Fluorinated conformationally restricted γ-aminobutyric acid aminotransferase inhibitors

Article abstract of DOI:10.1021/jm0608715

On the basis of the structures of several potent inhibitor molecules for γ-aminobutryric acid aminotransferase (GABA-AT) that were previously reported, six modified fluorine-containing conformationally restricted analogues were designed, synthesized, and tested as GABA-AT inhibitors. The syntheses of all six molecules followed from a readily synthesized ketone intermediate. Three of the molecules were found to be irreversible inhibitors of GABA-AT with comparable or larger k_inact/K₁ values than that of vigabatrin, a clinically used antiepilepsy drug, and the other three were reversible inhibitors. A possible mechanism for inactivation by one of the inactivators is proposed.

Full text of DOI:10.1021/jm0608715

7404
J. Med. Chem. 2006, 49, 7404-7412
Fluorinated Conformationally Restricted γ-Aminobutyric Acid Aminotransferase Inhibitors
Hejun Lu 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, Illinois 60208-3113
ReceiVed July 24, 2006
On the basis of the structures of several potent inhibitor molecules for γ-aminobutryric acid aminotransferase
(GABA-AT) that were previously reported, six modified fluorine-containing conformationally restricted
analogues were designed, synthesized, and tested as GABA-AT inhibitors. The syntheses of all six molecules
followed from a readily synthesized ketone intermediate. Three of the molecules were found to be irreversible
inhibitors of GABA-AT with comparable or larger kinact/KI values than that of vigabatrin, a clinically used
antiepilepsy drug, and the other three were reversible inhibitors. A possible mechanism for inactivation by
one of the inactivators is proposed.
There are two principal neurotransmitters involved in the
regulation of brain neuronal activity: γ-aminobutyric acid
(GABAa), one of the most widely distributed inhibitory neu-
rotransmitters, and L-glutamic acid, an excitatory neurotrans-
mitter.1 The GABA concentration is regulated by two pyridoxal
5′-phosphate (PLP)-dependent enzymes, L-glutamic acid decar-
boxylase (GAD), which catalyzes the conversion of L-glutamate
to GABA, and GABA aminotransferase (GABA-AT), which
degrades GABA to succinic semialdehyde (SSA) concomitant
with the conversion of R-ketoglutarate to L-glutamate.2 When
the concentration of GABA diminishes below a threshold level
in the brain, convulsions result;3 raising the brain GABA levels
terminates the seizure.4 A reduction in the concentrations of
GABA and of the enzyme GAD has been implicated not only
in the symptoms associated with epilepsy5,6 but also with several
other neurological diseases such as Huntington’s chorea,7,8
Parkinson’s disease,9,10 Alzheimer’s disease,11 and tardive
dyskinesia.12 GABA brain levels cannot be increased by admini-
stration of GABA because it does not cross the blood-brain
barrier. An approach that has been successful to increase brain
GABA levels is the use of a compound that crosses the blood-
brain barrier and then inhibits or inactivates GABA-AT, thereby
increasing the GABA concentration. Numerous competitive
inhibitors of GABA-AT, particularly compounds having a
similar backbone structure to GABA,13 show anticonvulsant
activity. A variety of mechanism-based inactivators14 of
GABA-AT15 also have been shown to be effective anticonvul-
sant agents. The most effective of the mechanism-based
inactivators as an anticonvulsant agent is 4-amino-5-hexenoic
acid (1, Figure 1; γ-vinyl-GABA),16 which has the commercial
generic name vigabatrin. This compound, which has high
potency,17 has been shown to be an effective treatment for
epilepsies that are resistant to other anticonvulsant drugs18 and
currently is prescribed in over 60 countries worldwide, but it
was not approved in the U.S. It also was found that vigabatrin
prevents cocaine addiction in rats and baboons.19 This activity
was observed with other forms of addiction, including nicotine
addiction20 and methamphetamine, alcohol, and heroin addic-
tions.21 Self-administration of cocaine by rats decreased or was
prevented by vigabatrin administration in a dose-dependent
fashion, without affecting the craving for food.22 GABA was
found to antagonize the extracellular dopamine levels respon-
sible for drug addiction, and by positron emission tomography
(PET) in primates, it was shown that vigabatrin, which causes
a rise in GABA levels, inhibits these drug-induced dopamine
increases.21 However, because vigabatrin is not approved in the
U.S., new GABA-AT inhibitors and inactivators are needed.
Conformationally restricted analogues of GABA are of
considerable interest among medicinal chemists.23 We previ-
ously reported the design and synthesis of a series of confor-
mationally restricted analogues of GABA. Among them,
(1R,4S)-4-aminocyclopent-2-enecarboxylic acid (2) was shown
to be a potent inhibitor and substrate of GABA-AT, but it was
not a time-dependent inactivator.24 (1S,3S)-3-Amino-4-methyl-
enecyclopentanecarboxylic acid (3) was found to inactivate
GABA-AT but only in the absence of 2-mercaptoethanol (indi-
cating that a reactive species was produced that was released
from the enzyme prior to inactivation), while (1S,3S)-3-amino-
4-difluoromethylenecyclopentanecarboxylic acid (4) is a much
more potent time-dependent inhibitor of γ-aminobutyric acid
aminotransferase, even in the presence of 2-mercaptoethanol
(indicating that any reactive species generated are not released
prior to inactivation).25 Compound 4 is 187 times more potent
of an inactivator of GABA-AT than vigabatrin, and the
corresponding E- and Z-monofluoro analogues of 3 also
inactivate GABA-AT but not with the same potency as 4. This
suggests an important significance for fluorine in the molecules.
Fluorine and fluoroalkyl substitutents have an important
electronic effect on the neighboring groups in a molecule and
also mimic hydrogen and alkyl groups, respectively.26 Therefore,
the introduction of one or more fluorine-containing groups into
molecules alters their physical properties, as well as their
biological activities.27 Fluorinated substrates also can be used
as mechanistic probes and inhibitors for obtaining information
about the catalytic mechanism of various enzymatic transforma-
tions.28 Consequently, it was thought that other fluorinated
conformationally restricted analogues of 2 and 3 should be
evaluated for their inhibitory activities. On the basis of that
hypothesis, we designed six new fluorine-containing confor-
mationally restricted vigabatrin analogues (5-10, Figure 2).
* To whom correspondence should be addressed. Address: Department
of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, IL
60208-3113. Phone: 847-491-5653. Fax: 847-491-7713. E-mail: agman@
chem.northwestern.edu.
a Abbreviations: CAN, ceric ammonium nitrate; DBDMH, 1,3-dibromo-
5,5-dimethylhydantoin; GABA, γ-aminobutyric acid; GABA-AT, γ-ami-
nobutyric acid aminotransferase; GAD, L-glutamic acid decarboxylase;
LiHMDS, lithium hexamethyldisilazide; MFSDA, methyl fluorosulfonyldi-
fluoroacetate; PMBCl, p-methoxybenzyl chloride; SSA, succinic semial-
dehyde dehydrogenase; TBAF, tetrabutylammonium fluoride, TPAP, tetra-
n-propylammonium perruthenate.
10.1021/jm0608715 CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/10/2006

γ-Aminobutyric Acid Aminotransferase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7405
Figure 1. Vigabatrin (1) and conformationally restricted analogues.
presence of NaH successfully gave the tosylates 23a and 23b,
respectively. Unfortunately, attempted elimination reactions of
23a and 23b with various bases always resulted in formation
of 21a and 21b, respectively, with only a trace amount of the
desired products detected.
An alternative synthesis of target molecule 6 (Scheme 4)
started with iodination of 16 with hydrazine and iodine followed
by elimination of one molecule of HI in the presence of
potassium tert-butoxide to give 17. Direct trifluoromethylation
of 17 with in situ generated CF3Cu from FSO2CF2CO2Me
(methyl fluorosulfonyldifluoroacetate, MFSDA) and CuI31 suc-
cessfully afforded 22a. Removal of the PMB group with CAN
followed by acidic hydrolysis gave 6.
Figure 2. New fluorinated conformationally restricted potential
inhibitors and inactivators of GABA-AT.
Scheme 1a
Compound 7 was synthesized from 17 using steps similar to
those used to prepare 6 (Scheme 5). A first attempt at
pentafluoroethylation of 17 with CF3CF2CO2Na/CuI at 140 °C
only resulted in decomposition of the substrate. Treatment of
17 with CF3CF2SiMe3/KF/CuI, however, afforded 22b in good
yields.
