Development of imidazole alkanoic acids as mGAT3 selective GABA uptake inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.01.042

A new series of potential GABA uptake inhibitors starting from of 1H-imidazol-4-ylacetic acid with the carboxylic acid side chain originating from different positions and varying in length have been synthesized and tested for the inhibitory potency at the

Full text of DOI:10.1016/j.ejmech.2011.01.042

European Journal of Medicinal Chemistry 46 (2011) 1483e1498
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Development of imidazole alkanoic acids as mGAT3 selective GABA uptake
inhibitors
*
Silke Hack, Babette Wörlein, Georg Höfner, Jörg Pabel, Klaus T. Wanner
Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-University Munich, Butenandtstr. 5-13, D-81377 Munich, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of potential GABA uptake inhibitors starting from of 1H-imidazol-4-ylacetic acid with the
carboxylic acid side chain originating from different positions and varying in length have been
synthesized and tested for the inhibitory potency at the four GABA uptake transporters mGAT1e4 stably
expressed in HEK cells. Further two bicyclic compounds with a rigidified carboxylic acid side chain were
Received 9 September 2010
Accepted 25 January 2011
Available online 3 February 2011
included in this study. The results of the biological tests indicated that most
alkenoic acid derivatives exhibit the highest potencies as GABA uptake inhibitors at mGAT3.
u-imidazole alkanoic and
Dedicated with the best wishes to Prof. Fritz
Eiden on occasion of his 85th birthday.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
GABA-uptake inhibitor
mGAT3
Imidazole
Antiepileptic
1. Introduction
subtypes mGAT1 and mGAT4 are mainly located in the CNS.
Thereby mGAT1 is widely distributed in the brain and predomi-
Since the initial isolation of
g
-aminobutyric acid (GABA) in brain
nantly found in the cerebral cortex, the cerebellum, the basal
ganglia, and the hippocampus [12,13]. The distribution of mGAT4 is
more restricted. Strong intensities are observed in certain brain-
stem nuclei, in the thalamus, in the hypothalamus, the retina, and
olfactory bulb [12,14]. The four subtypes also differ with respect to
their cellular and subcellular localization. Thus, mGAT1 is primarily
found on presynaptic neurons in the synapse and to a minor extent
on processes of astrocytes enveloping synapses. mGAT4 is
predominantly expressed on distal processes of astrocytes in direct
contact with GABAergic synapses [14,15]. mGAT2 is found to a large
extend on astrocytes, but outside the synaptic cleft of GABA
neurons [15,16]. mGAT3 has been found to be primarly located on
leptomeninges [15,17]. In very low level, it seems to be also present
throughout the brain on astrocytes and neurons at extrasynaptic
sites. The different carrier proteins vary also in their affinity to
GABA (1) [9]. mGAT1, mGAT3, and mGAT4 show high affinity,
whereas mGAT2 possesses low-affinity to GABA (1), but also is able
to carry betaine [7,12].
tissue in 1950 [1e3] and its identification as the major inhibitory
neurotransmitter in the central nervous system (CNS) in the 1960s
[4], about 40% of brain synapses were identified to be GABAergic. In
the same decade, the existence of an active transport system
deactivating the GABAergic signalling was discovered [5]. In 1986,
Radian et al. purified the first GABA transporter showing depen-
dence on Naþ and Clꢀ for transport [6]. By molecular cloning from
different species, including mice, rats, and humans, the existence of
four distinct subtypes of GABA transporters has been shown [7,8].
According to the nomenclature used for mice, the GABA transporter
subtypes are denoted as mGAT1, mGAT2, mGAT3, and mGAT4. In
contrast, GATs originating from other species including humans
and rats are termed GAT-1, BGT-1, GAT-2, and GAT-3 [9e11],
respectively.1 The GABA-transporters differ in their regional
distribution. In contrast to mGAT2 and mGAT3, which exist inside
and outside the brain [12,13], the prevalent GABA transporters
A number of CNS disorders such as epilepsy [18], Morbus Par-
kinson [19], and Chorea Huntington [20] are associated with
a reduced neurotransmission in the GABAergic system and can
potentially be alleviated by drugs that augment GABAergic neuro-
transmission [18,21,22]. Inhibition of the uptake by GABA trans-
porters and the associated increase of GABA concentration in the
synaptic cleft [23] has, for example, been realized with the cyclic
* Corresponding author. Tel.: þ49 89 2180 77249; fax: þ49 89 2180 77247.
E-mail address: klaus.wanner@cup.uni-muenchen.de (K.T. Wanner).
Since the biological activity of the compounds described in this paper was
evaluated using murine transporters, the mice nomenclature will preferably be
used in this publication. If test results obtained for GABA transporters of other
species are quoted, the nomenclature of the respective species will be given in
parenthesis.
1
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.042

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S. Hack et al. / European Journal of Medicinal Chemistry 46 (2011) 1483e1498
With potent and selective inhibitors of mGAT2, mGAT3, and
COOH
COOH
COOH
mGAT4 still lacking, the therapeutic potential of these GABA
transporters is not particularly clear so far. But anticonvulsant
activity has been demonstrated for the moderate mGAT4 selective
nipecotic acid derivative (S)-SNAP-5114 [28,30] (7, Table 1), the
poorly mGAT2 selective N-substituted 4-hydroxy-4-aryl-piperidine
derivative NNC 05-2045 [31] (8, Table 1) and the mGAT1/mGAT2
selective compound EF1502 [32] (9, Table 1).
H2N
GABA (1)
N
H
N
H
nipecotic acid (2)
guvacine (3)
Fig. 1. GABA (1) and cyclic GABA uptake inhibitors.
Up to date, only few selective inhibitors for mGAT3 are pub-
lished. The majority of compounds inhibiting this transporter are
also inhibitors of mGAT4 indicating that the binding sites of the
transporters mGAT3 and mGAT4 posses similar structural charac-
teristics [7]. SNAP-5294 (10, Table 1) developed by Murali Dhar
et al. [28] shows the best inhibitory potency at mGAT3 in combi-
nation with a reasonable selectivity for this transporter, which
mainly originates from the lipophilic side chain present in the
molecule [28].
COOH
COOH
COOH
N
N
N
O
N
S
S
In this publication, we present the syntheses and biological
evaluation of potential GABA uptake inhibitors based on imidazole
derivatives. The drug development started with 1H-imidazol-4-yl-
acetic acid (20, Fig. 3), a conformational restricted analogue of
GABA. First evidence of partial agonistic activity at GABAA-recep-
tors as well as at GABAC-receptors of the histamine metabolite 1H-
imidazol-4-ylacetic acid (20) was provided by Tunnicliff in 1998
[33]. Recently, Madsen et al. [34] reported on the influence of
various substituted 1H-imidazol-4-ylacetic acid derivatives on the
activity of GABAA-receptors and GABAC-receptors. In parallel, we
had found, when screening for new GABA uptake inhibitors
applying a test system based on stably expressed mGAT1emGAT4
in HEK cells [35], that, apart from effects on GABA receptors, 1H-
imidazol-4-ylacetic acid (20) inhibits also the GABA uptake.
Besides a moderate inhibitory effect at mGAT1 (pIC50 value of
3.208 ꢁ 0.121) the imidazole compound 20 exhibits a particularly
high inhibitory potency at mGAT3 (pIC50 value of 4.756 ꢁ 0.080).
With a subtype selectivity of >10:1 for mGAT3 compared to
mGAT1, we considered 1H-imidazol-4-ylacetic acid (20) a prom-
ising starting point for the development of mGAT3 selective GABA
uptake inhibitors.
SK&F-89976-A (4)
Tiagabine (5)
NO 711 (6)
Fig. 2. Cyclic GABA uptake inhibitors of mGAT1.
compounds (R,S)-nipecotic acid (2, Fig. 1) and guvacine (3, Fig. 1)
belonging to the first generation of GABA uptake inhibitors.
Although both compounds show in vitro high GABA uptake inhi-
bition without intrinsic activity at GABAA-receptors, the pharma-
cological usefulness of these compounds was limited as they lack
the capability to cross the bloodebrain barrier [24].
This obstacle was overcome by introducing characteristic
lipophilic side chains to the parent compounds like nipecotic
acid and guvacine leading to several GABA uptake inhibitors
such as SK&F-89976-A [25] (4, Fig. 2), Tiagabine [26] (5, Fig. 2),
or NO 711 [27] (6, Fig. 2) which, apart from being systemically
active, were found to be selective and, as compared to the parent
compounds, distinctly more potent inhibitors of mGAT1 [28].
This research culminated in the development of Tiagabine (5)
which is in clinical use for the therapy of epilepsy since 1997
classifying mGAT1 an approved drug target for the therapy of
this disease [29].
In order to determine the relationship between the structure
and the activity as well as the selectivity of imidazole derivatives
Table 1
GABA uptake inhibitors.
OMe
COOH
O
COOH
HO
N
N
N
HO
N
N
CH3
MeO
O
MeO
OMe
O
S
N
S
OMe
SNAP-5294 (10)
EF1502 (9)
(S)-SNAP-5114 (7)
NNC 05-2045 (8)
Compound
Uptake-inhibition IC50
mGAT1
(m
M)a
mGAT2
mGAT3
mGAT4
7 [30]
8 [31]
9 [32]
10 [28]
85 ꢁ 14
19 ꢁ 2
4
35 ꢁ 13
1.4 ꢁ 0.3
22
22 ꢁ 4
41 ꢁ 11
>300
3.0 ꢁ 0.3
15 ꢁ 4
>300
132 ꢁ 50
27 ꢁ 10%b
(hBGT-1)
51 ꢁ 4
142 ꢁ 21
(hGAT-1)
(rGAT-2)
(hGAT-3)
a
The IC50 values originate from literature. The references are given in parenthesis after the corresponding compound.
Percent inhibition at 100 mM.
b

S. Hack et al. / European Journal of Medicinal Chemistry 46 (2011) 1483e1498
1485
R
N
N
N
R
N
H
N
R
N
H
R =
COOH
CH2COOH
R =
COOH
R =
CH2COOH
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
CH2COOH
(CH2)2COOH
(CH2)3COOH
(CH2)4COOH
(CH2)5COOH
(CH2)6COOH
(CH2)7COOH
(CH2)2COOH
(CH2)2COOH
(CH2)3COOH
(CH2)4COOH
CH=CHCOOH (E)
(CH2)2CH=CHCOOH (E)
(CH2)3COOH
(CH2)4COOH
CH=CHCOOH (E)
CH=CHCOOH (Z)
(CH2)2CH=CHCOOH (E)
Fig. 3. Different monocyclic imidazole compounds.
with a carboxylic acid side chain as GABA uptake inhibitors, the
structure of 1H-imidazol-4-ylacetic acid (20, Fig. 3) was system-
atically modified. Thus, by changing the length and the position of
the side chain various analogues of 20 (11e32, Fig. 3) were
synthesized as potential GABA uptake inhibitors and characterized
with regard to their potency at and selectivity for the transporters.
For the 2-substituted imidazole derivatives the side chain was
extended from one C-atom (1H-imidazol-2-carboxylic acid (11))
up to five C-atoms (5-(1H-imidazol-2-yl)pentanoic acid (15)). In
addition, the unsaturated compounds (2E)-3-(1H-imidazol-2-yl)
acrylic acid (16), the Z-isomer (2Z)-3-(1H-imidazol-2-yl)acrylic
acid (17) as well as (2E)-5-(1H-imidazol-2-yl)pent-2-enoic acid
(18) were synthesized as structural variations of 13 and 15, and
evaluated for their biological activity to study the effects of the
side chain geometry on the potency of these compounds. To
complete the array of test compounds, carboxylic acid side chains
of different length were also attached to the 4-position of the
2. Chemistry
2.1. -Imidazole-2-yl alkanoic and alkenoic acids
1H-Imidazol-2-carboxylic acid (11) was prepared according to
literature [36] and 1H-imidazol-2-yl propanoic acid 13 by hydro-
lysis of 3-(1H-imidazol-2-yl)propanoic acid ethyl ester [37] with
NaOH in aqueous EtOH (yield 86%).
u
For the syntheses of the u-imidazole-2-yl alkanoic acids 12 and
14e18, the synthetic sequences outlined in Schemes 1e4 were
followed.
The synthesis of the 2-(1H-imidazol-2-yl)acetic acid hydro-
chloride (12∙HCl) was realized starting from 2-methyl-1-triphe-
nylmethyl-1H-imidazole (33) which was treated with nBuLi in THF
at ꢀ40 ꢂC and subsequently with carbon dioxide to provide the
acetic acid derivative 35. Subsequent removal of the protecting
group performed by refluxing 35 in 1 M HCl yielded 2-(1H-imida-
zol-2-yl)acetic acid hydrochloride (12∙HCl), albeit in low yield (11%,
Scheme 1).
imidazole core structure which resulted in
u-imidazole-4-yl
alkanoic and alkenoic acids 19e25. Attaching carboxylic acid side
chains at the ring nitrogen led to the
acids 26e32.
u
-imidazole-1-yl alkanoic
For the synthesis of the stereoisomeric propenoic acid deriva-
tives 16 and 17, first 1H-imidazol-2-carbaldehyde (36) was
N
N
N
Li
CO2
COOH
nBuLi
N
Tr
N
Tr
N
Tr
THF
-40 °C -> rt
THF , -40 °C
33
34
35
1 M HCl, Δ, 1 h
11%
.
N
HCl
COOH
N
H
12·HCl
Scheme 1. Synthesis of 12∙HCl (Tr ¼ Triphenylmethyl).

