Development of Positron Emission Tomography (PET) Radiotracers for the GABA Transporter 1 (GAT-1)

Article abstract of DOI:10.1021/acschemneuro.8b00183

In vivo PET imaging of the γ-aminobutyric acid (GABA) receptor complex has been accomplished using radiolabeled benzodiazepine derivatives, but development of specific presynaptic radioligands targeting the neuronal membrane GABA transporter type 1 (GAT-1) has been less successful. The availability of new structure-activity studies of GAT-1 inhibitors and the introduction of a GAT-1 inhibitor (tiagabine, Gabatril) into clinical use prompted us to reinvestigate the syntheses of PET ligands for this transporter. Initial synthesis and rodent PET studies of N-[¹¹C]methylnipecotic acid confirmed the low brain uptake of that small and polar molecule. The common design approach to improve blood-brain barrier permeability of GAT-1 inhibitors is the attachment of a large lipophilic substituent. We selected an unsymmetrical bis-aromatic residue attached to the ring nitrogen by a vinyl ether spacer from a series recently reported by Wanner and coworkers. Nucleophilic aromatic substitution of an aryl chloride precursor with [¹⁸F]fluoride was used to prepare the desired candidate radiotracer (R,E/Z)-1-(2-((4-fluoro-2-(4-[¹⁸F]fluorobenzoyl)styryl)oxy)ethyl)piperidine-3-carboxylic acid ((R,E/Z)-[¹⁸F]10). PET studies in rat showed no brain uptake, which was not altered by pretreatment of animals with the P-glycoprotein inhibitor cyclosporine A, indicating efflux by Pgp was not responsible. Subsequent PET imaging studies of (R,E/Z)-[¹⁸F]10 in rhesus monkey brain showed very low brain uptake. Finally, to test if the free carboxylic acid group was the likely cause of poor brain uptake, PET studies were done using the ethyl ester derivative of (R,E/Z)-[¹⁸F]10. Rapid and significant monkey brain uptake of the ester was observed, followed by a slow washout over 90 minutes. The blood-brain barrier permeability of the ester supports a hypothesis that the free acid function limits brain uptake of nipecotic acid-based GAT-1 radioligands, and future radiotracer efforts should investigate the use of carboxylic acid bioisosteres.

Full text of DOI:10.1021/acschemneuro.8b00183

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Article
Development of Positron Emission Tomography (PET)
Radiotracers for the GABA Transporter 1 (GAT-1)
Alexandra R Sowa, Allen F Brooks, Xia Shao, Bradford D Henderson, Phillip S. Sherman, Janna
Arteaga, Jenelle Stauff, Adam C Lee, Robert A Koeppe, Peter J. H. Scott, and Michael R. Kilbourn
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00183 • Publication Date (Web): 15 May 2018
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Development of Positron Emission Tomography (PET) Radiotracers for the GABA
Transporter 1 (GAT-1)
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Alexandra R. Sowa,1,2 Allen F. Brooks,1 Xia Shao,1 Bradford D. Henderson,1 Philip
Sherman,1 Janna Arteaga,1 Jenelle Stauff,1 Adam C. Lee,3 Robert A. Koeppe,1
Peter J. H. Scott1,2* and Michael R. Kilbourn1*
1. Department of Radiology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
2. Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48105, USA.
3. E.I. du Pont de Nemours and Company, DuPont Haskell Global Center for Health Sciences, P.O. Box
30, Newark, DE 19714, USA.
