New substrates and inhibitors of γ-aminobutyric acid aminotransferase containing bioisosteres of the carboxylic acid group: Design, synthesis, and biological activity

Article abstract of DOI:10.1016/j.bmc.2005.09.067

A series of potential substrates of γ-aminobutyric acid aminotransferase (GABA-AT) with lipophilic bioisosteres of the carboxylic acid group (2-7) were synthesized and tested. Most of the synthesized compounds showed substrate activities with GABA-AT; 1H-tetrazole-5-propanamine (6) was the best of those tested. The potential time-dependent inhibitor of GABA-AT, 1H-tetrazole-5-(α-vinyl-propanamine) (8), was designed based on the structures of 6 and the antiepilepsy drug vigabatrin (4-aminohex-5-enoic acid, 1). The synthesized compound 8 showed time-dependent inhibition of GABA-AT, but its potency is lower than that of vigabatrin. Methylation of the tetrazole group in 8 resulted in loss of time-dependent activity, suggesting that the tetrazole ring, the carboxylate bioisostere, exists in its deprotonated form in the enzyme active site.

Full text of DOI:10.1016/j.bmc.2005.09.067

Bioorganic & Medicinal Chemistry 14 (2006) 1331–1338
New substrates and inhibitors of c-aminobutyric
acid aminotransferase containing bioisosteres of the carboxylic
acid group: Design, synthesis, and biological activity
Hai Yuan 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 30 August 2005; revised 26 September 2005; accepted 26 September 2005
Available online 2 November 2005
Abstract—A series of potential substrates of c-aminobutyric acid aminotransferase (GABA-AT) with lipophilic bioisosteres of the
carboxylic acid group (2–7) were synthesized and tested. Most of the synthesized compounds showed substrate activities with
GABA-AT; 1H-tetrazole-5-propanamine (6) was the best of those tested. The potential time-dependent inhibitor of GABA-AT,
1H-tetrazole-5-(a-vinyl-propanamine) (8), was designed based on the structures of 6 and the antiepilepsy drug vigabatrin (4-amino-
hex-5-enoic acid, 1). The synthesized compound 8 showed time-dependent inhibition of GABA-AT, but its potency is lower than
that of vigabatrin. Methylation of the tetrazole group in 8 resulted in loss of time-dependent activity, suggesting that the tetrazole
ring, the carboxylate bioisostere, exists in its deprotonated form in the enzyme active site.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
To correct the deficiency of brain GABA and therefore
stop convulsions, an important approach is to use an
inhibitor of GABA-AT that is able to cross blood–brain
barrier.10 Inhibition of this enzyme increases the concen-
tration of GABA in the brain and could have therapeu-
tic applications in epilepsy as well as other neurological
disorders. One of the most effective in vivo time-depen-
dent inhibitors of GABA-AT is 4-amino-5-hexenoic
acid11 (Fig. 1, vigabatrin, 1), an anticonvulsant drug
marketed all over the world except in the U.S.
c-Aminobutyric acid aminotransferase1 (GABA-AT) is
a pyridoxal-50-phosphate (PLP)-dependent enzyme that
degrades the major inhibitory neurotransmitter c-ami-
nobutyric acid2 (GABA) in the central nervous system
(CNS, Scheme 1). GABA is important to several neuro-
logical disorders, including Parkinsonꢀs disease,3 Hun-
tingtonꢀs chorea,4 Alzheimerꢀs disease,5 and epilepsy,6
a central nervous system disease characterized by recur-
ring convulsive seizures. A deficiency of GABA in the
brain has been implicated as one cause for convulsions.7
In an effort to raise the concentration of GABA in the
brain, both direct injection and oral administration of
GABA have been studied. It was shown that injection
of GABA into the brain has an anticonvulsant effect,
but it is obviously not a practical method.8 Taking
GABA orally, however, is not effective because GABA
cannot cross the blood–brain barrier, a membrane pro-
tecting the CNS from xenobiotics in the blood.9
Previously, our group reported various analogues of vig-
abatrin as inhibitors of GABA-AT.12–14 All of these
inhibitors contain the hydrophilic carboxylic acid group.
However, lipophilicity is an important factor that
COO
O
H3N
COO
OOC
COO
PLP
PMP
O
H
H3N
COO
COO
Keywords: GABA-AT; Vigabatrin; Bioisostere; Tetrazole.
*
GABA-AT
Corresponding author. Tel.: +1 847 491 5653; fax: +1 847 491
7713; e-mail: Agman@chem.northwestern.edu
Scheme 1. GABA degradation by GABA-AT.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.067

1332
H. Yuan, R. B. Silverman / Bioorg. Med. Chem. 14 (2006) 1331–1338
O
lipophilicity.18 To optimize the carbon chain length, tet-
razole derivatives 6 and 7 with one and two additional
methylenes, respectively, were also made.
H2N
OH
1
Vigabatrin
Based on the structure of 6, compound 8, a tetrazole
bioisostere of the antiepilepsy drug vigabatrin, was syn-
thesized. N-Methyl tetrazole derivatives 9 and 10 were
also made and tested to determine the form of the tetra-
zole ring in the active site of the enzyme (Fig. 3). Herein
we report the syntheses and the enzymatic results with
these compounds.
Figure 1. Vigabatrin.
O
O
O
O O
S
H2N
H2N
N
H
OMe
H2N
N
CH3
N
H
H
4
2
3
2. Results
N
N
N
N
N
N
N
N
N
H2N
N
H2N
N
H
N
H2N
2.1. Search for efficient bioisosteres of the carboxylic acid
group with higher lipophilicities
H
H
7
6
5
Methyl b-alanylcarbamate (2) was made from N-Cbz-b-
alanine (11) as shown in Scheme 2. Compound 11 was
treated with oxalyl chloride to give acyl chloride 12,
which was allowed to react with methyl carbamate to
give methyl N-Cbz-b-alanylcarbamate (13).19 Catalytic
transfer hydrogenation20 using formic acid and 10% pal-
ladium on active carbon gave 2 in the form of a formate
salt.
