SAR studies of 3-cyclopropanecarbonyloxy-2-cyclohexen-1-one as inhibitors of 4-hydroxyphenylpyruvate dioxygenase

Article abstract of DOI:10.1016/S0968-0896(01)00322-4

Various 3-cyclopropanecarbonyloxy-2-cyclohexen-1-one 1 derivatives have been synthesized and tested as inhibitors of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) from pig liver. The inhibition results indicated that well-positioned dicarbonyl groups as well as the cyclopropyl group of 1 were essential for potent inhibition. Substitution at the 2-position of the ring system has a significant effect on inhibitor potency, while the 5-position can undergo substantial variations and retain inhibitor potency. In the compounds examined, 2-chloro substituted 12 is the best inhibitor of all with IC₅₀ of 15 nM, the rest of the synthesized analogues were less potent inhibitors than the parent compound.

Full text of DOI:10.1016/S0968-0896(01)00322-4

Bioorganic & Medicinal Chemistry 10 (2002) 685–690
SAR Studies of 3-Cyclopropanecarbonyloxy-2-cyclohexen-1-one
as Inhibitors of 4-Hydroxyphenylpyruvate Dioxygenase
Yung-Lung Lin, Chung-Shieh Wu, Shean-Woei Lin, Jian-Lin Huang,
Yang-Sheng Sun and Ding-Yah Yang*
Department of Chemistry, Tunghai University, 181, Taichung-Kang Rd. Sec.3, Taichung, Taiwan 40704, ROC
Received 25 June 2001; accepted 8 September 2001
Abstract—Various 3-cyclopropanecarbonyloxy-2-cyclohexen-1-one 1 derivatives have been synthesized and tested as inhibitors of
4-hydroxyphenylpyruvate dioxygenase (4-HPPD) frompig liver. The inhibition results indicated that well-positioned dicarbonyl
groups as well as the cyclopropyl group of 1 were essential for potent inhibition. Substitution at the 2-position of the ring system
has a significant effect on inhibitor potency, while the 5-position can undergo substantial variations and retain inhibitor potency. In
the compounds examined, 2-chloro substituted 12 is the best inhibitor of all with IC50 of 15 nM, the rest of the synthesized analo-
gues were less potent inhibitors than the parent compound. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
4-Hydroxyphenylpyruvate dioxygenase (4-HPPD)1 is an
important enzyme involved in the catabolism of tyrosine
and phenylalanine in most organisms which catalyzes
the conversion of 4-hydroxyphenylpyruvate to homo-
gentisate. The biotransformations catalyzed by 4-HPPD
require an oxidative decarboxylation 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 In the past 10 years, 4-
HPPD has been the target site to develop effective drug
therapy for the fatal hereditary disease tyrosinemia type
I3 which is characterized by a deficiency of fumaryl-
acetoacetase activity that leads to the accumulation of
hepatotoxic and nephrotoxic compounds like succiny-
lacetone.4 Inhibition of liver 4-HPPD activity will pre-
vent the formation of homogentisate, thereby blocking
tyrosine catabolism. Currently, 2-(2-(nitro-4-tri-
fluoromethylbenzoyl)-cyclohexane-1,3-dione5 (NTBC),
a potent 4-HPPD inhibitor with a triketone type struc-
ture, has been on the clinical trial as the first effective
drug therapy to treat tyrosinemia type I patients.
Recently, we reported6 the discovery of a new potent,
low molecular weight, non-triketone type 4-HPPD inhi-
bitor 3-cyclopropanecarbonyloxy-2-cyclohexen-1-one 1.
Inhibition studies indicated that compound 1 was a potent
4-HPPD inhibitor with IC50=30 nM, which is compar-
able to that of NTBC (IC50=40 nM). Here we report the
structure–activity relationships of the parent compound 1
as inhibitors of 4-HPPD in an effort to provide insights
into its mode of action at the molecular level.
Results and Discussion
The molecules designed and synthesized for SAR are
compounds 2-–16, shown in Figure 1, Scheme 1 and 3.