Abstract
In vivo PET imaging of the γꢀaminobutyric acid (GABA) receptor complex has been
accomplished using radiolabeled benzodiazepine derivatives, but development of
specific presynaptic radioligands targeting the neuronal membrane GABA transporter
type 1 (GATꢀ1) has been less successful. The availability of new structureꢀactivity
studies of GATꢀ1 inhibitors and the introduction of a GATꢀ1 inhibitor (tiagabine,
Gabatril®) into clinical use prompted us to reinvestigate the syntheses of PET ligands for
this transporter. Initial synthesis and rodent PET studies of Nꢀ[11C]methylnipecotic acid
confirmed the low brain uptake of that small and polar molecule. The common design
approach to improve bloodꢀbrain barrier permeability of GATꢀ1 inhibitors is the
attachment of a large lipophilic substituent. We selected an unsymmetrical bisꢀaromatic
residue attached to the ring nitrogen by a vinyl ether spacer from a series recently
reported by Wanner and coworkers. Nucleophilic aromatic substitution of an aryl
chloride precursor with [18F]fluoride was used to prepare the desired candidate
radiotracer (R,E/Z)ꢀ1ꢀ(2ꢀ((4ꢀfluoroꢀ2ꢀ(4ꢀ[18F]fluorobenzoyl)styryl)oxy)ethyl)piperidineꢀ3ꢀ
carboxylic acid ((R,E/Z)ꢀ[18F]10). PET studies in rat showed no brain uptake, which was
not altered by pretreatment of animals with the Pꢀglycoprotein inhibitor cyclosporine A,
indicating efflux by Pgp was not responsible. Subsequent PET imaging studies of
(R,E/Z)ꢀ[18F]10 in rhesus monkey brain showed very low brain uptake. Finally, to test if
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the free carboxylic acid group was the likely cause of poor brain uptake, PET studies
were done using the ethyl ester derivative of (R,E/Z)ꢀ[18F]10. Rapid and significant
monkey brain uptake of the ester was observed, followed by a slow washout over 90
minutes. The bloodꢀbrain barrier permeability of the ester supports a hypothesis that the
free acid function limits brain uptake of nipecotic acidꢀbased GATꢀ1 radioligands, and
future radiotracer efforts should investigate the use of carboxylic acid bioisosteres.
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Keywords
GABA, transporter, positron emission tomography, fluorineꢀ18.
Introduction
The amino acids γꢀaminobutyric acid (GABA (1), Figure 1) and glycine are the
predominant inhibitory amino acid neurotransmitters in the mammalian central nervous
system. Neurons presenting the biochemical features of GABAꢀergic neurons are
widespread throughout the CNS, comprising 20ꢀ30% of cortical neurons,1 and provide
the inhibitory balance to excitatory glutamatergic neurons. Reflecting this, dysfunction of
the GABA system has been implicated in numerous neurodevelopmental diseases,
such as seizure disorders (e.g., epilepsy), and psychiatric diseases such as
schizophrenia, Autism Spectrum Disorder (ASD), RETT syndrome, depression, and
anxiety disorders. Drugs that target the GABA system are widely prescribed (e.g.,
benzodiazepines, progabide, gabapentin, pregabalin) to manage these disorders.
Despite the importance of the GABAꢀergic system in health and disease, efforts to
develop in vivo agents for positron emission tomography (PET) or single photon
emission computed tomography (SPECT) imaging of specific sites on GABAꢀergic
neurons have been limited. Successful imaging agents have targeted the
benzodiazepine binding site on the GABAA receptor complex.2 Changes in radioligand
(e.g., [11C]flumazenil ([11C]FMZ)) binding to the benzodiazepine binding site are then
used as surrogate markers of alterations in concentrations of GABAA receptors, but as
those receptors are largely found in the postꢀsynaptic membranes (and are subject to
regulation by trafficking mechanisms3) the binding of radiolabeled benzodiazepines
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such as [11C]FMZ does not provide information on the concentration of presynaptic
GABAꢀergic neurons. Radioligand development efforts for other sites in the GABA
system receptor, such as GABAB receptors or the chloride ion channel of GABAA
receptor complexes have been less successful,4 and have not progressed to human
studies.
