July 2002
1009
1
3
Preparation of 17-Nor-17-oxo-steviol Methyl Ester (7) and 13,16-Seco-
3-oxo-steviol Methyl Ester (8) A solution of steviol methyl ester (1.00 g)
Table 2.
C No.
C-NMR Chemical Shifts (d) of Steviol and Its Derivatives
1
in dichloromethane (10 ml) was cooled to 0 °C and subjected to a passing
stream of oxygen that contain ozone for 10 h. The mixture was treated with
Zn–AcOH, then the solvent was removed in vacuo, and the residue was puri-
fied by chromatography (silica gel, Wakogel C-300) by gradient elution of
hexane to ethyl acetate. The fraction eluted with hexane : ethylacetate (1 : 1)
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
41.0 41.0 40.7 40.7 41.7 40.6
19.8 19.8 19.9 19.7 19.5 19.4
38.6 38.7 38.8 38.6 38.3 37.2
43.9 43.9 44.1 44.0 43.9 43.9
57.0 56.9 57.4 56.4 56.9 56.6
22.6 22.6 22.2 21.3 22.4 21.2
41.8 41.9 41.3 40.3 41.7 41.2
41.9 41.5 46.6 55.4 41.7 39.5
54.3 54.1 53.4 51.2 54.1 53.0
39.8 39.8 40.3 40.5 39.5 39.4
20.8 19.9 20.7 21.0 20.8 20.0
40.8 36.6 44.4 45.4 40.7 38.1
41.5 41.4 38.0
19.6 19.7 16.6
37.9 33.5 29.2
43.8 43.8 43.7
56.4 57.0 56.5
20.4 21.0 20.3
39.0 41.6 41.0
41.3 39.8 39.5
49.6 50.6 53.1
39.5 39.0 34.0
20.9 20.5 19.5
47.2 38.1 33.0
Ϫ1
afforded 16-ketone 7 (317 mg): mp 207—208 °C; IR (Nujol) cm : 3447
1
(
OH), 1744, 1721 (CϭO), 1237, 1208; H-NMR (pyridine-d ) d: 0.89 (3H,
5
s, C20-H ), 1.18 (3H, s, C18-H ), 1.74, 2.09 (each 1H, d, Jϭ2.1 Hz, C15-
3
3
H ), 3.66 (3H, s, –COOCH ). The fraction eluted with hexane : ethylacetate
2
3
(
(
1 : 2) afforded keto carboxylic acid 8 (218 mg): mp 170—172 °C; IR
Ϫ1
1
Nujol) cm : 1721, 1694 (CϭO), 1237, 1154, 972; H-NMR (pyridine-d )
5
d: 0.95 (3H, s, C20-H ), 1.15 (3H, s, C18-H ), 1.65 (1H, m, C14-H ), 2.44,
1
1
1
1
1
1
1
1
1
1
2
0
1
2
3
4
5
6
7
8
9
0
3
3
2
2
.93 (each 1H, m, C15-H ), 2.52 (1H, m, C12-H ), 3.66 (3H, s, –COOCH ).
