m/z (%):
264 [Mþ þ 1] (80), 220 (63), 91 (100). C14H17NO4 (263.3): calcd C
63.87, H 6.51, N 5.32; found C 63.50, H 6.66, N 5.25.
2 H, J ¼ 12.5 Hz). 13C NMR (CDCl3):
d
¼ 57.3 ppm, 66.1, 157.6. IR
n
¼ 2954 cmꢁ1
,
~
~
(KBr):
n
¼ 2994 cmꢁ1, 2833, 2709, 1769, 1583, 1202, 1026, 696. MS
(CI): m/z (%): 142 [Mþ þ 1 ꢁ HCl] (100). C4H8ClN5O (177.6): calcd C
27.05, H 4.54, N 39.43; found C 26.82, H 4.50, N 39.43.
5.1.12. 5-(Azetidin-3-yloxy)-1H-tetrazole hydrochloride (17a$HCl)
As described for compound 16a$HCl starting from 34 (0.22 g,
0.51 mmol) in 10 ml MeOH and 85 mg Pd(OH)2/C (40%). Yield:
85.5 mg (94%); colorless solid, mp 130 ꢂC. 1H NMR (MeOH-d4):
5.1.16. (RS)-Benzyl (2-tert-butoxycarbonylmethyl)azetidine-1-
carboxylate (23)
As described for compound 22 starting from TiCl4 (500
0.50 mmol, 1 M in CH2Cl2), 21 (250 mg, 1.00 mmol) in 2 ml
ml,

M.R. Faust et al. / European Journal of Medicinal Chemistry 45 (2010) 2453e2466
2463
CH2Cl2 and (1-tert-butoxy-1-vinyloxy)trimethylsilane (450 mg,
2.83 mmol). Purification by CC (n-heptane/EtOAc ¼ 80:20) afforded
compound 23. Yield: 168 mg (55%); colorless oil. TLC: Rf ¼ 0.22
then filtrated. The solvent was evaporated. Yield: 184 mg (61%);
colorless oil. 1H NMR (CDCl3):
d
¼ 1.45 ppm (s, 9 H), 2.04 (s, 3 H),
2.30e2.41 (m, 1 H), 2.51e2.61 (m, 1 H), 2.78 (dd, 1 H, J ¼ 17.1,
6.8 Hz), 2.95 (dd, 1 H, J ¼ 17.1, 7.2 Hz), 3.82 (ddd, 1 H, J ¼ 15.1, 9.7,
5.1 Hz), 3.92e4.01 (m, 1 H), 4.63 (qui, 1 H, J ¼ 7.6 Hz), 9.47 (s br, 2 H).
1
(n-heptane/EtOAc ¼ 80:20). H NMR (nitrobenzene d5, 140 ꢂC):
d
¼ 1.47 ppm (s, 9 H), 2.08e2.19 (m, 1 H), 2.34e2.45 (m, 1 H), 2.69
(dd, 1 H, J ¼ 15.4, 8.6 Hz), 2.94 (dd, 1 H, J ¼ 15.4, 4.5 Hz), 3.91e3.96
13C NMR (APT) (CDCl3):
d
¼ 22.5 ppm, 24.9, 28.0, 38.8, 42.3, 55.5,
¼ 3414 cmꢁ1, 2980, 1722, 1556, 1410,
~
n
(m, 2 H), 4.57e4.65 (m, 1 H), 5.16 (s, 2 H), 7.18e7.42 (m, 5 H). IR
81.8, 168.8, 177.2. IR (KBr):
(KBr):
¼ 2975 cmꢁ1, 2892, 1726, 1709, 1411, 1351, 1148, 753, 698.
1370, 1161, 1016, 845. MS (CI): m/z (%): 231 [Mþ þ 1] (16), 213 (100),
~
n
MS (CI): m/z (%): 306 [Mþ þ 1] (1), 250 (39), 206 (63), 91 (100).
C17H23NO4 (305.4): calcd C 66.86, H 7.59, N 4.59; found C 66.75, H
7.41, N 4.62.
172 (6), 145 (8), 130 (42), 116 (81).
