Photobleaching Activity of 2-(Phenylamino)methylidenecyclohexane-1,3-diones in Tobacco (Nicotiana tabacum) Cultured Cells

Article abstract of DOI:10.1021/jf960962i

A series of phenylalkylidenecyclohexane-1,3-dione derivatives were designed and synthesized as vinylogous analogs of phthalimides, which are known photobleaching herbicides. The bleaching activity of synthesized compounds was assayed with photomixotrophic tobacco cultured cells under either light or dark conditions. Both the chlorophyll and the carotenoid contents of the cells treated with the cyclic diones decreased to almost zero within 24 h under the light condition but not under the dark condition. This rapid emergence of bleaching activity with light is one of the most distinctive actions of Protox-inhibiting herbicides; however, the cyclic dione did not inhibit protoporphyrinogen oxidase in vitro. Thus, we concluded that the cyclic diones possessed a different herbicidal mode of action from Protox-inhibiting herbicide.

Full text of DOI:10.1021/jf960962i

2728
J. Agric. Food Chem. 1997, 45, 2728−2734
P h otoblea ch in g Activity of
2-(P h en yla m in o)m eth ylid en ecycloh exa n e-1,3-d ion es in Toba cco
(Nicotia n a ta ba cu m ) Cu ltu r ed Cells
J ing-Ming Wang,† Tadao Asami,*,‡ Fan-Sik Che,§ Noboru Murofushi,† and Shigeo Yoshida‡
Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-Ku, Tokyo 113, J apan,
The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi,
Saitama 351-01, J apan, and Nara Institute of Science and Technology (NAIST), Takayama 8916-5,
Ikoma-shi, Nara 630-01, J apan
A series of phenylalkylidenecyclohexane-1,3-dione derivatives were designed and synthesized as
vinylogous analogs of phthalimides, which are known photobleaching herbicides. The bleaching
activity of synthesized compounds was assayed with photomixotrophic tobacco cultured cells under
either light or dark conditions. Both the chlorophyll and the carotenoid contents of the cells treated
with the cyclic diones decreased to almost zero within 24 h under the light condition but not under
the dark condition. This rapid emergence of bleaching activity with light is one of the most distinctive
actions of Protox-inhibiting herbicides; however, the cyclic dione did not inhibit protoporphyrinogen
oxidase in vitro. Thus, we concluded that the cyclic diones possessed a different herbicidal mode of
action from Protox-inhibiting herbicide.
Keyw or d s: Photobleaching herbicide; tobacco cells; chlorophyll biosynthesis
Photobleaching herbicides, such as photosystem I
disrupting herbicides, carotenoid biosynthesis inhibi-
tors, and chlorophyll biosynthesis inhibitors, are widely
used to protect crops. The physiological characteristic
point of these herbicides is their requirement for pho-
toirradiation of plants to exhibit their herbicidal activity.
To design a new lead compound for photobleaching
herbicides, we focused our attention on chlorophyll
biosynthesis inhibitors because the physiological effects
of these inhibitors have been well investigated. The
primary site for the action of these herbicides is proto-
porphyrinogen oxidase (Protox) (Matringe et al., 1989a;
Camadro et al., 1989, 1991; Nicolaus et al., 1993), which
is the last enzyme that is common to the biosynthetic
pathways of both heme and chlorophylls. Plants treated
with these herbicides accumulate large amounts of
protoporphyrin IX (proto IX), which readily generates
radicals upon exposure to light and oxygen and induces
membrane damage (Matringe et al., 1988; Sandmann
et al., 1988). Although these herbicides belong to
different chemical classes, such as phthalimides and
diphenyl ethers, biochemical investigations have dem-
onstrated that these herbicides all bind with Protox
(Matringe et al., 1992; Versano et al., 1990), which
suggests that these different classes of Protox inhibitors
share some common structural features. The search for
a common structural feature of known Protox inhibitors
has revealed that they should contain at least two
aromatic or alicyclic rings (Matringe et al., 1989b; Sato
et al., 1987), which could be a key factor in discovering
other novel inhibitors. X-ray crystallography of some
Protox inhibitors has further revealed that the struc-
tures of such inhibitors mimic protoporphyrinogen IX
(protogen IX) in that the two rings of the inhibitors
correspond to the pyrrole rings of protogen IX (Nandi-
halli et al., 1992). On the basis of these findings, we
designed new compounds which can be considered
vinylogues of phthalimide herbicides, since some bio-
active compounds retain their activity even after modi-
fication into vinylogues (McFadden et al., 1993). We
named the series of compounds in this paper the “RWH
series”.
The sensitivity of photomixotrophic cultured cells to
the stress of potential herbicides is expected to be
intermediate between those of enzymes and whole
plants. Since cultured cells retain essential cellular
functions to keep growing, they may provide a conve-
nient system for assaying phytotoxic action at the
cellular level. In particular, cultured tobacco cells have
been reported to be a good material for surveying new
photosynthetic electron transport inhibitors and pho-
tobleaching herbicides because they are easy to handle
and highly sensitive to herbicides (Kohno et al., 1994;
Sato et al., 1991). In this study, we used photomix-
otrophic tobacco cells as a plant material to measure
the photobleaching activity and in vitro Protox inhibi-
tory activity of the synthesized chemicals.
RESULTS AND DISCUSSION
Str u ctu r e-Activity Rela tion sh ip s in th e RWH
Ser ies. There have been several reports that the
chemical substituents on the aromatic ring play a
significant role in the photobleaching activity of known
Protox inhibitors. On the basis of these studies, we
introduced various substituents onto the aromatic ring
of phenylaminomethylidene-1,3-cyclohexanediones. The
effects of the ring substituents of the anilide moiety on
photobleaching activity are listed in Table 1. Compound
1, which has no substituent on the phenyl ring, is used
below as a standard of this activity for a discussion of
structure-activity relationships.
(1) Effect of the Substituent on the Phenyl Ring Moiety
of the RWH Series. The introduction of a chlorine atom
(3), methoxyl group (12), or butyloxycarbonyl group (19)
to the 4-position of the aromatic ring of compound (1)
did not give a good result for an enhancement of its
* To whom correspondence should be addressed.
† The University of Tokyo.
‡ RIKEN.
§ NAIST.
S0021-8561(96)00962-4 CCC: $14.00
© 1997 American Chemical Society

New Photobleaching Compounds
J. Agric. Food Chem., Vol. 45, No. 7, 1997 2729
Ta ble 1. Effect of th e Su bstitu en ts on th e P h en yl Moiety
of Cycloh exa n ed ion e Der iva tives on Blea ch in g Activity
Ta ble 3. Effect of th e Cyclic Dion e Moiety on Blea ch in g
Activity
compound
pI50
28
31
32
33
4.8
nda
nd
compound no.
