Mode of action of 4-hydroxyphenylpyruvate dioxygenase inhibition by triketone-type inhibitors

Article abstract of DOI:10.1021/jm010568y

A series of 2-(2-nitrobenzoyl)cyclohexane-1,3-dione analogues (1 -9) were designed, synthesized, and evaluated for inhibition of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD), a key enzyme involved in the catabolism of tyrosine which catalyzes the conversion of 4-hydroxyphenylpyruvate to homogentisate. The correlations between the results of enzyme inhibition, ferric chloride tests, and the conformational analysis suggested that the tight binding between triketonetype inhibitors and 4-HPPD is likely due to chelation of the enzyme-bound ferric iron with the enol tautomer of 1,3-diketone moiety of the triketones. The presence of a 2-carbonyl group in the triketone is an essential structural feature for potent 4-HPPD inhibition. Modification of the 3-carbonyl group of triketone moiety to other functionality will reduce the overall planarity and thus prevent keto-enol tautomerization, resulting in a decrease or lack of inhibition activity.

Full text of DOI:10.1021/jm010568y

2222
J . Med. Chem. 2002, 45, 2222-2228
Mod e of Action of 4-Hyd r oxyp h en ylp yr u va te Dioxygen a se In h ibition by
Tr ik eton e-typ e In h ibitor s
Chung-Shieh Wu, J ian-Lin Huang, Yang-Sheng Sun, and Ding-Yah Yang*
Department of Chemistry, Tunghai University, 181, Taichung-Kang Road, Section 3, Taichung,
Taiwan, Republic of China 40704
Received December 17, 2001
A series of 2-(2-nitrobenzoyl)cyclohexane-1,3-dione analogues (1-9) were designed, synthesized,
and evaluated for inhibition of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD), a key enzyme
involved in the catabolism of tyrosine which catalyzes the conversion of 4-hydroxyphenylpyru-
vate to homogentisate. The correlations between the results of enzyme inhibition, ferric chloride
tests, and the conformational analysis suggested that the tight binding between triketone-
type inhibitors and 4-HPPD is likely due to chelation of the enzyme-bound ferric iron with the
enol tautomer of 1,3-diketone moiety of the triketones. The presence of a 2-carbonyl group in
the triketone is an essential structural feature for potent 4-HPPD inhibition. Modification of
the 3-carbonyl group of triketone moiety to other functionality will reduce the overall planarity
and thus prevent keto-enol tautomerization, resulting in a decrease or lack of inhibition
activity.
Sch em e 1. Reaction Catalyzed by 4-HPPD
In tr od u ction
4-Hydroxyphenylpyruvate dioxygenase (4-HPPD, EC
1.13.11.27) is an important enzyme involved in the
catabolism of tyrosine in most organisms.1 It catalyzes
the conversion of 4-hydroxyphenylpyruvate to homogen-
tisate and carbon dioxide in the presence of oxygen and
ferrous ion as shown in Scheme 1. The biotransforma-
tions catalyzed by 4-HPPD require an oxidative decar-
boxylation of the 2-oxoacid side chain of the substrate
with an accompanying hydroxylation of the aromatic
ring and a 1,2-migration of the carboxymethyl group.2
Clinically, a hereditary disease called tyrosinemia type
I3 is caused by the deficiency of fumarylacetoacetate
hydrolase activity which converts fumarylacetoacetate
to fumarate and acetoacetate in the tyrosine degrada-
tion pathway. Inhibition of 4-HPPD activity, an enzyme
preceding the furmarylacetoacetate hydrolase in the
same pathway, will provide an alternate treatment for
this fatal disease. Although the first effective drug
therapy for tyrosinemia has been discovered, that is,
2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1,3-di-
one4 (NTBC, a triketone-type compound as shown
below), the mode of action for this inhibition remains
unclear. Since NTBC is a strong competitive inhibitor
of 4-HPPD and the enzyme activity may be partly
reversed by ascorbate, it is reasonable to assume that
the favored keto-enol form mimics the ketoacid func-
tionality present in the substrate and is capable of
binding strongly to the ferric ion in the active site.5 Here
we report the synthesis, characterization, and structure-
activity relationships (SAR) of 2-(2-nitrobenzoyl)cyclo-
hexane-1,3-dione derivatives as inhibitors of 4-HPPD
in an effort to provide insights into its mode of action
at the molecular level.
Ch em istr y
The compounds designed and synthesized for SAR are
compounds 1-9 as shown in Figure 1. Since our
previous SAR studies6 of 2-o-substituted-benzoyl- and
2-alkanoylcyclohexane-1,3-dione analogues have dem-
onstrated that both the nitro group on benzene moiety
and the benzene ring of NTBC are critical on 4-HPPD
inhibition activity, the main objective of the present
study is to investigate the crucial role of triketone
functionality in NTBC at the molecular level. While
keeping both the 2-nitrobenzoyl moiety and the six-
membered ring intact, one of the carbonyl groups
present in the triketone-type inhibitors was either
selectively removed (compounds 2, 8, and 9) or modified
* Corresponding author. Telephone: 886-4-2359-7613. Fax: 886-4-
2359-0426. E-mail: yang@mail.thu.edu.tw.
