Aminomethyltetrazoles as potential inhibitors of the c-aminobutyric acid transporters mGAT1-mGAT4: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2011.08.039

1,5-Disubstituted and 5-monosubstituted aminomethyltetrazole derivatives derived from glycine were synthesized employing a TMSN3-modified variant of the Ugi reaction as a key step. All compounds were evaluated regarding their inhibitory potency and subtype selectivity at the four murine GABA transporter subtypes mGAT1-mGAT4. Though none of the 5-monosubstituted tetrazoles turned out to inhibit [3H]GABA uptake to a significant extent, the 1,5-disubstituted tetrazole derivatives displayed a distinct activity, especially at the GABA transport proteins mGAT2-mGAT4. Thus, a reasonable potent and selective inhibitor of mGAT3 was found. Additionally, two more compounds were identified as potent inhibitors of mGAT2. This is especially relevant, as up to date only few potent inhibitors of mGAT2 that do not affect mGAT1 are known.

Full text of DOI:10.1016/j.bmc.2011.08.039

Bioorganic & Medicinal Chemistry 19 (2011) 6492–6504
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Aminomethyltetrazoles as potential inhibitors of the c-aminobutyric acid
transporters mGAT1–mGAT4: Synthesis and biological evaluation
⇑
Eva S. Schaffert, Georg Höfner, 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:
1,5-Disubstituted and 5-monosubstituted aminomethyltetrazole derivatives derived from glycine were
synthesized employing a TMSN3-modified variant of the Ugi reaction as a key step. All compounds were
evaluated regarding their inhibitory potency and subtype selectivity at the four murine GABA transporter
subtypes mGAT1–mGAT4. Though none of the 5-monosubstituted tetrazoles turned out to inhibit
[3H]GABA uptake to a significant extent, the 1,5-disubstituted tetrazole derivatives displayed a distinct
activity, especially at the GABA transport proteins mGAT2–mGAT4. Thus, a reasonable potent and selec-
tive inhibitor of mGAT3 was found. Additionally, two more compounds were identified as potent inhib-
itors of mGAT2. This is especially relevant, as up to date only few potent inhibitors of mGAT2 that do not
affect mGAT1 are known.
Received 16 June 2011
Revised 18 August 2011
Accepted 19 August 2011
Available online 26 August 2011
Keywords:
GABA uptake
Tetrazole
Bioisosteric replacement
Ugi reaction
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
proteins have been identified as members of the SLC6 (solute
carrier 6) family. Genes coding for other Na+/ClÀ dependent trans-
Brain function is based on a fine-tuned balance of excitatory
and inhibitory signals that is maintained by numerous feedback-
mechanisms.1 An imbalance of excitation and inhibition may
underlie numerous neuropathological and psychiatric diseases of
the central nervous system (CNS). With approximately 30–40% of
porters, such as the transport proteins for serotonin, dopamine,
norepinephrine or glycine, have also been assigned to this
group.12,13
To date, four different subtypes of membrane bound GABA
transport proteins are known.14 When cloned from murine brain
cells these GABA transporters are termed mGAT1, mGAT2, mGAT3,
and mGAT4. The nomenclature suggested by the Human Genome
Organization (HUGO) as well as the nomenclature for GATs origi-
nating from other species including humans and rats, however, dif-
fers from the mouse nomenclature. Here, the corresponding GABA
all synapses being GABAergic,
c-aminobutyric acid (GABA, 1,
Fig. 1) is the major inhibitory neurotransmitter in the mammalian
brain.2,3 Thus, perturbations in GABA neurotransmission play a key
role in the pathophysiology of neurological disorders. For example,
a deficiency of GABA is related to epilepsy,4 Morbus Parkinson,5
Morbus Alzheimer,6 Huntington’s chorea,7 neuropathic pain,8
schizophrenia,9 and depression10—to name only a few.
transport proteins are denoted as GAT-1, BGT-1, GAT-2, and GAT-3,
14
respectively.
,
Consequently, virtually all receptors, metabolic enzymes and
transporters involved in GABAergic neurotransmission can be con-
sidered as valid targets when designing new CNS-active drugs. For
example, enhancement of the GABAergic signal transduction can
be effected either by direct receptor agonism or allosteric modula-
tion of the GABA receptors, as it is accomplished by benzodiaze-
pines or barbiturates.11 On the other hand, the GABAergic
neurotransmission can be enhanced by increasing the concentra-
tion of GABA in the synaptic cleft. This may be achieved by inhibi-
tion of enzymatic GABA degradation or by blocking specific high
affinity GABA transport proteins (GATs) responsible for the re-
moval of synaptic GABA.11 These GABA transporters are located
in the cell membranes of the presynaptic nerve terminals and also
in those of glial cells. The genes encoding the GABA-uptake
The four subtypes of GABA transporters differ in their expression
pattern and their pharmacological role. Whereas mGAT2 and
mGAT3 are also found in peripheral tissue, the most prevalent GABA
transporter subtypes, mGAT1 and mGAT4, are almost exclusively
located in the CNS.15,16 mGAT1 is widely distributed throughout
the brain with high densities found for example in the neocortex,
hippocampus, cerebellum, and the basal ganglia.13,16 The distribu-
tion of mGAT4, however, is more restricted.14 It has a strong expres-
sion in the spinal cord, the brain stem, the thalamus, and
hypothalamus.16 mGAT3 has been found to be primarily located
on the leptomeninges and in ependymal tissue.17 The mRNA encod-
ing mGAT2, was found mainly in the hippocampus, hypothalamus,
thalamus, substantia nigra, and the amygdala.15
In the present paper mouse nomenclature will be used, as the biological
characterization of the test compounds was performed using murine GABA
transporters.
⇑
Corresponding author. Tel.: +49 89 2180 77249; fax: +49 89 2180 77247.
E-mail address: klaus.wanner@cup.uni-muenchen.de (K.T. Wanner).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.039

E. S. Schaffert et al. / Bioorg. Med. Chem. 19 (2011) 6492–6504
6493
intrinsic activity at GABAA-receptors.23 Being relatively small, at
physiological pH zwitterionic molecules, these first GABA uptake
inhibitors, however, proved to be too polar to cross the blood–
brain barrier in sufficient quantities. For this reason, some 30 years
ago the first lipophilic derivatives of nipecotic acid and guvacine
have been developed.24 Addition of characteristic lipophilic side
chains to the parent compounds resulted in potent GABA uptake
inhibitors such as SK&F-89976-A (4),24 Tiagabine (5),25,26 or
NO-711 (6)27 (Fig. 2). These compounds were not only found to
be systemically active and considerably more potent than the
parent compounds, but also selective for the GABA transporter
subtype mGAT1.
Up to now, the majority of published GAT inhibitors target the
transporter subtype mGAT1. The number of effective inhibitors of
the other subtypes, however, is still small. A closer look at two of
the most potent mGAT2 inhibitors known to date, NNC 05-2090
(7)28 and the mGAT1/mGAT2 selective EF1502 (8),29 reveals that
neither is derived from a classical heterocyclic amino acid parent
structure (Fig. 3). Detailed pharmacophore models for mGAT2
inhibitors, however, are still lacking. For mGAT3 and mGAT4, too,
only few selective inhibitors have been published. Small amino
acids like b-alanine or (RS)-2,3-diaminopropionic acid are reported
to exhibit moderate affinity to GABA uptake transporters with
selectivity for mGAT3 and mGAT4.30 As for the lipophilic inhibitors
of mGAT3 and mGAT4, for example SNAP-5294 (9),31 or the
mGAT4 selective inhibitors (S)-SNAP-5114 (10)31 and compound
11,32 which is derived from the only moderately potent (S)-pyrrol-
idine-2-yl-acetic acid, subtype selectivity seems to originate
mainly from the structure of the lipophilic side chain attached to
the nitrogen of the parent structure (Fig. 3).
COOH
COOH
COOH
H2N
N
H
N
H
GABA (1)
2
3
Figure 1. GABA (1) and conformationally restricted GAT inhibitors.
While mGAT2 and mGAT3 are predominately located at extra-
synaptic sites,15,16 mGAT1 and mGAT4 have been found in close
proximity to the synapse or in the synapse itself. Thereby, mGAT1
is mainly assigned to neuronal transport, while it is assumed that
mGAT4 accounts predominantly for glial transport of GABA.16,18
Except for mGAT2, the GABA transport proteins show high affinity
to GABA. mGAT2 possesses only low affinity, but is also able to
carry betaine.16,19
Since the identification of the GABA transport system as phar-
macological target20,21, a number of acyclic and cyclic structures
have been tested as potential GAT inhibitors. Inhibitors of the first
generation, mainly analogs of GABA (1) itself, often showed low
selectivity for the GABA transporters.22 The conformationally re-
stricted amino acids nipecotic acid (2) and guvacine (3, Fig. 1) were
eventually found to be potent inhibitors of [3H] GABA uptake
demonstrating high in vitro GABA uptake inhibition without
COOH
COOH
COOH
N
N
N
With Tiagabine (5) in clinical use for the therapy of partial sei-
zures, mGAT1 represents an approved drug target.33 Yet, little is
known about the possible therapeutic relevance of mGAT2–
mGAT4. Potent inhibitors of these GABA transporter subtypes are
of fundamental importance for the elucidation of the physiological
function of these targets and, possibly, for the development of a
novel class of CNS-active drugs.19
O
S
N
S
SK&F-89976-A (4)24
Tiagabine (5)25,26
NO-711 (6)27
Figure 2. mGAT1 selective GABA uptake inhibitors.
COOH
N
O
N
HO
OH
OMe
N
H
3C
N
O
OMe
S
S
N
NNC 05-2090 (7)28
EF1502 (8)29
SNAP-5294 (9)31
COOH
COOH
MeO
MeO
N
N
O
O
MeO
MeO
OMe
OMe
(S)-SNAP-5114 (10)31
1132
Figure 3. Selected inhibitors of mGAT2–mGAT4.

