Conformationally-restricted vigabatrin analogs as irreversible and reversible inhibitors of γ-aminobutyric acid aminotransferase

Article abstract of DOI:10.1016/j.bmc.2004.07.065

Compounds that inhibit γ-aminobutyric acid aminotransferase exhibit anticonvulsant activity; vigabatrin is a known irreversible inhibitor of this enzyme and anticonvulsant drug. Conformationally-restricted, five-membered- and six-membered-ring vigabatrin analogs were synthesized and tested as inhibitors of γ-aminobutyric acid aminotransferase. Two monofluorinated compounds, 4 and 5, are time-dependent inhibitors of the enzyme, and their potencies are comparable to that of vigabatrin. Compounds 6 and 7 are weak reversible inhibitors.

Full text of DOI:10.1016/j.bmc.2004.07.065

Bioorganic & Medicinal Chemistry 12 (2004) 5719–5725
Conformationally-restricted vigabatrin analogs as irreversible
and reversible inhibitors of c-aminobutyric acid aminotransferase
Yue Pan, Kristi Calvert and Richard B. Silverman*
Department of Chemistry, Department of Biochemistry, Molecular Biology, and Cell Biology,
and Drug Discovery Program, Northwestern University, Evanston, IL 60208-3113, USA
Received 2 June 2004; accepted 2 July 2004
Available online 11 September 2004
Abstract—Compounds that inhibit c-aminobutyric acid aminotransferase exhibit anticonvulsant activity; vigabatrin is a known
irreversible inhibitor of this enzyme and anticonvulsant drug. Conformationally-restricted, five-membered- and six-membered-ring
vigabatrin analogs were synthesized and tested as inhibitors of c-aminobutyric acid aminotransferase. Two monofluorinated
compounds, 4 and 5, are time-dependent inhibitors of the enzyme, and their potencies are comparable to that of vigabatrin. Comp-
ounds 6 and 7 are weak reversible inhibitors.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
animals.7 However, because GABA does not cross the
blood–brain barrier,8 it cannot be used as an anticonvul-
sant drug.9 c-Aminobutyric acid aminotransferase
(GABA-AT, E.C. 2.6.1.19) is the enzyme that degrades
GABA, and inhibition of this enzyme increases the avail-
ability of GABA in the brain for therapeutic applications.
Epilepsy refers to an etiologically and clinically diverse
group of neurological disorders characterized by sponta-
neous, recurring, cerebral discharges called seizures.1 Con-
vulsions arise from a variety of reasons including heredity,
head trauma, brain tumors, heat stroke, acute intoxica-
tion, and labor.2,3 When the level of c-aminobutyric acid
(GABA) in the brain falls below a threshold level, convul-
sions occur.4 Raising the level of GABA in the brain has an
anticonvulsant effect,5,6 as seizures were stopped when
GABA was directly injected into the brain of convulsing
Vigabatrin (1) is a potent irreversible inhibitor of
GABA-AT10 and is used in the treatment of epilepsy.11
Vigabatrin inactivates GABA-AT through two mecha-
nistic pathways (Scheme 1): a Michael addition mecha-
nism (pathway a) and an enamine mechanism
Scheme 1. Inactivation of GABA-AT by vigabatrin.
*
Corresponding author. Tel.: +1 847 491 5653; fax: +1 847 491 7713; e-mail: Agman@chem.northwestern.edu
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.07.065

5720
Y. Pan et al. / Bioorg. Med. Chem. 12 (2004) 5719–5725
Figure 1. Conformationally-restricted vigabatrin analogs.
(pathway b).12 The advantages of conformationally-re-
stricted compounds are that conformation restriction
may increase the potency by stabilizing a biologically ac-
tive conformer (therefore reducing the entropic penalty
on binding to the enzyme), decrease degradation by
eliminating metabolized conformers, and improve selec-
tivity by eliminating bioactive conformers that give
undesired biological responses.13 Previously, our group
reported two conformationally-restricted five-mem-
bered-ring vigabatrin analogs (2 and 3, Fig. 1).14
Compound 2 showed time-dependent inhibition of
GABA-AT only in the absence of 2-mercaptoethanol,
which is an antioxidant used in enzyme assays; no inac-
tivation occurred when 2-mercaptoethanol was present,
probably because it trapped the released electrophilic
species responsible for inactivation. To increase the elec-
trophilicity of the reactive species with the goal of get-
ting a reaction to occur with GABA-AT prior to its
release into the medium, fluorines were substituted at
the terminus of the double bond. Although fluorine sub-
stitution decreases the potency of vigabatrin,15 the
difluorinated analog of 2, that is, 3, is a very potent irre-
versible inactivator of GABA-AT, even more effective
than vigabatrin in vitro.14 Here we report the E- and
Z-monofluorinated analogs of 2 (compounds 4 and 5).
Another strategy we utilized to optimize lead compound
2 was to synthesize 6 and 7, both of which are six-mem-
bered-ring vigabatrin analogs with an exocyclic double
bond. Because the orientation of the double bond is very
important to the activity and potency of GABA-AT
inactivators,16 these compounds, with the exocyclic dou-
ble bond oriented differently from that of 2, may be cov-
alently attached to the enzyme before being released
from the active site. We are also interested in 6 and 7
because the vigabatrin analogs containing a six-
membered-ring with an endocyclic double bond (8
and 9) are inhibitors; 8 is a weak irreversible inhibi-
tor
(Ki
2.3mM,
kinact
0.01minꢀ1
,
kinact/Ki
0.004mMꢀ1 minꢀ1), and 9 is a reversible GABA-AT
inhibitor (Ki 100lM).17 It would be intriguing if the
different orientation of the double bond resulted in
increased potency.