1486
S. Hack et al. / European Journal of Medicinal Chemistry 46 (2011) 1483e1498
O
O
P
EtO
EtO
OEt
OEt
O
N
N
N
OEt
N
O
NaH
NEt3
O
TrCl
+
+
N
Tr
N
Tr
N
H
O
DMF
rt, 48 h
75%
N
Tr
DME
H
H
1. Δ, 15 min
2. 60 °C, 1 h
69%
29%
36
37
38
39
40
1 M HCl
Δ, 4 h
1 M HCl
Δ, 4 h
82%
100%
OH
.
O
.
HCl
HCl
N
N
OH
N
H
N
H
O
16·HCl
17·HCl
Scheme 2. Synthesis of 16∙HCl and 17∙HCl (Tr ¼ Triphenylmethyl).
transformed into the N-triphenylmethyl protected derivative 38 by
treatment with triphenylmethyl chloride in the presence of NEt3
(yield 75%). When subjected to a HornereWadswortheEmmons
reaction analogous to a procedure published by Hunt [37], 38
yielded the isomeric mixture of the propenoic ethyl esters 39 [38]
and 40 which were isolated in pure form. Subsequent depro-
tection and hydrolysis afforded the test compounds 16$HCl and
17$HCl in high yields (Scheme 2).
For the synthesis of 4-(1H-imidazol-2-yl)butanoic acid hydro-
chloride (14∙HCl), 1-triphenylmethyl-1H-imidazole (41) [39] was
treated with nBuLi to generate 42, followed by 1-(3-iodopropyl)-4-
methyl-2,6,7-trioxabicyclo[2.2.2]octane (43) in THF providing 44 in
14% yield, which was converted to the target compound 14∙HCl by
heating in aqueous HCl (53%, Scheme 3).
The preparation of the test compounds 15 and 18 exhibiting a C-
5 carboxylic acid side chain was realized starting from 3-(1-tri-
phenylmethyl-1H-imidazol-2-yl)propanal (45) [37,40]. Upon
subjection of 45 to a HornereWadswortheEmmons reaction, the
unsaturated ester 46 was obtained. Hydrolysis of 46 carried out by
refluxing in 2 M HCl yielded the free, unsaturated amino acid
18∙HCl (70%) which upon catalytic hydrogenation over 5% PdeC in
MeOH provided the saturated 5-(1H-imidazol-2-yl)pentanoic acid
hydrochloride 15∙HCl (74%).
2.2.
u-Imidazole-4-yl alkanoic and alkenoic acids
In this series of compounds with the carboxylic acid side chain
originating from the 4-position of the imidazole ring, 1H-imidazol-
4-ylcarboxylic acid (19), 1H-imidazol-4-ylacetic acid (20), and (2E)-
3-(1H-imidazol-4-yl)acrylic acid (24) were commercially available.
3-(1H-imidazol-4-yl)propanoic acid (21) [41] and 4-(1H-imidazol-
4-yl)butanoic acid (22) [42e45] were synthesized according to
literature procedures.
For the synthesis of 5-(1H-imidazol-4-yl)pentanoic acid hydro-
chloride (23$HCl), a strategy analogous to that employed in the prep-
aration of 5-(1H-imidazol-2-yl)pentanoic acid hydrochloride (15$HCl)
was used. Thus, 3-(1-triphenylmehtyl-1H-imidazol-4-yl)propanal (47)
prepared from the N-protected 3-(1H-imidazole-4-yl)propanoic acid
methyl ester according to a literature procedure [46] was transformed
into (2E)-5-(1-triphenylmethyl-1H-imidazol-4-yl)pent-2-enoic acid
O
I
O
O
N
N
CH3
43
1.0 equiv. nBuLi
Li
N
Tr
N
Tr
THF, -15 °C
THF,
20 min
-10 °C -> 10 °C
17 h
14%
42
41
CH3
.
HCl
O
N
O
COOH
1 M HCl
N
O
N
H
Δ, 4 h
53%
N
Tr
44
14·HCl
Scheme 3. Synthesis of 14∙HCl (Tr ¼ Triphenylmethyl).

S. Hack et al. / European Journal of Medicinal Chemistry 46 (2011) 1483e1498
1487
O
P
O
EtO
EtO
OEt
.
N
OEt
HCl
O
NaH
N
N
2 M HCl
COOH
N
Tr
DME
50 °C, 3 h
96%
Δ, 4 h
70%
H
O
N
Tr
N
H
45
18·HCl
46
Pd-C, H2
MeOH
rt, 18 h
74%
.
HCl
N
COOH
N
H
15·HCl
Scheme 4. Synthesis of 15∙HCl and 18∙HCl (Tr ¼ Triphenylmethyl).
ethyl ester (48) via a HornereWadswortheEmmons reaction. Subse-
quent deprotection of the imidazole moiety and hydrolysis of the ethyl
ester group by heating 48 in a mixture of 2 M HCl and EtOH to reflux
yielded the free amino acid 25$HCl. Finally, the desired saturated
amino acid 23$HCl was obtained by catalytic hydrogenation over 5%
PdeC in MeOH in 72% yield (Scheme 5).
transporters, i.e. mGAT1, mGAT2, mGAT3, and mGAT4. These [3H]
GABA uptake assays were performed in a uniform manner using
HEK cell lines, each stably expressing one of the four GABA trans-
porter subtypes [30]. The potency of the tested compounds to
inhibit [3H]GABA uptake is given as pIC50 value. For test compounds
of low potency, unable to reduce the specific [3H]GABA uptake to
a value below 50% at a specific concentration (100
mM, 1 mM,
10 mM), no pIC50 values were determined. Instead, for these
compounds, the percentage of the remaining specific [3H]GABA
uptake at the specified concentration is given.
2.3.
u-Imidazole-1-yl alkanoic acids
Compounds 27 [47] and 28 [48] belonging to the third series of
In addition to the three series of 1H-imidazole derivatives with
different carboxylic acid side chains, a related series of homologous
u
-imidazole alkanoic acids with the alkanoic acid side chain orig-
inating from the 1-position of the imidazole ring were prepared
according to literature procedures. The syntheses of the remaining
u
-aminoalkanoic acids with chain length ranging from two to
seven carbons including GABA (1) was evaluated for their potencies
at the different GABA transporters. The data obtained for these
reference compounds are listed in Table 2, those for the imidazole
derivatives in Tables 3, 5, and 7.
u
-imidazole-1-yl alkanoic acids, compounds 26 as well as 29e31
and 32, were performed according or rather analogue to modified
literature procedures [49e51].
3. Results and discussion
3.1.1.
u
-Imidazole-2-yl alkanoic and alkenoic acids
-imidazole-2-yl alkanoic
For better legibility the potencies of
u
3.1. Biological evaluation
acids are, in addition, presented in a graphical manner as function
of the chain length (Fig. 4). As can be seen from Fig. 4 the
compounds are most potent at mGAT3 with 3-(1H-imidazol-2-yl)
propanoic acid hydrochloride (13$HCl) exhibiting an alkyl chain
The compounds of all three series of different imidazole alka-
noic and alkenoic acids were evaluated in [3H]GABA uptake assays
for their inhibitory potency at the four subtypes of GABA
O
P
O
EtO
EtO
O
O
OEt
N
N
N
NaH
H
2 M HCl
EtO
DME
50°C, 3 h
79%
EtOH
Δ, 4 h
88%
N
Tr
Tr
48
47
. HCl
HCl
.
HOOC
HOOC
N
N
Pd-C, H2
MeOH
rt, 18 h
N
H
N
H
25·HCl
72%
23·HCl
Scheme 5. Synthesis of 23$HCl and 25$HCl (Tr ¼ Triphenylmethyl).

1488
S. Hack et al. / European Journal of Medicinal Chemistry 46 (2011) 1483e1498
Table 2
pIC50 values of
u-aminoalkanoic acids at mGAT1emGAT4.
Compound
Gylcine
No.
mGAT1
mGAT2
mGAT3
mGAT4
49
50
1
51
52
53
59.8%a
2.59 ꢁ 0.03
5.14 ꢁ 0.09
4.13 ꢁ 0.09
80.8%
2.65 ꢁ 0.10
3.48 ꢁ 0.10
4.56 ꢁ 0.06
3.87 ꢁ 0.02
55.8%
2.87, 2.85
4.66 ꢁ 0.06
5.09 ꢁ 0.23
3.72 ꢁ 0.05
70.9%
2.51 ꢁ 0.12
4.46 ꢁ 0.13
5.18 ꢁ 0.13
3.30 ꢁ 0.04
65.1%
b
-Alanine
GABA
-Aminovalerianic acid
d
e
z
-Aminohexanoic acid
-Aminoheptanoic acid
107.0%
61.8%
96.4%
84.3%
If not stated otherwise the results are given as means of pIC50-values ꢁ SEM of three independent experiments each performed in triplicate, percentages represent specific
binding remaining in presence of 1 mM.
a
10 mM inhibitor.
length of three C-atoms inhibiting mGAT3 best. According to Fig. 4
(and Table 3), 13$HCl exhibits not only the highest affinity of the
tested compounds to the mGAT3 transporter with a pIC50 value of
4.54, but also good subtype selectivity. Notably, the efficacy of
compound 13$HCl at mGAT3 compared to mGAT4 is 10, compared
to mGAT2 18 and compared to mGAT1 even ꢃ34 times higher
(Table 4). Both, elongation and reduction of the chain length cause
lower inhibitory potencies (see Fig. 4). For example, 2-(1H-imida-
zol-2-yl)acetic acid hydrochloride (12$HCl) possesses lower inhib-
itory potency at mGAT3, but the potency remains still detectable as
does the subtype selectivity. Further shortening leads to 1H-
imidazol-2-carboxylic acid hydrochloride (11$HCl), which is then
devoid of any significant inhibitory potency (pIC50 3.0).
ꢄ
The obtained biological data demonstrate also the influence of the
presence of a double bond on the potency of the compounds. The
unsaturated, E-configured (2E)-3-(1H-imidazol-2-yl)acrylic acid
hydrochloride (16$HCl) possesses the same number of C-atoms as
3-(1H-imidazol-2-yl)propanoic acid hydrochloride (13$HCl), but
exhibits lower potency at mGAT3, though this is still significant (see
Table 4). In contrast, the Z-isomer (2Z)-3-(1H-imidazol-2-yl)acrylic
acid hydrochloride (17$HCl) is lacking any reasonable affinity at
mGAT3, but also at any other GABA transporter subtype. When the
Fig. 4. Potencies of imidazole-2-yl-compounds. (Lines are drawn between the data points only for better legibility and do not represent mathematical relations.)
Table 3
pIC50 values of
u-imidazole-2-yl alkanoic and alkenoic acids at mGAT1emGAT4.
Compound
No.
mGAT1
mGAT2
mGAT3
mGAT4
N
R
N
H
R =
COOH
CH2COOH
(CH2)2COOH
(CH2)3COOH
(CH2)4COOH
11$HCl
12$HCl
13$HCl
14$HCl
15$HCl
62.4%
75.3%
62.8%
62.3%
75.5%
63.8%
70.2%
98.6%
63.4%
3.51 ꢁ 0.03
3.29 ꢁ 0.10
3.36 ꢁ 0.03
3.86 ꢁ 0.12
4.54 ꢁ 0.15
3.71 ꢁ 0.04
3.49 ꢁ 0.06
3.28 ꢁ 0.19
53.4%
3.14 ꢁ 0.03
57.8%
CH=CHCOOH
16$HCl
17$HCl
73.3%
77.3%
92.2%
3.71 ꢁ 0.05
47.2%
(E)
CH=CHCOOH
102%
79.1%
93.6%
(Z)
3.33, 3.49a
3.20 ꢁ 0.13
53.5%
3.51 ꢁ 0.18
(CH2)2CH=CHCOOH
18$HCl
(E)
If not stated otherwise the results are given as means of pIC50-values ꢁ SEM of three independent experiments each performed in triplicate, percentages represent specific
binding remaining in presence of 1 mM inhibitor.
a
As only two independent experiments were performed, the results of the GABA uptake are given instead of the means of pIC50-values ꢁ SEM.

