Discovery of a potent, non-triketone type inhibitor of 4-hydroxyphenylpyruvate dioxygenase

Article abstract of DOI:10.1016/S0960-894X(00)00228-6

3-Cyclopropanecarbonyloxy-2-cyclohexen-1-one has been found to be a new, potent, low molecular weight non-triketone type inhibitor of 4-hydroxyphenylpyruvate dioxygenase with IC₅₀ value of 30 nM. Preliminary studies suggest that the two carbonyl groups present in the compound are crucial for the inhibition activity. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00228-6

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1297±1298
Discovery of a Potent, Non-Triketone Type Inhibitor of
4-Hydroxyphenylpyruvate Dioxygenase
Shean-Woei Lin, Yun-Loung Lin, Tzu-Ching Lin and Ding-Yah Yang*
Department of Chemistry, Tunghai Christian University, 181 Taichung-Kang Rd. Sec. 3, Taichung, Taiwan 40704, ROC
Received 31 January 2000; accepted 8 April 2000
AbstractÐ3-Cyclopropanecarbonyloxy-2-cyclohexen-1-one has been found to be a new, potent, low molecular weight non-trike-
tone type inhibitor of 4-hydroxyphenylpyruvate dioxygenase with IC50 value of 30 nM. Preliminary studies suggest that the two
carbonyl groups present in the compound are crucial for the inhibition activity. # 2000 Elsevier Science Ltd. All rights reserved.
2-(2-Nitro-4-tri¯uoromethylbenzoyl)-cyclohexane-1,3-
dione1 (NTBC), a triketone type compound, is the ®rst
e€ective drug therapy for the fatal hereditary disease
tyrosinemia type I,2 which is characterized by a de®-
ciency in the activity of fumarylacetoacetase, leading to
the accumulation of hepatotoxic and nephrotoxic com-
pounds like succinylacetone.3 Recent studies4 have
demonstrated that the primary site of action of NTBC is
4-hydroxyphenylpyruvate dioxygenase5 (HPPD, EC
hypothesis, other potent ACCase inhibitors may serve
as a good starting point to search for non-triketone type
potent HPPD inhibitors. In light of the information that
various enol ethers of allyloximes8 have been found to
be more e€ective and selective herbicides than cyclohex-
anedione class herbicides, we synthesized a series of alka-
noic acid 3-oxo-cyclohex-1-enyl esters 2a±g as potential
inhibitors of HPPD. Esters 2a±g were easily prepared by a
reaction coupling the various acyl chlorides 1a±g with 1,3-
cyclohexanedione in the presence of triethylamine in
methylene chloride in an ice bath, as shown in Scheme 1.
1.13.11.27),
a non-heme Fe(II)-dependent enzyme
involved in the catabolism6 of tyrosine and phenylala-
nine in most organisms which catalyzes the conversion
of 4-hydroxyphenylpyruvate to homogentisate. Inhibi-
tion of liver HPPD by NTBC will prevent the formation
of homogentisate, thereby blocking tyrosine catabolism.
Here, we report the discovery of a new, potent, low
molecular weight, non-triketone type HPPD inhibitor.
The synthesized compounds were evaluated in vitro for
inhibition activity against 4-hydroxyphenylpyruvate
dioxygenase from pig liver9 by the spectrophotometric
enol-borate method.10 The inhibition constants11 for the
reactions of these esters with HPPD are listed in Table 1.
The most potent inhibitor tested was 3-cyclopropane-
carbonyloxy-2-cyclohexen-1-one 2d with an IC50 of 30
nM, which is comparable to that of NTBC (IC50=40
nM). The fact that 2c decreased inhibition activity up to
140-fold relative to 2d suggests that the cyclopropyl
group present in 2d is important for potent HPPD inhi-
bition. Similarly, either reducing the carbon number of
the R group or increasing its bulkiness resulted in a
decrease in potency, indicating that HPPD inhibition by
this class of molecules appears to have higher steric and
structural demands on the alkyl group.
Our recent discovery7 that the herbicide sethoxydim
which targets the enzyme acetyl-CoA carboxylase
(ACCase) exhibited modest in vitro inhibition activity
against 4-hydroxyphenylpyruvate dioxygenase suggested
that the biological distinct HPPD and ACCase may share
at least some similar binding pockets in the enzyme active
site. Although more evidence is needed to con®rm this
In an e€ort to investigate the role played by the two
carbonyl groups of 2d in HPPD inhibition, we selectively
removed each of the carbonyl groups present in 2d and
tested the competence of the resulting compounds as
HPPD inhibitors as described above. The synthesis of
analogues 4 and 6 is shown in Scheme 2. Compound 4
*Corresponding author. Tel.: +886-4-359-0248; fax: +886-4-359-
0426; e-mail: yang@mail.thu.edu.tw
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00228-6