2. Results and discussion
2.1. Chemistry
The syntheses of the monofluorinated vigabatrin ana-
logs (4 and 5) started from 10 (Scheme 2).18,19 The fluoro-
sulfone20,21 11 was prepared in an 82% yield as an
inseparable mixture of the E- and Z-isomers. Although
aluminum amalgam has been used to reductively remove
the phenylsulfone moiety,22,23 it was unreactive toward
11. Magnesium combined with mercury chloride,24
however, produced the E-(12) and Z-(13) monofluoro
alkenes smoothly, the stereochemistry of which was as-
signed based on the presence or absence of a nuclear
Overhauser effect (NOE) between H-1 and the alkene
proton. For 12, a NOE was observed between H-1
and the alkene proton but for 13 it was not observed.
Subsequent oxidative deprotection of the PMB group
with ceric ammonium nitrate (CAN) and hydrolysis of
the lactam gave the corresponding conformationally-
restricted, monofluorinated vigabatrin analogs 4 and 5.
The syntheses of 6 and 7 (Scheme 3) started with a
Wittig reaction, which converted 16 to the alkene 17.
Compound 17 was then oxidized with tert-butyl
hydroperoxide under the catalysis of selenium dioxide
to give the trans-allylic alcohol 18 as the major product
(trans:cis 14:1, the configurations were assigned by 2D
Scheme 2. Syntheses of 4 and 5. Reagents and conditions: (a) PhSO2CH2F, (EtO)2POCl, LiHMDS, 82%; (b) Mg, HgCl2, 64% for 12 and 23% for 13;
(c) CAN, 42%; (d) HCl, 75%.

Y. Pan et al. / Bioorg. Med. Chem. 12 (2004) 5719–5725
5721
O
OH
NBocCOCO2Et
NHBoc
NH2 HCl
a
c
b
d
e
COOEt
COOEt
( )-18
COOEt
( )-19
COOH
( )-20
COOH
( )-6
COOEt
16
17
f
O
OC
OH
NBocCOCO2Et
i
NHBoc
NH2 HCl
NO2
g
h
j
COOEt
( )-22
COOEt
( )-23
COOH
( )-24
COOH
COOEt
( )-21
( )-7
Scheme 3. Syntheses of 6 and 7. Reagents and conditions: (a) Ph3PCH3Br, LiHMDS, 87%; (b) t-BuOOH, SeO2, 90%; (c) BocNHCOCO2Et, DIAD,
PPh3, 64%; (d) LiOH, 52%; (e) TFA, CH2Cl2, then HCl, 85%; (f) p-nitrobenzoic acid, DIAD, PPh3, 58%; (g) NaN3, MeOH, 40ꢁC, 64%;
(h) BocNHCOCO2Et, DIAD, PPh3, 55%; (i) LiOH, 36%; (j) TFA, CH2Cl2, then HCl, 80%.
NOESY experiments; for 22 there is a NOE between H-
1 and H-3).25 AMitsunobu reaction between 18 and N-
Boc ethyl oxamate gave 19,26 which was unstable and
hard to completely purify at this stage. Crude 19 was
subjected to hydrolysis to produce the Boc-protected
amino acid 20. Deprotection of the Boc group with
TFAfurnished 6. To synthesize 7, a Mitsunobu reaction
with p-nitrobenzoic acid followed by methanolysis in the
presence of sodium azide27 was used to carry out the
inversion of configuration of 18 to give the cis-alcohol
22. Compound 7 was synthesized from 22 using steps
similar to those used for 6.
mMꢀ1 minꢀ1 ) are comparable to that of vigabatrin
(kinact/Ki is 1.7mMꢀ1 minꢀ1). The inactivation mecha-
nisms of 4 and 5 are under investigation. Despite the sim-
ilarities in structure and potency between 4 and 5, the
inactivation mechanisms may be different, like the differ-
ence in mechanism that was reported for 25 and 26
(Fig. 3).28
Unfortunately, neither 6 nor 7 inactivated GABA-AT.
Also, no substrate activity was observed. Compound 6
was tested as a reversible inhibitor, and the IC50 was
1.7mM, while 7 was even weaker. It is possible that
the exocyclic methylene group affects the binding of
these compounds and as a result, the c-proton of 6
and 7 is too far away from Lys329 for abstraction,
which is necessary for both substrate and inactivator
properties.
2.2. Enzyme inhibition results
Both 4 and 5 exhibited concentration- and time-depend-
ent inhibition of pig brain GABA-AT in the presence of
2-mercaptoethanol (Fig. 2). Remarkably, substitution
with only one fluorine atom changed the activity of 2
dramatically. The Ki values for 4 (Ki 250lM) and 5 (Ki
530lM) are lower (better binding) than that for (S)-vig-
abatrin (Ki 1.3mM), but the rate constants for inactiva-
tion of 4 (kinact 0.25minꢀ1) and 5 (kinact 0.74minꢀ1) also
are lower than that for vigabatrin (kinact 2.2minꢀ1),
3. Conclusion
By fluorine substitution of 2, we discovered two potent,
conformationally-restricted, time-dependent inhibitors
of GABA-AT, although they are not as potent as the
difluorinated analog 3. Currently the inhibition mecha-
nisms are under investigation. The fact that none of 2,
therefore,
4
the
efficiency
)
constants
and 5
for
(kinact/Ki 1.4
(kinact/KI 1.0mMꢀ1 minꢀ1
12
10
8
Compound 4
Compound 5
6
4
2
0
0
0.002
0.004
0.006
0.008
0.01
0.012
1/[Inhibitor] (µM-1)
Figure 2. Determination of the inactivation constants for 4 and 5 at pH8.5, 25ꢁC.

