Structure of a steroid saponin from Digitalis ciliata

Article abstract of DOI:10.1007/s10600-007-0069-2

A new steroid glycoside was isolated from leaves of Digitalis ciliata (Scrophulariaceae) by fractionation of the total extracted substances. Its structure was determined as (25R)-5α-spirostan-3β-ol 3-O-β-D-glucopyranosyl-(1→3)[β-D-fucopyranosyl-(1→2)] -β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside based on chemical transformations, physical constants, and spectral data.

Full text of DOI:10.1007/s10600-007-0069-2

Chemistry of Natural Compounds, Vol. 43, No. 2, 2007
STRUCTURE OF A STEROID SAPONIN FROM Digitalis ciliata
1
2
L. N. Gvazava and V. S. Kikoladze
UDC 547.918
A new steroid glycoside was isolated from leaves of Digitalis ciliata (Scrophulariaceae) by fractionation of
the total extracted substances. Its structure was determined as (25R)-5α-spirostan-3β-ol 3-O-β-D-
glucopyranosyl-(1→3)[β-D-fucopyranosyl-(1→2)]-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside based
on chemical transformations, physical constants, and spectral data.
Key words: Digitalis ciliata, Scrophulariaceae, steroid glycoside, spirostane, tigogenin.
We have previously reported on steroid glycosides that are derivatives of gitogenin and diosgenin [1] and are isolated
from leaves of Digitalis ciliata (Scrophlariaceae). Herein the structure of a new glycoside (1), a derivative of tigogenin, is
proved.
Glycoside 1 had a positive Sannie—Lapin color reaction [2] and IR absorption characteristic of a spiroketal belonging
to the 25R-spirostane series [3].
O
(B)
( )
OH
OH
O
HO
O
O
HO
HO
OH
O
O
O
OH
O
O
HO
HO
Me
OH
O
HO
OH
1
Acid hydrolysis of 1 produced the aglycon, which was identified by its physical chemical constants, spectral data, and
comparison with an authentic sample of tigogenin [4].
Methanolysis of 1 and subsequent analysis of the sugars by GC [5] established that the carbohydrate part contained
galactose, glucose, and fucose in a 1:2:1 ratio.
+
+
The FAB mass spectrum of 1 gave key peaks with relative mass m/z 1071 [M + Na] , 925 [M + Na - deoxyhexose] ,
09 [M + Na - hexose] , and 439 [M + Na - tetraose] .
+
+
9
The 13C NMR spectrum (Table 1) recorded with full decoupling of protons contained 51 lines, of which 27 belonged
to the aglycon and, therefore, 24 to the sugars. Four lines were observed in the region of anomeric resonances. Five methyls
appeared at strong field. Obviously, the fifth methyl could belong only to one of the sugars. Thus, a comparison of the FAB
mass spectrum of tigogenin (m/z 439 [M + Na] ) and its reported 13C NMR spectrum [6] with those of 1 and GC data led to
the conclusion that the carbohydrate part consisted of four sugars, one of which was a deoxyhexose.
+
Methylation of 1 by the Hakomori method [7] produced its permethylated derivative. The completeness of the
methylation was monitored by the disappearance in the IR spectrum of hydroxyl absorptions and the presence in the FAB mass
spectrum of a peak for the molecular ion with m/z 1239 [M + Na]+.
1
) I. Kutateladze Institute of Pharmaceutical Chemistry, 0159, Tbilisi, ul. P. Sarajishvili, 36, e-mail:
liligvazava@yahoo.com; 2) P. Melikishvili Institute of Physical and Organic Chemistry, 0186, Tbilisi, ul. Jikia, 5. Translated
from Khimiya Prirodnykh Soedinenii, No. 2, pp. 135-137, March-April, 2007. Original article submitted January 10, 2007.
©
0
009-3130/07/4302-0162 2007 Springer Science+Business Media, Inc.
1
62