Figure 2. Designed GABA-AT substrates.
influences the ability of a compound to permeate the
blood–brain barrier.15 Therefore, we designed a series
of potential substrates of GABA-AT by replacing the car-
boxylic acid group with more lipophilic bioisosteres
(Fig. 2, 2–5). In order to fit the potential substrates con-
taining bioisosteric groups larger than the carboxylic acid
group of GABA into the small and constricted active site
of the enzyme,16 b-alanine, another natural substrate of
GABA-AT containing one less methylene group than
GABA,17 was selected as the parent structure.
Methyl b-alanylsulfonamide (3) was synthesized as
shown in Scheme 3. Protected b-alanine 11 was treated
with carbonyldiimidazole to give 14, which was allowed
to react with methanesulfonamide in the presence of 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) to afford 15.21
Deprotection of the Cbz group with 30% HBr in acetic
acid provided the desired 3 in the form of a hydrobro-
mide salt.
Compound 2 was selected because it contains an isoster-
ic functionality that is less acidic (pKa ꢀ8) than that of a
carboxylic group; compound 3 has a pKa value compa-
rable to that of a carboxylic acid. Compound 4 contains
an indole ring, which may be able to participate in a p-
cation interaction with Arg-192, the residue to which the
carboxylic acid group of GABA binds.16 Compound 5
was also considered because of the biological compati-
bility of its tetrazole group, which is a well-known bio-
Indole-5-methanamine (4) was prepared from 5-cyano-
indole (16) by reduction with LiAlH4 (Scheme 4).22
The synthesis of 1H-tetrazole-5-ethanamine (5) is shown
in Scheme 5. 3-Aminopropionitrile (17) was treated with
benzyl chloroformate and sodium hydroxide to give N-
Cbz-3-aminopropionitrile (18). The reaction of 18 with
sodium azide in the presence of triethylammonium
hydrochloride afforded N-Cbz-aminoethyltetrazole
(19).23 Deprotection provided desired compound 5.
isostere of
a
carboxylic acid but with higher
Me
N
N
N
N
N
N
N
N
H2N
H2N
N
N
N
H2N
N
H
Me
Preliminary substrate activity tests using [14C]-labeled a-
ketoglutarate (a-KG) showed that compounds 2, 3, and
5 are substrates for GABA-AT; 4, however, showed an
9
10
8
Figure 3. Designed GABA-AT inhibitors.
O
COCl
COCl , CH2Cl2
O
O
O
H2N OMe
, toluene
Z
COOH
N
Z
Z
N
Cl
N
N
OMe
H
80 0C (37%)
rt (99%)
H
H
H
11
12
13
O
O
10% Pd/C, HCOOH
MeOH, rt (78%)
H3N
N
OMe
H
HCOO-
2
Scheme 2. Synthesis of 2.

H. Yuan, R. B. Silverman / Bioorg. Med. Chem. 14 (2006) 1331–1338
1333
O
O O
N
N
S
N
N
O
O
O
O O
S
H2N CH3
, DBU, rt
Z
Z
Z
N
N
N
OH
N
N
CH3
H
(2 steps, 73%)
THF, reflux
H
H
H
N
11
14
15
O
O O
S
HBr/CH3COOH
rt (71%)
H3N
Br
N
CH3
H
3
Scheme 3. Synthesis of 3.
NC
to give azide 22, which was then reduced to 4-aminobu-
tyronitrile (24). The preparation of 6 from 24 was simi-
lar to that of 5 from 17. Compound 7 was synthesized in
a similar manner (Scheme 6).
H2N
LiAlH4, THF, rt
(45%)
N
N
H
H
16
4
Scheme 4. Synthesis of 4.
The substrate kinetic constants Km and kcat for the three
tetrazoles (5–7) were determined by Hanes and Woolf
plots.24 Compound 6, containing three methylene
groups, has the highest kcat/Km value, indicating that 6
is the most efficient GABA-AT substrate with the opti-
mal carbon chain length (Table 2).
a-KG conversion of only 0.1%, indicating that 4 has lit-
tle or no substrate activity (Table 1). Tetrazole deriva-
tive 5 was the most efficient of the synthesized
substrates. The tetrazole group was the best bioisostere
for the carboxylic acid group in this series.
2.3. GABA-AT inhibitors based on the structure of the
most efficient substrate
2.2. Optimization of the carbon chain length of the
tetrazole derivatives
Based on the structure of 6, a potential time-dependent
inhibitor of GABA-AT (8) was designed and synthe-
sized. The synthesis of a-vinyl-1H-tetrazole-5-propan-
amine (8) from 4,4-diethoxybutanenitrile (30) is shown
in Scheme 7. Deprotection of 30 gave the 4-oxobutane-
nitrile (31), which was treated with vinylmagnesium bro-
mide to give 32. The hydroxyl group in 32 was then
To determine the optimal carbon chain length of the tet-
razole derivatives, compounds 6 and 7 with one and two
additional methylene groups, respectively, than 5 were
synthesized. 1H-Tetrazole-5-propanamine (6) was syn-
thesized from 4-bromobutyronitrile (20) as shown in
Scheme 6. Compound 20 was treated with sodium azide
NaN3, Et3NH+Cl-
N
N
N
BnOCOCl, NaOH
rt, (98%)
N
Z
N
Z
N
H2N
N
N
toluene, 95 0C, (56%)
H
H
H
17
18
19
N
, CH3OH, 10% Pd/C
reflux, (63%)
N
N
N
H2N
H
5
Scheme 5. Synthesis of 5.
