2,6-difluorophenol as a bioisostere of a carboxylic acid: Bioisosteric analogues of γ-aminobutyric acid

Article abstract of DOI:10.1021/jm980435l

3-(Aminomethyl)-2,6-difluorophenol (6) and 4-(aminomethyl)-2,6- difluorophenol (7) were synthesized in eight and four steps, respectively, starting from 2,6-difluorophenol, to test the potential of the 2,6- difluorophenol moiety to act as a lipophilic bio

Full text of DOI:10.1021/jm980435l

J . Med. Chem. 1999, 42, 329-332
329
2,6-Diflu or op h en ol a s a Bioisoster e of a Ca r boxylic Acid : Bioisoster ic
An a logu es of γ-Am in obu tyr ic Acid
J ian Qiu, Scott H. Stevenson, Michael J . O’Beirne, and Richard B. Silverman*
Departments of Chemistry and of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University,
Evanston, Illinois 60208-3113
Received J uly 27, 1998
3-(Aminomethyl)-2,6-difluorophenol (6) and 4-(aminomethyl)-2,6-difluorophenol (7) were syn-
thesized in eight and four steps, respectively, starting from 2,6-difluorophenol, to test the
potential of the 2,6-difluorophenol moiety to act as a lipophilic bioisostere of a carboxylic acid.
Compounds 6 and 7 are potential bioisosteric analogues of γ-aminobutyric acid (GABA).
Substrate studies and inhibition studies were carried out with pig brain γ-aminobutyric acid
aminotransferase; 6 and 7 are very poor substrates, but both inhibit the enzyme, indicating
that the 2,6-difluorophenol moiety appears to be able to substitute for a carboxylic acid to
increase the lipophilicity of drug candidates.
In tr od u ction
nism-based inactivator6 of GABA aminotransferase,7
thereby increasing the brain concentration of GABA,
which controls the seizure. We thought that a more
lipophilic bioisostere of the carboxylic acid moiety might
be useful in the design of GABA mimics that could be
more effective at crossing the blood-brain barrier. If
this approach is successful, it could be applied as a
bioisostere for any molecule containing a carboxylic acid
group.
Given that the pKa of phenol (9.8) drops to 7.1 by 2,6-
difluoro substitution,8 at pH 8.5, the pH optimum for
GABA aminotransferase, 2,6-difluorophenol would be
completely ionized (4) and may mimic a carboxylate ion
(5). Since fluorine atoms are nearly as small as hydrogen
γ-Aminobutyric acid (GABA) is the major inhibitory
neurotransmitter in the mammalian central nervous
system (CNS).1 The receptors to which it binds (GABAA
and GABAB receptors), as well as the enzyme which
degrades it (GABA aminotransferase), have been targets
for drug design for decades.2 The concentration of GABA
in the brain is regulated principally by the enzyme that
biosynthesizes it, L-glutamate decarboxylase, and the
enzyme that degrades it, GABA aminotransferase
(GABA-AT). When the GABA levels in the brain fall
below a threshold level, convulsions begin.3 These
convulsions can be interrupted by injecting GABA
directly into the brain,4 but this is not a practical
solution for controlling seizures. Because GABA is a
small polar and hydrophilic molecule, it does not cross
the blood-brain barrier; therefore, it cannot serve as
an effective anticonvulsant agent. Several anticonvul-
sant drugs, however, that have structures similar to
that of GABA, such as valproic acid (1), gabapentin (2),
and vigabatrin (3), but which are slightly more lipo-
but are capable of hydrogen bonding,9 they may mimic
the carboxylate carbonyl oxygen. However, fluorine is
slightly lipophilic10 as, of course, is benzene, so the 2,6-
difluorophenol moiety should be a lipophilic isostere of
a carboxylic acid. To determine the viability of 4 as a
bioisostere of a carboxylic acid for drug design, 3-(ami-
nomethyl)-2,6-difluorophenol (6) and 4-(aminomethyl)-
2,6-difluorophenol (7) were synthesized as analogues of
GABA and were tested as substrates and inhibitors of
GABA aminotransferase.
philic, do cross the blood-brain barrier (gabapentin,
however, may be actively transported via an L-system
amino acid transporter5). The mechanism of action of 1
is not clear, 2 appears to bind to the R2δ subunit of a
calcium channel,5 and 3, after crossing the blood-brain
barrier into the brain, albeit poorly, acts as a mecha-
Ch em istr y
Compound 6 was synthesized by the route shown in
Scheme 1, starting from 2,6-difluorophenol (8). Nitration
of 8 at 20 °C gave 2,6-difluoro-4-nitrophenol (9).11 The
nitrophenol 9 was heated at reflux with CH3I and
* Address correspondence to this author at the Department of
Chemistry.