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The development of radiotracers intended as presynaptic neuronal markers has
targeted neuronal membrane and vesicular neurotransmitter transporters, resulting in
validated radiotracers for the neuronal membrane transporters (dopamine, serotonin,
norepinephrine and glycine) and vesicular transporters (monoamines and
acetylcholine).5
To date there are no radiotracers for the neuronal membrane GABA transporters. Our
prior efforts in the design and synthesis of radioligands for GABA transporters targeted
fluorineꢀ18 radiotracers based on the structure of CIꢀ966 (2), and yielded a potential
GATꢀ1
radioligand
((R,S)ꢀ1ꢀ[2ꢀ(4ꢀ[18F]fluorophenyl)(4ꢀfluorophenyl)]ꢀ
methoxyethyl]piperidineꢀ3ꢀcarboxylic acid ([18F]3) (Figure 1).6 Although exhibiting low
brain permeability, [18F]3 did have a heterogeneous in vivo brain distribution similar to
that obtained using [3H]tiagabine in vitro and ex vivo .7,8 These early studies were
encouraging, but the combination of (1) a still rudimentary understanding of the
pharmacology and brain distribution of the GABA transporters, (2) a lack of published
structureꢀactivity studies for GATꢀ1 inhibitors, (3) concerns over potential adverse
pharmacological effects of this family of GATꢀ1 inhibitors,9 and (4) difficult chemistry to
obtain the high specific activities needed for human studies led us to cease further effort
in development of GABA transporter inhibitor radioligands. Subsequent efforts by others
in syntheses of radiolabeled GABA transporter inhibitors (GATꢀ1 and GATꢀ3) for
imaging have also been unsuccessful.10
In the intervening 20 years, much has changed. The molecular biology and
pharmacology of GABA transporters is better characterized, including proposed tertiary
structures of potential binding sites for ligands.11 Advances in radiochemistry have
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provided routinely successful preparations of 11Cꢀ and Fꢀlabeled compounds at high
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specific activities (>1000 Ci/mmol). Structureꢀactivity relationships of GAT inhibitors and
their selectivity for the four forms of GAT (GATꢀ1, 2, 3 and 4) have been explored by
multiple investigators,12 and GATꢀ1 is now recognized as being predominantly located
on presynaptic neurons, with a minor population on astrocytes.13 Notably, there is a
large concentration of GATꢀ1 in human cortex (3400 fmol/mg protein, or 340 nM),14 a
concentration higher than many receptor sites that have been successfully imaged.15
Finally, and perhaps most importantly, the GATꢀ1 inhibitor tiagabine (Gabitril® (4),
Figure 1) has been shown safe for use in humans,16 and was FDA approved for clinical
use in 1997 as an adjunctive treatment for seizures. [3H]Tiagabine has also been used
for in vitro binding studies in rat and human brain tissues,7,14 including recent postꢀ
mortem studies that demonstrate significant changes in GATꢀ1 in aging17 and
schizophrenia.18 In succeeding years multiple additional molecular scaffolds have been
investigated, but all share a consistent structural design, that of a large lipophilic group
attached to the nitrogen in the ring of a small cyclic amino acid such as nipecotic acid
(5) (Figure 1) or guvacine (1,2,5,6ꢀtetrahydropyridineꢀ3ꢀcarboxylic acid). The added
lipophilicity is considered necessary, as it is known that nipecotic acid does not cross
the bloodꢀbrain barrier, likely due to its polar nature (cLogD7.4 = ꢀ2.3819) and zwitterionic
character.
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Despite these advances, there are still no PET (or SPECT) radiotracers known for
imaging presynaptic GABA neuron densities in the clinic, and we have therefore
revisited the development of radioligands for GABA transporters to allow studies of
GABAꢀergic innervation in human diseases. Herein we report the design, synthesis and
evaluation of a potential new PET radiotracer for the GATꢀ1 transporter.
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Figure 1. GABA (1), GATꢀ1 inhibitors and radiotracers (2ꢀ5) and [11C]PMP (6)
Results and Discussion
Our reꢀinvestigation of potential GATꢀ1 imaging agents began with an examination of
the in vivo properties of the simple Nꢀ[11C]methylated nipecotic acid ([11C]9, clogD7.4 = ꢀ
1.62), which was easily prepared by Nꢀ[11C]methylation of racemic ethyl nipecotate (7)
followed by immediate ester hydrolysis using 5M LiOH. PET imaging studies in rats
showed no brain uptake of [11C]9 (see Supporting Information, Figure S13), which was
not completely unexpected and confirmed the need for larger lipophilic Nꢀmethyl
substituents. As the presence of the free carboxylic acid likely was the reason for poor
bloodꢀbrainꢀbarrier (BBB) permeability of [11C]9, we next isolated and purified the
intermediate Nꢀ[11C]methylnipecotic acid ethyl ester ([11C]8, cLogD7.4 = ꢀ0.27).