5722
Y. Pan et al. / Bioorg. Med. Chem. 12 (2004) 5719–5725
over anhydrous Na2SO4. This solution was concentrated
under reduced pressure and purified by flash column
chromatography (hexanes–EtOAc 1:0–1:2), giving an
inseparable mixture of the E- and Z-isomers (3:1,
1
1.44g, 82%) as a colorless oil. H NMR for the major
product (400MHz, CDCl3) d 7.94 (d, 2H, J 8.0Hz),
7.72 (t, 1H, J 7.4Hz), 7.61 (t, 2H, J 7.6Hz), 7.33 (d,
2H, J 8.4Hz), 6.90 (d, 2H, J 8.8Hz), 5.24 (s, 1H), 4.77
(d, 1H, J 14.8Hz), 3.82 (s, 3H), 3.79 (d, 1H, J
14.8Hz), 3.00 (s, 1H), 2.49–2.66 (m, 2H), 2.10 (d, 1H,
J 9.2Hz), 1.63 (d, 1H, J 8.8Hz); HRMS (EI) M calcd
for C21H20NO4FS 401.1092, found 401.1088.
Figure 3. Previously prepared monofluorovigabatrin analogs.
6, and 7 inactivated GABA-AT suggests that the orien-
tation of the double bond still requires further tuning for
these conformationally-restricted vigabatrin analogs.
4. Experimental
4.2. E-(1S,4S)-6-Fluoromethylenyl-2-(40-methoxybenzyl)-
azabicyclo[2.2.1]heptan-3-one (12) and Z-(1S,4S)-6-
fluoromethylenyl-2-(40-methoxybenzyl)-azabicyclo-
[2.2.1]heptan-3-one (13)
All NMR spectra were recorded with a Varian Mercury
400MHz or a Varian Inova 500MHz NMR spectrome-
ter. 1H NMR chemical shifts are reported as d values in
ppm downfield from Me4Si as the internal standard in
CDCl3. For samples run in D2O, the HOD resonance
was set at 4.80ppm. For samples run in CD3OD, the
HCD resonance was set at 3.31ppm. 13C chemical shifts
are listed in ppm with the CDCl3 carbon peak set to
77.23ppm. For samples run in D2O, DSS was used as
the external standard. For samples run in CD3OD, the
CD3OD carbon peak was set to 49.15ppm. 19F chemical
shifts are listed in ppm downfield from CFCl3 as the
external standard for samples in CDCl3 and TFAas
the external standard for samples in D2O. Mass spectra
were obtained on a VG70-250SE (EI) or a Micromass
Quattro II (ESI) mass spectrometer. Column chroma-
tography was carried out with Merck silica gel 60
(230–400mesh). TLC was run with EM Science silica
gel 60 F254 preloaded glass plates. Melting points were
obtained with a Fisher–Johns melting point apparatus
and are not corrected. An Orion Research 702 pH meter
with a general combination electrode was used for pH
measurements. All enzyme assays were recorded with a
Perkin–Elmer Lambda 10 UV/vis spectrometer. Fluoro-
methyl phenylsulfone was purchased from TCI Amer-
ica. Vigabatrin was purchased from Sigma Chemical
Co. All other reagents were purchased from Aldrich
Chemical Co. and were used without purification. All
solvents were purchased from Fisher Scientific. Anhy-
drous THF was distilled from sodium metal under
nitrogen.
Compound 11 (1.70g, 4.24mmol) was dissolved in anhy-
drous methanol (40mL) under nitrogen and cooled in an
ice–salt bath. Magnesium turnings (1.00g, 41.7mmol)
and mercury(II) chloride (150mg, 0.55mmol) were
added. The solution was stirred for 2h, warmed to room
temperature and stirred overnight. To the reaction mix-
ture was added HCl (1N, 30mL). Methanol was evapo-
rated under reduced pressure, and the resulting aqueous
solution was extracted with ethyl acetate (3 · 30mL).
The organic layers were combined, washed with satu-
rated aqueous NaHCO3 solution (2 · 30mL) and brine
(2 · 30mL), and dried over anhydrous Na2SO4. After
concentration under reduced pressure, the residue was
purified by flash column chromatography (hexanes–
EtOAc 3:1), giving 12 (0.70g, 64%) and 13 (0.25g,
23%), both as white solids.
For 12: mp 108.0–110.0ꢁC; 1H NMR (500MHz, CDCl3)
d 7.15 (d, 2H, J 8.5Hz), 6.87 (d, 2H, J 8.5Hz), 6.65 (d,
1H, J 82.9Hz), 4.66 (d, 1H, J 15.0Hz), 3.83 (s, 1H), 3.81
(s, 3H), 3.72 (d, 1H, J 15.0Hz), 2.98 (s, 1H), 2.55 (dd,
1H, J 16.0, 2.5Hz), 2.36 (dd, 1H, J 16.0, 1.5Hz), 2.02
(d, 1H, J 8.0Hz), 1.53 (d, 1H, J 9.5Hz); 13C NMR
(100MHz, CDCl3) d 177.60, 158.97, 142.67 (d, J
255.4Hz), 129.19, 128.54, 120.40 (d, J 7.3Hz), 113.98,
59.37 (d, J 9.2Hz), 55.20, 44.89, 43.53, 40.77, 27.93;
HRMS (EI) M calcd for C15H16NO2F 261.1160, found
261.1159.