10.1021/jm980435l CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/24/1998

330 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Sch em e 1. Synthesis of 6a
Brief Articles
a
(a) HNO3, CH3COOH, 15-20 °C, 96%; (b) CH3I, K2CO3, acetone, 92%; (c) H2, 10% Pd/C, 99%; (d) I2, NaHCO3, water, 64%; (e) 1.
NaNO2, H2SO4, 0 °C, 2. Cu powder, ethanol, 82%; (f) CuCN, DMF, 140 °C, 88%; (g) 1. BH3, THF, reflux, 2. 6 N HCl, reflux, 3. 48% HBr,
CH3CO2H, reflux, 4. ion-exchange chromatography, HCl, 78%.
Sch em e 2. Synthesis of 7a
reducing reagents, such as LiAlH4,18 NaBH4/CoCl2,19
and BH3/THF.16 The best results were obtained with
BH3/THF. The final product 7 was purified by an AG
50W-X12 resin column eluting with HCl solution and
then by recrystallization from ethanol/ethyl acetate.
Resu lts a n d Discu ssion
Compounds 6 and 7 were tested as substrates for
GABA aminotransferase from pig brain20 by the radio-
chemical procedure of measuring the conversion of
[14C]R-ketoglutarate to [14C]glutamate.21 Both were poor
substrates; only (0.07 ( 0.01)% conversion for 6 and (0.5
( 0.04)% conversion for 7 resulted over a 22-h incuba-
tion, too slow to determine kinetic constants.
a
(a) 1. H2, 10% Pd/C, 2. HCl, 99%; (b) 1. NaNO2, HCl, 0 °C, 2.
Both compounds, however, were competitive inhibi-
tors of GABA aminotransferase, having Ki values of 6.3
( 0.2 and 11 ( 1 mM for 6 and 7, respectively. The Km
value for GABA under these conditions is 2.5 mM, so
there is little difference in the binding of these inhibitors
as compared with GABA.
These results suggest that the 2,6-difluorophenol
moiety mimics the carboxylic acid functionality in the
case of GABA binding to GABA aminotransferase and,
therefore, acts as a lipophilic bioisostere. However, it
would be very useful to determine if these analogues
also mimic GABA in other biological systems, such as
in their ability to bind to GABAA or GABAB receptors,
to act as GABA mimetics in a functional model, for
example, to induce ion current in oocytes, or to inhibit
the GABA transporter. If they also show these activities,
then this group should be a useful addition to the
growing list of bioisosteres in the design of other enzyme
inhibitor or receptor agonist/antagonists analogues.
CuCN, KCN, 50 °C, 40%; (c) 1. BH3, THF, reflux, 2. 6 N HCl,
reflux, 71%.
K2CO3 in dry acetone.12 After workup and purification
by flash chromatography, the anisole 10 was obtained
in 92% yield. Hydrogenation of 10 afforded the methoxy-
aniline 11 in 99% yield. The aniline 11 was treated with
Br2 in acetic acid at room temperature and I2 and K2-
13
CO3 in water to obtain a monohalogenated product.
The iodination reaction gave better results since only
one product (12) was obtained, whereas the bromination
reaction gave both mono- and disubstituted products,
which increased the complexity of the purification. The
iodoaniline 12 was treated with NaNO2 and diluted H2-
SO4 solution to generate the diazonium, which was
immediately reduced14 with Cu powder in ethanol to
afford the iodoanisole 13. The corresponding nitrile (14)
was produced in 88% yield by heating 13 at 140 °C with
Cu(I)CN in DMF in a Rosenmund-von Braun reac-
tion.15 The cyano analogue (14) was reduced with BH3
16
to generate the intermediate (aminomethyl)-2,6-difluo-
roanisole, which was then treated with refluxing 48%
HBr and acetic acid to afford the desired product 6.