Surprisingly, that ester also demonstrated no permeability into the rat brain (see
Supporting Information, Figure S13). That result was unexpected, given that compound
[11C]8 is identical in molecular weight and functional groups (Nꢀmethylpiperidine and
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carboxylic acid ester) to the radiotracer Nꢀ[11C]methylpiperidinꢀ4ꢀyl propionate
([11C]PMP ([11C]6, Figure 1), clogD7.4 = ꢀ0.81) that has been extensively utilized for PET
imaging of acetylcholinesterase in rat, primate and human brain. The poor BBB
permeability of [11C]8 remains unexplained.
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Scheme 1. Radiosynthesis of [11C]8 and [11C]9. Reagents and Conditions: i)
[11C]MeOTf, DMF, room temperature, 3 min; ii) 5M LiOH, 100 °C, 5 min ([11C]8 was
isolated in 2% nonꢀcorrected radiochemical yield (RCY) from 7; [11C]9 was isolated in
4% nonꢀcorrected RCY from 7).
We concluded from these initial studies that Nꢀmethylnipecotic acid derivatives were not
sufficiently drugꢀlike to penetrate the CNS (Table 1), and so turned our attention to
developing a radiotracer that was based upon nipecotic acid but with improved drugꢀlike
properties. We chose to avoid further derivatives of CIꢀ966, given its known toxicity and
the poor brain uptake of [18F]3.6,9 Therefore, we were attracted by a new series of GATꢀ
1 inhibitors reported by Wanner and colleagues in 2013.20 These compounds consisted
of a nipecotic acid core and an unsymmetrical bisꢀaromatic residue attached to the ring
nitrogen by a vinyl ether spacer, and we selected compound 10 as the lead because of
its potency in a functional [3H]GABA uptake assay (pIC50 = 5.73±0.14 ((E)ꢀ10) and
6.47±0.08 ((Z)ꢀ10)) and >100ꢀfold selectivity for GATꢀ1 over GATꢀ2, ꢀ3 and ꢀ4.20 The
functional GABA uptake assay is commonly used to screen GATꢀ1 inhibitors, but has
been demonstrated to consistently and significantly underestimate the binding affinities
(Ki or Kd values).21 For example, tiagabine exhibits a pIC50 = 6.88 (or 132 nM) for
inhibiting [3H]GABA uptake, but binding assays with [3H]tiagbine gave a Kd = 16 nM.14
Extending this relationship to lead compound 10 suggests that a pIC50 of 6.47 (340 nM)
would equate with an in vitro binding affinity of approximately 41 nM. This estimated in
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vitro affinity was then used to project a potential in vivo cortical binding potential (BP) for
compound 10, where BP = Bmax/Kd, yielding a value of 340 nM/41 nM = 8. Such a value
is higher than the oftenꢀproposed need for a BP > 5 to achieve successful in vivo human
imaging of specific brain binding for new radiotracers,22 and together with the medicinal
chemistry properties comparable to successful CNS drugs (Table 1)23 and amenability
for labeling with both carbonꢀ11 and fluorineꢀ18 made compound 10 attractive from a
radiotracer development perspective.
