Synthesis and evaluation of novel aromatic substrates and competitive inhibitors of GABA aminotransferase

Article abstract of DOI:10.1016/j.bmcl.2007.10.060

The design, synthesis, and evaluation of novel γ-aminobutyric acid aminotransferase (GABA-AT) inhibitors and inactivators can lead to the discovery of new GABA-related therapeutics. To this end, a series of aromatic amino acid compounds was synthesized to aid in the design of new inhibitors and inactivators of GABA-AT. All compounds were tested as competitive inhibitors of GABA-AT. The amino acids with benzylic amines were also tested as substrates for GABA-AT. It was found that these compounds were all poor competitive inhibitors of GABA-AT, but some were substrates of the enzyme, suggesting their utility as scaffolds for potential GABA-AT mechanism-based inactivators. Computer modeling was used to rationalize the substrate activity of the various compounds.

Full text of DOI:10.1016/j.bmcl.2007.10.060

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 3122–3125
Synthesis and evaluation of novel aromatic substrates
and competitive inhibitors of GABA aminotransferase
Michael D. Clift and Richard B. Silverman*
Department of Chemistry, Department of Biochemistry, Molecular Biology, and Cell Biology, and the Center for Drug
Discovery and Chemical Biology, Northwestern University, Evanston, IL 60208-3113, USA
Received 18 September 2007; revised 15 October 2007; accepted 16 October 2007
Available online 22 October 2007
Abstract—The design, synthesis, and evaluation of novel c-aminobutyric acid aminotransferase (GABA-AT) inhibitors and inacti-
vators can lead to the discovery of new GABA-related therapeutics. To this end, a series of aromatic amino acid compounds was
synthesized to aid in the design of new inhibitors and inactivators of GABA-AT. All compounds were tested as competitive inhib-
itors of GABA-AT. The amino acids with benzylic amines were also tested as substrates for GABA-AT. It was found that these
compounds were all poor competitive inhibitors of GABA-AT, but some were substrates of the enzyme, suggesting their utility
as scaffolds for potential GABA-AT mechanism-based inactivators. Computer modeling was used to rationalize the substrate activ-
ity of the various compounds.
Ó 2007 Elsevier Ltd. All rights reserved.
c-Aminobutyric acid aminotransferase (GABA-AT) cat-
alyzes the degradation of c-aminobutyric acid (GABA)
to succinic semialdehyde (SSA). Control of GABA
levels in the body has numerous therapeutic benefits.
Depleted levels of GABA, a major inhibitory neuro-
transmitter,1 have been shown to cause convulsions.2
Raising GABA levels in the brain has an anticonvulsant
effect;3–5 however, direct GABA administration is not
effective, as GABA cannot cross the blood brain bar-
rier.6 Disruption of GABA levels has also been impli-
cated in numerous neurological disorders, such as
Alzheimer’s disease,7 Parkinson’s disease,8 Huntington’s
disease,9 and senile dementia.10 Numerous strategies
exist to elevate GABA levels in the brain. The strategy
that we have taken involves inhibition or inactivation
of GABA-AT, the enzyme that is responsible for the
degradation of GABA into an inactive form.
(1) and by 2, a potent conformationally restricted
analogue of 111 (Fig. 1), was designed, synthesized,
and evaluated. The results of these studies will aid in
the design of future GABA-AT inhibitors and inactiva-
tors. Compound 2 is a potent irreversible inactivator of
GABA-AT, showing a potency that is 186 times greater
than that of 1.
The syntheses of the three 1,3-disubstituted aromatic
amino acids (8, 10, and 14), that were not commercially
available, are outlined in Scheme 1.
F
F
CO2H
H2N
CO2H
To enhance the lipophilicity of GABA analogues for
more favorable bioavailability, a series of aromatic com-
pounds, inspired by the anticonvulsant drug vigabatrin
H2N
2
1
Keywords: c-Aminobutyric acid; c-Aminobutyric acid aminotransfer-
ase; GABA-AT; Substrate of GABA-AT; Inhibitor; Computer mod-
eling; Aromatic amino acid.
H2N
CO2H
n
H2N
CO2H
m
n
m
*
Figure 1. Previously described GABA-AT inactivators (1 and 2) and the
new series of aromatic analogues investigated (m = 0, 1 and n = 0, 1).
Corresponding author. Tel.: +1 847 491 5653; fax: +1 847 491
7713; e-mail: Agman@chem.northwestern.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.060

