Selective mGAT2 (BGT-1) GABA uptake inhibitors: Design, synthesis, and pharmacological characterization

Article abstract of DOI:10.1021/jm301872x

β-Amino acids sharing a lipophilic diaromatic side chain were synthesized and characterized pharmacologically on mouse GABA transporter subtypes mGAT1-4. The parent amino acids were also characterized. Compounds 13a, 13b, and 17b displayed more than 6-fold selectivity for mGAT2 over mGAT1. Compound 17b displayed anticonvulsive properties inferring a role of mGAT2 in epileptic disorders. These results provide new neuropharmacological tools and a strategy for designing subtype selective GABA transport inhibitors.

Full text of DOI:10.1021/jm301872x

Brief Article
pubs.acs.org/jmc
Selective mGAT2 (BGT-1) GABA Uptake Inhibitors: Design, Synthesis,
and Pharmacological Characterization
Stine B. Vogensen,†,§ Lars Jørgensen,†,§ Karsten K. Madsen,† Nrupa Borkar,† Petrine Wellendorph,†
Jonas Skovgaard-Petersen,† Arne Schousboe,† H. Steve White,‡ Povl Krogsgaard-Larsen,†
and Rasmus P. Clausen*,†
†Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2100
Copenhagen, Denmark
‡Anticonvulsant Drug Development Program, Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah,
United States
S
* Supporting Information
ABSTRACT: β-Amino acids sharing a lipophilic diaromatic side chain were synthesized and characterized pharmacologically on
mouse GABA transporter subtypes mGAT1−4. The parent amino acids were also characterized. Compounds 13a, 13b, and 17b
displayed more than 6-fold selectivity for mGAT2 over mGAT1. Compound 17b displayed anticonvulsive properties inferring a
role of mGAT2 in epileptic disorders. These results provide new neuropharmacological tools and a strategy for designing subtype
selective GABA transport inhibitors.
inhibitor tiagabine [(R)-N-[4,4-bis(3-methyl-2-thienyl)-3-
butenyl]nipecotic acid, 1] are clinically effective anticonvulsant
drugs.4 Therapeutic application of GABA transporter inhibitors
seems particularly attractive, since it increases GABA neuro-
transmission only upon synaptic release.5 Increasing evidence
suggests that selective inhibitors are therapeutically useful in
other CNS disorders related to GABA hypofunction such as
anxiety, neuropathic pain, and insomnia.6−8
INTRODUCTION
The major inhibitory neurotransmitter in the central nervous
system (CNS), GABA (Chart 1), plays a fundamental role in the
■
Chart 1. Chemical Structures of GABA and Selected GABA
Transporter Inhibitors
Four different GABA transporter subtypes have been cloned,9
but the nomenclature of the GATs among species is inconsistent.
Rat and human GAT-1, BGT-1, GAT-2, and GAT-3 correspond
to mouse mGAT1, mGAT2, mGAT3, and mGAT4, respec-
tively.10 GABA transporters belong to the solute carrier 6 gene
family which also comprises transporters for the monoamine
neurotransmitters serotonin, dopamine, and norepinephrine.
Compared with the monoamine transporters, GABA trans-
porters are less pharmacologically explored mainly because of the
lack of potent and selective ligands targeting the different GAT
subtypes.11 Highly selective and potent inhibitors of GAT1 have
been developed, but the pharmacological function and
therapeutic potential of the non-GAT1 subtypes remain to be
established, stressing the need for selective inhibitors of these
subtypes.12
Tiagabine (1) (Chart 1) is a lipophilic derivative of the classical
inhibitor (R)-nipecotic acid (2), which like the areca nut alkaloid
guvacine (3), is a substrate for GABA transporters.9,12−14 The
lipophilic side chain of tiagabine enables blood−brain barrier
penetration, disables substrate properties, and induces GAT1
selectivity. Bioisosteric exchange of the carboxylic acid in 1 and 2
and redesign of the scaffold led to N-methyl-exo-THPO (4),
which is a selective inhibitor of glial [3H]GABA uptake.15 Later,
healthy state of the CNS.1 Abnormalities in the GABAergic
system have been implicated in the pathophysiology of a variety
of disorders, and reduction in GABA neurotransmission leads to
seizure activity suggesting a relation to epileptic disorders.2,3
Pharmacological interventions that elevate the synaptic level of
GABA have therefore received attention as therapeutic strategies
in the treatment of epileptic disorders. Thus, the GABA
transaminase inhibitor vigabatrin and the GABA transport
Received: December 19, 2012
Published: February 11, 2013
© 2013 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
a
combining the lipophilic side chain from tiagabine and 4 resulted
in EF1502 (5), endowed with a novel pharmacological GABA
transporter inhibitory profile.16,17 Whereas tiagabine is a highly
selective inhibitor of GAT1, EF1502 was shown to have a dual
preference for mGAT1 and mGAT2 over mGAT3 and mGAT4.