X
pI50
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
H
2-Et
4-Cl
4.1
4.2
3.4
4.1
3.5
4.8
4.4
4.5
4.8
4.7
5.0
<3
4.6
4.7
4.7
4.3
3.7
<4
4.0
4.7
5.8
4.8
5.8
4.2
2,3-Cl2
2,4-Cl2
2,5-Cl2
3,4-Cl2
3,5-Cl2
2,4,6-Cl3
2,4,6-F3
2,6-Cl2-3-Me
4-methoxy
2-methoxycarbonyl
2-ethyloxycarbonyl
2-n-butyloxycarbonyl
2-pentyloxycarbonyl
2-n-hexyloxycarbonyl
2-n-heptyloxycarbonyl
4-n-butyloxycarbonyl
2-chloro-5-methoxycarbonyl
2-chloro-5-n-propyloxycarbonyl
2-chloro-5-(2-n-propenyloxycarbonyl)
5-chloro-2-n-propyloxycarbonyl
4-chloro-3-n-propyloxycarbonyl
nd
34
nd
Ta ble 2. Effect of Str u ctu r a l Mod ifica tion of th e
Alk ylid en e Moiety on Blea ch in g Activity
a
nd, not detected.
ylidene derivatives (28-30) were active, the ethylidene
derivatives (25-27) showed no activity.
The cyclic 1,3-dione derivatives synthesized in this
study can be considered vinylogous phthalimide deriva-
tives, but their need for substituents on the phenyl
group for exhibition of bleaching activity differs from
phthalimide-type Protox inhibitors. Examination of the
structure-activity relationships of phthalimide-type
Protox inhibitors revealed the importance of both fluo-
rine at position 2 and chlorine at position 4 on the
phenyl ring (Wakabayashi, 1988). On the other hand,
1,3-cyclohexanedione derivative 29 which possesses
such a 2-fluoro-4-chlorophenyl ring is less active than
3,4-dichlorophenyl (7) or 4-methoxyphenyl (12) deriva-
tives, although in phthalimide herbicides 3,4-dichlo-
rophenyl or 4-methoxyphenyl derivatives are far less
active than the 2-fluoro-4-chlorophenyl derivative.
(3) Effect of Modification of the Cyclic Dione Moiety
of the RWH Series. Table 3 shows the effect of the
structure of the cyclic dione moiety on bleaching activity.
Among the compounds listed, only the cyclohexanedione,
28, shows this activity.
Our new chemicals consist of three parts; i.e., phenyl,
methylidene, and cyclic dione moieties. With regard to
the phenyl and alkylidene parts, we have demonstrated
their importance in photobleaching activity. Consider-
ing the structural variation in the phthalimide part of
so-called phthalimide herbicides, new inhibitors might
show this activity after structural modification of the
cyclic dione part. Table 3 shows the activities of several
cyclic dione derivatives. Although all of the compounds
share some common chemical structure, the cyclic dione
moieties are different. Among these compounds, only
the cyclohexane-1,3-dione derivative 28 is active. Thus,
we have shown that new inhibitors should have a
2-phenylaminomethylidene-5,5-dimethylcyclohexane-
1,3-dione structure for the expression of bleaching acti-
compound no.
Y
X
pI50
25
26
27
28
29
30
CH3
CH3
CH3
H
H
H
2,6-Cl2
2-F-4-Cl
2,6-Et2
2,6-Cl2
2-F-4-Cl
2,6-Et2
nda
nd
nd
4.8
4.2
3.6
a
nd, not detected.
photobleaching activity. This result suggests that a
monosubstitution at the 4-position of the aromatic ring
of compound 1 may have a negative effect on this
activity regardless of the electronic character of the
substituent. The introduction of two chlorine atoms
increased photobleaching activity, except for compound
5, and the introduction of three chlorine or fluorine
atoms to the 2-, 4-, and 6-positions on the phenyl ring
(9, 10) also increased this activity. Methoxy, ethoxy,
butyloxy, and pentyloxycarbonyl derivatives (13-16),
when substituted at position 2, are more active than 1,
whereas hexyloxy and heptyloxy derivatives (17, 18) are
less active than 1. This suggests that an alkoxyl group
of a certain length is required for this activity: e.g.,
between an ethyl and a butyl group should be suitable.
The most active compounds synthesized were 21 and
23, in which one chlorine atom and one propylcarbonyl
group were introduced into positions 2 and 5, and vice
versa, of the phenyl ring.
(2) Effect of Modification of the Alkylidene Moiety of
the RWH Series. Table 2 shows the role of alkylidene
substitution on bleaching activity. The result indicates
that the alkylidene moiety plays a significant role in
producing the bleaching activity. While the meth-

2730 J. Agric. Food Chem., Vol. 45, No. 7, 1997
Wang et al.
F igu r e 2. Effects of 21 analogue, S-23142, and Fluridone on
the chlorophyll content of tobacco cells (N. tabacum cv. Samsun
NN; cell line NI). Tobacco cells (6 days old) were placed into
new culture medium containing 50 µM of 21 analogue (9,
incubated in darkness; b, incubated in light: 30 µE/m2s), 100
µM Fluridone (0, incubated in light: 30 µE/m2s), and 1 µM
S-23142 (O, incubated in light: 30 µE/m2s). The cell density
was 1 mg fresh weight/mL of culture medium.
F igu r e 1. General synthetic route for phenylaminoalkylidene-
1,3-cyclohexanedione analogues.
vity. Next, we studied the mode of action of cyclohex-
anedione derivatives by using compound 21, the most
potent inhibitor among the compounds synthesized.
Exa m in a tion of th e Mech a n ism of Action of
Com p ou n d 21. The cyclic dione derivatives can be
considered vinylogous phthalimide herbicides, but the
structure-activity relationships of the substituents on
the phenyl ring and the dione portion are different from
those of phthalimide herbicides. There are several
series of photobleaching herbicides which include a
phenyl group, but their requirements for the substitu-
ents on the phenyl ring for potent activity show wide
variety. Although the structure-activity relationships
of the substituents on the phenyl ring and the dione
portion of the cyclic dione derivatives are also different
from those of phthalimide herbicides, the diones de-
creased the chlorophyll content of cultured cells under
illumination, which is similar to the effects of photo-
bleaching herbicides. On the basis of this finding, we
analyzed the contents for both chlorophyll and caro-
tenoids after treatment with the newly synthesized
compounds under light or dark conditions to study the
mechanism of action of these compounds using tobacco
cells treated with compound 21 at a final concentration
of 50 µM or S-23142 at 1 µM, respectively. In the light
condition, the chlorophyll content of cells treated with
compound 21 or S-23142 slightly decreased after 12 h.
After 24 h, the tobacco cells showed almost no chloro-
phyll, whereas in the dark condition there was no
change in the content of chlorophyll (Figure 2). Similar
results were seen for carotenoids (Figure 3). These
results indicate that (1) cyclic diones require illumina-
tion for the emergence of bleaching activity and (2) cyclic
diones show a quick response, like other known photo-
bleaching herbicides. On the basis of these results, we
consider that cyclic diones most likely have a mode of
action similar to those of photobleaching herbicides. To
confirm this point, activity of 21 was enzymatically
assayed.
