In the aglycone of 1, two spin systems H -1 through H -2, H-3, H -4, H-5, H -6, H -7, H-8, H-9, and H -11 to
2
2
2
2
2
2
H -12 in rings A, B, and C, and CH –CH –CH (H -15 through H -16 to H-17) in ring D were identified in the DQF-COSY
2
2
2
2
2
spectra. The connectivity of ring A, B, C, and D was assigned based on the interpretation of the HMBC data. The long-range
correlations from H-19 at ꢄ 9.97 ppm to C-1, C-5, C-9, and C-10, and from H -1 to C-9 indicated the connectivity of rings A
2
and B. The relationship between rings C and D was established by the observation of correlations from H -18 to C-12, C-13,
3
the oxygenated quaternary carbon at C-14 and C-17, as well as those observed from H -12 to C-17 and H -15 to C-8.
2
2
The ꢂ,ꢀ-unsaurated ꢆ-lactone was determined to be connected at C-17 by the HMBC correlation from H-17 to C-18,
1
3
C-20, C-21, and C-22. Moreover, the assigned C NMR chemical shifts and 2D NMR studies of the aglycone of 1 in CDCl3
are very similar to those of corotoxigenin [10, 11].
The presence of two sugar units in 1 was indicated by the presence of two anomeric proton signals at ꢄ 4.94 ppm for
H-1ꢅ and 4.32 ppm for H-1ꢅꢅ. Their spin systems were determined by the COSY correlations H-1ꢅ–H -2ꢅ–H-3ꢅ–H-4ꢅ–H-5ꢅ–H -6ꢅ
2
3
and H-1ꢅꢅ–H-2ꢅꢅ–H-3ꢅꢅ–H-4ꢅꢅ–H-5ꢅꢅ–H -6ꢅꢅ. Again the interglycosidic linkage and the point of attachment to the genin were
3
established by HMBC correlations from H-1ꢅ to C-3 and H-1ꢅꢅ to C-4ꢅ.
The HMBC correlation between the proton at 3.52 ppm and C-3ꢅꢅ (ꢄ 83.4 ppm) placed the methoxy group at C-3ꢅꢅ.
C
Therefore, the structure of 1 was determined as corotoxigenin-3-O-ꢀ-digitalopyranosyl-(1–4)-O-ꢀ-digitoxopyanoside.
Compound 2 gave a positive Liebermann–Burchard and Molisch test, indicating it to be a steroidal glycoside. It is
isolated as a white powder obtained from a CH Cl –MeOH mixture and as a major metabolite in the EtOAc soluble fraction.
2
2
It is worth mentioning that 2 was not isolated in its natural states but was prepared by the hydrolysis of its glycoside [12]. The
MS data indicated the formula C H O for compound 2, which is composed of two hexoses, one of them a 6-desoxyhexose,
3
4 56 12
an O-methyl group, and a pregnenetriol as aglycone. The resonance signals of the anomeric protons of both saccharide units
showed large homonuclear coupling constants, indicating the axial orientation of the anomeric protons. The 6-desoxyhexose
was identified as digitalose (3-O-methyl-6-desoxy-galactopyranose) due to the following findings: the triplet structure of the
proton resonance of H-2ꢅ (J = 9.97 Hz) indicated the axial orientation of H-1ꢅ, H-2ꢅ, and H-3ꢅ. The second hexose was identified
1
3
as glucopyranose due to the C shift values compared with literature [13].
The linkages of the interglycosidic bonds and the point of attachment of the saccharide chains to the aglycon were
established by an HMBC experiment. Correlations were observed between H-1ꢅ of digitalose (4.48 ppm, d) and C-20
(
81.26 ppm) of the genin, as well as between H-1ꢅꢅ (4.79 ppm) of the glucose and C-2ꢅ (74.9 ppm) of the digitalose. In order to
confirm the sugar sequences and aglycon structure, compound 2 was hydrolyzed in 5% HCl–MeOH to furnish the aglycon, as
shown by TLC, and D-glucose and D-digitalose.
+
The aglycon of 2 gave a molecular ion peak at m/z 352 [M(C H O ) + H O] in its ES-IMS: comparison of the
2
1
34
3
2
1H and 13C NMR and MS data with [13] indicated that the aglycone was pregn-5-ene-3ꢀ,16,20-triol. The C chemical shift
values of the aglycon (Table1) indicated the presence of a pregnene molecule. The decoupled 13C NMR and DEPT confirmed
the presence of a double bond due to a quaternary carbon (C-5) signal at 140.9 ppm and a methine (C-6) signal at 120.8 ppm.
The singlet methyls resonating at 12.9/0.73 ppm and 18.4/1.02 ppm were assigned to C-18/H-18 and C-19/H-19, respectively,
by comparison with literature data [13, 14]. For compound 2, two spin systems H -1 through H -2, H-3, and H -4 in ring A and
13
2
2
2
H-6, H -7, H-8, H-9, and H -11 to H -12 in ring B, and H-14 through H -15, H-16, H-17, H-20 to H -21 in ring D were
2
2
2
2
3
identified in the DQF-COSY spectra.
Based on literature [13, 15] and after comparison of the chemical shift C-16 (77.01 ppm) and C-20 (69.7 ppm), the
compound 2 have similar configuration to those of 16ꢂ, and 20(S). The aglycon was confirmed as 5ꢁ-pregnene-3ꢀ,16ꢂ,20(S)-
triol [16–19].
The structure of compound 2 was determined to be 5ꢁ-pregnene-3ꢀ,16ꢂ,20(S)-diol 20-O-[ꢀ-D-digitalopyranosyl
13
1
(
1ꢃ2)-ꢀ-D-glucopyranoside], and complete assignments of C and H of this compound are reported for the first time (Table 1).
EXPERIMENTAL
General Experimental Procedures. Melting points were obtained using a Buchi 510 melting point apparatus. Optical
rotation were recorded on a JASCO P-2000 polarimeter. NMR spectra were recorded with a Bruker AV-400 with a cryoprobe
1
for H, APT, COSY, HSQC, and HMBC. Chemical shift values are given in ꢄ (ppm) using the peak signals of the solvent
CDCl (ꢄ 7.28 and ꢄ 77.3) for 1 and CD OD (ꢄ 3.35; and ꢄ 49.3) or C D N (ꢄ 8.74, 7.58, and 7.22; and ꢄ 150.35,
3
H
C
3
H
C
5
5
H
C
1
35.91, and 123.87) for 2 as references, and coupling constants are reported in Hz. ESI-MS data were measured with a
867