This was interesting, since very few inhibitors have been found
that are truly selective for other subtypes than GAT1. One
example, NNC 05-2090 (6), is a moderately selective inhibitor of
the human orthologue of mGAT2 but also possesses some non-
GAT activity, limiting its usefulness in vivo.18,19 This result
points to a design strategy where the lipophilic side chain is
maintained and variations in the amino acid moiety are
introduced, as opposed to previous strategies where a very
large number of derivatives based on 1 and 2 were synthesized
with different side chains.12,17 We will here report the
continuation of this new strategy that has also been followed
by others.20,21
Scheme 1. Synthetic Pathway toward 11a−c and 13a−c
The unique pharmacological profile of EF1502 prompted a
larger study that disclosed some unique properties of this uptake
inhibitor. Not only was EF1502 a potent anticonvulsant but it
potentiated the anticonvulsant properties of tiagabine in a
synergistic manner without potentiating sedative effects of
tiagabine.17,22 The synergistic effect of EF1502 and tiagabine was
further demonstrated in studies on spontaneous electrographic
bursting (SB) in the medial entorhinal cortex of rats treated with
kainic acid.23 Whereas burst area and duration were reduced by
both compounds, only EF1502 reduced SB frequency.
Furthermore, the compounds exhibited mechanistic differences
in their ability to modulate the ataxia and anticonvulsant action of
the extrasynaptic GABAA receptor agonist gaboxadol.24 Thus, it
is tempting to speculate that the mGAT2 activity of EF1502
could be involved in the observed effects. On the other hand,
since the levels of mGAT2 are very low in the normal CNS and
no apparent seizure-related phenotype can be observed in
mGAT2 knockout mice,25 it cannot be excluded that EF1502
acts at another unknown target. Nevertheless, the level of
mGAT2 expression could be altered during diseased conditions.
To study the role of mGAT2 in the CNS, an mGAT2 selective
compound is therefore essential. We here report a rational
approach to further increase the mGAT2 selectivity of EF1502
by combining the lipophilic side chain of tiagabine with a series of
new conformationally restricted N-methylated β-amino acids.
a
Reagents and conditions: (a) (i) TMS azide, 1,4-dioxane, reflux, (ii)
H2O, (iii) HCl, EtOH, reflux; (b) 2-nitrobenzenesulfonyl chloride,
triethylamine, rt; (c) MeI, K2CO3, DMF, (d) PhSH, K2CO3, 40 °C;
(e) NaOH/H2O, 16 h; (f) 4,4-bis(3-methyl-2-thienyl)-3-butenyl
methylsulfonate, Li2CO3, isopropyl acetate, reflux; (g) NaOH,
EtOH, H2O.
a
Scheme 2. Synthetic Pathway toward 17a,b and 22
RESULTS AND DISCUSSION
■
Cyclic β-amino acids were obtained through Curtius rearrange-
ment of diacid anhydrides26,27 7a−c (Scheme 1) or through Pd-
catalyzed and uncatalyzed allylic substitution reactions (Scheme
2).
The Curtius rearrangements (Scheme 1) resulted in β-amino
ethyl esters 8a−c that were monomethylated using the
Fukuyama protocol.28 Thus, sulfonamides 9a−c were methy-
lated with methyl iodide followed by desulfonation with
thiophenol, yielding 10a−c. The ethyl esters 10a−c were
hydrolyzed by suspension in water to yield the amino acids
11a−c or monoalkylated with 4,4-bis(3-methyl-2-thienyl)but-3-
en-1-yl mesylate to yield 12a−c, which subsequently were
hydrolyzed with NaOH to yield the lipophilic diaromatic amino
acids 13a−c.
a
Reagents and conditions: (a) Ac2O, pyridine; (b) 20, triethylamine,
DCM or 20, PPh3, PdCl2·(CH3CN)2, triethylamine, DCM; (c)
NaOH, EtOH; (d) 4,4-bis(3-methylthien-2-yl)but-3-en-1-ol, DEAD,
PPh3, DCM; (e) PhSH, K2CO3, DMF; (f) PPh3, PdCl2·(CH3CN)2,
triethylamine, methylamine, DCM; (g) 4,4-bis(3-methyl-2-thienyl)-3-
butenyl methanesulfanate, K2CO3, DMF; (h) EtOH, H2O.