S. Hack et al. / European Journal of Medicinal Chemistry 46 (2011) 1483e1498
1489
Table 4
differences lack statistical significance (see Table 5). Notably, as
mentioned in the introduction, 20, starting point of our study,
shows, good mGAT3 selectivity compared to mGAT1 (35:1), but
compared to mGAT2 as well as mGAT4 the selectivity is distinctly
less pronounced (Table 6). The homologous compound with a side
chain of three C-atoms, 21, achieves more positive results, as the
mGAT3 selectivity against mGAT1, mGAT2, and mGAT4 is higher in
each case (see Table 6). But, this is not the case for the butanoic acid
derivative 22$HCl as compound 22 exhibits lower mGAT3 selec-
tivity in relation to mGAT1, mGAT2, and mGAT4 as compared to the
acetic acid derivative 20. Even more, compound 22$HCl lacks any
selectivity for mGAT3 in relation to mGAT2, the differences
between the pIC50 values for these transporters are missing
statistical significance.
Subtype selectivity and pIC50 values (mean ꢁ SEM, n ¼ 3) at mGAT3.
Compound pIC50
no.
Subtype selectivity
mGAT3:mGAT1 mGAT3:mGAT2 mGAT3:mGAT4
12$HCl
13$HCl
16$HCl
3.86 ꢁ 0.12
ꢃ7:1a
ꢃ34:1a
ꢃ7:1a
ꢃ7:1a
ꢃ7:1a
4.54 ꢁ 0.15
3.71 ꢁ 0.05
18:1
10:1
ꢃ7:1a
ꢃ7:1a
For compounds reducing [3H]GABA uptake to not less than 50% at 1 mM
a
a pIC50 ꢄ 3.0 is assumed.
chain length reaches five C-atoms, the saturated as well as the
unsaturated compounds, 15$HCl and 18$HCl, show neither good
affinities nor subtype selectivities.
Remarkably, the unsaturated analogue of 3-(1H-imidazol-4-yl)
propanoic acid (21), compound (2E)-3-(1H-imidazol-4-yl)acrylic acid
(24), is, in contrast to 21, devoid of any significant inhibitory potency
(pIC50 ꢄ 3.0, see Table 5). 5-(1H-Imidazol-4-yl)pentanoic acid hydro-
chloride (23$HCl) and (2E)-5-(1H-imidazol-4-yl)pent-2-enoic acid
hydrochloride (25$HCl) with further elongated side chain (five C-
atoms)exhibitonlylowaffinity tomGAT3andmisssubtypeselectivity.
The reduction of the chain length to one C-atom (1H-imidazol-4-
ylcarboxylic acid (19)) is accompanied with an even more serious loss
of potency, being now almost neglectable (pIC50 ꢄ 3.0, see Table 5).
3.1.2.
u-Imidazole-4-yl alkanoic and alkenoic acids
As shown in Fig. 5, wherein the potencies of
u
-imidazole-4-yl
alkanoic acids are presented in a graphical manner as function of
the chain length, also for this series of compounds the highest
potencies are observed for mGAT3 with 1H-imidazol-4-ylacetic
acid (20), 3-(1H-imidazol-4-yl)propanoic acid (21) and 4-(1H-imi-
dazol-4-yl)butanoic acid hydrochloride (22$HCl) representing the
most potent mGAT3 inhibitors. For these compounds the pIC50
values are declining with extension of the side chain from two to
four carbons from 4.76 to 4.60, but only formally, as the observed
Fig. 5. Potencies of
u-imidazole-4-yl alkanoic acids. (Lines are drawn between the data points only for better legibility and do not represent mathematical relations.)
Table 5
pIC50 values of test compounds at mGAT1emGAT4.
Compound
No.
mGAT1
mGAT2
mGAT3
mGAT4
H
N
N
R
R =
COOH
19
90.8%
91.1%
67.5%
85.5%
CH2COOH
(CH2)2COOH
(CH2)3COOH
(CH2)4COOH
20
21
22$HCl
23$HCl
3.21 ꢁ 0.12
3.99 ꢁ 0.05
3.19 ꢁ 0.06
4.56 ꢁ 0.08
3.61 ꢁ 0.03
4.76 ꢁ 0.08
4.64 ꢁ 0.15
4.60 ꢁ 0.17
3.44 ꢁ 0.09
4.33 ꢁ 0.01
4.09 ꢁ 0.15
4.28 ꢁ 0.08
64.7%
61.7%
3.38 ꢁ 0.14
3.38 ꢁ 0.07
24
54.8%a
66.6%a
105%a
95.8%a
CH=CHCOOH
(E)
(CH2)2CH=CHCOOH
25$HCl
3.91 ꢁ 01
3.42 ꢁ 0.14
3.60 ꢁ 0.05
3.68 ꢁ 0.04
(E)
If not stated otherwise the results are given as means of pIC50-values ꢁ SEM of three independent experiments each performed in triplicate, percentages represent specific
binding remaining in presence of 1 mM.
a
10 mM inhibitor.

1490
S. Hack et al. / European Journal of Medicinal Chemistry 46 (2011) 1483e1498
Table 6
Table 8
Subtype selectivity and pIC50 values (mean ꢁ SEM, n ¼ 3) at mGAT3.
Subtype selectivity and pIC50 (mean ꢁ SEM, n ¼ 3) at mGAT3.
Compound no. pIC50
Subtype selectivity
Compound pIC50
no.
Subtype selectivity
mGAT3:mGAT1 mGAT3:mGAT2 mGAT3:mGAT4
mGAT3:mGAT1 mGAT3:mGAT2 mGAT3:mGAT4
20
21
22$HCl
4.76 ꢁ 0.08
35:1
ꢃ44:1a
16:1
6:1
29:1
1:1
3:1
4:1
2:1
26
27
28
4.37 ꢁ 0.10
ꢃ23:1a
ꢃ48:1a
3:1
5:1
19:1
2:1
14:1
7:1
1:1
4.64 ꢁ 0.15
4.59 ꢁ 0.02
4.60 ꢁ 0.17
4.40 ꢁ 0.07
a
a
For compounds reducing [3H]GABA uptake to not less than 50% at 1 mM
For compounds reducing [3H]GABA uptake to not less than 50% at 1 mM
a pIC50 ꢄ 3.0 is assumed.
a pIC50 ꢄ 3.0 is assumed.
Imidazol-1-ylacetic acid (26) possessing one C-atom less than 3-
(1H-imidazol-1-yl)propanoic acid (27) shows slightly lower
potency at mGAT3, but still high mGAT3 subtype selectivity
compared to mGAT1 (ꢃ23:1) and mGAT4 (14:1) (Table 8). By
reaching an alkyl chain length of four, the mGAT3 selectivity
compared to the other three transporters is reduced. Thus, 4-(1H-
imidazol-1-yl)butanoic acid (28) having one C-atom more than 27,
shows slightly lower potency at mGAT3 (pIC50: 4.40 ꢁ 0.07) with
distinctly diminished subtype selectivity as compared to the
latter compound (see Table 8). Further elongation of the chain
length (compounds 29e32) causes reduced mGAT3-affinity in
combination with very low or even vanished subtype selectivity
(Fig. 7 and 8).
3.1.3.
u-Imidazole-1-yl alkanoic acids
Table 7 presents the pIC50 values of
u
-imidazol-1-yl alkanoic
acids which are, in addition, displayed in Fig. 6 in a graphical
manner as function of the chain length of the alkanoic acid residue.
As 1H-imidazole-1-carboxylic acid is unstable, the series of
compounds studied starts with 1H-imidazol-1-ylacetic acid (26)
having a chain length of two C-atoms. According to Fig. 6,
u-imi-
dazol-1-yl alkanoic acids having a chain length of two and three C-
atoms, respectively, exhibit higher affinity to the mGAT3 trans-
porter than to other subtypes, especially to mGAT1. 3-(1H-Imida-
zol-1-yl)propanoic acid (27) (three C-atoms) displays high potency
at mGAT3 as well as high mGAT3 selectivity compared to mGAT1
(ꢃ48:1) and good mGAT3 selectivity compared to mGAT2 (19:1)
(Table 8). Only the mGAT3:mGAT4 selectivity is moderate (7:1). 1H-
3.2. Synthesis and biological evaluation of bicyclic systems
Table 7
Most of the imidazole derivatives described above are
predominantly active as mGAT3 inhibitors. Among them, 1H-imi-
dazol-4-ylacetic acid (20) and its homologue 3-(1H-imidazol-4-yl)
propanoic acid (21) exhibit the highest potencies at mGAT3, iden-
tical within the limits of error. But 21 clearly surpasses the imid-
azole derivative 20 with a shorter side chain with respect to its
subtype selectivity in favour of mGAT3 compared to mGAT1 and
mGAT2. For this reason the conformationally restrained 4,5,6,7-
tetrahydro-1H-benzimidazol-4-carboxylic acid (54) and 4,5,6,7-
tetrahydro-1H-benzimidazol-5-carboxylic acid (55) were included
in this study as cyclic analogues of 20 and of 21, with the aim to
improve subtype selectivity.
pIC50 values of test compounds at mGAT1emGAT4.
Compound
No.
mGAT1
mGAT2
mGAT3
mGAT4
N
N
R
R =
CH2COOH
(CH2)2COOH 27
(CH2)3COOH 28
26
73.3%
72.0%
3.70 ꢁ 0.05 4.37 ꢁ 0.10 3.21 ꢁ 0.11
3.30 ꢁ 0.15 4.59 ꢁ 0.02 3.72 ꢁ 0.09
3.93 ꢁ 0.11 4.12 ꢁ 0.14 4.40 ꢁ 0.07 4.42 ꢁ 0.02
(CH2)4COOH 29$HCl 3.94 ꢁ 0.04 3.65 ꢁ 0.11 3.79 ꢁ 0.03 3.65 ꢁ 0.10
(CH2)5COOH 30$HCl 2.99 ꢁ 0.10 3.79 ꢁ 0.07 3.63 ꢁ 0.04 3.63 ꢁ 0.07
(CH2)6COOH 31$HCl 3.17 ꢁ 0.10 56.6%
3.32, 3.11a
56.2%
71.1%
58.6%
3.2.1. Synthesis of bicyclic systems
(CH2)7COOH 32$HCl 68.3%
79.3%
The synthesis of the ethyl ester derivative of the bicyclic 4,5,6,7-
tetrahydro-1H-benzimidazol-4-carboxylic acid (54) (see Fig. 7) was
performed according to methods described in literature [52]. The
subsequent hydrolysis of the ester function carried out in 1 M HCl at
100 ꢂC (3 h) afforded the amino acid 54∙HCl in 90% yield.
If not stated otherwise the results are given as means of pIC50-values ꢁ SEM of three
independent experiments each performed in triplicate, percentages represent
specific binding remaining in presence of 1 mM inhibitor.
As only two independent experiments were performed, the results of the GABA
uptake are given instead of the means of pIC50-values ꢁ SEM.
a
Fig. 6. Potencies of imidazole-1-yl derivatives. (Lines are drawn between the data points only for better legibility and do not represent mathematical relations.)

S. Hack et al. / European Journal of Medicinal Chemistry 46 (2011) 1483e1498
1491
rahydro-1H-benzimidazol-5-carboxylate (59). As attempts to purify
59 by CC remained unsuccessful, the reaction product containing
compound 59 was treated with sodium hydride and di-tert-butyl
dicarbonate to substitute the NH moiety of the imidazole ring which
was considered to be the origin of the encountered problem. The
resulting, 1:1 mixture of the two N-substituted regioisomers 57 and
58, could then, indeed, be easily purified by CC. Deprotection was
accomplished by treating the obtained 1:1 mixture of 57 and 58 using
trifluoroacetic acid in ethanol at room temperature providing pure
ethyl 4,5,6,7-tetrahydro-1H-benzimidazol-5-carboxylate (59) in high
yield (94%). The target compound 4,5,6,7-tetrahydro-1H-benzimid-
azole-5-carboxylic acid hydrochloride (55∙HCl) was finally obtained
in90% yield, afterheating 59inaqueous HCl to 100 ꢂC to hydrolysethe
ester function (Scheme 6).
COOH
HOOC
N
N
N
H
N
H
54
55
Fig. 7. Bicyclic compounds.
The synthesis of 4,5,6,7-tetrahydro-1H-benzimidazol-5-carbox-
ylic acid (55) was performed in analogy to the preparation of 54. The
required starting material ethyl 3-bromo-4-oxocyclohexanecarbox-
ylate (56) was prepared as described by Berger et al. [53]. Reaction of
56 with formamide at 150 ꢂC for 1.5 h yielded the ethyl 4,5,6,7-tet-
Fig. 8. Summary of the potencies of tested imidazole compounds at mGAT3. (Lines are drawn between the data points only for better legibility and do not represent mathematical
relations.)
1. HCONH2
O
O
O
Boc
150 °C, 1.5 h
Br
O
N
N
2. NaH, Boc2O
F3CCOOH
H3C
O
H3C
O
H3C
O
+
EtOH, rt, 4 h
94%
THF, 0 °C -> rt
44%
N
N
Boc
57
58
56
O
HCl
·
HOOC
N
N
H3C
O
1 M HCl
100 °C, 3 h
N
H
N
H
90%
59
55·HCl
Scheme 6. Synthesis of 55$HCl.