6494
E. S. Schaffert et al. / Bioorg. Med. Chem. 19 (2011) 6492–6504
R1
R1
As part of a research project aimed at the development of
N
N
N
H
2N
N
N
tetrazole analogs of amino acids as new inhibitors of the GABA
transport proteins mGAT1–mGAT4, a series of glycine-derived
5-aminomethyltetrazoles was synthesized. Bioisosteric replace-
ment of functional groups is a widely used technique in medicinal
chemistry for the generation of more potent, more selective or bet-
ter tolerated analogs of lead structures.34 Thereby, the 1H-tetrazole
moiety represents a popular substitute for carboxylic acids. Yet, lit-
tle is known about the effect of tetrazole analogous amino acids on
GABA uptake. In a report published as early as 1984, five tetrazole
amino acids (13–17), including the analogs of nipecotic acid (2),
GABA (1) and b-alanine (12), the compounds 13, 17 and 15 have
been tested regarding their impact on the GABAergic system
(Fig. 4). Their potency to inhibit the [3H]GABA uptake into synap-
N
N
N
R2
R2
N
H
N
N
H
N
N
R3
N
19
20
21
Figure 5.
derivatives of the general structure 21 were also included in the
present study (Fig. 5).
2. Results and discussion
2.1. Chemistry
tosomes from rat brain, however, was low (IC50 <100 l
M).35
For the assembly of the aminomethyltetrazole-structure, a
TMSN3-modified variant of the Ugi reaction was chosen. The term
Ugi reaction actually comprises a group of multi-component reac-
tions generally involving a carbonyl compound, an amine, an isoni-
trile and an acid component. Thereby, the nature of the
components may vary widely.36 When employing an azide salt,
or, more conveniently, trimethylsilyl azide as azide source, 1,5-
To the best of our knowledge, the influence of other tetrazole
amino acids on GABA uptake, as well as the effect of their lipophilic
derivatives is still unknown. Thus, it seemed worthwhile to further
investigate tetrazole analogs of various amino acids with regard to
their impact on the GABA transport system. As starting point for
the present study, glycine (18) was chosen. Glycine (18) is an
inhibitor of the non-GAT1 transporters with only low activity
(pIC50 mGAT2 2.65 0.10, mGAT3 2.88 0.02, mGAT4
2.51 0.12) and modest selectivity as compared to mGAT1 at
which it is devoid of any significant potency (59.8%, 10 mM, see
Table 1). Aim of this study was to investigate the influence of bio-
isosteric replacement of the carboxylic acid by the larger and more
lipophilic 1H-tetrazole on the activity of glycine (18) and its deriv-
atives at the GABA transport proteins. Here, we present the synthe-
sis of the glycine analog 1H-tetrazol-5-ylmethylamine (19) and of
several lipophilic derivatives (general structure 20), as well as their
biological evaluation regarding their inhibitory potency and sub-
type selectivity at the four murine GABA transport proteins
mGAT1–mGAT4. Additionally, the 1,5-disubstituted tetrazole
disubstituted a
-aminotetrazole derivatives are obtained.37–41
Thus, starting from paraformaldehyde (36) as carbonyl compo-
nent, this variant of the Ugi reaction offers direct access to the 1,5-
disubstituted a-aminomethyltetrazoles of the general structure 21
(Scheme 1). By employing appropriate substituted amines and iso-
nitriles, assembly of the aminomethyltetrazole and linkage with
lipophilic groups is possible in only one step. Cleavage of a suitable
protecting group in 1-position of the tetrazole ring eventually leads
to the amino acid analogs of the general structure 20. For this pur-
pose, we chose the 1,1,3,3-tetramethylbutyl (tmb)-group, which
has been described as a convenient protective group for the tetra-
zole moiety.42
N
N
N
NH
N
N
N
N
N
NH
H
N
2N
N
N
H
2N
N
N
N
H
N
H
N
H
N
N
N
H
N
H
N
H
13
14
15
16
17
Figure 4.
Table 1
Test results of selected reference compounds
Entry
Compound
mGAT1
mGAT2
mGAT3
mGAT4
H
2N
18
COOH
COOH
59.8%a (10 mM)
83.1%b (1 mM)
2.654 0.097a
60.7%b (1 mM)
2.884 0.023a
49.3%b (1 mM)
2.506 0.118a
69.7%b (1 mM)
1
H
12
2N
2
3
4
2.59 0.03a
3.48 0.11a
4.66 0.06a
4.46 0.13a
NNC 05-2090
7
(S)-SNAP-5114
10
4.99 0.06
(4.72)c
5.09 0.06
(5.85)c
4.93 0.03
(4.39)c
4.78 0.10
(4.82)c
4.07 0.09d
(3.41, hGAT-1)e
56% (100
l
M)d
5.29 0.04d
(4.68, rGAT-2)e
5.81 0.10d
(5.30, hGAT-3)e
(3.85, hBGT-1)e
If not stated otherwise, the results are given as pIC50 SEM of three independent experiments, each performed in triplicate. Percentages represent specific binding remaining
in presence of the inhibitor concentration given in parentheses. To allow a better comparison, all pIC50 values given have been determined employing our test system (see Ref.
52).
a
From Ref. 53; data determined employing our test system.
These data are given for a more convenient comparison with the test results presented in Table 2.
Data reported by Thomsen et al., using a protocol differing from our test system (see Ref. 28).
From Ref. 52; data determined employing our test system.
b
c
d
e
Data reported by Dhar et al., using a protocol differing from our test system (see Ref. 31).

E. S. Schaffert et al. / Bioorg. Med. Chem. 19 (2011) 6492–6504
6495
R1
R1
NH
N
a
56% - 99%
N
R3 NC
+
TMSN3
(CH2O)x
N
+
+
R2
N
R2
N
R3
36
22-32
33-35
37
21
Scheme 1. Reagents and conditions: (a) MeOH, 80 °C (MW), 1.5 h.
NH2
N
NH2
( )n
NH2
( )n
22 n = 0
23 n = 1
24 n = 2
25 n = 3
26 n = 1
27 n = 2
28
Boc
N
O
BocHN
NH2
NH2
N
H
N
H
29
30
31
32
Figure 6. Amine components employed in the TMSN3-modified Ugi reaction.
NC
of carbazole with 2-(3-bromopropyl)isoindole-1,3-dione with sub-
sequent hydrazinolyses gave 3-carbazol-9-ylpropylamine (28)46 in
91% yield.
NC
NC
33
34
35
2.1.2. Synthesis of the 1,5-disubstituted tetrazoles
The Ugi reaction was performed by simply mixing equimolar
amounts of paraformaldehyde (36), the respective amine 22–32,
the isonitrile 33–35 and TMS-azide (37) in MeOH and subjecting
the mixture to microwave irradiation for 1.5 h at 80 °C
(Scheme 1).à
When employing sterically less hindered primary amines, the
amount of the amine was doubled to suppress the formation of
byproducts, such as 38 (Fig. 8), resulting from a repetitive Ugi reac-
tion. Thus, regardless the nature of the amine or the isocyanide, the
desired 1,5-disubstituted tetrazoles of the general structure 21
were isolated with good to excellent yields ranging from 56% to
99%. The results of the TMSN3-modified Ugi reaction are summa-
rized in Fig. 9.
As mentioned above, to avoid a double Ugi reaction, the mono-
Boc-protected diamines 30 and 31 have been employed. Therefore,
for the syntheses of 62 and 63Á2HCl, subsequent to the Ugi reac-
tion, the Boc-groups had to be cleaved off. For compounds 59
and 60, both N-Boc- and tmb-protected, selective Boc-deprotection
could be achieved at room temperature, using hydrochloric acid in
ethanol. Thus, the amines 62 and 63Á2HCl were obtained in reason-
able to excellent yields (Scheme 2).
Figure 7. Isonitrile components employed in the TMSN3-modified Ugi reaction.
The focus was on amino acid analogs that are substituted with
lipophilic groups which have been identified to be beneficiary for
the pharmacokinetic profile as well as for the binding to GAT-pro-
teins. Hence, primarily the amines 22–28 bearing bis-aromatic
structures were chosen. By variation of the spacer length, the opti-
mum distance between the amino function and the aromatic moi-
ety should be determined.
Additionally, four more primary and secondary amines 29–32
were employed in the Ugi reaction, to investigate the influence of a
pure alkyl chain, additional rings and further functional groups on
GATinhibition. In caseof thediamines 30and31, themono-Boc-pro-
tected form was employed, to avoid a double Ugi reaction (Fig. 6).
For the 1,5-disubstituted tetrazoles 21, the substituent in
1-position of the tetrazole moiety was varied, too. In Ugi reactions,
variation of the substituent in 1-position of the resulting tetrazole
can be realized by employing variably substituted isonitriles. For
this purpose, apart from 1,1,3,3-tetramethylbutyl isonitrile (33),
cyclohexyl isocyanide (34) and benzyl isocyanide (35) were chosen
(Fig. 7).
2.1.3. Synthesis of the 5-monosubstituted tetrazoles
2.1.1. Synthesis of the amines
From the tmb-protected tetrazole derivatives 39, 42, 45, 48, 51,
54, 57, 58, and 61 the protective group at the tetrazole moiety was
cleaved off to obtain the amino acid analogs of the general struc-
ture 20. As described by Dömling et al., this is easily accomplished
in acidic medium.42 For the deprotection of tetrazoles mostly ele-
vated temperatures were needed, therefore the 1,5-disubstituted
The synthesis of not commercially available amines 25–28
could easily be accomplished following literature procedures or
using well-established reactions. Thus, 26 was synthesized accord-
ing to a two-step procedure of van der Bent et al.43 starting from
benzophenone (71% yield). Likewise, 4,4-diphenylbut-3-en-1-yla-
mine (27) was assembled starting from cyclopropyldiphenylcarbi-
nole according to reported procedures43,24,44 (50% over three
steps). Subsequent reduction of the double bond with triethylsi-
lane in CF3COOH yielded amine 2529,45 in 69% yield. The reaction
à
Similar results, however, could be obtained by stirring the reaction mixture under
reflux conditions.