The cycloalkane derivatives 17a,b (Scheme 2) were obtained
from the cyclic allylic alcohols 14a,b via acetylation29 to 15a,b
followed by allylic substitution with amine 20, yielding amino
esters 16a,b. The allylic substitution proceeded uncatalyzed but
was much faster when catalyzed by Pd. Ester hydrolysis of 16a,b
yielded the amino acids 17a,b. The amine 20 employed in allylic
substitutions was obtained from sulfonamide 18 and 4,4-bis(3-
methyl-2-thienyl)but-3-en-1-ol in a Mitsonubo reaction, yielding
19 that upon desulfonation with thiophenol gave 20.
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Table 1. Inhibitory Activity Patterns of Established and Currently Investigated mGAT Inhibitors in Primary Cultures of Neurons,
Astrocytes, and Cloned Mouse GABA Transporter
a
GABA uptake inhibition, IC50 (μM) (pIC50 SEM)
fold selectivity,
mGAT2/mGAT1
compd
GABA
astrocyte
neuron
mGAT1
mGAT2
mGAT3
mGAT4
bc
,
bc
,
bc
,
bc
,
bc
,
bc
,
8
32
17
51
15
>300
17
800
0.33
d
d
d
d
d
tiagabine (1)
0.18
0.36
0.8
300
0.0026
nipecotic acid
(2)
10 (1.0 0.09)
4 (0.61 0.42)
25 (1.39 0.05)
>1000
>3000
114 (2.06 0.05)
93 (1.97 0.06)
d
d
d
d
d
d
N-Me-exo-
THPO (4)
48
405
450
>3000
>3000
e
e
e
e
e
e
EF1502 (5)
11a
2
2
7
26
>300
>300
0.27
>300
>300
>3000
>3000
>3000
>500
>3000
>3000
>500
>3000
>3000
>500
11b
>300
>300
>3000
11c
>250
>250
>500
22
38 (1.58 0.17)
473 (2.68 0.17)
423 (2.63 0.06)
273 (2.44 0.06)
>300
134 (2.13 0.10)
36 (1.56 0.07)
50 (1.70 0.08)
144 (2.16 0.23)
50 (1.70 0.11)
45 (1.66 0.05)
>1000
>300
>1000
>300
3.2
13a
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
7.6
13b
281 (2.45 0.19)
>300
>300
>6
13c
40 (1.61 0.17)
17 (1.23 0.29)
307 (2.49 0.07)
>300
0.28
0.34
6.4
17a
>300
>300
17b
>300
286 (2.46 0.08)
a
IC50 values were calculated from inhibition curves. Each value is the mean (pIC50 SEM) of at least three independent experiments carried out in
b
c
d
e
triplicate. ND: not determined. Km. Reference 10. Reference 22. Reference 16.
The acetate 15b could be substituted with methylamine,
yielding ester 21 that upon hydrolysis gave amino acid 22.
Compound 16b could alternatively be obtained via mono-
alkylation of 21. Unfortunately, we were not able to synthesize
the related five-membered compound from 15a using the same
procedures.
All generated compounds were subjected to pharmacological
characterization using cell cultures stably overexpressing each of
the four recombinant mGATs to probe for subtype selectivity
(Table 1).
in 17a, which has the same selectivity profile as GABA toward
mGAT1 and mGAT2.
The in vitro pharmacological characterization revealed that the
new inhibitors 22, 13a, 13b, and 17b all displayed mGAT2
preference. 17b was selected for further in vivo characterization
in the audiogenic seizure (AGS) susceptible Frings mouse based
on the pharmacological profile and facile synthetic route. In
particular the introduction of the lipophilic side chain posed a
problem for large scale synthesis of 13a and 13b. This model of
reflex seizures is nondiscriminatory with respect to seizure type
and is used as a general model for identifying potential
anticonvulsant compounds.30 17b has a time to peak effect of
15 min and an ED50 of 20.4 mg/kg compared to a time to peak
effect of 60 min and an ED50 of 1.1 mg/kg for tiagabine (Table S1
in Supporting Information). To further investigate the role of
mGAT2 in seizure management, synergistic action between 17b
and tiagabine was probed. However, only an additive interaction
was observed (Table S2 and Figure S1, Supporting Information).
