Design, Synthesis, and Evaluation of Postulated Transient
Intermediate and Substrate Analogues as Inhibitors of
4-Hydroxyphenylpyruvate Dioxygenase
Yun-Loung Lin, Jian-Lin Huang, Chung-Shieh Wu, Hung-Ge Liu and Ding-Yah Yang*
Department of Chemistry, Tunghai University, 181, Taichung-Kang Rd. Sec. 3, Taichung, Taiwan 40704, Republic of China
Received 21 January 2002; accepted 23 April 2002
Abstract—An epoxybenzoquinone, 4-hydroxyphenoxypropionic acid, and 2-hydroxy-3-phenyl-3-butenoic acid derivatives have
been designed, synthesized, and evaluated for in vitro inhibition activity against 4-hydroxyphenylpyruvate dioxygenase (4-HPPD)
from pig liver by the spectrophotometric enol-borate method. The biological data demonstrated that neither epoxybenzoquinone
ester nor 2-hydroxy-3-phenyl-3-butenoic acid is an inhibitor of 4-HPPD. The most potent 4-HPPD inhibitor tested was 3-hydroxy-
4-phenyl-2(5H)-furanone with an IC
50
value of 0.5 mM, which may serve as a lead compound for further design of more potent
4-HPPD inhibitors. # 2002 Elsevier Science Ltd. All rights reserved.
4-Hydroxyphenylpyruvate dioxygenase (4-HPPD, EC
1.13.11.27)
1
is a non-heme Fe(II)-dependentenzyme
involved in the catabolism of tyrosine in most organ-
isms
2
as well as the biosynthesis of plastoquinones and
tocopherols in plants.
3
It catalyzes the conversion of
4-hydroxyphenylpyruvate (4-HPP, 1) and molecular
oxygen to homogentisate 2 and carbon dioxide, as
shown in Scheme 1.
Inhibition of 4-HPPD has recently become the focus of
considerable research interest because potent 4-HPPD
inhibitors could provide an alternative treatment for
life-threatening tyrosinaemia type I disease
4
and also
have the potential to serve as a new class of bleaching
herbicides for control of grass and broadleaf weeds.
5
For example, 2-benzoylcyclohexane-1,3-dione deriva-
tives, referred to as triketones,
6,7
have been found to be
competitive inhibitors of 4-HPPD with IC
50
values as
low as 40 nM. Recently, we discovered a new family of
compounds, alkanoic acid 3-oxo-cyclohex-1-enyl
esters,
8
which appear to be non-triketone type 4-HPPD
inhibitors. Evidence suggests that these 4-HPPD inhibi-
tors act as analogues
9
to the substrate 4-HPP. No tran-
sient intermediate 4-HPPD inhibitors have been
reported so far.
Although the mechanistic details of this enzyme cata-
lyzed reaction remain unclear, several lines of evidence
have been reported to support the involvement of the
arene oxide intermediate shown below.
For example, studies involving the incubation of the
unnatural substrate 3-thienylpyruvate with 4-HPPD
resulted in formation of the product 3-carboxymethyl-
3-thiolene-2-one,
10
which is best accounted for by the
proposal of an arene oxide intermediate. Furthermore,
excretion of the unusual amino acid hawkinsin
11
by
patients suffering from Hawkinsinuria, a genetic disease
attributed to production of defective 4-HPPD, also
supports the formation of a transient arene oxide inter-
mediate. Here, we report studies of the reactions of a
chemically synthesized putative transient intermediate
analogue 11 and series of substrate analogues 15a–b, 27
with 4-hydroxyphenylpyruvate dioxygenase from pig
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00291-3
Bioorganic & Medicinal Chemistry Letters 12 (2002) 1709–1713
Scheme 1.
*Corresponding author. Tel.: +886-4-2359-7613; fax: +886-4-2359-
0426; e-mail: yang@mail.thu.edu.tw