5.1.21. 1-Diphenylmethyl-3-[1-(4-methoxybenzyl)-1H-tetrazol-5-
yl]azetidin-3-ol (30)
5.1.17. (RS)-1-[(Benzyloxy)carbonyl]azetidin-2-ylacetic acid (24)
NaOH (1.25 ml, 2.5 mmol, 2 N) was added dropwise to 22
(0.26 g, 0.97 mmol) in 4 ml MeOH at 0 ꢂC. The reaction mixture
was allowed to warm to rt and was stirred for 72 h. The solvent
was removed in vacuo. The crude product was suspended in water
and extracted with Et2O (5 ml). The water layer was acidified
using HCl (2 N) and extracted with CH2Cl2 (3 ꢃ10 ml). The organic
layer was dried (MgSO4) and concentrated in vacuo. Purification
by CC (n-heptane/EtOAc ¼ 80:20) afforded compound 24. Yield:
205 mg (85%); colorless oil. TLC: Rf ¼ 0.13 (n-heptane/
1-(4-Methoxybenzyl)tetrazol (1826 mg, 9.60 mmol) was dis-
solved in 48 ml of THF/TMEDA (10:1). nBuLi (6.0 ml,1.6 M in hexane)
was added dropwise at ꢁ90 ꢂC to generate lithium compound 29
and the reaction mixture was stirred for 5 min. Following the addi-
tion of compound 28 (2278 mg, 9.60 mmol) in 10 ml THF and stirring
for 30 min, the mixture was warmed to rt. The reaction was
quenched with NH4Clsat (20 ml). The mixture was extracted with
EtOAc (3 ꢃ 100 ml), the combined organic layers were dried (MgSO4)
and concentrated in vacuo. Purification by CC (petrol ether/
EtOAc ¼ 70:30) afforded compound 30. Yield: 2577 mg (63%);
colorless solid, mp 143 ꢂC. TLC: Rf ¼ 0.26 (petrol ether/
EtOAc ¼ 80:20). 1H NMR (CDCl3):
d
¼ 2.00e2.12 ppm (m, 1 H),
2.40e2.51 (m, 1 H), 2.73 (dd, 1 H, J ¼ 16.2, 8.1 Hz), 2.98e3.11 (m, 1
EtOAc ¼ 70:30). 1H NMR (benzene-d6):
¼ 3.18 ppm (s, 3 H), 3.23 (d,
d
H), 3.88e4.00 (m, 2 H), 4.56e4.65 (m, 1 H), 5.09 (s, 2 H), 7.28e7.39
2 H, J ¼ 9.1 Hz), 3.42e3.49 (s br,1 H), 3.67 (d, 2 H, J ¼ 9.1 Hz), 4.31 (s, 1
~
(m, 5 H). IR (KBr):
n
¼ 3319 cmꢁ1, 2963, 1706, 1540, 1420, 1355,
H), 5.11 (s, 2 H), 6.52e6.58 (m, 2 H), 6.95e7.03 (m, 4 H), 7.05e7.13 (m,
1283, 1139, 751, 697. MS (CI): m/z (%): 250 [Mþ þ 1] (5), 206 (23),
91 (100). C13H15NO4 (249.3): calcd C 62.64, H 6.07, N 5.62; found C
63.07, H 6.07, N 5.73.
4 H), 7.32e7.38 (m, 4 H). IR (KBr):
n
¼ 3269 cmꢁ1, 3025, 2955, 2835,
~
1733, 1612, 1514, 1252, 1029, 746, 704. MS (CI): m/z (%): 428 [Mþ þ 1]
(21), 238 (8), 167 (79), 121 (100). C25H25N5O2 (427.5): calcd C 70.24, H
5.89, N 16.38; found C 70.07, H 5.97, N 16.26.
5.1.18. (RS)-Benzyl 2-cyanoazetidine-1-carboxylate (25)
TiCl4 (38 mg, 0.20 mmol) in 0.5 ml CH2Cl2 was added dropwise to
a mixture of 21 (50 mg, 0.20 mmol) and TMSCN (60 mg, 0.56 mmol)
in 1.5 ml CH2Cl2 at ꢁ70 ꢂC. The reaction mixture was stirred for 2 h
and allowed to warm up to rt. Workup and purification as described
for compound 22 afforded compound 25. Yield: 19.7 mg (46%);
colorless oil. TLC: Rf ¼ 0.07 (n-heptane/EtOAc ¼ 80:20). 1H NMR
5.1.22. 3-[1-(4-Methoxybenzyl)-1H-tetrazol-5-yl]azetidin-3-ol (31)
Compound 30 (0.21 g, 0.48 mmol) was dissolved in 10 ml AcOH.
Pd/C (82.0 mg, 40%) and TFA (1 ml) were added. The mixture was
stirred for 72 h at rt under hydrogen. Following filtration and
washing with water, the combined methanol and water phases were
concentrated and acidified with HCl (2 N). The water phase was
extracted with CH2Cl2 (3 ꢃ 10 ml). Following alkalization using
NaOH (2 N) and extraction with CH2Cl2 (3 ꢃ 10 ml), the combined
organic layers were dried (MgSO4) and concentrated in vacuo to
afford compound 31. Yield: 49.0 mg (39%); colorless solid, mp 158 ꢂC.
(CDCl3):
d
¼ 2.55e2.72 ppm (m, 2 H), 4.03 (ddd, J ¼ 9.0/8.6/6.4 Hz, 1
H), 4.13 (ddd, 1 H, J ¼ 9.0/8.3/5.9 Hz), 4.83 (dd, 1 H, J ¼ 9.0/5.9 Hz),
5.17 (s, 2 H), 7.30e7.41 (m, 5 H). 13C NMR (CDCl3):
d
¼ 21.5 ppm, 48.0,
¼ 2968 cmꢁ1
,
~
n
67.5, 117.5, 135.7, 155.0, 128.1, 128.3, 128.5. IR (KBr):
2895, 1715, 1409, 1349, 1136, 769, 698. MS (CI): m/z (%): 217 [Mþ þ 1]
(18), 190 (11), 91 (100). C12H12N2O2 (216.2): calcd C 66.65, H 5.59, N
12.95; found C 66.24, H 5.62, N 12.48.
1H NMR (CDCl3):
d
¼ 3.50e3.57 ppm (m, 2 H), 3.77 (s, 3 H,),
4.27e4.34 (m, 2 H), 5.67 (s, 2 H), 6.79e6.87 (m, 2 H), 7.15e7.22 (m, 2
H). IR (KBr):
¼ 3263 cmꢁ1, 2977, 2943, 1613, 1515, 1202, 1253,
~
n
1031, 846, 776. MS (CI): m/z (%): 262 [Mþ þ 1] (37), 121 (100). HRMS
(ESIþ) for C12H16N5O2: calcd 262.13039; found 262.1303. C12H15N5O2
(261.2): calcd C 55.16, H 5.79, N 26.80; found C 55.19, H 6.09, N 24.36.