F igu r e 3. Effects of 21 analogue, S-23142, and Fluridone on
the carotenoid content of tobacco cells. Tobacco cells (6 days
old) were cultured in the presence of 21 analogue at a final
concentration of 50 µM (9, incubated in darkness, b, incubated
in light: 30 µE/m2s), 100 µM Fluridone (0, incubated in light:
30 µE/m2s), and 1 µM S-23142 (O, incubated in light: 30 µE/
m2s). The cell density was 1 mg fresh weight/mL of culture
medium.
gous moiety at the 2-position which cause photobleach-
ing in photomixotrophic tobacco cells. Among the
compounds synthesized, 21 was the most active, with a
pI50 of 5.8 in our assay system. This activity is lower
than that of the typical photobleaching herbicide acy-
fluorfen (pI50 ) 8.1); however, the structures of the cyclic
diones are new among photobleaching herbicides and
their herbicidal mode of action is different from that of
Protox inhibitor. These findings imply that the RWH
series could be a good lead for new herbicides possessing
new modes of action. Considering the structural and
physiological features of the RWH series, there would
be some possibilities that it affects chlorophyll biosyn-
thesis. However, further studies on mode of action
should reveal its physiological role in plant cell. Further
studies on structure-activity relationships of the RWH
series will also reveal more active structures for new
herbicides.
P r otox Assa y w ith HP LC. Proto IX biosynthesized
from ALA in chloroplasts was analyzed by HPLC, and
the effect of Protox-inhibiting herbicides on the Proto
IX biosynthesis was examined. Protox inhibitor S-23142
(1 µM) inhibits Proto IX synthesis from ALA at Protox
in stromal extracts, while cyclic dione 21 (100 µM) has
no effect on Proto IX synthesis (Figure 4). This result
clearly indicates that the mode of action of 21 is different
from that of Protox inhibitor in spite of its structural
and physiological similarities to phthalimide herbicide.
In conclusion, we have synthesized novel compounds
carrying a cyclohexane-1,3-dione moiety with a vinylo-
MATERIALS AND METHODS
Cell Cu ltu r e Meth od s. A photomixotrophic culture of
tobacco (Nicotiana tabacum cv. Samsun NN; cell line NI)
(Takeda et al., 1990) was maintained in modified Linsmaier

New Photobleaching Compounds
J. Agric. Food Chem., Vol. 45, No. 7, 1997 2731
with a small piece of nylon mesh attached to a glass rod. The
entire isolation procedure could be accomplished in less than
5 min.
P r otox Assa y w ith HP LC. Assay Using ALA. Proto IX
synthesis from ALA was measured using the procedure
previously reported (Mito et al., 1991). The Protox activity of
intact chloroplasts was assayed using ALA as the substrate.
The intact chloroplasts were added to 500 mL of a reaction
medium (4 mM ALA, 0.5 mM sucrose, 1 mM MgCl2, 1 mM
EDTA, 20 mM TES, 10 mM HEPES, pH 7.5, 4 mM glu-
tathione, 0.6 mM NAD, 1.5 mM ATP, 2% BSA). The mixture
was incubated for 3 h at 25 °C in darkness, and the reaction
was stopped by addition of 1 mL of methanol. After centrifu-
gation at 10000g for 5 min, amount of Proto IX was measured
by HPLC. To measure the amount of Proto IX in the reaction
mixture, a C8 reverse phase column (Senshu Pak, C8-2251-
N, 6 mm × 250 mm, Senshu Science Co., Ltd., Tokyo, J apan)
was used with a solvent system of 85% methanol containing
10 mM ammonium acetate (pH 6.0) with a flow rate of 1 mL/
min. Proto IX detection was performed with a fluorescence
detecter (F-1000, Hitachi Ltd., Tokyo, J apan) with excitation
and emission wavelength settings at 420 and 630 nm, respec-
tively. The amount of Proto IX was estimated using Chroma-
tocorder-12 (Senshu Science Co., Ltd., Tokyo, J apan).
Syn t h esis of P h en yla m in om et h ylid en ecycloh exa n e-
1,3-d ion e Der iva tives. All melting points (mp) are uncor-
rected. 1H-NMR spectra were recorded on a Bruker AC-300
Plus spectrometer. Chemical shifts are expressed in ppm
downfield from TMS as an internal standard.
F igu r e 4. HPLC profiles of porphyrins synthesized by intact
protoplasts. Poryphyrin in the reaction mixture were separated
by reverse phase HPLC as described under Materials and
Methods. This experiment was done three times with similar
results.
and Skoog liquid medium with 3% (w/v) sucrose (Yamada et
al., 1978). The cells were subcultured at 25 ( 1 °C with
reciprocal shaking (100 rpm/min) under continuous light (ca.
30 µE/m2s) for 6 days.
All of the phenylaminomethylidenecyclohexane-1,3-dione
derivatives listed in Table 1 were prepared by the method
described before (Zacharias et al., 1974). The phenylaminom-
ethylidenecyclohexane-1,3-dione derivatives (B) were synthe-
sized by the condensation of 1,3-cyclohexanedione homologues
with triethyl formate and related anilines (Figure 1). The
alkylidene moiety was modified as described previously (Asami
et al., 1987). The condensation of 2-acetyl-1,3-diketones (D)
with anilines gave ethylidene derivatives, as shown in Figure
1. All of the procedures gave good yields.
Deter m in a tion of Ch lor op h yll Con ten t a n d Blea ch in g
Activity. The method used to determine chlorophyll content
was based on that of Arnon et al. (1949). The tobacco cells
collected by filtration were transferred into a test tube with
80% acetone solution (10 mL) and stored in a refrigerator (4
°C) overnight until all visible pigment had been extracted. The
absorbance values at 645 and 663 nm were measured with a
Beckman spectrophotometer (DU 640). The chlorophyll con-
tent was determined using Arnon’s equation. The chlorophyll
content in the treated tobacco cells was calculated as a
percentage of that in the control. All of the measurements
were performed in triplicate. The bleaching activity of all of
the compounds was determined as the concentration that
reduced the chlorophyll content by 50%. In tables, activity is
expressed by the pI50 value, which means -log10 [the concen-
tration (M) that reduced the chlorophyll content by 50%].
Gen er a l Syn t h et ic P r oced u r e for 2-P h en yla m in o-
m eth ylid en e-5,5-d im eth ylcycloh exa n e-1,3-d ion e (1).
A
1.44 g (10 mmol) amount of 5,5-dimethylcyclohexane-1,3-dione
(A) was added to 5 mL of triethyl formate and warmed to about
60 °C until all of the cyclohexane-1,3-dione was dissolved. A
0.93 g (10 mmol) amount of aniline was added with stirring.