Compound 7 was synthesized from 9 by reduction
with hydrogen in the presence of palladium on charcoal
to give 15 quantitatively (Scheme 2). The aminophenol
15 is very sensitive to air unless it is converted to the
HCl salt. 4-Cyano-2,6-difluorophenol (16) was obtained
in a 40% yield by a Sandmeyer reaction,17 in which the
amine HCl salt (15) was treated with NaNO2 and HCl
at 0 °C to generate the diazonium salt in situ and then
treated with Cu(I)CN and KCN at 50 °C. The final
reduction of 16 to 7 was attempted using various
Exp er im en ta l Section
Gen er a l Meth od s. Optical spectra and GABA-AT assays
were recorded on a Perkin-Elmer Lambda 10 UV/Vis spectro-
photometer. NMR spectra were recorded on a Varian Gemini
300 MHz, a Varian VXR 300 MHz, or a Varian Unity plus 400
NMR spectrometer. Chemical shifts are reported as δ values
in parts per million downfield from Me4Si as the internal
standard in CDCl3. IR spectra were taken with a Bio-Rad
FTS60. An Orion Research model 701 pH meter with a general
combination electrode was used for pH measurements. Mass
spectra were obtained on a VG Instrument VG70-250SE high-
resolution spectrometer with a Maspec data system. Elemental
analyses were performed by the Department of Geological
Sciences at Northwestern University. Melting points were

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 331
taken on a Fisher-J ohns melting point apparatus and are
uncorrected. Flash column chromatography 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.
R ea gen t s. All reagents were purchased from Aldrich
Chemical Co. and used without further purification except
anhydrous ether or tetrahydrofuran, which was distilled over
sodium metal under nitrogen, and anhydrous dichloromethane,
which was distilled over calcium hydride.
prewashed with ether) and ethanol (15 mL) were added to the
above reaction mixture. The resultant mixture was stirred and
heated at 75 °C for 1 h and then at 60 °C for 2 h. After being
cooled, the resultant aqueous solution was extracted with ether
(3 × 20 mL). The combined organic extracts were washed with
brine (25 mL) and dried over MgSO4. After removal of the
solvent, the residue was purified by flash chromatography on
silica gel eluting with hexane to afford the iodoanisole 13 (0.64
g, 82%) as colorless needles: mp 28-29 °C; TLC (hexanes-
ethyl acetate, 4:1) Rf 0.63; IR (CHCl3) 3010, 2946, 1652, 1575,
2,6-Diflu or o-4-n itr op h en ol (9). Following a published
procedure,11 2,6-difluorophenol (8) (5.0 g, 3.8 mmol) was
treated with fuming nitric acid (2 mL). After removal of
solvents under reduced pressure, the residue was purified by
recrystallization from hexane to give the nitrophenol 9 (6.40
g, 96%) as pale-yellow needles: mp 103-105 °C (lit.11 mp 105-
105.5 °C); TLC (hexanes-ethyl acetate, 4:1) Rf 0.15; IR (CHCl3)
3200b, 3002, 3104, 1612, 1516, 1347 cm-1; 1H NMR (CDCl3) δ
1
1487, 1453 cm-1; H NMR (CDCl3) δ 7.37 (m, 2H), 6.74 (dt, J
) 6.0, 1.6 Hz, 1H), 4.0 (s, 3H); 19F NMR (CDCl3) -106.9 (m),
-128.3 (m); 13C NMR (CDCl3) 158 (m) 157 (m), 138 (m), 132
(m), 115 (m), 111, 63 (m); EI-MS m/z 270, 229, 144, 131, 103,
77; HRMS calcd for C7H5F2IO M 269.9355, found M 269.9349.