13
TABLE 1. C NMR Spectral Data for 1 and Tigogenin [5]
1
C atom
Tigogenin, δ, ppm
1, δ, ppm
C atom
1, δ, ppm
J (CH)/Hz
1
2
3
4
5
6
7
8
9
37.5
32.6
70.5
39.1
45.1
29.1
32.5
35.4
54.6
35.9
21.3
40.3
40.8
56.6
32.1
81.1
63.1
16.7
12.5
42.0
15.0
109.3
31.9
29.3
30.6
66.9
17.2
37.3
30.0
77.5
34.8
44.8
29.0
32.4
35.3
54.5
36.0
21.3
40.2
40.8
56.5
32.2
81.1
63.1
16.6
12.4
42.0
15.0
109.2
31.8
29.3
30.6
66.9
17.3
Galactose
1
2
3
4
5
6
103.0
73.4
75.6
79.9
76.0
61.0
159.2
Glucose (A)
1
2
3
4
5
6
105.0
81.8
87.2
71.4
78.1
63.0
162.6
162.0
161.4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
Glucose (B)
1
2
3
4
5
6
104.8
75.4
78.2
71.7
78.3
62.8
Fucose
1
2
3
4
5
6
106.0
71.9
75.0
72.9
71.7
17.3
TABLE 2. PMR Spectral Data for the Carbohydrate Part of 1
Atom
Chemical shift, ppm, J/Hz
-O-Galactose
Atom
Chemical shift, ppm, J/Hz
Glucose (B)
3
H-1
H-2
H-3
H-4
H-5
H-6
4.87, J(1-2) = 7.6
4.32, J(2-3) = 9.1
4.12, J(3-4) = 3.2
4.54, J(4-5) < 2
4.09, m
H-1
H-2
H-3
H-4
H-5
H-6
5.34, J(1-2) = 7.8
4.04, J(2-3) = 8.8
4.15, J(3-4) = 8.8
4.26, J(4-5) = 9.0
3.83, m
*
*
Glucose (A)
Fucose
H-1
H-2
H-3
H-4
H-5
H-6
5.14, J(1-2) = 7.6
4.38, J(2-3) = 8.6
4.22, J(3-4) = 8.6
3.74, J(4-5) = 8.6
3.80, m
H-1
H-2
H-3
H-4
H-5
4.64, J(1-2) = 7.5
4.24, J(2-3) = 8.9
4.06, J(3-4) = 3.5
4.02, m
3.73, m
1.55 br.s
*
Me-6
______
*Signals could not be found on a 300 MHz spectrometer.
163

Tigogenin and the total methylated sugars were isolated from the reaction mixture after methanolysis of the
permethylate. GCidentified2,3,4,6-tetra-O-methyl-D-glucopyranose(terminal), 2,3,4-tri-O-methyl-D-fucopyranose(terminal),
4
,6-di-O-methyl-D-glucopyranose(disubstitutedatC-2andC-3),and2,3,6-tri-O-methyl-D-galactopyranose(substitutedatC-4).
The structure of the carbohydrate chain of 1 and its attachment site to the aglycon were established using PMR and
13C NMR data (Tables 1 and 2).
The PMR spectrum of 1 was solved bya series of selective homonuclear double resonance experiments using the usual
and difference versions. The SSCC that were found were consistent with the carbohydrate part of 1 containing D-glucose,
D-galactose, and 6-deoxy-D-galactose (fucose). The values that were found for the anomeric protons were consistent with
vicinal axial—axial couplings and indicated that all glycoside bonds of all sugars had the β-configuration [8].
The results ofthe GC analysis ofthe attachment sites in the carbohydrate agreed with experimental data ofthe observed
nuclear Overhauser effect (NOE). Thus, pre-irradiation of anomeric proton H-1 (δ 5.34) of glucose (B) markedly increased the
intensity of two signals, H-2 (4.04) of glucose (B) and H-3 (4.22) of glucose (A), by5.9 and 7.2%, respectively. Pre-irradiation
of H-1 of glucose (A) (5.14) caused an analogous reaction by H-2 of glucose (A) (4.38) and H-4 of galactose (4.54). Pre-
irradiation of H-1 of fucose (4.64) caused a reaction of H-2 of fucose (4.24) and H-2 of glucose (A) (4.38). Thus, fucose and
glucose (B) glycosylated glucose (A) at C-2 and C-3, respectively; glucose (A), galactose at C-4. This was consistent with the
paramagnetic shift of the signals for the glycosylated centers in the 13C NMR spectrum compared with those in the literature
[
6, 9] by ∆ ∼ 9.1 ppm for galactose C-4 and by ∆ ∼ 5.8 ppm and ∆ ∼ 9.0 ppm for glucose (A) C-2 and C-3.
A comparison of the chemical shifts for 1 and tigogenin [5] showed that C-3 had the largest paramagnetic shift
∆ = 7.0 ppm); C-2 and C-4, diamagnetic shifts of 2.6 and 4.3, respectively. This fact unambiguously indicated that C-3 was
(
the attachment site of the carbohydrate.
Finally, geminal heteronuclear SSCC J(CH) (Table 1) confirmed conclusivelythat the C-1 ORgroup ofall sugars was
equatorial, i.e., all glycosidic bonds had the β-configuration with the C -conformation for their rings [9].
1
4
1
Thus, 1is(25R),5α-spirostan-3β-ol3-O-β-D-glucopyranosyl-(1→3)-[β-D-fucopyranosyl-(1→2)]-β-D-glucopyranosyl-
(1→4)-β-D-galactopyranoside.
EXPERIMENTAL
General Comments. Kieselgel 60F254 (Merck) and Silufol UV 254 plates were used for TLC. Column
chromatography used silica gel (KSK, particle size <63 and 63-100 µm). The following solvent systems were used:
) CHCl :CH OH:H O (a, 65:15:2; b, 65:35:8), 2) CHCl :CH OH (a, 10:1; b, 50:1).
1
3
3
2
3
3
GC was performed on a Chrom-5 instrument. Monosaccharides were chromatographed as trimethylsilyl esters of
methylglycosides over a column (3 m × 4 mm) with Chromaton N-AW containing silicone SE-30 (5%), thermostat 190°C, He
carrier gas, flow rate 45 mL/min.
Methylglycosides of methylated sugars were prepared by boiling (4 h) methyl esters in absolute methanol containing
HCl (5%). The products were chromatographed over a column (1.2 m × 3 mm) with celite containing 1,4-polybutanediol
succinate (20%), thermostat 160°C, He carrier gas, flow rate 50 mL/min.
PMR and 13C NMR spectra were recorded on a AM-300 (Bruker) spectrometer at 300 MHz for H and 75 MHz for
1
13
C in pyridine-d with TMS internal standard.
5
Other data have been reported [1].
Isolation of 1. Preliminary processing of the total extracted substances has been described [1].
After extraction of diosgenin and gitogenin, fractions enriched with the studied glycoside were combined and
chromatographed over silica-gel columns using systems 1a and 1b. Fractions containing chromatographically homogeneous
(0.48 g) were collected. The overall yield calculated per air-dried weight of the raw material was 0.01%.
25R),5α-Spirostan-3β-ol 3-O-β-D-glucopyranosyl-(1→3)-[β-D-fucopyranosyl-(1→2)]-β-D-glucopyranosyl-(1→4)-
1
(
2
0
β-D-galactopyranoside. White crystalline powder, mp 266-269°C (ethanol), [α]
MS (m/z, %): 1071 (38) [M + Na] , 925 (100) [M + Na - deoxyhexose] , 909 (42) [M + Na - hexose] , 809 (12) [M + 2Na -
deoxyhexose + hexose)] , 439 (24) [M + Na - tetrose] . IR spectrum (KBr, , cm ): 3420, 2900, 1440, 1350, 1045, 975, 915,
90, 695. PMR spectrum (δ, J/Hz): 0.70 (3H, d, J = 5.5, Me-27), 0.82 (3H, s, Me-18), 0.87 (3H, s, Me-19), 1.12 (3H, d, J = 6.8,
Me-21), 3.50 (1H, dd, J = 10.5, 10.5, H -26), 3.58 (1H, dd, J = 10.5, 2.9, H -26), 3.68 (1H, m, H-3).
-62.5° (CHCl + MeOH, 1:1, c 0.8). FAB
D
3
+
+
+
+
+
-1
(
8
b
a
1
64