6496
E. S. Schaffert et al. / Bioorg. Med. Chem. 19 (2011) 6492–6504
lane and TFA. Subsequent purification by ion exchange chromatog-
raphy and treatment with an excess of HCl gave the hydrochloride
salt 19ÁHCl in 70% yield (Scheme 3).
N
N
N
N
2.2. Pharmacology
N
N
N
N
All 1,5-disubstituted and 5-monosubstituted tetrazole deriva-
tives were characterized regarding their inhibitory potency and
subtype selectivity at the four GABA transporter subtypes
N
mGAT1–mGAT4. This was accomplished using
a
uniform
[3H]GABA uptake assay on HEK cell lines, each stably expressing
one of the four GATs.52 The potency of the tested compounds 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
38
Figure 8. Product of a repetitive Ugi reaction.
specified concentration (100 lM, 1 mM, 10 mM), no pIC50 values
were determined. For these compounds, the percentage of the
remaining specific [3H]GABA uptake at the defined concentration
is given.
As reference compounds for the unsubstituted glycine analog
19ÁHCl, the amino acid glycine (18) and its homolog, b-alanine
(12), were chosen. The inhibitory effect of glycine (18) on
GABA-uptake is only moderate (Table 1, entry 1). By contrast,
b-alanine (12), shows good activity at mGAT3 and mGAT4, with
remarkable selectivity especially towards mGAT1 (Table 1,
entry 2).53
tetrazole derivatives 39, 42, 45, 48, 51, 54, 57, 58, and 61 were re-
fluxed in an excess of HClEtOH. This way, the amino acid analogs
64–72 were obtained in good to excellent yields ranging from
65% to 99% (cf. Fig. 10). By the same procedure, starting from 59
and 60, combined cleavage of the Boc- and tmb-groups could be
accomplished, resulting in 73Á2HCl49 and 74Á2HCl in 91% and 31%
yield, respectively (Fig. 10).
For the synthesis of the unsubstituted tetrazole amino acid
19ÁHCl50,51 the diphenylmethyl- and tetramethylbutyl protected
tetrazole derivative 39 was employed. Both protecting groups
could easily be removed by refluxing 39 in a mixture of triethylsi-
Compounds NNC 05-2090 (7) and (S)-SNAP-5114 (10) served as
references for inhibitors with pronounced activity at mGAT2 or
N
N
N
N
H
N
N
H
N
N
N
N
N
N
N
H
N
H
R
R
N
N
N
N
N
N
R
R
39a R = tmb
78%
70%
95%
42b
43b
44b
R = tmb
R = Cyclohexyl
R = Bn
94%
92%
86%
45b
46b
47b
R = tmb
R = Cyclohexyl
R = Bn
92% 48b
72% 49b
85% 50b
R = tmb
R = Cyclohexyl
R = Bn
99%
76%
70%
40a R = Cyclohexyl
41b R = Bn
N
N
N
N
N
H
N
N
H
N
N
N
N
N
N
H
R
R
N
N
N
R
51b
52b
53b
R = tmb
R = Cyclohexyl
R = Bn
92%
76%
59%
54b
55b
56b
R = tmb
R = Cyclohexyl
R = Bn
82%
98%
73%
57a
R = tmb
56%
R
N
N
N
N
N
N
N
N
H
X
N
N
58a X = O
79%
60a
61a
R1 = BocHNCH2CH2 80%
R1= CH3CH2CH2CH2 63%
59a X = NHBoc 82%
Figure 9. Results of the TMSN3-modified Ugi reaction (a: 1 equiv amine employed; b: 2 equiv amine employed; tmb: 1,1,3,3-tetramethylbutyl).

E. S. Schaffert et al. / Bioorg. Med. Chem. 19 (2011) 6492–6504
6497
N
N
1) HClEtOH
RT
N
N
N
N
H
N
N
N
N
N
N
Boc
2) NaOH
59
62
99%
H
2N
N
BocHN
N
N
H
N
H
N
N
HClEtOH
RT, 4 h
N
N
N
N
x 2 HCl
60
63· 2 HCl
43%
Scheme 2. Syntheses of 62 and 63Á2HCl.
N
N
H
N
N
N
N
N
N
H
N
( )n
N
H
N
N
N
H
N
N
H
( )n
N
H
64 · HCl
65 · HCl
66 · HCl
67 · HCl
n = 0
n = 1
n = 2
n = 3
89%
80%
65%
65%
68 · HCl n = 1
69 · HCl n = 2
81%
78%
70 · HCl 95%
H2N
N
N
N
N
N
H
N
N
N
N
H
N
H
X
N
H
N
H
N
N
N
71 · HCl47
89%
72 · HCl48
X = O
99%
91%
74 · 2 HCl
31%
73 · 2 HCl49 X = NH
Figure 10. Results of the acidic cleavage of the protecting groups. (See above-mentioned references for further information)
1) TES, TFA
reflux, 5 h
2) Amberlite IR-120
.
N
N
N
H
H
2N
N
N
x HCl
N
N
N
H
N
3) HCl
39
19 · HCl
70%
Scheme 3. Synthesis of 19ÁHCl.
mGAT3 and mGAT4, respectively. To allow a better comparability
of the test results obtained for the newly synthesized compounds
presented in Tables 2 and 3 with those of the reference compounds
in Table 1, the reference compounds were evaluated for their
inhibitory potencies at the individual GABA transporters employ-
ing the test system of our group, too. For 7 and 10 published results
are given in parentheses (Table 1). NNC 05-2090 (7) has been char-
acterized as mGAT2 selective inhibitor by Thomsen et al.28 This
selectivity, however, could not be reproduced applying the test
system established in our group. Still, NNC 05-2090 (7) has been
found to be a potent inhibitor of GABA uptake, albeit with no
reasonable selectivity for one of the transporter subtypes (Table 1,
entry 3). (S)-SNAP-5114 (10), originally published by Dhar et al.,31
shows high activity at mGAT3 (rGAT-2) and, especially, at mGAT4
(and hGAT-3, respectively), with a good subtype selectivity (>57:1)
for mGAT4, as compared to mGAT1 and mGAT2 (Table 1, entry 4).§
In Table 2 the results of the biological evaluation of the amino
acid analogs are summarized. The tetrazole analog of glycine,
§
For compounds reducing [3H]GABA uptake by not more than 50% at 1 mM a
pIC50 6 3 and at 100 lM a pIC50 6 4 is assumed.

6498
E. S. Schaffert et al. / Bioorg. Med. Chem. 19 (2011) 6492–6504
Table 2
Uptake inhibition at mGAT1–mGAT4
R
N
N
N
H
N
N
H
Entry
1
mGAT1 (%)
85.1a
mGAT2 (%)
59.4a
mGAT3 (%)
66.5a
mGAT4 (%)
78.9a
No.
R
19ÁHCl
H
2
3
4
5
6
64ÁHCl
65ÁHCl
66ÁHCl
67ÁHCl
68ÁHCl
69.7
48.2
100
94.2
85.8
53.2
89.3
82.0
94.2
63.5
72.6
75.1
46.2
85.4
71.2
100
82.0
80.5
87.4
79.6
7
8
69ÁHCl
70ÁHCl
86.1
86.3
76.9
81.6
73.8
83.6
101
78.7
N
9
71ÁHCl
105
65.3
74.0
71.5
10
74Á2HCl
81.7a
80.0a
86.3a
60.7a
H2N
N
N
N
X
N
H
N
Entry
mGAT1
mGAT2
mGAT3
mGAT4
No.
X
NH
O
11
12
73Á2HCl
90.0a
90.6a
75.4a
88.0a
102a
101a
72ÁHCl
87.8a
90.8a
Percentages represent specific binding remaining in presence of 100
lM inhibitor.
a
1 mM inhibitor.
19ÁHCl, gave results comparable to the amino acid 18 itself. Thus,
at mGAT2 and mGAT3 moderate inhibition of GABA-uptake was
measurable (Table 2, entry 1). The larger size of the tetrazole com-
pared to the carboxylic acid apparently did not result in a higher
activity, for example comparable to b-alanine (12). When it comes
to the lipophilic derivatives of 19, contrary to expectations, no en-
hanced activity was observed. Regardless the length of the spacer,
no beneficiary effect of the bis-aromatic substituents as compared
to a pure alkyl-residue was found (Table 2, entries 2–7, and 9). Nei-
ther did substitution with a carbazolylpropyl residue, which con-
tributes to the activity of NNC 05-2090 (10), result in an increase
in potency (Table 2, entry 8).
As far as the cyclic amino acid analogs 73Á2HCl and 72ÁHCl are
concerned, both showed only negligible activity at the four GABA

E. S. Schaffert et al. / Bioorg. Med. Chem. 19 (2011) 6492–6504
6499
Table 3
Uptake inhibition at mGAT1–mGAT4
R1
N
N
N
H
N
Entry
mGAT1 (%)
mGAT2
mGAT3
mGAT4
56.9%
R2
N
No.
R1
R2
1
2
39
85.6
54.8
81.4%
76.3%
4.22 0.11
4.61 0.09
40
41
4.54 0.05
3
81.1
84.5%
4.79 0.13
57.2%
4
5
42
43
83.3
59.1
63.5%
75.9%
53.8%
4.17 0.10
4.35 0.09
4.37 0.10
6
44
72.4
67.1%
4.35 0.09
52.3%
7
8
45
46
70.7
54.6
77.1%
74.8%
5.09 0.13
4.44 0.02
4.53 0.04
4.46 0.09
9
47
73.9
61.6%
51.7%
4.20 0.15
10
11
48
49
63.8
70.6
4.78 0.13
4.66 0.16
4.89 0.02
4.76 0.05
4.53b
43.2%
12
50
68.1
4.81 0.25
4.39c
49.6%
13
14
51
52
65.7
62.9
4.99 0.04
4.61 0.08
4.64b
4.74b
4.58b
4.29b
15
53
57.3
4.61 0.72
4.46b
4.38b
16
17
54
55
75.4
64.5
54.9%
69.5%
4.91 0.17
4.67 0.10
4.74 0.13
58.2%
18
19
56
69.4
75.1
84.1%
72.6%
53.5%
68.8%
53.1%
59.7%
57
61
N
20
21
83.3
133a
60.3%
142%a
49.0%
4.31b
63Á2HCl
71.1%a
92.5%a
H
2N
N
N
N
N
X
N
Entry
No.
mGAT1
mGAT2
mGAT3
mGAT4
R
X
O
R
22
23
58
62
73.4
90.2
99.0%
106.0%
63.3%
81.4%
90.5%
78.4%
NHBoc
If not stated otherwise, the results are given as pIC50 SEM of three independent experiments, each performed in triplicate. Percentages represent specific binding remaining
in presence of 100 M inhibitor.
l
a
1 mM inhibitor.
One experiment, performed in triplicate.
Mean of two independent experiments, each performed in triplicate.
b
c