A Wittig reaction of 16 with CHBrdPPh3, generated in situ
t
from bromomethyltriphenylphosphonium bromide and BuOK
a Reagents and conditions: (a) PMBCl, NaH, Bu4NI, DMF, 0 °C to room
temp, 2 h, 83%; (b) DBDMH, HOAc, room temp, 20 h, 97%; (c) Bu3SnH,
AIBN, PhH, reflux, 12 h, 95%; (d) K2CO3, MeOH, H2O, room temp, 2 h,
87%; (e) NMO, TPAP, DCM, room temp, 24 h, 70%.
afforded an E/Z mixture of bromomethylenes 25a and 25b,
which was easily separated by column chromatography on silica
gel. The conformations of the double bonds in the two isomers
were determined based on NOE experiments. Trifluorometh-
ylation of 25a and 25b with CF3Cu under similar conditions
used for 17 produced compounds 26a and 26b, respectively.
Removal of the PMB protecting group with CAN followed by
acidic hydrolysis gave 8 and 9 (Scheme 6).
Treatment of 16 with CBr4 and PPh3 in toluene afforded 28,
which was double-trifluoromethylated with in situ generated
CF3Cu to give 29. Removal of the PMB group with CAN,
followed by hydrolysis with 4 N HCl(aq) at 75 °C gave 10
(Scheme 7).
Compounds 5-7 are C-3 fluorine or fluoroalkyl substituted
analogues of 2, while 8-10 are trifluoromethylated analogues
of 3 and 4. The presence of strongly electron-withdrawing
fluorine and fluoroalkyl groups also may enhance binding
and/or reactivity of these compounds during GABA-AT turnover
based on a Michael addition mechanism.23a The results of this
investigation are reported here.
Results and Discussion
It is noteworthy that, compared to the compounds with an
exocyclic double bond (27a, 27b, and 30), the hydrolysis of
20, 24a, and 24b, which have endocyclic double bonds, was
found to be much easier. The reaction is usually completed in
1 h when the compounds are treated with 4 N aqueous HCl at
70 °C. Prolonged heating and stirring of these compounds under
these conditions resulted in major side reactions. No such side
reactions were observed from hydrolysis of 27a, 27b, and 30.
Enzyme Inhibition Results. Compounds 6, 8, and 9 showed
concentration and time-dependent inhibition of pig brain
GABA-AT in the presence of â-mercaptoethanol (Table 1).
Compounds 5, 7, and 10 showed only weak reversible inhibition
of GABA-AT in the presence of â-mercaptoethanol. None of
the three reversible inhibitor target molecules was more potent
than 2 or 4. However, the irreversible inhibitors were com-
parable to vigabatrin as inactivators of GABA-AT. It is
interesting that although 6 was designed to be a reversible
inhibitor of GABA-AT because a simple elimination of HF was
not initially apparent, it was found to be an irreversible inhibitor.
Compounds 5 and 7, which differ from 6 only by the length of
the fluoroalkyl chain, are reversible inhibitors of GABA-AT.
Although 8 and 9 are irreversible inhibitors of GABA-AT, as
expected, introduction of a second trifluoromethyl group makes
10 a weak reversible inhibitor.
Chemistry. Key intermediate 16 for the synthesis of all of
the analogues was synthesized according to a modification of a
procedure we reported previously29 (Scheme 1). Protection of
11 was realized by treatment with p-methoxybenzyl chloride
(PMBCl) in the presence of sodium hydride in DMF instead of
LiHMDS in THF as reported. This revised procedure turned
out to be much easier and gave higher yields of product.
The synthesis of 5 was first attempted by a direct halogen
exchange reaction of 17. However, treatment of 17 with
(PhSO2)2NF/nBuLi resulted in the ent-12 reduction product as
the major product. Conversion of 17 to organotin intermediate
18 followed by fluorine-metal exchange with XeF2 afforded
19 in a satisfactory yield (Scheme 2). Deprotection of 19 with
ceric ammonium nitrate (CAN) followed by acidic hydrolysis
gave 5.
Syntheses of target molecules 6 and 7 via elimination of an
alcohol or the corresponding tosylated derivative were unsuc-
cessful (Scheme 3). Treatment of 16 with TMSRf (Rf ) CF3
or CF3CF2) in the presence of a catalytic amount of TBAF
afforded tertiary alcohols 21a and 21b in excellent yields.30
However, direct dehydration of 21a or 21b with various
dehydration conditions failed to produce the desired products
22a and 22b. Activation of 21a and 21b with TsCl in the

7406 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Lu and SilVerman
Scheme 2a
t
a Reagents and conditions: (a) NH2NH2‚H2O, Et3N, EtOH, reflux, 1 h; (b) I2, Et3N, benzene, room temp, 2 h; (c) BuOK, ether, room temp, 20 h, 60%
in three steps; (d) Me3SnSnMe3, Pd(PPh3)4, PhMe, reflux, 30 min, 49%; (e) XeF2, AgOTf, 2,6-bis(tert-butyl)-4-methylpyridine, DCM, room temp, 8 min,
30%; (f) CAN, CH3CN, H2O, room temp, 2 h, 55%; (g) 4 N HCl(aq), 70 °C, 0.5-1 h, 64%.
Scheme 3a
Scheme 6a
a Reagents and conditions: (a) BrCH2PPh3.Br, BuOK, THF, -78 °C,
20 h, 72% E/Z; (b) MFSDA, CuI, DMF, HMPA, 75 °C, 20 h; 95%; (c)
CAN, CH3CN, H2O, room temp, 2 h, 76%; (d) 4 N HCl(aq), 70 °C, 10-12
h, 79%.
t
a Reagents and conditions: (a) TMSRf, TBAF(cat.), THF, room temp,
1 h, 95%; (b) TsCl, NaH, ether, 0 °C, 16 h, 79%.
Scheme 4a
Scheme 7a
a Reagents and conditions: (a) MFSDA, CuI, DMF, HMPA, 20 h, 75%;
(b) CNA, MeCN/H2O, room temp, 3 h, 61%; (c) 4 N HCl(aq), 70 °C, 0.5-1
h, 85%.
a Reagents and conditions: (a) CBr4, PPh3, toluene, reflux 22 h, 86%;
(b) MFSDA, CuI, DMF, HMPA, 75 °C, 50 h, 82%; (c) CAN, CH3CN,
H2O, room temp, 1 h, 56%; (d) 4 N HCl(aq), 70 °C, 10-12 h, 77%.
Scheme 5a
Table 1. Kinetic Constants for 5-10
kinact
KI
(mM)
kinact/KI
Ki
(mM)
compd
(min-1
)
(min-1 mM-1
)
a Reagents and conditions: (a) CF3CF2SiMe3/KF/CuI, NMO/DMF
(1/1), 75 °C, 24 h, 57%; (b) CAN, CH3CN, H2O, room temp, 2 h, 76%; (c)
4 N HCl(aq), 70 °C, 0.5-1 h, 82%.
5
6
7
8
9
1.3
2.8
0.68
4.11
0.17
6.96
2.89
39.23
7.74
0.18
0.37
Exhaustive dialysis of GABA-AT that was inactivated by 6,
8, and 9 against potassium pyrophosphate buffer (pH 8.5)
resulted in different extents of irreversibility of inhibition.
Compared to the control, 48% and 13% of enzyme activity was
recovered for 6 and 8, respectively, after 22 h of dialysis, while
the percentage of recovered enzyme activity for 9 continued to
increase with extended dialysis. These results suggest that at
least part of the adducts formed between the enzyme and 6 and
8 are stable, while at least one with 9 can be slowly hydrolyzed
upon dialysis. Incubation of 6, 8, and 9 with GABA-AT in the
presence of GABA resulted in a significant increase in their
10
4.2
vigabatrina
0.24
0.85
0.28
a From ref 25.
Lys329) or hydroxide (X ) HO-) results in 32, which can
eliminate fluoride ion to give a highly reactive exocyclic
difluoromethylene group32 conjugated to the PMP iminium (33).
