Design, synthesis, and evaluation of postulated transient intermediate and substrate analogues as inhibitors of 4-hydroxyphenylpyruvate dioxygenase

Article abstract of DOI:10.1016/S0960-894X(02)00291-3

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 IC₅₀ value of 0.5 μM, which may serve as a lead compound for further design of more potent 4-HPPD inhibitors.

Full text of DOI:10.1016/S0960-894X(02)00291-3

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1709–1713
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 IC50 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-
isms2 as well as the biosynthesis of plastoquinones and
tocopherols in plants.3 It catalyzes the conversion of
Scheme 1.
4-hydroxyphenylpyruvate (4-HPP, 1) and molecular
oxygen to homogentisate 2 and carbon dioxide, as
shown in Scheme 1.
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.
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 disease4 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 IC50 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 analogues9 to the substrate 4-HPP. No tran-
sient intermediate 4-HPPD inhibitors have been
reported so far.
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 hawkinsin11 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
*Corresponding author. Tel.: +886-4-2359-7613; fax: +886-4-2359-
0426; e-mail: yang@mail.thu.edu.tw
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00291-3

1710
Y.-L. Lin et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1709–1713
Scheme 2. Preparation of compound 11: (i) SOCl2, CH3OH; (ii) LiAlH4, Et2O; (iii) SOCl2, CHCl3; (iv) NaCN, DMSO; (v) 10% NaOH, EtOH;
(vi) CH2N2, Et2O; (vii) AgO, 6 N HNO3, acetone; (viii) H2O2, NaHCO3, CH3OH.
liver as part of our continuing effort to develop potent
inhibitors for this enzyme. The aim of incorporation of
a 1,4-diketo moiety on the ring system of epoxy-
benzoquinone ester 11 is to stabilize the arene oxide
structure and to keep the six-membered ring nearly pla-
nar as it is in the proposed arene oxide intermediate. In
the case of 4-hydroxyphenoxypropionic acid derivatives
15a–b, t heb-carbon atom of the natural substrate
4-HPP was replaced by oxygen and the a-keto fuction-
ality was modified to either a hydrogen atom or methyl
group. We expected the resulting analogues, shown
below, although structurally similar to 4-HPP, would
not be able to serve as substrates for 4-HPPD.
The synthetic route used to prepare compound 11 is
outlined in Scheme 2. Commercially available tri-
methoxybenzoic acid 3 was first methylated with thionyl
chloride in methanol, then reduced by LiAlH4 to give
the primary alcohol 5 with high yield. The alcohol 5 was
converted to the corresponding chloride 6, followed by
reacting with sodium cyanide in DMSO. The resulting
nitrile 7 was hydrolyzed under basic conditions to pro-
duce acid 8 with the desired carbon skeleton. Direct
oxidation of 8 with silver oxide resulted in decomposi-
tion of the starting material. This observation prompted
us to protect the acid functionality of 8 with diazo-
methane to furnish the methyl ester 9. Subsequentoxi-
dation13 of the benzene moiety of 9 with silver(II) oxide
and 6 N nitric acid to benzoquione derivative 10, fol-
lowed by selective epoxidation with hydrogen peroxide
in the presence of sodium bicarbonate afforded the tar-
getepoxybenzoquinone ester 11 with an overall yield of
15%.
An effective route for preparation of 4-hydroxy-
phenoxyacetic acid 15a and 2-(4-hydroxyphenoxy)-
propionic acid 15b is depicted in Scheme 3. It
commenced with Elbs persulfate oxidation14 of phenol
with potassium persulfate in aqueous solution to
4-hydroxyphenyl sulfate salt 12. The sulfate salt 12 was
then treated with proper 2-chloroacid ester in the pre-
sence of potassium carbonate in DMF/xylene to give
the 4-substituted phenyl sulfate salts 13a–c.15 Further
removal of the sulfate group by boiling 13a–c with ace-
tic acid afforded 4-hydroxyphenoxyacid esters 14a–c
with a 50–55% combined yield. A final alkaline hydro-
lysis16 of esters 14a–c in 10% NaOH solution yielded
15a and 15b, respectively.
As for the 3-phenyl-2-oxo-3-butenoic acid 27, we intro-
duced a methylene group at the C-3 position of 4-HPP.
The design of this analogue took into account the fact
that interaction of the ortho substituent of 4-HPP ana-
logue with some active site groups may result in rota-
tion of the aromatic ring away from the enzymatic
oxygenating agent to prevent catalysis.12 Similarly,
incorporation of an extra methylene group on the side
chain of 4-HPP may alter its molecular geometry. Thus,
this substrate modification is expected to have a major
effect on binding when incubated with the enzyme
4-HPPD.