Vinyl ether 2 was prepared by acid-catalyzed dehydration
of cyclohexanedione and cyclopropanemethanol in ben-
zene at 140 ꢀC for 6h. Ester 3 was synthesized in two steps
by first converting cyclohexanone to the corresponding
*Corresponding author. Tel.:+886-4-2359-7613; fax: 886-4-2359-
0426; e-mail: yang@mail.thu.edu.tw
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00322-4

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Y.-L. Lin et al. / Bioorg. Med. Chem. 10 (2002) 685–690
shown in Scheme 2 according to the literature proce-
dures,8 followed by the same coupling reaction men-
tioned above. Base-catalyzed self-condensation of the
commercially available diethyl acetonylsuccinate affor-
ded 5-ethoxycarbonyl-cyclohexane-1,3-dione,9 which
upon treatment with cyclopropanecarbonyl chloride
under basic conditions gave rise to compound 14 with
57% combined yield. The synthesis of compounds 14
and 15 was started by first introducing an aldehyde
functional group onto 1,3,5-trimethoxybenzene via the
Vilsmeier10 reaction as illustrated in Scheme 3. The
resulting aldehydes were then oxidized under Baeyer–
Villiger11 conditions to produce phenols 23a and 23b.
After coupling with cyclopropanecarbonyl chloride as
described above, the esters 24a and 24b were exposed to
silver oxide and aqueous 6 N nitric acid in acetone to
afford the final benzoquinones 15 and 16 with 48 and
35% overall yield, respectively.
Scheme 1. Synthesis of compounds 2 and 3: (i) p-TsOH, benzene,
reflux; (ii) Et3N, trimethylsilyl chloride, DMF, 120 ꢀC; (iii) potassium
t-butoxide, cyclopropanecarbonyl chloride, THF, ꢁ20 ꢀC.
All compounds described herein were tested for their
ability to inhibit the activity of 4-hydroxyphenyl-
pyruvate dioxygenase using the spectrophotometric enol
borate method described by Lindstedt and Rundgren.12
Results of the inhibition reactions of these compounds
with pig liver 4-HPPD are summarized in Table 1. The
importance of the position of the two carbonyl groups
of 1 was investigated by comparing analogues 2 and 3.
When the ring carbonyl group of 1 was removed to
form 3, the inhibition potency decreased 23-fold relative
to 1 indicating that the ring carbonyl group is essential
for activity. In addition, replacement of the cyclic a,b-
unsaturated keto moiety of 1 with a phenol derivative 7
also resulted in significant losses in potency. In the case
in which the ester carbonyl group of 1 was removed to
form 2, no enzyme inhibition was observed up to the
concentration of 0.2 mM. This result suggests the car-
bonyl oxygen atomon the ester functionality is crucial
for binding, presumably by chelating with ferric ion in
the enzyme active site.13 The role played by the sub-
stitution on the six-membered ring of 1 toward 4-HPPD
inhibition was explored by investigation of the inhibi-
tion activity of compounds 4–16. Contraction of the six-
membered ring to the five-membered ring of 1 to 5,
fusion a benzene ring to the 4- and 5-position of the ring
systemto a 4-hydroxycoumarin derivative 6, as well as
the dimethyl and ethoxycarbonyl substitutions at 5-
position of the ring systemof 1 to compounds 4 and 14
Scheme 2. Preparation of compounds 17–21.
trimethylsilyl enol ether,7 followed by treatment with
cyclopropanecarbonyl chloride in the presence of
potassium t-butoxide in THF at ꢁ20 ꢀC with 63%
overall yield. Compounds 4–7 were easily prepared in
one step by a standard coupling reaction of cyclopro-
panecarbonyl chloride in the presence of triethylamine
as a base in methylene chloride with 5,5-dimethylcyclo-
hexane-1,3-dione, cyclopentane-1,3-dione, resorcinol,
and 4-hydroxy-coumarin, respectively. Preparation of
compounds 8–13, however, called for first preparing the
appropriate cyclohexane-1,3-dione derivatives 17–21
Table 1. Inhibition constants for reactions of 1–16 with 4-HPPD
frompig liver by the enol borate method
Compound
IC50 (mM)a
Compound
IC50 (mM)
1
2
3
4
5
6
7
8
0.03 (7.52)b
NAc
0.7 (6.15)
0.33 (6.48)
0.07 (7.15)
0.11 (6.96)
NA
9
5.6 (ꢁ0.75)
135 (ꢁ2.13)
20 (ꢁ1.30)
0.015 (7.82)
0.028 (7.55)
0.1 (7.00)
10
11
12
13
14
15
16
25 (ꢁ1.40)
92 (ꢁ1.96)
30 (ꢁ1.48)
aAssay detail is described in Experimental. Values are the means of at
least three independent determinations.
bpIC50 unit is given in parentheses.
cNA, not active; IC50> 200 mM.