NaN3, DMSO, rt
PPh3, H2O, rt
H2N
N
N
N
Br
N
3( )n
( )n
( )n
(n = 3, 67%;
n = 4, 99%)
(n = 3, 83%;
n = 4, 83%)
22, n = 3
23, n = 4
24, n = 3
25, n = 4
20, n = 3
21, n = 4
NaN3, Et3NH+Cl-,
toluene, reflux
, CH3OH,
n = 3:
N
N
N
N
H
10% Pd/C, reflux (61%)
BnOCOCl, NaOH, rt
H
N
N
N
H2N
N
N
( )n
N
( )n
N
Z
( )n
Z
(n = 3, 50%;
n = 4, 52%)
n = 4: H2, CH3OH,
(n = 3, 99%;
n = 4, 92%)
H
H
10% Pd/C, rt (78%)
6, n = 3
7, n = 4
26, n = 3
27, n = 4
28, n = 3
29, n = 4
Scheme 6. Syntheses of 6 and 7.

1334
H. Yuan, R. B. Silverman / Bioorg. Med. Chem. 14 (2006) 1331–1338
to be 2.2 minꢁ1 and 2.6 mM, respectively. Therefore, 8 is
6.6 times less efficient (kinact/KI) than vigabatrin.
Table 1. Preliminary substrate activity test results
Compound
GABA
25%
2
3
4
5
Converted a-KGa
1.3%
6.4%
0.10%
20%
The in vivo potency of enzyme inhibition, however,
strongly depends on the efficiency of the inhibitor to per-
meate the blood–brain barrier, which is related to the
lipophilicity of the molecule.15 To determine an estimate
of lipophilicity of these compounds, the logP values were
calculated using ClogP software. The logP values calcu-
lated for 8 and vigabatrin are ꢁ0.47 and ꢁ2.217, respec-
tively, which indicates that 8 has considerably higher
lipophilicity and, therefore, higher potential permeability
of the blood–brain barrier compared to vigabatrin.
a Incubation time: 48 h, GABA-AT: 0.7 lM, substrate: 2.5 mM, a-KG:
2.9 mM.
Table 2. Kinetic constants for substrates 5–7
Compound Km (mM) kcat (minꢁ1
)
kcat/Km (mMꢁ1 minꢁ1
)
GABA
2.4
2.3
2.4
8.0
49
20.4
6.9
5
6
7
15.9
28.6
41.6
11.7
5.2
The tetrazole ring of 8 may exist either in a protonated
or deprotonated form in the active site of GABA-AT;
the deprotonated form mimics the carboxylate anion.
To determine the existence of the deprotonated form
of the tetrazole ring in the enzyme active site methyl tet-
razole derivatives 9 and 10, which cannot exist in a
deprotonated form, were synthesized as shown in
Scheme 8. The previously made compound 34 was treat-
ed with sodium hydride and iodomethane to give a mix-
ture of 35 and 36, which were separated by column
chromatography.26 Deprotection with 6 N HCl gave
the desired compounds 9 and 10.
converted to the phthalimide-protected amino group.
The reaction of nitrile 33 with sodium azide resulted in
tetrazole 34. Deprotection of 34 with 6 N HCl gave
the desired compound 8.
As expected, 8 showed time-dependent inhibition of
GABA-AT, and its kinetic constants kinact and KI were
determined by a Kitz and Wilson replot25 to be
0.73 minꢁ1 and 5.6 mM, respectively (Table 3). The ki-
nact and KI values of racemic vigabatrin were determined
Table 3. Kinetic constants for the time-dependent inhibitor 8 and
vigabatrin
Neither 9 nor 10 showed time-dependent inhibition of
GABA-AT in comparison with the corresponding tetra-
zole derivative 8 at the same concentration. Instead,
both 9 and 10 were found to be weak time-independent
inhibitors of GABA-AT with estimated IC50 values for
Compound KI (mM) kinact (minꢁ1
)
kinact/KI (mMꢁ1 minꢁ1
)
Vigabatrin 2.6
5.6
2.2
0.73
0.86
0.13
8
HCl/acetone, 00C
N
N
CH2=CHMgBr, THF
HO
-780C-rt, (31%)
32
EtO
N
O
(98%)
OEt
30
31
N
NaN3, Et3NH+Cl-, toluene
reflux, (60%)
N
N
PPh3, DIAD, phthalimide
THF, 00C-rt, (98%)
PhtN
N
PhtN
N
H
33
34
N
N
N
H
N
6N HCl, reflux
H2N
( 56%)
8
Scheme 7. Synthesis of 8.
Me
N
N
Me
N
N
N
N
N
6N HCl, reflux
(61%)
N
PhtN
PhtN
H2N
N
N
35
9
N
NaH, MeI, THF, 0 0C-rt
N
N
PhtN
+
N
(35, 27%; 36, 41%)
H
N
N
6N HCl, reflux
(67%)
N
N
H2N
34
N
N
Me
Me
36
10
Scheme 8. Syntheses of 9 and 10.

H. Yuan, R. B. Silverman / Bioorg. Med. Chem. 14 (2006) 1331–1338
1335
both greater than 10 mM. If the tetrazole ring of 8 were
3.2. Chemistry
active in its protonated form in the active site of the en-
zyme, 9 and 10 would have inhibited the enzyme to a
similar extent to that of 8. The significantly decreased
activities of 9 and 10 caused by methylation of the tetra-
zole, therefore, suggest that the tetrazole ring of 8 exists
in the deprotonated form in the enzyme active site.