5.1.19. (RS)-Benzyl 2-[2-(4-fluorophenyl)-2-oxoethyl]azetidine-1-
carboxylate (26)
As described for compound 25 starting from TiCl4 (75 mg,
0.40 mmol) in 0.1 ml CH2Cl2, 21 (100 mg, 0.40 mmol) and [1-
(4-fluorophenyl)vinyloxy]trimethylsilane (0.17 g, 0.8 mmol) in 4 ml
CH2Cl2. Workup and purification as described for compound 22
afforded compound 26. Yield: 76 mg (58%); colorless oil. TLC:
5.1.23. 5-(1-Diphenylmethylazetidin-3-yloxy)-1-(4-
methoxybenzyl)-1H-tetrazol (34)
Compound 32 (685 mg, 2.86 mmol) was dissolved in 25 ml THF.
NaH (110 mg, 4.58 mmol, 1.6 equiv) was added and the mixture was
stirred at rt until gas development ceased (approximately 2 h). A
solution of 33 (904.8 mg, 2.86 mmol) in 5 ml THF was added drop-
wise and the mixture was stirred for 4 h at rt. The reaction was
quenched with ice water (20 ml) and extracted with Et2O
(3 ꢃ 50 ml). The combined organic layers were dried (MgSO4) and
concentrated in vacuo. Purification by CC (petrol ether/
acetone ¼ 80:20). Yield: 471 mg (39%); highly viscous oil. TLC:
Rf ¼ 0.15 (petrol ether/acetone ¼ 80:20). 1H NMR (CDCl3):
Rf ¼ 0.09
(n-heptane/EtOAc ¼ 80:20).
1H
NMR
(CDCl3):
d
¼ 1.97e2.09 ppm (m, 1 H), 2.49e2.61 (m, 1 H), 3.24 (dd, 1 H,
J ¼ 16.7, 9.5 Hz), 3.27e3.86 (m, 1 H), 3.90e4.07 (m, 2 H), 4.74e4.82
(m, 1 H), 5.09 (s, 2 H), 7.05e7.14 (m, 2 H), 7.28e7.40 (m, 5 H),
~
7.87e8.01 (m, 2 H). IR (KBr):
n
¼ 2958 cmꢁ1, 2883, 1705, 1680,
1597, 1410, 1351, 1135, 834, 752, 698. MS (CI): m/z (%): 328 [Mþ þ 1]
(7), 177 (20), 91 (100). C19H18FNO3 (327.4): calcd C 69.71, H 5.54, N
4.28; found C 69.35, H 5.54, N 4.19.
d
¼ 3.13e3.19 ppm (m, 2 H), 3.68e3.74 (m, 2 H), 3.83 (s, 3 H), 4.39 (s,
5.1.20. (RS)-tert-Butyl azetidin-2-ylacetate hydroacetate (27)
20% Pd/C (80 mg) and acetic acid (0.2 ml) were added to
a solution of 20 (0.40 g, 1.3 mmol) in 5 ml MeOH. The mixture was
subjected to hydrogen under ambient pressure at rt for 16 h and
1 H), 5.25 (s, 2 H), 5.29e5.35 (m, 1 H), 6.90e6.93 (m, 2 H), 7.19e7.24
(m, 2 H), 7.27e7.32 (m, 6 H), 7.39e7.43 (m, 4 H). IR (KBr):
~
n
¼ 3025 cmꢁ1, 2954, 2835, 1609, 1563, 1514, 1250, 1029, 747, 704.
MS (CI): m/z (%): 428 [Mþ þ 1] (45), 222 (65), 167 (100). HRMS (EIþ)

2464
M.R. Faust et al. / European Journal of Medicinal Chemistry 45 (2010) 2453e2466
for C25H25N5O2: calcd 427.2008; found 427.1992. C25H25N5O2
(427.5): calcd C 70.24, H 5.89, N 16.38; found C 69.66, H 5.97, N 16.07.
heptane/EtOAc ¼ 20:80). Yield: 64 mg (20%); colorless oil. 1H NMR
(CDCl3):
d
¼ 2.65 ppm (t, 2 H, J ¼ 5.5 Hz), 3.08 (t, 2 H, J ¼ 5.5 Hz),
3.27e3.36 (m, 1 H), 3.34e3.40 (m, 2 H), 3.55e3.61 (m, 2 H), 3.72 (s,
5.1.24. Methyl azetidin-3-carboxylate (35) [50]
3 H), 3.79 (s, 9 H), 6.78e6.83 (m, 6 H), 7.28e7.33 (m, 6 H). IR (KBr):
~
n
Thionylchloride (20.5 g,172 mmol) was added to MeOH (50 ml) at
ꢁ60 ꢂC. After 30 min azetidin-3-carboxylic acid (250 mg, 2.47 mmol)
was added and the resulting mixture was stirred for 16 h at rt.
Removement of the solvent in vacuo afford compound 35 [50]. Yield:
¼ 2951 cmꢁ1, 2835, 1734, 1607, 1507, 1249,1174, 1034, 827, 583.
MS (CI): m/z (%): 333 (84), 227 (100). C29H33NO6 (491.6): calcd C
70.86, H 6.77, N 2.85; found C 70.59, H 6.99, N 2.89.
364 mg (97%); yellow solid.1H NMR (CD3OD):
d
¼ 3.72e3.77 ppm (m,
5.1.29. Methyl 1-(4,4-diphenylbut-3-en-1-yl)azetidin-3-ylacetate
(40b)
¼ 3443 cmꢁ1, 2922, 2618, 2460,
~
n
4 H), 4.19e4.31 (m, 4 H). IR (KBr):
1732,1586,1440,1383,1304,1214,1191,1110,1065,1006, 920, 821, 741.