6500
E. S. Schaffert et al. / Bioorg. Med. Chem. 19 (2011) 6492–6504
transport proteins (Table 2, entries 11–12). The diamine 74Á2HCl,
too, was not able to considerably reduce GABA uptake (Table 2, en-
try 10).
subtypes (Table 3, entries 21–23). These results suggest that for the
activity of 1,5-disubstituted aminomethyltetrazoles of the general
structure 21, a lipophilic side chain containing a bis-aromatic sub-
structure is necessary.
A totally different pharmacological profile was obtained for the
1,5-disubstituted tetrazole-derivatives. Although, just like the 5-
monosubstituted tetrazoles, all 1,5-disubstituted tetrazole deriva-
tives showed only low to marginal inhibition of GABA uptake at
mGAT1, considerable inhibition of the non-GAT1 transporters
was observed for some of the test compounds (Table 3). Thus,
the aminomethyltetrazole derivatives substituted with a diphenyl-
methyl group at the amine nitrogen, 39–41, were found to be mod-
erately active inhibitors of mGAT3 and mGAT4, with a slightly
higher potency at mGAT3 (Table 3, entries 1–3). Compounds 42–
44, bearing a diphenylethyl residue at the amine nitrogen, how-
ever, proved to be somewhat less potent as compared to 39–41
(Table 3, entries 4–6). When it comes to the diphenylpropyl-deriv-
atives, 45–47, pronounced differences in activity were observed
between the in 1-position differently substituted tetrazole deriva-
tives (Table 3, entries 7–9). Here, the tetramethylbutyl derivative
45 displayed the highest inhibitory potency at mGAT3, combined
with a reasonable subtype selectivity for this transporter subtype
3. Conclusion
In summary, 1,5-disubstituted and 5-monosubstituted tetrazole
derivatives derived from glycine (18) have been synthesized. All
compounds have been evaluated regarding their inhibitory po-
tency and subtype selectivity at the four murine GABA transporter
subtypes mGAT1–mGAT4. The results for the N-substituted scaf-
fold type 20 were far from satisfying. None of the amino acid ana-
logs was capable of inhibiting the [3H]GABA uptake by more than
approximately 50% at the highest concentration employed (1 mM
and 100 lM, respectively). But, interestingly, the 1,5-disubstituted
tetrazole derivatives belonging to structural type 21 displayed by
far higher activity, especially at the GABA transport proteins
mGAT2–mGAT4. Thereby a moderate to good selectivity against
the GABA transporter mGAT1 was observed. With 45, a reasonable
potent and selective inhibitor of mGAT3 was found. Additionally
50 and 51 were identified as potent inhibitors of mGAT2. This is
especially relevant, as up to date only few potent inhibitors of
mGAT2 that do not affect mGAT1 are known. Although no general
dependency from chain length or substituent in 1-position of the
tetrazole moiety could be determined, these compounds might
represent a good starting point for the development of a new class
of non-GAT1 selective GABA-uptake inhibitors.
(pIC50
5.09,
selectivity
mGAT3/mGAT1 = 12:1,
mGAT3/
mGAT4 = 4:1; Table 3, entry 7). Although the potency of 45 at
mGAT4 is approximately 20 times lower than the potency of (S)-
SNAP 5114 (10), its inhibitory effect on GABA-uptake at mGAT3
is comparable (Table 1, entry 4).
For 48, 49 and 50, which are substituted with a diphenylbutyl
residue at the amine nitrogen, in addition to a moderate to good
activity at the transporter subtypes mGAT3 and mGAT4, significant
inhibition of GABA uptake at mGAT2 was observed (Table 3, entries
10–12). With the tetramethylbutyl substituted tetrazole derivative
48, a compound with good activity at the non-GAT1 transporter
subtypes was identified (pIC50 mGAT2: 4.78, pIC50 mGAT3: 4.89,
pIC50 mGAT4: 4.53; Table 3 entry 10). Furthermore, good activity
at mGAT2 (pIC50 4.81) combined with a moderate selectivity to-
wards mGAT1, mGAT3, and mGAT4 (>3:1) was observed for the
benzyl derivative 50 (Table 3, entry 12).
As far as the unsaturated 51–53 and 54–56 are concerned, there
are distinct differences in the inhibitory potency compared to their
saturated analogs 45–47 and 48–50. Among the diphenylpropenyl
derivatives 51–53, especially compound 51 displayed remarkable
uptake inhibition at mGAT2 (pIC50 4.99; Table 3, entry 13), which
is comparable to the activity of NNC 05-2090 (7, pIC50 5.09; Table 1,
entry 3). But, in contrast to the results we obtained for NNC 05-
2090 (7), 51 showed good subtype selectivity as compared to
mGAT1 (selectivity mGAT2/mGAT1 P 10:1). For 52 and 53 moder-
ate inhibition of non-GAT1 transport proteins was found, but with-
out any preference for one of the three transporter subtypes
(Table 3, entries 14–15).
When it comes to the aminomethyltetrazole derivates substi-
tuted with a diphenylbutenyl residue, the activity at mGAT2 van-
ishes. Compound 56, bearing a benzyl group in 1-position of the
tetrazole, has only negligible activity at all four transporter sub-
types (Table 3, entry 18). By contrast, for 54 and 55 moderate to
good uptake inhibition at mGAT3 was observed (Table 3, entries
16–17). While 55 was found to be moderately active only at
mGAT3 (pIC50 4.67), 54 displayed good activity at mGAT3 and
mGAT4 (pIC50 mGAT3: 4.91, pIC50 mGAT4: 4.74).
Although it is substituted with a carbazolylpropyl residue,
which can also be found in NNC 05-2090 (7), scarcely any uptake
inhibition could be detected for 57 (Table 3, entry 19). Likewise,
61, with a butyl group attached to the amine nitrogen did not dis-
play particular activity at mGAT1–mGAT4 (Table 3, entry 20). Also
compounds 58, 62 and 63Á2HCl that completely lack a lipophilic
side chain but display additional rings or amino groups instead,
were found to be more or less inactive at all four GABA transporter
4. Experimental
4.1. Chemistry
4.1.1. General experimental
Anhydrous reactions were carried out in vacuum dried glass-
ware under argon atmosphere. Purchased reagents were used
without further purification. THF was freshly distilled from sodium
metal/benzophenone ketyl, and CH3OH from Mg prior to use. Other
solvents for extraction, recrystallization and column chromatogra-
phy were distilled before use. Reactions under microwave irradia-
tion were performed in sealed glass vials in a Biotage Initiator
laboratory microwave. Column chromatography (CC) was per-
formed as flash-column chromatography according to Still54 on sil-
ica gel 60 (0.035–0.070 mm, Acros and Merck). TLC plates were
made from silica gel 60 F254 (Merck). Detection was effected in
UV or by staining the TLC plates with 5% (NH4)6Mo7O24 Â 4H2O
and 0.2% Ce(SO4)2 Â 4H2O in 5% H2SO4. MPLC purification was per-
formed using a SepacoreÒ column B-685 (26 Â 230 mm) filled with
silica gel RP 18 as stationary phase. Solvents for MPLC were of p.a.
quality and degassed prior to use. Melting points were determined
on a Büchi 510 melting point apparatus or a Barnstead Electrother-
mal melting pot apparatus and are uncorrected. IR spectra were re-
corded on a Perkin Elmer FT-Infrared Spectrometer Paragon 1000.
Solids were measured as KBr pellets, oily samples as film on NaCl
plates. 1H and 13C NMR spectra were recorded at room tempera-
ture on a JEOL JNMR-ECP 400 or a JEOL JNMR-ECP 500 using TMS
as internal standard. Samples without TMS were adjusted to the
signal of the partially deuterated solvent. For samples in D2O, diox-
ane was added as internal standard. NMR spectra were analyzed
with the NMR software MestReNova, Version 5.1.0-2940 (Mestr-
elab Research S.L.). Mass spectra were measured on a Mass Spec-
trometer 5989 A with 59980 B particle beam LC/MS interface
(Hewlett Packard) or an Applied Biosystems LC–MS/MS-Mass
Spectrometer API 2000. High resolution mass spectrometry
(HRMS) was accomplished with a LTQ FT (ThermoFinnigan) or a

