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New pregnane glycosides from Stelmatocrypton khasianum
Steroids 67 (2002) 347–351
New pregnane glycosides from Stelmatocrypton khasianum Qingying Zhanga, Yuying Zhaoa,*, Bin Wanga, Rui Fengb, Xuehui Liua, Tieming Chenga a
Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University, Beijing 100083, People’s Republic of China b National Center of Biomedical Analysis, Beijing 100850, People’s Republic of China Received 13 June 2001; received in revised form 21 August 2001; accepted 27 August 2001
Abstract Four new pregnane glycosides, stelmatocryptonoside A, B, C, and D (1–4), were isolated from the stems of Stelmatocrypton khasianum. On the basis of chemical and spectral data, the structures of 1–4 were established as 3, 16␣-dihydroxy-pregn-5-en-20-one-16-O--Dglucopyranosyl-(132)-[-D-glucopyranosyl-(136)]--D-glucopyranosyl-(136)--D-glucopyranoside; 3, 20-dihydroxy-pregn-5-en-20-D-glucopyranosyl-(136)--D-glucopyranosyl-(136)--D-glucopyranosyl-(132)--D-digitalopyranoside; 3, 16␣-dihydroxy-pregn-5en-20-one-16-O--D-glucopyranosyl-(132)--D-glucopyranosyl-(136)--D-glucopyranoside; and 3, 16␣-dihydroxy-pregn-5-en-20one-16-O--D-glucopyranosyl-(136)--D-glucopyranosyl-(136)--D-glucopyranoside. This is the first report of pregnane glycosides with sugar chains linked at C-16 of the aglycone. © 2002 Published by Elsevier Science Inc. Keywords: Pregnane glycoside; Steroidal glycoside; Stelmatocrypton khasianum; Stelmatocryptonoside
1. Introduction Stelmatocrypton khasianum (Benth.) Bail. (Asclepiadaceae) is distributed in the Yunnan, Guizhou, and Sichuan Provinces of China and used in Chinese folk medicine for the treatment of colds, tracheitis, stomach aches, and rheumatic aches. Reports on the chemical constituents of S. khasianum are very few. In our previous investigations of this plant [1–3], triterpenoids, semiterpenoid, and benzoid derivatives were isolated. This paper deals with the isolation and structural elucidation of four new pregnane glycosides, stelmatocryptonoside A-D (1– 4). This is the first report of pregnane glycosides having sugar chains bound to the C-16 of the aglycone.
500 spectrometer. HRSI-MS were recorded on an APEX II mass spectrometer, FAB-MS were taken on a ZABspec mass spectrometer, and TOF-MS were recorded on a BIFLEX II MADLI-TOF mass spectrometer or a LDI1700 MADLI-TOF mass spectrometer. TLC was performed on silica gel GF254 (10⬃40 , Qingdao). Separation and purification were performed by column chromatography on macroporous resin D101 (nandai), silica gel (200⬃300 mesh, Qingdao), Sephadex LH-20 (Pharmacia), and RP C18 Silica gel (100 –200 mesh) (Ouya, Pharmacia). HPLC was carried out using (1) a Gilson automatic system for semi-preparative HPLC with an ˚ , 7.80 ⫻ 300 nm) or (2) a Unicom C18 column (5 m, 100 A waters 600 analytical HPLC system with a Phenomenex C18 ˚ , 4.6 ⫻ 250 nm). column (5 m, 100 A
2. Experimental procedures
2.2. Plant material
2.1. General method
Dried stems of S. khasianum were purchased from Xishuangbanna of Yunnan Province in October 1997. A voucher specimen was identified by Prof. HB Chen and deposited in the Herbarium of the Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University.