1298
S.-W. Lin et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1297±1298
In summary, we have discovered a new, potent, low
molecular weight, non-triketone type HPPD inhibitor.
This study revealed that 3-cyclopropane-carbonyloxy-2-
cyclohexen-1-one has potent HPPD inhibition activity,
comparable to that of NTBC. The preliminary SAR
studies demonstrated that the well-positioned dicar-
bonyl groups of 2d are essential for this potent inhibi-
tion. Thus, 3-cyclopropanecarbonyloxy-2-cyclohexen-1-
one or its derivatives has the potential to serve as a new
therapeutic agent to treat the fatal human disease tyr-
osinemia type I. Further SAR studies are currently
underway in our laboratory.
Scheme 1. Preparation of compounds 2a±g.
Table 1. Inhibition constants for reactions of 2a±g with HPPD from
pig liver by the enol borate assay method
Acknowledgements
Compound
R
IC50
(mM)a
Compound
R
IC50
(mM)a
Financial support of this research from the National
Science Council of the Republic of China is gratefully
acknowledged.
2a
2b
2c
2d
CH3
C2H5
CH(CH3)2
CH(CH2)2
3.62
0.11
4.16
0.03
2e
2f
2g
C(CH3)3
CH(CH2)5
C6H5
79.60
3.70
1.58
References and Notes
aMean of two determinations.
1. Lindstedt, S.; Holme, E.; Lock, E.; Hialmarson, O.;
Strandvik, B. Lancet 1992, 340, 813.
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3. Puntoni, R.; Merlo, F.; Fini, A.; Meazza, L.; Santi, L.
Lancet 1986, ii, 525.
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Provan, W. M.; Lock, E. A.; Smith, L. L. Toxicol. Appl.
Pharmacol. 1995, 133, 12.
5. (a) Scheparts, B.; Gurin, S. J. Biol. Chem. 1949, 180, 663.
(b) Vitol, M. J.; Vilks, S. R.; Zabaroversuska, I. M.; Maurinia,
K. A. Kokl. Akad. Nauk. SSSR 1970, 192, 908.
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Scheme 2. Synthesis of compounds 4 and 6: (a) p-TsOH, benzene,
re¯ux; (b) Et3N, trimethylsilyl chloride, DMF, 120 ꢀC; (c) potassium t-
butoxide, cyclopropane carbonyl chloride, THF, 20 ꢀC.
was prepared in one step by acid catalyzed dehydration
of cyclohexanedione and cyclopropanemethanol 3.
Compound 6 was synthesized in two steps by ®rst con-
verting the cyclohexanone to the corresponding tri-
methylsilyl enol ether 5,12 followed by treatment with
cyclopropane carbonyl chloride in the presence of
potassium t-butoxide in THF.13
11. The reaction mixtures contained 0.85 mL potassium phos-
phate/borate bu€er (prepared by adjusting the pH of 0.42 M
H3BO3 to 6.2 with a 0.17 M Na3PO4 solution), 0.06 mL 4-
hydroxyphenylpyruvic acid (1.8 mM, in 0.2 M Na3PO4 bu€er),
0.03 mL dichlorophenolindophenol (reduced form, prepared
by mixing 1 mL of 3.3 mM sodium dichlorophenolindophenol
in H2O and 0.16 M glutathione in 0.2 M sodium phosphate
bu€er), and 0.01 mL phenylpyruvate tautomerase (10U/mL,
Sigma). The above solution was equilibrated for 15 min, the
inhibition reaction of 2a±g, 4 and 6 with the enzyme HPPD
was evaluated by measuring the decrease in absorbance at 308
nm over a 15 min period following coadministration of vary-
ing concentrations of the inhibitors and HPPD.
12. Suginome, H.; Kondoh, T.; Gogonea, C.; Singh, V.; Goto,
H.; Osawa, E. J. Chem. Soc. Perkin Trans. 1 1995, 69.
13. Duhamel, P.; Cahard, D.; Poirier, J.-M. J. Chem. Soc.
Perkin Trans. 1 1993, 2509.
14. Prisbylla, M. P. B.; Onisko, B. C.; Shribbs, J. M.; Adams,
D. O.; Liu, Y.; Ellis, M. K.; Hawkes, T. R.; Mutter, L. C.
Proc. British Crop Prot. Conf.-Weeds 1993, 2, 731.
The inhibition results showed that analogue 6 was a
much less potent inhibitor than 2d with IC50 of 0.7 mM
and analogue 4 was not a HPPD inhibitor. When the
ring carbonyl group of 2d was removed to form 6, the
inhibition potency decreased 23-fold relative to 2d indi-
cating that this carbonyl group is essential for binding.
When the ester carbonyl group of 2d was removed to
form 4, no enzyme inhibition was observed up to the
concentration of 0.2 mM. This result implies the car-
bonyl oxygen atom on the ester functionality is crucial
for inhibition, presumably by chelating with ferrous ion
in the enzyme active site.14