10.1021/jm010568y CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/27/2002

4-HPPD Inhibitions by Triketone-type Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2223
was prepared by a coupling reaction of glutarimide with
2-nitrobenzoyl chloride in the presence of triethylamine
as a base in methylene chloride under reflux conditions
for 3 h with a 50% yield.
Next, compound 2, as shown in Scheme 3, was
generated by reacting 1-morpholinecyclohexene (pre-
pared by dehydration of cyclohexanone with morpho-
line)10 with 2-nitrobenzoyl chloride under basic condi-
tions followed by refluxing in an aqueous acid for 5 h.11
1
The ratio of enol/keto isomers, determined by H NMR
spectra, was found to be 97/3. Unfortunately but not
surprisingly, reacting 2-nitrobenzyl bromide with cyclo-
hexane-1,3-dione in triethylamine in CH2Cl2 gave the
major undesired vinyl ether 12 and minor desired
compound 8 in the ratio of 2/1. A similar coupling
reaction was used to generate 9 except glutarimide and
potassium carbonate were used instead of cyclohexane-
1,3-dione and triethylamine, as depicted in Scheme 4.
F igu r e 1. Compounds synthesized for SAR studies.
Resu lts a n d Discu ssion
(compounds 3-6) in order to eliminate the possibility
of keto-enol tautomerization. In addition, the central
carbon atom of the triketone moiety was replaced by a
nitrogen atom (compound 7) to force the 1,3-diketo group
into a more rigid as well as nonplanar conformation.
The inhibition results of these compounds with 4-HPPD
will identify the essential structural elements required
for the inhibition activity.
The synthetic route used to prepare compounds 1 and
3-7 is outlined in Scheme 2. The preparation of 1
started with the O-acylation of cyclohexane-1,3-dione by
2-nitrobenzoyl chloride to give enol ester 10, which on
treatment with cyanide ion and triethylamine rear-
ranged to the corresponding C-acyl isomer 1, according
to the published procedure.7 Methylation of 1 with
diazomethane in ethyl acetate at 0 °C afforded 4.
Compound 1 was converted quantitatively to vinyl
chloride 5 by treatment with an excess of oxalyl chloride
at room temperature.8 Base-promoted isomerization of
diketovinyl chloride 5 to ketodienol form 3 was per-
formed by treatment of 5 with potassium carbonate and
a catalytic amount of tetra-n-butylammonium iodide in
acetone under room temperature for 2 days with a 32%
yield. Selective reaction of the carbonyl group adjacent
to the nitrophenyl ring of 1 with hydroxylamine hydro-
chloride in the presence of sodium methoxide afforded
oxime 6 as reported in the literature.9 2-Acyl imide 7
With these potential 4-HPPD inhibitors available,
incubation experiments were conducted to determine
inhibition parameters of 1-9 by the spectrophotometric
enol borate method.12 The inhibition data for the reac-
tions of these compounds with pig liver 4-HPPD are
summarized in Table 1. All the synthesized compounds
were also tested with ferric chloride to observe any
colorimetric change. Only NTBC and 1-3 gave a posi-
tive characteristic purple color, while the rest of the
compounds gave negative results, as shown in Table 1.
According to the inhibition results, the potent inhibi-
tors included NTBC and 1-3 with an IC50 less than 1
µM. These four inhibitors also gave a positive test with
ferric chloride assay, which suggests they all can bind
tightly with enzyme-bound ferric ion via the enol
tautomer of the triketones. The rest of the synthesized
compounds were not only poor 4-HPPD inhibitors but
also gave negative ferric chloride tests, indicating the
lack of the critical triketone enol tautomer. These results
prompted us to consider whether deprotonation of the
enolic hydrogen at the C-3 position in the triketone
system could alter the molecular three-dimensional
orientation, thereby directly affecting the molecular
affinity with the target enzyme and resulting in differ-
ent inhibition activity. Thus, the structures of 1 and 4
were established by X-ray diffraction analysis. From the
Sch em e 2. Synthesis of Compounds 1 and 3-7

2224 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Sch em e 3. Preparation of Compound 2
Wu et al.