2
2
3
Preparation of 13,16-Seco-13-hydroxy-steviol Methyl Ester (9) and
Steviol Methyl Ester 8,13-Lactone (10) To a solution of 13,16-seco-13-
79.8 75.1 79.0 76.5 79.8 80.3 211.7 65.8 75.8
48.2 48.2 36.4 33.8 48.1 44.4
47.5 47.7 82.0 209.1 47.4 53.3
oxo-steviol methyl ester (8) (1.0 g) in 1-butanol (50 ml), NaBH (250 mg)
48.5 42.2 42.9
38.6 47.5 48.4
4
was added, and the solution was stirred at room temperature for 1 h. The
mixture was neutralized with 5%-HCl, the solvent was removed in vacuo,
and the residue was extracted with ethyl acetate (3ϫ20 ml). The combined
157.7 65.2 163.4 154.2 157.6 220.1 173.9 175.0 171.6
102.9 46.4 107.2 114.1 103.7
29.3 29.3 29.4 29.3 28.7 28.6
180.1 180.1 180.2 180.0 177.7 177.6 177.7 178.0 177.7
—
—
—
—
ethyl acetate layers were dried over anhydrous Na SO , and evaporated to
28.5 28.7 28.6
2
4
give a mixture of esters 9 and 10 as an oily product. The mixture was puri-
fied by chromatography on silica gel by a gradient elution of chloroform to
chloroform : methanol (7 : 3) to yield 13,16-seco ester 9 (540 mg) and lac-
15.9 16.1 16.3 15.9 15.2 15.9
51.5 51.2
16.4 18.3 16.0
51.2 51.1 51.2
–
OCH3
—
—
—
—
Ϫ1
tone 10 (270 mg). Compound 9: mp 150—154 °C; IR (Nujol) cm : 3460
1
(
(
OH), 1727 (CϭO), 1234, 1150 (–C–O); H-NMR (pyridine-d ) d: 0.97
5
3H, s, C20-H ), 1.15 (3H, s, C18-H ), 2.44, 2.93 (each 1H, d, Jϭ13.0 Hz,
3
3
C15-H ), 3.56 (3H, s, –COOCH ), 4.54 (1H, m, C13-H). Compound 10: mp
2
3
Ϫ1
3
5 °C for 2 weeks. The resulting white crystalline precipitate was isolated 149—151 °C; IR (Nujol) cm : 1238, 1184 (–C–O), 1720 (CϭO), 1730
1
from the culture medium, and recrystallized from methanol to yield pure ste- (CϭO, lactone). H-NMR (pyridine-d ) d: 0.75 (3H, s, C20-H ), 1.17 (3H, s,
5
3
viol (2) (5.5 g): mp 206—207 °C; Anal. Calcd for C H O : C, 75.43; H,
C18-H ), 1.96, 2.38 (each 1H, d, Jϭ12.9 Hz, C15-H ), 3.66 (3H, s,
2
0
30
3
3
2
9
.50. Found: C, 75.54; 9.41. The IR and NMR spectral data of steviol were
–COOCH ), 4.69 (1H, m, C13-H).
3
9
,10)
identical to those published in literature.
Forward Mutation Assay To assess the mutagenicity of steviol and its
Preparation of Steviol-16a,17-epoxide (3) To a solution of steviol chemically synthesized derivatives, we employed forward mutation assay
1.0 g) in dichloromethane (40 ml), m-chloroperbenzoic acid (660 mg) was using Salmonella typhimurium TM677 and 8-azaguanine (8-AG, Tokyo
(
1
1,12)
added, and the solution was stirred at room temperature for 1 h. The mixture
Kasei Kogyo) as the detection reagent, as proposed by Skopek et al.,
1
3)
14)
was treated with 10%-Na S O solution. The organic layers was washed with modified by Takagi et al., and reduced in size by the authors. In our ex-
2
2
3
5
%-NaHCO solution, then with water, dried over an hydrous Na SO , and
periment, each test samples (100 ml; sterilized by membrane filtration, Milli-
3
2
4
evaporated to give an oily product. The product was recrystallized from pore, pore size, 0.45 mm) and an equal volume of cultured bacteria suspen-
12)
ethyl acetate to afford epoxide 3 (854 mg): mp 207—210 °C; IR (Nujol) sion, which was diluted to OD600ϭ0.12 with ME culture medium after
Ϫ1
1
cm : 3255 (OH), 1715 (CϭO), 1245 (epoxide); H-NMR (pyridine-d ) d: three hours pre-incubation at 37 °C, were combined into a micro-titer plate
5
1
.20 (3H, s, C20-H ), 1.36 (3H, s, C18-H ), 1.81 (2H, s, C15-H ), 2.94 (2H, equipped with a plastic cover (96 holes, g-radiation sterilized, Corning), and
3
3
2
s, C17-H2).
