Synthesis of lyoniresinol with combined utilization of synthetic chemistry and biotechnological methods

Article abstract of DOI:10.1248/cpb.54.226

We have synthesized lyoniresinol with the combined utilization of synthetic chemistry and biotechnological methods, specifically using plant cell cultures as an "enzyme source."

Full text of DOI:10.1248/cpb.54.226

226
Notes
Chem. Pharm. Bull. 54(2) 226—229 (2006)
Vol. 54, No. 2
Synthesis of Lyoniresinol with Combined Utilization of Synthetic
Chemistry and Biotechnological Methods
a
Masumi TAKEMOTO,*,a Ayako FUKUYO,a Yoichi AOSHIMA,b and Kiyoshi TANAKA
a School of Pharmaceutical Sciences, University of Shizuoka; 52–1 Yada, Suruga-ku, Shizuoka 422–8526, Japan: and
b Shizuoka Tea Experiment Station; Kikugawa, Ogasa, Shizuoka 439–0002, Japan.
Received July 20, 2005; accepted October 26, 2005
We have synthesized lyoniresinol with the combined utilization of synthetic chemistry and biotechnological
methods, specifically using plant cell cultures as an “enzyme source.”
Key words lyoniresinol; plant cell culture; oxidative coupling; enzyme; peroxidase; dibenzylbutanolide
Lignans of the 4-aryltetralin series, e.g., lyoniresinol (1), ever, the addition of H2O2 to the reaction mixture resulted in
have attracted considerable attention recently for their antiox- red-brown darkening of the solvent which decreased the
idative and antimutagenic activities (Chart 1). Lignans are chemical yield. Recently, we have found that Camellia sinen-
generally accessible only through multistep syntheses and/or sis cell culture is an efficient source of POD as “reagents” in
direct isolation from the living plant. Lyoniresinol (1) was organic synthesis and that a huge amount of H2O2 is pro-
isolated via extraction of the Lyonia ovalifolia var. elliptica duced in plant cell cultures with the addition of foreign sub-
plant.1,2) Compound 1 was also obtained by enzymatic hy- strates.7) Thus we planned to construct the skeleton of 1
drolysis of lyoniresinol glucoside with cellulase.3)
using C. sinensis cell culture (as an enzyme source)-cat-
In this paper, we report the synthesis of 1 with the com- alyzed ring-closure reaction of dibenzylbutanolide (11) as
bined utilization of synthetic chemistry and biotechnological the target synthetic intermediate to cyclic product 12 via a
methods, specifically using plant cell cultures as an “enzyme hypothetical quinone methide intermediate (A), as shown in
source.” The biosynthetic pathway generally postulated for Chart 3.
the podophyllotoxins (lignans in general), as shown in Chart
2, involves enzyme-catalyzed oxidative coupling of diben- Results and Discussion
zylbutanolide to the cyclic lignan in the later steps of the
Butanolide (11) as the target synthetic intermediate for the
pathway (from 2 to 3).4,5) Such ring-closure reactions (phenol synthesis of 1 was prepared according to the method shown
oxidative coupling) are considered to be achieved through in Chart 4. Benzyl ether (5), which was synthesized from 4-
peroxidase (POD) enzymes present in the plant. Kutney et al. hydroxy-3,5-dimethoxybenzaldehyde (4) with benzyl chlo-
reported horseradish peroxidase (HRP)–H2O2 catalyzed ring- ride and potassium carbonate (reflux 6 h, 92%), was sub-
closure reactions with phenolic systems: enzyme-catalyzed jected to Stobbe condensation with dimethylsuccinate to give
ring-closure reaction of dibenzylbutanolides suitable for bio- the conjugated hemisuccinate 6 in 69% yield. Spectral data
transformation to lignans as potential intermediates.6) How- indicated that only one geometric isomer (probably cis) was
present, as expected, by comparison with the results reported
for an analogous reaction with piperonal.8) The resulting dou-
ble bond of the hemisuccinate 6 was reduced with magne-
sium in methanol to afford the unconjugated hemisuccinate 7
1
in 73% yield. The H-NMR spectrum of 7 showed proton
signals at C2, C3, and C7ꢀ as multiplets due to the creation
of a chiral center at C2. The reductive lactonization of the
Chart 1. (ꢁ)-Lyoniresinol (1)
hemisuccinate 7 was treated with lithium borohydride to af-
Chart 2. Proposed Biosynthetic Pathway of Podophyllotoxin
∗ To whom correspondence should be addressed. e-mail: takemoto@ys2.u-shizuoka-ken.ac.jp
© 2006 Pharmaceutical Society of Japan

February 2006
227
Chart 3
Table 1. Biotransformation of Dibenzylbutanolide 11 to Cyclic Product 12
by C. sinensis Cell Culture
Time
(h)
Product
12 (%)
Recovery
11 (%)
Entry
Plant age
1
2
3
4
5
6
7
20
30
42
52
30
30
30
24
24
24
24
1
3
22
8
10
0
0
0
10
0
86
42
19
Chart 4. Synthesis of Dibenzylbutanolide 11
ford butanolide (8) directly in 68% yield. The 1H-NMR spec-
trum of 8 showed two new methylene protons at C1ꢀ as dou-
blets of doublets at d 4.05 and d 4.35, respectively. Alkyla-
tion of 8 with the bromide 9 gave the dibenzylbutanolide 10
5
10
34
48
1
in 65% yield. The H-NMR spectrum of the product 10, in
comparison with that of 8, showed the loss of one signal of
C2 but the expected trans orientation of the two substituents
with respect to the lactone ring could not be confirmed owing
to the close proximity to the complex signals at C2 and C3.