N-alkylation according to GP3 starting from 37 (375 mg,
2.26 mmol) in 2.5 ml of DME, (4-bromo-1-phenylbut-1-enyl)
benzene (38b) (800 mg, 2.80 mmol, 1.25 equiv) in 2.5 ml of DME,
K2CO3 (1.25 g, 9.05 mmol, 4.0 equiv) and NaI (10 mg). Reaction
temperature: rt; reaction time: 120 h. CH2Cl2 (10 ml) was added.
The precipitate was filtered off and washed with CH2Cl2. The filtrate
was extracted with water (3 ꢃ 5 ml). The combined organic layers
were dried (MgSO4) and concentrated in vacuo. Purification by CC
(n-heptane/EtOAc ¼ 60:40 þ 1% NEtMe2). Yield: 204 mg (27%);
colorless oil. TLC: Rf ¼ 0.12 (n-heptane/EtOAc ¼ 60:40 þ 1%
MS (CI): m/z (%): 116 [Mþ þ 1-HCl] (100).
5.1.25. Methyl azetidin-3-ylacetate hydrochloride (37) [51]
Esterification according to GP4 starting from 2-(1-tert-butylox-
ycarbonylazetidine-3-yl)acetic acid (36) [40] (100 mg, 0.46 mmol)
in 2 ml MeOH and acetyl chloride (36 mg, 0.46 mmol). Purification
by CC (CH2Cl2/EtOH ¼ 50:50 þ 0.5% AcOH). Yield: 44 mg (58%);
colorless, hygroscopic solid. 1H NMR (CDCl3):
d
¼ 2.80 ppm (d, 2 H,
J ¼ 7.8 Hz), 3.25 (sep, 1 H, J ¼ 7.8 Hz), 3.70 (s, 3 H), 3.83e3.93 (m, 2
¼ 3422 cmꢁ1
,
NEtMe2). 1H NMR (CDCl3):
d
¼ 2.13 ppm (q, 2 H, J ¼ 7.6 Hz), 2.50 (t, 2
~
H), 4.22e4.32 (m, 2 H), 9.71 (s br, 2 H). IR (KBr):
n
3092, 2977, 2954, 1725, 1541, 1432, 1294, 1210, 994, 869. MS (CI): m/
H, J ¼ 7.6 Hz), 2.56 (d, 2 H, J ¼ 7.1 Hz), 2.72e2.82 (m, 3 H), 3.36e3.46
z (%): 130 [Mþ þ 1 ꢁ HCl] (100).
(m, 2 H), 3.64 (s, 3 H), 6.05 (t, 1 H, J ¼ 7.6 Hz), 7.14e7.38 (m, 10 H). IR
(KBr):
n
¼ 2950 cmꢁ1, 2814, 1737, 1496, 1437, 1193, 1173, 769, 702.
~
5.1.26. Methyl 1-(4,4-diphenylbut-3-en-1-yl)azetidin-3-
carboxylate (39b)
MS (CI): m/z (%): 336 [Mþ þ 1] (100), 142 (55). C22H25NO2 (335.5):
calcd C 78.77, H 7.51, N 4.18; found C 78.30, H 7.39, N 4.14.
N-alkylation according to GP3 starting from 35 (158 mg,
1.04 mmol) in 5 ml THF, 4-bromo-1,1-diphenylbuten (38b)
(300 mg, 1.04 mmol), K2CO3 (577 mg, 4.16 mmol) and NaI (5 mg,
0.03 mmol). The reaction mixture was refluxed for 48 h. Purifica-
tion by CC (acetone/n-heptane ¼ 30:70). Yield: 71 mg (21%);
5.1.30. Methyl 1-{2-[tris(4-methoxyphenyl)methoxy]ethyl}
azetidin-3-ylacetic acid (40d)
N-alkylation according to GP3 starting from 37 (246 mg,
1.48 mmol) in 2.5 ml of THF, 1-{(2-bromoethoxy)[bis(4-methox-
yphenyl)]methyl}-4-methoxybenzene (38d) (1.35 mg, 2.96 mmol)
in 2.5 ml of THF, K2CO3 (821 mg, 5.94 mmol), and NaI (10 mg).
Reaction temperature: rt; reaction time: 116 h. Workup as
described for compound 39c. Purification by CC (n-heptane/
EtOAc ¼ 60:40 þ 1% NEtMe2). Yield: 119 mg (16%); colorless oil. 1H
colorless oil. 1H NMR (CDCl3):
d
¼ 2.13 ppm (q, 2 H, J ¼ 7.4 Hz), 2.52
(t, 2 H, J ¼ 7.4 Hz), 3.18e3.23 (m, 2 H), 3.25 (qui, 1 H), 3.44e3.49 (m,
2 H), 3.69 (s, 3 H), 6.05 (t, 1 H, J ¼ 7.6 Hz), 7.14e7.38 (m, 10 H). IR
~
(KBr):
n
¼ 2950 cmꢁ1, 2837, 1736, 1493, 1436, 1199, 760, 701. MS
(CI): m/z (%): 322 [Mþ þ 1] (100). C21H23NO2 (321.42): calcd C 78.47,
H 7.21, N 4.36; found C 78.55, H 7.41, N 4.08.