E. S. Schaffert et al. / Bioorg. Med. Chem. 19 (2011) 6492–6504
6501
JMS GCmate II (Jeol). Micoranalytical data for carbon, hydrogen,
and nitrogen were determined on an Elementar Vario Micro Cube
or an Elementar Vario EL analyzer.
calcd for C22H28N5, 362.2340; found: 362.2340. Anal. calcd for C
73.10, H 7.53, N 19.37. Found: C 72.68, H 7.37, N 19.29.
4.1.3.3. (3,3-Diphenylpropyl)-[1-(1,1,3,3-tetramethylbutyl)-1H-
tetrazol-5-ylmethyl]amine (45) and (3,3-diphenylpropyl)-bis-
[1-(1,1,3,3-tetramethylbutyl)-1H-tetrazol-5-ylmethyl]amine
(38). Method A: According to GP1 starting from parafor-
maldehyde (58.6 mg, 1.95 mmol), 3,3-diphenylpropylamine
(805.6 mg, 3.812 mmol, 2 equiv), 1,1,3,3-tetramethylbutyl isocya-
4.1.2. General procedures
4.1.2.1. General procedure 1 (GP1). To a solution of paraformal-
dehyde (1.0 mmol) in MeOH (10.0 mL) the respective amine (1.0
or 2.0 mmol), the isocyanide (1.0 mmol) and TMSN3 (1.0 mmol)
were added. The resulting solution was stirred at 80 °C in the
microwave for 1.5 h. After addition of phosphate buffer solution
(1 M, pH 7) and water (approx. 10 mL each) the crude product
was extracted with CH2Cl2 (5 Â approx. 20 mL). The combined or-
ganic layers were dried (Na2SO4) and the solvent was removed in
vacuo. Further purification of the tetrazole product was performed
as specified.
nide (265.2 mg, 1.905 mmol, 334.0
lL) and TMSN3 (219.0 mg,
1.901 mmol, 250.0 L). After purification by CC (n-pentane/
l
EtOAc = 4:6) 45 was isolated as sole product.
Compound 45. Colorless oil (709.3 mg, 92%). Rf = 0.40 (n-pen-
tane/EtOAc = 4:6). IR: 3324, 3026, 2952, 2871, 1808, 1599, 1583,
1493, 1471, 1451, 1233, 1114, 751, 702 cmÀ1 1H NMR (500 MHz,
.
CDCl3) d = 0.76 (s, 9H, (CH3)3), 1.76 (s, 6H, NC(CH3)2), 1.78 (sbr
,
4.1.2.2. General procedure for the acidic cleavage of the tmb-
group (GP2). The protected tetrazole derivative was treated with
an excess (approx. 15–19 equiv) of HClEtOH (ꢀ1.25 M) and was re-
fluxed overnight (8–13 h). The solvent was removed in vacuo and
the oily residue was treated with HClether. In doing so, the product
precipitated as a colorless solid. After the precipitation was com-
plete, the product was filtered off and washed with Et2O, if
necessary.
1H, NH), 1.96 (s, 2H, CH2C(CH3)3), 2.28 (q, J = 7.5 Hz, 2H, CH2CH),
2.66 (t, J = 7.2 Hz, 2H, CH2CH2NH), 4.05 (t, J = 7.9 Hz, 1H, CH2CH),
4.08 (s, 2H, NCH2C), 7.14–7.19 (m, 2H, Har,para), 7.21–7.30 (m, 8H,
Har,meta, Har,ortho)
ppm. 13C NMR (125 MHz, CDCl3) d = 30.16,
30.85, 31.81, 35.95, 45.16, 48.43, 49.02, 53.42, 64.79, 126.47,
127.96, 128.72, 144.70, 153.52 ppm. M (C25H35N5) = 405.59. MS
(CI, CH5+) m/z (%): 406 (8, [M+H]+), 294 (100), 210 (10). HRMS
(EI, 70 eV): M+ calcd for C25H35N5, 405.28925; found: 405.29145.
Method B: When the synthesis of 45 was conducted according to
GP1 starting from paraformaldehyde (60.6 mg, 2.02 mmol), 3,3-
diphenylpropylamine (408.4 mg, 1.933 mmol, 1 equiv), 1,1,3,3-tet-
4.1.3. Individual procedures
4.1.3.1. Benzhydryl-(1-benzyl-1H-tetrazol-5-ylmethyl)amine
(41). According to GP1 starting from paraformaldehyde (57.1 mg,
ramethylbutyl isocyanide (265.2 mg, 1.905 mmol, 334.0
lL) and
1.90 mmol), benzhydrylamine (349.0 mg, 1.904 mmol, 328.0
benzyl isocyanide (223.2 mg, 1.905 mmol, 232.0 L) and TMS azide
(115.6 mg, 1.003 mmol, 132.0 L). Purification by CC (n-pentane/
l
L),
TMSN3 (219.0 mg, 1.901 mmol, 250.0 L), besides approx. 86% of
the Ugi product 45 the byproduct 38 occurred. 38 was isolated
by CC (n-pentane/EtOAc = 1:1).
l
l
l
EtOAc = 1:1) yielded a yellowish oil that was diluted in mobile
phase (approx. 2 mL). After ultrasonication of the solution for ap-
prox. 30 s the product precipitated as colorless solid and was iso-
lated by filtration.
Compound 38. Colorless solid. (49.4 mg, 9%); mp: 116–117 °C.
Rf = 0.71 (n-pentane/EtOAc = 1:1). IR: 3059, 3028, 2954, 2870,
1952, 1881, 1598, 1450, 1375, 1229, 1114, 1093, 771, 708 cmÀ1
.
1H NMR (500 MHz, CDCl3) d = 0.61 (s, 18H, (CH3)3), 1.63 (s, 12H,
NC(CH3)2), 1.78 (s, 4H, CH2C(CH3)3), 2.21–2.27 (m, 2H,
CH2CH(C6H5)2), 2.85–2.90 (m, 2H, NCH2CH2CH), 3.97 (t, J = 7.8 Hz,
1H, CH(C6H5)2), 4.34 (s, 4H, NCH2C), 7.05–7.11 (m, 2H, Har,para),
7.14–7.20 (m, 8H, Har,meta, Har,ortho) ppm. 13C NMR (100 MHz,
CDCl3) d = 30.19, 30.81, 31.76, 33.21, 47.97, 48.69, 53.17, 53.46,
Compound 41. Colorless solid (337.7 mg, 95%); mp 132 °C.
Rf = 0.63 (n-pentane/EtOAc). IR: 3026, 2817, 1964, 1825, 1583,
1532, 1492, 1456, 1111, 752, 707, 713, 700 cmÀ1 1H NMR
.
(400 MHz, CDCl3) d = 2.11 (sbr, 1H, NH), 3.90 (s, 2H, CH2NH), 4.80
(s, 1H, CH(C6H5)2), 5.53 (s, 2H, CH2C6H5), 7.01–7.09 (m, 2H,
Hortho,Bn), 7.21–7.38 (m, 13H, Har) ppm. 13C NMR (100 MHz, CDCl3)
d = 40.43, 51.14, 66.89, 127.45, 127.73, 127.77, 128.94, 128.99,
129.31, 133.67, 142.62, 153.48 ppm. M (C22H21N5) = 355.45. MS
(CI, CH5+) m/z (%): 356 (25, [M+H]+), 182 (20), 167 (100). HRMS
(ESI+): [M+H]+ calcd for C22H22N5, 356.1870. Found: 356.1862.
64.90, 126.57, 127.95, 128.78, 144.43, 152.20 ppm.
M
(C35H53N9) = 613.90. MS (CI, CH5+) m/z (%): 600 (25, [M+H]+), 488
(100), 376 (52), 113 (29). HRMS (EI, 70 eV): M+ calcd for
C35H53N9, 599.4424; found: 599.4457.
4.1.3.4.
hydrochloride
(3,3-Diphenylpropyl)-(1H-tetrazol-5-ylmethyl)amine
of 45 (142.0 mg,
4.1.3.2.
(1-Cyclohexyl-1H-tetrazol-5-ylmethyl)-(2,2-diphenyl-
(66ÁHCl). Deprotection
ethyl)amine (43). According to GP1 starting from paraformalde-
hyde (59.0 mg, 1.97 mmol), 2,2-diphenylethylamine (754.0 mg,
3.822 mmol), cyclohexyl isocyanide (208.1 mg, 1.906 mmol,
0.3501 mmol) according to GP2 using HClEtOH (ꢀ1.25 M, 10 mmol,
8.0 mL, 29 equiv). Reaction time: 8 h. After removal of the excess
solvent and addition of HCl in Et2O the formed precipitate was
washed with EtOAc.
237.0 lL) and TMSN3 (219.0 mg, 1.901 mmol, 250.0 lL). Purifica-
tion by CC (n-pentane/EtOAc = 4:6) and subsequent recrystalliza-
tion from CH2Cl2/n-pentane (3:7) yielded 43.
Compound 66. HCl. Colorless crystals (75.1 mg, 65%); mp 168 °C.
IR: 3440, 3059, 2957, 2758, 1959, 1869, 1598, 1493, 1493, 1450,
Compound 43. Colorless crystals (633.1 mg, 92%); mp 109 °C.
Rf = 0.37 (n-pentane/EtOAc = 4:6). IR: 3300, 3025, 2934, 2861,
1020, 749, 704 cmÀ1 1H NMR (500 MHz, CD3OD) d = 2.47–2.56
.
(m, 2H, CH2CH), 3.08–3.15 (m, 2H, CH2CH2CH), 4.07 (t, J = 7.9 Hz,
1H, CH(C6H5)2), 4.60 (s, 2H, CCH2NH), 7.17–7.23 (m, 2H, Har,para),
7.27–7.33 (m, 8H, Har,ortho, Har,meta) ppm. 13C NMR (125 MHz,
1946, 1599, 1494, 1449, 1438, 1105, 742, 696 cmÀ1 1H NMR
.
(500 MHz, CDCl3) d = 1.22–1.41 (m, 3H, CHCH2CH2CH2, CHCH2
CH2CH2,ax), 1.56 (sbr, 1H, NH), 1.69–1.77 (m, 1H, CHCH2CH2CH2,eq),
1.85–2.01 (m, 6H, CHCH2CH2CH2, CHCH2,axCH2CH2, CHCH2
CH2,eqCH2), 3.26 (d, J = 7.6 Hz, 2H, CH2CH), 4.09 (s, 2H, NCH2C),
4.14 (t, J = 7.6 Hz, 1H, CH2CH), 4.31 (tt, J = 10.9/4.3 Hz, 1H,
CHCH2CH2CH2), 7.18–7.23 (m, 6H, Har,ortho, Har,para), 7.25–7.31 (m,
4H, Har,meta) ppm. 13C NMR (125 MHz, CDCl3) d = 25.03, 25.46,
32.95, 42.40, 51.32, 54.02, 58.12, 126.93, 128.13, 128.90, 142.54,
152.27 ppm. M (C22H27N5) = 361.49. MS (CI, CH5+) m/z (%): 362
(100, [M+H]+), 210 (32), 194 (48), 112 (22). HRMS (ESI+): [M+H]+
CD3OD) d = 32.67, 41.93, 48.17, 49.84, 127.88, 128.73, 129.86,
+
144.68, 155.88 ppm. M (C17H19N5 Â HCl) = 329.83. MS (CI, CH5
)
m/z (%): 294 (24, [M+H]+), 212 (91), 145 (100), 103 (81). HRMS
(ESI+): [M+H]+ calcd for C17H20N5, 294.1713; found: 294.1713.
4.1.3.5. (4,4-Diphenylbutyl)-[1-(1,1,3,3-tetramethylbutyl)-1H-
tetrazol-5-ylmethyl]amine (48). According to GP1 starting from
paraformaldehyde (17.5 mg, 0.583 mmol), 4,4-diphenylbutyl-
amine
(225.1 mg,
0.9989 mmol),
1,1,3,3-tetramethylbutyl

