Synthesis and biological evaluation of aminomethylphenol derivatives as inhibitors of the murine GABA transporters mGAT1-mGAT4

Article abstract of DOI:10.1016/j.ejmech.2008.01.005

A series of N-substituted aminomethylphenol derivatives was synthesized by reductive amination. To study the inhibitory potency of the target compounds at the murine GABA transporters (mGAT1-mGAT4), a [³H]GABA uptake test system in a 96-well format based on HEK cells stably expressing mGAT1-mGAT4 was established and validated. Inhibitory potencies at mGAT1-mGAT4 in the micromolar range and a slight subtype selectivity for mGAT3 were observed for the synthesized aminomethylphenol derivatives. Among the compounds investigated 5-n-dodecylaminomethyl-2-methoxyphenol (21) was found to be most potent with an IC₅₀ value at mGAT3 of about 3 μM.

Full text of DOI:10.1016/j.ejmech.2008.01.005

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2404e2411
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of aminomethylphenol derivatives
as inhibitors of the murine GABA transporters mGAT1emGAT4
*
Andrea Kragler, Georg Hofner, Klaus T. Wanner
¨
Department of Pharmacy e Centre of Drug Research, Ludwig-Maximilians-Universita¨t Mu¨nchen, Butenandtstrasse 5-13, 81377 Munich, Germany
Received 20 July 2007; received in revised form 21 December 2007; accepted 8 January 2008
Available online 25 January 2008
Dedicated with best wishes to Prof. Herbert Mayr on the occasion of his 60th birthday.
Abstract
A series of N-substituted aminomethylphenol derivatives was synthesized by reductive amination. To study the inhibitory potency of the
target compounds at the murine GABA transporters (mGAT1emGAT4), a [3H]GABA uptake test system in a 96-well format based on HEK
cells stably expressing mGAT1emGAT4 was established and validated. Inhibitory potencies at mGAT1emGAT4 in the micromolar range
and a slight subtype selectivity for mGAT3 were observed for the synthesized aminomethylphenol derivatives. Among the compounds investi-
gated 5-n-dodecylaminomethyl-2-methoxyphenol (21) was found to be most potent with an IC50 value at mGAT3 of about 3 mM.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: GABA-uptake inhibitors; Antiepileptic; Murine GABA transporters; Aminomethylphenols
1. Introduction
its pharmacological usefulness. About a decade later the first
systemically active GABA uptake inhibitors, such as SK&
F-8997-A (4), tiagabine (5) or NO 711 (6) (Fig. 1) were
described possessing characteristic lipophilic side chains at-
tached to the ring nitrogen of nipecotic acid and guvacine
[1,5e7]. These compounds have been shown to be potent
and subtype selective inhibitors of GAT1 [8], possessing anti-
convulsant activity in animal models [9]. In the meantime tia-
gabine has been introduced successfully in the therapy of
epilepsy classifying GAT1 as an approved target [10]. The
therapeutic potential mediated by mGAT2, mGAT3 and
mGAT4 is less clear so far, but an anticonvulsant activity
has also been demonstrated for the moderately mGAT4 selec-
tive nipecotic acid derivative (S )-SNAP-5114 (7, Fig. 1), the
poorly mGAT2 selective N-substituted 4-hydroxy-4-arylpiper-
idine derivative NNC 05-2045 (8, Fig. 1), and the mGAT1/
mGAT2 selective compound EF1502 (N-[4,4-bis(3-methyl-2-
thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetra-
hydrobenzo[d]isoxazol-3-ol) [11,12].
g-Aminobutyric acid (GABA, 1, Fig. 1) is the major inhib-
itory neurotransmitter in the central nervous system. The ac-
tivity of GABA in the synaptic cleft is thought to be
terminated by reuptake in GABA-ergic neurons and astroglial
cells. Four distinct subtypes of sodium-dependent transporters
mediating the transport of GABA are known [1]. When cloned
from murine brain these GABA transporters are generally
termed mGAT1, mGAT2, mGAT3, and mGAT4 [2] (for the
different nomenclature for various species see Ref. [3]). In
the 1970s (RS )-nipecotic acid (2) and guvacine (3, Fig. 1)
were found to be inhibitors of GABA uptake in neuronal
and astroglial cell cultures [4]. This first generation of
GABA uptake inhibitors, however, lacked the capability to
cross the bloodebrain barrier in sufficient quantities, limiting
Abbreviations: GABA, g-aminobutyric acid; mGAT, murine GABA trans-
port protein; DPB, diphenylbutenyl; DPP, Diphenylpropyl; r.t., room tempera-
ture; TOE, 2-[tris-(4-methoxyphenyl)methoxy]ethyl.
Interestingly, none of the latter GAT inhibitors, neither
NNC 05-2045 nor EF1502, are deduced from classical hetero-
cyclic amino acids as parent structures. Aminomethylphenol
* Corresponding author. Tel.: þ49 89 2180 77249; fax: þ49 89 2180 77247.
E-mail address: klaus.wanner@cup.uni-muenchen.de (K.T. Wanner).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.01.005

A. Kragler et al. / European Journal of Medicinal Chemistry 43 (2008) 2404e2411
2405
COOH
COOH
COOH
in Fig. 2) seemed to be the best option as this method should
be highly flexible by allowing a wide variety of phenol derived
aldehydes and amines to be combined to yield the correspond-
ing aminomethylphenol derivatives.
H2N
N
H
N
H
GABA (1)
nipecotic acid (2)
guvacine (3)
COOH
2. Chemistry
COOH
N
COOH
N
N
The aminomethylphenol derivatives 21e33 depicted in
Fig. 2 and Table 2 were selected as target compounds. They
are derived from the hydroxybenzaldehydes 11e13 and, in or-
der to study the effect of the acidity of the phenol function, also
from the fluorinated hydroxybenzaldehydes 14e16. As pri-
mary amines for the reductive amination compounds 17e20
were selected covering prototypical substituents in addition
to aliphatic residues at known GAT inhibitors. For the reductive
amination, a mixture of the respective aldehydes (11e16) and
the respective primary amines (17e20) was first allowed to re-
O
N
S
S
Tiagabine (5)
NO 711 (6)
SK&F-89976-A (4)
OMe
˚
COOH
act for 20 h in the presence of a 3 A molecular sieve at room
HO
N
N
1. R-NH2 (17-20),
R1
Mol.sieves (3Å),
R1
H
solvent, 20h
O
MeO
OMe
HN
2. NaBH4/ EtOH, 2h
O
R2
N
(11-16)
(9,10, 21-33)
OMe
OH
OH
(S)-SNAP-5114 (7)
NNC 05-2045 (8)
OH
MeO
R2
CHO
CHO
CHO
R1
13
11
12
F
OH
OH
HN
HO
F
F
F
F
R1 = OH, R2 = H: (9) R1 = H,R2 = OH: (10)
CHO
CHO
CHO
F
Fig. 1. Structure of GABA as well as of various known GABA uptake
inhibitors.