Y.-L. Lin et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1709–1713
1711
An attempt to prepare 3-phenyl-2-oxo-3-butenoic acid
27 is outlined in Scheme 4. According to this strategy,
epoxy ester 17 was obtained by Darzens17 glycidic ester
condensation of acetophenone 16 with methyl chloro-
acetate in the presence of sodium methoxide. Acid-
catalyzed ring opening of 17 in benzene produced the
a-hydroxy ester 18, which was then hydrolyzed, under
basic conditions, to give a-hydroxy acid 19 with a 32%
combined yield. Subsequent oxidation of the a-hydroxy
group to the corresponding keto functionality proved to
be problematic. All attempts to effect oxidation,
employing a variety of oxidizing reagents, failed to pro-
duce the desired a-keto acid 27; presumably due to the
propensity of the alkenylbenzene moiety to undergo side
chain oxidation. Thus, a different methodology was
applied to synthesize the compound. Epoxidation of
a-hydroxy acid 19 with m-chloroperoxybenzoic acid in
methylene chloride under room temperature generated
diastereomeric epoxy alcohols 20 and 21, respectively.
Under acidic conditions, epoxy alcohol 21 with the
a-hydrogen atom anti to the epoxy oxygen rearranged
to the more stable a-keto lactone 22 via a transient
intermediate 27, which then tautomerized to enol 23 in
an aqueous solution. The proposed mechanism for the
formation of a-keto lactone 22 from epoxy alcohol 21 is
depicted in Scheme 5. It started with an acid-catalyzed
ring opening of the protonated epoxy alcohol 24 via a
hydride shiftto a-keto lactone 26. Subsequentdehydra-
tion of 26 yielded the desired 3-phenyl-2-oxo-3-butenoic
acid 27, which was highly accessible to the intramole-
cular 1,4-addition reaction, giving rise to the cyclized
a-keto lactone 22. Similar intramolecular cyclization
reactions have been reported in the literature.18 Evi-
dence supporting this proposed mechanism is provided
by monitoring the epoxidation reaction with proton
NMR spectra as shown in Figure 1. Spectrum (a) is the
partial 300 MHz H NMR spectrum of 19 after treat-
Scheme 3. Preparation of compounds 15a and 15b: (i) K2S2O8, H2O;
(ii) R1CHClCO2R2, K2CO3, DMF/xylene; (iii) AcOH, reflux; (iv) 10%
NaOH, reflux.
mentwiht 0.5 equiv of mCPBA in CDCl
for 2 h. The
3
three peaks at 5.57, 5.53, and 5.18 ppm are the absorp-
tion of the methylene and a-hydrogens of starting
material 19. The doublets at 3.42, 2.86 ppm and 3.28,
2.94 ppm indicate formation of the expected epoxides 20
and 21, respectively. The two other downfield peaks at
6.65 and 6.20 ppm are the distinct absorption peaks for
the methylene hydrogens of compound 27, which is
apparently generated from 21. Spectrum (b) shown in
Figure 1 is the partial H NMR spectrum of 19 after
treatment with 0.5 equiv of mCPBA in CDCl3 for 12 h.
While the intensity of the absorption peaks at 3.42 and
2.86 ppm remains unchanged, the near disappearance of
absorption peaks at 3.28 and 2.94 ppm suggests that
mostof 21 has rearranged to 27 under these reaction
conditions.
Scheme 4. Preparation of compound 23: (i) NaOMe, methyl chloro-
acetate, MeOH; (ii) p-TsOH, benzene; (iii) 2 M NaOH; (iv) mCPBA,
CH2Cl2; (v) H2O.
Scheme 5. Proposed mechanism for the formation 22 from 21.