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Typical value
for successful
Property
CNS drugs23
clogp
1.5ꢀ2.7
ꢀ1.65
ꢀ1.62
1.81
2.56
clogD7.4
0ꢀ3
tPSA
60ꢀ90
40.54 Å
143 g/mol
3
66.84 Å
415 g/mol
5
molecular weight
heteroatoms (O + N)
Acidic pKa
Basic pKa
≤400ꢀ600 g/mol
≤5
>4
3.22
3.27
<10
9.60
7.76
Table 1. Lead GATꢀ1 Inhibitor (10) for radiolabeling
We elected to prepare the fluorineꢀ18 labeled version of the lead compound ((R,E/Z)ꢀ
[18F]10) from the corresponding chlorinated precursor (R,E/Z)ꢀ16b using nucleophilic
aromatic substitution with [18F]fluoride. The synthetic pathway to access unlabeled
reference standard (R,E/Z)ꢀ10 and chloroꢀprecursor (R,E/Z)ꢀ16b was the same
(Scheme 2). Initially 4ꢀfluorophenol 11 was coupled with the corresponding acyl chloride
12 to provide the ester. Treating the intermediate ester with aluminum trichloride
promoted a Fries rearrangement to give an orthoꢀacylated phenol, and the phenol was
converted to triflate derivative 13. Concomitantly, 2ꢀ(vinyloxy)ethanꢀ1ꢀol 14 was
tosylated and used to alkylate nipecotate (R)ꢀ7 and generate (R)ꢀ15. A Pdꢀmediated
Heck crossꢀcoupling of aryl triflate 13 and nipecotic acid derivative (R)ꢀ15 yielded ester
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derivative (R)ꢀ16 as a mixture of the E and Z products.24 The chlorinated ester is the
desired precursor for radiolabeling ((R,E/Z)ꢀ16b), while saponification of the fluorinated
ester ((R,E/Z)ꢀ16a) generated unlabeled reference standard (R,E/Z)ꢀ10.
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Scheme 2. Synthesis of precursor (R,E/Z)ꢀ16b and reference standard (R,E/Z)ꢀ10.
Reagents and conditions: i) Et3N, CH2Cl2, reflux, 1 h (X = F, 77% X=Cl, 83%); ii) AlCl3,
200 °C, 30ꢀ40 min (X= F, 54%, X=Cl, 59%); iii) Tf2O, 2,6ꢀlutidine, CH2Cl2, 0 °C – rt, 36 h
(X= F, 59% X=Cl, 61% ); iv) TsCl, Et3N, DMAP, CH2Cl2, 48 h (65%); v) Et3N, (R)ꢀ7, rt,
16 h (50%); vi) Pd(OAc)2, Et3N, PPh3, DMF, 80 °C, 48 h (X= F, 20%, X=Cl, 48%); vii)
2M LiOH, EtOH, 0 °C – rt, 0.5 h (X = F, 46%).
Synthesis of the fluorineꢀ18 labeled compound ((R,E/Z)ꢀ[18F]10, clogD7.4 = 2.56) was
accomplished by subjecting chloroꢀprecursor (R,E/Z)ꢀ16b to standard SNAr conditions
o
([18F]KF and kryptofixꢀ2.2.2 (K2.2.2) in DMF, 130 C, 30 min) and saponification of the
ester with LiOH (Scheme 3). Purification (semiꢀpreparative HPLC) and reformulation
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(C18 cartridge) provided 9.3±3.3 mCi of (R,E/Z)ꢀ[18F]10 (1% nonꢀcorrected RCY, 100%
radiochemical purity and specific activity = 1702 Ci/mmol, n = 4; see Supporting
Information for full details of the radiosynthesis).
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Scheme 3. Radiosynthesis of (R,E/Z)ꢀ[18F]10 and (R,E/Z)ꢀ[18F]16a. Reagents and
conditions: i) [18F]KF, K2.2.2, DMF, 130 °C, 30 min; ii) LiOH, 100 °C, 15 min ((R,E/Z)ꢀ
[18F]10 and (R,E/Z)ꢀ[18F]16a were both isolated in 1% nonꢀcorrected RCY).
PET imaging studies using (R,E/Z)ꢀ[18F]10 were first conducted in rodents (Figure 2A).
