SAR studies of 2-o-substituted-benzoyl- and 2-alkanoyl-cyclohexane-1,3-diones as inhibitors of 4-hydroxyphenylpyruvate dioxygenase

Article abstract of DOI:10.1016/S0960-894X(00)00115-3

Inhibition studies of 4-hydroxyphenylpyruvate dioxygenase (HPPD) with various synthesized 2-o-substituted-benzoyl- and 2-alkanoyl-cyclohexane-1,3-diones suggest that the presence of a strongly electronegative group at the ortho position and the conformati

Full text of DOI:10.1016/S0960-894X(00)00115-3

Bioorganic & Medicinal Chemistry Letters 10 (2000) 843±845
SAR Studies of 2-o-Substituted-benzoyl- and 2-Alkanoyl-
cyclohexane-1,3-diones as Inhibitors of 4-Hydroxyphenylpyruvate
Dioxygenase
Yung-lung Lin, Chung-shieh Wu, Shean-woei Lin and Ding-yah Yang*
Department of Chemistry, Tunghai Christian University, 181 Taichung-Kang Rd. Sec.3, Taichung, 40704 Taiwan, ROC
Received 25 October 1999; accepted 1 February 2000
AbstractÐInhibition studies of 4-hydroxyphenylpyruvate dioxygenase (HPPD) with various synthesized 2-o-substituted-benzoyl-
and 2-alkanoyl-cyclohexane-1,3-diones suggest that the presence of a strongly electronegative group at the ortho position and the
conformation of the benzene ring moiety on the benzoylcyclohexane-1,3-dione inhibitors are crucial for potent HPPD inhibition.
# 2000 Elsevier Science Ltd. All rights reserved.
4-Hydroxyphenylpyruvate dioxygenase (HPPD)1 is an
important enzyme involved in the biosynthesis of plas-
toquinones and tocopherols in plants, as well as in the
catabolism of the aromatic acids phenylalanine and
tyrosine in most organisms. It catalyzes oxidative de-
carboxylation, hydroxylation of the aromatic ring, and
a 1,2-shift of a carboxymethyl group from 4-hydroxy-
phenylpyruvate 1 to homogentisate 2 in the presence of
oxygen, as shown in Scheme 1.
HPPD enzyme, demonstrated that a 2-benzoylethen-1-
ol substructure, depicted in Figure 2, is the minimum
substructure required for potent in vitro inhibition of
HPPD. Nevertheless, the function of the ``X'' sub-
stituent at the ortho position of the benzene ring moiety
was not elucidated.
In this study, we synthesized a series of 2-o-substituted-
benzoylcyclohexane-1,3-diones and tested their relative
competence as inhibitors of 4-hydroxyphenylpyruvate
dioxygenase in an e€ort to investigate the role played by
the ortho substituent on triketone inhibitors in HPPD
inhibition. The synthesis of ortho substituted 2-ben-
zoylcyclohexane-1,3-diones is outlined in Scheme 2.
Preparations of 5a±h were accomplished by the cyanide
catalyzed isomerization of enol esters 4a±h derived from
the reaction of the corresponding benzoyl chloride 3a±h
with cyclohexane-1,3-dione using triethylamine as a base
in CH2Cl2, as described in the literature.6 The 2-o-¯uoro-
benzoylcyclohexane-1,3-dione was not prepared since
the consequent elimination of hydrogen ¯uoride pro-
ceeds relatively smoothly under the reaction conditions
to yield the nucleophilic intramolecular heterocyclized
product exclusively.7
While the detailed mechanism of the reaction catalyzed
by HPPD remains unclear, increasing evidence2 suggests
that HPPD is the target site of certain bleaching herbi-
cides that contain the 2-benzoylcyclohexane-1,3-dione
moiety, referred to as triketones. Inhibition of HPPD
activity by triketone herbicides decreases tocopherol and
plastoquinone levels in plants, indirectly reducing phy-
toene desaturation and leading to development of bleach-
ing symptoms. The structures of two representative
triketones 2-(2-nitro-4-tri¯uoromethylbenzoyl)-cyclohex-
ane-1,3-dione (NTBC)3 and 2-(2-chloro-4-methane-sulfo-
nylbenzoyl)-cyclohexane-1,3-dione (sulcotrione)4 are
shown in Figure 1. Both NTBC and sulcotrione were
found to be competitive HPPD inhibitors with IC50
values of 40 and 45 nM, respectively.
Inhibition of 4-hydroxy-phenylpyruvate dioxygenase by
2-o-substituted-benzoylcyclohexane-1,3-diones was stu-
died. Varying concentrations of 5a±h were incubated
with HPPD puri®ed from pig liver8 in the presence of the
natural substrate 4-hydroxyphenylpyruvate. The enzyme
activity was monitored by the spectrophotometric enol-
borate method as described by Lindstedt.9 The inhibition
In 1997, Lee and co-workers,5 after screening a wide
range of triketone analogues and isosteres against the
*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)00115-3