Ta ble 1. Inhibition Constant for Reactions of 1-9 with
4-HPPD, Results of Ferric Chloride Tests, and Predicted
Torsional Angles
F igu r e 2. X-ray structure of compound 1. ORTEP diagram
showing the crystallographic atom-numbering scheme. Tor-
sional angle of O(3)-C(7)-C(6)-C(5) ) 1.60°.
compd
IC50 (µM)a
F eCl3 testb
torsional anglec (deg)
NTBC
0.04 ( 0.01
0.16 ( 0.06
0.70 ( 0.15
0.53 ( 0.10
52 ( 2
+
0.3
1.4 (1.6)
0.5
1
2
3
4
5
6
7
8
9
+
+ +
+
0.5
-
59.8 (58.2)
61.2
11.1
29.2
-
64 ( 9
-
50 ( 4
60 ( 7
126 ( 12
250 ( 28
-
-
-
-
-
a
All data presented are averages of at least two separate
b
experiments. Degree of colorimetric change (high, ++; moderate,
+; no change, -). c Predicted torsional angle of O(8)-C(7)-C(2)-
C(3) as labeled in compound 1. dValues in the parentheses are
obtained from X-ray structures.
X-ray crystallographic analysis shown in Figure 2,
compound 1 in the crystal exists only as the cis-enol
tautomer with an intramolecular hydrogen bond. In this
tautomer, the enol hydrogen is covalently bound to the
oxygen atom that is remote from the phenyl group,
which is similar to the structure of benzoyldimedone
reported recently by Borisov.13 It is clear that the 1,3-
diketone and the 2-carbonyl moiety of 1 are coplanar
and conjugated by hydrogen bonding of the C-3 enolic
hydrogen to the oxygen atom of the C-2 carbonyl group
with a torsional angle of 1.6°. If one of the carbonyl
groups in the triketone is modified, however, the inhibi-
tion potency of the resulting compound decreases sub-
stantially. For example, when the C-3 enolic oxygen of
F igu r e 3. X-ray structure of compound 4. ORTEP diagram
showing the crystallographic atom-numbering scheme. Tor-
sional angle of O(3)-C(7)-C(6)-C(5) ) 58.2°.
compound 1 was methylated to vinyl ether 4 or chlori-
nated to vinyl chloride 5, their IC50 values increased to
52 and 64 µM, respectively. The solid-state conformation
of 4 from an X-ray diffraction study indicates that the
orientation of the 2-aroyl group is forced out of the plane
of the 1,3-diketone system with a torsional angle of
58.2°, as shown in Figure 3. This deformation from
planarity is due to the dipolar repulsions between the
C-2 carbonyl group and the C-1 carbonyl group as well
as the C-3 oxygen atom. Similar observations have been
Sch em e 4. Synthesis of Compounds 8 and 9

4-HPPD Inhibitions by Triketone-type Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2225
that compound 6 gave a negative ferric chloride test and
was a poor 4-HPPD inhibitor with IC50 ) 50 µM
supported our assumption. The foregoing studies indi-
cated that the absence of the C-3 enolic oxygen of 1,3-
triketone results in dipolar repulsion, which is relieved
in part by the out-of-plane orientation, thereby disturb-
ing the overall planarity of the triketone system. Ac-
cordingly, the dipolar repulsion and consequent skeletal
deformation might be responsible for the lack of 4-HPPD
inhibition in 4 and obvious decrease in 5.
When vinyl chloride 5 was treated with potassium
carbonate in acetone under room temperature, it isomer-
ized to ketodienol 3 with IC50 ) 0.53 µM. The presence
of the typical signal of the enolic hydrogen absorption
peak at 16.43 ppm in 1H NMR spectra as well as a
positive test with ferric chloride provided evidence of 3
in the enolized form. The fact that the inhibition potency
of 3 increased 120-fold relative to 5 and there was only
a 4-fold decrease in the inhibition potency of 2 compared
to 1 suggest that the 3-carbonyl group of the triketone
type inhibitors may not play a significant role in
4-HPPD inhibition. On the other hand, when the
2-carbonyl group was removed to give 8, 9 or modified
to obtain oxime 6, none of them showed potent 4-HPPD
inhibition activity. These results can be easily explained
since no ligands were available in these compounds to
interact with the ferric ion in the active site and thus
resulted in a weak affinity with the enzyme. Taken
together, these results demonstrate that the presence
of the 2-carbonyl group, but not the 3-carbonyl group,
is crucial for potent 4-HPPD inhibition, presumably by
chelation with the enzyme active site ferric iron. Finally,
the failure of 7 as a 4-HPPD inhibitor is consistent with
the results that the enol form as well as the coplanar
structure of the triketone is required for potent 4-HPPD
inhibition, even though compound 7 also contains the
same triketone functionality as NTBC.