To our surprise however, there was very little brain uptake of (R,E/Z)ꢀ[18F]10 in the rat
brain. To test if the lack of brain uptake was due to the actions of Pꢀglycoprotein (Pgp),
an efflux transporter at the bloodꢀbrain barrier,25 we repeated imaging of a rat following
preꢀtreatment with 50 mg/kg cyclosporine A, as we have done previously for other
radiotracers.25b However, this had no effect on brain uptake of (R,E/Z)ꢀ[18F]10 (Figure
2B). Finally, to test if the BBB impermeability was due to the free carboxylic acid
functionality, we isolated and purified the intermediate ester ((R,E/Z)ꢀ[18F]16a, clogD7.4
= 4.11) but PET studies again showed no brain uptake (Figure 2C).
A
B
C
2.40
2.00
1.60
1.20
0.80
0.40
0
= rat
brain
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Figure 2. Baseline rodent imaging with (R,E/Z)ꢀ[18F]10 (A), imaging of rodents preꢀ
treated with 50 mg/kg cyclosporine A 60 min prior to injection of (R,E/Z)ꢀ[18F]10 (B), and
baseline rodent imaging with (R,E/Z)ꢀ[18F]16A (C). Images are summed sagittal images
0−90 min postꢀinjection of the radiotracer.
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With the hypothesis that (R,E/Z)ꢀ[18F]10 possesses properties consistent with BBB
permeability (Table 1), the lack of brain uptake of both the ester and the free carboxylic
acid into the rat brain was unexpected. As there is always the possibility of species
differences in distribution and metabolism of drugs and radiotracers, we undertook
evaluation of imaging studies with (R,E/Z)ꢀ[18F]10 and (R,E/Z)ꢀ[18F]16a in rhesus
monkey brain. While there were some discernible points of retention of (R,E/Z)ꢀ[18F]10
in the monkey brain (Figure 3A), the levels were deemed too low for (R,E/Z)ꢀ[18F]10 to
be useful for imaging GATꢀ1 activity in the CNS. In contrast to the result in rats,
administration of the ester (R,E/Z)ꢀ[18F]16a gave significant brain uptake of radioactivity
in cortex, thalamus, striatum and cerebellum (Figure 3B, 3C). The species differences
between rodents and primates could be due to differences in esterase expression, as
the hydrolysis of certain esters is known to be much faster in rat than monkey.26
Although (R,E/Z)ꢀ[18F]16a clearly enters the brain, this uptake cannot be assumed to
correspond to specific binding to GATꢀ1, as it has been shown in a number of studies
into GABA and related compounds that a free acid is essential for activity and that
esters are not active.27 Moreover, even if ester [18F]16a were to have affinity for GATꢀ1
or was behaving as a proꢀdrug and being hydrolyzed in the brain to acid [18F]10, we
anticipated that the inability to determine whether the PET images were due to ester
[18F]16a or acid [18F]10 would thwart any attempts at quantitative analysis.
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Figure 3. Baseline nonhuman primate imaging with (R,E/Z)ꢀ[18F]10 (A), (R,E/Z)ꢀ
[18F]16a (B) and regional time−radioactivity curves for (R,E/Z)ꢀ[18F]16a (C). PET images
are summed sagittal images 0−90 min postꢀinjection of the radiotracer.
Our results of low brain uptake of (R,E/Z)ꢀ[18F]10 are consistent with those obtained for
[18F]3 and [123I]iodotiagabine, previously tested as GATꢀ1 radioligands.6,28 Together, the
radiotracers evaluated to date span a range of clogD7.4 values (ꢀ1.62 – +4.11) and do
not appear to be Pgp substrates. A common component of all of them is the nipecotic
acid moiety, suggesting that the free carboxylic acid functionality limits BBB
permeability: however, a carboxylic acid group in a molecule does not disqualify it from
crossing the BBB and entering the CNS, as exemplified by the radiotracer
[11C]bexarotene (clogD7.4 = 3.32) that has been used successfully for PET imaging of
retinoid X receptors.29 More likely, the difficulty with using nipecotic acid derivatives as
imaging agents lies in the ability of such compounds to form highly polar zwitterions.
