5292
J . Med. Chem. 2003, 46, 5292-5293
Letters
Design , Syn th esis, a n d Biologica l Activity
of a Diflu or o-Su bstitu ted ,
Con for m a tion a lly Rigid Viga ba tr in
An a logu e a s a P oten t γ-Am in obu tyr ic
Acid Am in otr a n sfer a se In h ibitor
Yue Pan, J ian Qiu, and Richard B. Silverman*
Department of Chemistry, Department of Biochemistry,
Molecular Biology, and Cell Biology, and
Drug Discovery Program, Northwestern University,
Evanston, Illinois 60208-3113
F igu r e 1. Comparison of the activities of vigabatrin and 14
with GABA-AT at pH 6.5, 25 °C.
Received J uly 29, 2003
Abst r a ct : Previously it was found that a conformationally
rigid analogue (2) of the epilepsy drug vigabatrin (1) did not
inactivate γ-aminobutyric acid aminotransferase (GABA-AT).
A cyclic compound with an exocyclic double bond (6) was
synthesized and was found to inactivate GABA-AT, but only
in the absence of 2-mercaptoethanol. The corresponding dif-
luoro-substituted analogue (14) was synthesized and was
shown to be a very potent time-dependent inhibitor, even in
the presence of 2-mercaptoethanol.
incubation mixture, no inactivation occurred (data not
shown). A possible mechanism accounting for this
phenomenon is shown in Scheme 3. It is likely that 6 is
only a substrate for GABA-AT. After formation of 7, the
double bond is not reactive enough, so this intermediate
is not trapped by the enzyme, but rather is released
from the active site in the form of an R,â-unsaturated
ketone (8). In the presence of 2-mercaptoethanol, a
reactive nucleophile, 8 is trapped to form 9, giving no
inactivation of the enzyme. In the absence of 2-mercap-
toethanol, however, 8 may return to the active site of
the enzyme and become covalently attached to the
enzyme (10), leading to the enzyme’s inactivation.
According to the definition of mechanism-based enzyme
inactivators,8 6 is not a mechanism-based inactivator
because inactivation does not occur prior to the release
of the active species from the active site.
γ-Aminobutyric acid (GABA) is the major inhibitory
neurotransmitter in the mammalian central nervous
system.1 When the level of GABA in the brain falls
below a threshold level, convulsions occur.2 Compounds
that inhibit γ-aminobutyric acid aminotransferase
(GABA-AT), the enzyme that degrades GABA, exhibit
anticonvulsant activity. One of the most effective inac-
tivators of GABA-AT is the antiepilepsy drug vigabatrin
(1),3 which inactivates the enzyme by two pathways: a
Michael addition mechanism (Scheme 1, pathway a) and
an enamine mechanism (pathway b).4
Because 6 did not inactivate GABA-AT, we designed
the corresponding, more reactive, difluoro-substituted
analogue 14. As a result of fluorine’s similar size to
hydrogen and its high electronegativity, a much more
reactive intermediate than 7 is expected. Prior to its
release, this species may be sufficiently reactive to
become covalently attached to GABA-AT, leading to its
inactivation.
Previously, 2, a conformationally rigid vigabatrin
analogue, was synthesized.5 Surprisingly, 2 was not a
GABA-AT inactivator but was a very good substrate
The synthesis of 14 (Scheme 4) started from 11.7,9
A
Horner-Wadsworth-Emmons reaction with diethyl
(difluoromethyl)phosphonate inserted the 1,1-difluoro-
methylene moiety in 12, which then underwent depro-
tection with ceric ammonium nitrate to give the lactam
13. Hydrolysis of 13 gave 14.
Compound 14 was found to be a very potent GABA-
AT inactivator, even in the presence of 2 mM 2-mer-
captoethanol (Figure 1). Because of its high potency,
kinact and KI values could not be determined accurately
under optimal conditions (pH 8.5, 25 °C), where the
enzyme exhibits maximum activity. When the concen-
tration of 14 was comparable to that of the enzyme, the
with a specificity constant almost five times greater
than that of GABA. It was later determined by computer
modeling6 that the endocyclic double bond is not in the
right orientation for Michael addition (pathway a,
Scheme 1), nor is it an effective enamine for enzyme
inactivation. Therefore 6, which has an exocyclic double
bond, was designed. The synthesis (Scheme 2) started
from 3.7 An addition reaction with (trimethylsilyl)-
methylmagnesium chloride followed by elimination
furnished 5. Deprotection of the benzyl group and
hydrolysis of the lactam gave the amino acid 6.
Interestingly, inactivation of GABA-AT was observed
with 6, but when 2-mercaptoethanol was added to the
k
inact/KI value for 14 was determined to be 186 times
greater than that for (S)-vigabatrin! To obtain more
accurate data, 14 and vigabatrin were compared under
nonoptimal conditions. Even at 0 °C in pH 8.5 buffer
no pseudo first-order kinetics were observed for 14. At
* Address correspondence to this author at the Department of
Chemistry. Phone: (847) 491-5653. FAX: (847) 491-7713. Email:
Agman@chem.northwestern.edu.
10.1021/jm034162s CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/11/2003
Pan, Yue