4.1. E/Z-(1S,4S)-6-(10-Fluoro-10-phenylsulfonyl)methyl-
enyl-2-(40-methoxybenzyl)-azabicyclo[2.2.1]heptan-3-one
(11)
For 13: mp 51.0–53.0ꢁC; 1H NMR (500MHz, CDCl3) d
7.21 (d, 2H, J 8.5Hz), 6.87 (d, 2H, J 8.5Hz), 6.54 (d, 1H,
J 84.9Hz), 4.67 (d, 1H, J 15.0Hz), 4.36 (s, 1H), 3.81 (s,
3H), 3.67 (d, 1H, J 15.0Hz), 2.96 (s, 1H), 2.43 (d, 1H, J
14.0Hz), 2.21 (d, 1H, J 15.0Hz), 1.97 (d, 1H, J 9.5Hz),
1.48 (d, 1H, J 9.5Hz); 13C NMR (100MHz, CDCl3) d
177.80, 159.11, 142.53 (d, J 250.9Hz), 129.77, 128.79,
120.59 (d, J 11.9Hz), 114.04, 57.46 (d, J 2.7Hz), 55.38,
45.30, 44.35, 39.74, 27.45 (d, J 4.6Hz); HRMS (EI) M
calcd for C15H16NO2F 261.1160, found 261.1155.
To anhydrous THF (20mL) under nitrogen was added
fluoromethyl phenylsulfone (1.18g, 6.77mmol) and
diethyl chlorophosphate (0.98mL, 6.78mmol). At
ꢀ78ꢁC, lithium bis(trimethylsilyl)amide (1.0M in
THF, 13.1mL, 13.1mmol) was slowly added over
0.5h. After 1h, a solution of 10 (1.07g, 4.37mmol) in
anhydrous THF (10mL) was slowly added via cannula.
The solution was then warmed to room temperature and
stirred overnight. After being quenched with saturated
aqueous ammonium chloride solution (30mL), THF
was evaporated, and the resulting solution was extracted
with ethyl acetate (3 · 30mL). The organic layers were
combined, washed with brine (2 · 30mL), and dried
4.3. E-(1S,4S)-6-Fluoromethylenyl-2-azabicyclo[2.2.1]-
heptan-3-one (14)
Compound 12 (482mg, 1.85mmol) was dissolved in
acetonitrile (10mL). To this solution was added a

Y. Pan et al. / Bioorg. Med. Chem. 12 (2004) 5719–5725
5723
solution of ceric ammonium nitrate (3.04g, 5.55mmol)
in water (5mL). After being stirred at room temperature
for 1.5h, the reaction mixture was diluted with ethyl ace-
tate (100mL), washed with saturated aqueous NaHCO3
solution (2 · 40mL) and brine (2 · 40mL), and dried
over anhydrous Na2SO4. After concentration under
reduced pressure, the residue was purified by column
chromatography (hexanes–EtOAc 1:1) to give 14 as a
white solid (110mg, 42%). Mp 119.0–121.0ꢁC; 1H
NMR (400MHz, CDCl3) 6.83 (d, 1H, J 83.2Hz), 5.48
(br s, 1H), 4.15 (s, 1H), 2.90 (s, 1H), 2.60 (d, 1H, J
16.8Hz), 2.39 (d, 1H, J 15.6Hz), 2.15 (d, 1H, J
9.2Hz), 1.61 (d, 1H, J 9.2Hz); 13C NMR (100MHz,
CDCl3) d 181.06, 142.66 (d, J 254.6Hz), 122.93 (d, J
7.3Hz), 56.62 (d, J 10.1Hz), 44.70, 42.72, 27.59; 19F
NMR (376MHz, CDCl3) d ꢀ2.75 (d, J 83.6Hz); HRMS
(EI) M calcd for C7 H8NOF 141.0584, found 141.0582.
51.97, 44.51, 36.37, 32.27 (d, J 5.4Hz); 19F NMR
(376MHz, D2O) d ꢀ50.47 (d, J 82.5Hz); HRMS (EI)
M calcd for C7H10NO2F 159.0690, found 159.0688.
Anal. Calcd for C7H11NO2FCl: C, 42.98; H, 5.67; N,
7.16. Found: C, 42.61; H, 5.40; N, 6.95.
4.7. Ethyl 4-methylenyl-1-cyclohexanecarboxylate (17)
To a 500 mL, three-neck flask was added methyltriphen-
ylphosphonium bromide (18.4g, 51.5mmol) and anhy-
drous THF (240mL) under N2. A t ꢁ0C, LiHMDS
(1.0M in THF, 56.0mL, 56.0mmol) was slowly added
over 1h. The solution was then stirred for 0.5h before
16 (7.95g, 46.8mmol) was slowly added. The resulting
solution was warmed to room temperature and stirred
overnight. Asaturated aqueous ammonium chloride
solution (100mL) was added, THF was evaporated,
and the aqueous solution was extracted with EtOAc
(3 · 100mL). The organic layers were combined, washed
with brine (2 · 100mL), and dried over anhydrous
Na2SO4. After being concentrated under reduced pres-
sure, the residue was purified with a silica gel plug (hex-
anes–EtOAc 7:1) to give a light yellow oil (6.8g, 87%).