This should lead to rapid nucleophilic attack,32 either by the
enzyme (pathway a) or by water (pathway b) to give 34 or 36,
respectively. Fluoride ion elimination and hydrolysis would give
35 (inactivation) or 38 (turnover). Inactivation by 8 and 9 may
be related to the mechanism of inactivation proposed for 4.25
To test this mechanism, fluoride ion release was monitored
during inactivation. GABA-AT was incubated with all six
compounds for 2 h, and the fluoride ion concentration was
monitored. With 6, 270 fluoride ions were released per enzyme
t
1/2 of inactivation (slower rate of inactivation), indicating active-
site binding to GABA-AT by these compounds.32
A possible mechanism for inactivation of GABA-AT by 6 is
shown in Scheme 8. Initial Schiff base formation with the PLP
followed by tautomerization gives 31. Michael addition to this
trifluoromethyl-activated Michael acceptor33 by Lys329 (X )

γ-Aminobutyric Acid Aminotransferase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7407
Scheme 8
dimer inactivated. This suggests that possibly 90 molecules of
6 are turned over for each inactivation event (partition ratio of
89). Possibly, the activated intermediate 33 undergoes reaction
with water (pathway b) 89 times for each attack by an active
site residue (pathway a). Depending on what enzyme residue
reacts with 33, the carboxylic acid derivative product formed
may or may not be stable to conditions of dialysis, leading to
partial reactivation of the enzyme during dialysis. For 7, 109
fluoride ions were released, and for the other four compounds,
less than 6 fluoride ions were released during inactivation.
Enzyme activity was monitored concomitantly with fluoride
ion release during inactivation of GABA-AT by 6. A good
correlation was observed (Figure 3), which suggests that
inactivation occurs as a consequence of fluoride ion release.
When GABA-AT was substituted by human albumin, no
fluoride ion was released, indicating that the presence of a
protein is not responsible for the fluoride ion release.
Figure 4 shows the time-dependent increase in fluoride ion
concentration when GABA-AT was incubated with 7, a weak
reversible inhibitor. The fluoride ion concentration reached it
highest level in 2-3 h. No fluoride ion was released when 7
was incubated with albumin, indicating a GABA-AT-dependent
reaction. This suggests that the corresponding reactive inter-
mediate (related to 33 in Scheme 8) only undergoes hydrolysis
(possibly because of steric hindrance by the trifluoromethyl
group of 7 that replaces the fluoride in 6).
Figure 4. Enzyme activity and fluoride ion release during inac-
tivation of GABA-AT with 7 (2 mM) as a function of time: fluoride
ion concentration when GABA-AT was incubated with 7 (diamonds);
remaining relative enzyme activity compared to control when
GABA-AT was incubated with 7 (squares).
Conclusion
Two series of fluorinated compounds were designed as
potential inhibitors and inactivators of GABA-AT based on the
high potency of 4. The kinact/KI value for 8 (0.37 mM-1 min-1
)
Figure 3. Enzyme activity and fluoride ion release during inactivation
of GABA-AT with 6 (2 mM) as a function of time: fluoride ion
concentration when GABA-AT was incubated with 6 (diamonds);
fluoride ion concentration when human albumin was incubated with 6
(squares); remaining relative enzyme activity compared to control when
GABA-AT was incubated with 6 (triangles).
is greater than that for the epilepsy drug vigabatrin (0.28 mM-1
min-1); that for 6 (0.16 mM-1 min-1) and for 9 (0.18 mM-1
min-1) are slightly lower. None of these, however, is as effi-
cient an inactivator as 4 (52 mM-1 min-1). Compounds 5-7
were designed as reversible inhibitors, so it was unexpected that

7408 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Lu and SilVerman
(1S,4R)-2-(4-Methoxybenzyl)-6-trimethylstannanyl-2-azabicyclo-
[2.2.1]hept-5-en-3-one (18). Compound 17 (41 mg, 0.115 mmol)
was dissolved in anhydrous toluene (4 mL) under argon, followed
by addition of Pd(PPh3)4 (20 mg, 0.017 mmol) and Me3SnSnMe3
(45 mg, 0.139 mmol). A light-yellow solution formed, which was
heated to reflux under argon and stirred for 1 h. Toluene was
evaporated under vacuum, and the residue was purified via column
chromatography on silica gel (hexanes/EtOAc, 3:1) to give 18 (25
6 was a potent time-dependent inactivator. It appears that there
is more to the effective binding of compounds to GABA-AT
than just the fluorine atoms. Other structures that more closely
resemble 4 are being constructed.
Experimental Section
General Methods and Reagents. Optical spectra and
1
GABA-AT assays were recorded on a Perkin-Elmer Lambda 10
mg, 56%) as a colorless oil. H NMR (500 MHz, CDCl3) δ 7.14
1
UV/vis spectrophotometer. H NMR spectra were recorded on a
(2H, d, J ) 8.5 Hz), 6.88 (2H, d, J ) 8.5 Hz), 6.85 (1H, m), 4.67
(1H, d, J ) 15.0 Hz), 4.13 (1H, s), 3.81 (3H, s), 3.47 (1H, s), 3.41
(1H, d, J ) 15.0 Hz), 2.24 (1H, d, J ) 7.5 Hz), 2.00 (1H, d, J )
8.0 Hz), 0.16 (9H, s). 13C NMR (CDCl3, 125.6 MHz) δ 179.83,
159.03, 153.07, 148.19, 129.26, 129.12, 114.12, 66.20, 58.27, 55.68,
55.34, 47.56, -9.28. MS m/z (EI, DCM): 389.0 (M)+, 374.0 (M
- CH3)+, 211.0 (M - PMB - CONH - CH3)+, 121.0 (PMB)+.
HRMS calcd for C17H23O2N116Sn (M)+: 389.0741. Found: 389.0749.
HRMS calcd for C16H20O2N116Sn (M - CH3)+: 374.0506. Found:
374.0504.
Varian Gemini 300 MHz NMR spectrometer. Chemical shifts are
reported as δ values in parts per million downfield from TMS as
the internal standard in CDCl3. For samples run in D2O, the HOD
resonance was arbitrarily set at 4.60 ppm. CClF3 was selected as
an external standard with δ 0.0 ppm for 19F NMR. Melting points
were determined on a Fisher-Johns melting point apparatus and
are uncorrected. Combustion analyses were performed by Oneida
Research Laboratories, NY. High-resolution mass spectra and
accurate mass spectra were obtained on a VG-70-250SE high-
resolution spectrometer and a Micromass Quattro II LC/MS
spectrometer. An Orion Research model 702A pH meter with a
general combination electrode was used for pH measurements.
Fluoride ion concentration measurements were obtained using an
Orion Research model 702A pH meter with an Orion Research
model 96-09 combination fluoride electrode. Flash column chro-
matography was carried out with Merck silica gel 60 (230-400
mesh ASTM). TLC was run with EM Science silica gel 60 F254
precoated glass plates.
(1S,4R)-6-Fluoro-2-(4-methoxybenzyl)-2-azabicyclo[2.2.1]hept-
5-en-3-one (19). AgOTf (48 mg, 0.187 mmol) was dissolved in
dry DCM under argon. Compound 18 (73.4 mg, 0.187 mmol) and
2,6-di-tert-butyl-4-methylpyridine (19 mg, 0.094 mmol) in dry
DCM (1 mL) were added via syringe, followed by immediate
addition of XeF2 (38 mg, 0.225 mmol) in DCM. The resulting
reaction mixture was shielded from light and stirred at room
temperature for 15 min. The reaction mixture was then partitioned
between saturated aqueous NaHCO3 and CHCl3, and the aqueous
layer was further extracted with CHCl3 (20 mL). The combined
CHCl3 solutions were washed with brine (20 mL) and dried over
anhydrous MgSO4. The CHCl3 was removed under vacuum, and
the residue was purified via column chromatography on silica gel
(hexanes/EtOAc, 3:1) to give 19 (20 mg, 30%) as a light-yellow
oil. 1H NMR (400 MHz, CDCl3) δ 7.15 (2H, d, J ) 8.8 Hz), 6.86
(2H, d, J ) 8.4 Hz), 5.57 (1H, s), 4.48 (1H, d, J ) 14.8 Hz), 4.08
(1H, d, J ) 14.8 Hz), 3.89 (1H, s), 3.80 (1H, s), 3.24 (1H, s), 2.33
- 2.38 (2H, m). 19F NMR (376 MHz, CDCl3) δ -119.72 (1F, s).
13C NMR (CDCl3, 100.63 MHz) δ 177.01, 173.97, 159.20, 129.68,
128.27, 114.11, 105.37 (d, J ) 7.3 Hz), 62.64, 57.16, 55.33, 49.62,
47.12. MS m/z (EI, DCM): 247.1 (M)+, 163.1, 121.1 (PMB)+.
HRMS calcd for C14H14O2NF (M)+: 247.1003. Found: 247.1003.