Figure 1. Various compounds synthesized for SAR studies.

Y.-L. Lin et al. / Bioorg. Med. Chem. 10 (2002) 685–690
687
Figure 2. General SAR for 3-cyclopropanecarbonyloxy-2-cyclohexen-
1-one inhibitors.
propyl group of 3-cyclopropanecarbonyloxy-2-cyclo-
hexen-1-one were required for potent 4-HPPD
inhibition. Substitution at the 2-position of ring system
has a significant effect on inhibitor potency, while the 5-
position can undergo substantial variations and retain
inhibitor potency. The ability to vary the 5-position of
compound 1 should become important as this class of 4-
HPPD inhibitors undergo further development, as it
may be possible to vary the in vivo profile of these
inhibitors without affecting inhibitor potency. The
results of our studies will provide an alternate treatment
for the fatal disease tyrosinemia type I.
Scheme 3. Preparation of compounds 15 and 16: (i) POCl3, DMF; (ii)
H2O2, H2SO4, MeOH; (iii) cyclopropanecarbonyl chloride, Et3N,
CH2Cl2; (iv) AgO, 6 N HNO3, acetone.
all have moderate effects on inhibition potency, but
modifications at the 2-position of the ring system gave
more variable results. When the 2-substituents were a
chlorine or bromine atom, the resulting 12 or 13 were as
potent as 1, whereas bulkier and less electronegative
substituents like methyl (9) or aryl groups (10 and 11)
led to a major reduction of activity. These results
implied that both the steric and electrostatic effects of
these substituents at the 2-position of the ring system
might promote a conformational change to give a lower
binding affinity for the 4-HPPD enzyme. Furthermore,
the observation of 3-order of magnitude reduction of
inhibition potency when the ester bond of 1 was replaced
with the amide functionality might be attributed to the
resulting conformational-constrained structure 8 that
prevents tight binding with the enzyme active site.
Experimental
General
Melting points were determined on a Mel-Temp melting
point apparatus in open capillaries and are uncorrected.
Ultraviolet-visible spectroscopy was performed on a
Hewlett-Packard 8453 spectrophotometer. High resolu-
tion FAB-MS was measured with a JEOL JMS-SX/SX
1
Analogue 15 explored the effect of an additional oxo
group at C-4 position when the six-membered ring was
replaced by a benzoquinone moiety. In this case, inhi-
bition potency of 15 decreased 833-fold (IC50=25 mM)
as compared to 1, and 227-fold as compared to 6. The
results indicate that incorporation of a small but
strongly electronegative atomlike oxygen at 4-position
of the ring systemof 1 is highly detrimental to the inhi-
bition activity, whereas incorporation of a large, planar,
but less electronegative aromatic moiety like benzene at
4- and 5-positions of the ring (6) is not. We reasoned
that the presence of a C-4 oxo group in the ring system
might interfere with either of the two carbonyl groups in
the molecule to chelate with the active site ferric ion and
thus result in a weak affinity with the enzyme 4-HPPD,
although more investigation is needed to confirm this
assumption. Finally, further incorporation of a methoxy
substituent on the 6-position of the benzoquinone moiety
(16) resulted in little improvement on inhibition potency.
102A spectrometer. H NMR spectra were recorded at
300 MHz on a Varian VXR300 spectrometer. Chemical
shifts were reported in ppmon the d scale relative to
internal standard (tetramethylsilane, or appropriate
solvent peaks) with coupling constants given in hertz.
1H NMR multiplicity data were denoted by s (singlet), d
(doublet), t (triplet), q (quartet), m(multiplet), and b
(broad). Flash chromatography was performed in col-
umns of various diameters with Merck silica gel (230–
400 mesh ASTM 9385 kieselgel 60H) by elution with the
solvents reported. Analytical thin-layer chromato-
graphy (TLC) was carried out on Merck silica gel 60F-
254 plates (0.2 mm) and developed with the solvents
mentioned. All new compounds exhibited satisfactory
spectroscopic and analytical data.