Optical rotations were recorded on an AA-IOR Automatic Polarimeter. Melting points were determined on a X4-A micromelting point apparatus and are uncorrected. NMR spectra were recorded on an INOVA-600 spectrometer or an INOVA-
2.3. Extraction and isolation procedure * Corresponding author. Tel.: ⫹86-10-62091592; fax:⫹ 86-1062015584. E-mail address: [email protected] (Y. Zhao). 0039-128X/02/$ – see front matter © 2002 Published by Elsevier Science Inc. PII: S 0 0 3 9 - 1 2 8 X ( 0 1 ) 0 0 1 8 7 - 8
Air-dried, powdered stems of S. khasianum (9.5 kg) were percolated with 95% EtOH. After evaporation of the sol-
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Q. Zhang et al. / Steroids 67 (2002) 347–351
Table 1 13 C-NMR data of compounds 1, 3, and 4 (C5D5N) Carbon
VIa
1
3
4
Carbon
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Glc(1) 1 2 3
37.32 31.74 71.73 42.40 140.91 121.33 31.86 31.66 50.03 36.71 21.94 38.99 44.60 56.61 32.16 81.64 71.73 14.63 19.56 208.22 31.74
37.62 32.48 71.17 43.34 141.84 120.89 31.96 31.49 50.19 36.79 20.88 38.86 44.86 54.48 33.73 80.78 72.10 14.65 19.47 208.27 32.93
37.62 32.46 71.22 43.34 141.74 120.86 31.90 31.43 50.17 36.74 20.89 38.67 44.77 54.41 33.56 80.53 71.89 14.59 19.44 207.85 32.46
37.58 32.49 71.27 43.31 141.86 120.75 31.90 31.38 50.12 36.74 20.79 38.66 44.85 54.39 33.40 80.58 71.92 14.54 19.41 208.41 32.49
104.88 75.11 78.31
104.67 76.35 78.23
104.70 76.38 78.32
4 5 6 glc(2) 1 2 3 4 5 6 glc(3) 1 2 3 4 5 6 glc(4) 1 2 3 4 5 6
a
1
3
4
70.48 76.28 69.02
70.92 76.53 69.57
70.89 76.84 69.09
102.62 84.82 78.07 70.58 78.31 69.37
102.75 85.01 78.00 70.81 78.12 62.40
104.94 75.04 78.22 71.09 76.84 69.73
105.43 75.39 78.36 71.60 78.36 62.71
106.55 76.38 78.12 71.22 78.86 62.24
105.41 75.04 78.15 71.47 78.32 62.57
106.64 76.54 78.25 71.28 78.82 62.34
VI ⫽ 16 ␣-methyoxy-pregnenolone.
vent, the residues were suspended in H2O and extracted sequentially with petroleum ether, ethyl ether, and n-BuOH. The BuOH extracts (370 g) were subjected to silica gel column chromatography and eluted with CHCl3/MeOH in order of increasing MeOH concentration (100:0– 60:40) to give 520 fractions. Fractions 266–520 were separated on a D101 macroporous resin column and eluted sequentially with 100% water, 30% EtOH, 50% EtOH, 70% EtOH, and 95% EtOH. The fraction from the 30% EtOH elution was subjected to column chromatography on Silica gel using CHCl3/MeOH (4:1–1:1) as a gradient eluent to afford 145 fractions. Fractions 41– 62 were isolated using a RP C18 and Sephadex LH-20 column and finally, semipreparative HPLC (50% MeOH, 2.5 ml/min) to give 3 (24 mg). Fractions 63– 80 were subjected to RP C18 and Sephadex LH-20 column chromatography and finally, semipreparative HPLC (50% MeOH, 2.5 ml/min) to yield 4 (15 mg). Fractions 81– 89 were chromatographed on a RP C18 and Sephadex LH-20 column to afford 2 (80 mg). Fractions 90–104 were subjected to RP C18 and Sephadex LH-20 column chromatography to yield 1 (400 mg).
using CHCl3/CH3OH/H2O/HOAc (30:12:4:6) as the development system. 2.5. Stelmatocryptonoside A (1) 25 White powder, mp 193⬃195°C; [␣]D ⫽ ⫺31.6 (H2O); FAB-MS (posit.) m/z: 1003.2 [M ⫹ Na]⫹; HRSI-MS (posit.) m/z: 981.4457 [M ⫹ H]⫹ (calcd. for C45H73O23: 981.4537), 819.4000 [M ⫺ glc]⫹, 657.3572 [M ⫺ 2glc]⫹, 495.2934 [M ⫺ 3glc]⫹, 333.2428 [M ⫺ 4glc]⫹; For the 1H NMR (500 MHz) and 13C NMR (125 MHz) data, see Table 1 and Table 2.
2.6. Stelmatocryptonoside B (2) 25 White powder, mp 184⬃186°C; [␣]D ⫽ ⫺44.1 (H2O); TOF-MS (posit.) m/z: 1003.49 [M ⫹ K]⫹, 987.49 [M ⫹ Na]⫹; HRSI-MS (posit.) m/z: 987.4755 [M ⫹ Na]⫹ (calcd. for C46H76O21Na: 987.4771); For the 1H NMR (500 MHz) and 13C NMR (125 MHz) data, see Table 3.