The mixture was warmed to 125-130 °C, and stirring was
continued for 10 min. The reaction mixture was concentrated
under a vacuum and washed twice with saturated aqueous
sodium hydrogen carbonate. The residue was recrystallized
with n-hexane-ethyl acetate twice to give 1.96 g of 1 (79%).
Mp 136-139 °C. 1H-NMR (CDCl3/TMS, ppm): δ 1.10 (6H, s),
2.45 (2H, s), 2.47 (2H, s), 7.18-7.46 (5H, m), 8.64 (1H, d, J )
13.8 Hz), 12.90 (1H, br). Anal. Calcd for (%) C15H17NO2: C,
74.05; H, 7.04; N, 5.76; O, 13.15. Found: C, 74.11; H, 7.08;
N, 5.78; O, 13.20.
Compounds 2-24 and 28-34 were prepared in the same
manner as 1 using the corresponding aniline instead of the
aniline.
2-(2-E t h ylp h en yl)a m in om et h ylid en e-5,5-d im et h ylcy-
cloh exa n e-1,3-d ion e (2). Mp 107-109 °C. 1H-NMR (CDCl3/
TMS, ppm): δ 1.10 (6H, s), 1.15 (3H, t, J ) 7.5 Hz), 2.44 (2H,
s), 2.46 (2H, q, J ) 7.5 Hz), 2.48 (2H, s), 7.07-7.31 (4H, m),
8.17 (1H, d, J ) 13.7 Hz), 12.47 (1H, br). Anal. Calcd for (%)
C17H21NO2: C, 75.25; H, 7.80; N, 5.16; O, 11.79. Found: C,
75.31; H, 7.96; N, 5.31; O, 11.88.
2-(4-Ch lor op h en yl)a m in om eth ylid en e-5,5-d im eth ylcy-
cloh exa n e-1,3-d ion e (3). Mp 192-195 °C. 1H-NMR (CDCl3/
TMS, ppm): δ 1.09 (6H, s), 2.43 (2H, s), 2.47 (2H, s), 7.20 (2H,
dd, J 1 ) 6.9, J 2 ) 2.1 Hz), 7.38 (2H, dd, J 1 ) 6.9, J 2 ) 2.1 Hz),
8.55 (1H, d, J ) 13.4 Hz), 12.85 (1H, br). Anal. Calcd for (%)
C15H16NO2Cl: C, 64.87; H, 5.81; N, 5.04; O, 11.52; Cl, 12.76.
Found: C, 64.84; H, 5.88; N, 5.11; O, 11.52; Cl, 12.77.
2-(2,3-Dich lor op h en yl)a m in om eth ylid en e-5,5-d im eth -
ylcycloh exa n e-1,3-d ion e (4). Mp 180-182 °C. 1H-NMR
(CDCl3/TMS, ppm): δ 1.09 (6H, s), 2.44 (2H, s), 2.49 (2H, s),
7.24-7.38 (3H, m), 8.57 (1H, d, J ) 13.1 Hz), 13.22 (1H, br).
Ca r oten oid Deter m in a tion . The method used to deter-
mine carotenoids was based on that of Lambert and Bo¨ger
(Lambert et al., 1983) with slight modifications. The tobacco
cells were collected by filtration and washed with distilled
water twice. Ten milligrams (fresh weight) of tobacco cells
were treated with 5 mL of 6% KOH in methanol (w/v) and
incubated at 65 ˚C for 20 min until the pigment had been
extracted. The methanol layer was then extracted with
n-hexane-diethyl ether (9:1 v/v). The absorbance values at
445 nm were measured with a Beckman spectrophotometer
(DU 640).
Isola tion of In ta ct Ch lor op la sts. Spinach chloroplasts
were isolated by the method of Mills and J oy (1980) with slight
modifications. The leaves (50 g) were cut into small pieces,
directly immersed in 150 mL of ice-cooled chloroplast extrac-
tion medium (330 mM sorbitol, 50 mM Tricine [N-tris-
(hydroxymethyl)methylglycine]-KOH, pH 7.9, 2 mM EDTA, 1
mM MgCl2, and 0.1% BSA), and homogenized for 5 s with a
Polytron (PT 10/35; Kinematica, Basel, Switzerland). After
filtration through four layers of gauze, 30 mL of the homoge-
nate was poured into each 50 mL centrifuge tube and was
underlayed with 14 mL of Percoll medium [40% (v/v) Percoll,
330 mM sorbitol, 50 mM Tricine, pH 7.9, and 0.1% BSA].
Chloroplasts were pelleted by centrifugation at 2500g for 1
min in an LC-121 centrifuge (Tomy Seiko Co., Ltd., Tokyo,
J apan) equipped with a TS-7 swing-out rotor. The superna-
tant and then the Percoll layer were removed by aspiration,
and the pellet was gently resuspended in appropriate medium

2732 J. Agric. Food Chem., Vol. 45, No. 7, 1997
Wang et al.
Anal. Calcd for (%) C15H15NO2Cl2: C, 57.71; H, 4.84; N, 4.49;
O, 10.25; Cl, 22.71. Found: C, 57.77; H, 4.83; N, 4.51; O, 10.37;
Cl, 22.76.
2-(2-Bu tyloxyca r bon ylp h en yl)a m in om eth ylid en e-5,5-
d im eth ylcycloh exa n e-1,3-d ion e (15). Mp 128-129 °C. 1H-
NMR (CDCl3/TMS, ppm): δ 0.92 (3H, t, J 2 ) 6.9 Hz), 1.09 (6H,
s), 1.34-1.45 (2H, m), 1.39-1.89 (2H, m), 2.44 (2H, s), 2.50
(2H, s), 4.44 (2H, t, J 1 ) 7.4 Hz), 7.26 (2H, m), 7.60 (2H, m),
8.13 (1H, dd, J 1 ) 8.3 Hz, J 2 ) 1.1 Hz), 8.69 (1H, d, J ) 13.8
Hz), 13.92 (1H, br). Anal. Calcd for (%) C20H25NO4: C, 69.95;
H, 7.34; N, 4.08; O, 18.64. Found: C, 70.02; H, 7.28; N, 4.12;
O, 18.58.
2-(2-P en tyloxyca r bon ylp h en yl)a m in om eth ylid en e-5,5-
d im eth ylcycloh exa n e-1,3-d ion e (16). Mp 107-109 °C. 1H-
NMR (CDCl3/TMS, ppm): δ 0.94 (3H, t, J ) 7.4 Hz), 1.10 (6H,
s), 1.36-1.41 (4H, m), 1.79 (2H, dd, J 1 ) 6.9, J 2 ) 7.4 Hz),
2.44 (2H, s), 2.50 (2H, s), 4.43 (2H, t, J ) 6.9 Hz), 7.25 (1H,
m), 7.59 (2H, m), 8.12 (1H, dd, J 1 ) 8.3 Hz, J 2 ) 1.1 Hz), 8.68
(1H, d, J ) 13.8 Hz), 13.85 (1H, br). Anal. Calcd for (%)
C21H27NO4: C, 70.56; H, 7.61; N, 3.92; O, 17.90. Found: C,
70.42; H, 7.48; N, 3.99; O, 18.02.