The present study thus defers us from confirming that the
previously reported observed synergistic interaction between
tiagabine and EF1502 in the AGS-susceptible Frings mouse is
distinctive of mGAT2 activity. This indicates that the new
selective mGAT2 inhibitor 17b possesses a different pharmaco-
logical effect or apparent mechanism of action than EF1502
when investigated in combination with tiagabine. At this point,
differences in stability and pharmacokinetic properties of 17b
compared to EF1502 cannot be ruled out, and further studies are
needed to assess the origin of the observed differences. It would
be interesting to see what effect 17b has on SB in the medial
entorhinal cortex of rats treated with kainic acid.23 Thus, further
investigation into the mechanistic details of action of 17b and
analogues at mGAT2 in vitro and in vivo are warranted.
In addition, to investigate the inhibitory activity at native
transporters and glial over neuronal selectivity, the three isosteric
analogues of N-Me-exo-THPO, 11a, 11b, and 22, were examined
in primary cortical cultures of mouse neurons and astrocytes.
Compounds 11a and 11b turned out to be inactive (IC50 > 300
μM and 3000 μM, respectively) at both recombinant and native
transporters, The unsaturated analogue 22 displayed some
activity being almost 10-fold glia-selective and, interestingly, with
a 3-fold preference for mGAT2 vs mGAT1 and a larger
preference over -3 and -4 (Table 1). This is the first example of a
small conformationally restricted GABA analogue displaying
preference for mGAT2. The norbornene amino acid 11c
displayed very weak inhibitory effects in the primary cell cultures,
but no effects were detected at the recombinant transporter
subtypes (IC50 > 500 μM for all subtypes). By contrast,
modification of the inactive saturated cyclic compounds 11a and
11b with the 4,4-bis(3-methyl-2-thienyl)-3-butenyl side chain
yielded 13a and 13b, both of which showed notable inhibitory
activity and selectivity for mGAT2 (Table 1). Interestingly,
replacing the saturated ring of 13 with a norbornene results in
13c, which showed inhibitory effects at mGAT1 instead. When
the cycloalkene derivative 22 was N-substituted with a
diaromatic lipophilic side chain (17b), the potency was
maintained and selectivity was doubled. As a novel mGAT2-
selective compound, 17b is close to equipotent with GABA at
mGAT2 and more than an order of magnitude less potent than
GABA at mGAT1. At mGAT3 and mGAT4, the relative activity
was even lower. Further decreasing the ring size in 17b resulted
CONCLUSION
■
We have reported the design, synthesis, and pharmacological
characterization of a new series of β-amino acids and their
derivatives containing the 4,4-bis(3-methyl-2-thienyl)-3-butenyl
side chain of tiagabine. This series of compounds demonstrates
how variations in the amino acid part of tiagabine can lead to
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Journal of Medicinal Chemistry
Brief Article
selective inhibitors of GABA transporter subtypes other than
mGAT1. Our design strategy has led to the identification of
several compounds with mGAT2-selective in vitro effects. We
believe that these selective compounds can be useful
pharmacological tools in elucidating the role of mGAT2 in the
CNS and that the design strategy may be explored further to
obtain subtype selective GABA uptake inhibitors.
ACKNOWLEDGMENTS
■
We thank the Lundbeck Foundation, the Novo Nordisk
Foundation, and the Carlsberg Foundation for financial support.
ABBREVIATIONS USED
■
AGS, audiogenic seizure; SB, spontaneous electrographic
bursting
EXPERIMENTAL SECTION
■
REFERENCES
■
Purity of the tested compounds was determined by elemental analysis
and/or HPLC analysis to be >95%.
(1) Enna, S. J.; McCarson, K. E. The Role of GABA in the Mediation
and Perception of Pain. In GABA; Enna, S. J., Ed.; Elsevier: San Diego,
CA, 2006; pp 1−27.