Enzyme purification and assay
4-HPPD was purified frompig liver by the method of
Hamilton14 with a specific activity of 0.1 mmol minꢁ1
mgꢁ1. The protein concentration was determined by
BCA method. Routine assay of the enzyme utilized the
spectrophotometric enol borate method of Lindstedt
and Rundgren.12 The typical assay mixtures contained
0.85 mL potassium phosphate/borate buffer (prepared
by adjusting the pH of 0.42 M H3BO3 to 6.2 with a
0.17 M Na3PO4 solution), 0.06 mL 4-hydroxyphenyl-
Conclusion
As a result of this investigation, we now have a general
understanding of the SAR for this class of 4-hydro-
xyphenylpyruvate dioxygenase inhibitors, as depicted in
Figure 2. The two carbonyl groups as well as the cyclo-

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Y.-L. Lin et al. / Bioorg. Med. Chem. 10 (2002) 685–690
pyruvic acid (1.8 mM, in 0.2 M Na3PO4 buffer), 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 sodiumphosphate
buffer), 0.01 mL phenylpyruvate tautomerase (10 U/mL,
Sigma). The above solution was equilibrated for about
15 min (monitored at 308 nm), then 4-HPPD to be tes-
ted 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 1–16 with the enzyme 4-HPPD was evaluated by
measuring the decrease in absorbance at 308 nm over a
15min period following coadministration of varying con-
centration of the inhibitors and 4-HPPD. The IC50 values
were determined by fitting the data to the equation vi=vo/
(1+[inhibitor]/IC50, where vi is the rate of absorbance
change at a given inhibitor concentration and vo the rate
of absorbance change in the absence of inhibitor, and
converted into pIC50 units for modeling SAR.
dione (700 mg, 5 mmol) with triethylamine (1.4 mL,
10 mmol) in dry methylene chloride (30 mL). The mix-
ture was stirred at roomtemperature for 1 h, then
washed with water, diluted hydrochloric acid, saturated
aqueous sodiumhydrogen carbonate, and water. The
organic phase was dried with MgSO4 and evaporated to
dryness in vacuo to give the crude enol ester, which was
further purified by flash chromatography to give the
desired product.
3-Cyclopropanecarbonyloxy-5,5-dimethyl-2-cyclohexen-1
- one (4). This compound was obtained as a colorless
liquid with a 87% yield. Rf=0.30 (25% EtOAc/hex-
1
anes). H NMR (CDCl3) d 5.91 (s, 1H, 2-H), 2.42 (s,
2H, 6-H), 2.27 (s, 2H, 4-H), 1.75–1.68 (m, 1H, CH),
1.13–1.10 (m, 2H, CH2), 1,11 (s, 6H, 5-Me), 1.04–1.00
(m, 2H, CH2). HRMS: calcd for C12H16O3 208.1100,
found 208.1092.
3-Cyclopropanecarbonyloxy-2-cyclopenten-1-one (5)
3-Cyclopropylmethoxy-2-cyclohexen-1-one (2). To
a
solution of 1,3-cyclohexanedione (166 mg, 1.48 mmol) in
benzene (30 mL) was added cyclopropanemethanol
(107mg, 1.48mmol) and catalytic amounts of p-toluene-
sulfonic acid. The resulting mixture was heated to 140 ꢀC.
The by-product water was removed by the Dean–Stark
trap. The reaction was completed within 6 h, then
quenched with water. The vinyl ether was extracted with
ethyl acetate, dried in MgSO4, and concentrated in vacuo
to obtain the crude product, which was further purified by
column chromatography to give a colorless liquid with a
This compound was obtained as a colorless liquid with
a 70% yield. Rf=0.17 (50% EtOAc/hexanes). 1H NMR
(CDCl3) d 6.20 (s, 1H, 2-H), 2.79–2.75 (m, 2H, 5-H),
2.47–2.43 (m, 2H, 4-H), 1.81–1.76 (m, 1H, CH), 1.19–
1.16 (m, 2H, CH2), 1.11–1.06 (m, 2H, CH2). HRMS:
calcd for C9H10O3 166.0630, found 166.0635.
4-Cyclopropanecarbonyloxy-coumarin (6). This com-
pound was obtained as a brown liquid with a 75% yield.