2-(2-Hexyloxyca r bon ylp h en yl)a m in om eth ylid en e-5,5-
d im eth ylcycloh exa n e-1,3-d ion e (17). Mp 108-109 °C. 1H-
NMR (CDCl3/TMS, ppm): δ 0.91 (3H, t, J 2 ) 6.9 Hz), 1.09 (6H,
s), 1.33-1.43 (8H, m), 1.81 (2H, dd, J 1 ) 7.4, J 2 ) 6.9 Hz),
2.46 (2H, s), 2.52 (2H, s), 4.45 (2H, t, J ) 6.8 Hz), 7.28 (1H,
m), 7.62 (2H, m), 8.13 (1H, dd, J 1 ) 8.5 Hz, J 2 ) 1.2 Hz), 8.71
(1H, d, J ) 13.0 Hz), 14.15 (1H, br). Anal. Calcd for (%)
C22H29NO4: C, 71.13; H, 7.88; N, 3.78; O, 17.28. Found: C,
71.22; H, 7.76; N, 3.88; O, 17.58.
2-(H ep t yloxyca r b on ylp h en yl)a m in om et h ylid en e-5,5-
d im eth ylcycloh exa n e-1,3-d ion e (18). Mp 108-110 °C. 1H-
NMR (CDCl3/TMS, ppm): δ 0.90 (3H, t, J ) 6.7 Hz), 1.09 (6H,
s), 1.22-1.40 (10H, m), 1.79 (2H, dd, J 1 ) 7.4, J 2 ) 6.9 Hz),
2.44 (2H, s), 2.50 (2H, s), 4.43 (2H, t, J ) 6.8 Hz), 7.28 (1H,
m), 7.62 (2H, m), 8.13 (1H, dd, J 1 ) 8.4 Hz, J 2 ) 1.2 Hz), 8.69
(1H, d, J ) 13.0 Hz), 13.95 (1H, br). Anal. Calcd for (%)
C23H31NO4: C, 71.66; H, 8.11; N, 3.63; O, 16.60. Found: C,
71.58; H, 7.98; N, 3.68; O, 16.48.
2-(2,4-Dich lor op h en yl)a m in om eth ylid en e-5,5-d im eth -
ylcycloh exa n e-1,3-d ion e (5). Mp 185-186 °C. 1H-NMR
(CDCl3/TMS, ppm): δ 1.10 (6H, s), 2.45 (2H, s), 2.50 (2H, s),
7.34 (1H, J 1 ) 2.5 Hz, J 2 ) 9.1 Hz), 7.43 (1H, d, J ) 8.9 Hz),
7.49 (1H, d, J ) 2.2 Hz), 8.55 (1H, d, J ) 13.1 Hz), 13.22 (1H,
br). Anal. Calcd for (%) C15H15NO2Cl2: C, 57.71; H, 4.84; N,
4.49; O, 10.25; Cl, 22.71. Found: C, 57.75; H, 4.82; N, 4.71;
O, 10.67; Cl, 21.76.
2-(2,5-Dich lor op h en yl)a m in om eth ylid en e-5,5-d im eth -
ylcycloh exa n e-1,3-d ion e (6). Mp 128-130 °C. 1H-NMR
(CDCl3/TMS, ppm): δ 1.10 (6H, s), 2.45 (2H, s), 2.50 (2H, s),
7.13 (1H, dd, J 1 ) 8.4 Hz, J 2 ) 2.4 Hz), 7.38 (1H, d, J 2 ) 2.2
Hz), 7.48 (1H, d, J 1 ) 8.5 Hz), 8.54 (1H, d, J ) 13.0 Hz), 13.11
(1H, br). Anal. Calcd for (%) C15H15NO2Cl2: C, 57.71; H, 4.84;
N, 4.49; O, 10.25; Cl, 22.71. Found: C, 57.74; H, 4.86; N, 4.51;
O, 10.22; Cl, 21.78.
2-(3,4-Dich lor op h en yl)a m in om eth ylid en e-5,5-d im eth -
ylcycloh exa n e-1,3-d ion e (7). Mp 175-177 °C. 1H-NMR
(CDCl3/TMS, ppm): δ 1.10 (6H, s), 2.44 (2H, s) 2.48 (2H, s),
7.18-7.38 (2H, m), 8.55 (1H, d, J ) 13.0 Hz), 12.88 (1H, br).
Anal. Calcd for (%) C15H15NO2Cl2: C, 57.71; H, 4.84; N, 4.49;
O, 10.25; Cl, 22.71. Found: C, 57.68; H, 4.88; N, 4.55; O, 10.33;
Cl, 21.82.
2-(3,5-Dich lor op h en yl)a m in om eth ylid en e-5,5-d im eth -
ylcycloh exa n e-1,3-d ion e (8). Mp 157-159 °C. 1H-NMR
(CDCl3/TMS, ppm): δ 1.10 (6H, s), 2.44 (2H, s), 2.48 (2H, s),
7.28-7.44 (2H, m), 8.55 (1H, d, J ) 13.0 Hz), 12.88 (1H, br).
Anal. Calcd for (%) C15H15NO2Cl2: C, 57.71; H, 4.84; N, 4.49;
O, 10.25; Cl, 22.71. Found: C, 57.75; H, 4.66; N, 4.55; O, 10.42;
Cl, 22.70.
2-(2,4,6-Tr ich lor op h e n yl)a m in om e t h ylid e n e -5,5-d i-
m eth ylcycloh exa n e-1,3-d ion e (9). Mp 139-141 °C. 1H-
NMR (CDCl3/TMS, ppm): δ 1.10 (6H, s), 2.43 (2H, s), 2.49 (2H,
s), 7.43 (2H, s), 8.48 (1H, d, J ) 13.0 Hz), 12.16 (1H, br). Anal.
Calcd for (%) C15H14NO2Cl3: C, 51.97; H, 4.07; N, 4.04; O, 9.23;
Cl, 30.68. Found: C, 51.90; H, 4.11; N, 4.12; O, 9.10; Cl, 30.81.
2-(2,4,6-Tr iflu or op h en yl)a m in om eth ylid en e-5,5-d im e-
th ylcycloh exa n e-1,3-d ion e (10). Mp 151-153 °C. 1H-NMR
(CDCl3/TMS, ppm): δ 1.10 (6H, s), 2.44 (2H, s), 2.49 (2H, s),
6.83 (2H, t, J ) 8.3 Hz), 8.55 (1H, d, J ) 13.1 Hz), 12.86 (1H,
br). Anal. Calcd for (%) C15H14NO2F3: C, 60.61; H, 4.75; N,
4.71; O, 10.76; F, 19.17. Found: C, 60.35; H, 4.67; N, 4.64; O,
10.67; F, 19.30.