2.4. Acidic hydrolysis on TLC
2.7. Stelmatocryptonoside C (3)
A sample (1 mg) was dissolved in 1 ml MeOH and loaded on a TLC plate. The plate was suspended over a solution of 10 ml 6N HCl at a temperature of 60°C for 30 min. After hydrolysis, HCl absorbed by the silica gel on the plate was evaporated. Then, the plate was chromatographed
25 White powder, mp 207⬃209°C; [␣]D ⫽ ⫺33.3 (H2O); ⫹ TOF-MS (posit.) m/z: 841.4 [M ⫹ Na] ; HRSI-MS (posit.) m/z: 841.3815 [M ⫹ Na]⫹ (calcd. for C39H62O18Na: 841.3828); For the 1H NMR (500 MHz) and 13C NMR (125 MHz) data, see Table 1 and Table 2.
Q. Zhang et al. / Steroids 67 (2002) 347–351 Table 2 1 H-NMR data of compounds 1, 3, and 4 (C5D5N) Proton Aglycone 3-H 4-H 6-H 16-H 17-H 18-Me 19-Me 21-Me Glc(1) 1-H 2-H 3-H 4-H 5-H 6-H Glc(2) 1-H 2-H 3-H 4-H 5-H 6-H Glc(3) 1-H 2-H 3-H 4-H 5-H 6-H Glc(4) 1-H 2-H 3-H 4-H 5-H 6-H
1
3
4
3.78 (m) 2.57 (2H, brs) 5.26 (m) 5.19 (brt, 7.2 Hz) 2.93 (d, 6.4 Hz) 0.59 (s) 0.88 (s) 2.46 (s)
3.81 2.58 5.28 5.24 2.91 0.61 0.91 2.33
4.88 (d, 7.5 Hz) 4.03 (t, 7.5 Hz) 4.19 (t, 7.0 Hz) 4.43 (t, 7.0 Hz) 3.92 (m) 4.54, 4.20 (m)
4.91 (d, 7.5 Hz) 4.05 (m) 4.20 (m) 4.40 (m) 4.01 (m) 4.32, 4.60 (m)
4.82 (d, 7.5 Hz)
5.05 (d, 7.8 Hz) 3.91 (t, 7.8 Hz) 4.27 (m) 4.27 (m) 4.02 (m) 4.72, 4.29 (m)
5.19 (d, 8.0 Hz) 4.07 (m) 4.09 (t, 7.6 Hz) 4.22 (m) 4.01 (m) 4.40, 4.48 (m)
5.01 (d, 7.5 Hz)
4.98 (d, 7.3 Hz) 4.03 (t, 7.3 Hz) 4.20 (m) 4.20 (m) 3.89 (m) 4.49, 4.37 (m)
5.29 (d, J ⫽ 7.5 Hz) 4.07 (m) 4.16 (m) 4.20 (m) 3.81 (m) 4.34, 4.60 (m)
5.06 (d, 7.5 Hz)
(m) (2H, brs) (m) (brt, 7.0 Hz) (d, 6.5 Hz) (s) (s) (s)
3.80 2.59 5.32 5.19 2.92 0.59 0.92 2.41
(m) (2H, brs) (m) (brt, 7.5 Hz) (d, 6.5 Hz) (s) (s) (s)
5.12 (d, 7.6 Hz) 4.02 (t, 7.8 Hz) 4.10 (m) 4.10 (m) 3.89 (m) 4.57, 4.33 (m)
2.8. Stelmatocryptonoside D (4) 25 White powder, mp 216⬃220°C (dec.); [␣]D ⫽ ⫺39.1 ⫹ (H2O); TOF-MS (posit.) m/z: 841.0 [M ⫹ Na] ; HRSI-MS (posit.) m/z: 841.3820 [M ⫹ Na]⫹ (calcd. for C39H62O18Na: 841.3828); For the 1H NMR (500 MHz) and 13C NMR (125 MHz) data, see Table 1 and Table 2.