(1S,4R)-6-Fluoro-2-azabicyclo[2.2.1]hept-5-en-3-one (20). This
compound was synthesized from 19 using general procedure II (see
All reagents were purchased from Aldrich Chemical Co. and
were used without further purification except anhydrous ether and
tetrahydrofuran (THF), which were distilled from sodium metal
under nitrogen and anhydrous dichloromethane, which was distilled
from calcium hydride.
(1S,4R)-6-Iodo-2-(4-methoxybenzyl)-2-azabicyclo[2.2.1]hept-
5-en-3-one (17). Compound 16 (0.246 g, 1.0 mmol)29 was dissolved
in anhydrous ethanol (5 mL) and was added via syringe to a solution
of hydrazine hydrate (51% of hydrazine, 1.02 mL, 21.0 mmol) and
Et3N (2.18 mL, 15.7 mmol) in anhydrous ethanol (5 mL) while
stirring. The resulting colorless solution was heated to reflux and
stirred under argon for 1 h. The reaction mixture was then
evaporated under vacuum to give the crude hydrazone product as
a colorless oil, which was used directly in the next step without
further purification. I2 (0.508 g, 2.0 mmol) was dissolved in
anhydrous benzene (6 mL) and was added dropwise into a solution
of the above hydrazone and Et3N (1.1 mL, 8.0 mmol) in anhydrous
benzene (5 mL) with stirring at room temperature. The resulting
brown suspension was stirred at room temperature under argon for
1.5 h. Water (20 mL) was added, and the resulting two layers were
separated; the aqueous layer was further extracted with ether (20
mL × 3). The combined organic layers were washed with aqueous
HCl (0.5 N, 10 mL), water (10 mL), saturated aqueous NaHCO3
(20 mL), and brine (20 mL × 2) and dried over MgSO4. The solvent
was removed under vacuum, and the resulting crude product was
purified via column chromatography on silica gel (hexanes/EtOAc,
9:1) to give the diiodo intermediate as a light-brown oil, which
was dissolved in anhydrous ether and treated by dropwise addition
1
below) in a 55% yield. H NMR (400.168 MHz, CDCl3) δ 6.41
(1H, s), 5.59 (1H, s), 4.12 (1H, s), 3.10 (1H, s), 2.44 (2H, s). 19F
NMR (376.492 MHz, CDCl3) δ -120.59 (1F, s). 13C NMR (CDCl3,
125.614 MHz) δ 184.95, 175.63 (d, JFC ) 304.9 Hz), 105.88, 59.52,
58.49, 49.45. MS m/z (CI, DCM): 128.1 (M + 1)+, 85.3 (M -
CON)+. HRMS calcd for C6H7ONF: 128.0506. Found: 128.0511.
(1R,4S)-4-Amino-3-fluorocyclopent-2-enecarboxylic Acid (5).
This compound was synthesized from 20 using general procedure
III in a yield of 64% as a light-yellow solid, mp 201.0-202.5 °C.
1H NMR (D2O, 499.511 MHz) δ 5.68 (1H, s), 4.45 (1H, d, J )
2.9 Hz), 3.66 (1H, d, J ) 4.0 Hz), 2.83 (1H, dt, J ) 14.5 Hz, 9.0
Hz), 2.20 (1H, dt, J ) 14.5 Hz, 4.5 Hz). 19F NMR (D2O, 376.493
MHz) δ -128.97 (1F, d, J ) 4.5 Hz). 13C NMR (D2O, 125.614
t
MHz) δ 177.18, 157.61 (d, JFC ) 280.2 Hz), 109.21 (d, JFC
)
of two equiv of BuOK at room temperature; the resulting brown
12.1 Hz), 51.37 (d, JFC ) 22.2 Hz), 42.57 (d, JFC ) 8.2 Hz), 29.32
(d, JFC ) 4.0 Hz). MS m/z (EI, MeOH): 145.1 (M)+, 126.1, 100.1
(M - CO2H)+. HRMS calcd for C6H8O2NF: 145.0534. Found:
145.0530. Anal. (C6H8O2NF‚0.5H2O) C, H, N.
suspension was stirred overnight. Water was added, and the two
layers were separated. The aqueous layer was further extracted with
ether, and the combined ether solutions were washed with brine
and dried over anhydrous MgSO4. Ether was removed under
vacuum, and the resulting crude product was purified via column
chromatography on silica gel (hexanes/EtOAc, 9:1) to give 17 as
(1S,4S,6SR)-6-Hydroxy-6-trifluoromethyl-2-(4′-methoxybenzyl)-
azabicyclo[2.2.1]heptan-3-one (21a). To a solution of 16 (0.123
g, 0.5 mmol) in dry THF (4 mL) was added TMSCF3 (0.107 mg,
0.75 mmol). Tetrabutylammonium fluoride (TBAF) (1.0 M solution
in THF, 50 µL, 0.05 mmol) was added dropwise. The resulting
mixture was stirred under nitrogen for 2 h and was concentrated
under vacuum. The residue was purified by column chromatography
(hexanes/EtOAc, 2:1 to 1:1), giving 21a (0.149 g, 94%) as a white
1
a light-green oil (213 mg, 60%). H NMR (400 MHz, CDCl3) δ
7.19 (2H, d, J ) 8.5 Hz), 6.96 (1H, s), 6.89 (2H, d, J ) 8.5 Hz),
4.53 (1H, d, J ) 14.5 Hz), 4.06 (1H, s), 4.01 (1H, d, J ) 14.5 Hz),
3.82 (3H, s), 3.36 (1H, s), 2.31 (1H, d, J ) 7.5 Hz), 2.22 (1H, d,
J ) 7.5 Hz). 13C NMR (100 MHz, CDCl3) δ 178.50, 159.36,
144.04, 129.84, 128.73, 114.32, 98.91, 71.65, 58.13, 56.33, 55.38,
46.83. MS m/z (ESI, MeOH): 378 (M + Na)+. HRMS calcd for
C14H14O2NI: 355.0064. Found: 355.0057.
1
solid. H NMR (400 MHz, CDCl3) δ 7.19 (2H, d, J ) 8.4 Hz),
6.87 (2H, d, J ) 8.4 Hz), 5.00 (1H, d, J ) 14.8 Hz), 4.56 (1H, br.

γ-Aminobutyric Acid Aminotransferase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7409
s), 4.04 (1H, d, J ) 14.4 Hz), 3.81 (3H, s), 3.74 (1H, s), 2.87 (1H,
s), 2.33 (1H, dd, J ) 14.0 Hz, 3.6 Hz), 1.80-1.91 (3H, m). 19F
NMR (376 MHz, CDCl3) δ -78.19 (3F, s). MS m/z (EI, DCM):
316.1 (M + 1)+, 315.1 (M)+, 121.1 (CH3OC6H4CH2)+. HRMS
calcd for C15H16O3NF3: 315.1070. Found: 315.1077.
(M + 1)+, 163.1 (M - PMBN)+, 121.0 (PMB)+. HRMS calcd for
C15H14O2NF3: 297.0971. Found: 297.0970.
(1S,4R)-6-Trifluoromethyl-2-azabicyclo[2.2.1]hept-5-en-3-
one (24a). General Procedure II. Ceric ammonium nitrate (CAN;
1.38 g, 2.525 mmol) in water (3 mL) was added to a solution of
22a (150 mg, 0.505 mmol) in acetonitrile (6 mL). The resulting
red solution was stirred at room temperature for 1.5 h. The reaction
mixture was diluted with ethyl acetate, washed with saturated
aqueous NaHCO3 and then brine, and dried over anhydrous MgSO4.
The solution was concentrated under vacuum, and the residue was
purified via column chromatography on silica gel (hexanes/EtOAc,
(1S,4S,6SR)-6-Hydroxyl-6-pentafluoroethyl-2-(4′-methoxy-
benzyl)azabicyclo[2.2.1]heptan-3-one (21b). This compound was
1
synthesized by the same method as for 21a. H NMR (400 MHz,
CDCl3) δ 7.17 (2H, d, J ) 8.8 Hz), 6.87 (2H, d, J ) 7.6 Hz), 4.98
(1H, d, J ) 15.2 Hz), 4.97 (1H, s), 4.01 (1H, d, J ) 14.8 Hz), 3.86
(1H, s), 3.80 (3H, s), 2.84 (1H, s), 2.42 (1H, d, J ) 13.6 Hz),
1.78-1.89 (3H, m). 19F NMR (376 MHz, CDCl3) δ -79.02 (3F,
s), -120.08 (2F, ab, J ) 277.5 Hz). MS m/z (EI, DCM): 366.1
(M + 1)+, 365.1 (M)+, 121.1 (CH3OC6H4CH2)+. HRMS calcd for
C16H16O3NF5: 365.1050. Found: 365.1045.