3.2.1. N-Cbz-b-alanyl chloride (12). To a solution of N-
Cbz-b-alanine (11, 1.0 g, 4.5 mmol) in dry methylene
chloride (10 mL) was added 2.0 M oxalyl chloride solu-
tion in methylene chloride (20 mL, 40 mmol). The mix-
ture was stirred at 25 ꢁC for 4 h. Evaporation of the
solvents gave 12 as a yellow oil (1.07 g, 99%). 1H
NMR (400 MHz, CDCl3) d 7.4 (s, 5H), 5.1 (s, 2H), 3.5
(t, 2H, J = 6.0 Hz), 3.2 (t, 2H, J = 5.2 Hz).
2.4. Conclusion
In summary, a series of substrates of GABA-AT con-
taining bioisosteres of the carboxylic acid group with
higher lipophilicity were synthesized and tested. Most
of the synthesized compounds were shown to be sub-
strates of GABA-AT; the tetrazole derivative was the
most efficient. The tetrazole group, therefore, was select-
ed as the carboxylate bioisostere for a potential time-de-
pendent inhibitor of the enzyme related to the structure
of vigabatrin. The optimal carbon chain length of the
tetrazole derivatives was determined. Compound 8, a
more lipophilic analogue of the antiepilepsy drug viga-
batrin, showed time-dependent inhibition of GABA-
AT. Its potency is similar to that of vigabatrin (one-sixth
the potency), and it is more lipophilic (ClogP value).
Therefore, it is potentially more easily able to cross
the blood–brain barrier, although this potential has yet
to be determined. N-Methyl tetrazole derivatives 9 and
10 were found to be only weak time-independent inhib-
itors of GABA-AT, indicating that deprotonation of the
tetrazole ring may be important for the molecule to bind
in the enzyme active site.
3.2.2. N-Cbz-methyl-b-alanylcarbamate (13). Methyl car-
bamate (0.68 g, 9 mmol) was added to a solution of 12
(1.0 g, 4.4 mmol) in dry toluene (5 mL) at room temper-
ature. The mixture was heated at 80 ꢁC for 6 h, cooled,
diluted with ethyl acetate (35 mL), and washed with
water (2 · 30 mL) and brine (1 · 30 mL). The organic
layer was dried with Na2SO4, filtered, and concentrated.
The product was crystallized from ethyl acetate/hexanes
1
to afford 13 as a white solid (0.46 g, 37%). H NMR
(400 MHz, CDCl3) d 7.3 (s, 5H), 5.1 (s, 2H), 3.8 (s,
3H), 3.5 (t, 2H, J = 5.6 Hz), 3.0 (t, 2H, J = 5.2 Hz).
13C NMR (100 MHz, CDCl3) d 73.6, 156.5, 152.2,
136.6, 128.7, 128.3, 128.2, 66.9, 53.4, 36.9, 36.1.
3.2.3. Methyl b-alanylcarbamate (2). A mixture of meth-
yl N-Cbz-b-alanylcarbamate (13, 0.056 g, 0.2 mmol),
10% Pd/C (0.05 g), formic acid (88%, 0.15 mL), and
methanol (7 mL) was stirred for 2 h at room tempera-
ture. The catalyst was removed by filtration through a
Celite bed. The filtrate was concentrated to give the for-
1
mate salt of 2 as a white solid (0.030 g, 78%). H NMR
(400 MHz, D2O) d 8.4 (s, 1H), 3.8 (s, 3H), 3.2 (t, 2H,
J = 6.0 Hz), 3.0 (t, 2H, J = 5.6 Hz). 13C NMR
(100 MHz, D2O) d 172.7, 169.2, 153.4, 53.3, 34.7, 33.1.
HRMS (ESI): calculated for C5H11N2O3 (M+H)+:
147.0770. Found: 147.0773.
3. Experimental
3.1. General
All chemicals, reagents and solvents were purchased
from commercial sources (e.g., Sigma–Aldrich, Fisher
Scientific, etc.) where available. Tetrahydrofuran was
distilled over sodium metal under N2, and dichlorometh-
ane was distilled over calcium hydride under N2. Mois-
ture sensitive reactions were carried out in oven-dried
glassware, cooled under a N2 atmosphere. Flash chro-
matography was performed with Merck silica gel 60
(230–400 mesh). Cation-exchange chromatography was
performed on Dowex 50 resin (BioRad AG50W-X8,
100–200 mesh). 1H and 13C NMR spectra were collected
on Varian Mercury 400 MHz and Inova 500 MHz
NMR spectrometers in the Analytical Service Laborato-
ry at Northwestern University. High resolution mass
spectra were obtained on a Finnigan MAT900XL mass
spectrometer (EI) in the Analytical Services Laboratory
at Northwestern University and on Micromass 70-VSE
(EI) and Micromass Q-Tof Ultima (ESI) mass spec-
trometers in the Mass Spectrometry Laboratory at the
University of Illinois. Elemental analyses were obtained
from Atlantic Microlab, Inc. (Norcross, GA). Enzyme
assays were recorded on a Perkin–Elmer Lambda 10
UV–vis spectrophotometer. Radioactivity was measured
by liquid scintillation counting using a Packard Tri-
Carb 2100TR counter and Packard Ultima Gold XR
scintillation cocktail.
3.2.4. Methyl N-Cbz-b-alanylsulfonamide (15). A solu-
tion of 11 (2.23 g, 10 mmol) in dry THF (20 mL) was
added dropwise to a stirred solution of carbonyldiimi-
dazole (1.62 g, 10 mmol) in dry THF (20 mL) under
N2. The mixture was stirred for 30 min, refluxed for
30 min, and allowed to cool to room temperature. Meth-
yl sulfonamide (0.95 g, 10 mmol) was added in one por-
tion, and the mixture was stirred for 10 min before a
solution of DBU (1.52 g, 10 mmol) in dry THF
(10 mL) was added dropwise. The resulting mixture
was stirred overnight and poured into ice-cold 1 N
HCl (200 mL). The formed precipitate was filtered,
washed with water, and dried to give 15 as a white solid
(2.2 g, 73%). 1H NMR (400 MHz, CDCl3) d 7.4 (s, 5H),
5.1 (s, 2H), 3.5 (tetra, 2H, J = 6.0 Hz), 3.3 (s, 3H), 2.6 (t,
2H, J = 4.8 Hz).