1712
Y.-L. Lin et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1709–1713
Figure 1. 1H NMR spectra (300 MHz) of 19 after treatment with 0.5 equiv of mCPBA in CDCl3 for (a) 2 h, (b) 12 h. The doublets at 3.42, 2.86 ppm
and 3.28, 2.94 ppm in (a) indicate formation of the epoxides 20 and 21. The decreased intensity at 3.28 and 2.94 ppm and increased intensity at 6.65
and 6.20 ppm in (b) indicated that 21 has rearranged to 27.
With these potential 4-HPPD inhibitors available, incu-
bation experiments were conducted to determine inhi-
bition parameters of 10, 11, 14a–c, 15a–b, 17–19 and 23
by the spectrophotometric enol borate method.19 The
inhibition data for the reactions of these compounds
with pig liver 4-HPPD are listed in Table 1. Unfortu-
nately, both epoxybenzoquinone ester 11 and benzo-
quinone ester 10 were found to be poor 4-HPPD
inhibitors with IC50 of 500 and 150 mM, respectively.
This result implied that the presence of the two oxo
groups in the ring system is detrimental to the inhibition
potency. Further efforts to develop transient arene
oxide mimics as potential 4-HPPD inhibitors should try
to avoid the benzoquinone functionality. In the case of
4-hydroxyphenoxypropionic acid derivatives, the best
synthesized 4-HPP analogue tested was compound 15a
Table 1. Inhibition constants for reactions of 10, 11, 14a–c, 15a–b,
17–19 and 23 with 4-HPPD from pig liver by the enol borate method
Compd
R1
R2
IC50 (mM)a
10
11
—
—
H
H
Me
H
Me
—
—
—
—
—
—
Me
Et
Et588
—
—
—
—
150
500
223
14a
14b
14c
15a
15b
17
18
19
23
73
6
93
120
>900
>900
0.5
—
—
aMean of two determinations.
with an IC50 of 6 mM. The facthtatcompound
15b
with the enzyme active site iron. Finally, compound 23
was found to be a potent 4-HPPD inhibitor, with an
IC50 value of 0.5 mM. It is likely that this conformation-
restricted substrate analogue possesses a a-hydroxyl
lactone functionality which can chelate strongly with
the enzyme active site iron. In fact, compound 23 did
give a positive ferric chloride test. Thus, compound 23
may share a similar mode of action with the triketone
type 4-HPPD inhibitors,20 although further studies are
needed to elucidate this point.
decreased inhibition activity up to 15-fold less relative
to 15a suggested that this type of 4-HPPD inhibitor
appears to have higher functional or structural demands
on the a-substituent of the 4-hydroxyphenoxyacetic
acid. Additionally, more than a 12-fold decrease in
potency by introducing the ester functionality on 14a–b
relative to 15a indicated that the terminal carboxylate
group is crucial for binding, presumably by chelating
with ferrous ion in the enzyme active site. For com-
pounds 18–19, no inhibition activity was observed up to
a concentration of 0.9 mM. This result suggests the
2-keto functionality of 4-HPP is crucial for chelating
In summary, a postulated reactive intermediate ana-
logue 11 was designed and synthesized as a potential