F
16
NH2
15
14
derivatives such as compounds 9 and 10 (Fig. 1) represent an-
other class of non-amino acid derived GABA uptake inhibi-
tors. First reported in the early 1980s, they inhibit neuronal
[3H]GABA and glial [3H]b-alanine uptake [13]. As these
uptake assays were performed with rat cortex e heterologous
expression of GABA transporters was not possible at that time e
the full subtype selectivities of these compounds are yet to be
determined. To examine these questions further we decided to
synthesize a series of N-substituted aminomethylphenol
derivatives and to determine their inhibitory potencies at
mGAT1emGAT4. In addition, our study was designed to
reveal the impact of the position and the acidity of the OH-
function as well as the influence of the lipophilic residues
attached to the nitrogen on the inhibitory potency of their ami-
nomethylphenyl derivatives at mGAT1emGAT4. To this end
a synthetic strategy based on reductive amination (as outlined
NH2
17 (nC12H25-NH2)
18 (DPB-NH2)
NH2
NH2
O
MeO
OMe
20 (DPP-NH2)
OMe
19 (TOE-NH2)
Fig. 2. Structure and synthesis of aminomethylphenol derivatives investigated
as GAT inhibitors.

2406
A. Kragler et al. / European Journal of Medicinal Chemistry 43 (2008) 2404e2411
temperature. Following filtration over silica gel and evapora-
tion, the reaction product was then dissolved in ethanol and
treated with sodium borohydride at room temperature for 2 h.
After purification by column chromatography or recrystalliza-
tion the desired secondary amines 21e33 were obtained in
moderate to good yields (Table 2).
may be compared with restrictions, the pIC50 value of 5.57
(i.e. 2.7 mM) obtained for compound 21 at mGAT3 (Fig. 3)
suggests that this new GABA uptake inhibitor is (to the best
of our knowledge) one of the most potent for this transporter
reported to date.
The other aminomethylphenol derivatives derived from
2-hydroxybenzaldehyde, 3-hydroxybenzaldehyde, and 3-hy-
droxy-4-methoxybenzaldehyde possessing DPB (22e24),
DPP (26 and 27), or TOE moiety (28e30), respectively,
3. Biological evaluation
were also found to display
a reasonable potency at
The inhibitory potencies of all the compounds prepared in
this way, the known aminomethylphenols 9 and 10, that had
been resynthesized for this study, as well as the new aminome-
thylphenols 21e33, were determined at the four murine
GABA transporter subtypes mGAT1emGAT4. The study
was performed as [3H]GABA uptake assays in a 96-well plate
format, based on stably transfected HEK cells. With this new
test system we significantly improved a former assay based on
a smaller 24-sample format and employing only transiently
mGAT1emGAT4 expressing COS and HEK cells suspended
in single test tubes [14]. In order to assess the reliability of
the new test system it was validated by a characterisation of
the well-known GAT inhibitors 2e7 (Fig. 1 and Table 1).
The results obtained in our new [3H]GABA uptake assay
were in excellent agreement with those obtained in the earlier
system [14] with regard to both the rank order of potency and
the subtype selectivity of the GAT inhibitors confirming the
validity of the new test system.
mGAT1emGAT4 (pIC50 values between 4.1 and 5.1, i.e.
IC50 values between w80 and 8 mM). In most cases a slight
subtype selectivity for mGAT3 was detected as well. Surpris-
ingly, the fluorinated aminomethylphenol derivatives (25, 31
and 33) were rather poor inhibitors at mGAT1emGAT4.
The fluorine substituted compound 32, however, appeared to
be an exception, being moderately potent at mGAT3 (pIC50
value 4.99; i.e. IC50 value 10.2 mM; see Table 2).
Unambiguous structureeactivity relationships could neither
be deduced for the position of the hydroxy group in the phenol
moiety nor for the substituent attached to the nitrogen. The lat-
ter observation may cause some surprise as previous studies
have shown that the potency and subtype selectivity at
GAT1 of heterocyclic amino acids such as nipecotic acid (2)
and basic structures not representing heterocyclic amino acids
such as 4-N-methylamino-4,5,6,7-tetrahydrobenzo[d]isoxazol-
3-ol have increased significantly on addition of the DPB-resi-
due [8,15]. The same is true for nipecotic acid (2) and the
TOE-residue with regard to their activity at mGAT4 [16].
4. Results and discussion
The N-dodecylsubstituted aminomethylphenols 9 and 10,
which have been already described as inhibitors of neuronal
[3H]GABA and glial [3H]b-alanine uptake in rat cortex [13],
were found to inhibit GABA uptake at mGAT1emGAT4
with a reasonable potency at all subtypes and to display a slight
subtype selectivity for mGAT3. 5-n-Dodecylaminomethyl-2-
methoxyphenol (21) was slightly more potent at each of the
four murine GABA transporters than the aforementioned N-
dodecylsubstituted aminomethylphenols 9 and 10. Although
the potencies of inhibitors obtained from different test systems
5. Conclusion
Several of the newly synthesized N-substituted aminome-
thylphenol derivatives turned out to be moderately potent in-
hibitors of the murine GABA transporters mGAT1emGAT4
displaying a slight preference for mGAT3. The aminomethyl-
phenol derivatives with a fluorinated phenol moiety that were
designed for their enhanced acidity showed comparatively
weak inhibitory potencies. The position of the hydroxy func-
tion, the introduction of an additional methoxy group in the
Table 1
GABA uptake inhibition for known GABA uptake inhibitors at mGAT1emGAT4 stably expressed in HEK
Compound
mGAT1
mGAT2
mGAT3
mGAT4
(RS )-Nip.a.a ((RS )-2)
(R)-Nip.a.a ((R)-2)
(S )-Nip.a.a ((S )-2)
Guvacine (3)
4.88 ꢀ 0.07
5.07 ꢀ 0.02
4.13 ꢀ 0.05
4.87 ꢀ 0.07
6.16 ꢀ 0.05
6.88 ꢀ 0.12
6.83 ꢀ 0.06
4.07 ꢀ 0.09
3.10 ꢀ 0.09
3.28 ꢀ 0.05
3.12 ꢀ 0.12
3.31 ꢀ 0.03
3.43 ꢀ 0.07
100 mM/52%b
3.20 ꢀ 0.09
100 mM/56%b
4.64 ꢀ 0.07
4.71 ꢀ 0.04
3.71 ꢀ 0.04
4.59 ꢀ 0.05
3.71 ꢀ 0.04
100 mM/64%b
3.62 ꢀ 0.04
5.29 ꢀ 0.04
4.70 ꢀ 0.07
4.79 ꢀ 0.05
3.51 ꢀ 0.06
4.59 ꢀ 0.05
3.56 ꢀ 0.06
100 mM/73%b
3.07 ꢀ 0.05
5.81 ꢀ 0.10
SK&F-89976-A (4)
Tiagabine (5)
NO 711 (6)
(S )-SNAP-5114 (7)
The compounds’ inhibitory potencies are given as pIC50 values (mean ꢀ S.E.M., N ¼ 3)c
a
Nipecotic acid.