3-Cya n o-2,6-d iflu or oa n isole (14). Compound 13 (0.37 g,
1.42 mmol) was stirred with CuCN (0.18 g, 2.03 mmol) in DMF
(15 mL) and heated to 120 °C for 18 h. Then the mixture was
heated at 140 °C for an additional 4 h. The resultant mixture
was diluted with water (40 mL) and extracted with ether (4 ×
25 mL). The combined organic phases were washed with brine
and dried over MgSO4. After removal of the solvent under
reduced pressure, the residue was purified by flash chroma-
tography on silica gel eluting with hexanes-ethyl acetate (4:
1) to afford the cyanoanisole 14 (0.2 g, 83%) as a colorless
solid: mp 41-42 °C; TLC (hexanes-ethyl acetate, 4:1) Rf 0.36;
IR (KBr disk) 3073, 2996, 2960, 2241(sharp), 1618, 1580, 1476
cm-1; 1H NMR (CD3OD) δ 7.29 (ddd, J ) 9.1, 6.6, 5.4 Hz, 1H),
7.10 (ddd, J ) 9.9, 9.0, 1.6 Hz, 1H), 4.0 (s, 3H); 19F NMR (CD3-
OD) -121.0 (m), -117.4 (m); 13C NMR (CDCl3) 159.8 (d, J )
256 Hz), 158.1 (d, J ) 258 Hz), 138.6 (m), 127.6 (dd, J ) 17.5,
9.1 Hz), 114.6 (m), 99.8 (d, J ) 13.7 Hz), 63.1 (m); EI-MS m/z
169, 154, 126, 100, 88, 75; HRMS calcd for C7H7F2NO M
169.0339, found 169.0332.
3-(Am in om eth yl)-2,6-diflu or oph en ol Hydr och lor ide (6).
BH3‚THF (1 M, 12 mL, 12 mmol) was added carefully to a
solution of 14 (0.18 g, 1.06 mmol) in anhydrous THF (10 mL).
The resultant solution was stirred and heated at reflux for 10
h. After the mixture had cooled, 6 N HCl was carefully added
to the solution and heating at relux was continued for 30 min.
The resultant solution was evaporated in vacuo to afford
3-(aminomethyl)-2,6-difluoroanisole hydrochloride: 1H NMR
(D2O) δ 7.18 (m, 1H), 7.10 (m, 1H), 4.2 (s, 2H), 3.9 (s, 3H).
Without purification, the 3-(aminomethyl)-2,6-difluoroani-
sole hydrochloride was added to a solution of 48% HBr (4 mL)
and HOAc (4 mL) and heated at 120 °C for 12 h. After removal
of all solvents in vacuo, the resultant residue was purified by
ion-exchange chromatography (AG 50W-X8) eluting with a
gradient between 0.1 and 3 N HCl to give the desired HCl salt
6 as a colorless solid (0.18 g, 84%): mp >173 °C dec; IR (KBr
disk) 3320b, 3075, 3019, 2927, 2811, 1627, 1612, 1548, 1494
cm-1; 1H NMR (CD3OD) δ 6.95 (m, 2H), 4.13 (s, 2H); 19F NMR
(CDCl3) -134.9, -129.5; 13C NMR (D2O) 153.5 (d, J ) 206 Hz),
151.8 (d, J ) 236 Hz), 133.8 (t, J ) 16.6 Hz) 121.6 (dd, J )
9.1, 3.8 Hz), 117.4 (d, J ) 9.9 Hz), 112.9 (dd, J ) 18.9, 3.7
Hz), 37.8 (d, J ) 3.8 Hz). Anal. (C7H8ClF2NO) C, H, N.
7.92 (m, 2H), 6.0 (s, 1H, -OH); 19F NMR (CDCl3) -131.5; 13
C
NMR (CDCl3) 151 (dd, J ) 246.5, 6.1 Hz), 140.4 (m), 109.8
(dd, J ) 17.5, 9.1 Hz), 102.5 (C1, m); EI-MS m/z 175, 159, 145,
129, 101, 81; HRMS calcd for C6H3F2NO3 M 175.0081, found
M 175.0081.