3.2.5. Methyl b-alanylsulfonamide (3). To 15 (1.0 g,
3.3 mmol) was added a solution of hydrogen bromide
in acetic acid (30%, 10 g) with stirring. After 20 min,
the mixture was slowly diluted to 100 mL with diethyl
ether, and the liquids were decanted. The solid was
resuspended in ether (100 mL) and stirred, and the sus-
pension filtered and washed with ether to give 3 as a
1
white solid (0.54 g, 71%). H NMR (400 MHz, D2O) d

1336
H. Yuan, R. B. Silverman / Bioorg. Med. Chem. 14 (2006) 1331–1338
3.33 (s, 3H), 3.29 (t, 2H, J = 6.0 Hz), 2.85 (t, 2H,
J = 6.4 Hz). 13C NMR (100 MHz, D2O) d 171.8, 40.8,
34.5, 32.5. HRMS (ESI): calculated for C4H11N2O3S
(M+H)+: 167.0490. Found: 167.0497.
3.2.10. 4-Azidobutanenitrile (22). Sodium azide (1.2 g,
18.5 mmol) was added to a solution of 4-bromobutyro-
nitrile (20, 1.8 g, 12.0 mmol) in DMSO (20 mL). After
18 h of stirring at room temperature, water (40 mL)
was added, and the solution was extracted with diethyl
ether. Evaporation of the solvent gave 20 as a light yel-
3.2.6. Indole-5-methanamine (4). To an ice-cold 1.0 M
solution of LiAlH4 in THF (18 mL, 0.018 mol) was add-
ed dropwise under N2 a solution of 5-cyanoindole (16,
1.56 g, 0.011 mol) in dry THF (25 mL). After the addi-
tion was complete, the mixture was allowed to warm
to room temperature and was stirred overnight. The
resulting mixture was cooled in an ice bath, and excess
LiAlH4 was quenched with 10% NaOH. The product
was extracted with ethyl acetate and dried over anhy-
drous magnesium sulfate. The solvent was removed by
rotary evaporation to give the crude product (1.1 g),
which was recrystallized from ethyl acetate/hexanes to
give crystalline 5 (0.7 g, 45%). 1H NMR (400 MHz,
DMSO-d6) d 11.0 (s, 1H), 7.5 (s, 1H), 7.3 (d, 2H,
J = 8.4 Hz), 7.0 (d, 1H, J = 8.4 Hz), 6.4 (s, 1H), 3.8 (s,
2H), 2–3 (br, 2H). HRMS (EI): calculated for
C9H10N2 (M+): 146.0838. Found: 146.0835.
1
low oil (0.9 g, 67%). H NMR (400 MHz, CDCl3) d 3.5
(t, 2H, J = 6.0 Hz), 2.5 (t, 2H, J = 7.2 Hz), 1.90 ꢀ 1.95
(m, 2H).
3.2.11. 4-Aminobutanenitrile (24). Triphenylphosphine
(2.11 g, 8.0 mmol) and water (0.2 mL) were added to
22 (0.9 g, 8.0 mmol) dissolved in THF (10 mL). After
18 h at room temperature, the solvent was removed by
rotary evaporation. Ethyl acetate (30 mL) was added
to the crude product, and the desired compound was
extracted with 1 N HCl (30 mL). The aqueous phase
was basified to pH 12 with 10% NaOH and was extract-
ed with ethyl acetate (2 · 30 mL). Evaporation of the
solvent gave 24 as an oil (0.56 g, 83%). 1H NMR
(400 MHz, CDCl3) d 2.9 (t, 2H, J = 6.8 Hz), 2.4, (t,
2H, J = 7.2 Hz), 1.75–1.82 (m, 2H).
3.2.7. N-Cbz-3-aminopropionitrile (18). 3-Aminopropio-
nitrile 17 (0.56 g, 8.0 mmol) was suspended in water
(10 mL) and THF (10 mL). The pH was adjusted to
9.0 by addition of NaOH (0.2 g, 5 mmol). Benzyl chlo-
roformate (1.7 g, 10 mmol) was added dropwise over
2 h at 20–25 ꢁC to the resulting clear solution, and the
pH was kept constant at 9.0 by addition of aqueous
NaOH (4 M, 2.5 mL). The mixture was stirred for 1 h
at pH 9.0, extracted with ethyl acetate, and dried with
Na2SO4. The solvents were removed by rotary evapora-
3.2.12. 1H-Tetrazole-5-propanamine (6). The synthetic
procedure from 24 to 6 (4 mmol scale, 30% for three
steps) is similar to that from 17 to 5. 1H NMR
(400 MHz, D2O): d 2.91–2.99 (m, 4H), 2.02–2.10 (m,
2H). 13C NMR (100 MHz, D2O) d 161.9, 38.9, 25.9,
21.5. Anal. Calcd for C4H10ClN5Æ0.4H2O: C, 28.13; H,
6.37; N, 41.00. Found: C, 28.53; H, 6.02; N, 40.81.
3.2.13. 1H-Tetrazole-5-butanamine (7). The synthetic
procedure from 21 to 7 (12 mmol scale, 31% for five
steps) is similar to that from 20 to 6. 1H NMR
(500 MHz, D2O) d 2.93–2.98 (m, 2H), 2.84–2.88 (m,
2H), 1.72–1.78 (m, 2H), 1.57–1.62 (m, 2H). 13C NMR
(126 MHz, D2O) d 163.1, 39.3, 26.2, 24.9, 23.6. Anal.