[4,4-Bis(3-methylthien-2-yl)but-3-enyl](2-carboxycyclohex-
2-enyl)methylammonium Chloride (17b). To a solution of 6-{[4,4-
bis(3-methylthien-2-yl)but-3-enyl]methylamino}cyclohex-1-enecar-
boxylic acid ethyl ester 16b (1.82 g, 4.2 mmol) in ethanol (15 mL) was
added 12 M NaOH (1.2 mL). The solution was stirred for 3 days at
room temperature. Upon evaporation of ethanol the aqueous phase was
adjusted to neutral pH with aqueous HCl (1 M) and extracted with
DCM. The combined organic phase was evaporated and the residue
dissolved in ethyl acetate followed by extraction with aqueous HCl (1
M). The combined aqueous phase was washed with diethyl ether and
then extracted with DCM. The combined organic phase was washed
with brine and dried (MgSO4). Evaporation and recrystallization
(acetone) gave [4,4-bis(3-methylthien-2-yl)but-3-enyl](2-carboxycy-
clohex-2-enyl)methylammonium chloride (620 mg, 33%) as a white
solid. Mp 103−110 °C. 1H NMR (CD3OD): δ 7.39 (1H, d, J = 5.0 Hz),
7.27−7.25 (1H, m), 7.17 (1H, d, J = 5.0 Hz), 6.93 (1H, d, J = 5.0 Hz),
6.79 (1H, d, J = 5.0 Hz), 6.06 (1H, bs), 4.81−4.76 (1H, m), 3.33−3.03
(2H, m), 2.91−2.35 (9H, m), 2.29−2.21 (2H, m), 2.05 (3H, s), 1.99
(3H, s). 13C NMR (CD3OD): δ 166.63, (154.96), 154.44, 139.50,
(137.52), 137.28, 135.85, 135.21, 133.31, 132.51, 131.41, 131.18,
128.42, 126.31, 124.75, (72.81), 69.78, 55.48, (51.47), 40.09, (36.12),
32.96, (32.40), 26.73, 24.63, 23.28, 15.14, 14.68. Six carbons give rise to
two peaks (peaks in parentheses) due to the stereocenter at the
protonated amine. Anal. Calcd for C22H28ClNO2S2·0.5H2O: C, 59.11;
H, 6.54; N, 3.13. Found: C, 59.43; H, 6.50; N, 3.42.
(2) Treiman, D. M. GABAergic mechanisms in epilepsy. Epilepsia
2001, 42 (Suppl. 3), 8−12.
(3) Ashton, H.; Young, A. H. GABA-ergic drugs: exit stage left, enter
stage right. J. Psychopharmacol. 2003, 17, 174−178.
̈
(4) Angehagen, M.; Ben-Menachem, E.; Ronnback, L.; Hansson, E.
̈
̈
Novel mechanisms of action of three antiepileptic drugs, vigabatrin,
tiagabine, and topiramate. Neurochem. Res. 2003, 28, 333−340.
(5) Madsen, K. K.; Clausen, R. P.; Larsson, O. M.; Krogsgaard-Larsen,
P.; Schousboe, A.; White, H. S. Synaptic and extrasynaptic GABA
transporters as targets for anti-epileptic drugs. J. Neurochem. 2009, 109,
139−144.
(6) Urbano, M. R.; Spiegel, D. R.; Laguerta, N.; Shrader, C. J.; Rowe, D.
F.; Hategan, L. F. Gabapentin and tiagabine for social anxiety: a
randomized, double-blind, crossover study of 8 adults. Primary Care
Companion J. Clin. Psychiatry 2009, 11, 123.
(7) Walsh, J. K.; Randazzo, A. C.; Stone, K.; Eisenstein, R.; Feren, S. D.;
Kajy, S.; Dickey, P.; Roehrs, T.; Roth, T.; Schweitzer, P. K. Tiagabine is
associated with sustained attention during sleep restriction: evidence for
the value of slow-wave sleep enhancement? Sleep 2006, 29, 433−443.
(8) Finnerup, N. B.; Baastrup, C.; Jensen, T. S. Anticonvulsants in the
Management of Chronic Pain. In Clinical Pain Management: A Practical
Guide; Lynch, M. E., Craig, K. D., Peng, P. W. H.Wiley-Blackwell:
Oxford, U.K., 2010; pp 121−127.