Remaining [3H]GABA uptake in the presence of 100 mM test compound as compared to a control without inhibitor.
b
c
Data show the pIC50 (ꢁlog IC50) for inhibition of [3H]GABA in uptake experiments performed as described in Section 6. Each value represents a mean ꢀ
S.E.M. of three independent experiments (with triplicate samples for each data point).

A. Kragler et al. / European Journal of Medicinal Chemistry 43 (2008) 2404e2411
2407
mGAT1
mGAT2
100
50
0
100
50
0
-7
-6
-5
-4
log (c test compound 21 / M)
-3
-7
-6
-5
-4
log (c test compound 21 / M)
-3
mGAT3
mGAT4
100
50
0
100
50
0
-7
-6
log (c test compound 21 / M)
-5
-4
-3
-7
-6
log (c test compound 21 / M)
-5
-4
-3
Fig. 3. Inhibition of [3H]GABA uptake by compound 21 at mGAT1emGAT4 stably expressed in HEK cells. Each inhibition curve with its data points (mean ꢀ
S.E.M. from triplicate samples) shows one representative [3H]GABA uptake experiment (out of three independent experiments) performed for mGAT1, mGAT2,
mGAT3 and mGAT4 as described in Section 6.2.2.
phenol moiety or variations of the lipophilic residues attached
to the amino group in all aminomethylphenol derivatives stud-
ied produced only marginal variations of the inhibitory poten-
cies at all four murine GABA transporters. Though potency
and subtype selectivity at the individual GABA transporters
has still to be improved, this class of GABA uptake inhibitors
could nevertheless represent an interesting starting point, espe-
cially for the development of new mGAT3 inhibitors.
Table 2
Physical data and inhibitory potency of aminomethylphenol derivatives (given as pIC50 values; mean ꢀ S.E.M., N ¼ 3) at mGAT1emGAT4c
Productx Starting material
Aldehydey Aminez
Physical data
GABA uptake inhibition (pIC50 values)
Mp (ꢂC) Yield (%) Empirical formula Analysis mGAT1
mGAT2
mGAT3
mGAT4
9
11
12
13
11
12
13
14
11
12
11
12
13
14
15
16
nC12H25eNH2
nC12H25eNH2
e
e
e
C19H33NO
C19H33NO
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
4.23 ꢀ 0.16
4.68 ꢀ 0.11
4.91 ꢀ 0.11
4.48 ꢀ 0.03
4.54 ꢀ 0.06
4.73 ꢀ 0.02
4.54 ꢀ 0.03
4.82 ꢀ 0.08
4.90 ꢀ 0.07
4.84 ꢀ 0.04
4.75 ꢀ 0.05
4.71 ꢀ 0.05
4.73 ꢀ 0.09
5.25 ꢀ 0.15
5.57 ꢀ 0.04
5.11 ꢀ 0.07
5.10 ꢀ 0.07
4.92 ꢀ 0.02
4.20 ꢀ 0.07b
4.60 ꢀ 0.16
5.06 ꢀ 0.04
4.83 ꢀ 0.03
4.68 ꢀ 0.02
4.64 ꢀ 0.09
10
21
22
23
24
25
26
27
28
29
30
31
32
33
e
nC12H25eNH2 76e77
22
66
C20H35NO2
C23H23NO
C23H23NO
DPBeNH2
DPBeNH2
DPBeNH2
DPBeNH2
DPPeNH2
DPPeNH2
TOEeNH2
TOEeNH2
TOEeNH2
TOEeNH2
TOEeNH2
TOEeNH2
Oil
129e130 57
81e82 70
160e161 31
Oil 69
144e145 69
C24H25NO2
C23H21F2NO
C22H23NO
100 mM/70%a 100 mM/82%a 100 mM/66%a 100 mM/76%a
4.28 ꢀ 0.07b
4.06 ꢀ 0.11b
4.57 ꢀ 0.09
4.36 ꢀ 0.10
4.59 ꢀ 0.06
4.74 ꢀ 0.05
4.32 ꢀ 0.03
4.93 ꢀ 0.04
4.89 ꢀ 0.02
4.68 ꢀ 0.08
4.61 ꢀ 0.05
4.42 ꢀ 0.07
5.08 ꢀ 0.02
5.02 ꢀ 0.09
5.05 ꢀ 0.09
4.60 ꢀ 0.03
4.30 ꢀ 0.10b
4.79 ꢀ 0.04
5.11 ꢀ 0.10
4.84 ꢀ 0.08
C22H23NO
Oil
62e63
122e123 75
199e200 54
151e152 56
128e129 62
86
80
C31H33NO5
C31H33NO5
C32H35NO6
C31H31F2NO5
C31H31F2NO5
C31H31F3NO5
100 mM/59%a 100 mM/64%a 100 mM/51%a 100 mM/67%a
4.07 ꢀ 0.10b
100 mM/54%a 4.99 ꢀ 0.04
100 mM/82%a
100 mM/87%a 100 mM/89%a 100 mM/56%a 100 mM/64%a
x,y,z: Compound numbers of products and starting material according to Fig. 2.
a
Remaining [3H]GABA uptake in the presence of 100 mM test compound as compared to a control without inhibitor.
R2 obtained for curve fitting <0.8 (mean of three inhibition curves).
b
c
Data show the pIC50 (ꢁlog IC50) for inhibition of [3H]GABA in uptake experiments performed as described in Section 6.2.2. Each value represents a mean ꢀ
S.E.M. of three independent experiments (with triplicate samples for each data point); examples for representative inhibition curves obtained for test compound 21
are shown in Fig. 3.