Calcd for C5H12ClN5Æ0.5H2O: C, 32.18; H, 7.02; N,
37.52. Found: C, 32.39; H, 6.76; N, 37.29.
1
tion to give crude 18 (1.6 g, 98%) as an oil. H NMR
(400 MHz, DMSO-d6) d 7.3 (s, 5H), 5.0 (s, 2H),
3.22 ꢀ 3.27 (m, 2H), 2.6 (t, 2H, J = 6.4 Hz).
3.2.8. N-Cbz-1H-tetrazole-5-ethanamine (19). The mix-
ture of N-Cbz-3-aminopropionitrile 18 (0.26 g,
1.3 mmol),
triethylamine
hydrochloride
(0.38 g,
4 mmol), and sodium azide (0.26 g, 4 mmol) in toluene
(10 mL) was heated to 95–100 ꢁC for 24 h. After cooling,
the product was extracted with water (20 mL). The sepa-
rated aqueous layer was acidified with 1 N HCl to pH 1.5
to precipitate the produced tetrazole. The formed precip-
itate was filtered, washed with 1 N HCl, and dried under
reduced pressure to give 19 (0.17 g, 56%) as a white solid.
1H NMR (400 MHz, CD3OD) d 7.3 (s, 5H), 5.0 (s, 2H),
3.5 (t, 2H, J = 4.8 Hz), 3.1 (d, 2H, J = 5.6 Hz).
3.2.14. 4-Oxobutanenitrile (31). A mixture of 4,4-diet-
hoxybutanenitrile (30, 0.95 g, 6.0 mmol), acetone
(30 mL), and 6 N HCl (12 mL) was stirred at 0 ꢁC for
9 h. After the reaction was complete, the mixture was
concentrated to approximately 2 mL and was extracted
with chloroform (4 · 10 mL). The combined organic
phase was dried with sodium sulfate. The solvent was re-
moved by rotary evaporation to give crude 31 as an oil
(0.49 g, 98%). 1H NMR (400 MHz, CDCl3) d 9.8 (s,
1H), 2.9 (t, 2H, J = 7.2 Hz), 2.6 (t, 2H, J = 7.2 Hz).
3.2.9. 1H-Tetrazole-5-ethanamine (5). A mixture of 19
(0.17 g, 0.7 mmol), 10% Pd/C (0.10 g), cyclohexene
(4 mL), and methanol (6 mL) was refluxed overnight.
The catalyst was removed by filtration through a Celite
bed. The solvent was removed by rotary evaporation to
give crude 5 as a white solid, which was purified by cat-
ion-exchange chromatography (AGꢂ 50W-X8, eluting
with 0.15 N HCl) to give pure 5 in the form of a hydro-
chloride salt (0.05 g, 63%). 1H NMR (400 MHz, D2O) d
3.3 (t, 2H, J = 6.0 Hz), 3.1 (t, 2H, J = 6.0 Hz). 13C NMR
(100 MHz, D2O) d 159.0, 38.3, 22.7. Anal. Calcd for
C3H8ClN5Æ0.2H2O: C, 23.52; H, 5.53; N, 45.73. Found:
C, 23.93; H, 5.42; N, 45.52.
3.2.15. 4-Hydroxy-5-hexenenitrile (32). A 1.0 M solution
of vinylmagnesium bromide (5.9 mL, 5.9 mmol) in THF
was added dropwise to a solution of crude 31 (0.49 g,
5.9 mmol) in dry THF (10 mL) at ꢁ78 ꢁC. The mixture
was stirred at ꢁ78 ꢁC for 1 h and was further stirred at
room temperature overnight. Saturated aqueous NH4Cl
(15 mL) was added with stirring to the turbid solution
chilled in an ice bath. The aqueous phase was extracted
with ethyl acetate (3 · 15 mL), and the combined organ-
ic extracts were washed with water (10 mL) and brine
(2 · 10 mL), dried with sodium sulfate, and concentrat-
ed under vacuum to give crude 32 as a yellow oil. The

H. Yuan, R. B. Silverman / Bioorg. Med. Chem. 14 (2006) 1331–1338
1337
crude product was purified by chromatography on silica
gel (ethyl acetate/hexanes, 4:6) to give a colorless oil
(0.20 g, 31%). H NMR (400 MHz, CDCl3) d 5.82–5.90
(m, 1H), 5.3 (d, 1H, J = 17.6 Hz), 5.2 (d, 1H,
J = 10.0 Hz), 2.46–2.57 (m, 2H) 1.78–1.95 (m, 2H). 13C
NMR (100 MHz, CDCl3) d 139.5, 116.5, 71.3, 32.3, 13.6.
NaH (60% in mineral oil, 0.034 g, 0.75 mmol) dissolved
in dry THF (5 mL) was added dropwise over 20 min.
The mixture was stirred for an additional 10 min, and
iodomethane (0.11 g, 0.75 mmol) was added. After the
mixture was stirred at room temperature for 2 h, H2O
(20 mL) was added, and the resulting mixture was
extracted with EtOAc (2 · 20 mL). The combined
organic phases were washed with H2O (2 · 20 mL) and
brine (2 · 20 mL), dried with Na2SO4, and the solvents
were removed by rotary evaporation. The resulting mix-
ture of 35 and 36 was separated by column chromatog-
raphy on silica gel (EtOAc/hexanes, 4:6). Evaporation of
the faster eluting fractions gave 35 as an oil (0.047 g,
27%). 1H NMR (400 MHz, CDCl3) d 7.73–7.85 (m,
4H), 6.22–6.28 (m, 1H), 5.30 (d, 1H, J = 17.2 Hz), 5.23
(d, 1H, J = 10.4 Hz), 4.82 (tetra, 1H, J = 8.0 Hz), 4.25
(s, 3H), 2.91 (tetra, 2H, J = 8.0 Hz), 2.56–2.61 (m,
1H), 2.45–2.55 (m, 1H). Evaporation of the slower elut-
1
3.2.16. 4-Phthalimido-5-hexenenitrile (33). A solution of
32 (0.35 g, 3.1 mmol), triphenylphosphine (0.87 g,
3.3 mmol), and phthalimide (0.50 g, 3.3 mmol) in dry
THF (15 mL) was stirred at 0 ꢁC under N2 for 10 min.