(9) Borden, L. A. GABA transporter heterogeneity: pharmacology and
cellular localization. Neurochem. Int. 1996, 29, 335−356.
(10) Schousboe, A.; Kanner, B. I. GABA Transporters: Functional and
Pharmacological Properties. In Glutamate and GABA Receptors and
Transporters: Structure, Function and Pharmacology; Egebjerg, J.,
Schousboe, A., Krogsgaard-Larsen, P., Eds.; Taylor & Francis: London,
2002; pp 337−349.
(11) Kristensen, A. S.; Andersen, J.; Jørgensen, T. N.; Sørensen, L.;
Eriksen, J.; Loland, C. J.; Strømgaard, K.; Gether, U. SLC6
neurotransmitter transporters: structure, function, and regulation.
Pharmacol. Rev. 2011, 63, 585−640.
[3H]GABA Uptake. [3H]GABA uptake in primary cultures of
astrocytes, neurons, and recombinant cell systems was investigated
essentially as previously described.16 The incubations were carried out at
37 °C in phosphate buffered saline containing [3H]GABA (1 μCi/mL)
and 1 μM GABA and increasing concentrations of inhibitor. The
incubations were terminated after 3 min using an Elx50 automated strip
washer (BioTek, VT, U.S.). The cells were solubilized directly in
Microscint-20, and radioactivity was counted in a TopCount microplate
scintillation counter from Packard (Boston, MA, U.S.). Inhibition curves
were analyzed using Prism 5.04 (GraphPad Software, San Diego, CA,
U.S.). The concentration−inhibition curves generated in the
[3H]GABA uptake assay were fitted by nonlinear regression.
(12) Høg, S.; Greenwood, J. R.; Madsen, K. B.; Larsson, O. M.;
Frølund, B.; Schousboe, A.; Krogsgaard-Larsen, P.; Clausen, R. P.
Structure−activity relationships of selective GABA uptake inhibitors.
Curr. Top. Med. Chem. 2006, 6, 1861−1882.
(13) Krogsgaard-Larsen, P.; Falch, E.; Larsson, O. M.; Schousboe, A.
GABA uptake inhibitors: relevance to antiepileptic drug research.
Epilepsy Res. 1987, 1, 77−93.
(14) Krogsgaard-Larsen, P.; Frølund, B.; Frydenvang, K. GABA uptake
inhibitors. Design, molecular pharmacology and therapeutic aspects.
Curr. Pharm. Des. 2000, 6, 1193−1209.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental data for 8a−c to 22, complete description of in
vitro and in vivo pharmacological methods, and additional tables
and figure. This material is available free of charge via the Internet
at http://pubs.acs.org.
(15) Falch, E.; Perregaard, J.; Frølund, B.; Søkilde, B.; Buur, A.;
Hansen, L. M.; Frydenvang, K.; Brehm, L.; Bolvig, T.; Larsson, O. M.;
Sanchez, C.; White, H. S.; Schousboe, A.; Krogsgaard-Larsen, P.
Selective inhibitors of glial GABA uptake: synthesis, absolute stereo-
chemistry, and pharmacology of the enantiomers of 3-hydroxy-4-amino-
4,5,6,7-tetrahydro-1,2-benzisoxazole (exo-THPO) and analogues. J.
Med. Chem. 1999, 42, 5402−5414.
(16) 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. Selective inhibitors of GABA
uptake: synthesis and molecular pharmacology of 4-N-methylamino-
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +45 35 33 65 66. E-mail: rac@sund.ku.dk.
Author Contributions
§These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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Brief Article
4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol analogues. Bioorg. Med. Chem.
2005, 13, 895−908.
(17) Clausen, R. P.; Frølund, B.; Larsson, O. M.; Schousboe, A.;
Krogsgaard-Larsen, P.; White, H. S. A novel selective gamma-
aminobutyric acid transport inhibitor demonstrates a functional role
for GABA transporter subtype GAT2/BGT-1 in the CNS. Neurochem.
Int. 2006, 48, 637−642.
(18) Thomsen, C.; Sørensen, P. O.; Egebjerg, J. 1-(3-(9H-carbazol-9-
yl)-1-propyl)-4-(2-methoxyphenyl)-4-piperidinol, a novel subtype
selective inhibitor of the mouse type II GABA-transporter. Br. J.
Pharmacol. 1997, 120, 983−985.