3. Results and discussion Stelmatocryptonoside A (1) was isolated as a white powder. The FAB-MS spectrum gave a quasi-molecular ion peak at m/z 1003.2 [M ⫹ Na]⫹, and the HRSI-MS exhibited a quasi-molecular ion peak at m/z 981.4557 [M ⫹ H]⫹ (calcd. for C45H73O23: 981.4537), from which the molecular formula C45H72O23 was deduced. Compound 1 showed positive Liebermann–Burchard and Molish reactions. The 1H NMR spectrum of 1 showed signals for methyl groups at ␦
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0.59 (3H, s), 0.88 (3H, s), and 2.46 (3H, s), one trisubstituted olefinic proton at ␦ 5.26 (1H, m), and four anomeric protons at ␦ 5.12 (1H, d, J ⫽ 7.8 Hz), 5.05 (1H, d, J ⫽ 7.8 Hz), 4.98 (1H, d, J ⫽ 7.3 Hz), and 4.88 (1H, d, J ⫽ 7.8 Hz). The 13C NMR spectrum revealed the presence of a pair of characteristic olefinic carbons at ␦ 120.89 and 141.84, one carbonyl carbon at ␦ 208.27, and four anomeric carbons at ␦ 106.64, 102.62, 105.43, and 104.88. All of the above data, together with the biogenesis relationship, suggested that 1 was a steroidal glycoside with the skeleton of pregn-5-en-20-one. The signals at ␦ 5.19 (1H, d, J ⫽ 7.2 Hz) and 3.78 (1H, m) in the 1H NMR spectrum and the corresponding signals at ␦ 80.78 and 71.17 in the 13C NMR spectrum indicated the presence of two oxygenated groups in the aglycone. In the 13 C NMR spectrum, the signals for the aglycone moiety of 1 were in good agreement with those of 16␣-methyoxypregnenolone (VI) (3-hydroxy-16 ␣ -methoxy-pregn-5-en20-one) [4] (Table 1), except for the absence of the signal for the methoxy group, indicating that the aglycone of 1 was 3, 16␣-dihydroxy-pregn-5-en-20-one. This was ultimately confirmed by the results of one- and two-dimensional NMR spectra. The stereochemistry of 16␣-OH was further determined to be ␣ configuration by the NOE correlation between H-16 (␦ 5.19) and H-18 (␦ 0.59). Acid hydrolysis of 1 on TLC produced glucose, which was identical to an authentic sample. The signals of anomeric protons and carbons in the 1H and 13C NMR spectra showed that 1 was a tetraglycoside consisting of four glucose residues with -anomeric configuration. One and two-dimensional NMR techniques (COSY, DEPT, HMQC, TOCSY, TOCSY-1D, and HMBC) permitted assignments of all the 1H and 13C NMR signals for the aglycone and sugar moieties of 1 (Table 1 and Table 2). In the HMBC spectrum, significant correlations were observed between ␦ 4.88 (glc(1) H-1) and ␦ 80.78 (aglycone C-16), ␦ 5.05 (glc(2) H-1) and ␦ 69.02 (glc(1) C-6), ␦ 4.98 (glc(3) H-1) and ␦ 69.37 (glc(2) C-6), and ␦ 5.15 (glc(4) H-1) and ␦ 84.82 (glc(2) C-2), suggesting that the positions of the glycosidic linkages were as shown in Fig. 1. The glucoses linked together at C-2, C-6, C-6, and the sugar chain bound to C-16 of the genin. The linkages were further proved by the results of the NOESY-1D spectrum (Fig. 1). Based on these observations, the structure of 1 was unambiguously established as 3,16␣-dihydroxy-pregn-5-en-20-one-16-O-D-glucopyranosyl-(132)-[-D-glucopyranosyl-(136)]-D-glucopyranosyl-(136)--D-glucopyranoside Stelmatocrytonoside B (2) was obtained as a white powder. The TOF-MS spectrum showed quasi-molecular ion peaks at m/z 1003.49 [M ⫹ K]⫹, 987.49 [M ⫹ Na]⫹, and the HRSI-MS gave a quasi-molecular ion peak at m/z 987.4755 [M ⫹ Na]⫹ (calcd. for C46H76O21Na: 987.4771), from which the molecular formula C46H76O21 was deduced. Compound 2 showed positive Liebermann–Burchard and Molish reactions. In the 1H NMR spectrum, 2 showed signals for three methyl groups at ␦ 0.61 (3H, s), 0.97 (3H, s) and 1.44 (3H, d, J ⫽ 5.5 Hz), a trisubstituted olefinic