1
Rf=0.39 (50% EtOAc/hexanes). H NMR (CDCl3) d
1
90% yield. Rf=0.54 (40% EtOAc/hexanes). H NMR
7.66 (d, J=7.8 Hz, 1H, Ar H), 7.59 (t, J=8.2 Hz, 1H,
Ar H), 7.38–7.28 (m, 2H, Ar H0s), 6.51 (s, 1H, 3-H),
1.99–1.59 (m, 1H, CH), 1.29–1.19 (m, 2H, CH2), 1.18–
1.15 (m, 2H, CH2). HRMS: calcd for C13H10O4
230.0579, found 230.0570.
(CDCl3) d 5.17 (s, 1H, 2-H), 3.54 (d, J=6.9Hz, 2H,
OCH2), 2.32–2.29 (m, 2H, 6-H), 2.23–2.20 (m, 2H, 4-H),
1.87–1.83 (m, 2H, 5-H), 1.10–1.02 (m, 1H, OCH2CH),
0.52–0.47 (m, 2H, CH2), 0.20–0.17 (m, 2H, CH2).
HRMS: calcd for C10H14O2 166.0994, found 166.0989.
3-Cyclopropanecarbonyloxy-phenol (7). This compound
was obtained as a light blue liquid with a 92% yield.
Rf=0.44 (25% EtOAc/hexanes). H NMR (CDCl3) d
7.18 (t, J=8.4 Hz, 1H, Ar H), 6.68–6.62 (m, 2H, Ar
H0s), 6.57 (s, 1H, Ar H), 5.90 (bs, 1H, OH), 1.85–1.80
(m, 1H, CH), 1.17–1.14 (m, 2H, CH2), 1.05–1.00 (m,
2H, CH2). HRMS: calcd for C10H10O3 178.0630, found
178.0636.
2-Cyclopropanecarbonyloxy-1-cyclohexene (3). A solu-
tion of potassium t-butoxide (560 mg, 5 mmol) in THF
(5 mL) was added to a stirred solution of 1-cyclohex-
enyloxy-trimethylsilane7 (850 mg, 5 mmol) in THF
(10 mL) at ꢁ78 ꢀC under nitrogen and the mixture was
stirred at this temperature for 1 h. After this, the tem-
perature was increased to ꢁ20 ꢀC, then a solution of
cyclopropanecarbonyl chloride (523 mg, 5 mmol) in
THF (5 mL) was added to it. After 15 min, the mixture
was quenched with saturated aqueous NaHCO3 and
extracted with Et2O. The extract was dried and evapo-
rated under reduced pressure and the crude product was
purified by flash chromatography on silica gel to give a
colorless liquid with a 70% yield. Rf=0.25 (100% hex-
anes). 1H NMR (CDCl3) d 6.59 (t, J=1.9 Hz, 1H, 2-H),
2.57–2.49 (m, 4H, 3-H, 6-H), 2.09–2.03 (m, 2H, 4-H),
1.80–1.75 (m, 1H, CH), 1.65–1.50 (m, 2H, 5-H), 1.14–
1.11 (m, 2H, CH2), 1.00–0.97 (m, 2H, CH2). HRMS:
calcd for C10H14O2 166.0994, found 166.0985.
1
N-(3-Oxo-cyclohex-1-enyl)-cyclopropanecarboxamide (8).
This compound was obtained from 3-amino-2-cyclo-
hexen-1-one15 17 as a brown solid with a 65% yield. Mp
177–178 ꢀC. Rf=0.40 (100% EtOAc). 1H NMR
(CDCl3) d 7.18 (bs, 1H, NH), 6.48 (s, 1H, 2-H), 2.62 (t,
J=6.0 Hz, 2H, 6-H), 2.38 (t, J=6.3 Hz, 2H, 4-H), 2.21–
2.10 (m, 2H, 5-H), 1.49–1.41 (m, 2H, CH), 1.10–1.07
(m, 2H, CH2), 0.91–0.87 (m, 2H, CH2). HRMS: calcd
for C10H13NO2 179.0947, found 179.0957.