1492
S. Hack et al. / European Journal of Medicinal Chemistry 46 (2011) 1483e1498
3.2.2. Biological evaluation of bicyclic systems
alkenoic acid derivatives inhibited GABA uptake with the highest
potency at mGAT3.
In addition to 54∙HCl and 55∙HCl, two commercially available
substances, 1H-benzimidazol-4-carboxylic acid hydrochloride
(60$HCl) and 1H-benzimidazol-5-carboxylic acid (61) were evalu-
ated for their inhibitory potency at mGAT1, mGAT2, mGAT3, and
mGAT4. The results obtained in the biological test are illustrated in
Table 9.
Though the
u-imidazole alkanoic and alkenoic acid derivatives
studied do not reach the affinity of GABA (1), compounds exhibiting
good mGAT3 inhibitory potency in combination with good subtype
selectivity, in contrast to GABA (1), were found. Of all synthesized
substances, 3-(1H-imidazol-2-yl)propanoic acid hydrochloride
(13$HCl), 1H-imidazol-4-ylacetic acid (20), 3-(1H-imidazol-4-yl)
propanoic acid (21), and 3-(1H-imidazole-1-yl)propanoic acid (27)
exhibited the best inhibitory potency at mGAT3 associated with
a preference for this transporter (see Fig. 8).
Table 9
pIC50 values of
u-aminoalkanoic acids at mGAT1emGAT4.
Within a given series of
u-imidazole alkanoic acids elongation
Compound
No.
mGAT1
mGAT2
mGAT3
78.2%a
mGAT4
68.2%a
or reduction of the length of the side chain resulted in less potent or
selective compounds. Furthermore, integration of the alkyl side
chain into a bicyclic ring system led to ineffective compounds.
Although the starting compound 1H-imidazol-4-ylacetic acid
(20) (pIC50 mGAT1: 3.21 ꢁ 0.12; pIC50 mGAT2: 3.99 ꢁ 0.05; pIC50
mGAT3: 4.76 ꢁ 0.08; pIC50 mGAT4: 4.33 ꢁ 0.01) as well as the
chain-extended analogue 3-(1H-imidazol-4-yl)propanoic acid (21)
(pIC50 mGAT1: 61.7% 1 mM; pIC50 mGAT2: 3.19 ꢁ 0.06; pIC50 mGAT3:
4.64 ꢁ 0.15; pIC50 mGAT4: 4.09 ꢁ 0.15) showed great inhibitory
potency at mGAT3 in combination with a better mGAT3 selectivity,
especiallycompared to mGAT1 and, for compound 21, also compared
to mGAT2 than the amino acid GABA (1), the preference compared to
mGAT4 still remains low (see Table 10). 3-(1H-imidazol-2-yl)prop-
anoicacid(13)(mGAT1:62.8%1mM;pIC50 mGAT2:3.28ꢁ 0.19;pIC50
mGAT3: 4.54 ꢁ 0.15; pIC50 mGAT4: 3.51 ꢁ0.03) and 3-(1H-imidazol-
1-yl)propanoic acid (27) (mGAT1: 72.0% 1 mM; pIC50 mGAT2:
3.30 ꢁ 0.15; pIC50 mGAT3: 4.59 ꢁ 0.02; pIC50 mGAT4: 3.72 ꢁ 0.09)
possesswithinthelimitsoferroridentical orslightlylowerinhibitory
potency than 1H-imidazol-4-ylacetic acid (20). However, while the
mGAT3 selectivity of compounds 13$HCl and 27 compared to mGAT1
COOH
N
60$HCl
95.9%a
94.2%a
N
H
HOOC
N
61
108%a
59.0%
74.2%a
60.8%
103%a
97.6%
67.8%a
81.6%
N
H
COOH
N
54$HCl
N
H
HOOC
N
55$HCl
85.0%
65.4%
81.5%
82.4%
N
H
If not stated otherwise the results are given as means of pIC50-values ꢁ SEM of three
independent experiments each performed in triplicate, percentages represent
is similar to that of the u-imidazole-2-yl alkanoic acids 20 and 21, the
specific binding remaining in presence of 1 mM.
a
mGAT3:mGAT2 preference is, compared to the regio isomer 21,
reduced, but still considerably better than that of the starting
compound 20. Especially worth-mentioning is the improved
mGAT3:mGAT4 selectivity, which is 7:1 and 10:1, respectively, and
thus better than that of compounds 20 and 21.
Up to date, only few selective inhibitors of mGAT3 are published.
In this study two compounds, 3-(1H-imidazol-2-yl)propanoic
acid$hydrochloride (13$HCl) and 3-(1H-imidazol-1-yl)propanoic
acid (27), were characterized, which might represent, due to their
good inhibitory potencies at mGAT3 in combination with an, even
for mGAT4, improved subtype selectivity, good starting points for
the development of GABA uptake inhibitors of mGAT3 with high
potency and subtype selectivity.
100 mM inhibitor.
As shown in Table 9, the bicyclic compounds 4,5,6,7-tetrahydro-
1H-benzimidazol-4-carboxylic acid hydrochloride (54$HCl) and
4,5,6,7-tetrahydro-1H-benzimidazol-5-carboxylic acid hydrochloride
(55$HCl) are devoid of any reasonable potency at any of the four
GABA transport proteins. This applies to the fully unsaturated bicyclic
systems as well, the inhibitory potency of 1H-benzimidazol-4-
carboxylic acid hydrochloride (60$HCl) and 1H-benzimidazol-5-
carboxylic acid (61) is very low or even negligible.
4. Conclusion
5. Experimental
In summary, new series of GABA uptake inhibitors based on
the structure of 1H-imidazol-4-ylacetic acid (20) modified by
changing the length and the position of the side chain have been
synthesized and tested on stably expressed mGAT1emGAT4 in
5.1. Materials and methods
Solvents were p.a. quality and freshly distilled before use.
Purchased reagents were used without further purification. TLC
plates were made from silica gel 60 F254 on aluminium sheet
(Merck). Column chromatography (CC) was carried out using Merck
silica gel 60 (mesh 0.040e0.063 mm) as stationary phase. As strong
basic ion exchange resin Amberlite IRA 410, 20e50 mesh (Fa. Fluka)
and as strong acidic ion exchange resin Amberlite IR 120 were used.
Melting points: m.p. (uncorrected) were determined with a Büchi
512 Melting Point apparatus. NMR spectroscopy: 1H NMR spectra
were recorded at rt with a JNMR-GX (JEOL, 400 or 500 MHz) using
TMS as internal standard and integrated with the NMR software
Nuts (2D Version 5.097, Acorn NMR, 1995). IR spectroscopy: FT-IR
Spectrometer 410 (Jasco); samples were measured as KBr-pellets.
Mass spectrometry (MS): Mass Spectrometer 5989 A with 59980 B
HEK cells. Most of the synthesized
u-imidazole alkanoic and
Table 10
Subtype selectivity and pIC50 values (mean ꢁ SEM, n ¼ 3) at mGAT3.
Compound no. pIC50
Subtype selectivity
mGAT3:mGAT1 mGAT3:mGAT2 mGAT3:mGAT4
1
5.14 ꢁ 0.09
1:1
3:1
18:1
6:1
1:1
10:1
3:1
13$HCl
20
21
4.54 ꢁ 0.15
4.76 ꢁ 0.08
4.64 ꢁ 0.15
4.59 ꢁ 0.02
ꢃ34:1a
35:1
ꢃ44:1a
29:1
19:1
4:1
7:1
27
ꢃ48:1a
a
For compounds reducing [3H]GABA uptake to not less than 50% at 1 mM
a pIC50 ꢄ 3.0 is assumed.

S. Hack et al. / European Journal of Medicinal Chemistry 46 (2011) 1483e1498
1493
particle beam LC/MS interface (Hewlett Packard) or Applied Bio-
systems LC-MS/MS-Mass Spectrometer API 2000; analysis was
carried out using chemical ionization (CHþ5 ) or electron impact ioni-
zation. High resolution mass spectrometry (HRMS): JEOL MS-Station
JMS-700, FAB (Xenon, 6 KV, MBA, reference PEG), LTQ FT (Thermo
Finnigan).Elementaryanalysis:ElementaranalysatorRapid(Heraeus).
1H NMR (CD3OD):
CH[4H-Imid.]); 126 MHz 13C NMR (CD3OD):
(CH[5C-Imid.], CH[4C-Imid.]), 142.6 (C[2C-Imid.]), 169.1 (COO); MS (EI,
70 eV): m/z (%) ¼ 126 (5) [Mþ], 82 (100); MS (CI, CHþ5 ): m/z (%) ¼ 127
(13) [Mþþ1], 83 (100); HMRS (FAB, NBA) calcd. for C5H7N2O2:
127.0508; found 127.0483.
d
¼ 4.19 (s, 2H, CH2), 7.25 (s, 2H, CH[5H-Imid.]
,
d
¼ 32.1 (CH2), 120.4
5.1.1. General procedure 1 (GP1)
5.1.9. 3-(1H-Imidazol-2-yl)propanoic acid hydrochloride(13∙HCl)
To 3-(1H-imidazol-2-yl)propanoic acid ethyl ester [37] (68 mg,
0.40 mmol) in EtOH (0.5 mL) NaOH (2 M, 1.5 mmol, 0.75 mL) was
added. The mixture was stirred for 48 h at rt before a pH-value of
1e2 with HCl (0.5 M) was adjusted. After concentration, the crude
product was purified using a strong basic ion exchange resin.
PdeC (10%, 40.0 mg) was added to the alkenoic acid (1.0 equiv)
solved in MeOH. After hydrogenation for 18 h at rt and room
pressure, the catalyst was removed and the filtrate was evaporated
and dried with MgSO4. Purification was realized using a strong
basic ion exchange resin.
Colourless crystals; Yield: 61 mg (86%); mp 221 ꢂC. IR (KBr)
cmꢀ1: 3085, 2968, 2727,1725,1619,1507,1429,1212,1188; 500 MHz
1H NMR (CD3OD):
¼ 3.21 (m, 2H, CH2COO), 3.30 (m, 2H,
CH2CH2COO), 7.41 (s, 2H, CH[4H-Imid.], CH[5H-Imid.]); 126 MHz 13C
NMR (CD3OD):
¼ 23.6 (CH2CH2COO), 31.0 (CH2CH2COO),119.0 (CH
n
5.1.2. General procedure 2 (GP2)
The imidazole derivative (1.0 equiv) was dissolved in HCl (1 M).
After 4 h reflux, the reaction solution was washed three times with
CH2Cl2. The combined organic layers were extracted with H2O.
Then, the combined aqueous layers were concentrated.
d
d
[
4C-Imid.], CH[5C-Imid.]), 147.7 (C[2C-Imid.]), 173.7 (COO); MS (EI, 70 eV):
m/z (%) ¼ 140 (16) [Mþ], 95 (100); MS (CI, CHþ5 ): m/z (%) ¼ 141 (100)
5.1.3. General procedure 3 (GP3)
[Mþþ1]; HMRS (EI) calcd. for C6H8N2O2: 140.0586; found 140.0583.
At 0 ꢂC triethylphosphonoacetate (1.3 equiv) was treated with
NaH (1.0 equiv) suspended in DME. The reaction solution was
warmed up to rt and subsequently stirred for the given time. The
imidazole derivative (1.0 equiv) solved in DME was added. The
reaction solution was warmed up to the given temperature and
stirring was continued for the given time. Having evaporated the
solvent the residue was purified by CC (EtOAc/n-Pentane ¼ 1/1).
5.1.10. 4-(1H-Imidazol-2-yl)butyric acid hydrochloride (14∙HCl)
1-Triphenylmethyl-2-[3-(4-methyl-2,6,7-trioxabicyclo[2.2.2]
octan-1-yl)propyl]-1H-imidazole (44) (150 mg, 0.312 mmol) was
stirred in methanolic H2SO4 (1.1 mol, 0.30 mL) for 20 min at rt.
After removing the solvent in vacuum, the residue was dissolved
in HCl (2 M, 6 mmol, 3 mL) and refluxed for 4 h. The aqueous
phase was washed three times with CH2Cl2 followed by con-
centration. Purification was achieved with a strong basic ion
exchange resin.
5.1.4. General procedure 4 (GP4)
Imidazole (2.0 equiv) in acetone was treated with K2CO3
(6.0 equiv), NaI (40 mg, 0.27 mmol) and the respective bro-
moalkanoic acid methyl ester (1.0 equiv) or bromoalkanenitrile
(1.0 equiv). After the given time at rt, the reaction mixture was
filtered, evaporated, and purified by CC.
Colourless oil; Yield: 31.3 mg (52.6%); IR (KBr)
3106, 2983, 1715, 1621, 1416, 1198; 500 MHz 1H NMR (CD3OD):
¼ 2.07 (m, 2H, CH2CH2COO), 2.41 (m, 2H, CH2COO), 3.05 (m, 2H,
CH2CH2CH2COO), 7.43 (s, 2H, CH[4H-Imid.], CH[4H-Imid.]); 100 MHz 13
NMR (CD3OD):
n
cmꢀ1: 3418,
d
C
d
¼ 23.7 (CH2CH2COOH), 26.0 (CH2CH2CH2COOH),
5.1.5. General procedure 5 (GP5)
33.5 (CH2COOH), 120.1 (CH[5C-Imid.], CH[4C-Imid.]), 142.5 (C[2C-Imid.]),
175.8 (COOH); MS (EI, 70 eV): m/z (%) ¼ 154 (4) [Mþ], 95 (100); MS
(CI, CHþ5 ): m/z (%) ¼ 155 (100) [Mþþ1],137 (16); HMRS (EI) calcd. for
C7H10N2O2: 154.0742; found 154.0741. Anal. Calcd. for
C7H10N2O2∙HCl (190.63)þ 0.25H2O (195.13) (%): C 43.09, H 5.94, N
14.35. Found: C 43.46, H 6.36, N 14.77.
The carboxylic acid ester was dissolved in NaOH (1 M) and
stirred for the given time at rt before a pH-value of 6 with HCl
(0.5 M) was adjusted and the solution was concentrated.
5.1.6. General procedure 6 (GP6)
Imidazole (2.0 equiv) in THF was treated with NaH (2.0 equiv).
After 3 h at rt, the respective bromoalkanoic acid ester (1.0 equiv)
was added and stirring was continued for the given time. Then, the
reaction solution was filtered, evaporated, and purified by CC.
5.1.11. 5-(1H-Imidazol-2-yl)pentanoic acid hydrochloride (15∙HCl)
According to GP1: (2E)-5-(1H-imidazol-2-yl)pent-2-enoic acid
hydrochloride (18∙HCl) (79 mg, 0.39 mmol), MeOH (2 mL).
Colourless crystals; Yield: 65.4 mg (73.7%); mp 173 ꢂC. IR (KBr)
5.1.7. General procedure 7 (GP7)
n
cmꢀ1: 3434, 2920, 1700, 1616, 1404, 1209; 400 MHz 1H NMR
The carboxylic acid ethyl ester (1.0 equiv) was dissolved in HCl
(1 M or 2 M) and warmed up to 100 ꢂC for 3 h followed by
concentration.
(D2O):
d
¼ 1.55e1.63 (m, 2H, CH2CH2CH2), 1.73e1.81 (m, 2H,
CH2CH2CH2), 2.38 (t, J ¼ 6.9 Hz, 2H, CH2COO), 3.00 (t, J ¼ 7.4 Hz,
2H, CCH2CH2), 7.30 (s, 2H, CH[5H-Imid.], CH[4H-Imid.]); 100 MHz 13C
NMR (D2O):
d
¼ 23.5 (CH2), 24.9 (CH2), 26.7 (CH2), 33.3 (CH2COO),
5.1.8. 2-(1H-Imidazol-2-yl)acetic acid hydrochloride (12∙HCl)
nBuLi (1.6 M in hexane, 15.8 mmol, 9.80 mL) was added to
118.4 (CH[5C-Imid.], CH[4C-Imid.]), 148.0 (C[2C-Imid.]), 178.5 (COO); MS
(EI, 70 eV): m/z (%) ¼ 168 (7) [Mþ], 95 (100); MS (CI, CH5þ): m/z
(%) ¼ 169 (100) [Mþþ1], 151 (10). HMRS (EI) calcd. for C8H12N2O2:
168.0899; found 168.0899. Anal Calcd. for C8H12N2O2∙HCl
(204.66) þ 0.2H2O (208.26) (%): C 46.14, H 6.49, N 13.45. Found C
46.39, H 6.28, N 13.16.
a
solution of 2-methyl-1-triphenylmethyl-1H-imidazole (33)
(1.62 mg, 15.0 mmol) suspended in THF (25 mL) at ꢀ40 ꢂC. After the
reactionmixturewasstirred for1 h atrt, itwascooledagainto ꢀ40ꢂC
and solid carbon dioxide (5 g) was added. After the mixture was
stirred for 1 h, H2O (1 mL) was added and it was warmed up to rt. The
solvent was evaporated and the residue was dissolved in HCl (0.5 M,
0.4 mol, 200 mL) and refluxed for 1 h. After cooling to rt, the reaction
mixture was washed three times with Et2O and concentrated. The
residue was purified using a strong basic ion exchange resin.
5.1.12. (2E)-3-(1H-Imidazol-2-yl)acrylic acid hydrochloride
(16$HCl)
According to GP2: (2E)-3-(1-Triphenylmethyl-1H-imidazol-2-
yl)acrylic acid ethyl ester (39) (408 mg, 1.00 mmol), HCl (1 M,
10 mmol, 10 mL).
Colourless crystals; Yield: 260 mg (11%); mp 95 ꢂC. IR (KBr)
cmꢀ1: 3386, 3147, 2988, 1727, 1620, 1352, 1290, 1218, 613; 500 MHz
n