1
1:1) to give 24a (54 mg, 61%) as a white solid. H NMR (500
MHz, CDCl3) δ 7.07 (1H, s), 6.15 (1H, br. s), 4.51 (1H, s), 3.42
(1H, s), 2.54 (1H, d, J ) 8.0 Hz), 2.43 (1H, d, J ) 8.0 Hz). 19F
NMR (376 MHz, CDCl3) δ -66.42 (3F, s). 13C NMR (CDCl3, 125.6
MHz) δ 182.3, 144.3 (m), 141.0 (q, JFC ) 5.5 Hz), 122.4 (q, JFC
) 268.2 Hz), 60.1, 58.6, 53.7. MS m/z (CI, MeOH): 178.3 (M +
1)+, 115.2. HRMS calcd for C7H7ONF3 (M + H): 178.0474.
Found: 178.0481.
(1S,4R,6S)-Toluene-4-sulfonic Acid 2-(4-Methoxybenzyl)-3-
oxo-6-trifluoromethyl-2-azabicyclo[2.2.1]hept-6-yl Ester (23a).
NaH in mineral oil (14 mg, 60%, 0.3 mmol) was washed with
anhydrous pentane, suspended in dry ether (3 mL), and cooled to
0 °C in an ice bath. Compound 21a (52 mg, 0.16 mmol) in dry
ether (1 mL) was then added dropwise via syringe. The resulting
white suspension was stirred at 0 °C for 15 min under argon
followed by addition of TsCl (64 mg, 0.3 mmol) in dry ether (1
mL). The resulting white suspension was allowed to slowly warm
up to room temperature and was stirred under argon for 16 h. Water
was added, and the two layers were separated. The aqueous layer
was further extracted with ether (10 mL × 3), and the combined
ether solutions were washed with brine (10 mL × 2) and
concentrated under vacuum. The residue was separated by flash
column chromatography on silica gel (hexanes/EtOAc, 1:1) to give
(1R,4S)-4-Amino-3-trifluoromethylcyclopent-2-enecarboxyl-
ic Acid (6). General Procedure III. Compound 24a (44 mg, 0.249
mmol) was dissolved in 4 N aqueous HCl (5 mL), and the resulting
clear solution was heated to 70-75 °C and stirred for 40-45 min.
The resulting colorless clear solution was extracted with EtOAc (3
mL × 2) and evaporated to dryness under high vacuum to give the
crude product, which was further purified via ion exchange
chromatography (0.15 N aqueous HCl as eluant) to give 6 as a
1
white solid (49 mg, 85%), mp 195.0-196.0 °C. H NMR (D2O,
400.169 MHz) δ 6.96 (1H, s), 4.69 (1H, s), 3.92 (1H, s), 2.87-
2.95 (1H, m), 2.29-2.35 (1H, m). 19F NMR (D2O, 376.493 MHz)
δ -64.35 (3F, s). 13C NMR (D2O, 125.614 MHz) δ 175.12, 142.54
1
23a (58 mg, 79%) as a white solid. H NMR (500 MHz, CDCl3)
(q, JFC ) 4.6 Hz), 130.82 (q, JFC ) 33.7 Hz), 121.62 (q, JFC
)
δ 7.82 (2H, d, J ) 8.0 Hz), 7.40 (2H, d, J ) 6.0 Hz), 7.04 (2H, d,
J ) 6.4 Hz), 6.82 (2H, d, J ) 6.4 Hz), 4.75 (1H, d, J ) 12.0 Hz),
3.84 (1H, s), 3.78 (3H, s), 3.43 (1H, d, J ) 12.0 Hz), 3.09 (1H, d,
J ) 12.0 Hz), 2.96 (1H, s), 2.47 (3H, s), 2.40 (1H, dd, J ) 12.4
Hz, 2.8 Hz), 1.81-1.84 (2H, m). 19F NMR (376 MHz, CDCl3) δ
-74.41 (3F, s). 13C NMR (CDCl3, 125.6 MHz) δ 175.17, 159.24,
145.72, 134.22, 130.13, 129.54, 128.30, 127.76, 123.16 (q), 114.21,
63.32, 55.31, 45.83, 45.03, 39.13, 31.11, 21.80.
269.7 Hz), 53.64, 48.80, 32.11. MS m/z (EI, MeOH): 196.1 (M)+,
179.1 (M - OH)+, 159.1. HRMS calcd for C7H9O2NF3: 196.0580.
Found: 196.0576. Anal. (C7H9O2NF3) C, H, N.
(1S,4R)-6-Pentafluoroethyl-2-(4-methoxybenzyl)-2-azabicyclo-
[2.2.1]hept-5-en-3-one (22b). A 5 mL screw-capped Pyrex tube
was charged with CuI (19 mg, 0.10 mmol), KF (14.5 mg, 0.25
mmol), and a stirring bar. The tube was dried with a heat gun under
vacuum for 20 min and filled with argon. Compound 17 (18 mg,
0.05 mmol) in anhydrous NMP (1 mL) was added via syringe to
the above reaction tube followed by TMSC2F5 (48 mg, 0.25 mmol)
in anhydrous DMF (1 mL). The resulting suspension was heated
to 80 °C and stirred vigorously for 48 h. The reaction mixture was
then evaporated under high vacuum, and the residue was purified
via column chromatography on silica gel (hexanes/EtOAc, 3:1) to
(1S,4R,6S)-Toluene-4-sulfonic Acid 2-(4-Methoxybenzyl)-3-
oxo-6-pentafluoroethyl-2-azabicyclo[2.2.1]hept-6-yl Ester (23b).
This compound was synthesized from 21b similar to the synthesis
1
of 23a. H NMR (400 MHz, CDCl3) δ 7.84 (2H, d, J ) 8.0 Hz),
7.40 (2H, d, J ) 8.8 Hz), 7.04 (2H, d, J ) 8.4 Hz), 6.83 (2H, d,
J ) 8.0 Hz), 4.95 (1H, d, J ) 15.6 Hz), 3.90 (1H, s), 3.78 (3H, s),
3.54 (1H, d, J ) 14.8 Hz), 3.29 (1H, d, J ) 15.6 Hz), 2.96 (1H, s),
2.48-2.51 (4H, m), 1.85 (1H, d, J ) 11.2 Hz), 1.79 (1H, d,
J ) 10.4 Hz). 19F NMR (376 MHz, CDCl3) δ -78.99 (3F, s),
-116.41 (2F, ab). 13C NMR (CDCl3, 125.6 MHz) δ 174.98, 159.22,
145.86, 133.85, 130.04, 129.62, 127.98, 114.21, 63.21, 55.33,
46.13, 44.59, 30.77, 21.87. MS m/z (EI, MeOH): 519.0 (M)+, 520.0
(M + 1)+, 365 (M - CH3C6H4SO2)+, 121.0. HRMS calcd for
C23H22O5NF5S: 519.183. Found: 519.1135.
1
give 22b (10 mg, 57%) as a light-yellow oil. H NMR (400.168
MHz, CDCl3) δ 7.17-7.19 (3H, m), 6.89 (2H, d, J ) 8.8 Hz),
4.80 (1H, d, J ) 15.2 Hz), 4.26 (1H, s), 3.82 (3H, s), 3.60 (1H, s),
3.52 (1H, d, J ) 14.4 Hz), 2.40 (1H, d, J ) 6.4 Hz), 2.32 (1H, d,
J ) 8.0 Hz). 19F NMR (376.49 MHz, CDCl3) δ -84.12 (3F, s),
-113.67 (2F, ab, Jab ) 279.4 Hz). 13C NMR (CDCl3, 125.6 MHz)
δ 177.59, 159.30, 144.14 (t, JCF ) 8.0 Hz), 143.03 (t, JCF ) 26.0
Hz), 129.51, 128.21, 118.81 (dt, JCF ) 285.4 Hz, 37.8 Hz), 114.24,
61.90, 59.79, 55.32, 54.54, 46.64. MS m/z (EI, DCM): 347.1 (M)+,
121.0 (PMB)+. HRMS calcd for C16H14O2NF5: 347.0939. Found:
347.0933.