2-(2,6-Dich lor o-3-m et h ylp h en yl)a m in om et h ylid en e-
5,5-d im eth ylcycloh exa n e-1,3-d ion e (11). Mp 90-92 °C. 1H-
NMR (CDCl3/TMS, ppm): δ 1.10 (6H, s), 2.40 (3H, s) 2.43 (2H,
s), 2.49 (2H, s), 7.13 (1H, d, J ) 8.3 Hz), 7.31 (1H, d, J ) 8.3
Hz), 8.50 (1H, d, J ) 13.2 Hz), 12.63 (1H, br). Anal. Calcd
for (%) C16H17NO2Cl2: C, 58.91; H, 5.25; N, 4.29; O, 9.81; Cl,
21.47. Found: C, 59.14; H, 5.31; N, 4.15; O, 9.81; Cl, 21.70.
2-(4-Meth oxyp h en yl)a m in om eth ylid en e-5,5-d im eth yl-
cycloh exa n e-1,3-d ion e (12). Mp 101-102 °C. 1H-NMR
(CDCl3/TMS, ppm): δ 1.09 (6H, s), 2.42 (2H, s), 2.47 (2H, s),
3.97 (3H, s), 6.95-6.98 (2H, m), 7.18-7.21 (1H, m), 7.40-7.43
(1H, m), 8.65 (1H, d, J ) 14.0 Hz), 13.02 (1H, br). Anal. Calcd
for (%) C16H19NO3: C, 70.31; H, 7.01; N, 5.12; O, 17.56.
Found: C, 70.22; H, 6.94; N, 5.08; O, 17.39.
2-(2-Meth oxyca r bon ylp h en yl)a m in om eth ylid en e-5,5-
d im eth ylcycloh exa n e-1,3-d ion e (13). Mp 176-177 °C. 1H-
NMR (CDCl3/TMS, ppm): δ 1.09 (6H, s), 2.45 (2H, s), 2.51 (2H,
s), 4.04 (3H,s), 7.26-7.27 (1H, m), 7.61-7.63 (2H, m), 8.13 (1H,
m), 8.70 (1H, d, J ) 13.7 Hz), 13.12 (1H, br). Anal. Calcd for
(%) C17H19NO4: C, 67.76.; H, 6.36; N, 4.65; O, 21.24. Found:
C, 67.68; H, 6.28; N, 4.52; O, 21.57.
2-(2-Eth oxyca r bon ylp h en yl)a m in om eth ylid en e-5,5-d i-
m eth ylcycloh exa n e-1,3-d ion e (14). Mp 156-157 °C. 1H-
NMR (CDCl3/TMS, ppm): δ 1.10 (6H, s), 1.42 (3H, t, J ) 7.3
Hz), 2.45 (2H, s), 2.51 (2H, s), 4.52 (2H, q, J ) 7.2 Hz), 7.24-
7.28 (1H, m), 7.61-7.63 (2H, m), 8.12 (1H, m), 8.70 (1H, d, J
) 13.7 Hz), 13.10 (1H, br). Anal. Calcd for (%) C18H21NO4:
C, 68.55; H, 6.71; N, 4.44; O, 20.29. Found: C, 68.42; H, 6.73;
N, 4.48; O, 20.34.
2-(4-Bu tyloxyca r bon ylp h en yl)a m in om eth ylid en e-5,5-
d im eth ylcycloh exa n e-1,3-d ion e (19). Mp 150-151 °C. 1H-
NMR (CDCl3/TMS, ppm): δ 0.98 (3H, t, J ) 7.3 Hz), 1.10 (6H,
s), 1.44-1.49 (2H, m), 1.69-1.78 (2H, m), 2.44 (2H, s), 2.48
(2H, s), 4.32 (2H, t, J 1 ) 6.6 Hz), 7.29 (2H, dd, J 1 ) 1.9 Hz, J 2
) 6.9 Hz), 8.07 (2H, dd, J 1 ) 1.9 Hz, J 2 ) 6.9 Hz), 8.63 (1H, d,
J ) 13.4 Hz), 12.86 (1H, br). Anal. Calcd for (%) C20H25NO4:
C, 69.95; H, 7.34; N, 4.08; O, 18.64. Found: C, 69.89; H, 7.39;
N, 4.03; O, 18.65.
2 -(2 -C h l o r o -5 -m e t h o x y c a r b o n y l p h e n y l )a m i n o -
m eth ylid en e-5,5-d im eth ylcycloh exa n e-1,3-d ion e (20). Mp
159-160 °C. 1H-NMR (CDCl3/TMS, ppm): δ 1.11 (6H, s), 2.46
(2H, s), 2.52 (2H, s), 3.96 (3H, s), 7.56 (1H, d, J ) 8.4 Hz),
7.85 (1H, dd, J 1 ) 8.4 Hz, J 2 ) 1.9 Hz), 8.17 (1H, d, J ) 1.9
Hz), 8.68 (1H, d, J ) 13.0 Hz), 13.29 (1H, br). Anal. Calcd
for (%) C17H19NO4Cl: C, 60.63; H, 5.69; N, 4.16; O, 19.00; Cl,
10.53. Found: C, 60.42; H, 5.48; N, 4.08; O, 18.88; Cl, 10.44.
2-(2-C h lo r o -5-p r o p y lo x y c a r b o n y lp h e n y l)a m i n o -
m eth ylid en e-5,5-d im eth ylcycloh exa n e-1,3-d ion e (21). Mp
104-105 °C. 1H-NMR (CDCl3/TMS, ppm): δ 1.03 (3H, t, J )
7.4 Hz), 1.11 (6H, s), 1.82 (2H, tq, J 1 ) 7.4 Hz, J 2 ) 6.8 Hz),
2.46 (2H, s), 2.51 (2H, s), 4.32 (2H, t, J ) 6.8 Hz), 7.53 (1H, d,
J ) 8.4 Hz), 7.83 (1H, dd, J 1 ) 8.4 Hz, J 2 ) 1.9 Hz), 8.13 (1H,
d, J ) 1.9 Hz), 8.68 (1H, d, J ) 13.0 Hz), 13.29 (1H, br). Anal.
Calcd for (%) C19H23NO4Cl: C, 62.55; H, 6.35; N, 3.84; O, 17.54;
Cl, 9.72. Found: C, 62.67; H, 6.52; N, 3.88; O, 17.43; Cl, 9.89.