2408
A. Kragler et al. / European Journal of Medicinal Chemistry 43 (2008) 2404e2411
6. Experimental section
6.1. Chemistry
TLC: Rf ¼ 0.27 (petroleum ether/EtOAc ¼ 6/4); 1H NMR
(CDCl3): d ¼ 5.19 (s, 1H, OH), 6.83 (m, 1H, Haromat), 10.25 (s,
1H, CHO); IR: ~n ¼ 3078, 1672, 1504, 1249, 1121, 1048 cmꢁ1
;
MS: m/z (%): 177 (100) [M þ 1]þ, 145 (5), 127 (5); HRMS
6.1.1. General
(70 eV) calcd for C7H3F3O2: 176.0085, found: 176.0129.
Tetrahydrofuran was distilled from sodium under nitrogen.
All other solvents for column chromatography and recrystalliza-
tion were distilled prior to use. Purchased chemicals were used
without further purification. TLC plates were made from silica
gel 60 F254 on aluminium sheets (Merck). Flash column chroma-
tography (CC) was carried out with Merck silica gel 60 (mesh
0.040e0.063 mm) as stationary phase. Melting points: m.p.
6.1.3.2.
2-[Tris-(4-methoxyphenyl)methoxy]ethylamine
(19). Tris-(4-methoxyphenyl)methanol [18] (2.80 g, 8.0 mmol,
1 equiv.) and 0.15 ml conc. H2SO4 in dry toluene (15 ml) were
heated for 5 min at 65 ꢂC. After addition of N-hydroxyethylph-
thalimide (2.29 g, 12.0 mmol, 1.5 equiv.) the mixture was stirred
at r.t. for 3 h. CH2Cl2 (10 ml) and H2O (10 ml) were added. The
solid material was filtered off. The filtrate was extracted three
times with CH2Cl2. The combined organic layers were dried
over MgSO4 and concentrated in vacuo. The residue was purified
by CC (petroleum ether/EtOAc ¼ 6/4) to give 2-{2-[Tris-(4-me-
thoxyphenyl)methoxy]ethyl}isoindol-1,3-dione. Yield: 4.19 g
(76%);yellow powder, m.p. 105e106 ꢂC; TLC: Rf ¼ 0.27(petro-
leum ether/EtOAc ¼ 6/4); 1H NMR (CDCl3): d ¼ 3.35 (t,
J ¼ 5.6 Hz, 2H, CH2CH2), 3.74 (s, 9H, OCH3), 3.90 (t,
J ¼ 5.6 Hz, 2H, CH2CH2), 6.71e6.84 (m, 6H, Haromat), 7.22e
7.26 (m, 6H, Haromat), 7.71e7.85 (m, 4H, Haromat); IR: ~n ¼
2953, 2834, 1712, 1508, 1390, 1248, 1176, 1032 cmꢁ1; MS
(EI, 70 eV) m/z (%): 523 (4) [Mþ], 333 (100), 174 (12), 135
(8); C32H29NO6 (523.59).
(uncorrected) was determined with a Buchi 510 Melting Point
¨
apparatus. NMR spectroscopy: 1H NMR spectra were recorded
at room temperature with a JNMR-GX (400 MHz, Jeol) using
TMS as internal standard and integrated with the NMR software
Nuts (2D Version 5.097, Acorn NMR, 1995). IR spectroscopy:
FT-IR Spectrometer 1600 and Paragon 1000 (Perkin Elmer);
oils were measured as film, solid samples as KBr pellets. Mass
spectrometry: Mass Spectrometer 5989 Awith 59,980 B particle
beam LC/MS interface (Hewlett Packard); analysis was carried
out using chemical ionization (CHþ5 ) unless stated otherwise.
HRMS was obtained on JMS GCMate II (Jeol). Elementary
analysis: Elementaranalysator Rapid (Heraeus).
6.1.2. General procedures
To 2-{2-[Tris-(4-methoxyphenyl)methoxy]ethyl}isoindol-
1,3-dione (2.1 g, 4.0 mmol, 1 equiv.) hydrazine-hydrate
(2.0 g in 40 ml MeOH, 40.0 mmol, 10 equiv.) was added.
The solution was stirred at r.t. for 7 h. The solid material
was filtered off, and the filtrate was concentrated in vacuo.
H2O (40 ml) was added, and the mixture was extracted four
times with CH2Cl2. The combined organic layers were dried
over MgSO4, filtered and concentrated in vacuo to give 19.
Yield: 1.10 g (70%); yellow oil; TLC: Rf ¼ 0.24 (CH2Cl2/
MeOH/NH3 (25%) ¼ 100/2/1); 1H NMR (CDCl3): d ¼ 2.86
(t, J ¼ 5.4 Hz, 2H, CH2CH2), 3.13 (t, J ¼ 5.5 Hz, 2H,
CH2CH2), 3.79 (s, 9H, OCH3), 6.83 (m, 6H, Haromat), 7.32
(m, 6H, Haromat); IR: ~n ¼ 3380, 2932, 2835, 1607, 1504,
1302, 1249, 1175, 1035 cmꢁ1; MS (EI, 70 eV) m/z (%): 393
(2) [Mþ], 333 (100), 243 (5), 135 (11); C19H33NO (291.48).
6.1.2.1. General procedure 1 (GP1). To a solution of the hy-
droxybenzaldehyde (1.0 equiv.) and the primary amine
(1.0 equiv.) in EtOH (10 ml mmolꢁ1) molecular sieve (3 A)
˚
was added and the mixture was stirred at r.t. for 20 h. After fil-
tration over silica gel the solvent was evaporated. The resulting
residue was dissolved in EtOH (10 ml mmolꢁ1), NaBH4
(1.2 equiv.) was added and the solution was stirred at r.t. for
2 h. Then EtOH was evaporated and the obtained residue was
dissolved in EtOAc (10 ml mmolꢁ1) and washed three times
with brine. The organic layer was separated, dried (MgSO4),
and concentrated in vacuo. Products were purified by CC.
6.1.2.2. General procedure 2 (GP2). To a solution of the hy-
droxybenzaldehyde (1.0 equiv.) and the primary amine
(1.0 equiv.) in EtOH (10 ml mmolꢁ1) molecular sieve (3 A)
˚
was added and the mixture stirred at r.t. for 20 h. After filtra-
tion over silica gel the solvent was evaporated. The resulting
residue was dissolved in EtOH (10 ml mmolꢁ1), NaBH4
(1.2 equiv.) was added and the solution was stirred at r.t. for
2 h. Then EtOH was evaporated almost completely and H2O
(10 ml) was added, and the precipitate was recrystallized
from a suitable solvent.
6.1.3.3. 5-n-Dodecylaminomethyl-2-methoxyphenol (21). Ac-
cording to GP1 from 3-hydroxy-4-methoxybenzaldehyde
(13) (1.52 g, 10.0 mmol) and n-dodecylamine (17) (4.63 g,
25.0 mmol). CC: (CH2Cl2/MeOH/NH3 (25%) ¼ 90/9/1).