To remove the benzyl group, 10 was hydrogenated over Pd
on carbon to give bis(hydroxybenzyl)butanolide 11 in 93%
yield.
Chart 5
Next, POD-catalyzed oxidative coupling of 11 was exam-
ined with C. sinensis cell culture9) which produced POD proton signal at C4 (a doublet at 4.10 ppm with coupling
[15.5 units (U)/ml].7) The oxidative coupling reaction of the constant of 11.6 Hz).
dibenzylbutanolide 11 to cyclic product 12 was performed in
Next, we performed a large-scale biotransformation with a
small-scale experiments (250 mg of 11) with freely sus- bioreactor using C. sinensis cell culture under optimum con-
pended cells in the stationary phase after 30 d of incubation ditions. The bioreactor was fitted with a peristaltic pump and
(50 g of cells and 200 ml of broth). To derive optimum condi- a filter. Substrate 11 (1 g in 8 ml ethanol) was added to the
tions for the biotransformation of 11 to 12 using C. sinensis, freely suspended C. sinensis cell culture (200 g of cells and
we evaluated the biological and reaction parameters (the age 800 ml of broth, 30 d old) in the bioreactor for 10 h to afford
of C. sinensis). Oxidative coupling was performed using 12 in 62% yield.
cells of various ages (20—52 d old), as shown in Table 1 (en-
Finally, 12 thus obtained was reduced with lithium alu-
tries 1—4). The optimum age of C. sinensis was 30 d (entry minum hydride (LAH) in THF to give 1 in 62% yield as
2). A longer reaction time (24 h) promoted the decomposi- shown in Chart 5. The structure of synthetic lyoniresinol (1)
1
tion of 12. Then the reaction was carried out for different was confirmed by a comparison of the H-NMR and FAB-
times using C. sinensis (30 d) (entries 5—7). The optimum MS data with that reported.3)
reaction time was 10 h (entry 7). It was concluded that 30-d-
old C. sinensis and a reaction time of 10 h would give opti- Conclusion
mum yields of 12. The structure of cyclic product 12 was
In this study, we used C. sinensis cell cultures, in which
1
confirmed by analysis of the H-NMR spectrum, which re- cell wall peroxidases rapidly metabolize a huge amount of
vealed only aromatic proton singlets (d 6.30 and 6.51 for H2O2 produced by the addition of foreign substrates. We suc-
protons at C2ꢀ, C6ꢀ, and C8). The trans relationship of the ceeded in the oxidative coupling of the dibenzylbutanolide
lactone ring (stereochemistry of C3 and C4) was confirmed 11 with C. sinensis cell culture in the absence of foreign hy-
from the proton signal at C3 (doublets of doublets at drogen peroxide as a cofactor. The development of enzymes
2.40 ppm with coupling constants of 15.4 and 11.6 Hz) and for oxidation reactions aimed at green chemistry is very im-

228
Vol. 54, No. 2
solid was subjected to silica gel column chromatography using CH2Cl2–
MeOH (40 : 1) to afford the hemisuccinate 6 (5.6 g, 69%) as an amber resin
that solidified at 0 °C (mp 122—130 °C). Anal. Calcd for C21H22O7: C,
65.28; H, 5.74. Found: C, 65.26; H, 5.65. H-NMR (CDCl3) d: 3.62 (2H, s,
3-H), 3.82 (6H, s, –OMe), 3.85 (3H, s, –COOMe) , 5.05 (2H, s, –OCH2Ph),
portant. HRP is a commercially available metalloporphyrin
oxidative enzyme and has been established as an effective
biocatalyst for organic reactions using H2O2. However, in the
oxidative coupling of dibenzylbutanolide, the addition of
1
H2O2 to the reaction decreases chemical yield. This synthetic 6.64 (2H, s, 2ꢀ-H and 6ꢀ-H), 7.26—7.49 (5H, m, –OCH2Ph), 7.85 (1H, s, 7ꢀ-
H).