shaken at 37 °C for 2 h. The incubated mixture (25 ml) was transferred to a
Preparation of 15a-Hydroxy-steviol (4) To a solution of steviol (500 test tube equipped with an aluminum cap, which was sterilized at 180 °C for
mg) in dioxane (30 ml) and water (10 ml), SeO (20 mg) and aqueous H O
4 h. Due to the low solubility of the test samples in 5% dimethylsulfoxide
2
2
2
(
36%, 2 ml) were added, and the solution was stirred at 60 °C for 2 h. The (DMSO), the top dose of each sample was varied between 100 and 200
mixture was evaporated and extracted with ethyl acetate (3ϫ30 ml). The mg/100 ml of the solvent. Test samples were successively diluted five-fold by
combined ethyl acetate layer were dried over anhydrous Na SO and evapo- addition of the same amount of 5% DMSO. For the analyses of mutagenic
2
4
rated to give an oily product. The product was recrystallized from CHCl3 : compounds that required enzymatic activation, S9 mixture (a mixture of co-
MeOH (99 : 1) to give alcohol 4 (395 mg): mp 271—275 °C; IR (Nujol) factors and S9 obtained from homogenates of livers of rats to induce the cy-
Ϫ1
1
cm : 3468, 3382 (OH), 1698 (CϭO); H-NMR (pyridine-d ) d: 1.27 (3H, s, tochrome P-450 mono-oxygenase system) was added to the cultured bacteria
5
C20-H ), 1.37 (3H, s, C18-H ), 4.29 (1H, s, C15-H), 5.67, 5.74 (each 1H, d,
Jϭ1.8 Hz, C17-H ). MS m/z: 334 (M ), 316, 301, 270, 255, 237, 221, 209,
suspension. Soft agar (2.5 ml) containing 8-AG (1 mg) was added to the test
tube and spread over a minimum glucose agar plate. Each measurement was
made in triplicate at each sample dose. Benzo[a]pyrene (BaP) and 2-(2-
3
3
ϩ
2
1
89.
Preparation of 15-Oxo-steviol (5) To a solution of 15-hydroxy-steviol furyl)-3-(5-nitro-2-furyl)acrylamide (AF2) were used as the positive con-
4) (500 mg) in pyridine (20 ml), pyridinium chlorochromate (PCC) trols for ϩS9 and ϪS9 analyses, respectively. Sterilized 5% DMSO was
490 mg) was added, and the solution was stirred at room temperature for 4 used as the negative control for both ϩS9 and ϪS9 analyses. We calculated
(
(
h. The mixture was poured into ice-water (30 ml) and extracted with diethyl the specific mutagenicity (expressed as mutagenicity of 1 mg test samples
ether (3ϫ30 ml). The combined diethyl ether layers were washed with brine, per plate) at the slope where the liner dose-response curve was observed,
dried over anhydrous Na SO , and evaporated to give an oily product. The using the least square method, at the dose range with no direct toxicity of
2
4
product was recrystallized from diethyl ether to give 15-ketone 5 (144 mg): samples. Mutagenicity was determined to be positive in the case where the
Ϫ1
1
mp 216—218 °C; IR (Nujol) cm : 3518 (OH), 1720, 1697 (CϭO); H- number of mutants was more than double that of the negative control and to
NMR (pyridine-d ) d: 1.22 (3H, s, C20-H ), 1.34 (3H, s, C18-H ), 5.47, 6.27 be negative (below detection limit) where the number was less than double.
5
3
3
ϩ
(
each 1H, d, Jϭ1.8 Hz, C17-H ); MS m/z: 332 (M ), 314, 304, 286, 268, The mutation ratio was calculated by the following formula: mutation ratio
2
2
53.
Preparation of Steviol Methyl Ester (6) To a diazomethane–diethyl negative control)/numbers of colonies for viable cells.
ether solution (12 ml), steviol (1.00 g) was added. The mixture was shaken
and allowed to stand overnight at room temperature. The mixture was con- References
(/mg sample/plate)ϭ(number of colonies of mutantsϪnumber of colonies for
centrated and the residue was recrystallized from hexane–diethyl ether to
1) Wood H. B., Allerton R., Diehl H. W., Fletcher H. G., J. Org. Chem.,
Ϫ1
give ester 6 (930 mg): mp 113—115 °C; IR (Nujol) cm : 3483, 3426 (OH),
20, 875—883 (1955).
1
1
(
732, 1709 (CϭO); H-NMR (pyridine-d ) d: 0.87 (3H, s, C20-H ), 1.17
2) Akashi H., Yokoyama Y., Shokuhinkougyo, 10, 34—43 (1975).
3) Xili L., Chengjiany B., Eryi X., Fed. Chem. Toxicol., 30, 957—965
(1992).
5
3
3H, s, C18-H ), 3.62 (3H, s, –COOCH ); Anal. Calcd for C H O : C,
3
3
21 32
3
7
5.86; H, 9.70, Found: C, 75.77; H, 9.62.