Selective mGAT2 (BGT-1) GABA Uptake Inhibitors: Design

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.

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INTRODUCTION The major inhibitory neurotransmitter in the central nervous system (CNS), GABA (Chart 1), plays a fundamental role in the

inhibitor tiagabine [(R)-N-[4,4-bis(3-methyl-2-thienyl)-3butenyl]nipecotic acid, 1] are clinically effective anticonvulsant drugs.4 Therapeutic application of GABA transporter inhibitors seems particularly attractive, since it increases GABA neurotransmission 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 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, respectively.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 transporters 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,

Chart 1. Chemical Structures of GABA and Selected GABA Transporter Inhibitors

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 © 2013 American Chemical Society

Received: December 19, 2012 Published: February 11, 2013 2160

dx.doi.org/10.1021/jm301872x | J. Med. Chem. 2013, 56, 2160−2164

Journal of Medicinal Chemistry

Brief Article

Scheme 1. Synthetic Pathway toward 11a−c and 13a−ca

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 nonGAT 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 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.

Scheme 2. Synthetic Pathway toward 17a,b and 22a

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RESULTS AND DISCUSSION Cyclic β-amino acids were obtained through Curtius rearrangement of diacid anhydrides26,27 7a−c (Scheme 1) or through Pdcatalyzed 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 methylated 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-3en-1-yl mesylate to yield 12a−c, which subsequently were hydrolyzed with NaOH to yield the lipophilic diaromatic amino acids 13a−c. 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

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)-3butenyl methanesulfanate, K2CO3, DMF; (h) EtOH, H2O.

yielded the amino acids 17a,b. The amine 20 employed in allylic substitutions was obtained from sulfonamide 18 and 4,4-bis(3methyl-2-thienyl)but-3-en-1-ol in a Mitsonubo reaction, yielding 19 that upon desulfonation with thiophenol gave 20. 2161



Brief Article

Table 1. Inhibitory Activity Patterns of Established and Currently Investigated mGAT Inhibitors in Primary Cultures of Neurons, Astrocytes, and Cloned Mouse GABA Transporter GABA uptake inhibition, IC50 (μM)a (pIC50 ± SEM) compd GABA tiagabine (1) nipecotic acid (2) N-Me-exoTHPO (4) EF1502 (5) 11a 11b 11c 22 13a 13b 13c 17a 17b

astrocyte

neuron

mGAT1

mGAT2

mGAT3

mGAT4

8b,c 0.18d 10 (1.0 ± 0.09)

32b,c 0.36d 4 (0.61 ± 0.42)

17b,c 0.8d 25 (1.39 ± 0.05)

51b,c 300d >1000

15b,c >300d 114 (2.06 ± 0.05)

17b,c 800 93 (1.97 ± 0.06)

48d

405d

450d

>3000d

>3000d

>3000d

2e >300 >300 >250 38 (1.58 ± 0.17) ND ND ND ND ND

2e >300 >300 >250 473 (2.68 ± 0.17) ND ND ND ND ND

7e >3000 >3000 >500 423 (2.63 ± 0.06) 273 (2.44 ± 0.06) >300 40 (1.61 ± 0.17) 17 (1.23 ± 0.29) 307 (2.49 ± 0.07)

26e >3000 >3000 >500 134 (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)

>300e >3000 >3000 >500 >1000 >300 281 (2.45 ± 0.19) >300 >300 >300

>300e >3000 >3000 >500 >1000 >300 >300 >300 >300 286 (2.46 ± 0.08)

fold selectivity, mGAT2/mGAT1 0.33 0.0026

0.27

3.2 7.6 >6 0.28 0.34 6.4

a IC50 values were calculated from inhibition curves. Each value is the mean (pIC50 ± SEM) of at least three independent experiments carried out in triplicate. ND: not determined. bKm. cReference 10. dReference 22. eReference 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 monoalkylation 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 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 mGAT2selective 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

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 pharmacological 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.

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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 2162



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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.

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ABBREVIATIONS USED AGS, audiogenic seizure; SB, spontaneous electrographic bursting

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EXPERIMENTAL SECTION

REFERENCES

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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.

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ACKNOWLEDGMENTS

We thank the Lundbeck Foundation, the Novo Nordisk Foundation, and the Carlsberg Foundation for financial support.

Purity of the tested compounds was determined by elemental analysis and/or HPLC analysis to be >95%. [4,4-Bis(3-methylthien-2-yl)but-3-enyl](2-carboxycyclohex2-enyl)methylammonium Chloride (17b). To a solution of 6-{[4,4bis(3-methylthien-2-yl)but-3-enyl]methylamino}cyclohex-1-enecarboxylic 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-carboxycyclohex-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. [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.

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Brief Article

AUTHOR INFORMATION

Corresponding Author

*Phone: +45 35 33 65 66. E-mail: [email protected]. Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest. 2163



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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 gammaaminobutyric 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-9yl)-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 052090, not acting preferentially on GAT-1. Epilepsy Res. 1997, 28, 51−61. (20) Fülep, G. H.; Hoesl, C. E.; Höfner, 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-aminoacid 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-Nitrobenzenesulfonamides 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 building 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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Selective mGAT2 (BGT-1) GABA Uptake Inhibitors: Design

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