method has some advantageous features such as mild reac-
(ꢀ)-2-(4-Benzyloxy-3,5-dimethoxybenzyl)butanedioic Acid 1-Methyl
Ester (7) The hemisuccinate 6 (5.2 g, 13.5 mmol) was added to a suspen-
sion of magnesium shavings (5.0 g, 208 mmol) in dry methanol (100 ml)
tions, easy work-up, and safety; therefore it is a valuable al-
ternative to the oxidative coupling by HRP.
under Ar. After a few minutes of stirring, the reaction vessel was immersed
Experimental
in an ice bath and stirred at 0 °C for 5 h. The suspension was acidified with
hydrochloric acid 6 M and the remaining solids were removed by filtration.
The filtrate was extracted with dichloromethane (3ꢂ50 ml), washed with
brine (100 ml), dried over MgSO4, filtered, and evaporated in vacuo. The
residue was subjected to silica gel column chromatography using CH2Cl2–
MeOH (40 : 1) to afford the hemisuccinate 7 (3.8 g, 73%). The analytical
sample was recrystallized from AcOEt–hexane as a yellow powder (mp
102—105 °C). Anal. Calcd for C21H24O7: C, 64.94; H, 6.23. Found: C,
All melting points were determined on a Yanagimoto micromelting point
apparatus without correction. 1H-NMR spectra were recorded on a JEOL
JNM-EX 270 FT NMR (270 MHz). Chemical shifts (d) are given in ppm
with tetramethylsilane as an internal standard, and coupling constants (J) are
given in Hz. FAB-MS were recorded on a JEOL JMS-SX 102 mass spec-
trometer, and HR-MS on a JEOL JMS-DX 300 mass spectrometer. Thin-
layer chromatography (TLC) was performed on silica gel (Kieselgel 60F254
on aluminum sheets, Merck). All compounds were located by spraying the
TLC plates with a 10% solution of phosphomolybdic acid in ethanol and
heating it on a hot plate. Preparative TLC was performed on preparative
layer chromatography plates (Kieselgel 60F254, 2 and 0.5 mm, Merck). Col-
umn chromatography was performed on silica gel (Kieselgel 60, 70—230
mesh, Merck).
1
64.72; H, 5.72. H-NMR (CDCl3) d: 2.49 (1H, dd, Jꢃ17.0, 4.6 Hz, 3-H),
2.65—2.76 (2H, m, 2-H, 3-H), 2.97—3.10 (2H, m, 7ꢀ-H), 3.68 (3H, s,
–COOMe), 3.80 (6H, s, –OMe ), 4.98 (2H, s, –OCH2Ph), 6.35 (2H, s, 2ꢀ-H,
6ꢀ-H), 7.28—7.49 (5H, m, –OCH2Ph).
3-(4-Benzyloxy-3,5-dimethoxybenzyl)butanolide (8) Lithium borohy-
dride (294 mg, 5.7 mmol) in dry THF (50 ml) was carefully added to a solu-
tion of the hemisuccinate 7 (3.3 g, 8.5 mmol) in THF (80 ml) during reflux
under Ar. The solution was stirred for 4 h during reflux and then cooled to
room temperature. Water (2 ml) and hydrochloric acid 6 M (3 ml) were added
and the solution was stirred at room temperature for 15 h. The bulk of the
solvent was removed by evaporation in vacuo and the resultant mixture was
extracted with ether (50 ml). The organic extract was washed with saturated
sodium bicarbonate (3ꢂ20 ml) and water (20 ml) before being dried over
MgSO4, filtered, and evaporated in vacuo. The residue was subjected to sil-
ica gel column chromatography using CH2Cl2–MeOH (20 : 1) to afford b-
butanolide 8 (2.0 g, 68%). The analytical sample was recrystallized from
AcOEt–hexane as a white powder (mp 104—108 °C). Anal. Calcd for
Cultivation of C. sinensis Cells Suspensions of C. sinensis cells were
subcultured every 14 d by transferring a 2-week culture (10 ml) into B510)
medium (80 ml) containing 2,4-D (1.25 mg/l) and 5% sucrose (pH 5.8) on a
rotary shaker (110 rpm) at 25 °C in the dark.