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Table 3 NMR spectral data of compound 2 (C5D5N) Position
7a
2
␦H 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Digi 2 3 4 5 6 ⫺OCH3 Glc(1) 1 2 3 4 5 6 Glc(2) 1 2 3 4 5 6 Glc(3) 1 2 3 4 5 6 a
5.40 (m)
0.67 1.02 3.75 1.61 4.69 4.86
(s) (s) (brq, 6.2 Hz) (d, 6.2 Hz) (d, 7.8 Hz) (dd, 7.8, 9.7 Hz)
1.49 (d, 6.4 Hz) 3.51 (s) 5.26 (d, 7.6 Hz)
5.37 (d, 7.8 Hz)
␦C
␦C
DEPT
37.76 32.61 71.23 43.47 141.77 121.31 32.17 31.92 50.42 36.90 21.05 39.07 41.52 58.06 26.86 24.45 56.63 12.62 17.40 81.72 23.19 104.19 76.21 85.38 68.36 72.19 17.32 56.67 104.40 75.23 77.77 71.23 77.28 69.95 105.17 75.45 78.19 71.90 78.19 62.94
37.74 32.53 71.32 43.39 141.68 121.37 32.54 31.87 50.32 36.83 21.09 39.13 41.51 58.07 26.88 24.44 56.58 12.60 19.59 81.62 23.24 104.29 76.14 85.26 68.22 70.65 17.32 56.58 104.29 75.14 78.08 71.32 77.19 69.85 105.13 75.45 77.04 71.96 76.68 69.96 105.52 75.14 78.21 71.41 78.34 62.54
CH2 CH2 CH CH2 C CH CH2 CH CH C CH2 CH2 C CH CH2 CH2 CH CH3 CH3 CH CH3 CH CH CH CH CH CH3 CH3 CH CH CH CH CH CH2 CH CH CH CH CH CH2 CH CH CH CH CH CH2
␦H
3.80 (m) 2.57 (2H, m) 5.36 (m)
0.78 (m)
1.09 (m) 1.62 (m)
0.98 (m) 0.61 (s) 0.97 (s) 3.71 (brq, 6.5 Hz) 1.54 (d, 5.5 Hz) 4.66 (d, 7.5 Hz) 4.76 (dd, 7.5, 10.0 Hz) 3.57 (dd, 3.1, 7.5 Hz) 3.94 (m) 3.62 (brq, 7.0 Hz) 1.44 (d, 7.0 Hz) 3.46 (s) 5.21 (d, 7.5 Hz) 3.91 (t, 7.5 Hz) 4.32 (m) 4.22 (m) 3.88 (m) 4.35, 4.80 (m) 5.25 (d, 8.0 Hz) 3.98 (m) 4.10 (m) 4.12 (m) 3.83 (m) 4.35, 4.69 (m) 5.01 (d, 8.0 Hz) 4.00 (t, 8.0 Hz) 4.17 (m) 4.20 (m) 3.86 (m) 4.32, 4.45 (m)
7 ⫽ 3,20-dihydroxy-pregn-5-en-20--D-glucopyranosyl-(136)--D-glucopyranosyl-(132)--D-digitalopyranoside.
proton at ␦ 5.36 (1H, m). The 13C NMR spectrum of 2 showed signals for a pair of characteristic olefinic carbons at ␦ 121.37 and 141.68. All of the above evidence suggested that 2 was a steroidal glycoside with the skeleton of pregn5-ene. The 13C NMR spectrum of aglycone showed two oxygenated carbon signals at ␦ 81.62 and 71.22, which suggested the presence of two oxygenerated groups in the aglycone moiety of 2. Acidic hydrolysis of 2 on TLC give glucose and digitalose. The signals at ␦ 5.25 (1H, d, J ⫽ 8.0 Hz), 5.21 (1H, d, J ⫽ 7.5 Hz), 5.01 (1H, d, J ⫽ 8.0 Hz), and
4.66 (1H, d, J ⫽ 7.5 Hz) in the 1H NMR spectrum and those at ␦ 105.52, 105.13, and 104.29 in the 13C NMR spectrum among which the signal at ␦ 104.29 was due to two anomeric carbons based on the analysis of HMQC, indicated that 2 was a tetraglycoside with -anomeric configurations. Comparison of the 13C NMR data of 2 with those of a known pregnane triglycoside, compound 7 [5] (Table 3) showed that all of the signals due to the aglycone, the digitalose and the glucose moieties of glc(1) and glc(2), were almost identical with those of 7. The exception was the presence of a
Q. Zhang et al. / Steroids 67 (2002) 347–351
351
Fig. 1. The main correlations of HMBC and NOESY-1D of compound 1.