3-Cyclopropanecarbonyloxy-2-methyl-2-cyclohexen-1-
one (9). This compound was obtained from 2-methyl-
cyclohexane-1,3-dione16 18 as a colorless liquid with a
General procedure for preparation of compounds 4–16,
24a, and 24b
1
87% yield. Rf=0.36 (25% EtOAc/hexanes). H NMR
(CDCl3) d 2.53–2.51 (m, 2H, 6-H), 2.46–2.42 (m, 2H, 4-
H), 2.04–1.99 (m, 2H, 5-H), 1.79–1.75 (m, 1H, CH),
1.67 (s, 3H, 2-CH3), 1.34–1.11 (m, 2H, CH2), 1.03–1.00
The cyclopropanecarbonyl chloride (523 mg, 5 mmol)
was added to a solution of 5,5-dimethylcyclohexane-1,3-

Y.-L. Lin et al. / Bioorg. Med. Chem. 10 (2002) 685–690
689
(m, 2H, CH2). HRMS: calcd for C11H14O3 194.0943,
found 194.0940.
product was purified by column chromatography to
give a gray solid with a yield of 81%. Mp 114–115 ꢀC.
Rf=0.48 (40% EtOAc/hexanes). H NMR (CDCl3) d
1
2 - p - Nitrophenyl - 3 - cyclopropanecarbonyloxy - 2 - cyclo-
hexen-1-one (10). This compound was obtained from 2-
p-nitrophenyl-cyclohexane-1,3-dione17 19 as a yellow
solid with a 75% yield. Mp 92–93 ꢀC. Rf=0.33 (40%
10.32 (s, 1H, CHO), 7.33 (s, 1H, Ar H), 6.50 (s, 1H, Ar
H), 3.98 (s, 3H, OMe), 3.93 (s, 3H, OMe), 3.88 (s, 3H,
OMe). HRMS: calcd for C10H12O4 196.0736, found
196.0740.
EtOAc/hexanes). 1H NMR (CDCl3)
d 8.21 (d,
J=9.0 Hz, 2H, Ar H0s), 7.31 (d, J=9.0 Hz, 2H, Ar H0s),
2.75 (t, J=6.0 Hz, 2H, 6-H), 2.62 (t, J=6.0 Hz, 2H, 4-
H), 2.18 (quintet, J=6.0 Hz, 2H, 5-H), 1.55–1.49 (m,
1H, CH), 0.90–0.85 (m, 4H, CH2CH2). HRMS: calcd
for C16H15NO5 301.0951, found 301.0946.
2,5-Dimethoxyphenol (23a). To a solution of commer-
cially available 2,5-dimethoxybenzaldehyde (196 mg,
1.00 mmol) was added dropwise 30% hydrogen perox-
ide (0.23 mL, 2.00 mmol) and sulfuric acid (0.05 mL).
The resulting mixture was then stirred at room tem-
perature for 2 h. After completion of the reaction, water
was added to the mixture and the solution was neu-
tralized to pH 7. The product was extracted with ethyl
acetate twice. The combined organic extracts were dried
over MgSO4, filtered, and concentrated. The resulting
syrup was purified by column chromatography to give a
yellow liquid with a yield of 65%. Rf=0.45 (40%
2 - o - Nitrophenyl - 3 - cyclopropanecarbonyloxy - 2 - cyclo-
hexen-1-one (11). This compound was obtained from 2-
o-nitrophenyl-cyclohexane-1,3-dione17 as a yellow solid
with a 85% yield. Mp 82–83 ꢀC. Rf=0.42 (40% EtOAc/
1
hexanes). H NMR (CDCl3) d 8.11 (dd, J=8.1, 1.5 Hz,
1H, Ar H), 7.61 (dt, J=7.5, 1.5 Hz, 1H, Ar H), 7.50 (dt,
J=8.4, 1.8 Hz, 1H, Ar H), 7.20 (dd, J=7.5, 1.8 Hz, 1H,
Ar H), 2.88–2.58 (m, 4H, 4-H, 6-H), 2.24–2.13 (m, 2H,
5-H), 1.53–1.45 (m, 1H, CH), 0.89–0.75 (m, 4H,
CH2CH2). HRMS: calcd for C16H15NO5 301.0951,
found 301.0935.
EtOAc/hexanes). 1H NMR (CDCl3)
d 6.78 (d,
J=8.7 Hz, 1H, Ar H), 6.56 (d, J=3.0 Hz, 1H, Ar H),
6.38 (dd, J=8.7, 3.0 Hz, 1H, Ar H), 5.67 (bs, 1H, OH),
3.84 (s, 3H, OMe), 3.75 (s, 3H, OMe). HRMS: calcd for
C8H10O3 154.0630, found 154.0634.