844
Y. Lin et al. / Bioorg. Med. Chem. Lett. 10 (2000) 843±845
Scheme 1.
Table 1. Inhibition constants for reactions of 5a±h with HPPD from
pig liver by the enol-borate method
Compound
X
IC50 (mM)a
Compound
X
IC50 (mM)a
NTBC
5a
5b
5c
5d
Ð
H
Cl
Br
I
0.04
11.20
0.50
0.56
0.76
5e
5f
5g
5h
NO2
CH3
CF3
0.16
3.75
0.25
11.70
Figure 1.
OMe
aMean of two determinations.
inhibitors might be crucial for potent HPPD inhibition,
although more evidence is needed to support this
assumption.
Figure 2.
In 1995, Crouch and co-workers10 demonstrated
that 4-hydroxyphenylpyruvate dioxygenase also dis-
plays a-ketoisocaproate dioxygenase (a-KICD) activ-
ity. a-KICD11 is also an internal keto-acid dependent
dioxygenase responsible for the oxidative decarboxyla-
tion and hydroxylation of a-ketoisocaproate (6) to b-
hydroxyisovalerate (7), as shown in Scheme 3. Further
studies12 have indicated that HPPD and a-KICD are
actually the same enzyme, that is, both 4-hydroxy-
phenylpyruvate (1) and a-ketoisocaproate (6) serve as
natural substrates for HPPD.
constants for the reactions of 5a±h with HPPD are listed
in Table 1.
The inhibition results indicated that the presence of a
strongly electronegative group like chloro, tri¯uoromethyl,
or nitro at the ortho position of the benzene ring of trike-
tones enhances the inhibition potency. A maximum 68-
fold increase in potency was observed when a nitro
group replaced the ortho hydrogen atom. When the
ortho hydrogen was replaced with a methyl or methoxy
group, however, no signi®cant increase in inhibition
potency was detected. It is assumed that the electrostatic
interaction between the relatively electronegative atom
(group) at the ortho position and the nearby enzyme
active site residue induces a rotation of the benzene ring,
with the resulting triketone conformation mimicking the
structure of the enzyme±substrate complex to tightly
bind with the enzyme. Thus, the active conformation
adopted by the benzene ring moiety of the triketone
In light of these ®ndings and subsequent comparisons of
the molecular structures of both HPPD enzyme sub-
strates and triketone type inhibitors, we speculate that 2-
isopropylcarbonylcyclohexane-1,3-dione (8c), illustrated
in Figure 3, is a potential HPPD inhibitor. Further
structure±activity relationship studies of various 2-alka-
noyl-cyclohexane-1,3-diones may not only discover new
potent HPPD inhibitors but also shed light on the mode
Scheme 2. (i) Et3N, CH2Cl2; (ii) Et3N, KCN, CH2Cl2.
Scheme 3.

Y. Lin et al. / Bioorg. Med. Chem. Lett. 10 (2000) 843±845
845
Table 2. Inhibition constants for reactions of 8a-e with HPPD from
pig liver by the enol-borate method
Compound
R
IC50 (mM)a
8a
8b
8c
8d
8e
CH3
11.2
17.8
93.3
6.0
CH2CH3
CH(CH3)2
CH(CH2)2
CH(CH2)5
364.5
aMean of two determinations.
negative group at the ortho position and the conformation
of the benzene ring moiety of the 2-benzoylcyclohexane-
1,3-diones are both crucial for potent HPPD inhibition.
The information provided in this study may allow us to
better de®ne the speci®c binding characteristics for
inhibitors of the enzyme HPPD and may further reveal
mechanistic details of this intriguing enzyme. Further
characterization of the mechanism of action of the tri-
ketone-type HPPD inhibitors at a molecular level is
being actively pursued in our laboratory.
Figure 3.
of action of the benzene ring moiety of the existing 2-
benzoylcyclohexane-1,3-dione inhibitors.
Several 2-alkanoylcyclohexane-1,3-diones (8a±e), shown
in Figure 4, were then prepared as described earlier.
Attempts to synthesize 2-t-butylcarbonylcyclohexane-1,3-
dione from the corresponding enol ester by either cyanid-
catalyzed isomerization or aluminum chloride-catalyzed
Fries rearrangement were unsuccessful, presumably due
to low reactivity of the bulky substrate. Similar ®ndings
have been reported in the literature.13
Acknowledgements
Financial support of this study was received from the
National Science Council of the Republic of China.
Subsequently, the synthesized compounds 8a±d were
evaluated in vitro for inhibition activity against HPPD,
and the biological data are listed in Table 2. The results
show that 2-alkanoylcyclohexane-1,3-diones were gen-
erally less potent HPPD inhibitors than 2-benzoylcy-
clohexane-1,3-diones. For instance, compound 8c was
almost 9-fold less potent than the corresponding com-
pound 5a. Furthermore, when the isopropyl group on
8c was replaced by the cyclopropyl group, the inhibition
potency of the resulting 8d was up to 15 times higher.
The major di€erence between the isopropyl group of 8c
and cyclopropyl group of 8d when bound to the enzyme
active site is their conformation. This result supports
our assumption that the conformation of the benzene
ring moiety plays an important role in potent HPPD
inhibition. If both an ortho substituent and the benzene
ring conformation of 2-benzoylcyclohexane-1,3-dione
inhibitors are essential for tight binding, perhaps the
mode of action of triketones in HPPD inactivation is
more complex than simply acting as analogues to the
substrate 4-hydroxyphenylpyruvate as previously sur-
mised.14 Further studies are needed to elucidate this
point.
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In summary, inhibition studies of 4-hydroxy-
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Figure 4.