Yield: 697 mg (22%); colourless crystals, m.p. 76e77 ꢂC;
TLC: Rf ¼ 0.35 (CH2Cl2/MeOH/NH3 (25%) ¼ 90/9/1); 1H
NMR (CDCl3): d ¼ 0.88 (t, J ¼ 7 Hz, 3H, CH3), 1.25e1.31
(m, 18H, 9ꢃCH2), 1.54 (t, J ¼ 7 Hz, 2H, NHCH2CH2), 2.62
(t, J ¼ 7 Hz, 2H, ArCH2NHCH2), 3.72 (s, 2H, ArCH2NH),
3.88 (s, 3H, OCH3), 6.69e7.15 (m, 3H, Haromat); IR: ~n ¼
3318, 2916, 2852, 2353, 1504, 1284, 1223, 1131,
1034 cmꢁ1; MS m/z (%): 322 (100) [M þ 1]þ, 166 (7), 137
(27); C20H35NO2 (321.50).
6.1.3. Individual procedures
6.1.3.1. 2,4,6-Trifluoro-3-hydroxybenzaldehyde (16). In anal-
ogy to literature [16] from 1.33 g (9.0 mmol) 2,4,6-trifluorophe-
nol, 1.086 g (10.0 mmol) trimethylsilyl chloride, and 0.715 g
(10.5 mmol) imidazole. CC (petroleum ether/EtOAc ¼ 6/4).
Yield: 230 mg (22%); orange crystals, m.p. 158e159 ꢂC;
6.1.3.4.
2-[(4,4-Diphenylbut-3-enylamino)methyl]phenol
(22). According to GP1 from 2-hydroxybenzaldehyde (11)

A. Kragler et al. / European Journal of Medicinal Chemistry 43 (2008) 2404e2411
2409
(122 mg, 1.0 mmol) and 4,4-diphenylbut-3-enylamine (18)
(260 mg, 1.0 mmol) [5,18,19]. CC: (petroleum ether/EtOAc/
NEtMe2 ¼ 50/50/1). Yield: 216 mg (66%); yellow oil; TLC:
Rf ¼ 0.42 (petroleum ether/EtOAc/NEtMe2 ¼ 50/50/1); 1H
NMR (CDCl3): d ¼ 2.38 (q, J ¼ 6.9 Hz, 2H, NHCH2CH2), 2.78
(t, J ¼ 6.9 Hz, 2H, NHCH2CH2), 3.92 (s, 2H, ArCH2NH), 6.05
(t, J ¼ 6.9 Hz, 1H, CH]), 6.76e7.38 (m, 14H, Haromat); IR: ~n
¼ 3321, 3053, 2843, 1589, 1493, 1258, 1104 cmꢁ1; MS m/z
(%): 330 (100) [M þ 1]þ, 236 (3), 224 (5), 136 (15), 107 (5);
C23H23NO (329.44).
(q, J ¼ 7.5 Hz, 2H, NHCH2CH2), 2.66 (t, J ¼ 7.5 Hz, 2H,
NHCH2CH2), 3.91 (s, 2H, ArCH2), 3.99 (t, J ¼ 7.5 Hz, 1H,
NHCH2CH2CH ), 6.75e7.28 (m, 14H, Haromat); IR: ~n ¼ 3334,
3024, 2913, 2850, 1590, 1450, 1260, 1104 cmꢁ1; MS m/z
(%): 318 (100) [M þ 1]þ, 212 (13), 107 (17); C22H23NO
(317.43).
6.1.3.9. 3-[(3,3-Diphenylpropylamino)methyl]phenol (27). Ac-
cording to GP1 from 3-hydroxybenzaldehyde (12) (122 mg,
1.0 mmol) and 3,3-diphenylpropylamine (20) (230 mg,
1.0 mmol). CC: (CH2Cl2/MeOH/NEtMe2 ¼ 100/3/3). Yield:
219 mg (69%); colourless crystals, m.p. 144e145 ꢂC; TLC:
Rf ¼ 0.33 (CH2Cl2/MeOH/NEtMe2 ¼ 100/3/3); 1H NMR
(CDCl3): d ¼ 2.29 (q, J ¼ 7.5 Hz, 2H, NHCH2CH2), 2.62 (t,
J ¼ 7.5 Hz, 2H, NHCH2CH2), 3.62 (s, 2H, ArCH2), 3.98 (t,
J ¼ 7.5 Hz, 1H, NHCH2CH2CH ), 6.61e7.26 (m, 14H, Haromat);
6.1.3.5.
3-[(4,4-Diphenylbut-3-enylamino)methyl]phenol
(23). According to GP1 from 3-hydroxybenzaldehyde (12)
(122 mg, 1.0 mmol) and 4,4-diphenylbut-3-enylamine (18)
(260 mg, 1.0 mmol) [5,18,19]. CC: (CH2Cl2/MeOH/NH3
(25%) ¼ 100/5/1). Yield: 186 mg (57%); colourless crystals,
m.p. 129e130 ꢂC; TLC: Rf ¼ 0.29 (CH2Cl2/MeOH/NH3
(25%) ¼ 100/5/1); 1H NMR (CDCl3): d ¼ 2.38 (q,
J ¼ 7.5 Hz, 2H, NHCH2CH2), 2.78 (t, J ¼ 7.5 Hz, 2H,
NHCH2CH2), 3.92 (s, 2H, ArCH2NH), 6.06 (t, J ¼ 7.5 Hz,
1H, CH]), 6.75e7.38 (m, 14H, Haromat); IR: ~n ¼ 3282,
2856, 2573, 1580, 1481, 1343, 1278, 1102 cmꢁ1; MS m/z
(%): 330 (100), 136 (25), 107 (4); C23H23NO (329.44).
IR: ~n ¼ 3446, 3278, 3023, 2905, 2848, 1598, 1450, 1275 cmꢁ1
;
MS (EI, 70 eV) m/z (%): 317 (32) [Mþ], 136 (57), 107 (100);
C22H23NO (317.43).