Biotransformation of 11 with C. sinensis Cell Cultures Compound 11
(250 mg) in ethanol (2 ml) was added to the freely suspended C. sinensis
(50 g of cells and 200 ml of broth, pH 5.8). The mixture was shaken at 25 °C
on a rotary shaker (110 rpm) in the dark. Upon termination of the reaction,
the incubation mixture was filtered, and the filtered cells were washed with
AcOEt. The filtrates and washings were combined and extracted with
AcOEt. The AcOEt layer was washed with brine, dried over MgSO4, and
concentrated in vacuo. The residue was subjected to silica gel column chro-
matography with hexane–AcOEt (2 : 1). The reaction time and the chemical
yield are listed in Table 1.
4-Benzyloxy-3,5-dimethoxybenzaldehyde (5) A suspension of 4-hy-
droxy-3,5-dimethoxybenzaldehyde (4, 4.5 g, 24.6 mmol), potassium carbon-
ate (3.7 g, 28.5 mmol), benzyl chloride (3.7 g, 29.5 mmol), sodium iodide
(1.0 g, 6.5 mmol), and ethanol (250 ml) was refluxed for 6 h while stirring
with a mechanical stirrer. Water (10 ml) was added and the ethanol removed
in vacuo. The slurry was poured into a mixture of 1 M sodium hydroxide
(20 ml) and ice (8 g). The solids were filtered off, washed with ice-cold water
(3ꢂ20 ml), and dried in vacuo. The residue was subjected to silica gel col-
umn chromatography using hexane–AcOEt (7 : 1), hexane–AcOEt (5 : 1),
and hexane–AcOEt (4 : 1) to afford 5 (6.2 g, 92%). 1H-NMR (CDCl3) d: 3.90
(6H, s, –OMe), 5.13 (2H, s, –OCH2Ph), 7.11—7.48 (7H, m, aromatic), 9.86
(1H, s, –CHO).
4-Benzyloxy-3,5-dimethoxybenzyl Bromide (9) Sodium borohydride
(0.9 g, 24.6 mmol) was carefully added to a solution of benzyl ether 5 (6.0 g,
22.0 mmol) in EtOH (200 ml). The mixture was stirred for 4 h. Aqueous
NH4Cl (50 ml) was added and the solution was extracted with ether
(2ꢂ100 ml). The ether extracts were dried over MgSO4, filtered, and evapo-
rated in vacuo to afford benzyl alcohol (5.7 g, 95%). PBr3 (1.8 g, 6.7 mmol)
was carefully added to a solution of benzyl alcohol (1.8 g, 6.6 mmol) in ether
(200 ml) under Ar at 0 °C. The mixture was stirred for 1 h at 0 °C. NaHCO3
was added and the solution was neutralized. The solution was extracted with
ether (2ꢂ100 ml). The ether extracts were dried over MgSO4, filtered, and
1
C20H22O5: C, 70.16; H, 6.48. Found: C, 70.03; H, 6.52. H-NMR (CDCl3)
d: 2.30 (1H, dd, Jꢃ17.5, 6.6 Hz, 2-H), 2.55—2.92 (4H, m, 2-H, 2ꢀ-H, 3ꢀ-H),
3.81 (6H, s, –OMe), 4.05 (1H, dd, Jꢃ5.9, 9.2 Hz, 1ꢀ-H), 4.35 (1H, dd, Jꢃ
6.9, 9.2 Hz, 1ꢀ-H), 4.99 (2H, s, –OCH2Ph), 6.34 (2H, s, 5ꢀ-H, 9ꢀ-H), 7.26—
7.49 (5H, m, –OCH2Ph).