1H NMR (400MHz, CDCl3) d 4.50 (s, 2H), 3.99 (q,
2H, J 6.8Hz), 2.27–2.33 (m, 1H), 2.20 (d, 2H, J
13.6Hz), 1.84–1.95 (m, 4H), 1.44 (dq, 2H, J 12.4,
4.4Hz), 1.11 (t, 3H, J 6.4Hz); 13C NMR (100MHz,
CDCl3) d 175.15, 147.46, 107.85, 60.13, 42.58, 33.66,
30.17, 14.25.
4.4. Z-(1S,4S)-6-Fluoromethylenyl-2-azabicyclo[2.2.1]-
heptan-3-one (15)
This was prepared as a colorless oil using the same pro-
cedure as 14 in 43% yield from 13. 1H NMR (400MHz,
CDCl3) d 6.47 (d, J 85.6Hz, 1H), 5.93 (br s, 1H), 4.61 (s,
1H), 2.88 (s, 1H), 2.47 (dd, 1H, J 15.0, 2.0Hz), 2.25 (d,
1H, J 15.0Hz), 2.12 (d, 1H, J 9.5Hz), 1.58 (d, 1H, J
9.5Hz); 13C NMR (125MHz, CDCl3) d 180.64, 141.77
(d, J 251.3Hz), 122.74 (d, J 11.2Hz), 54.42 (d, J
4.4Hz), 44.74, 41.66, 26.80 (d, J 5.4Hz); 19F NMR
(376MHz, CDCl3) d ꢀ0.35 (d, J 84.7Hz); HRMS (EI)
M calcd for C7H8NOF 141.0584, found 141.0585.
4.8. trans-Ethyl 3-hydroxy-4-methylenyl-1-cyclohexane-
carboxylate (18)
4.5. E-(1S,3S)-3-Amino-4-fluoromethylenyl-1-cyclopent-
anoic acid, hydrochloride (4)
To CH2Cl2 (250mL) was added selenium dioxide
(0.30g, 2.7mmol) and tert-butyl hydroperoxide (5.0–
6.0M solution in decane, 20mL). Asolution of 17
(6.8g, 40mmol) in CH2Cl2 (100mL) was slowly added,
and the resulting solution was stirred at room tempera-
ture for 7h. After CH2Cl2 was evaporated, the solution
was concentrated under high vacuum to remove excess
t-BuOOH and decane. The residue was purified by flash
column chromatography (hexanes–EtOAc 3:1) to give a
light yellow oil (6.7g, 90%). 1H NMR (400MHz,
CDCl3) d 4.82 (s, 1H), 4.72 (s, 1H), 4.29 (t, 1H, J
3.6Hz), 4.06 (q, 2H, J 7.6Hz), 2.80–2.86 (m, 1H), 2.38
(dt, 1H, J 12.4, 4.0Hz), 2.13 (dt, 1H, J 13.6, 4.4Hz),
1.95–2.00 (m, 1H), 1.87–1.91 (m, 1H), 1.74–1.77 (m,
1H), 1.55 (dq, 1H, J 10.8, 4.4Hz), 1.18 (t, 3H, J
7.6Hz); 13C NMR (100MHz, CDCl3) d 175.64, 149.01,
109.52, 71.22, 60.55, 37.77, 36.74, 29.92, 29.60, 14.43;
HRMS (EI) M calcd for C10H16O3 184.1094, found
184.1097.
To compound 14 (38.5mg, 27.3lmol) was added HCl
(4N, 4mL). The solution was heated to 70ꢁC and stirred
for 10h. It was cooled, washed with ethyl acetate
(2 · 4mL), and evaporated under reduced pressure to
give a white solid, which was recrystallized with ethanol
and ethyl ether to give white crystals (40mg, 75%). Mp
1
200.0–202.0ꢁC (dec); H NMR (400MHz, D2O) d 6.96
(d, 1H, J 80.4Hz), 4.36 (s, 1H), 3.08 (t, 1H, J 7.6Hz),
2.93 (d, 1H, J 17.2Hz), 2.72 (d, 1H, J 16.0Hz), 2.51 (t,
1H, J 6.8Hz), 2.01–2.08 (m, 1H); 13C NMR (100MHz,
D2O) d 181.42, 149.82 (d, J 257.3Hz), 123.57 (d, J
12.8Hz), 53.20 (d, J 9.2Hz), 44.02, 36.78, 32.03 (d, J
1.9Hz); 19F NMR (376MHz, D2O) d ꢀ48.59 (d, J
78.7Hz); HRMS (EI)
M calcd for C7H10NO2F
159.0690, found 159.0687. Anal. Calcd for
C7H11NO2FCl: C, 42.98; H, 5.67; N, 7.16. Found: C,
42.65; H, 5.36; N, 6.81.