2-(2-Ch lor o-5-p r op e n yloxyca r b on ylp h e n yl)a m in o-
m eth ylid en e-5,5-d im eth ylcycloh exa n e-1,3-d ion e (22). Mp
106-107 °C. 1H-NMR (CDCl3/TMS, ppm): δ 1.10 (6H, s), 2.46
(2H, s), 2.51 (2H, s), 4.86 (2H, dt, J 1 ) 5.9 Hz, J 2 ) 1.5 Hz),
5.32 (1H, dd, J 1 ) 10.4 Hz, J 2 ) 1.5 Hz), 5.43 (1H, dd, J 1
)
17.6 Hz, J 2 ) 1.6 Hz), 5.9-6.3 (1H, m), 7.55 (1H, d, J ) 8.4
Hz), 7.85 (1H, dd, J 1 ) 8.4 Hz, J 2 ) 1.8 Hz), 8.15 (1H, J ) 1.8
Hz), 8.67 (1H, d, J ) 13.0 Hz), 13.16 (1H, br). Anal. Calcd
for (%) C19H21NO4Cl: C, 62.90; H, 5.83; N, 3.86; O, 17.64; Cl,
9.77. Found: C, 62.72; H, 5.75; N, 3.92; O, 17.47; Cl, 10.00.
2-(5-C h lo r o -2-p r o p y lo x y c a r b o n y lp h e n y l)a m i n o -
m eth ylid en e-5,5-d im eth ylcycloh exa n e-1,3-d ion e (23). Mp
210-211 °C. 1H-NMR (CDCl3/TMS, ppm): δ 1.03 (3H, t, J )
7.4 Hz), 1.11 (6H, s), 1.82 (2H, tq, J 1 ) 7.4 Hz, J 2 ) 6.8 Hz),

New Photobleaching Compounds
J. Agric. Food Chem., Vol. 45, No. 7, 1997 2733
2.45 (2H, s), 2.51 (2H, s), 4.34 (2H, t, J ) 6.8 Hz), 7.52 (1H, d,
J ) 8.3 Hz), 7.82 (1H, dd, J 1 ) 8.4 Hz, J 2 ) 2.0 Hz), 8.13 (1H,
d, J ) 2.0 Hz), 8.15 (1H, J ) 1.9 Hz), 8.64 (1H, d, J ) 13.1
Hz), 13.10 (1H, br). Anal. Calcd for (%) C19H23NO4Cl: C,
62.55; H, 6.35; N, 3.84; O, 17.54; Cl, 9.72. Found: C, 62.47;
H, 6.28; N, 3.46; O, 17.66; Cl, 9.62.
in dry 1,2-dichloroethane (50 mL). The mixture was stirred
for 1 h at room temperature and then poured into a mixture
of ice (20 g) and concentrated hydrochloric acid (20 g). The
organic phase was washed with water, dried with anhydrous
sodium sulfate, and evaporated in vacuo. The residue was
dissolved in a minimum quantity of ether, treated with
concentrated hydrochloric acid, and extracted with ether. The
ether solution was dried with anhydrous sodium sulfate and
concentrated in vacuo, and the residue was recrystallized from
hexane.
2-(4-C h lo r o -3-p r o p y lo x y c a r b o n y lp h e n y l)a m i n o -
m eth ylid en e-5,5-d im eth ylcycloh exa n e-1,3-d ion e (24). Mp
84-86 °C. 1H-NMR (CDCl3/TMS, ppm): δ 1.02 (3H, t, J )
7.4 Hz), 1.11 (6H, s), 1.82 (2H, tq, J 1 ) 7.4 Hz, J 2 ) 6.8 Hz),
2.46 (2H, s), 2.52 (2H, s), 4.35 (2H, t, J ) 6.8 Hz), 7.53 (1H, d,
J ) 8.4 Hz), 7.86 (1H, dd, J 1 ) 8.4 Hz, J 2 ) 1.8 Hz), 8.12 (1H,
d, J ) 1.8 Hz), 8.68 (1H, d, J ) 13.0 Hz), 13.31 (1H, br). Anal.
Calcd for (%) C19H23NO4Cl: C, 62.55; H, 6.35; N, 3.84; O, 17.54;
Cl, 9.72. Found: C, 62.57; H, 6.12; N, 3.99; O, 17.68; Cl, 9.48.
2-(2,6-Dich lor op h en yl)a m in om eth ylid en e-5,5-d im eth -
ylcycloh exa n e-1,3-d ion e (28). Mp 135-138 °C. 1H-NMR
(CDCl3/TMS, ppm): δ 1.10 (6H, s), 2.43 (2H, s), 2.50 (2H, s),
7.18 (1H, dd, J 1 ) 7.7 Hz, J 2 ) 8.5 Hz), 7.41 (2H, d, J ) 8.2
Hz), 8.55 (1H, d, J ) 13.1 Hz), 12.65 (1H, br). Anal. Calcd
for (%) C15H15NO2Cl2: C, 57.71; H, 4.84; N, 4.49; O, 10.25; Cl,
22.71. Found: C, 57.75; H, 4.82; N, 4.71; O, 10.47; Cl, 22.72.
2-(2-F lu or o-4-ch lor op h en yl)a m in om eth ylid en e-5,5-d i-
m eth ylcycloh exa n e-1,3-d ion e (29). Mp 173-176 °C. 1H-
NMR (CDCl3/TMS, ppm): δ 1.09 (6H, s), 2.43 (2H, s), 2.47 (2H,
s), 7.21-7.37 (3H, m), 8.53 (d, 1H, J ) 13.2 Hz), 12.98 (1H,
br). Anal. Calcd for (%) C15H15NO2ClF: C, 60.92; H, 5.11; N,
4.74; O, 10.82; Cl, 11.99; F, 6.42. Found: C, 60.77; H, 5.10;
N, 4.73; O, 10.88; Cl, 11.92; F, 6.35.
Gen er a l Syn th etic P r oced u r e for 2-(2,6-Dich lor op h e-
n yla m in o)et h ylid en e-5,5-d im et h ylcycloh exa n e-1,3-d i-
on e (25). 2-Acyl-1,3-cyclohexanedione (D, 10 mmol) was
dissolved in toluene (100 mL), and 2,6-dichloroaniline (10
mmol) was added. After the reaction mixture had been
refluxed for 12 h, the toluene was removed by concentration
in vacuo. The residue was washed with saturated aqueous
sodium hydrogen carbonate and extracted with ethyl acetate.
The organic phase was dried with anhydrous Na2SO4. After
removal of the solvent, the residue was chromatographed on
silica gel by eluting with hexane-ethyl acetate (7:3) and ethyl
acetate. Concentration of the ethyl acetate eluate under
reduced pressure followed by recrystallization of residue from
1
ethanol-H2O gave 124 mg (4%) of (25). Mp 134-136 °C. H-
NMR (CDCl3/TMS, ppm): δ 1.10 (6H, s), 2.37 (3H, s), 2.45 (2H,
s), 2.49 (2H, s), 7.18 (1H, dd, J 1 ) J 2 ) 7.6 Hz), 7.41 (2H, d, J
) 8.2 Hz), 15.01 (1H, br). Anal. Calcd for (%) C16H17NO2Cl2:
C, 58.91; H, 5.25; N, 4.29; O, 9.81; Cl, 21.74. Found: C, 58.78;
H, 5.22; N, 4.32; O, 9.89; Cl, 21.76.