1494
S. Hack et al. / European Journal of Medicinal Chemistry 46 (2011) 1483e1498
Colourless crystals; Yield: 175 mg (100%); mp 163 ꢂC. IR (KBr)
cmꢀ1: 2884, 2753, 1714, 1603, 1444, 1375, 1337, 1248, 1192, 1141,
1117; 500 MHz 1H NMR (CD3OD):
n
1.00 mmol) [46], DME (1 mL), 50 ꢂC, 3 h (2E)-5-(1H-Imidazol-4-yl)
pent-2-enoic acid hydrochloride (25∙HCl): According to GP2: (2E)-
5-(1-Triphenylmethyl-1H-imidazol-4-yl)pent-2-enoic acid ethyl
ester (48) (171 mg, 0.350 mmol) solved in HCl (2 M, 4 mmol, 2 mL)
and, differing to GP2, EtOH (0.5 mL).
d
¼ 6.88 (d, J ¼ 16.2 Hz, 1H,
CHCOO), 7.51 (d, J ¼ 16.2 Hz, 1H, CHCHCOO), 6.87 (d, J ¼ 1.2 Hz, 1 H,
CH[5H-Imid.]), 7.69 (d, J ¼ 1.2 Hz, 2 H, CH[4H-Imid.]); 126 MHz 13C NMR
(CD3OD):
d
¼ 122.5 (CH[4C-Imid.], CH[4C-Imid.]),124.8 (CH), 130.0 (CH),
Colourless crystals; Yield: 63 mg (88%); mp 170 ꢂC. IR (KBr)
n
140.8 (C[2C-Imid.]), 167.2 (COO); MS (EI, 70 eV): m/z (%) ¼ 138 (42)
[Mþ], 94 (100); MS (CI, CHþ5 ): m/z (%) ¼ 139 (100) [Mþþ1], 121 (15);
HMRS (EI) calcd. for C5H6N2O2: 138.0421; found 138.0429.
cmꢀ1: 3430, 3090, 3007, 2825, 2604, 2362, 1694, 1646, 1617, 1476,
1416, 1294, 1228, 1181; 500 MHz 1H NMR (CD3OD):
d
¼ 2.61 (m, 2H,
CH2CH), 2.92 (t, J ¼ 7.2 Hz, 2H, CCH2CH2), 5.86 (dt, J ¼ 15.6/1.3 Hz,
1H, CHCOO), 6.93 (dt, J ¼ 15.6/8.6 Hz, 1H, CH2CH), 6.35 (s, 1H, CH[5H-
Imid.]), 8.80 (s, 1H, CH[2H-Imid.]); 100 MHz 13C NMR (CD3OD):
d
¼ 23.3
5.1.13. (2Z)-3-(1H-imidazol-2-yl)acrylic acid hydrochloride
(17$HCl)
(CCH2CH2), 31.0 (CH2CH), 116.5 (CH[5C-Imid.]), 123.6 (CHCOO), 133.7
(CH[2C-Imid.]), 134.2 (C[4C-Imid.]), 147.1 (CH2CH), 168.9 (COO); MS (EI,
70 eV): m/z (%) ¼ 166 (7) [Mþ], 121 (46), 81 (100); MS (CI, CHþ5 ): m/z
(%) ¼ 167 (100) [Mþþ1], 149 (21); HMRS (EI) calcd. for C8H10N2O2:
166.0742; found 166.0770.
According to GP2: (2Z)-3-(1-Triphenylmethyl-1H-imidazol-2-
yl)acrylic acid ethyl ester (40) (408 mg, 1.00 mmol), HCl (1 M,
10 mmol, 10 mL).
Colourless crystals; Yield: 143 mg (82%); mp 182e185 ꢂC. IR
(KBr)
1141, 1117; 500 MHz 1H NMR (CD3OD):
CHCOO), 7.04 (d, J ¼ 12.0 Hz,1H, CHCHCOO), 7.70 (s, 2H, CH[4H-Imid.],
CH[5H-Imid.]); 126 MHz 13C NMR (CD3OD):
¼ 122.2 (CH[4C-Imid.], CH
n
cmꢀ1: 2884, 2753, 1714, 1603, 1444, 1375, 1337, 1248, 1192,
5.1.17. 1H-Imidazol-1-ylacetic acid hydrochloride (26∙HCl)
1H-Imidazol-1-ylacetic acid methyl ester.
Synthesis was performed according to a modified literature
procedure [50].
d
¼ 6.55 (d, J ¼ 12.0 Hz, 1H,
d
[
5C-Imid.]), 123.8 (CH), 128.7 (CH), 140.8 (C[2C-Imid.]), 169.3 (COO); MS
(EI, 70 eV): m/z (%) ¼ 138 (45) [Mþ], 94 (100); MS (CI, CH5þ): m/z
(%) ¼ 139 (100) [Mþþ1], 121 (16); HMRS (ESI) calcd. for C6H7N2O2:
139.0508; found 139.0503.
According to GP4: Imidazole (1.36 g, 20.0 mmol), acetone
(15 mL), K2CO3 (8.29 g, 60.0 mmol), NaI (40 mg, 0.27 mmol), 2-
bromoacetic acid methyl ester (1.53 g, 10.0 mmol), 7 h, CC (CH2Cl2/
C2H5OH ¼ 9/1).
Colourless crystals; Yield: 1.08 g (77%); mp 55 ꢂC. IR (KBr)
n
5.1.14. (2E)-5-(1H-Imidazol-2-yl)pent-2-enoic acid hydrochloride
(18∙HCl)
cmꢀ1: 3386, 3112, 2985, 2956, 1748, 1626, 1511, 1294, 1221;
500 MHz 1H NMR (CDCl3):
¼ 3.72 (s, 3H, COOCH3), 4.65 (s, 2H,
CH2COO), 6.89 (s, 1H, CH[5H-Imid.]), 7.03 (s, 1H, CH[4H-Imid.]), 7.43 (s,
1H, CH[2H-Imid.]); 126 MHz 13C NMR (CD3OD):
d
According to GP2: (2E)-5-(1-Triphenylmethyl-1H-imidazol-2-
yl)pent-2-enoic acid ethyl ester (46) (294 mg, 0.670 mmol), HCl
(1 M, 3 mmol, 3 mL).
d
¼ 53.1 (CH3), 122.1
Colourless crystals; Yield: 95 mg (70%); mp 170 ꢂC. IR (KBr)
n
(CH[5C-Imid.]), 128.6 (CH[4C-Imid.]), 139.5 (CH[2C-Imid.]), 170.1 (COO);
MS (EI, 70 eV): m/z (%) ¼ 140 (18) [Mþ], 81 (100); MS (CI, CH5þ): m/z
(%) ¼ 141 (100) [Mþþ1]; HMRS (EI) calcd. for C6H8N2O2: 140.0586;
found 140.0596.
cmꢀ1: 3431, 2928, 2835, 2365, 1719, 1606, 1580, 1507, 1461, 1295,
1250; 500 MHz 1H NMR (D2O):
¼ 2.60 (m, 2H, CH2CH), 3.07 (t,
J ¼ 7.3 Hz, 2H, CH2CH2), 5.74 (dt, J ¼ 15.7 Hz, 1.4 Hz, 1H, CHCOO),
6.82 (dt, J ¼ 15.7 Hz, 7.0 Hz, 1H, CH2CH), 7.19 (s, 2H, CH[5H-Imid.]
CH[4H-Imid.]); 126 MHz 13C NMR (D2O):
d
NMR, IR and melting point (lit.: 55e56 ꢂC) are according to
literature values [54].
,
d
¼ 24.0 (CH2CH2CH), 29.2
1H-Imidazol-1-ylacetic acid hydrochloride (26∙HCl).
Synthesis was performed analogue to a modified literature
procedure [49].
According to GP5: 1H-Imidazol-1-ylacetic acid methyl ester
(100 mg, 0.700 mmol), NaOH (1 M, 1 mmol, 1 mL).
Colourless crystals; Yield: 114 mg (100%); mp 216 ꢂC (lit.:
(CH2CH2CH), 118.7 (CH[5C-Imid.], CH[4C-Imid.]), 123.0 (CHCOO), 146.7
(CH[2C-Imid.]), 147.3 (CH2CH), 170.4 (COO). MS (EI, 70 eV): m/z
(%) ¼ 166 (5) [Mþ], 121 (100), 81 (86); MS (CI, CHþ5 ): m/z (%) ¼ 167
(100) [Mþþ1], 149 (13); Anal. Calcd. for C8H10N2O2∙HCl
(202.64) þ 0.25H2O (207.14) (%): C 46.39, H 5.60, N 13.52. Found C
46.13, H 5.30, N 13.33.
193e195 ꢂC [55]); IR (KBr)
n
cmꢀ1: 3129, 3067, 2951, 2846, 2360,
2340,1732,1581,1404,1226; 500 MHz 1H NMR (CD3OD):
2H, CH2COOH), 7.60 (s, 1H, CH[5H-Imid.]), 7.65 (s, 1H, CH[4H-Imid.]),
9.00 (s, 1H, CH[2H-Imid.]); 126 MHz 13C NMR (D2O):
d
¼ 5.18 (s,
5.1.15. 5-(1H-Imidazol-4-yl)pentanoic acid hydrochloride (23∙HCl)
According to GP1: (2E)-5-(1H-imidazol-4-yl)pent-2-enoic acid
hydrochloride (25∙HCl) (101 mg, 0.50 mmol), MeOH (3 mL).
d
¼ 50.6 (CH2),
120.7 (CH[5C-Imid.]), 124.8 (CH[4C-Imid.]), 137.9 (CH[2C-Imid.]), 169.2
(COOH); MS (EI, 70 eV): m/z (%) ¼ 126 (80) [Mþ], 81 (100); MS (CI,
CHþ5 ): m/z (%) ¼ 127 (100) [Mþþ1].
Colourless crystals; Yield: 75.4 mg (74%); mp 173 ꢂC. IR (KBr)
n
cmꢀ1: 3462, 3096, 3024, 2865, 2362, 1699, 1624, 1476, 1338, 1269,
1228, 1203; 500 MHz 1H NMR (CD3OD):
d
¼ 1.65e1.73 (m, 4H,
NMR, IR are according to literature values [55].
CH2CH2CH2CH2), 2.35 (t, J ¼ 6.9 Hz, 2H, CH2COO), 2.75 (t,
J ¼ 7.4 Hz, 2H, CCH2CH2), 7.32 (s, 1H, CH[5H-Imid.]), 8.79 (s, 1H,
5.1.18. 5-(1H-Imidazol-1-yl)pentanoic acid (29)
CH[2H-Imid.]); 100 MHz 13C NMR (CD3OD):
d
¼ 24.4 (CCH2), 24.5
5-(1H-Imidazol-1-yl)pentanoic acid ethyl ester.
Synthesis was performed according to a modified literature
procedure [49].
(CH2), 28.3 (CH2), 33.6 (CH2COO), 116.1 (CH[5C-Imid.]), 134.0 (CH[2C-
Imid.]), 134.8 (C[4C-Imid.]), 176.5 (COO); MS (EI, 70 eV): m/z (%) ¼ 168
(2) [Mþ], 151 (3), 95 (100); MS (CI, CHþ5 ): m/z (%) ¼ 169 (100)
[Mþþ1], 151 (16); HMRS (EI) calcd. for C8H12N2O2: 168.0899;
found 168.0872.
According to GP6: Imidazole (1.36 g, 20.0 mmol), THF (20 mL),
NaH (510 mg, 20.0 mmol), 5-bromovalerianic acid ethyl ester
(2.00 g, 10.0 mmol, 1.58 mL), 16 h, CC (CH2Cl2/C2H5OH ¼ 95/5).
Colourless oil; Yield: 542 mg (30%); IR (KBr)
n
cmꢀ1: 3400, 3112,
2940, 2363, 1729, 1509, 1452, 1374, 1230, 1188; 500 MHz 1H NMR
(CD3OD):
5.1.16. (2E)-5-(1H-Imidazol-4-yl)pent-2-enoic acid hydrochloride
(25∙HCl)
(2E)-5-(1-Triphenylmethyl-1H-imidazol-4-yl)pent-2-enoic acid
ethyl ester (48): [60] According to GP3: Triethylphosphonoacetate
(0.29 g,1.3 mmol, 0.26 mL), NaH (26 mg,1.0 mmol), DME (2 mL),1 h,
3-(1-triphenylmethyl-1H-imidazol-2-yl)propanal (47) (367 mg,
d
¼ 1.22 (t, J ¼ 7.2 Hz, 3H, COOCH2CH3), 1.53e1.60 (m, 2H,
CH2), 1.78e1.84 (m, 2H, CH2), 2.34 (t, J ¼ 7.4 Hz, 2H, CH2COO), 4.04
(t, J ¼ 7.1 Hz, 2H, NCH2), 4.10 (q, J ¼ 7.2 Hz, 2H, COOCH2), 6.96 (s, 1H,
CH[5H-Imid.]), 7.13 (s, 1H, CH[4H-Imid.]), 7.65 (s, 1H, CH[2H-Imid.]);
126 MHz 13C NMR (CD3OD):
d
¼ 14.5 (COOCH2CH3), 22.9 (CH2), 31.5