4.6. Z-(1S,3S)-3-Amino-4-fluoromethylenyl-1-cyclopent-
anoic acid, hydrochloride (5)
4.9. cis-Ethyl 3-(N-Boc-N-ethyloxamate)-4-methylenyl-1-
cyclohexanecarboxylate (19)
This was prepared as white crystals using the same pro-
cedure as 4 in 75% yield from 15. Mp 212.0–214.0ꢁC
Compound 18 (0.81g, 4.4mmol), triphenylphosphine
(1.73g, 6.6mmol), and N-Boc ethyl oxamate (1.43g,
6.59mmol) were added to anhydrous THF (20mL) un-
der nitrogen. At 0ꢁC, DIAD (1.28mL, 6.50mmol) was
slowly added over 5min. The solution was then heated
to reflux for 36h. After being cooled, the solution was
concentrated under reduced pressure, and the residue
1
(dec); H NMR (400MHz, D2O) d 6.84 (d, J 82.4Hz,
1H), 4.51 (s, 1H), 3.01 (q, 1H, J 8.0Hz), 2.71 (dd, 1H,
J 16.0, 8.0Hz), 2.61–2.63 (m, 1H), 2.51 ꢀ2.58 (m, 1H),
1.96–2.03 (m, 1H); 13C NMR (100MHz, D2O) d
180.67, 149.14 (d, J 254.5Hz), 122.07 (d, J 7.3Hz),

5724
Y. Pan et al. / Bioorg. Med. Chem. 12 (2004) 5719–5725
was purified by flash column chromatography (hexanes–
EtOAc 5:1) to give the product as a colorless oil (1.08g,
64%). H NMR (400MHz, CDCl3) d 4.97 (s, 1H), 4.85
(s, 1H), 4.64 (s, 1H), 4.35 (q, 2H, J 7.6Hz), 4.14 (q,
2H, J 7.6Hz), 2.44–2.57 (m, 3H), 2.05–2.19 (m, 4H),
1.50 (s, 9H), 1.37 (t, 3H, J 7.6Hz), 1.26 (t, 3H, J
7.6Hz); MS (CI) M + H calcd for C19H30NO7 384,
found 384.2, 324.2, 284.2, 167.1.
warmed to room temperature and stirred for 36h. After
concentration under reduced pressure, the residue was
purified by flash column chromatography (hexanes–
EtOAc 7:1) to give the product as a colorless oil
1
1
(1.25g, 58%). H NMR (400MHz, CDCl3) d 8.29 (dd,
4H, J 22.8, 8.4Hz), 5.52 (dd, 1H, J 10.4, 4.4Hz), 4.92
(s, 1H), 4.90 (s, 1H), 4.03–4.17 (m, 2H), 2.66–2.73 (m,
1H), 2.60 (dt, 1H, J 14.0, 3.6Hz), 2.42 (d, 1H, J
12.4Hz), 2.21 (dt, 1H, J 12.8, 3.6Hz), 2.05–2.08 (m,
1H), 1.92 (q, 1H, J 11.6Hz), 1.66 (dd, 1H, J 12.0,
4.4Hz), 1.23 (t, 3H, J 7.6Hz); 13C NMR (100MHz,
CDCl3) d 173.65, 163.38, 150.52, 144.44, 135.44,
130.71, 123.54, 107.07, 74.17, 60.59, 40.96, 35.14,
4.10. cis-3-(Boc-amino)-4-methylenyl-1-cyclohexanoic
acid (20)
Compound 19 (1.08g, 4.22mmol) was dissolved in THF
(15mL) and cooled to 0ꢁC. An aqueous solution of
LiOH (0.40g in 15mL water) was added, and the result-
ing solution was stirred at room temperature for 4h.
THF was evaporated, and the aqueous solution was
washed with EtOAc (2 · 10mL), acidified with HCl
(1N) to pH1, and extracted with EtOAc (3 · 10mL).
The organic layers were combined and dried over anhy-
drous Na2SO4. After concentration under reduced
pressure, the residue was purified by column chro-
matography (hexanes–EtOAc–HOAc 6:1:0.7) to give a
colorless oil (0.37g, 52%). 1H NMR (400MHz, CD3OD)
d 4.99 (s, 1H), 4.77 (s, 1H), 3.99 (d, 1H, J 7.6Hz), 2.60 (t,
1H, J 12.0Hz), 2.47–2.50 (m, 1H), 1.96–2.16 (m, 4H),
1.52 (d, 1H, J 4.0Hz), 1.45 (s, 9H); 13C NMR
(100MHz, CD3OD) d 178.32, 157.90, 149.10, 106.23,
80.22, 53.54, 44.07, 37.92, 34.81, 31.52, 28.94; HRMS
(CI) M + H calcd for C13H22NO4 256.1543, found
256.1540.
32.34, 29.45, 14.23; HRMS (EI)
C17H19O6N 333.1207, found 333.1205.
M
calcd for
4.13. cis-Ethyl 3-hydroxy-4-methylenyl-1-cyclohexane-
carboxylate (22)
Compound 21 (4.00g, 21.7mmol) was dissolved in
methanol (100mL). Sodium azide (5.65g, 86.9mmol)
was added, and the solution was heated to 40ꢁC for
5.5h. Water (100mL) was added, and methanol was
evaporated under reduced pressure. The resulting aque-
ous solution was extracted with EtOAc (3 · 80mL). The
organic layers were combined, washed with brine
(2 · 80mL), and dried over Na2SO4. After being concen-
trated under reduced pressure, the residue was purified
by column chromatography (hexanes–EtOAc 6:1) to
1
give a colorless oil (1.42g, 64%). H NMR (400MHz,
CDCl3) d 4.99 (s, 1H), 4.81 (s, 1H), 4.06–4.16 (m, 3H),
2.77 (br s, 1H), 2.56 (tt, 1H, J 10.8, 3.6Hz), 2.47 (dt,
1H, J 13.2, 2.8Hz), 2.31 (d, 1H, J 12.4Hz), 1.97–2.08
(m, 2H), 1.45–1.56 (m, 2H), 1.25 (t, 3H, J 6.8Hz);
4.11. cis-3-Amino-4-methylenyl-1-cyclohexanoic acid,
hydrochloride (6)
13C NMR (100MHz, CDCl3)
d 174.73, 149.69,
105.44, 71.17, 60.66, 42.01, 38.70, 32.62, 29.98, 14.28;
HRMS (EI) M calcd for C10H16O3 184.1094, found
184.1091.