6.1.3.10. 2-({2-[Tris-(4-methoxyphenyl)methoxy]ethylamino}-
methyl)phenol (28). According to GP1 from 2-hydroxyben-
zaldehyde (11) (92 mg, 0.75 mmol) and 2-[tris-(4-
methoxyphenyl)methoxy]ethylamine
(19)
(295 mg,
6.1.3.6. 5-[(4,4-Diphenylbut-3-enylamino)methyl]-2-methoxy-
phenol (24). According to GP1 from 3-hydroxy-4-methoxy-
benzaldehyde (13) (152 mg, 1.0 mmol) and 4,4-diphenylbut-
3-enylamine (18) (260 mg, 1.0 mmol) [5,18,19]. CC:
(CH2Cl2/MeOH/NH3 (25%) ¼ 100/3/1) Yield: 251 mg
(70%); colourless crystals, m.p. 81e82 ꢂC; TLC: Rf ¼ 0.16
(CH2Cl2/MeOH/NH3 (25%) ¼ 100/3/1); 1H NMR (CDCl3):
d ¼ 2.35 (q, J ¼ 7.5 Hz, 2H, NHCH2CH2), 2.73 (t,
J ¼ 7.5 Hz, 2H, NHCH2CH2), 3.65 (s, 2H, ArCH2NH), 3.87
(s, 3H, OCH3), 6.07 (t, J ¼ 7.5 Hz, 1H, CH]), 6.77e7.36
(m, 13H, Haromat); IR: ~n ¼ 3287, 2928, 2857, 1503, 1437,
1276, 1224, 1131, 1028 cmꢁ1; MS m/z (%): 360 (100)
[M þ 1]þ, 166 (69), 137 (55); C24H25NO (359.47).
0.75 mmol). CC: (petroleum ether/EtOAc/NEtMe2 ¼ 65/35/
2). Yield: 323 mg (86%); glassy solid; TLC: Rf ¼ 0.18 (petro-
leum ether/EtOAc/NEtMe2 ¼ 65/35/2); 1H NMR (CDCl3):
d ¼ 2.82 (t, J ¼ 5.1 Hz, 2H, CH2CH2), 3.27 (t, J ¼ 5.1 Hz,
2H, CH2CH2), 3.78 (s, 9H, OCH3), 3.94 (s, 2H, ArCH2),
6.76e7.31 (m, 16H, Haromat); IR: ~n ¼ 3430, 2931, 2836,
1608, 1506, 1250, 1175, 1034 cmꢁ1; MS m/z (%): 333
(100), 227 (16), 168 (22), 107 (34); C31H33NO5 (499.61).
6.1.3.11. 3-({2-[Tris-(4-methoxyphenyl)methoxy]ethylamino}-
methyl)phenol (29). According to GP1 from 3-hydroxybenzal-
dehyde (12) (92 mg, 0.75 mmol) and 2-[tris-(4-
methoxyphenyl)methoxy]ethylamine
(19)
(295 mg,
0.75 mmol). CC: (EtOAc/NEtMe2 ¼ 100/2). Yield: 298 mg
(80%); colourless crystals, m.p. 62e63 ꢂC; TLC: Rf ¼ 0.12
(EtOAc/NEtMe2 ¼ 100/2); 1H NMR (CDCl3): d ¼ 2.83 (t,
J ¼ 5.4 Hz, 2H, CH2CH2), 3.27 (t, J ¼ 5.4 Hz, 2H, CH2CH2),
3.67 (s, 2H, ArCH2), 3.77 (s, 9H, OCH3), 6.71e7.32 (m,
16H, Haromat); IR: ~n ¼ 3407, 2931, 2834, 1607, 1508,
1249, 1175, 103 cmꢁ1; MS m/z (%): 333 (22), 227 (83), 168
(100), 145 (21), 127 (22), 107 (24); C31H33NO5$0.5H2O
(508.62).
6.1.3.7. 4-[(4,4-Diphenylbut-3-enylamino)methyl]-2,6-difluor-
ophenol (25). According to GP2 from 3,5-difluoro-4-hydroxy-
benzaldehyde (14) (158 mg, 1.0 mmol) [17] and 4,4-
diphenylbut-3-enylamine (18) (260 mg, 1.0 mmol) [5,18,19].
Recrystallization from EtOH. Yield: 112 mg (31%); colourless
crystals, m.p. 160e161 ꢂC; TLC: Rf ¼ 0.28 (CH2Cl2/MeOH/
NH3 (25%) ¼ 90/10/1); 1H NMR (CDCl3): d ¼ 2.41 (q,
J ¼ 7.2 Hz, 2H, NHCH2CH2), 2.80 (t, J ¼ 7.2 Hz, 2H,
NHCH2CH2), 3.69 (s, 2H, ArCH2), 6.07 (t, J ¼ 7.2 Hz, 1H,
CH]), 6.77 (d, J ¼ 7.8 Hz, 2H, Haromat), 7.13e7.39 (m,
10H, Haromat); IR: ~n ¼ 3442, 3024, 2362, 1612, 1505, 1445,
1334, 1013 cmꢁ1; C23H21F2NO (346.42).
6.1.3.12. 5-({2-[Tris-(4-methoxyphenyl)methoxy]ethylamino}-
methyl)-2-methoxyphenol (30). According to GP1 from 3-hy-
droxy-4-methoxybenzaldehyde (13) (114 mg, 0.75 mmol) and
2-[tris-(4-methoxyphenyl)methoxy]ethylamine (19) (295 mg,
0.75 mmol). CC: (EtOAc/NEtMe2 ¼ 100/2). Yield: 297 mg
(75%); colourless crystals, m.p. 122e123 ꢂC; TLC: Rf ¼ 0.12
(EtOAc/NEtMe2 ¼ 100/2); 1H NMR (CDCl3): d ¼ 2.80 (t,
J ¼ 5.4 Hz, 2H, CH2CH2), 3.23 (t, J ¼ 5.4 Hz, 2H, CH2CH2),
3.68 (s, 2H, ArCH2), 3.78 (s, 9H, OCH3), 3.88 (s, 3H, OCH3),
6.80e7.32 (m, 15H, Haromat); IR: ~n ¼ 3431, 2954, 2834, 1608,
6.1.3.8. 2-[(3,3-Diphenylpropylamino)methyl]phenol (26). Ac-
cording to GP1 from 2-hydroxybenzaldehyde (11) (122 mg,
1.0 mmol) and 3,3-diphenylpropylamine (20) (230 mg,
1.0 mmol). CC: (petroleum ether/EtOAc/NEtMe2 ¼ 70/30/3).
Yield: 220 mg (69%); yellow oil; TLC: Rf ¼ 0.33 (petroleum
1
ether/EtOAc/NEtMe2 ¼ 70/30/3); H NMR (CDCl3): d ¼ 2.30

2410
A. Kragler et al. / European Journal of Medicinal Chemistry 43 (2008) 2404e2411
1507, 1247, 1177, 1096, 1036 cmꢁ1; MS m/z (%): 333 (40), 257
(40), 225 (45), 198 (36), 137 (92), 105 (100); C32H35NO6
(519.55).