S. Hack et al. / European Journal of Medicinal Chemistry 46 (2011) 1483e1498
1495
(CH2), 34.4 (CH2COO), 47.6 (NCH2), 61.5 (COOCH2), 120.5 (CH[5C-
Imid.]), 129.1 (CH[4C-Imid.]), 138.4 (CH[2C-mid.]), 174.9 (COO); MS (EI,
70 eV): m/z (%) ¼ 196 (13) [Mþ], 152 (35), 96 (100); MS (CI, CHþ5 ): m/
z (%) ¼ 197 (100) [Mþþ1]; HMRS (EI) calcd. for C10H16N2O2:
196.1212; found 196.1207.
d
¼ 17.0 (CH2), 25.1 (CH2), 25.5 (CH2), 28.1 (CH2), 30.5 (CH2CN), 46.7
(NCH2), 118.7 (CN), 119.5 (CH[5C-Imid.]), 129.5 (CH[4C-Imid.]), 137.0 (CH
[
2C-Imid.]); MS (EI, 70 eV): m/z (%) ¼ 177 (16) [Mþ],137 (21), 82 (100);
MS (CI, CHþ5 ): m/z (%) ¼ 178 (100) [Mþþ1]; HMRS (EI) calcd. for
C10H15N3: 177.1266; found 177.1271.
5-(1H-Imidazol-1-yl)pentanoic acid (29)
Synthesis was performed analogue to a modified literature
procedure [49].
NMR and IR are according to literature values [56].
7-(1H-Imidazol-1-yl)heptanoic acid hydrochloride (31∙HCl).
Synthesis was performed analogue to a modified literature
procedure [51].
According to GP5: 5-(1H-Imidazol-1-yl)pentanoic acid ethyl
ester (197 mg,1.00 mmol), NaOH (1 M,1.25 mmol,1.25 mL). Differing
to GP5, EtOH (1 mL) was added. 48 h. After concentration, the crude
product was purified using a strong acidic ion exchange resin.
To 7-(1H-imidazol-1-yl)heptanenitrile (354 mg, 2.00 mmol) in
EtOH (3 mL) was added KOH (10 M, 10 mmol, 1.0 mL). Then, the
reaction mixture was warmed up to 85 ꢂC for 5 h. Having evapo-
rated the solvent, the residue was purified using a strong basic ion
exchange.
Colourless crystals; Yield: 163 mg (97%); mp 164 ꢂC. IR (KBr)
n
cmꢀ1: 3442, 3128, 2952, 2359, 1716, 1516, 1381, 1326, 1283, 1241,
1202, 1098; 500 MHz 1H NMR (CD3OD):
d
¼ 1.54e1.60 (m, 2H, CH2),
Colourless crystals; Yield: 190 mg (41%); mp 132 ꢂC. IR (KBr)
cmꢀ1: 3130, 3062, 2939, 2854, 1718, 1572, 1541, 1402, 1286, 1226,
1179, 1082; 500 MHz 1H NMR (CD3OD):
n
1.81e1.96 (m, 2H, CH2), 2.29 (t, J ¼ 7.3 Hz, 2H, CH2COO), 4.07 (t,
J ¼ 7.1 Hz, 2H, NCH2), 7.03 (s, 1H, CH[5H-Imid.]), 7.20 (s, 1H, CH[4H-
d
¼ 1.34e1.44 (m, 4H, CH2),
Imid.]), 7.82 (s, 1H, CH[2H-Imid.]); 126 MHz 13C NMR (CD3OD):
d
¼ 23.3
1.57e1.64 (m, 2H, CH2), 1.88e1.94 (m, 2H, CH2), 2.29 (t, J ¼ 7.3 Hz,
2H, CH2COO), 4.26 (t, J ¼ 7.3 Hz, 2H, NCH2), 7.57 (s, 1H, CH[5H-Imid.]),
7.67 (s, 1H, CH[4H-Imid.]), 8.96 (s, 1H, CH[2H-Imid.]); 126 MHz 13C NMR
(CH2), 31.5 (CH2), 35.1 (CH2COO), 48.0 (NCH2), 120.9 (CH[5C-Imid.]),
128.0 (CH[4C-Imid.]), 138.1 (CH[2C-Imid.]), 178.1 (COOH); MS (EI,
70 eV): m/z (%) ¼ 168 (39) [Mþ], 96 (42), 82 (100); MS (CI, CHþ5 ): m/z
(%) ¼ 169 (100) [Mþþ1]; HMRS (EI) calcd. for C8H12N2O2: 168.0899;
found 168.0889.
(CD3OD):
d
¼ 24.2 (CH2), 25.4 (CH2), 27.9 (CH2), 29.4 (CH2), 33.1
(CH2COO), 49.0 (NCH2),119.6 (CH[5C-Imid.]),121.6 (CH[4C-Imid.]),145.0
(CH[2C-Imid.]), 175.9 (COOH); MS (EI, 70 eV): m/z (%) ¼ 196 (10) [Mþ],
195 (72), 82 (100); MS (CI, CHþ5 ): m/z (%) ¼ 197 (100) [Mþþ1]; HMRS
(EI) calcd. for C10H17N2O2: 197.1290; found 197.1284.
Melting point (lit.: 135e136.5 ꢂC) is according to literature value
[57].
NMR, IR and melting point (lit.: 165e166.5 ꢂC) are according to
literature values [56].
5.1.19. 6-(1H-Imidazol-1-yl)hexanoic acid hydrochloride (30∙HCl)
Synthesis was performed according to a modified literature
procedure [49].
5.1.21. 8-(1H-Imidazol-1-yl)octanoic acid hydrochloride (32∙HCl)
8-(1H-Imidazol-1-yl)octanoic acid methyl ester.
Synthesis was performed analogue to a modified literature
procedure [49].
6-(1H-Imidazol-1-yl)hexanoic acid methyl ester: According to
GP6: Imidazole (1.36 g, 20.0 mmol), THF (20 mL), NaH (510 mg,
20.0 mmol), 6-bromohexanoic acid ethyl ester (2.09 g, 10.0 mmol),
72 h, CC (CH2Cl2/C2H5OH ¼ 9/1); 6-(1H-Imidazol-1-yl)hexanoic
acid hydrochloride: According to GP5: 6-(1H-Imidazol-1-yl)hex-
anoic acid methyl ester (117 mg, 0.600 mmol), NaOH (1 M,
0.75 mmol, 0.75 mL). Differing to GP5, MeOH (0.5 mL) was added.
24 h.
According to GP4: Imidazole (1.36 g, 20.0 mmol), acetone
(15 mL), K2CO3 (8.29 g, 60.0 mmol), NaI (40 mg, 0.27 mmol), 8-
bromooctanoic acid methyl ester (2.37 g, 10.0 mmol) 16 h, CC
(CH2Cl2/C2H5OH ¼ 95/5).
Colourless oil; Yield: 1.72 g (77%); IR (KBr)
n
cmꢀ1: 3409, 3111,
Colourless crystals; Yield: 124 mg (95%); mp 126 ꢂC (lit.:
2934, 2858, 1734, 1509, 1438, 1362, 1230, 1199, 1173, 1080; 500 MHz
139e140 ꢂC[56]); IR (KBr)
n
cmꢀ1: 3128, 3061, 2943, 2860, 2360,
1H NMR (CDCl3):
CH2), 1.71e1.77 (m, 2H, CH2), 2.27 (t, J ¼ 7.3 Hz, 2H, CH2), 3.64 (s, 3H,
COOCH3), 3.89 (t, J ¼ 7.3 Hz, 2H, NCH2), 6.87 (s,1H, CH[5H-Imid.]), 7.02
(s, 1H, CH[4H-Imid.]), 7.42 (s, 1H, CH[2H-Imid.]); 126 MHz 13C NMR
d
¼ 1.22e1.32 (m, 6H, CH2), 1.55e1.61 (m, 2H,
2342, 1715, 1574, 1541, 1403, 1306, 1291, 1236, 1180; 500 MHz 1H
NMR (CD3OD):
d
¼ 1.32e1.40 (m, 2H, CH2), 1.63e1.69 (m, 2H, CH2),
1.88e1.95 ( m, 2H, CH2), 2.32 (t, J ¼ 7.3 Hz, 2H, CH2COO), 4.27 (t,
J ¼ 7.1 Hz, 2H, NCH2), 7.57 (s, 1H, CH[5H-Imid.]), 7.67 (s, 1H, CH[4H-
(CDCl3):
d
¼ 24.7 (CH2), 26.3 (CH2), 28.7 (CH2), 28.8 (CH2), 31.0
Imid.]), 8.97 (s, 1H, CH[2H-Imid.]); 126 MHz 13C NMR (D2O):
d
¼ 23.8
(CH2), 33.9 (CH2COO), 47.0 (NCH2), 51.4 (COOCH3), 118.7 (CH[5C-
Imid.]), 129.4 (CH[4C-Imid.]), 137.0 (CH[2C-Imid.]), 174.1 (COO); MS (EI,
70 eV): m/z (%) ¼ 224 (18) [Mþ], 223 (92), 82 (100); MS (CI, CHþ5 ): m/
z (%) ¼ 225 (100) [Mþþ1]; HMRS (EI) calcd. for C12H20N2O2:
224.1525; found 224.1550.
(CH2), 25.0 (CH2), 29.1 (CH2), 33.6 (CH2COO), 49.3 (NCH2), 119.9 (CH
[
5C-Imid.]), 122.0 (CH[4C-Imid.]), 134.5 (CH[2C-Imid.]), 178.9 (COO); MS
(EI, 70 eV): m/z (%) ¼ 182 (17) [Mþ],181 (60), 82 (100); MS (CI, CHþ5 ):
m/z (%) ¼ 183 (100) [Mþþ1]; HMRS (EI) calcd. for C9H14N2O2:
182.1055; found 182.1065.
NMR and IR are according to literature values [56].
8-(1H-Imidazol-1-yl)octanoic acid hydrochloride (32∙HCl).
Synthesis was performed analogue to a modified literature
procedure [49].
According to GP5: 8-(1H-Imidazol-1-yl)octanoic acid methyl
ester (224 mg, 1.00 mmol), NaOH (1 M, 1.5 mmol, 1.5 mL), 24 h.
NMR and IR are according to literature values [56].
5.1.20. 7-(1H-Imidazol-1-yl)heptanoic acid hydrochloride (31∙HCl)
7-(1H-Imidazol-1-yl)heptanenitrile.
Synthesis was performed analogue to a modified literature
procedure [49].
Colourless crystals; Yield: 241 mg (98%); mp 152 ꢂC. IR (KBr)
n
According to GP4: Imidazole (1.36 g, 20.0 mmol), acetone
(15 mL), K2CO3 (8.29 g, 60.0 mmol), NaI (40 mg, 0.27 mmol), 7-
bromoheptanenitrile (1.92 g, 10.0 mmol, 1.51 mL), 16 h, CC (CH2Cl2/
C2H5OH ¼ 95/5).
cmꢀ1: 3127, 2939, 2854, 2360, 1713, 1573, 1542, 1403, 1362, 1295,
1220, 1177, 1082; 500 MHz 1H NMR (CD3OD):
d
¼ 1.35e1.39 (m, 6H,
CH2), 1.57e1.61 (m, 2H, CH2), 1.89e1.92 (m, 2H, CH2), 2.29 (t,
J ¼ 7.3 Hz, 2H, CH2COO), 4.22 (t, J ¼ 7.3 Hz, 2H, NCH2), 7.54 (s, 1H, CH
Colourless oil; Yield: 926 mg (52%); IR (KBr)
n
cmꢀ1: 3397, 3113,
[5H-Imid.]), 7.64 (s, 1H, CH[4H-Imid.]), 8.95 (s, 1H, CH[2H-Imid.]);
2245, 1639, 1511, 1462, 1231, 1109, 1081; 500 MHz 1H NMR (CDCl3):
126 MHz 13C NMR (CDCl3):
d
¼ 24.5 (CH2), 25.7 (CH2), 28.3 (CH2),
d
¼ 1.30e1.35 (m, 2H, CH2), 1.44e1.50 (m, 2H, CH2), 1.61e1.67 (m,
28.5 (CH2), 29.7 (CH2), 33.4 (CH2COO), 49.2 (NCH2), 119.8 (CH[5C-
Imid.]), 122.0 (CH[4C-Imid.]), 135.0 (CH[2C-Imid.]), 176.2 (COO); MS (EI,
70 eV): m/z (%) ¼ 209 (30) [Mþ], 195 (13), 82 (100); HMRS (EI) calcd.
for C11H18N2O2: 209.1290; found 209.1294.
2H, CH2), 1.77e1.82 (m, 2H, CH2), 2.33 (t, J ¼ 7.0 Hz, 2H, CH2CN),
3.94 (t, J ¼ 7.0 Hz, 2H, NCH2), 6.89 (s, 1H, CH[5H-Imid.]), 7.02 (s, 1H, CH
[
4H-Imid.]), 7.45 (s, 1H, CH[2H-Imid.]); 126 MHz 13C NMR (CDCl3):