In summary, we have designed and prepared a series
of 2-(2-nitrobenzoyl)cyclohexane-1,3-dione (1) deriva-
tives. SAR studies within this series indicate that the
enol form and coplanar structure of the triketone are
absolutely required in order to chelate with the enzyme-
bound ferric iron. The correlations between the results
of enzyme inhibition, ferric chloride tests, and confor-
mational analysis suggest that the tight binding be-
tween triketone-type inhibitors and 4-HPPD is likely
due to chelation of the enzyme-bound ferric iron with
the enol tautomer of 1,3-diketone moiety of the tri-
ketones (Figure 5). The presence of the 2-carbonyl group
in the triketone is an essential feature for potent
4-HPPD inhibition, whereas the 3-carbonyl group is not.
Modification of the 3-carbonyl group of the triketone
moiety to another functionality will reduce the overall
planarity and thus prevent keto-enol tautomerization,
resulting in decrease or loss of inhibition activity.
Furthermore, the ferric chloride assay may serve as a
simple and easy screening test for further development
of triketone type 4-HPPD inhibitors. Understanding the
mode of action of triketone type 4-HPPD inhibitors will
certainly aid the design of new 4-HPPD inhibitors for
use in tyrosinemia treatment.
F igu r e 4. UV spectra of compounds 1 and 4. Compound 1:
heavy line, λmax ) 262 nm. Compound 4: light line, λmax ) 255
nm.
reported in the literature.14 Additionally, the bond
length of 1.445 Å between the C-2 and the 2-carbonyl
carbon in 1 indicates extensive conjugation in the
triketone system, while that in 4 is shown to be 1.487
Å, suggesting its single bond character. This observation
implies that the conformation of triketone-type com-
pounds can be controlled or regulated by protonation
or deprotonation of the enolic hydrogen at the C-3
position. Furthermore, the UV spectral data of 1 and 4,
shown in Figure 4, also supported their observed con-
formational differences. The longest wavelength absorp-
tion band for 1 was at 262 nm, indicating full conjuga-
tion in the triketone system. With 4, the corresponding
absorption bands shifted to 255 nm. This blue shift of
the absorption band indicates a loss of conjugation
because of the dipolar repulsions between the C-2
carbonyl group and the C-1 carbonyl group as well as
the C-3 oxygen atom. Moreover, the distinctly different
polarity of 1 (Rf ) 0.59, 60% EtOAc/hexanes) and 4 (Rf
) 0.17, 60% EtOAc/hexanes) also resulted possibly from
their conformational differences.
To obtain a further understanding of the SARs of
triketone analogues, we performed conformational
searches for compounds 1-7 using Insight II software.15
The values of the resulting torsional angles [O(8)-C(7)-
C(2)-C(3)] of stable three-dimensional structures are
shown in Table 1. Clearly, in the cases in which the
3-hydroxyl groups of the triketones were replaced or
modified, the resulting compounds (3-5 and 7) tend to
have higher torsional angles values ranging from 29 to
61° compared to those potent inhibitors (NTBC and
1-3), which can undergo keto-enol tautomerization
with torsional angles of 0-2°. These results are consis-
tent with the hypothesis that the dipolar repulsions
between the C-2 carbonyl group and the other two
oxygen atoms on the ring system cause deformation of
the triketone from planarity and result in poor affinity
to the enzyme 4-HPPD. Furthermore, the solid-state
conformation and torsional angle data for compounds
1 and 4 from X-ray structure are similar to the compu-
tational data. The only exception is compound 6 with
the predicted torsional angle of 11.1°. Although the
conformation of 6 is relatively close to planarity based
on the molecular modeling studies, we speculate that
the presence of the oxime functionality prevents its
chelating with the enzyme active site ferric ion, and
therefore results in low affinity with 4-HPPD. The fact
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Mel-Temp
melting point apparatus in open capillaries and are uncor-

2226 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Wu et al.
F igu r e 5. Model predicting the binding mode of NTBC to 4-HPPD. The chelation of the enzyme bound iron (purple) with enol
tautomer of 1,3-tiketone is indicated with a dashed line. See Experimental Section for modeling detail.
rected. Elemental analysis was performed using a Heraeus
CHN-OS rapid element analyzer. Ultraviolet-visible spec-
troscopy was performed on a Hewlett-Packard 8453 spectro-
photometer. 1H and 13C NMR spectra were recorded at 300
and 75 MHz on a Varian VXR300 spectrometer. Chemical
shifts were reported in parts per million on the δ scale relative
to an internal standard (tetramethylsilane, or appropriate
solvent peaks) with coupling constants given in hertz. 1H NMR
multiplicity data are denoted by s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), and b (broad). Analytical
thin-layer chromatography (TLC) was carried out on Merck
silica gel 60G-254 plates (25 mm) and developed with the
solvents mentioned. Flash chromatography was performed in
columns of various diameters with Merck silica gel (230-400
mesh ASTM 9385 kieselgel 60H) by elution with the solvent
systems indicated in the parentheses after the Rf values.
Solvents, unless otherwise specified, were reagent grade and
distilled once prior to use. All new compounds exhibited
satisfactory spectroscopic and analytical data.