M. D. Clift, R. B. Silverman / Bioorg. Med. Chem. Lett. 18 (2008) 3122–3125
3123
c
a
b
CO2Me
CO2Me
CO2Me
CO2Me
N3
NHCbz
Br
3
4
5
6
d
e
6
CO2H
CO2H
.
NHCbz
NH2 HCl
7
8
f
e
7
CO2Me
CO2H
.
NHCbz
NH2 HCl
9
10
e
f
g
CO2H
CO2Me
H2N
.
CbzHN
H2N
CO2H
CbzHN
CO2H
HCl
13
14
12
11
Scheme 1. Synthesis of the 1,3-disubstituted aromatic amino acids. Reagents and conditions: (a) NBS, hm, MeCN; (b) NaN3, EtOH, reflux; (c) i—H2,
Pd–C, MeOH; ii—CbzCl, TEA, THF, 0 °C to rt; (d) NaOH, MeOH; (e) 6 M HCl, reflux; (f) CbzCl, H2O, Na2CO3, 0 °C to rt; (g) i—SOCl2, reflux;
ii—CH2N2, TEA, ether, 0 °C; iii—cat. AgOBz, TEA, MeOH.
The commercially unavailable 1,2-disubstituted aro-
matic amino acids (16, 20, and 25) were synthesized
as shown in Scheme 2. The chemistry applied to
the synthesis of intermediate 6 proved applicable to
the synthesis of amino acid 25, but different synthetic
strategies were required in the cases of 16 and 20.
All of the compounds were tested as competitive inhib-
itors of GABA-AT using a coupled enzyme assay,12 ex-
cept for 16. Compound 16 underwent cyclization to 15
under the conditions of the assays and was, therefore,
omitted from testing. It was found that 8, 10, 11, 14,
20, 25, and 26 (Fig. 2) all showed IC50 values that ex-
a
c
e
O
N
H
H2N
.HCl
16
CO2H
15
b
a
H2N
.HCl
CO2H
O
NC
CO2Me
NC
CO2H
N
H
18
17
21
19
20
f
d
CO2Me
N3
Br
CO2Me
CO2Me
HN
24
23
O
22
a
24
CO2H
H2N
.HCl
25
Scheme 2. Synthesis of the 1,2-disubstituted aromatic amino acids. Reagents and conditions: (a) 6 M HCl, reflux; (b) MeI, NaHCO3, DMF; (c) H2,
Pd–C, AcOH/MeOH (1:1), then TEA, MeOH; (d) NBS, hm, MeCN; (e) NaN3, EtOH, reflux; (f) H2, Pd–C, MeOH, then TEA, MeOH.

3124
M. D. Clift, R. B. Silverman / Bioorg. Med. Chem. Lett. 18 (2008) 3122–3125
CO2H
CO2H
CO2H
H2N
CO2H
H2N
.HCl
.HCl
.HCl
NH2
NH2
8
11
14
10
CO2H
CO2H
H2N
H2N
.HCl
H2N
.HCl
CO2H
26
25
20
Figure 2. Amino acids that were tested as inhibitors and substrates of GABA-AT.
Table 1. Substrate activities of compounds shown in Fig. 2 relative to
GABA
Compound
Relative substrate activity (%)
GABA
8
10
100.00
3.42 ( 0.82)
0.78 ( 0.06)
0.00
11
14
0.00
0.00a
20
25
5.65 ( 0.86)
0.00
26
Numbers represent relative rates of turnover. Standard deviations are
also shown.
a Transamination was observed, but it was found to be insignificant.
ceeded 5.0 mM concentrations. Because of the relatively
poor potency of these compounds, the exact IC50 values
were not determined.
All of the compounds were subsequently tested as sub-
strates for GABA-AT. The substrate activities (relative
to the rate of GABA turnover) for all of the com-
pounds are shown in Table 1. The fact that 11, 14,
and 26 showed no transamination was anticipated,
as these compounds lack protons alpha to the amino
group and, therefore, cannot undergo oxidation at
the necessary position. It is clear that the two best
substrates are 8 and 25. This most likely results from
their similarity to the structure of bound GABA in
terms of the relative positions of the acid and amine
functionalities. The automated flexible docking pro-
gram FlexX was employed to perform docking calcu-
lations for 8, 10, 20, and 25 using the crystallographic
structure of the homodimers of pig liver GABA-AT in
complex with vigabatrin 1 (PDB code: 1OHW).13 The
FlexX docking models show that none of carboxylic
groups of these compounds can exactly mimic the
binding mode of the carboxylic group of vigabatrin,
as illustrated in Fig. 3. However, compared with 10
and 20, the carboxylic groups of 8 and 25 are more
similar to that of bound vigabatrin. The poor turn-
over of 10 may result from the fact that there are
too many carbon atoms between the polar groups
and, therefore, the carboxylic acid group of 10 cannot
be accommodated by the active site of GABA-AT
(Fig. 3a).
Figure 3. (a) The superimposition of the FlexX docking conformations
of 8, 10, and 25 and the binding conformation of vigabatrin from the
crystallographic structure (PDB code: 1OHW). The 8–PLP adduct, the
10–PLP adduct, and the 25–PLP adduct are shown in green, red, and
purple, respectively. (b) The superimposition of the FlexX docking
conformations of 20 and the binding conformation of vigabatrin from
the crystallographic structure (PDB code: 1OHW). Lys329 is shown in
green. Phe351 and Thr353, which are from the other monomer of the
homodimer, are shown in magenta. All of the hydrogen atoms were
omitted for clarity. Vigabatrin and PLP from the crystal structure are
shown in cyan. The 20–PLP adduct is shown in gray.

M. D. Clift, R. B. Silverman / Bioorg. Med. Chem. Lett. 18 (2008) 3122–3125
3125
Compound 20 has the same chain length as GABA so it
References and notes
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analysis indicates that the rigidity of the aromatic ring
prevents the carboxylate from engaging in a critical salt
bridge interaction with Arg-12913 present in the active
site (Fig. 3b). This could explain why 25 undergoes
transamination and 20 does not; the additional methy-
lene unit in 25 allows the carboxylate to assume an
appropriate conformation that facilitates binding by
GABA-AT and subsequent turnover.14
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Acknowledgements
The authors are grateful to the National Institutes of
Health (GM66132) for financial support of this re-
search. We thank Dr. Haitao Ji for carrying out the
computer modeling and Dr. Graham Lawton and Dr.
Wenxin Gu for helpful discussions.
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code: 1OHW) in complex with the docking ligands (the 8–
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the 25–PLP adduct) and the crystallographic vigabatrin–
PLP adduct are accessible from the supporting
information.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2007.10.060.