6502
E. S. Schaffert et al. / Bioorg. Med. Chem. 19 (2011) 6492–6504
isonitrile (69.9 mg, 0.502 mmol, 88.0
l
L) and trimethylsilyl azide
H
ar,ortho), 7.20–7.40 (m, 8H, Har) ppm. 13C NMR (100 MHz, CDCl3)
(57.8 mg, 0.502 mmol, 66.0
EtOAc = 1:1) gave a colorless oil.
l
L). Purification by CC (n-pentane/
d = 29.92, 30.63, 31.61, 44.18, 48.34, 53.15, 64.67, 125.91, 127.42,
127.48, 127.51, 128.19, 128.29, 129.67, 139.22, 141.77, 144.66,
153.04 ppm. M (C25H33N5) = 403.58. MS (CI, CH5+) m/z (%): 404
(3, [M+H]+), 191 (31), 208 (10), 193 (100). HRMS (EI, 70 eV): M+
calcd for C25H33N5, 403.27360; found: 403.27476.
Compound 48. Colorless oil (202.9 mg, 96%). Rf = 0.61 (n-pen-
tane/EtOAc = 35:65). IR: 3329, 3025, 2950, 1948, 1806, 1673,
1599, 1493, 1451, 1231, 1114, 749, 702 cmÀ1 1H NMR (500 MHz,
.
CDCl3) d = 0.76 (s, 9H, (CH3)3), 1.46–1.55 (m, 2H, CH2CH2CH2),
1.78 (s, 6H, NC(CH3)2), 1.96 (s, 2H, CH2C(CH3)3), 2.07–2.13 (m,
2H, CH2CH(C6H5)2), 2.34 (sbr, 1H, NH), 2.74 (t, J = 7.1 Hz, 2H,
HNCH2CH2), 3.89 (t, J = 7.8 Hz, 1H, CH(C6H5)2), 4.13 (s, 2H,
CCH2NH), 7.14–7.18 (m, 2H, Har,para), 7.20–7.24 (m, 4H, Har,ortho),
7.24–7.29 (m, 4H, Har,meta) ppm. 13C NMR (125 MHz, CDCl3)
d = 28.18, 29.91, 30.64, 31.60, 33.15, 44.71, 49.50, 51.14, 53.20,
4.1.3.9. (3,3-Diphenylallyl)-(1H-tetrazol-5-ylmethyl)amine hydro-
chloride (68ÁHCl). Deprotection of 51 (195.6 mg, 0.4846 mmol)
according to GP2 using HClEtOH (ꢀ1.25 M, 7.5 mmol, 6.0 mL,
15 equiv). Reaction time 8 h.
Compound 68. HCl. Pale yellow solid (128.8 mg, 81%); mp 142 °C
dec. IR: 3433, 3057, 2944, 2735, 2647, 2468, 2345, 1773, 1627,
64.67, 126.16, 127.78, 128.44, 144.79, 153.05 ppm.
M
1446, 1110, 1053, 759, 706 cmÀ1 1H NMR (500 MHz, CD3OD)
.
(C26H37N5) = 419.62. MS (CI, CH5+) m/z (%): 420 (9, [M+H]+), 308
(100). HRMS (EI, 70 eV): M+ calcd for C26H37N5, 419.30490; found:
419.30567.
d = 3.94 (d, J = 7.1 Hz, 2H, CH2CH@), 4.61 (s, 2H, HNCH2C), 6.20
(tbr, J = 7.1 Hz, 1H, CH@C), 7.15–7.20 (m, 2H, Har,ortho), 7.25–7.30
(m, 2H, Har,ortho), 7.30–7.35 (m, 3H, Har,meta, Har,para), 7.38–7.47
(m, 3H, Har,meta
d = 41.28, 47.78, 117.59, 128.75, 129.51, 129.53, 129.70, 129.86,
, Har,para)
ppm. 13C NMR (125 MHz, CD3OD)
4.1.3.6. (1-Benzyl-1H-tetrazol-5-ylmethyl)-(4,4-diphenylbutyl)
amine (50). According to GP1 starting from paraformaldehyde
(15.0 mg, 0.500 mmol), 4,4-diphenylbutylamine (205.4 mg,
130.61, 139.22, 142.03, 151.44, 155.84 ppm.
M
(C17H17N5 Â
HCl) = 327.82. MS (CI, CH5+) m/z (%): 292 (6, [M+H]+), 193 (100),
113 (40), 101 (60). HRMS (ESI+): [M+H]+ calcd for C17H18N5,
292.1556; found: 292.1556.
0.9115 mmol), benzyl isocyanide (58.7 mg, 0.501 mmol, 61.0
l
L)
and TMS azide (57.8 mg, 0.502 mmol, 66.0
lL). Purification by CC
(n-pentane/EtOAc = 1:1).
Compound 50. Pale yellow oil (139.2 mg, 70%). Rf = 0.16 (n-pen-
4.1.3.10. (4,4-Diphenylbut-3-en-1-yl)-[1-(1,1,3,3-tetramethylbu-
tyl)-1H-tetrazol-5-ylmethyl]amine (54). According to GP1 start-
ing from paraformaldehyde (15.3 mg, 0.509 mmol), 4,4-
diphenylbut-3-en-1-ylamine (225.0 mg, 1.008 mmol), 1,1,3,3-tet-
tane/EtOAc = 1:1). IR: 3331, 3060, 3026, 2934, 2859, 1952, 1812,
1599, 1494, 1452, 1360, 1115, 725, 702 cmÀ1 1H NMR (500 MHz,
.
CDCl3) d = 1.36–1.43 (m, 2H, CH2CH2CH2), 1.64 (sbr, 1H, NH),
1.99–2.06 (m, 2H, CH2CH), 2.56 (t, J = 7.1 Hz, 2H, HNCH2CH2),
3.84 (t, J = 7.8 Hz, 1H, CH(C6H5)2), 3.90 (s, 2H, CCH2NH), 5.62 (s,
2H, CH2C6H5), 7.14–7.22 (m, 8H, Har), 7.24–7.34 (m, 7H, Har)
ppm. 13C NMR (100 MHz, CDCl3) d = 28.21, 33.07, 42.22, 49.33,
ramethylbutyl isocyanide (69.9 mg, 0.502 mmol, 88.0
l
L) and
TMSN3 (57.8 mg, 0.502 mmol, 66.0
lL). Purification by CC (n-pen-
tane/EtOAc = 1:1) yielded 54.
Compound 54. Pale yellow oil (171.9 mg, 82%). Rf = 0.32 (n-pen-
51.05, 51.11, 126.21, 127.70, 127.74, 128.48, 128.84, 129.11,
tane/EtOAc = 1:1). IR: 3329, 3022, 2952, 2917, 1950, 1597, 1444,
1113, 761, 700 cmÀ1 1H NMR (500 MHz, CDCl3) d = 0.75 (s, 9H,
.
+
133.52, 144.72, 153.15 ppm. M (C25H27N5) = 397.53. MS (CI, CH5
)
m/z (%): 398 (100, [M+H]+), 238 (15). HRMS (EI, 70 eV): M+ calcd
(CH3)3), 1.79 (s, 7H, NC(CH3)3, NH), 1.97 (s, 2H, CH2C(CH3)3), 2.36
(q, J = 7.1 Hz, 2H, CH2CH), 2.82 (t, J = 7.0 Hz, 2H, CH2CH2NH), 4.11
(s, 2H, NCH2C), 6.10 (t, J = 7.4 Hz, 1H, CH2CH), 7.15–7.19 (m, 2H,
for C25H27N5, 397.22665; found: 397.22747.
4.1.3.7. (4,4-Diphenylbutyl)-(1H-tetrazol-5-ylmethyl)amine
hydrochloride (67ÁHCl). According to GP2 starting from 48
(190.2 mg, 0.4533 mmol) in HClEtOH (ꢀ1.25 M, 8.8 mmol, 7.0 mL,
19 equiv). The reaction mixture was refluxed for 8 h.
Har,ortho), 7.19–7.32 (m, 6H, Har), 7.33–7.39 (m, 2H, Har,meta) ppm.
13C NMR (125 MHz, CDCl3) d = 29.94, 30.20, 30.66, 31.61, 44.67,
49.51, 53.24, 64.69, 126.46, 127.08, 127.21, 128.11, 128.27,
129.82, 139.87, 142.34, 143.61, 153.21 ppm.
M (C26H35N5) =
Compound 67. HCl. Colorless solid (101.4 mg, 65%); mp 154–
156 °C dec IR: 3081, 3024, 2954, 2767, 2414, 1954, 1718, 1597,
417.60. MS (CI, CH5+) m/z (%): 418 (15, [M+H]+), 306 (100), 112
(36). HRMS (EI, 70 eV): M+ calcd for C26H35N5, 417.28925; found:
417.28989. C26H35N5 Anal. calcd for C, 74.78; H, 8.45; N, 16.77.
Found: C, 74.60; H, 8.70; N, 16.79.
1492, 1450, 1023, 745, 703 cmÀ1 1H NMR (400 MHz, CD3OD)
.
d = 1.65–1.77 (m, 2H, CCH2CH2CH2N), 2.12–2.23 (m, 2H,
CCH2CH2CH2N), 3.14–3.25 (m, 2H, CCH2CH2CH2N), 3.97 (t,
J = 7.9 Hz, 1H, CH(C6H5)2), 4.57 (s, 2H, CCH2NH), 7.11–7.21 (m,
2H, Har,para), 7.22–7.33 (m, 8H, Har,meta, Har,ortho) ppm. 13C NMR
(100 MHz, CD3OD) d = 25.97, 33.36, 41.92, 49.11, 52.15, 127.47,
128.87, 129.64, 145.81, 155.80 ppm. M (C18H21N5 Â HCl) = 343.86.
MS (CI, CH5+) m/z (%): 308 (100, [M+H]+), 193 (39). HRMS (ESI+):
[M+H]+ calcd for C18H22N5, 308.1869; found: 308.1868.
4.1.3.11. (4,4-Diphenylbut-3-en-1-yl)-(1H-tetrazol-5-ylmethy-
l)amine hydrochloride (69ÁHCl). According to GP2 starting from
54 (91.7 mg, 0.220 mmol) and HClEtOH (ꢀ1.25 M, 3.0 mL, 3.8 mmol,
17 equiv). Reaction time: 9 h.
Compound 69. HCl. Colorless solid (58.3 mg, 78%); mp 158 °C.
IR: 3055, 2927, 2754, 2684, 1556, 1457, 2362, 1956, 1889, 1598,
1555, 1443, 1053, 866, 765, 703 cmÀ1
.
1H NMR (500 MHz,
4.1.3.8. (3,3-Diphenylallyl)-[1-(1,1,3,3-tetramethylbutyl)-1H-tet-
razol-5-ylmethyl]amine (51). According to GP1 starting from
paraformaldehyde (59.3 mg, 1.98 mmol), 3,3-diphenylallylamine
(807.0 mg, 3.856 mmol), 1,1,3,3-tetramethylbutyl isocyanide
CD3CD2OD) d = 2.59–2.67 (m, 2H, CH2CH), 3.30–3.37 (m, 2H,
NCH2CH2), 4.62 (s, 2H, CCH2NH), 6.12 (t, J = 7.3 Hz, 1H, CH@C),
7.15–7.19 (m, 2H, Har,ortho), 7.19–7.27 (m, 5H, Har), 7.31–7.36 (m,
1H, Har,para), 7.38–7.43 (m, 2H, Har,meta) ppm. 13C NMR (125 MHz,
CD3CD2OD) d = 27.47, 41.62, 48.34, 122.86, 128.08, 128.31,
128.36, 128.92, 129.40, 130.41, 140.28, 142.68, 146.71,
155.28 ppm. M (C18H19N5 Â HCl) = 341.85. MS (ESI+) m/z: 611
([2 M+H]+), 306 ([M+H]+). HRMS (ESI+): [M+H]+ calcd for
(265.2 mg, 1.905 mmol, 334.0
lL) and trimethylsilyl azide
(219.0 mg, 1.901 mmol, 250.0 L). Purification by CC (n-pentane/
l
EtOAc = 35:65 und 1:1) yielded 91% of 51.
Compound 51. Yellow oil (701.7 mg, 91%). Rf = 0.38 (n-pentane/
EtOAc = 1:1). IR: 3321, 3022, 2954, 1953, 1678, 1598, 1492, 1444,
C18H20N5, 306.1713; found: 306.1711.
1234, 1114, 759, 701 cmÀ1 1H NMR (400 MHz, CDCl3) d = 0.76 (s,
.
9H, (CH3)3), 1.79 (s, 6H, NC(CH3)3), 1.97 (s, 2H, CH2C(CH3)3), 2.41
(sbr, 1H, NH), 3.47 (d, J = 6.9 Hz, 2H, HNCH2CH), 4.11 (s, 2H,
CCH2NH), 6.19 (t, J = 6.9 Hz, 1H, CH2CH = C), 7.14–7.18 (m, 2H,
4.1.3.12. (3-Carbazol-9-ylpropyl)-1-[1-(1,1,3,3-tetramethylbu-
tyl)-1H-tetrazol-5-ylmethyl]amine (57). According to GP1 start-
ing from paraformaldehyde (30.4 mg, 1.01 mmol), 3-carbazol-9-