En zym e P u r ifica tion a n d Assa y. 4-HPPD was purified
from pig liver by the method of Hamilton16 with a specific
activity of 0.1 µmol min-1 mg-1. The protein concentration was
determined by the BCA method. Routine assay of the enzyme
utilized the spectrophotometric enol borate method of Lind-
stedt and Rundgren.12 The typical assay mixtures contained
0.85 mL of potassium phosphate/borate buffer (prepared by
adjusting the pH of 0.42 M H3BO3 to 6.2 with a 0.17 M Na3-
PO4 solution), 0.06 mL of 4-hydroxyphenylpyruvic acid (1.8
mM, in 0.2 M Na3PO4 buffer), 0.03 mL of dichlorophenolindo-
phenol (reduced form, prepared by mixing 1 mL of 3.3 mM
sodium dichlorophenolindophenol in H2O and 0.16 M gluta-
thione in 0.2 M sodium phosphate buffer), and 0.01 mL of
phenylpyruvate tautomerase (10U/mL, Sigma). The above
solution was equilibrated for about 15 min (monitored at 308
nm); then the 4-HPPD to be tested was added (0.05 mL
solution). For calculation of dioxygenase activity the change
in absorbance between the 8th and 10th min was used. The
inhibition reaction of NTBC and 1-9 with the enzyme 4-HPPD
was evaluated by measuring the decrease in absorbance at 308
nm over a 15 min period following coadministration of varying
concentrations of the inhibitors and 4-HPPD. The IC50 values
were determined by fitting the data to the equation vi ) v0/(1
+ [inhibitor]/IC50), where vi is the rate of absorbance change
at a given inhibitor concentration and v0 the rate of absorbance
change in the absence of inhibitor.
were performed by means of Insight II software using the
CFF91 force field. Modeling to predict the binding mode of
NTBC to 4-HPPD (Figure 5) was carried out on a SGI R5000
workstation. Building and calculations were done with Insight
II software according to the following protocol. First, the
hydrogen atoms were added to the structure of one monomer.
Second, the energy minimization via Discover took place with
the following specifications: consistent valence force field
(CVFF),17 10 Å cutoff distance, implicit solvent, and distance-
dependent dielectric constant ꢀ ) 4r. Third, the acetate
molecule and three water molecules (wat5, wat25, and wat88)
bound in the active site were removed and one NTBC molecule
was included. Fourth, the ligand and the protein side chains
in a spherical domain centered on the iron atom and defined
by a 10 Å radius were submitted to a 1000 K simulated
annealing procedure; five constraints corresponding to the iron
coordination bonds with His161, His240, Glu322, and the two
oxygen atoms of the 1,3-diketone moiety of the NTBC molecule
were set in the range 1.8-2.3 Å. Finally, the best energy
structure was energy minimized without any aggregate and
without constraints.
3-Hyd r oxy-2-(2-n itr oben zoyl)cycloh ex-2-en -1-on e (1).
This compound was prepared by the literature procedure6 to
give a light yellow solid with a yield of 85%; mp 135-137 °C.
Rf ) 0.53 (25% EtOAc/hexanes). 1H NMR (CDCl3): δ 16.61 (bs,
1H, OH), 8.19 (dd, J ) 8.4, 1.2 Hz, 1H, Ar H), 7.69 (td, J )
7.5, 1.2 Hz, 1H, Ar H), 7.56 (td, J ) 8.4, 1.5 Hz, 1H, Ar H),
7.23 (dd, J ) 7.2, 1.5 Hz, 1H, Ar H), 2.77 (t, J ) 6.3 Hz, 2H,
6-CH2), 2,34 (t, J ) 6.3 Hz, 2H, 4-CH2), 2.00 (quintet, J ) 6.3
Hz, 2H, 5-CH2). 13C NMR (CDCl3): δ 197.6 (C-1), 195.5 (C-3),
193.8 (CdO), 145.4, 136.4, 134.0, 129.5, 126.6, 123.5 (Ar C’s),
112.7 (C-2), 37.4 (C-6), 31.7 (C-4), 19.0 (C-5). IR (KBr): v 3200-
2800 (OH), 1666 (CdO), 1581 (CdO), 1562, 1345 (NO2) cm-1
.
Anal. (C13H11NO5) C, H, N.