(19) Dalby, N. O.; Thomsen, C.; Fink-Jensen, A.; Lundbeck, J.;
Søkilde, B.; Man, C. M.; Sørensen, P. O.; Meldrum, B. Anticonvulsant
properties of two GABA uptake inhibitors NNC 05-2045 and NNC 05-
2090, not acting preferentially on GAT-1. Epilepsy Res. 1997, 28, 51−61.
(20) Fulep, G. H.; Hoesl, C. E.; Hofner, G.; Wanner, K. T. New highly
̈
̈
potent GABA uptake inhibitors selective for GAT-1 and GAT-3 derived
from (R)- and (S)-proline and homologous pyrrolidine-2-alkanoic acids.
Eur. J. Med. Chem. 2006, 41, 809−824.
(21) Schaffert, E. S.; Hofner, G.; Wanner, K. T. Aminomethyltetrazoles
as potential inhibitors of the gamma-aminobutyric acid transporters
mGAT1-mGAT4: synthesis and biological evaluation. Bioorg. Med.
Chem. 2011, 19, 6492−6504.
(22) White, H. S.; Watson, W. P.; Hansen, S. L.; Slough, S.; Perregaard,
J.; Sarup, A.; Bolvig, T.; Petersen, G.; Larsson, O. M.; Clausen, R. P.;
Frølund, B.; Falch, E.; Krogsgaard-Larsen, P.; Schousboe, A. First
demonstration of a functional role for central nervous system betaine/
gamma-aminobutyric acid transporter (mGAT2) based on synergistic
anticonvulsant action among inhibitors of mGAT1 and mGAT2. J.
Pharmacol. Exp. Ther. 2005, 312, 866−874.
(23) Smith, M. D.; Saunders, G. W.; Clausen, R. P.; Frølund, B.;
Krogsgaard-Larsen, P.; Larsson, O. M.; Schousboe, A.; Wilcox, K. S.;
White, H. S. Inhibition of the betaine-GABA transporter (mGAT2/
BGT-1) modulates spontaneous electrographic bursting in the medial
entorhinal cortex (mEC). Epilepsy Res. 2008, 79, 6−13.
(24) Madsen, K. K.; Ebert, B.; Clausen, R. P.; Krogsgaard-Larsen, P.;
Schousboe, A.; White, H. S. Selective GABA transporter inhibitors
tiagabine and EF1502 exhibit mechanistic differences in their ability to
modulate the ataxia and anticonvulsant action of the extrasynaptic
GABA(A) receptor agonist gaboxadol. J. Pharmacol. Exp. Ther. 2011,
338, 214−219.
(25) Lehre, A. C.; Rowley, N. M.; Zhou, Y.; Holmseth, S.; Guo, C.;
Holen, T.; Hua, R.; Laake, P.; Olofsson, A. M.; Poblete-Naredo, I.;
Rusakov, D. A.; Madsen, K. K.; Clausen, R. P.; Schousboe, A.; White, H.
S.; Danbolt, N. C. Deletion of the betaine-GABA transporter (BGT1;
slc6a12) gene does not affect seizure thresholds of adult mice. Epilepsy
Res. 2011, 95, 70−81.
(26) Kricheldorf, H. R. Synthesis and polymerization of beta-amino-
acid N-carboxyanhydrides. Makromol. Chem. 1973, 173, 13−41.
(27) Kricheldorf, H. R. Reactions with silyl azides. 10. Synthesis of
alicyclic beta-amino acids and their derivatives. Justus Liebig Ann. Chem.
1975, 1387−1393.
(28) Fukuyama, T.; Jow, C. K.; Cheung, M. 2-Nitrobenzenesulfona-
mides and 4-nitrobenzenesulfonamidesexceptionally versatile means
for preparation of secondary-amines and protection of amines.
Tetrahedron Lett. 1995, 36, 6373−6374.
(29) Takano, S.; Yamane, T.; Takahashi, M.; Ogasawara, K.
Enantiocomplementary synthesis of functionalized cycloalkenol build-
ing blocks using lipase. Tetrahedron: Asymmetry 1992, 3, 837−840.
(30) White, H. S.; Patel, S.; Meldrum, B. S. Anticonvulsant profile of
MDL 27,266: an orally active, broad-spectrum anticonvulsant agent.
Epilepsy Res. 1992, 12, 217−226.
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