(1S,4R)-6-Trifluoromethyl-2-(4-methoxybenzyl)-2-azabicyclo-
[2.2.1]hept-5-en-3-one (22a). General Procedure I. Methyl fluo-
rosulfonyldifluoroacetate (MFSDA; 48 mg, 0.25 mmol) in anhy-
drous DMF (1 mL) was added dropwise via syringe to a suspension
of 17 (35.5 mg, 0.1 mmol) and CuI (23 mg, 0.12 mmol) in
anhydrous DMF (2 mL) and HMPA (1 mL) at 75 °C under argon
over a period of 1 h. The resulting suspension was stirred at 75 °C
under argon for 72 h and evaporated under high vacuum. The
residue was purified via column chromatography on silica gel
(hexanes/EtOAc, 3:1) to give 22a (22.1 mg, 75%) as a light-brown
oil. 1H NMR (500 MHz, CDCl3) δ 7.18 (2H, d, J ) 8.5 Hz), 7.11
(1H, s), 6.89 (2H, d, J ) 8.5 Hz), 4.77 (1H, d, J ) 15.0 Hz), 4.23
(1H, s), 3.82 (3H, s), 3.56 (1H, s), 3.54 (1H, d, J ) 15.0 Hz), 2.40
(1H, d, J ) 8.0 Hz), 2.32 (1H, d, J ) 8.0 Hz). 19F NMR (376.49
MHz, CDCl3) δ -64.69 (3F, s). 13C NMR (CDCl3, 125.6 MHz) δ
178.04, 159.30, 143.97 (q, JCF ) 36.6 Hz), 141.52 (q, JCF ) 5.4
Hz), 129.54, 128.26, 122.44 (q, JCF ) 267.8 Hz), 114.25, 61.35,
59.31, 55.35, 54.06, 46.81. MS m/z (EI, MeOH): 297.1 (M)+, 298.1
(1S,4R)-6-Pentafluoroethyl-2-azabicyclo[2.2.1]hept-5-en-3-
one (24b) was synthesized from 22b in a yield of 74% using general
1
procedure II. H NMR (500 MHz, CDCl3) δ 7.14 (1H, s), 6.64
(1H, br. s), 4.52 (1H, s), 3.43 (1H, s), 2.52 (1H, d, J ) 7.0 Hz),
2.41 (1H, d, J ) 7.0 Hz). 19F NMR (376 MHz, CDCl3) δ -84.45
(3F, s), -116.41 (2F, ab, Jab ) 276.3 Hz, ∆V ) 268.h Hz). 13C
NMR (CDCl3, 125.6 MHz) δ 182.28, 143.40-143.78 (m, 2C),
111.80-122.15 (m, 2C, C2F5), 60.72, 59.23, 54.13. MS m/z (CI,
DCM): 228.0 (M + 1)+, 185.3 (M - CON)+, 165.3 (M - CONH
- F)+. HRMS calcd for C16H14O2NF5: 347.0939. Found: 347.0933.
(1R,4S)-4-Amino-3-pentafluoroethylcyclopent-2-enecarboxy-
lic acid (7) was synthesized using general procedure III from 24b
as a white solid in a yield of 82%, mp 187.0-188.5 °C. 1H NMR
(D2O, 499.511 MHz) δ 7.00 (1H, s), 4.71 (1H, d, J ) 3.5 Hz),

7410 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Lu and SilVerman
(q, JFC ) 5.5 Hz), 122.93 (q, JFC ) 270.1 Hz), 110.02 (q, JFC
34.8 Hz), 56.67, 43.58, 41.49, 32.23. MS m/z (EI, DCM): 192 (M
+ 1)+, 191 (M)+, 79.1. HRMS calcd for C8H8ONF3: 191.0553.
Found: 191.0559.
(E)-(1S,4R)-6-(2,2,2-Trifluoroethylidene)-2-azabicyclo[2.2.1]-
heptan-3-one (27b) was synthesized in a 74% yield from 26b using
general procedure II. 1H NMR (400 MHz, CDCl3) δ 6.57 (1H, s),
5.77 (1H, d, J ) 7.6 Hz), 4.13 (1H, s), 2.87 (1H, s), 2.65 (1H, d,
J ) 16.0 Hz), 2.48 (1H, d, J ) 16.8 Hz), 2.14 (1H, d, J ) 9.6 Hz),
1.62 (1H, d, J ) 9.6 Hz). 19F NMR (376 MHz, CDCl3) δ -61.08
(3F, m). 13C NMR (CDCl3, 125.6 MHz) δ 180.27, 151.69 (q, JFC
) 4.6 Hz), 123.20 (q, JFC ) 270.1 Hz), 110.82 (q, JFC ) 35.7 Hz),
60.10, 44.60, 41.52, 30.53. MS m/z (EI, DCM): 192 (M + 1)+,
191 (M)+, 163 (M - CO)+, 79. HRMS calcd for C8H8ONF3:
191.0553. Found: 191.0553.
(1S,3S)-(Z)-3-Amino-4-(2,2,2-trifluoroethylidene)cyclopen-
tanecarboxylic acid (8) was synthesized in a 66% yield as a white
solid from 27a using general procedure III; mp 212.0-214.0 °C.
1H NMR (D2O, 499.511 MHz) δ 5.97 (1H, q, J ) 7.5 Hz), 4.37
(1H, s), 3.09-3.16 (2H, m), 2.92-2.95 (1H, m), 2.56-2.62 (1H,
m), 1.98 (1H, q, J ) 12.5 Hz). 19F NMR (D2O, 376.493 MHz) δ
-60.78 (3F, d, JHF ) 6.0 Hz). 13C NMR (D2O, 125.614 MHz) δ
177.93, 150.92 (q, JFC ) 5.0 Hz), 123.11 (q, JFC ) 270.2 Hz),
114.77 (q, JFC ) 35.3 Hz), 53.76, 40.69, 32.95, 32.43. MS m/z
(EI, MeOH): 209.1 (M)+, 208.1 (M - 1)+, 189.1 (M - H2O -
2)+, 164.1 (M - CO2H)+. HRMS calcd for C8H10O2NF3: 209.0658.
Found: 277.0656. Anal. (C8H10O2NF3‚0.25H2O) C, H, N.
(1S,3S)-(Z)-3-Amino-4-(2,2,2-trifluoroethylidene)cyclopen-
tanecarboxylic acid (9) was synthesized from 27b in a 79% yield
as a white solid using general procedure III, mp 206.0-207.5 °C.
1H NMR (D2O, 499.749 MHz) δ 6.09 (1H, qd, J ) 9.0 Hz, 1.5
Hz), 4.65 (1H, s), 3.02-3.09 (1H, m), 2.91-2.92 (2H, m), 2.57-
2.63 (1H, m), 2.13-2.18 (1H, m). 19F NMR (D2O, 376.493 MHz)
δ -60.37 (3F, d, JHF ) 7.9 Hz). 13C NMR (D2O, 125.673 MHz) δ
178.64, 149.42 (q, JFC ) 5.4 Hz), 120.43 (q, JFC ) 269.3 Hz),
116.73 (q, JFC ) 34.3 Hz), 50.15, 40.74, 37.00, 34.44. MS m/z
(EI, MeOH): 210.1 (M + 1)+, 209.1 (M)+, 191.1 (M - H2O)+,
164.1 (M - CO2H)+. HRMS calcd for C8H10O2NF3: 209.0664.
Found: 277.0675. Anal. (C8H10O2NF3‚0.25H2O) C, H, N.
(1S,4R)-6-Dibromomethylene-2-(4-methoxybenzyl)-2-azabi-
cyclo[2.2.1]heptan-3-one (28). Compound 16 (0.246 g, 1.0 mmol),
carbon tetrabromide (0.498 g, 1.5 mmol), and triphenylphosphine
(0.787 g, 3.0 mmol) were dissolved in anhydrous toluene (10 mL),
and the resulting light-yellow suspension was stirred while refluxing
under argon for 22 h. The reaction mixture was then filtered, washed
with benzene, and evaporated to give the crude product, which was
purified via column chromatography on silica gel (hexanes/EtOAc,
1:1) to give 28 (377 mg, 94%) as a light-yellow oil. 1H NMR (500
MHz, CDCl3) δ 7.21 (2H, m), 6.85 (2H, m), 4.72 (1H, d, J ) 12.5
Hz), 4.20 (1H, s), 3.73-3.78 (4H, m), 3.03 (1H, s), 2.45 (1H, d, J
) 16.5 Hz), 2.29 (1H, d, J ) 16.5 Hz), 2.02 (1H, s), 1.63 (1H, s).