2-(2-Nitr oben zoyl)cycloh ex-1-en ol (2). A solution of 1-mor-
pholinocyclohexene (1.0 g, 5.98 mmol, prepared by the reported
literature procedure10) and triethylamine (0.83 mL, 5.98 mmol)
in chloroform (25 mL) was cooled to -10 °C, and a solution of
2-nitrobenzoyl chloride (1.1 g, 5.98 mmol) in 10 mL of
chloroform was added dropwise. The mixture was stirred at
room temperature overnight. Then, 2.0 mL of 4 N hydrochloric
acid was, added and the solution was refluxed for 5 h. The
organic layer was separated and washed with water (to pH
5). The aqueous layer was neutralized and extracted with
chloroform. The combined organic extracts were dried, con-
centrated, and purified by column chromatography to give a
white solid with a yield of 65%; mp 90-92 °C. Rf ) 0.59 (20%
Molecu la r Mod elin g. The atomic coordinates of 4-HPPD
were obtained from the Protein Data Bank maintained by the
Research Collaboratory for Structural Bioinformatics (RCSB)
under the accession code 1cjx. The construction of molecular
models, structure optimization, and conformational analysis
1
EtOAc/hexanes). H NMR (CDCl3): δ 15.05 (s, 1H, OH), 8.19
(dd, J ) 8.1, 1.2 Hz, 1H, Ar H), 7.74 (td, J ) 8.4, 1.2 Hz, 1H,
Ar H), 7.59 (td, J ) 8.1, 1.5 Hz, 1H, Ar H), 7.36 (dd, J ) 8.4,

4-HPPD Inhibitions by Triketone-type Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2227
1.5 Hz, 1H, Ar H), 2.46 (t, J ) 6.6 Hz, 2H, 6-CH2), 1.94 (t, J )
5.7 Hz, 2H, 3-CH2), 1.75-1.68 (m, 2H, 5-CH2), 1.60-1.56 (m,
2H, 4-CH2). 13C NMR (CDCl3): δ 195.0 (CdO), 182.0 (C-1),
145.4, 134.7, 134.2, 129.9, 127.9, 124.3 (Ar C’s), 106.7 (C-2),
30.6 (C-6), 24.4 (C-3), 22.5 (C-4), 21.3 (C-5). â-Diketone:enol
ratio: 97:3. IR (KBr): v 3200-2800 (OH), 1620 (CdO), 1574,
1344 (NO2) cm-1. Anal. (C13H13NO4) C, H, N.
2H, 5-CH2). 13C NMR (CDCl3): δ192.3 (C-1), 166.8 (CdN),
164.1 (C-3), 148.1, 133.0, 132.0, 131.6, 124.9, 121.5 (Ar C’s),
113.5 (C-2), 38.9 (C-6), 22.2 (C-4), 21.3 (C-5). IR (KBr): v 3100-
2900 (OH), 1669 (CdO), 1545, 1351 (NO2) cm-1. Anal.
(C13H12N2O5) C, H, N.
1-(2-Nitr oben zoyl)p ip er id in e-2,6-d ion e (7). To a solution
of glutarimide (500 mg, 4.42 mmol) in methylene chloride (20
mL) was added triethylamine (0.65 mL, 4.66 mmol), and then
dropwise 2-nitrobenzoyl chloride (freshly prepared and dis-
solved in 10 mL of CH2Cl2, 820 mg, 4.42 mmol) at ice-bath
temperature. The mixture was refluxed for 3 h, and the
reaction was then quenched with water. The product was
extracted twice with methylene chloride. The combined organic
extracts were dried over MgSO4, filtered, and concentrated.
The resulting crude product was purified by column chroma-
tography to give a white solid with a yield of 50%; mp 155-
3-Ch lor o-2-(2-n itr oben zoyl)-1,3-cycloh exa d ien -1-ol (3).
To a solution of vinyl chloride 5 (500 mg, 1.80 mmol) in acetone
(10 mL) was added potassium carbonate (500 mg, 3.6 mmol)
and a catalytic amount of tetra-n-butylammonium iodide (50
mg, 0.14 mmol) at room temperature. The resulting mixture
was stirred at that temperature for 2 h. After acetone was
removed under reduced pressure, the residue was dissolved
in water. The aqueous layer was neutralized and extracted
with ethyl acetate. The combined organic extracts were dried
over MgSO4, filtered, and concentrated, and the residue was
purified by column chromatography to give a light brown solid
with a yield of 32%; mp 80-82 °C (dec.). Rf ) 0.81 (10% EtOAc/
1
156 °C. Rf ) 0.21 (50% EtOAc/hexanes). H NMR (CDCl3): δ
7.90-7.66 (m, 4H, Ar H’s), 2.72 (t, J ) 6.6 Hz, 4H, 3-CH2,
5-CH2), 2.04 (quintet, J ) 6.6 Hz, 2H, 4-CH2). 13C NMR
(CDCl3): δ 171.1 (CdO), 166.6 (CdO), 148.2, 133.1, 132.9,
130.9, 129.3, 124.0 (Ar C’s), 32.8 (C3, 5), 16.8 (C-4). IR (KBr):
v 1755 (CdO), 1695 (CdO), 1576, 1337 (NO2) cm-1. Anal.
(C12H10N2O5) C, H, N.