2,6-Diflu or o-4-n itr oa n isole (10). K2CO3 (3.5 g, 22.8 mmol)
was added to 9 (2.0 g, 11.4 mmol) and CH3I (4.6 g, 32.4 mmol)
in dry acetone (15 mL). The mixture was stirred and heated
at reflux for 6 h. After removal of acetone under reduced
pressure, the resultant residue was washed with ether (50
mL). The organic solution was dried over anhydrous MgSO4
and evaporated under reduced pressure. The residue was
purified by flash chromatography on silica gel eluting with
hexane/ether (4:1) to give the desired anisole 10 as a colorless
crystalline solid (2.04 g, 94%): mp 33-35 °C (lit.11 mp 33-
35.5 °C); TLC (hexanes-ethyl acetate, 4:1) Rf 0.78; IR (CHCl3)
1
3095, 3014, 2964, 1614, 1534, 1506 cm-1; H NMR (CDCl3) δ
1
7.85 (m, 2H), 4.17 (s, 3H); F NMR (CDCl3) -124.9 (m); 13C
NMR (CDCl3) 154 (dd, J ) 246.5, 6.1 Hz), 143 (t, J ) 12.9
Hz), 142.0 (m), 110 (dd, J ) 18.9, 9.8 Hz), 62.8; EI-MS m/z
189, 159, 143, 128, 113, 100, 81; HRMS calcd for C7H5F2NO3
M 189.0237, found M 189.0238.
3,5-Diflu or o-4-m eth oxya n ilin e (11). Compound 10 (2.96
g, 15.7 mmol) in ethyl acetate (15 mL) was hydrogenated over
a catalytic amount of 10% Pd/charcoal until no more hydrogen
was consumed. After removal of the catalyst by filtration, the
solvent was evaporated under reduced pressure to give the
pure aniline 11 (2.42 g, 99%) as a colorless solid: mp 72-74
°C; TLC (hexanes-ethyl acetate, 4:1) Rf 0.18; IR (CHCl3) 3095,
1
3014, 2964, 1614, 1534, 1506 cm-1; H NMR (CDCl3) δ 6.20
(m, 2H), 3.75 (s, 3H); 19F NMR (CDCl3) -129.2 (d, J ) 9.3 Hz);
13C NMR (CDCl3) 154 (dd, J ) 244, 7.5 Hz), 142 (m), 99 (dd,
J ) 17, 9.0 Hz), 62.4 (t, J ) 2.47 Hz); EI-MS m/z 159, 144,
116; HRMS calcd for C7H7F2NO M 159.0496, found M 159.0496.
3,5-Diflu or o-2-iod o-4-m et h oxya n ilin e (12). Compound
11 (0.8 g, 5.0 mmol) was stirred with iodine (1.6 g, 6.4 mmol)
and NaHCO3 (9.0 g, 9.0 mmol) in water (15 mL) at room
temperature overnight. The resultant mixture was extracted
with ethyl acetate (4 × 25 mL). The combined organic extracts
were washed with 20% Na2S2O3 (30 mL) and brine (25 mL)
and were dried over anhydrous NaSO4. After removal of
solvents, the residue was purified by flash chromatography
on a silica gel column eluting with hexanes-ethyl acetate (6:
1) to afford the iodoaniline 12 (0.90 g, 64%) as a colorless
solid: mp 50-51 °C; TLC (hexanes-ethyl acetate, 4:1) Rf 0.31;
IR (CHCl3) 3440, 3379, 3296, 2947, 1630, 1570 cm-1; 1H NMR
(CDCl3) δ 6.38 (dd, J ) 10, 1.8 Hz, 1H), 3.86 (s, 3H); 19F NMR
(CDCl3) -105.7 (d, J ) 7.8 Hz), -129.2 (t, J ) 10 Hz); 13C
NMR (CDCl3) 158 (dd, J ) 253, 6.8 Hz), 157 (dd, J ) 233, 8.3
Hz), 144.2 (dd, J ) 6.1, 12.9 Hz), 129.2 (m), 98.2 (dt, J ) 20.5,
2.3 Hz), 63.6 (d, J ) 19.7 Hz, OCH3); EI-MS m/z 285, 270, 143,
115, 88; HRMS calcd for C7H6F2INO M 284.9464, found M
284.9463.
4-Am in o-2,6-d iflu or op h en ol (15). 2,6-Difluoro-4-nitro-
phenol (9) (0.52 g, 0.30 mmol) was dissolved in ethanol and
hydrogenated over 10% Pd/charcoal catalyst (20 mg) for 2 h.