37 ꢂC. Transfections with linearised plasmids (mGAT1,
mGAT3 and mGAT4: ScaI, mGAT2: MfeI) were performed us-
ing FuGENE6 (Roche, Mannheim, Germany) according to
manufacturer’s protocol: About 3.5ꢃ106 HEK cells were
plated in normal DMEM. On the next day the cells were
treated with a mixture of 40 ml FuGENE6 and 13.3 mg
cDNA (mGAT1, mGAT3, mGAT4) or 60 ml FuGENE6 and
10.0 mg cDNA (mGAT2). Two days after transfection the cells
were split in DMEM supplemented with 10% fetal calf serum
and 100 U mlꢁ1 penicillin, 100 mg mlꢁ1 streptomycin and
500 mg mlꢁ1 geneticin (selection medium). Having reached
a confluence of about 30% stably transfected cells were plated
1/10,000 in selection medium. About two weeks later single
colonies of stably transfected cells were isolated and further
cultivated in selection medium.
6.1.3.13. 2,6-Difluoro-4-({2-[tris-(4-methoxyphenyl)methoxy]
ethylamino}methyl)phenol (31). According to GP2 from 3,5-di-
fluoro-4-hydroxybenzaldehyde (14) (158 mg, 1.0 mmol) and 2-
[tris-(4-methoxyphenyl)methoxy]ethylamine (19) (394 mg,
1.0 mmol). Reduction with NaBH4 in isopropanol instead of
EtOH as solvent, recrystallization from EtOH/Et2O (3/1). Yield:
144 mg (54%); white powder, m.p. 199e200 ꢂC; TLC: Rf ¼ 0.30
(CH2Cl2/MeOH/NH3 (25%) ¼ 90/10/1); 1H NMR (CDCl3):
d ¼ 2.86 (t, J ¼ 5.3 Hz, 2H, CH2CH2), 3.33 (t, J ¼ 5.3 Hz,2H,
CH2CH2), 3.58 (s, 2H, ArCH2), 6.67 (d, J ¼ 8.4 Hz, 2H, Haromat),
6.84 (d, J ¼ 6.6 Hz, 6H, Haromat), 7.29 (d, J ¼ 6.6 Hz, 6H,
Haromat); IR: ~n ¼ 3446, 2951, 2834, 2360, 1608, 1508, 1463,
1327, 1252, 1176, 1035 cmꢁ1; C31H31F2NO5 (535.59).
6.2.2. [3H]GABA uptake
Cells grown in 145 cm2 plates to a confluence of 70e90%
were treated with 4 ml trypsin/EDTA for about 30 s. After-
wards 8 ml normal medium was added and the resulting cell
suspension centrifuged 5 min at 500g. The cells were washed
for three times in phosphate buffered saline (137 mM NaCl,
2.7 mM KCl, 8 mM Na2HPO4, 1.75 mM KH2PO4, pH 7.4)
by centrifugation as described above and finally resuspended
in Krebs buffer (2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM
KH2PO4, 4.7 mM KCl, 11 mM glucose, 25 mM Tris,
119 mM NaCl, pH 7.2). The uptake assays were performed
with aliquots of the resulting cell suspension (about
100,000 cells per well for mGAT1, mGAT3, mGAT4 and
about 200,000 cells per well for mGAT2) in a total volume
of 250 ml in 96 well 2.2 ml polyethylene deep well plates (Ab-
gene, Epsom, UK). The cells were equilibrated for 25 min in
Krebs buffer in the presence of the test compound at 37 ꢂC
in a gently shaking water bath. Due to the poor solubility of
the test compounds all samples contained 1% DMSO. (The
highest concentration investigated was 100 mM. Since many
of the test compounds tended to precipitate at this concentra-
tion it cannot be ruled out absolutely that the inhibition assays
are affected by precipitation at 100 mM of the test compounds.
However, the inhibition curves obtained gave no indication of
any distorting effects.) After addition of 25 ml of a solution
containing [3H]GABA (3 TBq mmol, Amersham Biosciences,
Freiburg, Germany) and unlabeled GABA in Krebs (final con-
centration 8 nM [3H]GABA and 32 nM unlabeled GABA for
mGAT1, mGAT3, mGAT4 and 20 nM [3H]GABA and 20
nM unlabeled GABA for mGAT2) the cells were incubated
for further 4 min (mGAT1, mGAT3, mGAT4) or 10 min
(mGAT2), respectively. The incubation was stopped by filtra-
tion through Whatman GF/C filters pre-soaked for 1 h in 0.9%
NaCl by means of a Brandell MWXR-96TI cell harvester
(Brandell, Gaithersburg, MD, USA) under reduced pressure
(not below 250 mbar). The filters were rinsed four times
with cold 0.9% NaCl and subsequently transferred to 96-
well sample plates (Perkin Elmer LAS, Boston, MA, USA).
After addition of 200 ml Rotiszint Eco Plus (Roth, Karlsruhe,
Germany) per well the radioactivity was determined in a mi-
crobeta liquid scintillation counter (Perkin Elmer LAS,
6.1.3.14. 2,6-Difluoro-3-({2-[tris-(4-methoxyphenyl)methoxy]
ethylamino}methyl)phenol (32). According to GP2 from 2,4-
difluoro-3-hydroxybenzaldehyde (15) (158 mg, 1.0 mmol) [17]
and
2-[tris-(4-methoxyphenyl)methoxy]ethylamine
(19)
(394 mg, 1.0 mmol). Recrystallization from EtOH/ether (3/1).
Yield: 158 mg (56%); white powder, m.p. 151e152 ꢂC; TLC:
Rf ¼ 0.30 (CH2Cl2/MeOH/NH3 (25%) ¼ 90/10/1); 1H NMR
(CDCl3): d ¼ 2.83 (t, J ¼ 5.2 Hz, 2H, CH2CH2), 3.28
(t, J ¼ 5.2 Hz, 2H, CH2CH2), 3.72 (s, 2H, ArCH2), 3.78 (s,
9H, OCH3), 6.71e7.32 (m, 14H, Haromat); IR: ~n ¼ 3440,
2930, 2835, 1608, 1509, 1252, 1175, 1034 cmꢁ1
;
C31H31F2NO5$0.25H2O (540.19).