1
hexanes). H NMR (CDCl3): δ 16.43 (s, 1H, OH), 8.16 (d, J )
6.9 Hz, 1H, Ar H’s), 7.70-7.58 (m, 2H, Ar H’s), 7.42 (dd, J )
7.2, 1.5 Hz, 1H, Ar H), 5.84 (t, J ) 5.4 Hz, 1H, 4-CH), 2.66 (t,
J ) 7.2 Hz, 2H, 6-CH2), 2.40 (td, J ) 7.2, 5.4 Hz, 2H, 5-CH2).
13C NMR (CDCl3): δ 189.2 (CdO), 181.7 (C-1), 147.1, 136.8,
133.2, 131.3, 130.7, 124.4 (Ar C’s), 128.6 (C-4), 124.9 (C-3),
107.3 (C-2), 33.1 (C-6), 27.0 (c-5). Anal. (C13H10ClNO4) C, H,
N.
2-(2-Nitr oben zyl)-3-h yd r oxycycloh ex-2-en on e (8). To a
solution of 1,3-cyclohexadione (1.0 g, 8.93 mmol) in methylene
chloride (30 mL) was added triethylamine (1.25 mL, 8.89
mmol) and 2-nitrobenzyl bromide (2.0 g, 9.26 mmol) at 0 °C.
The resulting mixture was stirred at room temperature for 1
day. After the reaction was completed, the products were
washed with water. The combined organic extracts were dried
over MgSO4, filtered, and concentrated, and the residue was
purified by column chromatography to give a major white solid
3-(2-nitrobenzyloxy)-2-cyclohexen-1-one (12) with a yield of
3-Meth oxy-2-(2-n itr oben zoyl)-2-cycloh exen -1-on e (4).
To a solution of 1 (1.0 g, 3.85 mmol) in ethyl acetate (25 mL)
was added dropwise diazomethane (3.85 mmol, dissolved in
ether, generated by Diazald) at 0 °C. The resulting solution
was allowed to stand at that temperature until the precipita-
tion of the product was complete. After filtration, the crude
product was recrystalized in ethyl acetate to give a white solid
with a yield of 89%; mp 137-138 °C. Rf ) 0.38 (100% EtOAc).
1H NMR (CDCl3): δ 7.85 (d, J ) 8.1 Hz, 1H, Ar H), 7.65-7.53
(m, 3H, Ar H’s), 3.91 (s, 3H, OMe), 2.70 (t, J ) 6.3 Hz, 2H,
4-CH2), 2.34 (t, J ) 6.3 Hz, 2H, 6-CH2), 2.03 (quintet, J ) 6.3
Hz, 2H, 5-CH2). 13C NMR (CDCl3): δ 195.2 (CdO), 190.4 (Cd
O), 180.4 (C-3), 147.1, 137.4, 133.0, 130.4, 129.7, 123.2 (Ar C’s),
118.9 (C-2), 56.9 (OMe), 36.5 (C-6), 26.2 (C-4), 19.8 (C-5). Anal.
(C14H13NO5) C, H, N.
1
59%; mp 71-72 °C. Rf ) 0.68 (50% EtOAc/hexanes). H NMR
(CDCl3): δ 8.16 (d, J ) 7.8 Hz, 1H, Ar H), 7.70-7.66 (m, 2H,
Ar H’s), 7.55-7.20 (m, 1H, Ar H), 5.49 (s, 1H, 2-CH), 5.33 (s,
2H, benzylic H’s), 2.53 (t, J ) 6.3 Hz, 2H, 4-CH2), 2.39 (t, J )
6.3 Hz, 2H, 6-CH2), 2.04 (quintet, J ) 6.3 Hz, 2H, 5-CH2). A
minor desired white solid 8 with a yield of 31%; mp 170-171
1
°C. Rf ) 0.83 (50% EtOAc/hexanes). H NMR (acetone-d6): δ
7.80 (dd, J ) 8.1, 1.5 Hz, 1H, Ar H), 7.51 (td, J ) 7.5, 1.2 Hz,
1H, Ar H), 7.39-7.31 (m, 2H, Ar H’s), 3.87 (s, 2H, benzylic
H’s), 2.46 (t, J ) 6.3 Hz, 2H, 6-CH2), 2.05 (t, J ) 6.3 Hz, 2H,
4-CH2), 1.98 (quintet, J ) 6.3 Hz, 2H, 5-CH2). 13C NMR
(CDCl3): δ 199.3 (CdO), 176.5 (C-3), 146.8, 133.8, 131.2, 128.8,
128.5, 125.0 (Ar C’s), 103.7 (C-2), 66.9 (benzylic C), 36.5 (C-6),
28.5 (C-4), 21.0 (C-5). IR (KBr): v 2930-3065 (OH), 1723 (Cd
O), 1576, 1378 (NO2) cm-1. Anal. (C13H13NO4) C, H, N.