13C NMR (126 MHz, CDCl3) δ 177.41, 159.15, 145.83, 129.45,
128.51, 114.04, 81.58, 64.27, 55.28, 45.66, 44.49, 40.84, 36.61.
MS m/z (ESI, MeOH): 399.9 (M)+, 401.9 (M + 2)+, 404 (M +
4)+, 322.0 (M - Br)+, 293.9 (M - CH3OC6H4)+. HRMS calcd for
C15H15O2NBr2: 398.9464. Found: 398.9460.
3.92 (1H, s), 2.85-2.91 (1H, m), 2.29-2.33 (1H, m). 19F NMR
(D2O, 376.493 MHz) δ -84.60 (3F, t, J ) 18.8 Hz), -113.07 (2F,
ab, J ) 283.9 Hz, ∆ ) 62.5 Hz). 13C NMR (D2O, 125.614 MHz)
δ 174.89, 145.37 (t, JFC ) 7.2 Hz), 129.63 (t, JFC ) 25.0 Hz),
109.70-119.59 (2C, m), 54.61, 49.27, 32.10. MS m/z (EI,
MeOH): 246.1 (M + 1)+, 245.1 (M)+, 200.1 (M - CO2H)+, 176.1.
HRMS calcd for C8H8O2NF5: 245.0470. Found: 245.0475. Anal.
(C8H8O2NF5‚1.5H2O) C, H, N.
)
(1S,4R)-6-Bromomethylene-2-(4-methoxybenzyl)-2-azabicyclo-
[2.2.1]heptan-3-one (25). (Bromomethyl)triphenylphosphonium
bromide (0.567 g, 1.3 mmol) was suspended in dry THF (4 mL),
t
cooled to -78 °C, and then treated by addition of BuOK (1.0 M
in THF, 1.2 mL, 1.2 mmol). The resulting brown suspension was
stirred at -78 °C for 1.5 h and then treated with 16 (0.246 g, 1.0
mmol) in dry THF (1 mL). The resulting light-brown mixture was
allowed to gradually warm to room temperature and was stirred
overnight. The reaction was quenched via addition of water, and
the mixture was extracted with dichloromethane (10 mL × 3). The
combined organic layers were washed with brine (20 mL × 2) and
dried over anhydrous MgSO4. The solvent was removed under
vacuum, and the residue was purified via column chromatography
on silica gel (hexanes/EtOAc, 1:1) to give 25a (Z-isomer, 100 mg,
31%) as a light-yellow oil and 25b (E-isomer, 131 mg, 41%) as a
white solid.
1
25a: H NMR (500 MHz, CDCl3) δ 7.26 (2H, d, J ) 8.0 Hz),
6.88 (2H, d, J ) 8.0 Hz), 6.01 (1H, s), 4.76 (1H, d, J ) 15.0 Hz),
4.35 (1H, s), 3.81 (3H, s), 3.70 (1H, d, J ) 15.0 Hz), 3.02 (1H, s),
2.47 (1H, d, J ) 16.0 Hz), 2.31 (1H, d, J ) 15.5 Hz), 1.97 (1H, d,
J ) 9.5 Hz), 1.54 (1H, d, J ) 10.0 Hz). 13C NMR (126 MHz,
CDCl3) δ 177.51, 159.15, 143.88, 129.64, 128.94, 114.01, 97.82,
61.88, 55.31, 45.81, 44.36, 39.78, 32.77. MS m/z (ESI, MeOH):
344.6 (M + Na)+, 346.5 (M + 2 + Na)+. HRMS calcd for
C15H16O2NBr: 321.0359. Found: 321.0359.
1
25b: H NMR (500 MHz, CDCl3) δ 7.14 (2H, d, J ) 8.5 Hz),
6.86 (2H, d, J ) 8.5 Hz), 6.10 (1H, s), 4.62 (1H, d, J ) 15.0 Hz),
3.86 (1H, s), 3.80 (3H, s), 3.79 (1H, d, J ) 15.0 Hz), 2.97 (1H, s),
2.43 (1H, dt, J ) 16.5 Hz, 3.0 Hz), 2.26 (1H, d, J ) 16.5 Hz),
2.06 (1H, d, J ) 9.5 Hz), 1.60 (1H, d, J ) 9.5 Hz). 13C NMR (126
MHz, CDCl3) δ 177.79, 159.19, 144.28, 129.42, 128.53, 114.15,
100.00, 63.46, 55.33, 44.89, 43.95, 41.16, 33.49. MS m/z (ESI,
MeOH): 344.6 (M + Na)+, 346.2 (M + 2 + Na)+. HRMS calcd
for C15H16O2NBr: 321.0359. Found: 321.0359.
(Z)-(1S,4R)-2-(4-Methoxybenzyl)-6-(2,2,2-trifluoroethylidene)-
2-azabicyclo[2.2.1]heptan-3-one (26a) was synthesized in a 95%
yield from 25a using general procedure I (reaction time, 20 h). 1H
NMR (400 MHz, CDCl3) δ 7.18 (2H, d, J ) 8.8 Hz), 6.88 (2H, d,
J ) 8.4 Hz), 5.68 (1H, q, J ) 8.0 Hz), 4.81 (1H, d, J ) 14.8 Hz),
4.44 (1H, s), 3.82 (1H, s), 3.58 (1H, d, J ) 15.2 Hz), 2.95 (1H, s),
2.57 (1H, d, J ) 17.2 Hz), 2.41 (1H, d, J ) 17.2 Hz), 2.06 (1H, d,
J ) 8.8 Hz), 1.58 (1H, d, J ) 10.4 Hz). 19F NMR (376.49 MHz,
CDCl3) δ -58.02 (3F, m). MS m/z (EI, DCM): 311.1 (M)+, 312.1
(M + 1)+, 121.0 (PMB)+. HRMS calcd for C16H16O2NF3: 311.1128.
Found: 311.1133.
(E)-(1S,4R)-2-(4-Methoxybenzyl)-6-(2,2,2-trifluoroethylidene)-
2-azabicyclo[2.2.1]heptan-3-one (26b) was synthesized in 95%
yield from 25b using general procedure I (reaction time, 20 h). 1H
NMR (400 MHz, CDCl3) δ 7.15 (2H, d, J ) 8.8 Hz), 6.87 (2H, d,
J ) 8.4 Hz), 5.51 (1H, q, J ) 7.2 Hz), 4.57 (1H, d, J ) 14.4 Hz),
3.90 (1H, d, J ) 15.2 Hz), 3.86 (1H, s), 3.80 (3H, s), 2.98 (1H, s),
2.62 (1H, dq, J ) 17.2 Hz, 2.4 Hz), 2.48 (1H, dq, J ) 17.2 Hz, 2.4
Hz), 2.05 (1H, d, J ) 9.2 Hz), 1.56 (1H, d, J ) 10.0 Hz). 19F
NMR (376.49 MHz, CDCl3) δ -60.98 (3F, m). MS m/z (EI,
DCM): 311.1 (M)+, 312.1 (M + 1)+, 121.0 (PMB)+. HRMS calcd
for C16H16O2NF3: 311.1128. Found: 311.1133.
(1S,4R)-2-(4-Methoxybenzyl)-6-(2,2,2-trifluoro-1-trifluoro-
methylethylidene)-2-azabicyclo[2.2.1]heptan-3-one (29) was syn-
thesized from 28 in an 82% yield using general procedure I (50 h).
1H NMR (400 MHz, CDCl3) δ 7.15 (2H, d, J ) 8.8 Hz), 6.88 (2H,
d, J ) 8.8 Hz), 4.82 (1H, d, J ) 15.2 Hz), 4.62 (1H, s), 3.81 (3H,
s), 3.65 (1H, d, J ) 15.2 Hz), 3.03 (1H, s), 2.82 (1H, d, J )
18.4 Hz), 2.73 (1H, d, J ) 18.4 Hz), 2.12 (1H, d, J ) 10.4 Hz),
1.63 (1H, d, J ) 10.4 Hz). 19F NMR (376.49 MHz, CDCl3) δ
-57.12 to -57.03 (3F, m), -59.97 to -59.91 (3F, m). MS m/z
(EI, DCM): 379.1 (M)+, 380.1 (M + 1)+, 136.1 (PMB + NH)+,
121.0 (PMB)+. HRMS calcd for C16H16O2NF3: 379.1001. Found:
379.1003.