1496
S. Hack et al. / European Journal of Medicinal Chemistry 46 (2011) 1483e1498
NMR, IR and melting point (lit.: 153e154 ꢂC) are according to
trioxabicyclo[2.2.2]octane (43) (0.904 g, 3.00 mmol) solved in THF
literature values [49,56,57].
(9 mL) was added. The reaction mixture was stirred for 25 min at
ꢀ25 ꢂC, 3 h at 0 ꢂC, and 14 h at 10 ꢂC. The reaction was stopped by
adding H2O (1 mL) and the solvent was evaporated. The residue was
resolved in CH2Cl2 and washed three times with H2O. The
combined organic layers were dried over MgSO4. Purification was
realized by CC (n-Pentane/EtOAc/NEt3 ¼ 9/3/0.5).
5.1.22. 1-Triphenylmethyl-1H-imidazol-2-carbaldehyde (38)
1H-Imidazol-2-carbaldehyde (36) (1.98 g, 20.0 mmol) in DMF
(20 mL) was treated with NEt3 (4.10 g, 40.0 mmol, 5.62 mL). The
mixture was stirred for 15 min at rt before triphenylmethyl chloride
(37) (6.26 g, 22.0 mmol), suspended in DMF (10 mL), was added.
After stirring for 48 h the solution was diluted with EtOAc (20 mL/
mmol) and washed with brine, sat. Na2CO3-solution and H2O. The
organic layers were dried over MgSO4, filtered and concentrated.
The crude product was purified by CC (n-Pentane/EtOAc ¼ 6/4).
Colourless crystals; Yield: 195 mg (13.5%); mp 215 ꢂC. IR (KBr)
n
cmꢀ1: 3418, 2874,1492,1448,1398,1266,1190, 1131,1055; 500 MHz
1H NMR (CDCl3):
d
¼ 0.74 (s, 3H, CH3), 1.33 (t, J ¼ 7.7 Hz, 2H,
CH2CH2CH2C), 1.61 (m, 2H, CH2CH2CH2), 1.85 (t, J ¼ 8.0 Hz, 2H,
CH2CH2CH2C), 3.75 (s, 6H, OCH2), 6.67 (d, J ¼ 1.4 Hz,1H, CH[5H-Imid.]),
6.95 (d, J ¼ 1.4 Hz,1H, CH[4H-Imid.]), 7.11e7.13 (m, 6H, CHar), 7.30e7.32
Colourless crystals; Yield: 5.59 g (81%); mp 190 ꢂC. IR (KBr)
n
cmꢀ1: 3397, 3065, 2845, 1708, 1491, 1442, 1396, 1356, 1242, 1169;
500 MHz 1H NMR (CD3OD):
¼ 7.01 (s, 1H, CH[5H-Imid.]), 7.09e7.11
(m, 6H, CHar), 7.24e7.36 (m,10H, CHar, CH[4H-Imid.]), 9.21 (s,1H, CHO);
126 MHz 13C NMR (CDCl3):
(m, 9H, CHar); 100 MHz 13C NMR (CDCl3):
d
¼ 16.9 (CH3), 23.4
d
(CH2CH2CH2C), 32.5 (CH2CH2CH2), 38.2 (CH2CH2CH2C), 74.7 (OCH2),
75.2 (C(C6H5)3), 111.1 (Cq), 123.3 (CH[5C-Imid.]), 128.2 (CH[4C-Imid.]),
130.0 (CHar), 130.3 (CHar), 132.3 (CHar), 144.9 (C[2C-Imid.]), 152.8 (Cq);
MS (EI, 70 eV): m/z (%) ¼ 480 (100) [Mþ], 467 (12), 455 (40), 443 (55);
HMRS (EI) calcd. for C31H32N2O3: 480.2413; found 480.2401.
d
¼ 76.9 (C(C6H5)3), 126.9 (CHar), 128.1
(CHar), 129.6 (CH[4C-Imid.], CH[5C-Imid.]), 142.1 (C[2C-Imid.]), 145.6 (Cq),
178.6 (CHO); MS (CI, CHþ5 ): m/z (%) ¼ 338 (1) [Mþþ1], 243 (100), 167
(109); MS (FAB, NBA): m/z (%) ¼ 339.2 (5) [Mþþ1], 243.2 (100),165.1
(11); HMRS (ESI) calcd. for C23H18N2ONa1: 361.1317; found 361.1313.
NMR and melting point (lit.: 189e190 ꢂC) are according to
literature values [58].
5.1.25. (2E)-5-(1-Triphenylmethyl-1H-imidazol-2-yl)pent-2-enoic
acid ethyl ester (46)
According to GP3: Triethylphosphonoacetate (0.20 g,
0.91 mmol, 0.18 mL), NaH (18 mg, 0.70 mmol), DME (2 mL), 30 min,
3-(1-triphenylmethyl-1H-imidazole-2-yl)propanal (45) (256 mg,
0.70 mmol), DME (1 mL), 55 ꢂC, 4 h.
5.1.23. (2E)-3-(1-Triphenylmethyl-1H-imidazol-2-yl)acrylic acid
ethyl ester (39) and (2Z)-3-(1-triphenylmethyl-1H-imidazol-2-yl)
acrylic acid ethyl ester (40)
Colourless crystals; Yield: 294 mg (96%); mp 122 ꢂC. IR (KBr)
n
At 0 ꢂC triethylphosphonoacetate (3.0 g,13.0 mmol, 2.68 mL) was
added to NaH (278 mg, 11.0 mmol) suspended in DME (10 mL). The
reaction solution was warmed up to rt and subsequently stirred for
1 h. 1-Triphenylmethyl-1H-imidazol-2-carbaldehyde (38) (3.38 g,
10.0 mmol) solved in DME (14 mL) was added. After 15 min at reflux
conditions the reaction solution was cooled down to 60 ꢂC and
stirring was continued for 2 h. Having extracted the solution with
CH2Cl2, purification was realized by CC (EtOAc/n-Pentane ¼ 1/1).
39: Colourless crystals; Yield: 2.80 mg (69%); mp 198 ꢂC. IR (KBr)
cmꢀ1: 3408, 3057, 2977, 2926, 1711, 1656, 1489, 1447, 1405, 1365,
1315, 1274, 1234, 1203, 1157; 500 MHz 1H NMR (CDCl3):
d
¼ 1.26 (t,
J ¼ 7.1 Hz, 3H, CH3), 2.04e2.06 (m, 4H, CH2CH2CH), 4.13 (q,
J ¼ 7.1 Hz, 2H, CH2CH3), 5.50 (dt, J ¼ 15.6/1.6 Hz, 1H, CHCOO), 6.61
(dt, J ¼ 15.6/6.6 Hz, 1H, CH2CH), 6.71 (d, J ¼ 1.4 Hz, 1H, CH[5H-Imid.]),
6.97 (d, J ¼ 1.4 Hz, 1H, CH[4H-Imid.]), 7.12e7.14 (m, 6H, CHar),
7.32e7.35 (m, 9H, CHar); 100 MHz 13C NMR (CDCl3):
d
¼ 16.5 (CH3),
31.4 (CH2CH2), 32.4 (CH2CH2CH), 62.4 (CH2CH3), 77.0 (C(C6H5)3),
123.6 (CHCOO), 123.8 (CH[5C-Imid.]), 127.9 (CH[4C-Imid.]), 130.2 (CHar),
130.4 (CHar), 132.1 (CHar), 144.7 (C[2C-Imid.]), 150.2 (CH2CH), 151.2
(Cq), 168.9 (COO); MS (CI, CHþ5 ): m/z (%) ¼ 437 (2) [Mþþ1], 243
(100),195 (13),167 (7). HMRS (ESI) calcd. for C29H29N2O2: 437.2229;
found 437.2224. Anal. Calcd. for C29H28N2O2 (436.56) (%): C 79.79, H
6.46, N 6.42. Found C 79.87, H 6.48, N 6.37.
n
cmꢀ1: 29911721,1606,1490,1445,1424,1227,1203,1128; 400 MHz
1H NMR (CDCl3):
d
¼ 1.13 (t, J ¼ 7.1 Hz, 3H, CH3), 4.00 (q, J ¼ 7.1 Hz, 2H,
CH2CH3), 6.53 (d, J ¼ 15.2 Hz, 1H, CHCOO), 6.70 (d, J ¼ 15.2 Hz, 1H,
CHCHCOO), 6.87 (d, J ¼ 1.2 Hz, 1H, CH[5H-Imid.]), 7.10e7.14 (m, 7H,
CHar, CH[4H-Imid.]), 7.31e7.35 (m, 9H, CHar); 100 MHz 13C NMR
(CDCl3):
d
¼ 14.1 (CH2CH3), 60.0 (CH2CH3), 75.1 (C(C6H5)3), 119.3
(CH), 124.2 (CH[5CꢀImid.]), 128.1 (CHar), 128.2 (CHar, CH[4C-Imid.]),
129.8 (CHar),132.5 (CHar),142.3 (C[2CꢀImid.]),145.0 (Cq),166.3 (COO);
MS (CI, CHþ5 ): m/z (%) ¼ 409 (1) [Mþþ1], 243 (100), 167 (26); HMRS
(EI) calcd. for C27H24N2O2Na1: 431.1736; found 431.1732.
NMR is according to the literature value [38].
5.1.26. 4,5,6,7-Tetrahydro-1H-benzimidazol-4-carboxylic acid
hydrochloride (54∙HCl)
According to GP7: 4,5,6,7-Tetrahydro-1H-benzimidazol-4-
carboxylic acid ethyl ester (23.0 mg, 0.118 mmol) [52], HCl (1 M,
1.0 mmol, 1.0 mL).
40: Colourless crystals; Yield: 1.20 mg (29%); mp 193 ꢂC. IR (KBr)
Colourless crystals; Yield: 23.0 mg (96%); mp 200e205 ꢂC. IR
n
cmꢀ1: 2991, 1721, 1606, 1490, 1445, 1424, 1227, 1203, 1128;
(KBr)
n
cmꢀ1: 3364, 3128, 3026, 2932, 1712, 1647, 1292, 1235;
500 MHz 1H NMR (CDCl3):
d
¼ 1.24 (t, J ¼ 7.1 Hz, 3H, CH3), 4.19 (q,
400 MHz 1H NMR (D2O):
d
¼ 1.94e1.73 (m, 2H, CH2CH2CHCOO),
J ¼ 7.1 Hz, 2H, CH2CH3), 5.39 (d, J ¼ 12.0 Hz, 1H, CHCOO), 5.68 (d,
2.17e2.01 (m, 2H, CH2CHCOO), 2.70e2.52 (m, 2H, CH2CNH), 3.87 (t,
J ¼ 6.0 Hz, 1H, CHCOO), 8.48 (s, 1H, H[2H-Imid.]); 100 MHz 13C NMR
J ¼ 12.0 Hz, 1H, CHCHCOO), 6.79 (s, 1H, CH[5H-Imid.]), 7.05 (s, 1H, CH
[
4H-Imid.]), 7.15e7.17 (m,6H, CHar), 7.31e7.33 (m, 9H, CHar); 100 MHz
(D2O):
d
¼ 19.34 (CH2CNH or CH2CH2CHCOO), 19.45 (CH2CNH or
13C NMR (CDCl3):
d
¼ 14.1 (CH3), 60.4 (CH2CH3), 75.3 (C(C6H5)3),
CH2CH2CHCOO), 25.33 (CH2CHCOO), 37.36 (CHCOO), 123.86 (C[4C-
Imid.]), 129.24 (C[5C-Imid.]), 131.72 (C[2C-Imid.]), 175.73 (COO); MS (CI,
CHþ5 ): m/z (%) ¼ 167 (100, Mþ), 121 (26); HMRS (EI) calcd. for
C8H10N2O2: 166.0742; found 166.0759. Anal. Calcd. for
C8H10N2O2$HClþ0.5H2O (211.65) (%): C 45.40, H 5.71, N 13.24.
Found C 45.77, H 5.91, N 13.11.
121.8 (CH), 122.7 (CH[5C-Imid.]), 127.3 (CHar), 127.8 (CHar), 128.0
(CHar), 130.1 (CH[4C-Imid.]),142.2 (C[2C-Imid.]),144.0 (Cq), 166.8 (COO);
MS (CI, CHþ5 ): m/z (%) ¼ 409 (1) [Mþþ1], 243 (100), 167 (7); HMRS
(ESI) calcd. for C27H24N2O2Na1: 431.1736; found 431.1733.
5.1.24. 2-[3-(4-Methyl-2,6,7-trioxa-bicyclo[2.2.2]octan-1-yl)
propyl]-1-triphenylmethyl-1H-imidazole (44)
5.1.27. 1-Tertbutyl 6-ethyl 4,5,6,7-tetrahydro-1H-benzimidazol-1,5-
dicarboxylate (57) and 1-tertbutyl 6-ethyl 4,5,6,7-tetrahydro-1H-
benzimidazol-1,6-dicarboxylate (58)
A solution of formamide (3.40 g, 75.5 mmol, 3.0 mL) and ethyl
3-bromo-4-oxocyclohexanecarboxylate (56) (820 mg, 3.29 mmol)
nBuLi (1.5 M, 3.0 mmol, 2.0 mL) was added at ꢀ15 ꢂC to a solu-
tion of 1-triphenylmethyl-1H-imidazole (41) (0.931 g, 3.00 mmol)
in THF (36 mL) and the mixture was stirred at rt for 30 min. After
cooling the mixture to ꢀ10 ꢂC, 1-(3-iodopropyl)-4-methyl-2,6,7-