Compounds 26 and 27 were prepared in the same manner
as 25 by using the corresponding anilines instead of the 2,6-
dichloroaniline.
2-(2-F lu or o-4-ch lor op h en yla m in o)et h ylid en e-5,5-d i-
m eth ylcycloh exa n e-1,3-d ion e (26). Mp 189-192 °C. 1H-
NMR (CDCl3/TMS, ppm): δ 1.10 (6H, s), 2.40 (3H, s), 2.45 (2H,
s), 2.49 (2H, s), 7.11-7.65 (3H, m), 14.94 (1H, br). Anal. Calcd
for (%) C16H17NO2ClF: C, 62.04; H, 5.53; N, 4.52; O, 10.33;
Cl, 11.44; F, 6.13. Found: C, 62.05; H, 5.59; N, 4.53; O, 10.20;
Cl, 11.33; F, 6.25.
2-(2,6-Dieth ylp h en yla m in o)eth ylid en e-5,5-d im eth ylcy-
cloh exa n e-1,3-d ion e (27). Mp 81-83 °C. 1H-NMR (CDCl3/
TMS, ppm): δ 1.10 (6H, s), 1.15 (6H, t, J ) 7.6 Hz), 2.43 (2H,
s), 2.48 (2H, s), 2.61 (4H, q, J ) 7.6 Hz), 7.15 (1H, dd, J 1 ) J 2
) 7.6 Hz), 7.28 (2H, d, J ) 8.2 Hz), 8.43 (1H, d, J ) 13.8 Hz),-
12.66 (1H, br). Anal. Calcd for (%) C20H27NO2: C, 76.64; H,
8.68; N, 4.47; O, 10.21. Found: C, 76.42; H, 8.67; N, 4.54; O,
9.98.
2-(2,6-Dieth ylp h en yl)a m in om eth ylid en e-5,5-d im eth yl-
cycloh exa n e-1,3-d ion e (30). Mp 115-119 °C. 1H-NMR
(CDCl3/TMS, ppm): δ 1.10 (6H, s), 1.20 (6H, t, J ) 7.6 Hz),
2.40 (2H, s), 2.48 (2H, s), 2.62 (4H, q, J ) 7.6 Hz), 7.16-7.29
(3H, m), 8.13 (d, 1H, J ) 13.8 Hz), 12.47 (1H, br). Anal. Calcd
for (%) C19H25NO2: C, 76.64; H, 8.68; N, 4.47; O, 10.21.
Found: C, 76.42; H, 8.67; N, 4.54; O, 9.98.
5-(2,6-Dich lor oph en yl)am in om eth yliden e-2,2-dim eth yl-
1,3-d ioxa n e-4,6-d ion e (31). Mp 128-130 °C. 1H-NMR
(CDCl3/TMS, ppm): δ 1.77 (6H, s), 7.15 (1H, dd, J 1 ) J 2 ) 7.6
Hz), 7.54 (2H, d, J ) 8.2 Hz), 8.62 (1H, d, J ) 13.1 Hz), 11.05
(1H, br). Anal. Calcd for (%) C13H11NO4C12: C, 49.39; H, 3.51;
N, 4.43; O, 20.24; Cl, 22.43. Found: C, 49.53; H, 3.49; N, 4.39;
O, 13.88; Cl, 22.09.
6-Meth yl-3-(2,6-dich lor oph en yl)am in om eth yliden e-2H,-
3H-p yr a n -2,4-d ion e (32). Mp 171-175 °C. 1H-NMR (CDCl3/
TMS, ppm): δ 2.19 (3H, s), 5.79 (1H, s), 7.19 (1H, dd, J 1 ) J 2
) 7.6 Hz), 7.52 (2H, d, J ) 8.2 Hz), 8.70 (0.7H, d, J ) 13.1
Hz), 8.77 (0.3H, d, J ) 13.1 Hz), 13.46 (1H, br). Anal. Calcd
for (%) C13H9NO3C12: C, 52.38; H, 3.04; N, 4.7; O, 16.1; Cl,
23.78. Found: C, 52.43; H, 3.09; N, 4.75; O, 16.21; Cl, 23.78.
5-(2,6-Dich lor oph en yl)am in om eth yliden e-1,3-dim eth yl-
2,4,6-p yr im id in etr ion e (33). Mp 203-207 °C. 1H-NMR
(CDCl3/TMS, ppm): δ 3.37 (3H, s), 3.39 (3H, s), 7.13 (1H, dd,
J 1 ) J 2 ) 7.6 Hz), 7.50 (2H, d, J ) 8.2 Hz), 8.66 (1H, d, J ) 17
Hz), 11.85 (1H, br). Anal. Calcd for (%) C13H11N3O3Cl2: C,
47.58; H, 3.38; N, 12.8; O, 14.63; Cl, 20.61. Found: C, 47.46;
H, 3.36; N, 12.77; O, 14.45; Cl, 20.46.
2-(2,6-Dich lor oph en yl)am in om eth yliden ecyclopen tan e-
1,3-d ion e (34). Mp 145-147 °C. 1H-NMR (CDCl3/TMS,
ppm): δ 2.59-2.72 (4H, m), 7.15 (1H, dd, J 1 ) J 2 ) 7.6 Hz),
7.78 (2H, d, J ) 8.2 Hz), 8.17 (1H, br). Anal. Calcd for (%)
C11H7NO3Cl2: C,48.56; H, 2.59; N, 5.15; O, 17.64; Cl, 26.06.
Found: C, 48.78; H, 2.44; N, 5.16; O, 17.53; Cl, 26.17.
Gen er a l Syn th etic P r oced u r e for 3-Acyloxy-2-cyclo-
h exen on es (C). Acyl chloride (11 mmol) was added to a
stirred solution of cyclohexane-1,3-dione (A, 10 mmol) and
pyridine (0.79 g, 10 mmol) in dry chloroform (50 mL). The
mixture was stirred at room temperature for 1 h, and then
washed with water, diluted hydrochloric acid, saturated aque-
ous sodium hydrogen carbonate, and water. The organic phase
was dried with anhydrous Na2SO4 and evaporated to dryness
in vacuo to give the pure enol ester (B), which was submitted
to the isomerization reaction without further purification.
Gen er a l Syn th etic P r oced u r e for 2-Acyl-1,3-cycloh ex-
a n ed ion es (D). The enol ester (B, 10 mmol) was added to a
stirred suspension of anhydrous aluminum chloride (20 mmol)
ABBREVIATIONS USED
Protox, protoporphyrinogen oxidase; Protogen IX,
protoporphyrinogen IX; Proto IX, protoporphyrin IX;
RWH, a name for a series of compounds reported.
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