Acid Hydrolysis. Glycoside 1 (50 mg) was dissolved in aqueous methanol (10 mL, 50%) containing conc. H SO
2
4
(
0.4 mL) and boiled for 10 h. The resulting precipitate was filtered off and recrystallized from ethanol to afford the aglycon
2
0
−1
(22 mg), C H O , mp 200-202°C, [α]
-69° (CHCl , c 0.5). IR spectrum (KBr, ν, cm ): 3400 (OH), 1045, 980, 961, 920,
2
7
44
3
D
+
3
13
9
01, 860. FAB MS (m/z): 439 [M + Na] . These data, the C NMR spectrum, and TLC with an authentic sample identified
the aglycon as tigogenin.
Methanolysis of 1. Glycoside 1 (10 mg) was dissolved in absolute methanol (4 mL) containing HCl (5%), boiled for
2 h, cooled, and treated with an equal volume of water. The resulting precipitate was filtered off and shown to be identical
with tigogenin (TLC, system 2a). The solid was neutralized with Ag CO , filtered off, and evaporated to dryness. GC detected
1
2
3
D-galactose, D-glucose, and D-fucose in a 1:2:1 ratio.
Methylation of 1. Glycoside 1 (100 mg) was dissolved in DMSO (10 mL), slowly treated with NaH (100 mg), stirred
vigorously for 1 h, slowly treated with CH I (1.5 mL), stirred another 3 h, poured into water (0.1 L), and extracted with CHCl
3
3
(
5 × 10 mL). The combined CHCl extracts were treated with sodium thiosulfate and evaporated to dryness. The solid was
3
chromatographed over a silica-gel column (system 2b). Recrystallization from methanol produced the permethylated product
88 mg) which was subjected to acid hydrolysis as described above. The resulting precipitate of the aglycon was separated and
identified as tigogenin.
An aqueous solution of the methylated sugars was boiled for 6 h, after which the reaction mixture was neutralized with
(
anion-exchanger EDE-10P and taken to dryness. The methylglycosides that were described above in the discussion were
identified by GC.
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1
2
3
4
5
6
7
8
9
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1
0.
165