To 20 (78mg, 0.31mmol) was added CH2Cl2 (4mL) and
TFA(4mL). The solution was stirred at room tempera-
ture for 4h. The solvent was then evaporated under re-
duced pressure, and the residue was dissolved in HCl
(1.0N, 5mL). After evaporation under high vacuum,
HCl (1.0N, 5mL) was added and evaporated two more
times to give a white solid. The solid was recrystallized
from ethanol/ethyl ether to give the product as white
crystals (50mg, 85%). Mp 241.5–243.5ꢁC; 1H NMR
(400MHz, CD3OD) d 5.00 (s, 1H), 4.81 (s, 1H), 3.81
(d, 1H, J 11.6Hz), 2.71 (dt, 1H, J 12.4, 3.2Hz), 2.54
(d, 1H, J 14.0Hz), 2.41 (d, 1H, J 12.4Hz), 2.14–2.26
(m, 2H), 1.55 (q, 1H, J 12.4Hz), 1.44 (dq, 1H, J 12.4,
4.4Hz); 13C NMR (100MHz, CD3OD) d 177.23,
145.44, 107.79, 53.14, 42.64, 35.76, 34.62, 31.29; HRMS
(EI) M calcd for C8H13NO2 155.0941, found 155.0940.
Anal. Calcd for C8H14NO2Cl: C, 50.13; H, 7.37; N,
7.31. Found: C, 49.91; H, 7.00; N, 7.08.
4.14. trans-Ethyl 3-(N-Boc-N-ethyloxamate)-4-methyl-
enyl-1-cyclohexanecarboxylate (23)
This was prepared using the same procedure as 19 in
55% yield from 22. 1H NMR (400MHz, CDCl3) d
5.49 (s, 1H), 4.74 (s, 1H), 4.53 (s, 1H), 4.28 (q, 2H, J
7.2Hz), 4.09 (q, 2H, J 7.6Hz), 2.84 (s, 1H), 2.43 (td,
1H, J 12.0, 5.6Hz), 2.33 (d, 1H, J 15.2Hz), 2.12–2.21
(m, 2H), 1.94–1.98 (m, 2H), 1.43 (s, 9H), 1.30 (t, 3H, J
7.2Hz), 1.19 (t, 3H, J 7.2Hz); MS (CI) M + H calcd
for C19H30NO7 384, found 384.2, 324.2, 284.2, 167.1.
4.15. trans-3-(Boc-amino)-4-methylenyl-1-cyclohexanoic
acid (24)
This was prepared using the same procedure as 20 in
36% yield from 23. 1H NMR (400MHz, CD3OD) d
4.79 (s, 1H), 4.75 (s, 1H), 4.20 (s, 1H), 2.78 (t, 1H, J
4.4Hz), 2.30–2.34 (m, 1H), 2.20–2.25 (m, 1H), 2.00–
2.05 (m, 1H), 1.85–1.89 (m, 1H), 1.71–1.78 (m, 2H),
1.45 (s, 9H); 13C NMR (100MHz, CD3OD) d 178.58,
157.86, 149.30, 108.25, 80.22, 52.59, 40.04, 36.40,
31.94, 30.64, 28.92; HRMS (CI) M + H calcd for
C13H22NO4 256.1543, found 256.1541.
4.12. cis-Ethyl 3-(40-nitrobenzoyloxy)-4-methylenyl-1-
cyclohexanecarboxylate (21)
Compound 18 (1.19g, 6.47mmol), triphenylphosphine
(2.20g, 8.40mmol), and p-nitrobenzoic acid (1.43g,
8.56mmol) were added to anhydrous THF (30mL)
under nitrogen. At 0ꢁC, DIAD (1.65mL, 8.38mmol)
was slowly added over 15min. The solution was then

Y. Pan et al. / Bioorg. Med. Chem. 12 (2004) 5719–5725
5725
4. Tower, D. B. In GABA in Nervous System Function;
Roberts, E., Chase, T. N., Tower, D. B., Eds.; Raven:
New York, 1975.
4.16. trans-3-Amino-4-methylenyl-1-cyclohexanoic acid,
hydrochloride (7)
5. Gale, K. Epilepsia 1989, 30(Suppl. 3), S1–11.
6. Hayashi, T. J. Physiol. 1959, 145, 570–578.
7. Van Gelder, N. M.; Elliott, K. A. C. J. Neurochem. 1958,
3, 139–143.
8. Meldrum, B. S.; Horton, R. W. In Epilepsy; Harris, P.,
Mawdsley, C., Eds.; Churchill Livingston: Edinburgh,
1974.
9. Krogsgaard-Larsen, P. J. Med. Chem. 1981, 24,
1377–1383.
10. Lippert, B.; Metcalf, B. W.; Jung, M. J.; Casara, P. Eur. J.
Biochem. 1977, 74, 441–445.