NMR (benzene-d6):
d
¼ 2.36 ppm (d, 2 H, J ¼ 7.8 Hz), 2.58 (t, 2 H,
J ¼ 5.8 Hz), 2.60e2.70 (m, 1 H), 2.82e2.86 (m, 2 H), 3.27 (s, 3 H),
3.28 (t, 2 H, J ¼ 5.8 Hz), 3.31 (s, 9 H), 3.35e3.39 (m, 2 H), 6.78e6.73
5.1.27. Methyl 1-[4,4-bis(3-methylthiophen-2-yl)but-3-en-1-yl]
azetidine-3-carboxylate (39c)
(m,
6 H), 7.56e7.61 (m, 6
H). 13C APT NMR (benzene-d6):
N-alkylation according to GP3 starting from 35 (200 mg,
1.32 mmol) in 2 ml THF, 2-[4-bromo-1-(3-methylthien-2-yl)but-1-
enyl]-3-methylthiophene (38c) (844 mg, 2.58 mmol) in 2 ml THF,
K2CO3 (950 mg, 6.9 mmol) and NaI (5 mg, 0.03 mmol). The reaction
mixture was refluxed for 16 h and subsequently stirred for 32 h at
rt. CH2Cl2 (4 ml) was added. The precipitate was filtered off and
washed with CH2Cl2. The filtrate was concentrated in vacuo. Puri-
fication by CC (n-heptane/EtOAc ¼ 80:20 þ 1% NEtMe2). Yield:
242 mg (51%); yellowish oil. TLC: Rf ¼ 0.08 (n-heptane/
d
¼ 28.1 ppm, 38.4, 50.9, 54.7, 59.7, 61.1, 63.1, 86.3, 113.4, 130.3,
137.6, 158.9, 172.2. IR (KBr):
¼ 2951 cmꢁ1, 2834, 1735, 1607, 1507,
~
n
1249, 1175, 1035, 827, 583. MS (CI): m/z (%): 506 [Mþ þ 1] (68), 474
(100). C30H35NO6 (505.6): calcd C 71.27, H 6.98, N 2.77; found C
71.38, H 7.47, N 2.69.
5.1.31. (RS)-tert-Butyl 1-(4,4-diphenylbut-3-en-1-yl)azetidin-2-
ylacetate (41b)
N-alkylation according to GP3 starting from 27 (183 mg,
0.788 mmol) in 2.0 ml of THF, (4-bromo-1-phenylbutenyl)benzene
(38b) (452 mg, 1.59 mmol) in 2.0 ml of THF, K2CO3 (444 mg,
3.21 mmol) and NaI (5 mg). Reaction temperature: rt; reaction
time: 120 h. The solvent was evaporated. The crude product was
extracted with CH2Cl2 (3 ꢃ 20 ml). The combined organic layers
were washed with water (10 ml), dried (MgSO4), and concentrated
in vacuo. Purification by CC (n-heptane/EtOAc ¼ 80:20 þ 1%
NEtMe2). Yield: 104 mg (35%); colorless oil. 1H NMR (CDCl3):
EtOAc ¼ 80:20 þ 1% NEtMe2). 1H NMR (benzene-d6):
d
¼ 1.96 ppm
(s, 3 H), 1.97 (s, 3 H), 2.12 (q, 2 H, J ¼ 7.1 Hz), 2.25 (t, 2 H, J ¼ 7.1 Hz),
2.92e3.00 (m, 1 H), 3.13e3.23 (m, 4 H), 3.29 (s, 3 H), 6.10 (t, 1 H,
J ¼ 7.1 Hz), 6.57 (d, 1 H, J ¼ 5.4 Hz), 6.61 (d, 1 H, J ¼ 5.4 Hz), 6.75 (d, 1
~
H, J ¼ 5.4 Hz), 6.86 (d, 1 H, J ¼ 5.4 Hz). IR (KBr):
n
¼ 2949 cmꢁ1
,
2835, 1736, 1436, 1364, 1199, 1174, 712. MS (CI): m/z (%): 362
[Mþ þ 1] (17), 128 (100). C19H23NO2S2 (361.5): calcd C 63.12, H 6.41,
N 3.87, S 17.74; found C 62.83, H 6.38, N 3.87, S 17.65.
d
¼ 1.36 ppm (s, 9 H), 1.72e1.82 (m, 1 H), 1.83e1.91 (m, 1 H),
5.1.28. Methyl 1-{2-tris(4-methoxyphenyl)methoxy]ethyl}
azetidine-3-carboxylate (39d)
N-alkylation according to GP3 starting from 35 (100 mg,
0.662 mmol) in 1.5 ml of CH2Cl2, 1-{(2-bromoethoxy)[bis(4-
2.14e2e28 (m, 3 H), 2.36 (dd, 1 H, J ¼ 15.0, 7.3 Hz), 2.39e2.46 (m, 1
H), 2.50 (dd, 1 H, J ¼ 15.0, 5.8 Hz), 2.68 (dt, 1 H, J ¼ 11.0, 7.3 Hz),
3.14e3.21 (m, 1 H), 3.27e3.26 (m, 1 H), 6.17 (t, 1 H, J ¼ 7.3 Hz),
7.02e7.14 (m, 4 H), 7.17e7.22 (m, 4 H), 7.30e7.34 (m, 2 H). 13C NMR
methoxyphenyl)]methyl}-4-methoxybenzene
0.76 mmol) in 1.5 ml of CH2Cl2 and K2CO3 (228 mg, 1.65 mmol).
Reaction temperature: rt; reaction time: 48 h. Purification by CC (n-
(38d)
(347 mg,
(benzene-d6):
128.3, 130.1, 140.5, 142.5, 143.1, 170.4. IR (KBr):
2929, 1725, 1493, 1444, 1367, 1153, 761, 701. MS (CI): m/z (%): 378
d
¼ 23.9, 27.8, 28.7, 42.8, 51.6, 58.3, 62.8, 79.4, 126.8,
¼ 2975 cmꢁ1
,
~
n

M.R. Faust et al. / European Journal of Medicinal Chemistry 45 (2010) 2453e2466
2465
[Mþ þ 1] (19), 322 (39), 262 (3), 184 (100), 128 (57). C25H31NO2
5.1.35. 1-(4,4-Diphenylbut-3-en-1-yl)-3-(4-methoxyphenyl)
azetidin-3-ol (18b)
(377.5): calcd C 79.54, H 8.28, N 3.71; found C 79.18, H 8.38, N 3.69.