trans-2-(4-Benzyloxy-3,5-dimethoxybenzyl)-3-(4-benzyloxy-3,5-
dimethoxybenzyl)butanolide (10) To a solution of diisopropylamine
(1.3 g, 13.0 mmol) in dry THF (20 ml) at ꢄ78 °C was added n-BuLi (7.7 ml,
11.0 mmol) and stirring was continued for 30 min. b-Butanolide 8 (3.0 g,
8.0 mmol) in THF (15 ml) was added, and the bright yellow solution was
stirred for 90 min prior to the addition of the bromide 9 (3.4 g, 10.0 mmol)
in THF (15 ml) and further stirred for 16 h at ꢄ78 °C. The solution was
warmed to 0 °C, acidified with hydrochloric acid 1 M, and extracted with
dichloromethane (2ꢂ100 ml). The combined organic extracts were washed
with water (80 ml), dried over MgSO4, filtered, and evaporated in vacuo. The
residue was subjected to silica gel column chromatography using
hexane–AcOEt (1 : 1) to afford dibenzylbutanolide 10 (3.3 g, 65%) as a yel-
low oil. 1H-NMR (CDCl3) d: 2.42—2.65 (4H, m, 2-H, 3-H, 6-H), 2.95 (2H,
dd, Jꢃ14.0, 8.4 Hz, 5-H), 3.76 (6H, s, –OMe), 3.78 (6H, s, –OMe), 3.88
(2H, dd, Jꢃ9.0, 8.4 Hz, 4-H), 4.97 (2H, s, –OCH2Ph), 4.99 (2H, s, –OCH2Ph),
6.19, 6.38 (2H each, s, s, 2ꢀ-H, 6ꢀ-H, 2ꢅ-H, 6ꢅ-H), 7.24—7.47 (10H, m,
–OCH2Ph). FAB-MS m/z 598 (Mꢆ), HR-MS m/z: 598.2589 (Calcd for
C36H38O8: 598.2567).
trans-2-(3,5-Dimethoxy-4-hydroxybenzyl)-3-(4-hydroxy-3,5-dimethoxy-
benzyl)butanolide (11) Palladium-on-charcoal (5%, 1.5 g) and 10 (1.4 g,
2.3 mmol) were suspended in AcOEt–EtOH (1 : 3) (30 ml) and stirred under
hydrogen at atmospheric pressure for 3.5 h. The catalyst was filtered off and
the solvent evaporated in vacuo to yield bis(hydroxybenzyl)butanolide 11
(0.9 g, 93%) as an amorphous white solid. The analytical sample was recrys-
tallized from diethyl ether/petroleum ether as a white powder (mp 60—
63 °C). FAB-MS m/z 418 (Mꢆ), HR-MS m/z: 418.1642 (Calcd for C22H26O8:
1
evaporated in vacuo to afford the benzyl bromide 9 (1.4 g, 61%). H-NMR
(CDCl3) d: 3.83 (6H, s, –OMe), 4.46 (2H, s, –CH2Br), 5.00 (2H, s, –OCH2Ph),
6.61 (2H, s, 2-H, 6-H), 7.26—7.50 (5H, m, –OCH2Ph).
2-(4-Benzyloxy-3,5-dimethoxybenzylidene)butanedioic Acid 1-Methyl
Ester (6) Sodium methoxide (4.6 g, 85.0 mmol) was carefully added to dry
methanol (20 ml) under Ar. A solution of benzyl ether 5 (5.7 g, 21.0 mmol)
in dimethyl succinate (4.3 g, 29.0 mmol) was added dropwise over 40 min
during reflux. After an additional 4.5 h of stirring during reflux, the bulk of
the solvent was removed in vacuo. The suspension was cooled to 0 °C and
acidified with hydrochloric acid 6 M. The solids were removed by filtration
and the filtrate was extracted with dichloromethane (2ꢂ50 ml). The solids
were added to the organic extract, washed with brine (100 ml), dried over
MgSO4, filtered, and evaporated in vacuo to yield an oily yellow solid. The
1
418.1628). H-NMR (CDCl3) d: 2.42—2.63 (4H, m, 2-H, 3-H, 6-H), 2.91
(2H, dd, Jꢃ5.6, 11.6 Hz, 5-H), 3.82—3.85 (13H, m, –OMe, 4-H), 4.21 (1H,
dd, Jꢃ6.9, 8.9 Hz, 4-H), 5.42 (2H, s, –OH), 6.18 (2H, s, 2ꢅ-H, 6ꢅ-H), 6.32
(2H, s, 2ꢀ-H, 6ꢀ-H).