2-Chloro-3-cyclopropanecarbonyloxy-2-cyclohexen-1-one
(12). This compound was obtained from 2-chloro-
cyclohexane-1,3-dione18 20 as a yellow liquid with a
2, 4, 5-Trimethoxyphenol (23b). This compound was
obtained as a white solid with a yield of 69% following
the procedure of 23a. Mp 60–61 ꢀC. Rf=0.40 (50%
1
1
75% yield. Rf=0.23 (25% EtOAc/hexanes). H NMR
EtOAc/hexanes). H NMR (CDCl3) d 6.61 (s, 1H, Ar
(CDCl3) d 2.71–2.62 (m, 2H, 6-H), 2.60–2.58 (m, 2H, 4-
H), 2.11–2.03 (m, 2H, 5-H), 1.84–1.79 (m, 1H, CH),
1.26–1.18 (m, 2H, CH2), 1.16–1.03 (m, 2H, CH2).
HRMS: calcd for C10H11ClO3 214.0397, found 214.0403.
H), 6.58 (s, 1H, Ar H), 5.28 (bs, 1H, OH), 3.85 (s, 3H,
OMe), 3.84 (s, 3H, OMe), 3.82 (s, 3H, OMe). HRMS:
calcd for C9H12O4 184.0736, found 184.0739.
1 - Cyclopropanecarbonyloxy - 2,5 - dimethoxybenzene
(24a). This compound was obtained as a yellow liquid
with a yield of 92%. Rf=0.60 (40% EtOAc/hexanes).
1H NMR (CDCl3) d 6.89 (d, J=8.7 Hz, 1H, Ar H), 6.72
(dd, J=8.7, 3.0 Hz, 1H, Ar H), 6.65 (d, J=3.0 Hz, 1H,
Ar H), 3.38 (s, 3H, OMe), 3.75 (s, 3H, OMe), 1.91–1.83
(m, 1H, CH), 1.21–1.16 (m, 2H, CH2), 1.05–0.98 (m,
2H, CH2). HRMS: calcd for C12H14O4 222.0892, found
222.0880.
2-Bromo-3-cyclopropanecarbonyloxy-2-cyclohexen-1-one
(13). This compound was obtained from 2-bromo-
cyclohexane-1,3-dione19 21 as a yellow liquid with a
1
50% yield. Rf=0.26 (25% EtOAc/hexanes). H NMR
(CDCl3) d 2.71–2.61 (m, 4H, 4-H, 6-H), 2.12–2.04 (m,
2H, 5-H), 1.84–1.76 (m, 1H, CH), 1.23–1.20 (m, 2H,
CH2), 1.09–1.05 (m, 2H, CH2). HRMS: calcd for
C10H11BrO3 257.9891, found 257.9886.
3-Cyclopropanecarbonyloxy-5-ethoxycarbonyl-2-cyclo-
hexen-1-one (14). This compound was obtained from 5-
1 - Cyclopropanecarbonyloxy - 2,4,5 - trimethoxybenzene
(24b). This compound was obtained as a colorless
liquid with a yield of 75%. Rf=0.47 (50% EtOAc/hex-
ethoxycarbonyl-cyclohexane-1,3-dione9 as
liquid with a 67% yield. Rf=0.35 (35% EtOAc/hex-
a
yellow
1
anes). H NMR (CDCl3) d 6.64 (s, 1H, Ar H), 6.60 (s,
1
anes). H NMR (CDCl3) d 5.96 (s, 1H, 2-H), 4.18 (q,
1H, Ar H), 3.87 (s, 3H, OMe), 3.85 (s, 3H, OMe), 3.82
(s, 3H, OMe), 1.92–1.84 (m, 1H, CH), 1.19–1.03 (m, 2H,
CH2), 1.02–0.99 (m, 2H, CH2). HRMS: calcd for
C13H16O5 252.0998, found 252.0993.
J=7.2 Hz, 2H, OCH2CH3), 3.15–3.05 (m, 1H, 5-H),
2.93–2.55 (m, 4H, 4-H, 6-H), 1.77–1.71 (m, 1H, CH),
1.27 (t, J=7.2 Hz, 3H, OCH2CH3), 1.14–1.12 (m, 2H,
CH2), 1.06–1.01 (m, 2H, CH2). HRMS: calcd for
C13H16O5 252.0998, found 252.1002.