E. S. Schaffert et al. / Bioorg. Med. Chem. 19 (2011) 6492–6504
6503
ylpropylamine (225.8 mg, 1.007 mmol), 1,1,3,3-tetramethylbutyl
isonitrile (139.7 mg, 1.004 mmol, 176.0 L) and TMSN3
L). Purification by CC (n-pentane/
3 mL) and extracted with Et2O (3 Â 4 mL). The combined organic
phases were dried (Na2SO4) and evaporated to dryness, to yield a
colorless solid.
l
(115.6 mg, 1.004 mmol, 132.0
EtOAc = 6:4).
l
Compound 62. Colorless solid (46.0 mg, 99%); mp 89–91 °C. IR:
Compound 57. Colorless solid (236.3 mg, 56%); mp 153 °C.
Rf = 0.16 (n-pentane/EtOAc = 6:4). IR: 3316, 3053, 2957, 1897,
3302, 2947, 2806, 1464, 1394, 1229, 1120, 823, 806 cmÀ1 1H
.
NMR (500 MHz, CDCl3) d = 0.82 (s, 9H, (CH3)3), 1.66 (sbr, 1H, NH),
1.84 (s, 6H, (NC(CH3)2), 2.09 (s, 2H, CH2C(CH3)3), 2.48 (sbr, 4H,
NCH2CH2NH), 2.86 (t, J = 4.8 Hz, 4H, NCH2CH2NH), 3.82 (s, 2H,
CCH2N) ppm. 13C NMR (125 MHz, CDCl3) d = 29.62, 30.81, 31.66,
45.77, 52.86, 53.35, 54.41, 65.66, 150.92 ppm. M (C14H28N6) =
280.42. MS (CI, CH5+) m/z (%): 281 (32, [M+H]+), 169 (100). HRMS
(EI, 70 eV): M+ calcd for C14H28N6, 280.2376; found: 280.2400.
1626, 1593, 1484, 1454, 1326, 837, 754, 727 cmÀ1 1H NMR
.
(500 MHz, CDCl3) d = 0.66 (s, 9H, (CH3)3), 1.69 (s, 6H, NC(CH3)2),
1.70 (sbr, 1H, NH) 1.83 (s, 2H, CH2C(CH3)3), 2.02 (p, J = 6.7 Hz, 2H,
CH2CH2CH2), 2.62 (t, J = 6.7 Hz, 2H, CH2CH2NH), 4.00 (s, 2H,
CCH2NH), 4.38 (t, J = 6.7 Hz, 2H, CH2NC), 7.15 (ddd, J = 7.8/5.1/
2.8 Hz, 2H, NCCHCHCH), 7.34–7.41 (m, 4H, NCCH, NCCHCH), 8.02
(dt, J = 7.8/0.9 Hz, 2H, NCCCH) ppm. 13C NMR (125 MHz, CDCl3)
d = 29.54, 30.21, 30.84, 31.81, 40.68, 45.38, 47.03, 53.41, 64.67,
108.84, 119.08, 120.57, 123.05, 125.87, 140.63, 153.61 ppm. M
(C25H34N6) = 418.59. MS (CI, CH5+) m/z (%): 419 (52, [M+H]+), 307
(100). HRMS (EI, 70 eV): M+ calcd for C25H34N6, 418.2845; found:
418.2849. Anal. calcd for C, 71.74; H, 8.19; N, 20.08. Found: C,
71.38; H, 8.23; N, 19.86.
4.1.3.16. 1-(1H-Tetrazol-5-ylmethyl)piperazine dihydrochloride
(73Á2HCl). Compound 59 (185.4 mg, 0.4872 mg) was treated with
HClEtOH (ꢀ1.25 M, 18.8 mmol, 15.0 mL, 19 equiv) and stirred for
29 h at 40 °C. In doing so, 73Á2HCl precipitated and could be iso-
lated by filtration.
Compound 73. 2HCl. Colorless solid (107.3 mg, 91%); mp 236 °C
dec IR: 3423, 3017, 3000, 2949, 2726, 2366, 2345, 1563, 1458,
4.1.3.13.
(3-Carbazol-9-ylpropyl)-(1H-tetrazol-5-ylmethyl)a-
1430, 1388, 1210, 1165, 1063, 1026, 968, 858 cmÀ1 1H NMR
.
mine hydrochloride (70ÁHCl). Deprotection of 57 (156.6 mg,
0.3693 mmol) according to GP2 in HClEtOH (ꢀ1.25 M, 6.3 mmol,
5.0 mL, 17 equiv). Reaction time 3 h reflux, 16 h rt. In deviation from
GP2, the product was purified by prep. MPLC (RP18, CH3CN/
H2O = 1:9–10:0, UV 220 nm). After chromatographic purification
the product was dissolved in an excess of HClEtOH. The solvent was
removed in vacuo and 68ÁHCl was obtained as a pale yellow solid.
Compound 70. HCl. Pale yellow solid (120.7 mg, 95%); mp 98–
101 °C (dec.). IR: 3184, 2948, 2811, 1450, 1911, 1771, 1625,
(500 MHz, D2O) d = 3.41–3.48 (m, 4H, CH2CH2), 3.48–3.56 (m, 4H,
CH2CH2), 4.63 (s, 2H, CCH2N) ppm. 13C NMR (125 MHz, D2O)
d = 42.13, 49.30, 49.64, 152.49 ppm. M (C6H12N6 Â 2HCl) = 241.12.
MS (CI, CH5+) m/z (%): 169 (100, [M+H]+), 145 (17), 127 (18). HRMS
(ESI+): [M+H]+ calcd for C6H13N6, 169.1196; found: 169.1197.
The TFA-salt 73Á2TFA is described in literature.49
4.1.3.17.
1H-Tetrazol-5-ylmethylamine
hydrochloride
(19ÁHCl)50,51. Triethylsilane (0.73 g, 6.3 mmol, 1.0 mL, 11 equiv)
was slowly added to a solution of 39 (220.8 mg, 0.5848 mmol) in
TFA (4.0 mL). The reaction mixture was refluxed for 4.5 h. After
cooling to rt, H2O (10 mL) was added and the aqueous phase was
washed with Et2O (3 Â 10 mL). The crude product was purified
by ion exchange chromatography (Amberlite IRA 410, strongly ba-
sic and (Amberlite IR 120, strongly acidic ion exchange resin).
Compound 19. HCl. 43.4 mg (70%). Colorless solid; mp: 138 °C
(Lit: 143 °C50, 138–141 °C51). IR: 3422, 3161, 1718, 1636, 1404,
1595, 1548, 1483, 1453, 1325, 1231, 1222, 1042, 743, 722 cmÀ1
.
1H NMR (500 MHz, CD3OD) d = 2.29–2.39 (m, 2H, CH2CH2CH2),
3.17–3.27 (m, 2H, CH2CH2NH), 4.50–4.59 (m, 4H, NCH2C,
CH2CH2N), 7.22 (t, J = 7.3 Hz, 2H, NCCHCHCH), 7.47 (t, J = 7.6 Hz,
2H, NCCHCH), 7.56 (d, J = 7.6 Hz, 2H, NCCHCHCH), 8.09 (d,
J = 7.5 Hz, 2H, NCCH) ppm. 13C NMR (125 MHz, CD3OD) d = 26.88,
40.61, 41.92, 46.98, 109.81, 120.38, 121.32, 124.31, 127.05,
141.55, 155.72 ppm. M (C17H18N6 Â HCl) = 342.83. MS (EI, 70 eV)
m/z (%): 306 (38, [M]+), 180 (89), 66 (100). HRMS (ESI+): [M+H]+
calcd for C17H19N6, 307.1666; found: 307.1664.
1091, 945, 801 cmÀ1 1H NMR (400 MHz, D2O) d = 4.63 (s, 2H,
.
CH2) ppm. 13C NMR (100 MHz, D2O) d = 33.61, 153.40 ppm. M
(C2H5N5 Â HCl) = 135.56. MS (FAB+) m/z (%): 100 ([M+H]+). HRMS
(FAB+): [M+H]+ ber. für C2H6N5, 100.0623; gef. 100.0618.
4.1.3.14. 4-[1-(1,1,3,3-Tetramethylbutyl)-1H-tetrazol-5-ylmethyl]
piperazine-1-carboxylic acid-tert-butylester (59). According to
GP1 starting from paraformaldehyde (57.2 mg, 1.91 mmol), 1-
Boc-piperazine (352.1 mg, 1.890 mmol), 1,1,3,3-tetramethylbutyl
4.2. GABA uptake assays
isocyanide (266.0 mg, 1.910 mmol, 335.0
lL) and TMSN3
[3H]GABA uptake was determined as reported.52
Acknowledgments
(219.9 mg, 1.908 mmol, 251.0
lL). Purification by CC (n-pentane/
EtOAc = 1:1) gave 59.
Compound 59. Colorless solid (586.3 mg, 82%); mp 129 °C.
Rf = 0.36 (n-pentane/EtOAc = 1:1). IR: 2982, 2962, 1683, 1432,
For technical support we thank Daniel Merk, Gerd Bauschke,
Silke Duensing-Kropp, Ljiljana Galogaza and Keum-Ja Pankau.
1365, 1277, 1182, 1128, 999, 766 cmÀ1 1H NMR (500 MHz, CDCl3)
.
d = 0.83 (s, 9H, C(CH3)3), 1.46 (s, 9H, OC(CH3)3), 1.85 (s, 6H,
NC(CH3)2), 2.10 (s, 2H, CH2C(CH3)3), 2.50 (sbr, 4H, NCH2CH2NCO),
3.42 (sbr, 4H, CH2NCO), 3.88 (s, 2H, CCH2N) ppm. 13C NMR
(125 MHz, CDCl3) d = 28.57, 29.91, 30.98, 31.86, 43.35, 52.49,
Supplementary data
Supplementary data (Experimental data for compounds 25, 39,
40, 42, 44, 46, 47, 49, 52, 53, 55, 56, 58, 60, 61, 63. 2HCl, 64ÁHCl,
65ÁHCl, 71ÁHCl, 72ÁHCl and 74. 2HCl) associated with this article
can be found, in the online version, at doi:10.1016/j.bmc.
2011.08.039.
53.08,
53.68,
65.82,
80.14,
150.90,
154.85 ppm.
M
(C19H36N6O2) = 380.54. MS (CI, CH5+) m/z (%): 381 (81, [M+H]+),
325 (38), 269 (84), 270 (12), 213 (100). HRMS (EI, 70 eV): M+ calcd
for C19H36N6O2, 380.2900; found: 380.2901. Anal. calcd for C,
59.97; H, 9.54; N, 22.08. Found: C, 59.86; H, 9.44; N, 22.14.
References and notes
4.1.3.15. 1-[1-(1,1,3,3-Tetramethylbutyl)-1H-tetrazol-5-ylmethyl]
piperazine (62). Compound 59 (63.1 mg, 0.166 mmol) was trea-
ted with HCl (ꢀ0.5 M in EtOH, 1.0 mL, 0.5 mmol) and stirred at
rt. After a reaction time of 7 h the solvent was removed in vacuo.
The resulting crude product was taken up in aqueous NaOH (2 M,
1. Foster, A. C.; Kemp, J. Curr. Opin. Pharmacol. 2006, 6, 7.
2. Beleboni, R. O.; Carolino, R. O. G.; Pizzo, A. B.; Castellan-Baldan, L.; Coutinho-
Netto, J.; dos Santos, W. F.; Coimbra, N. C. Cell. Mol. Neurobiol. 2004, 24, 707.
3. Bowery, N. G.; Smart, T. G. Br. J. Pharmacol. 2006, 147(S1), S109.
4. Treiman, D. M. Epilepsia 2001, 42(Suppl. 3), 8.