Fig. 3. The structures of compounds 3 and 4.
set of additional signals corresponding to a terminal glucose moiety in the spectra of 2 and the downshift of the signals due to the C-6 of the glc(2) moiety of 2. Thus, it was deduced that the aglycone of 2 was 3, 20-dihydroxy-pregn-5-en, the same as that of 7, and the sugar moieties of 2 consisted of a digitalose and three glucose residues. In the HMBC spectrum, significant correlations were observed between ␦ 4.66 (dig H-1) and ␦ 81.62 (aglycone C-20), ␦ 5.21 (glc(1) H-1) and ␦ 76.14 (dig C-2), ␦ 5.25 (glc(2) H-1) and ␦ 69.85 (glc(1) C-6), and ␦ 5.01 (glc(3) H-1) and ␦ 69.96 (glc(2) C-6), suggesting that the positions of the glycosidic linkages were as shown in Fig. 2. Consequently, the structure of 2 was established as 3, 20dihydroxy-pregn-5-en-20--D-glucopyranosyl-(136)--Dglucopyranosyl-(136)--D-glucopyranosyl-(132)--Ddigitalopyranoside. Stelmatocryptonoside C (3) was obtained as a white powder. Its molecular formula, C39H62O18, was determined from HRSI-MS data. Compound 3 showed positive Liebermann-Burchard and Molish reactions. A comparison of the 13 C NMR data of 3 with those of 1 showed that the 13C NMR data of 3 were very similar to those of 1 except for the absence of the signals for a glucose residue, and no glycosidation shift at C-6 of the glc(2) moiety was observed in the
spectra of 3 (Table 1). On the basis of these observations, the structure of 3 was determined to be 3, 16␣-dihydroxypregn-5-en-20-one-16-O--D-glucopyranosyl-(132)--Dglucopyranosyl-(136)--D-glucopyranoside (Fig. 3), which was unambiguously confirmed by 2D NMR spectra data. Stelmatocryptonoside D (4) was isolated as a white powder, and its molecular formula, C39H62O18, was deduced from HRSI-MS data. Compound 4 showed positive Liebermann–Burchard and Molish reactions. A comparison of the 13 C NMR data of the sugar moieties of 4 with those of 1 showed that the 13C NMR data of 4 were very similar to those of 1 except for the absence of the signals for a glucose residue, and no glycosidation shift at C-2 of the glc(2) moiety was observed in the spectra of 4 (Table 1). On the basis of these observations, the structure of 4 was established as 3, 16␣-dihydroxy-pregn-5-en-20-one-16-O--Dglucopyranosyl-(136)--D-glucopyranosyl-(136)--Dglucopyranoside (Fig. 3), which was unambiguously confirmed by 2D NMR spectra data.
Acknowledgement This work was supported by the National Natural Science Foundation of China.
References
Fig. 2. The main correlations of the HMBC spectrum of compound 2.
[1] Zhang QY, Zhao YY, Cheng TM, Cui YX, Liu XH. A new triterpenoid from Stelmatocrypton khasianum. J Asian Nat Prod Res 2000;2:81– 6. [2] Zhang QY, Zhao YY, Ma LB, Cheng TM. Chemical constituents of Stelmatocrypton khasianum. J Chin Pharm Sci 1999;8:237– 40. [3] Zhang QY, Zhao YY, Cheng TM, Cui YX, Liu XH. Studies on chemical constituents of Stelmatocrypton khasianum. J Chin Materia Medica 2000;25:101–3. [4] Terada S, Hayashi K, Mitsuhoshi H. On the structure of Stephanthraniline C. Tetrahedron Lett 1978;23:1995– 8. [5] Itokowa H, Xu JP, Takeyak K. Pregnane glycosides from an antitumor fraction of Periploca sepium. Phytochemistry 1988;27:1173–9. [6] Zhao PP, Li BM, He LY. Studies on the method of combined sugars in glycosides. Acta Pharmaceutica Sinica 1987;22:70 – 4.