A solution of diisopropyl azodicarboxylate (DIAD,
0.66 g, 3.3 mmol) in THF (8 mL) was added dropwise
over 20 min. The mixture was stirred at room tempera-
ture for 3 h. After the solvent was removed by rotary
evaporation, the crude product was purified by chroma-
tography on silica gel (ethyl acetate/hexanes, 1:9) to give
a mixture of 33 and diisopropyl hydrazodicarboxylate, a
by-product formed from DIAD (1.19 g, ꢀ3:2 m/m,
97%). 1H NMR (400 MHz, CDCl3) d 7.72–7.85 (m,
4H), 6.14–6.23 (m, 1H), 5.34 (d, 1H, J = 17.2 Hz), 5.26
(d, 1H, J = 10.4 Hz) 4.8 (tetra, 1H, J = 5.6 Hz), 2.27–
2.48 (m, 4H).
1
ing fractions gave 36 as an oil (0.071 g, 41%). H NMR
(400 MHz, CDCl3) d 7.74–7.86 (m, 4H), 6.21–6.30 (m,
1H), 5.32 (d, 1H, J = 17.6 Hz) 5.27 (d, 1H, J = 10.0 Hz),
4.86 (tetra, 1H, J = 5.6 Hz), 3.97 (s, 3H), 2.84–2.90 (m,
2H), 2.66–2.74 (m, 1H), 2.51–2.55 (m, 1H).
3.2.17. 5-(3-Phthalimido-4-pantenyl)-1H-tetrazole (34).
The mixture of 33 and diisopropyl hydrazodicarboxy-
late prepared above (0.88 g, 2.6 mmol) was added to a
solution of triethylamine hydrochloride (0.76 g, 8 mmol)
and sodium azide (0.52 g, 8 mmol) in toluene (15 mL).
After 18 h of stirring at 95–100 ꢁC, the cooled product
was extracted with water (20 mL). The separated aque-
ous layer was acidified with 10% HCl to pH 1.5 to salt
out the produced tetrazole. The formed precipitate was
filtered and dried to give 34 as a light brown solid
3.2.20. 2-Methyl-2H-tetrazole-5-(a-vinyl-propanamine)
(9) and 1-methyl-1H-tetrazole-5-(a-vinyl-propanamine)
(10). The deprotection procedures from 35 and 36 to 9
and 10, respectively, are similar to that from 34 to 8.
Compound 9 (0.016 g, 61%). 1H NMR (400 MHz,
D2O) d 5.74 ꢀ 5.81 (m, 1H), 5.35–5.40 (m, 2H), 4.25
(s, 3H), 3.76 (s, 1H), 2.87–2.91 (m, 2H), 2.14–2.22 (m,
1H), 2.02 ꢀ 2.10 (m, 1H). 13C NMR (400 MHz, D2O)
d 165.1, 132.2, 122.1, 53.5, 33.6, 30.1, 20.9. HRMS
(EI): calculated for C7H13N5 (M+): 167.1171. Found:
167.1160. Compound 10 (0.027 g, 67%). 1H NMR
(400 MHz, D2O) d 5.63–5.71 (m, 1H), 5.29–5.33 (m,
2H), 3.84 (s, 3H), 3.76–3.79 (m, 1H), 2.80–2.84 (m,
2H), 2.13–2.17 (m, 1H), 2.00–2.05 (m, 1H). 13C NMR
(100 MHz, D2O) d 155.2, 131.9, 122.5, 53.4, 33.6, 28.6,
19.0. HRMS (EI): calculated for C7H13N5 (M+):
167.1171. Found: 167.1158.
1
(0.44 g, 60%). H NMR (400 MHz, CDCl3) d 7.8 (dd,
4H, J = 35.2 Hz, 2.4 Hz) 6.22–6.29 (m, 1H), 5.27 (d,
1H, J = 4.4 Hz), 5.24 (d, 1H, J = 3.2 Hz) 4.72 (s, 1H),
3.19–3.22 (m, 1H), 2.78–2.84 (m, 1H), 2.62–2.71 (m,
1H), 2.18–2.22 (m, 1H).
3.2.18. 1H-Tetrzole-5-(a-vinyl-propanamine) (8). To a
solution of 6 N HCl (20 mL) was added 34 (0.2 g,
1 mmol), and the mixture was refluxed for 6 h. The mix-
ture was washed with ethyl acetate (2 · 20 mL). Evapo-
ration of the solvent gave crude 8 as a yellow oil. To
remove the trace amount of phthalic acid, the crude
product was purified by cation-exchange chromatogra-
phy (AGꢂ 50W-X8, eluting with 0.2 N HCl) to give 8
in the form of a hydrochloride as a colorless oil. The
hydrochloride was loaded on a second cation-exchange
column, eluted with water followed by 0.15 N ammoni-
um hydroxide to give the free amine form of 8 as a white
3.3. Enzymatic tests
3.3.1. Enzyme and assays. GABA-AT (1.88 mg/mL, spe-
cific activity 2.73 unit/mg) was purified from pig brain
by the procedure described by Churchich and Moses.27
Succinic semialdehyde dehydrogenase (SSDH) was puri-
fied from GABAse, a commercially available mixture of
SSDH and GABA-AT, using the procedure of Jeffery
et al.28 GABA-AT activity was assayed using a pub-
lished method with modifications.29 The final assay solu-
tion consists of 11 mM GABA, 1.1 mM NADP+,
5.3 mM a-KG, 2 mM b-mercaptoethanol, and excess
SSDH in 50 mM potassium pyrophosphate buffer,
pH 8.5. The change in UV absorbance at the wavelength
of 340 nm caused by the formation of NADPH is pro-
portional to the GABA-AT activity.