N-alkylation according to GP3 starting from 18a (183 mg,
1.02 mmol) in 5.0 ml of THF, (4-bromo-1-phenylbutenyl)benzene
(38b) (293 mg, 1.02 mmol), K2CO3 (422 mg, 3.06 mmol), and NaI
(5 mg, 0.03 mmol). Reaction temperature: rt; reaction time: 48 h.
Workup as described for compound 39c. Purification by (n-heptane/
EtOAc ¼ 50:50 þ 5% NEtMe2). Yield: 230 mg (58%); colorless solid,
5.1.32. (RS)-tert-Butyl 1-[4,4-bis(3-methylthien-2-yl)but-3-en-1-
yl]azetidin-2-ylacetate (41c)
N-alkylation according to GP3 starting from 27 (200 mg,
0.86 mmol) in 2.0 ml of THF, 2-[4-bromo-1-(3-methylthien-2-yl)
butenyl]-3-methylthiophene (38c) (573 mg, 1.75 mmol) in 2.0 ml
THF, K2CO3 (484 mg, 3.51 mmol), and NaI (5 mg, 0.03 mmol).
Reaction temperature: rt; reaction time: 48 h. Workup as described
for compound 39c. Purification by (n-heptane/EtOAc ¼ 90:10 þ 1%
NEtMe2). Yield: 57 mg (16%); yellowish oil. 1H NMR (benzene-d6):
mp 53 ꢂC.1H NMR (CDCl3):
d
¼ 2.22 ppm (q, 4 H, J ¼ 7.4 Hz), 2.64 (t, 2
H, J ¼ 7.3 Hz), 3.00 (s br, 1 H), 3.36 (d, 2 H, J ¼ 8.7 Hz), 3.55 (d, 2 H,
J ¼ 8.7 Hz), 3.80 (s, 3 H), 6.09 (t, 1 H, J ¼ 7.6 Hz), 6.87e6.92 (m, 2 H),
~
7.15e7.38 (m, 10 H), 7.40e7.45 (m, 2 H). IR (KBr):
n
¼ 3052 cmꢁ1
,
d
¼ 1.37 ppm (s, 9 H),1.71e1.82 (m,1 H),1.83e1.92 (m,1 H),1.99 (s, 3
2947, 2832, 1610, 1581, 1513, 1442, 1245, 1173, 1032, 830, 758, 701.
MS (CI): m/z (%): 386 [Mþ þ 1] (34), 370 (100), 236 (79), 176 (25).
C26H27NO2 (385.5): calcd C 81.01, H 7.06, N 3.63; found C 80.89, H
7.17, N 3.64.
H), 2.00 (s, 3 H), 2.14e2.26 (m, 3 H), 2.36 (dd, 1 H, J ¼ 15.2, 7.4 Hz),
2.37e2.47 (m, 1 H), 2.54 (dd, 1 H, J ¼ 15.2, 5.8 Hz), 2.59e2.69 (m, 1
H), 3.15e3.21 (m, 1 H), 3.27e3.37 (m, 1 H), 6.17 (t, 1 H, J ¼ 7.2 Hz),
6.59 (d, 1 H, J ¼ 5.1 Hz), 6.62 (d, 1 H, J ¼ 5.1 Hz), 6.76 (d, 1 H,
J ¼ 5.1 Hz), 6.87 (d, 1 H, J ¼ 5.1 Hz). 13C NMR APT (benzene-d6):
5.1.36. 1-[4,4-Bis(3-methylthien-2-yl)but-3-en-1-yl]-3-
(4-methoxyphenyl)azetidin-3-ol (18c)
d
¼ 14.5 ppm, 14.9, 24.2, 28.1, 28.9, 43.1, 51.8, 58.0, 63.0, 79.6, 123.0,
~
n
124.6, 129.7, 131.3, 133.7, 134.4, 170.4. IR (KBr):
¼ 2920 cmꢁ1
,
N-alkylation according to GP3 starting from 18a (200 mg,
1.12 mmol) in 4.0 ml of THF, 2-[4-bromo-1-(3-methylthien-2-yl)
butenyl]-3-methylthiophene (38c) (548 mg, 1.67 mmol), K2CO3
(463 mg, 3.4 mmol), and NaI (5 mg, 0.03 mmol). Reaction temper-
ature: reflux; reaction time: 24 h. Workup as described for
compound 39c. Purification by (n-heptane/EtOAc ¼ 60:40 þ 1%
NEtMe2). Yield: 247 mg (52%); colorless solid, mp 93 ꢂC. 1H NMR
2851, 1728, 1641, 1560, 1458, 1367, 1156, 711. MS (CI): m/z (%): 418
[Mþ þ 1] (27), 362 (4), 184 (100), 128 (8). C23H31NO2S2 (417.6): calcd
C 66.15, H 7.48, N 3.35, S 15.35; found C 66.24, H 7.40, N 3.33, S
15.06.