6.1.3.15. 2,4,6-Trifluoro-3-({2-[tris-(4-methoxyphenyl)methox-
y]ethylamino}methyl)phenol (33). According to GP1 from
2,4,6-trifluoro-3-hydroxybenzaldehyde
(16)
(175 mg,
1.0 mmol) and 2-[tris-(4-methoxyphenyl)methoxy]ethylamine
(19) (394 mg, 1.0 mmol). CC: (CH2Cl2/MeOH ¼ 100/3)
Yield: 342 mg (62%); white powder, m.p. 128e129 ꢂC;
1
TLC: Rf ¼ 0.30 (CH2Cl2/MeOH ¼ 100/3); H NMR (CDCl3):
d ¼ 2.88 (t, J ¼ 5.1 Hz, 2H, CH2CH2), 3.32 (t, J ¼ 5.1 Hz,
2H, CH2CH2), 3.70 (s, 2H, ArCH2), 3.77 (s, 9H, OCH3),
6.46e7.35 (m, 13H, Haromat); IR: ~n ¼ 3436, 2934, 2836,
1608, 1506, 1250 cmꢁ1; C31H31F2NO5$H2O (553.60).
6.2. Biological evaluation
6.2.1. Cell culture and stable transfection
All the media, sera, additives and antibiotics for cell culture
were purchased from PAA (Colbe, Germany). The following
¨
vector constructs were used to express the murine GABA
transporters as previously described [14]: pRC-CMV2 con-
taining the cDNA for mGAT1 (obtained from Prof. H. Lud-
¨
dens) and pcDNA3.1(þ) for mGAT2, mGAT3, and mGAT4
(cDNA for these transporters was obtained from Prof. N. Nel-
son). HEK cells were cultured in Dulbecco’s modified Eagles
medium (DMEM) supplemented with 10% fetal calf serum,
100 U mlꢁ1 penicillin and 100 mg mlꢁ1 streptomycin (normal
DMEM) in a humidified atmosphere (92% air, 8% CO2) at

A. Kragler et al. / European Journal of Medicinal Chemistry 43 (2008) 2404e2411
2411
[5] F.E. Ali, W.E. Bondinell, P.A. Dandridge, J.S. Frazee, E. Garvey,
G.R. Girare, C. Kaiser, T.W. Ku, J.L. Lafferty, G.I. Moonsammy,
H.-J. Oh, J.A. Rush, P.E. Setler, O.D. Stringer, J.W. Venslavsky,
B.W. Volpe, L.M. Yunger, C.L. Zirkle, J. Med. Chem. 28 (1985) 653e
660.
Boston, MA, USA). Non-specific uptake was defined in paral-
lel experiments with 10 mM NO 711 (mGAT1) or 1 mM
GABA (mGAT2, mGAT3, mGAT4) and was subtracted from
total uptake (no inhibitor) to yield specific uptake. IC50 values
were determined by non-linear regression using Prism 4 (‘‘one
site competition’’; bottom and top were fixed to 0 and 100%,
respectively). The results are expressed as means ꢀ S.E.M. of
at least three separate experiments, each carried out in tripli-
cate. Protein was determined in an aliquot of the final cell sus-
pension according to Bradford [20] using BSA as a standard.
[6] C. Braestrup, E.B. Nielsen, U. Sonnewald, L.J.S. Knutsen,
K.E. Andersen, J.A. Jansen, K. Frederiksen, P.H. Andersen,
P.A. Mortensen, P.D. Suzdak, J. Neurochem. 54 (1990) 639e647.
[7] P.D. Suzdak, K. Frederiksen, K.E. Andersen, P.O. Sorensen,
L.J.S. Knutsen, E.B. Nielsen, Eur. J. Pharmacol. 223 (1992) 189e198.
[8] L.A. Borden, T.G.M. Dhar, K.E. Smith, R.L. Weinshank, T.A. Branchek,
C. Gluchowski, Eur. J. Pharmacol. 269 (1994) 219e224.
[9] P.D. Suzdak, Drugs Future 18 (1993) 1129e1136.
[10] B.S. Meldrum, A.G. Chapman, Epilepsia 40 (Suppl. 9) (1999) S2eS6.
[11] N.O. Dalby, Neuropharmacology 39 (2000) 2399e2407.
[12] H.S. White, W.P. Watson, S.L. Hansen, S. Slough, J. Perregaard,
A. Sarup, T. Bolvig, G. Petersen, O.M. Larssson, R.P. Clausen,
B. Frolund, P. Krogsgaard-Larsen, A. Schousboe, J. Pharmacol. Exp.
Ther. 312 (2005) 866e874.
Acknowledgement
For technical support we thank Silke Duensing-Kropp and
Ljiljana Galogaza. We thank Monika Simon for assistance
with editing and proofreading. We also thank Prof. Dr. H. Lud-
¨
dens for providing the cDNA of mGAT1 and Prof. Dr. N. Nel-
son for providing the cDNAs of mGAT2, mGAT3 and
mGAT4.
[13] R. Breckenridge, S. Nicholson, A. Nicol, C. Suckling, B. Leigh,
L. Iversen, Biochem. Pharmacol. 30 (1981) 3045e3049.
[14] A. Kragler, G. Hofner, K.T. Wanner, Eur. J. Pharmacol. 519 (2005) 43e47.
¨
[15] R.P. Clausen, E.K. Moltzen, J. Perregaard, S.M. Lenz, C. Sanchez,
E. Falch, B. Frolund, T. Bolvig, A. Sarup, O.M. Larsson, A. Schousboe,
P. Krogsgaard-Larsen, Bioorg. Med. Chem. 13 (2005) 895e908.
[16] T.G.M. Dhar, L. Borden, S. Tyagarajan, K.E. Smith, T.A. Branchek,
R.L. Weinshank, C. Gluchowski, J. Med. Chem. 37 (1994) 2334e2342.
[17] S. Songca, R. Bonnett, C.S. Maes, S. Afr. J. Chem. 50 (1997) 40e47.
[18] A. van der Bent, A.G.S. Blommaert, C.T.M. Melman, A.P. Ijzerman,
I. van Wijngaarden, W. Soudijn, J. Med. Chem. 35 (1992) 1042e1049.
[19] P.M. Cardiola, H. van der Goot, H. Timmerman, Eur. J. Med. Chem. 27
(1992) 571e579.
References
[1] L.A. Borden, Neurochem. Int. 29 (1996) 335e356.
[2] Q.-R. Liu, B. Lopez-Corcuera, S. Mandiyan, H. Nelson, N. Nelson,
J. Biol. Chem. 268 (1993) 2106e2112.
[3] N.O. Dalby, Eur. J. Pharmacol. 479 (2003) 127e137.
[4] P. Krogsgaard-Larsen, E. Falch, O.M. Larsson, A. Schousboe, Epilepsy
Res. 1 (1987) 77e93.
[20] M. Bradford, Anal.Biochem. 72 (1976) 248e254.