1
solid (0.086 g, 56%). H NMR (400 MHz, D2O) d 5.72–
5.81 (m, 1H), 5.38–5.45 (m, 2H), 3.80 (br s, 1H), 3.00–
3.07 (m, 2H), 2.24–2.32 (m, 1H), 2.09–2.19 (m, 1H).
13C NMR (400 MHz, D2O) d 155.1, 131.5, 122.3, 53.2,
29.1, 19.0. Anal. Calcd for C6H11N5Æ0.4H2O: C, 44.93;
H, 7.42; N, 43.66. Found: C, 44.93; H, 7.38; N, 43.56.
3.2.19. 2-Methyl-2H-tetrazole-5-(a-vinyl-N-phthaloylpro-
panamine) (35) and 1-methyl-1H-tetrazole-5-(a-vinyl-N-
phthaloylpropanamine) (36). A solution of 34 (0.167 g,
0.6 mmol) in dry THF (10 mL) was cooled to 0 ꢁC.
3.3.2. Substrate activities of 2–7. Potential substrates 2–7
of varying concentrations (1–5 mM) were incubated with
GABA-AT (17.1 lM, 5–7 lL) at 25 ꢁC in 50 mM potas-
sium pyrophosphate buffer, pH 8.5, containing 2 mM b-

1338
H. Yuan, R. B. Silverman / Bioorg. Med. Chem. 14 (2006) 1331–1338
mercaptoethanol and 2.9 mM [5-14C]2-ketoglutarate
(0.1 mCi/mmol) in a total volume of 100 lL. After incu-
bation (48 h for the preliminary test, 1 h for determina-
tion of kinetic constants), the mixture was quenched
with trichloroacetic acid. The resulting [14C]glutamate
was isolated by cation-exchange chromatography, and
the DPM (disintegration per minute) value was mea-
sured. Controls consisted of the entire incubation mix-
ture with substrates omitted. The substrate kinetic
constants kcat and Km were determined by the method
of Hanes and Woolf.24
Tower, D. B., Eds.; Raven Press: New York, 1976; pp
479–485.
4. Perry, T. L.; Hansen, S.; Kloster, M. New Engl. J. Med.
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5. Aoyage, T.; Wada, T.; Nagai, M.; Kojima, F.; Harada, S.;
Takeuchi, T.; Takahashi, H.; Kirokawa, K.; Tsumita, T.
Chem. Pharm. Bull. 1990, 38, 1748–1749.
6. Gale, K. Epilepsia 1989, 30, 1–11.
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8. Purpura, D. P.; Girando, M.; Smith, T. A.; Callan, D. A.;
Groundfest, J. J. Neurochem. 1959, 3, 238–268.
9. Tower, D. B. In GABA in Nervous System Function;
Roberts, E., Chase, T. N., Tower, D. B., Eds.; Raven
Press: New York, 1976; pp 461–478.
4. Time-dependent inhibition of GABA-AT by 8
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2413–2421.
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5292–5293.
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4539.
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15. Pardridge, W. M. J. Neurovirol. 1999, 5, 556–569.
16. Storici, P.; Capitani, G.; De Biase, D.; Moser, M.; John,
R. A.; Jansonius, J. N.; Schirmer, T. Biochemistry 1999,
38, 8628–8634.
GABA-AT (17.1 lM, 25 lL) was incubated with 8
(120 lL final volume, 1–2 mM) at 25 ꢁC in 50 mM potas-
sium pyrophosphate buffer solution, pH 8.5, containing
2 mM a-ketoglutarate and 2 mM b-mercaptoethanol.
Aliquots (20 lL) were withdrawn at timed intervals and
were added immediately to the assay solution (575 lL)
followed by the addition of excess SSDH (5 lL). The reac-
tion rates were measured by a UV–vis spectrophotometer
at 340 nm. Racemic vigabatrin was tested under the same
conditions. A Kitz and Wilson replot25 was used to deter-
mine the kinetic constants kinact and KI.
17. Jeremiah, S.; Povey, S. Ann. Hum. Genet. 1981, 45, 231–236.
18. Kraus, J. L.; Faury, P.; Charvet, A. S.; Camplo, M. Res.
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2001, 66, 6988–6993.
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1653–1656.
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1995, 60, 7997–8001.
23. Koguro, K.; Oga, T.; Mitsui, S.; Orita, R. Synthesis 1998,
10, 910–914.
5. Time-independent inhibition of GABA-AT by 9 and 10
GABA-AT (17.1 lM, 5 lL) was assayed for its activity
at 25 ꢁC with varying concentrations (1–10 mM) of 9
and 10. The percentage of remained enzyme activity
was obtained by comparison to that of an untreated en-
zyme control. The logarithm of the percentage of re-
mained activity is plotted versus the concentration of
the inhibitors to calculate IC50 values.
24. (a) Woolf, B., cited by Haldane, J. B. S.; Stern, K. G.
Algemeine Chemie der Enzyme; Steinkopf: Dresden, 1932;
pp 119–120; (b) Hanes, C. S. Biochem. J. 1932, 26, 1406–
1421.
Acknowledgment
The authors are grateful to the National Institutes of
Health (Grant GM66132) for financial support of this
research.
25. Kitz, R.; Wilson, I. B. J. Biol. Chem. 1962, 237, 3245–
3249.
26. Balle, T.; Perregaard, J.; Larsen, A. K.; Ramirez, M. T.;
Soby, K. K.; Liljefors, T.; Andersen, K. Bioorg. Med.
Chem. 2003, 11, 1065–1078.
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