This was prepared using the same procedure as 6 in 80%
yield from 24. Mp 203.0–205.0ꢁC; H NMR (400MHz,
1
CD3OD) 5.08 (s, 1H), 4.90 (s, 1H), 4.01–4.04 (m, 1H),
2.91 (t, 1H, J 4.8Hz), 2.38–2.43 (m, 1H), 2.25–2.36 (m,
2H), 2.01–2.05 (m, 1H), 1.78–1.84 (m, 2H); 13C NMR
(100MHz, CD3OD) d 177.18, 145.09, 110.48, 52.36,
39.36, 34.30, 31.89, 30.22; HRMS (EI) M calcd for
C8H13NO2 155.0941, found 155.0940. Anal. Calcd for
C8H14NO2Cl: C, 50.13; H, 7.37; N, 7.31. Found: C,
49.95; H, 7.07; N, 7.00.
11. Jung, M. J.; Lippert, B.; Metcalf, B. W.; Bohlen, P.;
Schechter, P. J. J. Neurochem. 1977, 29, 797–802.
12. Nanavati, S. M.; Silverman, R. B. J. Am. Chem. Soc. 1991,
113, 9341–9349.
4.17. Time-dependent inhibition of GABA-AT by 4 and 5
GABA-AT was isolated and purified from pig brain by a
modified procedure.29 Incubation solutions (100lL)
contained the enzyme (20lL, 1.84mg/mL), potassium
pyrophosphate buffer (60lL, 50mM, pH8.5), a-keto-
glutarate (10lL, 16mM in 50mM potassium pyrophos-
phate buffer, pH8.5), 2-mercaptoethanol (2mM), and 4
or 5. The concentrations of 4 and 5 were 100, 125, 250,
and 500lM. At timed intervals, aliquots (20lL) from
the incubation solution were added to the assay solution
(575lL, 50mM potassium pyrophosphate buffer con-
taining 5.3mM of a-ketoglutarate, 11mM of GABA,
1.1mM of NADP+, and 4.8mM of 2-mercaptoethanol)
with excess succinic semialdehyde dehydrogenase
(SSDH). Rates were measured spectrophotometrically
at 340nm, and the logarithm of the remaining activity
(percentage) was plotted against time for each concen-
tration to determine the half-life. Then a secondary plot
of half-life versus the reciprocal of the inhibitor concen-
tration was obtained to determine the kinact/Ki.
13. Freidinger, R. M.; Veber, D. F.; Perlow, D. S. Science
1980, 210, 656–658.
14. Pan, Y.; Qiu, J.; Silverman, R. B. J. Med. Chem. 2003, 46,
5292–5293.
15. Kolb, M.; Barth, J.; Heydt, J.-G.; Jung, M. J. J. Med.
Chem. 1987, 30, 267–272.
16. Choi, S.; Storici, P.; Schirmer, T.; Silverman, R. B. J. Am.
Chem. Soc. 2002, 124, 1620–1624.
17. Choi, S.; Silverman, R. B. J. Med. Chem. 2002, 45,
4531–4539.
18. Qiu, J.; Silverman, R. B. J. Med. Chem. 2000, 43, 706–
720.
19. Palmer, C. F.; Parry, K. P.; Roberts, S. M.; Sik, V.
J. Chem. Soc., Perkin Trans. 1 1992, 8, 1021–1028.
20. McCarthy, J. R.; Matthews, D. P.; Paolini, J. P. Org.
Synth. 1995, 72, 216–224.
21. McCarthy, J. R.; Huber, E. W.; Le, T.-B.; Laskovics,
F. M.; Matthews, D. P. Tetrahedron 1996, 52, 45–
58.
22. Inbasekaran, M.; Peet, N. P.; McCarthy, J. R.; LeTour-
neau, M. E. J. Chem. Soc., Chem. Commun. 1985, 10,
678–679.
23. McCarthy, J. R.; Matthews, D. P.; Edwards, M. L.;
Stemerick, D. M.; Jarvi, E. T. Tetrahedron Lett. 1990, 31,
5449–5452.
24. Toyota, A.; Ono, Y.; Kaneko, C.; Hayakawa, I. Tetra-
hedron Lett. 1996, 37, 8507–8510.
25. Arigoni, D.; Vassela, A.; Sharpless, K. B.; Jensen, H. P.
J. Am. Chem. Soc. 1973, 95, 7917–7919.
26. Berree, F.; Michelot, G.; Le Corre, M. Tetrahedron Lett.
1998, 39, 8275–8276.
Acknowledgements
We are grateful to the National Institutes of Health
(NS15703 and GM66132) for financial support of this
research.
References and notes
´
27. Gomez-Vidal, J. A.; Forrester, M. T.; Silverman, R. B.
Org. Lett. 2001, 3, 2477–2479.
28. Silverman, R. B.; Bichler, K. A.; Leon, A. J. J. Am. Chem.
Soc. 1996, 118, 1253–1261.
29. Koo, Y. K.; Nandi, D.; Silverman, R. B. Arch. Biochem.
Biophys. 2000, 374, 248–254.
1. Wyke, B. Principles of Neurology; Elsevier: London, 1969.
2. Biemond, A. Brain Diseases; Elsevier: London, 1970.
3. Karlsson, A.; Fonnum, F.; Malthe-Sorrensen, D.; Storm-
Mathisen J. Biochem. Pharmacol. 1974, 23, 3053–
3061.