5.1.33. (RS)-tert-Butyl 1-{2-[tris(4-methoxyphenyl)methoxy]ethyl}
azetidin-2-ylacetate (41d)
(benzene-d6):
d
¼ 1.98 ppm (s, 3 H), 2.00 (s, 3 H), 2.25 (q, 2 H,
N-alkylation according to GP3 starting from 27 (300 mg,
1.29 mmol) in 6.0 ml of THF, 1-{(2-bromoethoxy)[bis(4-methox-
yphenyl)]methyl}-4-methoxybenzene (38d) (1.6 g, 3.4 mmol),
K2CO3 (1.21 g, 8.75 mmol), and NaI (10 mg, 0.060 mmol). Reaction
temperature: rt; reaction time: 48 h. Workup as described for
compound 39c. Purification by (n-heptane/EtOAc ¼ 90:10 þ 1%
NEtMe2). Yield: 179 mg (25%); colorless oil. 1H NMR (CDCl3):
J ¼ 7.2 Hz), 2.43 (t, 2 H, J ¼ 7.2 Hz), 3.15 (d, 2 H, J ¼ 8.4 Hz), 3.34 (s, 3
H), 3.46 (d, 2 H, J ¼ 8.4 Hz), 6.16 (t, 1 H, J ¼ 7.2 Hz), 6.58 (d, 1 H,
J ¼ 5.1 Hz), 6.62 (d, 1 H, J ¼ 5.1 Hz), 6.76 (d, 1 H, J ¼ 5.1 Hz),
~
6.84e6.89 (m, 3 H), 7.59e7.64 (m, 2 H). IR (KBr):
n
¼ 3412 cmꢁ1
,
2930, 2833, 1611, 1514, 1456, 1247, 1177, 1034, 832, 713. MS (CI): m/z
(%): 426 [Mþ þ 1] (12), 408 (4), 192 (100). C24H27NO2S2 (425.6):
calcd C 67.73, H 6.39, N 3.29, S 15.07; found C 67.78, H 6.32, N 3.29, S
14.96.
d
¼ 1.37 (s, 9 H, C(CH3)3), 1.80e1.91 (m, 1 H, CH2CH2CHN), 1.91e2.00
(m, 1 H, CH2CH2CHN), 2.40 (dd, 1 H, J ¼ 15.2/7.4 Hz, CHCH2CO),
2.52e2.63 (m, 2 H, CHCH2CO, NCH2CH2O), 2.73e2.81 (m, 1 H,
NCH2CH2CH), 2.84e2.91 (m, 1 H, NCH2CH2O), 3.31 (s, 9 H, PhOCH3),
3.22e3.37 (m, 11 H, NCH2CH2O, PhOCH3), 3.42e3.52 (m, 2 H,
NCH2CH2CH, CHCH2CO), 6.77e6.84 (m, 6 H, CHarCO), 7.52e7.58 (m,
5.1.37. 3-(4-Methoxyphenyl)-1-{2-[tris(4-methoxyphenyl)
methoxy]ethyl}azetidin-3-ol (18d)
N-alkylation according to GP3 starting from 18a (100 mg,
0.56 mmol) in 5.0 ml of THF, 1-{(2-bromoethoxy)[bis(4-methox-
yphenyl)]methyl}-4-methoxybenzene (38d) (0.26 mg, 0.56 mmol),
K2CO3 (231 mg, 1.68 mmol), and NaI (5 mg, 0.03 mmol). Reaction
temperature: reflux; reaction time: 48 h. Workup as described for
compound 39c. Purification by (n-heptane/EtOAc ¼ 60:40 þ 5%
NEtMe2). Yield: 92 mg (25%); colorless solid, mp 64 ꢂC. 1H NMR
6 H, CHarCC). 13C NMR APT (benzene-d6):
d
¼ 24.7 (CH2CH2CHN),
28.0 (C(CH3)3), 42.9 (CHCH2CO), 53.4 (NCHCH2CH2), 54.6 (PhOCH3),
58.3 (NCH2CH2O), 63.1 (NCH), 63.2 (NCH2CH2O), 113.2 (CHarCO),
130.1 (CHarCC), 137.4 (Cq), 158.7 (Cq), 170.5 (C ¼ O). IR (KBr):
~
n
¼ 2930 cmꢁ1, 2832, 1726, 1608, 1508, 1251,1175, 1153, 1037, 828.
MS (CI, CHþ5 ): m/z (%): 333 (100), 227 (9). C33H41NO6 (547.7): calcd C
(CDCl3):
d
¼ 2.77 ppm (t, 2 H, J ¼ 5.7 Hz), 3.15 (t, 2 H, J ¼ 5.7 Hz),
72.37, H 7.55, N 2.56; found C 72.94, H 8.17, N 2.54.
3.50 (d, 2 H, J ¼ 8.7 Hz), 3.73 (d, 2 H, J ¼ 8.7 Hz), 3.77 (s, 9 H), 3.81 (s,
3 H), 6.78e6.83 (m, 6 H), 6.90e6.95 (m, 2 H), 7.30e7.35 (m, 6 H),
~
5.1.34. 1-(3-Carbazol-9-ylpropyl)-3-[1-(4-methoxybenzyl)-1H-
tetrazol-5-yl]azetidin-3-ol (42e)
7.47e7.52 (m, 2 H). IR (KBr):
n
¼ 3396 cmꢁ1, 2931, 2833, 1608,
1582, 1508, 1463, 1248, 1175, 1033, 827, 582. MS (CI): m/z (%): 180
[Mþ þ 1] (60), 162 (100), 151 (17). C34H37NO6 (555.7): calcd C 73.49,
H 6.71, N 2.52; found C 73.29, H 6.93, N 2.36.