After addition of concentrated HCl (0.5 mL) and removal of
the catalyst by filtration, the residue was purified by recrys-
tallization from ethanol/ether to give the aminophenol‚HCl salt
15 as a gray solid (0.54 g, 99%): mp >230 °C dec; IR (CHCl3)
1
3200b, 3180, 3045, 2887, 1565, 1537 cm-1; H NMR (DMSO)
δ 7.55 (m, 2H); 19F NMR (CDCl3) -131.5; 13C NMR (D2O) 153.5
(dd, J ) 244, 7.6 Hz), 134.5 (t, J ) 100 Hz), 121.8 (t, J ) 122
Hz), 108.7 (dd, J ) 17.4, 9.1 Hz); EI-MS m/z 145, 124, 116,
97, 70, 36; HRMS calcd for C6H5F2NO M 145.0339, found M
145.0339.
4-Cya n o-2,6-d iflu or op h en ol (16). Sodium nitrate (0.51 g,
7.4 mmol) in water (10 mL) was added dropwise to an ice-cold
solution of 15 (1.2 g, 6.7 mmol) and concentrated HCl (3 mL)
in water (8 mL) until the resultant solution was tested for
excess nitrous acid with KI-starch paper. Then the diazotized
2,6-Diflu or o-3-iod oa n isole (13). NaNO2 (0.3 g, 4.3 mmol)
in water (5 mL) was added to a solution of 12 (0.82 g, 2.9 mmol)
and concentrated H2SO4 (1 mL) in ice-cold water (15 mL). After
the mixture was stirred for 30 min, Cu powder (0.2 g,

332 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
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(10) For example, the log P for benzene is 2.13 and for fluorobenzene
is 2.27; the log P for phenol is 1.46 and for p-fluorophenol is
1.77; the log P for phenylacetic acid is 1.41 and for o-fluorophen-
ylacetic acid is 1.50. Devillers, J .; Domine, D.; Guillon, C.;
Bintein, S.; Karcher, W. Prediction of Partition Coefficients (log
solution was carefully neutralized with sodium carbonate and
was added to a suspended solution of KCN (2.1 g, 33.5 mmol)
and CuCN (2.4 g, 26.8 mmol) in water (15 mL) at room
temperature. The resultant solution was gradually heated to
50 °C. After being stirred for 2 h, the resultant solution was
extracted with CHCl3 (4 × 25 mL). The combined organic
extracts were washed with brine, dried over sodium sulfate,
and evaporated under reduced pressure. The residue was
purified by flash chromatography on silica gel eluting with
hexane/ethyl acetate (4:1) to give the cyano product 16 (0.51
g, 40%) as a colorless solid: mp 89-92 °C; TLC (hexane-ethyl
acetate, 4:1) Rf 0.10; IR (KBr disk) 3200b, 2245, 1624, 1603,
1
1527, 1448 cm-1; H NMR (CDCl3) δ 7.28 (m, 2H); 19F NMR
(CDCl3) -132; 13C NMR (D2O) 157 (d, J ) 244 Hz), 140, 118
(m), 116 (m); EI-MS m/z 155, 154, 126, 100, 88, 75; HRMS
calcd for C7H3F2NO M 155.0183, found M 155.0195.
4-(Am in om eth yl)-2,6-diflu or oph en ol Hydr och lor ide (7).
BH3‚THF (1 M, 6.4 mL, 6.4 mmol) was added carefully to a
solution of 16 (0.25 g, 1.61 mmol) in anhydrous THF (15 mL).
The resultant solution was stirred and heated to reflux for 10
h. After the mixture had cooled, 6 N HCl was carefully added
to the solution, and heating was continued at reflux for 30 min.
The resultant solution was evaporated in vacuo. The residue
was purified by ion-excahnge chromatography (AG 50W-X8),
eluting with a gradient between 0.1 and 3 N HCl, to give the
desired (aminomethyl)phenol 7 (0.22 g, 71%) as a colorless
solid: mp >176 °C dec; IR (KBr disk) 3300b, 3100, 2733, 1610,
1574, 1534 cm-1 1H NMR (CD3OD) δ 7.07 (m, 2H) 4.02 (s,
;
2H); 19F NMR (CDCl3) -132 (m); 13C NMR (D2O) 153 (d, J )
243 Hz), 134.2 (m), 124.7 (m), 113.7 (m), 43.2. Anal. (C7H8-
ClF2NO) C, H, N.
En zym es a n d Assa ys. Pig brain GABA aminotransferase
(specific activity 3.9 units/mg), GABAse, and succinic semial-
dehyde dehydrogenase were obtained and assayed as previ-
ously described.21
P
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Deter m in a tion of Su bstr a te Activity for 6 a n d 7.