The development of GATꢀ1 imaging agents thus remains unsuccessful, and may
present different challenges than the requirements for therapeutic drug applications.
Tiagabine may in fact achieve low brain uptake, which would be consistent with its
pharmacology in rodents30 and need for careful dosing in human subjects including the
requirement for an extensive titration period.31 Whereas, low brain uptake of a
therapeutic drug such as tiagabine can be countered by administering repeated doses
of the drug over several weeks to build up brain concentrations until the desired efficacy
is achieved, for radiotracers used in PET or SPECT rapid brain uptake at sufficient
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levels must be achieved to allow external imaging and quantification. This apparent
mismatch between the pharmacokinetic properties of GATꢀ1 inhibitors such as tiagabine
and CIꢀ966 and the requirements for an in vivo imaging radiotracer offers a plausible
explanation for why our efforts to image GATꢀ1 have been unsuccessful to date, but
also points to a possible solution: carboxylic acid bioisosterism. If the carboxylic acid
function is necessary for high affinity binding to the GATꢀ1, but limits BBB permeability,
then the replacement by an alternative functional group that mimics the size, volume,
electronic and physicochemical properties of the carboxylic acid might be considered.
Fortunately for our purposes, many bioisosteres of the carboxylic acid group have been
developed,32 and there is precedent for applying them to GABA and its analogs.33,34 Our
efforts to design 2nd generation GATꢀ1 PET radiotracers containing carboxylic acid
bioisosteres are ongoing and will be the subject of a future report.
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Conclusions
The attachment of a large lipophilic substituent to a small cyclic amino acid such as
nipecotic acid remains the only successful design strategy for high affinity GATꢀ1
inhibitors. This has resulted in a useful clinical GATꢀ1 inhibitor (tiagabine, Gabatril®);
unfortunately, as demonstrated by the latest fluorineꢀ18 labeled radioligand reported
here and consistent with prior efforts, such molecules show very low brain uptake and
are not useful for in vivo imaging. Converting the free acid function to an ester resulted
in good uptake into the rhesus monkey brain, supporting that the acid function may be
largely responsible for the bloodꢀbrain barrier impermeability, possibly due to formation
of a zwitterionic species. As the acid group is necessary for high affinity binding it
cannot be simply removed or esterified, and future efforts in GATꢀ1 radiotracer design
will investigate carboxylic acid bioisosteres in an attempt to improve brain uptake.
Methods
Details of experimental procedures as well as associated analytical data can be found in
the Supporting Information.
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Associated Content
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Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website
at DOI: XXX. Experimental procedures and spectral data for all novel compounds
synthesized, as well as procedures for radiochemistry and in vivo PET imaging.
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Author Information
Corresponding Authors
*Email: pjhscott@umich.edu
*Email: mkilbour@umich.edu
ORCID
Alexandra R. Sowa: 0000ꢀ0003ꢀ3991ꢀ171X.
Allen F. Brooks: 0000ꢀ0003ꢀ3773ꢀ3024.
Peter J. H. Scott: 0000ꢀ0002ꢀ6505ꢀ0450.
Michael R. Kilbourn: 0000ꢀ0003ꢀ4100ꢀ4676.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript.
Funding
Funding from the National Institutes of Health (R21NS086758) and the University of
Michigan (Rackham Preꢀdoctoral Fellowship and College of Pharmacy) is gratefully
acknowledged.
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The authors declare no competing financial interests.
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Abbreviations
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BBB, blood−brain barrier; CNS, central nervous system; FMZ, flumazenil; GABA, γꢀ
aminobutyric acid; GATꢀ1, GABA Transporter 1; HPLC, highꢀperformance liquid
chromatography; K2.2.2, kryptofixꢀ2.2.2; PET, positron emission tomography; Pgp, Pꢀ
glycoprotein transporter; RCY, radiochemical yield; SPECT, single photon emission
computed tomography.
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Petersen, H., Suzdak, P. D., Swedberg, M. D. (1999) Synthesis of novel GABA uptake
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Graphical Abstract
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