6504
E. S. Schaffert et al. / Bioorg. Med. Chem. 19 (2011) 6492–6504
5. Ishiwari, K.; Mingot, S.; Correa, M.; Trevitt, J. T.; Carlson, B. B.; Salamone, J. D. J.
Neurosci. Methods 2004, 140, 39.
6. Lanctôt, K. L.; Herrmann, N.; Mazzotta, P.; Khan, L. R.; Ingber, N. Can. J.
Psychiatry 2004, 49, 439.
28. Thomsen, C.; Sørensen, P. O.; Egebjerg, J. Br. J. Pharmacol. 1997, 120, 983.
29. Clausen, R. P.; Moltzen, E. K.; Perregaard, J.; Lenz, S. M.; Sanchez, C.; Falch, E.;
Frølund, B.; Bolvig, T.; Sarup, A.; Larsson, O. M.; Schousboe, A.; Krogsgaard-
Larsen, P. Bioorg. Med. Chem. 2005, 13, 895.
7. Zádori, D.; Geisz, A.; Vámos, E.; Vécsei, P. K. Pharmacol. Biochem. Behav. 2009,
94, 148.
8. Daemen, M. A. R. C.; Hoogland, G.; Cijntje, J.-M.; Spincemaille, G. H. Neurosci.
Lett. 2008, 444, 112.
9. Cherlyn, S. Y. T.; Woon, P. S.; Liu, J. J.; Ong, W. Y.; Tsai, G. C.; Sim, K. Neurosci.
Biobehav. Rev. 2010, 34, 958.
10. Kalueff, A. V.; Nutt, D. J. Depress. Anxiety 2007, 24, 495.
30. Kragler, A.; Höfner, G.; Wanner, K. T. Eur. J. Pharmacol. 2005, 519, 43.
31. Dhar, T. G. M.; Borden, L. A.; Tyagarajan, S.; Smith, K. E.; Branchek, T. A.;
Weinshank, R. L.; Gluchowski, C. J. Med. Chem. 1994, 37, 2334.
32. Fülep, G. H.; Hoesl, C. E.; Höfner, G.; Wanner, K. T. Eur. J. Med. Chem. 2006, 41,
809.
33. Bialer, M.; Johannessen, S. I.; Kupferberg, H. J.; Levy, R. H.; Perucca, E.; Tomson,
T. Epilepsy Res. 2007, 73, 1.
´
11. Czapinski, P.; Blaszczyk, B.; Czuczwar, S. J. Curr. Top. Med. Chem. 2005, 5, 3.
34. Langdon, S. R.; Ertl, P.; Brown, N. Mol. Inf. 2010, 29, 366.
35. Schlewer, G.; Wermuth, C.-G. Eur. J. Med. Chem. 1984, 19, 181.
36. Marcaccini, S.; Torroba, T. Nat. Protoc. 2007, 2, 632.
12. Hediger, M. A.; Romero, M. F.; Peng, J.-B.; Rolfs, A.; Takanaga, H.; Bruford, E. A.
Pflugers Arch. 2004, 447, 465.
13. Chen, N.-H.; Reith, M. E. A.; Quick, M. W. Pflugers Arch. 2004, 447, 519.
14. Madsen, K. K.; White, H. S.; Schousboe, A. Pharmacol. Ther. 2010, 125, 394.
15. Dalby, N. O. Eur. J. Pharmacol. 2003, 479, 127.
37. Ugi, I.; Meyr, R.; Fetzer, U.; Steinbrückner, C. Angew. Chem. 1959, 71, 386.
38. Ugi, I.; Steinbrückner, C. Angew. Chem. 1960, 72, 267.
39. Ugi, I.; Steinbrückner, C. Chem. Ber. 1961, 94, 734.
16. Palacín, M.; Estévez, R.; Bertran, J.; Zorzano, A. Physiol. Rev. 1998, 78, 969.
17. Conti, F.; Zuccarello, L. V.; Barbaresi, P.; Minelli, A.; Brecha, N. C.; Melone, M. J.
Comp. Neurol. 1999, 409, 482.
18. Madsen, K. K.; Clausen, R. P.; Larsson, O. M.; Krogsgaard-Larsen, P.; Schousboe,
A.; White, H. S. J. Neurochem. 2009, 109(Suppl.1), 139.
19. Schousboe, A.; Madsen, K. K.; White, H. S. Future Med. Chem. 2011, 3, 183.
20. Iversen, L. L.; Neal, M. J. J. Neurochem. 1968, 15, 1141.
21. Iversen, L. L.; Johnston, G. A. R. J. Neurochem. 1971, 18, 1939.
22. N’Goka, V.; Schlewer, G.; Linget, J.-M.; Chambon, J.-P.; Wermuth, C.-G. J. Med.
Chem. 1991, 34, 2547.
23. Kulig, K.; Szwaczkiewicz, M. Mini-Rev. Med. Chem. 2008, 8, 1214.
24. Ali, F. E.; Bondinell, W. E.; Dandridge, P. A.; Frazee, J. S.; Garvey, E.; Girard, G. R.;
Kaiser, C.; Ku, T. W.; Lafferty, J. J.; Moonsammy, G. I.; Oh, H.-J.; Rush, J. A.;
Setler, P. E.; Stringer, O. D.; Venslavsky, J. W.; Volpe, B. W.; Yunger, L. M.; Zirkle,
C. L. J. Med. Chem. 1985, 28, 653.
40. Bienayme, H.; Bouzid, K. Tetrahedron Lett. 1998, 39, 2735.
41. Nixey, T.; Kelly, M.; Semin, D.; Hulme, C. Tetrahedron Lett. 2002, 43, 3681.
42. Dömling, A.; Beck, B.; Magnin-Lachaux, M. Tetrahedron Lett. 2006, 47, 4289.
43. Van der Bent, A.; Blommaert, G. S.; Melman, C. T. M.; Ijzerman, A. P.; van
Wijngaarden, I.; Soudijn, W. J. Med. Chem. 1992, 35, 1042.
44. Caldirola, P. M.; van der Goot, H.; Timmermann, H. Eur. J. Med. Chem. 1992, 27,
571.
45. Shah, J. R.; Mosier, P. D.; Roth, B. L.; Kellog, G. E.; Westkaemper, R. B. Bioorg.
Med. Chem. 2009, 17, 6496.
46. Park, J. W.; Song, H. J.; Cho, Y. J.; Park, K. K. J. Phys. Chem. C 2007, 111, 18605.
47. Satzinger, G. Liebigs Ann. Chem. 1960, 638, 159.
48. Combs, A.P.; Takvorian, A.; Zhu, W.; Sparks, R.B. PCT Int. Appl. WO 2007075598
A2 20070705, 2007.
49. Edmonson, S. D.; Berger, R.; Chang, L.; Cholandrea, V. J.; Hale, J. J.; Harper, B.;
Kar, N. F.; Li, B.; Moriello, G. J.; Moyes, C. R.; Sha, D.; Shen, D.-M.; Wang, L.;
Wendt, H. R.; Zhu, C. PCT Int. Appl. WO 201102569 A1 20110303, 2011.
50. Behringer, H.; Kohl, K. Chem. Ber. 1956, 89, 2648.
51. Moderhack, D.; Goos, K.-H.; Preu, L. Liebigs Ann. Chem. 1989, 7, 689.
52. Kragler, A.; Höfner, G.; Wanner, K. T. Eur. J. Med. Chem. 2008, 43, 2404.
53. Hack, S.; Wörlein, B.; Pabel, J.; Höfner, G.; Wanner, K. T. Eur. J. Med. Chem. 2011,
46, 1483.
25. Braestrup, C.; Nielsen, E. B.; Sonnewald, U.; Knutsen, L. J. S.; Andersen, K. E.;
Jansen, J. A.; Frederiksen, K.; Andersen, P. H.; Mortensen, A.; Suzdak, P. J.
Neurochem. 1990, 54, 639.
26. Andersen, K. E.; Braestrup, C.; Grønwald, F. C.; Jargensen, A. S.; Nielsen, E. B.;
Sonnewald, U.; Serrensen, P. O.; Suzdak, P. D.; Knutsen, L. J. S. J. Med. Chem.
1993, 36, 1716.
27. Suzdak, P. D.; Frederiksen, K.; Andersen, K. E.; Sørensen, P. O.; Knutsen, L. J. S.;
Nielsen, E. B. Eur. J. Pharmacol. 1992, 223, 189.
54. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923.