Y.-L. Lin et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1709–1713
1713
4-HPPD inhibitor. The biological data demonstrated
that epoxybenzoquinone 11 is nota 4-HPPD inhibiot r,
with an IC50 of more than 500 mM. Several 4-hydroxy-
phenoxypropionic acid and 2-hydroxy-3-phenyl-3-bute-
noic acid derivatives were also prepared as structure-
based 4-HPPD inhibitors. The inhibition results suggest
3-hydroxy-4-phenyl-2(5H)-furanone 23 is the best inhi-
bitor tested, with an IC50 value of 0.5 mM. Further
investigation toward development of more potent
4-HPPD inhibitors as a new class of bleaching herbi-
cides based on the 3-hydroxy-4-phenyl-2(5H)-furanone
template are currently underway in our laboratory.
4. Hall, M. G.; Wilks, M. F.; Provan, W. M.; Eksborg, S.;
Lumholtz, B. Br. J. Clin. Pharmacol. 2001, 52, 169.
5. Pallett, K. E.; Cramp, S. M.; Little, J. P.; Veerasekaran, P.;
Crudace, A. J.; Slater, A. E. Pest. Manag. Sci. 2001, 57, 133.
6. Ellis, M. K.; Whitfield, A. C.; Gowans, L. A.; Auton, T. R.;
Provan, W. M.; Lock, E. A.; Smith, L. L. Toxicol. Appl.
Pharmacol. 1995, 133, 12.
7. Schulz, A.; Ort, O.; Beyer, P.; Kleinig, H. FEBS 1993, 318, 162.
8. (a) Lin, S. W.; Lin, Y. L.; Lin, T. C.; Yang, D. Y. Bioorg.
Med. Chem. Lett. 2000, 10, 1297. (b) Lin, Y. L.; Wu, C. S.;
Lin, S. W.; Huang, J. L.; Sun, Y. S.; Yang, D. Y. Bioorg. Med.
Chem. 2002, 10, 685.
9. Prisbylla, M. P.; Onisko, B. C.; Shribbs, J. M.; Adams,
D. O.; Liu, Y.; Ellis, M. K.; Hawkes, T. R.; Mutter, L. C.
Proc. Brighton Crop Protection Conf. Weeds 1993, 2, 731.
10. Forbes, B. J. R.; Hamilton, G. A. Bioorg. Chem. 1994, 22,
343.
Acknowledgements
11. Niederwieser, A.; Motsovic, A.; Neuhauser, F.; Wetzel, E.
J. Chromatogr. 1978, 146, 207.
12. Pascal, R. A.; Oliver, M. A.; Chen, Y. C. J. Biochemistry
1985, 24, 3158.
13. Snyder, C. D.; Rapoport, H. J. Am. Chem. Soc. 1972, 94, 227.
14. (a) Elbs, K. J. Prakt. Chem. 1893, 48, 179. (b) Behrman,
E. J. Org. React. 1986, 35, 421.
The financial assistance provided by National Science
Council of Republic of China is thankfully acknow-
ledged. The authors also thank the reviewers for their
suggestions and encouragement.
15. Watson, K. G.; Serban, A. Aust. J. Chem. 1995, 48, 1503.
16. Moser, C. M. J. Am. Chem. Soc. 1950, 72, 1413.
17. Darzens, G. Compt. Rend. 1904, 139, 1214.
18. Mageswaran, S.; Ollis, W. D.; Southam, D. A.; Suther-
land, I. O.; Thebtaranonth, Y. J. Chem. Soc., Perkin Trans. 1
1981, 1969.
References and Notes
1. (a) Scheparts, B.; Gurin, S. J. Biol. Chem. 1949, 180, 663.
(b) Vitol, M. J.; Vilks, S. R.; Zabarovska, I. M.; Maurinia,
K. A. Kokl. Akad. Nauk SSSR 1970, 192, 908.
2. Bender, D. A. In Amino Acid Metabolism, 2nd ed.; John
Wiley & Sons: London, 1985; p. 208.
19. Lindstedt, S.; Rundgren, M. Biochim. Biophys. Acta 1982,
704, 66.
3. Goodwin, T. W.; Mercer, E. I. In Introduction to Plant
Biochemistry, 2nd ed.; Pergamon: Oxford, 1983; p 458.
20. Wu, C. S.; Huang, J. L.; Sun, Y. S.; Yang, D. Y. J. Med.
Chem. In press.