S. Hack et al. / European Journal of Medicinal Chemistry 46 (2011) 1483e1498
1497
was heated at 150 ꢂC for 2 h. The solution was then diluted with
H2O and washed with Et2O. Having adjusted a pH-value of 8e9,
the solution was extracted with CHCl3. The combined organic
layers were dried over MgSO4 and concentrated. The obtained
residue was added to a stirred suspension of NaH (91.0 mg,
3.66 mmol) and THF (12 mL) at 0 ꢂC. After 15 min at 0 ꢂC, the
solution was warmed up to rt for 1 h. Again, the solution was
cooled to 0 ꢂC, di-tert-butyl dicarbonate (720 mg, 3.33 mmol) was
added and it was stirred 18 h at rt. Then, the reaction mixture was
washed three times with H2O and brine. The combined aqueous
layers were extracted with CH2Cl2 before the organic layers were
concentrated and dried over MgSO4. Purification was realized by
CC (EtOAc ¼ 100). Ratio: 57:58 ¼ 0.53:0.47.
125.48 (C[4C-Imid.]), 126.50 (C[5C-Imid.]), 131.22 (C[2C-Imid.]), 178.14
(COO); MS (EI, 70 eV): m/z (%) ¼ 166 (40) [Mþ], 119 (35), 94 (100);
HMRS (EI) calcd. for C8H10N2O2: 166.0742; found 166.0765; Anal.
Calcd. for C8H10N2O2 (166.18) (%): C 46.05, H 5.64, N 5.83, N 13.43.
Found C 46.21, H 5.44, N 13.16.
NMR and melting point (lit.: 248e250 ꢂC) are according to
literature values [59].
6. Pharmacological methods
6.1. [3H]GABA uptake
[3H]GABA uptake was studied as described (A. Kragler, G. Höf-
ner, K. T. Wanner, synthesis of aminomehtylphenol derivatives as
inhibitors of the murine GABA transporters mGAT1emGAT4, Eur. J.
Med. Chem., 43, 2404e2411, 2008).
Colourless oil; Yield: 438 mg (44%); IR (KBr)
2858, 1751, 1733, 1479, 1372, 1302, 1279, 1257, 1165, 1127, 1063,
1034; 500 MHz 1H NMR (CDCl3):
n
cmꢀ1: 2980, 2936,
d
¼ 1.26 (t, J ¼ 7.1 Hz, 3H, CH3),1.59
(d, J ¼ 3.1 Hz, 9H, C(CH3)3), 1.96e1.82 (m, 1H, CH2CH2CHCOO),
2.23e2.13 (m, 1H, CH2CH2CHCOO), 3.18e2.54 (m, 5H, CHCOO,
CH2CH2CHCOO, CCH2CHCOO), 4.20e4.10 (m, 2H, CH2CH3), 7.95 (d,
References
J ¼ 3.4 Hz, 1H, H[2H-Imid.]); 100 MHz 13C NMR (CDCl3):
d
¼ 14.22
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(CH3), 14.24 (CH3), 21.97 (CH2CH2CHCOO), 23.15 (CCH2CHCOO),
25.43 (CH2CH2CHCOO or CCH2CHCOO), 25.57 (CH2CH2CHCOO or
CCH2CHCOO), 25.61 (CH2CH2CHCOO or CCH2CHCOO), 26.77
(CH2CH2CHCOO), 27.96 (C(CH3)3), 27.97 (C(CH3)3), 39.69 (CHCOO),
39.69 (CHCOO), 60.62 (CH2CH3), 60.67 (CH2CH3), 85.11 (C(CH3)3),
85.21 (C(CH3)3), 124.12 (C[4C-Imid.]), 124.98 (C[4C-Imid.]), 136.57 (C[2C-
Imid.]), 136.60 (C[2C-Imid.]), 137.20 (C[5C-Imid.]), 138.12 (C[5C-Imid.]),
147.74 (NCOO), 147.79 (NCOO), 174.66 (CHCOO), 174.81 (CHCOO);
MS (EI, 70 eV): m/z (%) ¼ 294 (8) [Mþ], 221 (18), 194 (50) 165 (36),
120 (100); MS (CI, CHþ5 ): m/z (%) ¼ 295 (42) [Mþþ1], 239 (100), 195
(88); HMRS (EI) calcd. for C12H22N2O2: 294.1580; found 294.1570.
[12] M. Palacín, R. Estévez, J. Bertran, A. Zorzano, Physiol. Rev. 78 (1998) 969e1054.
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519e531.
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J. Comp, Neurology 409 (1999) 482e494.
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Acad. Sci. U S A 75 (1978) 6319e6323.
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1716e1725.
5.1.28. 4,5,6,7-Tetrahydro-1H-benzimidazol-5-carboxylic acid ethyl
ester (59)
TFA (6.14 g, 53.85 mmol, 4 mL) was added to a stirred solution of
57 and 58 (58 mg, 0.20 mmol) in EtOH (1 mL). The mixture was
stirred for 4 h at rt before a pH-value of 9e10 with solid Na2CO3 and
NaOH (2 M) was adjusted. After extraction with CH2Cl2, the
combined organic layers were dried over MgSO4 and concentrated.
Colourless crystals; Yield: 51.6 mg (94%); mp 145e150 ꢂC. IR (KBr)
n
cmꢀ1: 3149, 3045, 2925, 2858, 2822, 2674, 1716, 1665, 1446, 1383,
1299,1206,1172,1132;500MHz1HNMR (CDCl3):
¼ 1.27 (t, J¼ 7.1 Hz,
3H, CH3), 2.00 (ddt, J ¼ 14.2/9.6/5.9 Hz,1H, CH2CH2CHCOO), 2.25 (ddt,
14.2/5.1/3.0 Hz, 1H, CH2CH2CHCOO), 2.79e2.63 (m, 2H,
d
J
¼
CH2CH2CHCOO), 2.87e2.79 (m, 1H, CHCOO), 2.92 (d, J ¼ 6.9 Hz, 2H,
CCH2CHCOO), 4.17 (q, J ¼ 7.1 Hz, 2H, CH2CH3), 7.97 (s, 1H, H[2H-Imid.]);
125 MHz 13C NMR (CDCl3):
d
¼ 14.17 (CH3), 20.07 (CH2CH2CHCOO),
23.68 (CCH2CHCOO), 25.24 (CH2CH2CHCOO), 39.35 (CHCOO), 60.99
(CH2CH3), 127.25 (C[4C-Imid.]), 127.73 (C[5C-Imid.]), 132.17 (C[2C-Imid.]),
173.91 (COO); MS (CI, CHþ5 ): m/z (%) ¼ 195 (100) [Mþþ1], 120 (4);
HMRS (EI) calcd. for C10H14N2O2: 194.1055; found 194.1100.
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5.1.29. 4,5,6,7-Tetrahydro-1H-benzimidazol-5-carboxylic acid
hydrochloride (55$HCl)
According to GP7: 59 (51.6 mg, 0.266 mmol), HCl (2 M, 6.0 mmol,
3.0 mL).
Colourless crystals; Yield: 48.8 mg (90%); mp 248e250 ꢂC. IR
(KBr)
n
cmꢀ1: 3424, 3150, 3007, 2855, 1707, 1654, 1497, 1459, 1412,
1331, 1246, 1190, 1087; 500 MHz 1H NMR (D2O):
d
¼ 2.00 (ddt,
J ¼ 13.8/9.3/7.0 Hz, 1H, CH2CH2CHCOO), 2.21 (ddt, J ¼ 13.8/5.6/
3.3 Hz, 1H, CH2CH2CHCOO), 2.69 (t, J ¼ 6.0 Hz, 2H, CH2CH2CHCOO),
2.85 (ddt, J ¼ 16.2/8.0/1.6 Hz, 2H, CCH2CHCOO), 2.92 (dd, J ¼ 16.2/
5.5 Hz, 1H, CCH2CHCOO), 3.02e2.95 (m, 1H, CHCOO), 8.43 (s, 1H,
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H[2H-Imid.]); 125 MHz 13C NMR (D2O):
d
¼ 18.19 (CH2CH2CHCOO),
21.80 (CCH2CHCOO), 23.89 (CH2CH2CHCOO), 38.06 (CHCOO),

1498
S. Hack et al. / European Journal of Medicinal Chemistry 46 (2011) 1483e1498
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