(3,5-Dimethoxy-4-hydroxyphenyl)-6,8-dimethoxy-7-hydroxy-3-hydrox-
ymethyl-1,2,3,4-tetrahydro-2-naphthoic Acid -Lactone (12) Com-
g
pound 11 (1 g in 8 ml ethanol) was added to the freely suspended C. sinensis

February 2006
229
cell culture (200 g of cells and 800 ml of broth, 30 d old) for 10 h in a biore-
actor fitted with a peristaltic pump and a filter. The mixture was reacted at
25 °C (110 rpm) in the dark. Upon termination of the reaction, the incuba-
tion mixture was filtered, and the filtered cells were washed with AcOEt.
The filtrates and washings were combined and extracted with AcOEt. The
AcOEt layer was washed with brine, dried over MgSO4, and concentrated in
vacuo. The residue was subjected to silica gel column chromatography with
hexane–AcOEt (1 : 2) to afford 12 (617 mg) in 62% yield. FAB-MS m/z 416
(Mꢆ). HR-MS m/z: 416.1463 (Calcd for C22H24O8: 416.1471). 1H-NMR
(CDCl3) d: 2.40 (1H, dd, Jꢃ15.4, 11.6 Hz, 3-H), 2.70—2.95 (2H, m, 1-H, 2-
H), 3.10—3.20 (1H, m, 1-H), 3.83 (6H, s, –OMe), 3.91 (6H, s, –OMe), 3.98
(1H, dd, Jꢃ11.6, 7.3 Hz, 2a-H), 4.10 (1H, d, Jꢃ11.6 Hz, 4-H), 4.26 (1H, dd,
Jꢃ11.6, 7.3 Hz, 2a-H), 5.37 (1H, br s, OH), 5.41 (1H, br s, OH), 6.30 (2H,
s, 2ꢀ-H, 6ꢀ-H), 6.51 (1H, s, 8-H).
Lyoniresinol (1) A solution of 12 (500 mg, 1.2 mmol) in THF (10 ml)
was added to a suspension of LiAlH4 (36.3 mg, 0.96 mmol) in THF (15 ml)
at 0 °C, and the mixture was stirred at room temperature for 1 h. The mixture
was extracted with ether (50 ml). The organic extract was evaporated in
vacuo. The residue was subjected to silica gel column chromatography using
hexane–AcOEt (1 : 2) to afford 1 (313 mg, 62%) as a white amorphous solid.
FAB-MS m/z 420 (Mꢆ). HR-MS m/z: 420.1796 (Calcd for C22H28O8:
420.1784). 1H-NMR (CDCl3): d: 1.72 (1H, m, 2H), 1.90 (1H, m, 3-H),
2.53—2.71 (2H, m, 1-H2), 3.29 (3H, s, OMe), 3.50—3.77 (4H, m, 2a-H2,
3a-H2), 3.78 (6H, s, OMe), 3.87 (3H, s, OMe), 3.99 (1H, d, Jꢃ7.6 Hz, 4-H),
5.44 (2H, br s, OH), 6.34 (2H, s, 2ꢀ-H, 6ꢀ-H), 6.44 (1H, s, 8H).
References
1) Hostettler F. D., Seikel M. K., Tetrahedron, 25, 2325—2337 (1969).
2) Sakakibara J., Ina H., Yasue M., Yakugaku Zasshi, 94, 1377—1383
(1974).
3) Shibuya H., Takeda Y., Zhang R., Tanitame A., Tsai Y., Kitagawa I.,
Chem. Pharm. Bull., 40, 2639—2646 (1992).
4) Kutney J. P., Synlett, 1991, 11—19 (1991).
5) Umezawa T., Davin L. B., Lewis N. G., Biochem. Biophys. Res. Com-
mun., 171, 1008—1014 (1990).
6) Kutney J. P., Du X., Naidu R., Stoynov N. M., Takemoto M., Heterocy-
cles, 42, 479—484 (1996).
7) Takemoto M., Aoshima Y., Stoynov N., Kutney J. P., Tetrahedron Lett.,
43, 6915—6917 (2002).
8) El-Assal L. S., El-Wahhab S. A. M., J. Chem. Soc., 849—852 (1960).
9) Takemoto M., Achiwa K., Chem. Pharm. Bull., 49, 639—641 (2001).
10) Gamborg O. L., Miller R. A., Ojima K., Exp. Cell Res., 1968, 151—
158 (1968).