(Z)-(1S,4R)-6-(2,2,2-Trifluoroethylidene)-2-azabicyclo[2.2.1]-
heptan-3-one (27a) was synthesized in 74% yield from 26a using
general procedure II. 1H NMR (400 MHz, CDCl3) δ 6.30 (1H, s),
5.58 (1H, q, J ) 8.0 Hz), 4.61 (1H, s), 2.83 (1H, s), 2.55 (1H, d,
J ) 17.2 Hz), 2.36 (1H, d, J ) 16.4 Hz), 2.18 (1H, dd, J ) 10.4
Hz, 1.2 Hz), 1.63 (1H, d, J ) 9.6 Hz). 19F NMR (376 MHz, CDCl3)
δ -58.28 (3F, m). 13C NMR (CDCl3, 125.6 MHz) δ 179.92, 152.44
(1S,4R)-6-(2,2,2-Trifluoro-1-trifluoromethylethylidene)-2-aza-
bicyclo[2.2.1]heptan-3-one (30) was synthesized from 29 in a 56%
1
yield using general procedure II. H NMR (400 MHz, CDCl3) δ

γ-Aminobutyric Acid Aminotransferase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7411
5.62 (1H, s), 4.78 (1H, s), 2.96 (1H, s), 2.83 (1H, s), 2.74 (1H, s),
2.29 (1H, s), 1.71 (1H, s). 19F NMR (376 MHz, CDCl3) δ -57.32
(3F, s), -60.25 (3F, s). MS m/z (EI, DCM): 260.1 (M + 1)+, 259.1
(M)+, 189.1 (M - CF3)+. HRMS calcd for C9H7ONF6: 259.0426.
Found: 259.0434.
0 µL in control, 6, 8, or 9 in doubly distilled H2O, 0.2 M), and
GABA solution (0 µL (control), 17.6 and 35.2 µL). The concentra-
tions of GABA in the incubation solutions were 0 (control), 3, and
6 mM, respectively. At time intervals, aliquots (27 µL) from the
incubation solution were added to the assay solution (568 µL) with
excess succinic semialdehyde dehydrogenase (SSDH). Rates were
measured spectrophotometrically at 340 nm, and the remaining
activity (percentage) was determined against the control and plotted
(1S,3S)-3-Amino-4-(2,2,2-trifluoro-1-trifluoromethylethylidene)-
cyclopentanecarboxylic acid (10) was synthesized from 30 in a
77% yield as a white solid using general procedure III; mp 198.0-
1
versus time to determine the t1/2
.
199.5 °C. H NMR (D2O, 400.169 MHz) δ 4.92 (1H, s), 3.29-
Fluoride Ion Released During Inhibition of GABA-AT by
5-10. GABA-AT (25 µL) was incubated for 3 h with each of the
six compounds 5-10 (0.8 mM) in 50 mM potassium pyrophosphate
buffer, pH 8.5, containing 2.0 mM â-mercaptoethanol and 0.64 mM
R-ketoglutarate in a total volume of 250 µL at 25 °C. A control
containing no enzyme (25 µL of buffer was added instead) was
also run. The fluoride ion concentration (200 µL) of each sample
mixed with 1.00 mL of total ionic strength adjusting buffer (TISAB
low level), 790 µL of potassium pyrophosphate buffer, and 10 µL
of diluted standard fluoride solution (2.38 × 10-4 M), was measured
with a fluoride ion electrode.
Fluoride Ion Release during Inhibition of GABA-AT by 6
and 7 with Time. GABA-AT (120 µL) was incubated with 6 or 7
(12 µL, 0.2 M in doubly distilled water) in 50 mM potassium
pyrophosphate buffer, pH 8.5, containing 2.0 mM â-mercapto-
ethanol and 0.64 mM R-ketoglutarate in a total volume of 1.2 mL
at 25 °C. Another two samples containing no GABA-AT but serum
albumin or just potassium pyrophosphate buffer (control) were also
run. At time intervals, 200 µL of each sample was removed and
mixed with 1.00 mL of total ionic strength adjusting buffer (TISAB
low level), 790 µL of potassium pyrophosphate buffer, and 10 µL
of diluted standard fluoride solution (2.38 × 10-4 M). The fluoride
ion concentration was measured with a fluoride ion electrode.
3.34 (1H, m), 3.16-3.20 (2H, m), 2.57-2.65 (1H, m), 2.20-2.27
(1H, m). 19F NMR (D2O, 376.493 MHz) δ -59.97 (3F, q, J ) 9.0
Hz), -60.47 (3F, q, J ) 9.0 Hz). 13C NMR (D2O, 100.749 MHz)
δ 178.08, 158.14, 122.40, 119.16-119.67 (m), 52.56 (d, JFC ) 2.3
Hz), 40.73, 36.31 (q, JFC ) 3.8 Hz), 33.68. MS m/z (EI, MeOH):
278.1 (M + 1)+, 277.1 (M)+, 259.1 (M - H2O)+, 232.0 (M -
CO2H)+. HRMS calcd for C9H9O2NF6: 277.0538. Found: 277.0545.
Anal. (C9H9O2NF6‚H2O) C, H, N.
Enzyme and Assays. GABA aminotransferase was isolated from
pig brains by a known procedure.34 Succinic semialdehyde dehy-
drogenase (SSDH) was isolated from GABAse, a commercially
available mixture of SSDH and GABA aminotransferase, by
inactivation of the GABA aminotransferase with gabaculine as
described previously.35 GABA activity assays were carried out using
a modification of the coupled assay developed by Scott and
Jakoby.36 The assay solution contains 11 mM GABA, 5.3 mM
R-ketoglutarate, 1.1 mM NADP+, and 5 mM â-mercaptoethanol
in 100 mM potassium pyrophosphate, pH 8.5, and excess SSDH.
By use of this assay, the change in absorbance at 340 nm indicates
production of NADPH, which is directly proportional to the activity
of GABA aminotransferase.
Time-Dependent Inactivation of GABA Aminotransferase.
Incubation solutions (200 µL) contained enzyme (30 µL), potassium
pyrophosphate buffer (volume dependent on volume of inhibitor,
50 mM, pH 8.5), R-ketoglutarate (20 µL, 16 mM in 50 mM
potassium pyrophosphate buffer, pH 8.5), 2-mercaptoethanol (2.0
mM), and various volumes of the inhibitor solutions (6, 8, and 9,
0.2 M in doubly distilled H2O). The concentration of inhibitors
was calculated on the basis of a total volume of 200 µL. At time
intervals, aliquots (27 µL) from the incubation solution were added
to the assay solution (568 µL) with excess succinic semialdehyde
dehydrogenase (SSDH). Rates were measured spectrophotometri-
cally at 340 nm, and the logarithm of the remaining activity
(percentage) was plotted against time for each concentration to
determine the half-time. Then a secondary plot of half-time versus
the reciprocal of the inhibitor concentration was obtained to
determine the kinact and KI values.
Exhaustive Dialysis of 6, 8, and 9 Inactivated GABA-AT with
Potassium Pyrophosphate Buffer. Incubation solutions (200 µL)
contained enzyme (30 µL), potassium pyrophosphate buffer (147
µL, 50 mM, pH 8.5), R-ketoglutarate (20 µL, 16 mM in 50 mM
potassium pyrophosphate buffer, pH 8.5), 2-mercaptoethanol (2.0
mM), and the inhibitor solutions (6, 8, and 9, 3.0 µL, 0.2 M in
doubly distilled H2O). A control solution (200 µL) containing
enzyme (30 µL), potassium pyrophosphate buffer (150 µL, 50 mM,
pH 8.5), R-ketoglutarate (20 µL, 16 mM in 50 mM potassium
pyrophosphate buffer, pH 8.5), 2-mercaptoethanol (2.0 mM), and
no inhibitor solution was also prepared. The incubation solution
was allowed to stand at room temperature for about 1 h to ensure
that most of the enzyme was inactivated. The incubation solutions
and the control solution were injected into a Slide-A-Lyzer dialysis
cassette (extra strength, 10000 MWCO, 0.5-3.0 mL capacity),
respectively, and stirred in a 1 L beaker containing a stirring bar
and 800 mL of 50 mM potassium pyrophosphate buffer (pH 8.5).
The buffer was changed every 4 h, at which point an aliquot was
taken out to assay.
Acknowledgment. The authors are grateful to the National
Institutes of Health (Grant GM66132) for financial support of
this research.
Supporting Information Available: Results from combustion
analyses. This material is available free of charge via the Internet
at http://pubs.acs.org.
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