2-Cyclopropanecarbonyloxy-1,4-benzoquinone (15). To a
solution of 24a (127 mg, 0.64 mmol) in acetone was
added silver (II) oxide (372 mg, 3.00 mmol), and 6 N
nitric acid (1 mL) dropwise at room temperature. The
reaction was finished within 1 h. After acetone was
removed under reduced pressure, the residue was dis-
solved in water. The aqueous layer was neutralized with
sodiumbicarbonate and extracted with ether. The com-
bined organic extracts were dried over MgSO4, filtered,
concentrated, and the residue was purified by column
2, 4, 5-Trimethoxybenzaldehyde (22b). To a solution of
1, 2, 4-trimethoxybenzene (336 mg, 2.00 mmol) in DMF
(10 mL) was added dropwise phosphorus oxychloride
(307 mg, 2.00 mmol) at room temperature. The resulting
mixture was stirred at that temperature for 1 h. After
completion of the reaction, monitored by TLC, 100 mL
of water and 10% NaOH solution (20 mL) was added to
the solution. The precipitate was then filtered and the

690
Y.-L. Lin et al. / Bioorg. Med. Chem. 10 (2002) 685–690
chromatography to give a yellow solid with a yield of
80%. Mp 173–175 ꢀC. Rf=0.55 (40% EtOAc/hexanes).
1H NMR (CDCl3) d 7.26 (s, 1H, Ar H), 6.87 (s, 1H, Ar
H), 6.70 (s, 1H, Ar H), 1.91–1.84 (m, 1H, CH), 1.27–
1.20 (m, 2H, CH2), 1.15–1.09 (m, 2H, CH2). HRMS:
calcd for C10H8O4 192.0422, found 192.0428.
2. Jefford, C. W.; Cadby, P. Experimentia 1981, 37, 1134.
3. Lindblad, B.; Lindstedt, S.; Steen, G. Proc. Natl. Acad. Sci.
U.S.A. 1977, 74, 4641.
4. Puntoni, R.; Merlo, F.; Fini, A.; Meazza, L.; Santi, L.
Lancet 1986, ii, 525.
5. Lindstedt, S.; Holme, E.; Lock, E.; Hialmarson, O.;
Strandvik, B. Lancet 1992, 340, 813.
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2-Cyclopropanecarbonyloxy-5-methoxy-1,4-benzoquinone
(16). This compound was obtained as a yellow solid
with a yield of 83% following the procedure of 15. Mp
85–87 ꢀC. Rf=0.43 (50% EtOAc/hexanes). 1H NMR
(CDCl3) d 6.54 (s, 1H, Ar H), 5.95 (s, 1H, Ar H), 3.86 (s,
3H, OMe), 1.90–1.84 (m, 1H, CH), 1.26–1.20 (m, 2H,
CH2), 1.13–1.08 (m, 2H, CH2). HRMS: calcd for
C11H10O5 222.0528, found 222.0533.
7. Suginome, H.; Kondoh, T.; Gogonea, C.; Singh, V.; Goto,
H.; Osawa, E. J. Chem. Soc., Perkin Trans. 1, 1995, 69.
8. Montes, I. F.; Burger, U. Tetrahedron Lett. 1996, 37, 1007.
9. Wu, B.; Bai, D. J. Org. Chem. 1997, 62, 5978.
10. Vilsmeier, A.; Haack, A. Chem. Ber. 1927, 60, 119.
11. Baeyer, A.; Villiger, V. Chem. Ber. 1902, 35, 3013.
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704, 66.
13. Crouch, N. P.; Adlington, R. M.; Baldwin, J. E.; Lee, M.-
H.; MacKinnon, C. H. Tetrahedron 1997, 53, 6993.
14. Buckthal, D. J.; Roche, P. A.; Moorehead, T. J.; Forbes,
B. J. R.; Hamilton, G. A. Methods in Enzymol. 1987, 142, 132.
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1197.
Acknowledgements
This work was supported by the National Science
Council of the Republic of China.
16. Barrack, S. A.; Okamura, W. H. J. Org. Chem. 1986, 51,
3201.
17. Sole, D.; Bosch, J.; Bonjoch, J. Tetrahedron 1996, 52,
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References and Notes
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