N-alkylation according to GP3 starting from 30 (275 mg,
0.92 mmol) in 5.0 ml of THF, 9-(3-bromopropyl)-9H-carbazole
(38e) (266 mg, 0.92 mmol, 1.0 equiv), K2CO3 (382 mg, 2.76 mmol,
3 equiv), and NaI (10 mg). Reaction temperature: rt; reaction time:
96 h. Workup as described for compound 39c. Purification by
(petrol ether/acetone ¼ 60:40). Yield: 149.5 mg (35%); colorless
5.1.38. 1-(3-Carbazol-9-ylpropyl)-3-(4-methoxyphenyl)azetidin-3-
ol (18e)
N-alkylation according to GP3 starting from 18a (1.00 g,
5.58 mmol) in 10 ml of THF, 9-(3-bromopropyl)-9H-carbazole (38e)
(1.61 mg, 5.58 mmol), K2CO3 (2.31 g, 16.7 mmol), and NaI (10 mg,
0.07 mmol). Reaction temperature: rt; reaction time: 72 h. Workup
as described for compound 39c. Purification by (n-heptane/
EtOAc ¼ 60:40 þ 1% NEtMe2). Yield: 1150 mg (53%); colorless solid,
solid, mp 155 ꢂC. 1H NMR (CDCl3):
d
¼ 1.90 ppm (qui, 2 H, J ¼ 7.1 Hz),
2.50 (t, 2 H, J ¼ 7.1 Hz), 3.14 (d, 2 H, J ¼ 9.4 Hz), 3.54 (d, 2 H,
J ¼ 9.4 Hz), 3.71 (s, 3 H), 4.33 (t, 2 H, J ¼ 7.1 Hz), 5.39 (s, 2 H),
6.64e6.69 (m, 2 H), 7.21 (t, 2 H, J ¼ 7.5 Hz), 7.35 (d, 2 H, J ¼ 8.0 Hz),
~
7.42 (t, 2 H, J ¼ 7.5 Hz), 8.06 (d, 2 H, J ¼ 8.0 Hz). IR:
n
¼ 3309 cmꢁ1
,
3054, 2934, 2829, 1612, 1594, 1514, 1484, 1452, 1326, 1251, 1176,
1031, 825, 751, 724. MS (CI): m/z (%): 469 [Mþ þ 1] (1), 279 (43), 121
(100). C27H28N6O2 (468.6): calcd C 69.21, H 6.02, N 17.94; found C
69.24, H 6.13, N 17.51.
mp 47 ꢂC. 1H NMR (benzene-d6):
d
¼ 1.52 ppm (qui, 2 H, J ¼ 6.5 Hz),
1.90e2.02 (s br, 1 H), 2.10 (t, 2 H, J ¼ 6.5 Hz), 2.96 (d, 2 H, J ¼ 7.9 Hz),
3.34 (s, 3 H), 3.41 (d, 2 H, J ¼ 7.9 Hz), 3.98 (t, 2 H, J ¼ 6.5 Hz),
6.88e6.92 (m, 2 H), 7.23 (t, 2 H, J ¼ 7.3 Hz), 7.35 (d, 2 H, J ¼ 8.3 Hz),

2466
M.R. Faust et al. / European Journal of Medicinal Chemistry 45 (2010) 2453e2466
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7.42 (t, 2 H, J ¼ 7.3 Hz), 7.59e7.63 (m, 2 H), 8.06 (d, 2 H, J ¼ 8.3 Hz).
13C NMR (benzene-d6):
d
¼ 28.8 ppm, 41.8, 56.4, 57.7, 70.7, 73.0,
110.7, 115.5, 120.8, 122.2, 125, 127.4, 128.0, 138.7, 142.6, 161.0. IR:
~
n
¼ 3380 cmꢁ1, 3048, 2932, 2829, 1606, 1596, 1512, 1484, 1452,
1326, 1246, 1030, 830, 748, 723. MS (CI); m/z (%): 387 [Mþ þ 1]
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77.69, H 6.78, N 7.25; found C 77.78, H 6.93, N 7.13.
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5.1.39. Preparation of subcellular membrane suspensions
Two subcellular membrane pellets, termed bfcP2B (from bovine
frontal cortex)a and bbsP2C (from bovine brain stem)b, respectively,
were prepared according to literature.[54] Their suspensions were
prepared and measured as described by Bradford. [56] cfcP2B (from
calf frontal cortex)c and cbsP2C (from calf brain stem)d, pfcP2B
(from porcine frontal cortex)e and pbsP2C (from porcine brain
stem)f were applied alternatively instead of bfcP2B and bbsP2C,
respectively.
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Aliquots of about 50e100
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acid and a test compound in 200 l of buffer (119 mM NaCl, 2.5 mM
m
g protein bfcP2B (alternatively
mM aminooxyacetic
m
CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 4.7 mM KCl, 11 mM glucose
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l of 12.5 nM [3H] GABA and 25
the sample was incubated at 37 ꢂC for 4 min. The incubation was
terminated by filtration in a Brandel M-24R Harvester through
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5.1.41. Inhibition of GAT-3 mediated GABA-uptake
Aliquots of about 50e100
cbsP2C or pbsP2C) were preincubated with 10
acid, 10 M NNC-711 and a test compound in 200
m
g protein bbsP2C (alternatively
M aminooxyacetic
l of buffer
m
m
m
(119 mM NaCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4,
4.7 mM KCl, 11 mM glucose and 25 mM Tris-HCl pH 7.2) for 10 min
at 37 ꢂC. The addition of 25
GABA was followed.
m ml of 1 mM
l of 50 nM [3H] GABA and 25
Acknowledgment
Financial support of this work by the Deutsche For-
schungsgemeinschaft, the Fonds der Chemischen Industrie and the
BMBF is greatly appreciated.
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