Substrate activity was determined in duplicate by a radio-
chemical assay, measuring the conversion of [U-14C]R-keto-
glutarate to [U-14C]L-glutamate, as previously described.21
Compound 6 (7.3 mM) or 7 (6.9 mM) was incubated in
potassium phosphate buffer (100 mM, pH 7.4) at 25 °C for 22
h with GABA aminotransferase (0.02 unit) in the presence of
[U-14C]R-ketoglutarate (4.7 mM, 120 µCi/mmol) and â-mer-
captoethanol (5 mM). A control experiment also was carried
out with the entire incubation mixture except substrate.
In h ibition of GABA Am in otr a n sfer a se by 6 a n d 7. The
competitive inhibition of GABA aminotransferase by 6 and 7
was carried out and analyzed as previously described.22 Pig
brain GABA aminotransferase (0.004 unit) was incubated at
25 °C with R-ketoglutarate (5 mM), NADP+ (1 mM), â-mer-
captoethanol (5.0 mM), excess succinic semialdehyde dehy-
drogenase, and varying amounts of 6, 7, and GABA in
potassium phosphate buffer (100 mM) at pH 7.4. Initial rates
were measured spectrophotometrically, and kinetic parameters
were derived from Dixon23 and Cornish-Bowden24 plots with
four different concentrations of 6 and 7 (0.0, 3.5, 5.8, and 8.2
mM) for every concentration of GABA (0.68, 1.7, and 3.5 mM).
Both analogues are competitive inhibitors of GABA amino-
transferase.
(13) Brewster, R. Q. p-Iodoaniline. Organic Syntheses; Wiley: New
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(14) Wallingford, V. H.; Krueger, P. A. m-Iodobenzoic Acid. Organic
Syntheses; Wiley: New York, 1943; Collect. Vol. II, pp 353-355.
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Halides. Chem. Rev. 1987, 87, 779-794.
(16) Brown, H. C.; Choi, Y. M.; Narasimhan, S. Improved Procedure
for Borane-Dimethyl Sulfide Reduction of Nitriles. Synthesis
1981, 605-606.
(17) Clarke, H. T.; Read, R. R. o-Tolunitrile and p-Tolunitrile. Organic
Syntheses; Wiley: New York, 1941; Collect. Vol. I, pp 514-516.
(18) Cimino, G.; Gavagnin, M.; Sodano, G.; Spinella, A.; Strazzullo,
G. Revised Structure of Bursatellin. J . Org. Chem. 1987, 52,
2301-2303.
(19) Osby, J . O.; Heinzman, S. W.; Ganem, B. Studies on the
Mechanism of Transition-Metal-Assisted Sodium-Borohydride
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1986, 108, 67-72.
(20) (a) Churchich, J . E.; Moses, U. 4-Aminobutyrate Aminotrans-
ferase. The Presence of Nonequivalent Binding-Sites. J . Biol.
Chem. 1981, 256, 1101-1104. (b) Biase, D. D.; Barna, D.; Bossa,
F.; Pucci, P.; J ohn, R. A. Chemistry of the Inactivation of
4-Aminobutyrate Aminotransferase by the Antiepileptic Drug
Vigabatrin. J . Biol. Chem. 1991, 266, 20056-20061.
(21) Silverman, R. B.; Levy, M. A. 3-Substituted 4-Aminobutanoic
Acids: Substrates for 4-Aminobutyric Acid-R-Ketoglutaric Acid
Aminotransferase. J . Biol. Chem. 1981, 256, 11565-11568.
(22) Silverman, R. B.; Nanavati, S. M. Selective Inhibition of γ-Ami-
nobutyric acid Aminotransferase by (3R,4R), (3S,4S) and (3R,4S),
(3S,4R)-4-Amino-5-fluoro-3-phenylpentanoic Acids. J . Med. Chem.
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Ack n ow led gm en t. The authors are grateful to the
National Institutes of Health (Grant NS15703) for
financial support of this research.
Refer en ces
(23) Dixon, M. The Determination of Enzyme Inhibitor Constants.
Biochem. J . 1953, 55, 170-171.
(24) Cornish-Bowden, A. Fundamentals of Enzyme Kinetics; Portland
Press: London, 1995; pp 102-108.
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