1-(2-Nitr oben zyl)p ip er id in e-2,6-d ion e (9). To a solution
of glutarimide (500 mg, 4.42 mmol) in acetone (10 mL) was
added 2-nitrobenzyl bromide (955 mg, 4.42 mmol), potassium
carbonate (800 mg, 5.79 mmol), and a catalytic amount of
tetra-n-butylammonium iodide (50 mg, 0.14 mmol). The re-
sulting mixture was stirred at room temperature for 24 h. After
acetone was removed under reduced pressure, the residue was
dissolved in water. The aqueous layer was neutralized with
saturated ammonium chloride solution and extracted with
ethyl acetate. The combined organic extracts were dried over
MgSO4, filtered, and concentrated. The residue was purified
by column chromatography to give a white solid with a yield
of 92%; mp 92-93 °C. Rf ) 0.26 (50% EtOAc/hexanes). 1H NMR
(CDCl3): δ 7.96 (dd, J ) 8.4, 1.2 Hz, 1H, Ar H), 7.53 (td, J )
7.8, 1.2 Hz, 1H, Ar H), 7.39 (td, J ) 8.4, 1.2 Hz, Ar H), 7.14
(dd, J ) 7.8, 0.9 Hz, 1H, Ar H), 5.32 (s, 2H, N-CH2), 2.74 (t, J
) 6.6 Hz, 4H, 3, 5-CH2), 2.04 (quintet, J ) 6.6 Hz, 2H, 4-CH2).
13C NMR (CDCl3): δ 172.3 (C-2), 148.8, 133.2, 132.3, 127.9,
127.7, 124.9 (Ar C’s), 39.8 (benzylic C), 32.7 (C-3, 5), 17.0 (C-
4). IR (KBr): v 1731 (CdO), 1578, 1378 (NO2) cm-1. Anal.
(C12H12N2O4) C, H, N.
3-Ch lor o-2-(2-n itr oben zoyl)-2-cycloh exen -1-on e (5). A
solution of 1 (1.0 g, 3.84 mmol) in oxalyl chloride (5 mL) was
stirred at room temperature for 3 h. The excess oxalyl chloride
was removed at reduced pressure. The residue was dissolved
in methylene chloride. The solution obtained was washed with
saturated sodium bicarbonate solution, and then with water,
and dried with magnesium sulfate. The solvent was removed
on a rotary evaporator to obtain a crude product, which was
then recrystallized in ethyl acetate to give quantitatively a
yellow solid; mp 120-121 °C. Rf ) 0.35 (40% EtOAc/hexanes).
1H NMR (CDCl3): δ 7.84 (d, J ) 8.7 Hz, 1H, Ar H), 7.71-7.64
(m, 3H, Ar H’s), 2.88 (t, J ) 6.3 Hz, 2H, 4-CH2), 2.50 (t, J )
6.3 Hz, 6-CH2), 2.12 (quintet, J ) 6.3 Hz, 2H, 5-CH2). 13C NMR
(CDCl3): δ 193.4 (C-1), 188.7 (CdO), 159.1 (Ar C), 148.2 (C-
3), 136.3 (Ar C), 133.3 (C-2), 132.8, 132.4, 130.6, 123.7 (Ar C’s),
36.8 (C-6), 35.4 (C-4), 21.3 (C-5). IR (KBr): v 1687 (CdO), 1678
(CdO), 1536, 1366 (NO2), 762 (C-Cl) cm-1. Anal. (C13H10
ClNO4) C, H, N.
-
2-[(2-Nitr oph en yl)-1-h ydr oxyim in om eth yl)]-3-h ydr oxy-
cycloh ex-2-en on e (6). To a solution of 1 (500 mg, 1.94 mmol)
in water (30 mL) was added hydroxylamine hydrochloride
(NH2OH HCl, 272 mg, 3.91 mmol), and sodium methoxide (211
mg, 3.91 mmol) at 0 °C. The mixture was stirred at room
temperature for 2 days. After completion of the reaction, the
solution was neutralized to pH 7. The product was extracted
twice with methylene chloride. The combined organic extracts
were dried over MgSO4, filtered, and concentrated. The result-
ing crude product was purified by column chromatography to
give a white liquid with a yield of 75%; mp 113-115 °C. Rf )
0.48 (40% EtOAc/hexanes). 1H NMR (CDCl3): δ 8.17-8.14 (m,
1H, Ar H), 7.76-7.71 (m, 3H, Ar H’s), 3.01 (t, J ) 6.0 Hz, 2H,
4-CH2), 2.53 (t, J ) 6.0 Hz, 6-CH2), 2.20 (quintet, J ) 6.0 Hz,
Ack n ow led gm en t. This work was supported by the
National Science Council of the Republic of China.
Su p p or tin g In for m a tion Ava ila ble: X-ray crystallo-
graphic data and structure refinement and bond lengths and

2228 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
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