New pregnane glycosides from Stelmatocrypton khasianum Qingying Zhanga, Yuying Zhaoa,*, Bin Wanga, Rui Fengb, Xuehui Liua, Tieming Chenga a
Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University, Beijing 100083, People’s Republic of China b National Center of Biomedical Analysis, Beijing 100850, People’s Republic of China Received 13 June 2001; received in revised form 21 August 2001; accepted 27 August 2001
Abstract Four new pregnane glycosides, stelmatocryptonoside A, B, C, and D (1–4), were isolated from the stems of Stelmatocrypton khasianum. On the basis of chemical and spectral data, the structures of 1–4 were established as 3, 16␣-dihydroxy-pregn-5-en-20-one-16-O--Dglucopyranosyl-(132)-[-D-glucopyranosyl-(136)]--D-glucopyranosyl-(136)--D-glucopyranoside; 3, 20-dihydroxy-pregn-5-en-20-D-glucopyranosyl-(136)--D-glucopyranosyl-(136)--D-glucopyranosyl-(132)--D-digitalopyranoside; 3, 16␣-dihydroxy-pregn-5en-20-one-16-O--D-glucopyranosyl-(132)--D-glucopyranosyl-(136)--D-glucopyranoside; and 3, 16␣-dihydroxy-pregn-5-en-20one-16-O--D-glucopyranosyl-(136)--D-glucopyranosyl-(136)--D-glucopyranoside. This is the first report of pregnane glycosides with sugar chains linked at C-16 of the aglycone. © 2002 Published by Elsevier Science Inc. Keywords: Pregnane glycoside; Steroidal glycoside; Stelmatocrypton khasianum; Stelmatocryptonoside
1. Introduction Stelmatocrypton khasianum (Benth.) Bail. (Asclepiadaceae) is distributed in the Yunnan, Guizhou, and Sichuan Provinces of China and used in Chinese folk medicine for the treatment of colds, tracheitis, stomach aches, and rheumatic aches. Reports on the chemical constituents of S. khasianum are very few. In our previous investigations of this plant [1–3], triterpenoids, semiterpenoid, and benzoid derivatives were isolated. This paper deals with the isolation and structural elucidation of four new pregnane glycosides, stelmatocryptonoside A-D (1– 4). This is the first report of pregnane glycosides having sugar chains bound to the C-16 of the aglycone.
500 spectrometer. HRSI-MS were recorded on an APEX II mass spectrometer, FAB-MS were taken on a ZABspec mass spectrometer, and TOF-MS were recorded on a BIFLEX II MADLI-TOF mass spectrometer or a LDI1700 MADLI-TOF mass spectrometer. TLC was performed on silica gel GF254 (10⬃40 , Qingdao). Separation and purification were performed by column chromatography on macroporous resin D101 (nandai), silica gel (200⬃300 mesh, Qingdao), Sephadex LH-20 (Pharmacia), and RP C18 Silica gel (100 –200 mesh) (Ouya, Pharmacia). HPLC was carried out using (1) a Gilson automatic system for semi-preparative HPLC with an ˚ , 7.80 ⫻ 300 nm) or (2) a Unicom C18 column (5 m, 100 A waters 600 analytical HPLC system with a Phenomenex C18 ˚ , 4.6 ⫻ 250 nm). column (5 m, 100 A
2. Experimental procedures
2.2. Plant material
2.1. General method
Dried stems of S. khasianum were purchased from Xishuangbanna of Yunnan Province in October 1997. A voucher specimen was identified by Prof. HB Chen and deposited in the Herbarium of the Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University.
Optical rotations were recorded on an AA-IOR Automatic Polarimeter. Melting points were determined on a X4-A micromelting point apparatus and are uncorrected. NMR spectra were recorded on an INOVA-600 spectrometer or an INOVA-
2.3. Extraction and isolation procedure * Corresponding author. Tel.: ⫹86-10-62091592; fax:⫹ 86-1062015584. E-mail address: [email protected] (Y. Zhao). 0039-128X/02/$ – see front matter © 2002 Published by Elsevier Science Inc. PII: S 0 0 3 9 - 1 2 8 X ( 0 1 ) 0 0 1 8 7 - 8
Air-dried, powdered stems of S. khasianum (9.5 kg) were percolated with 95% EtOH. After evaporation of the sol-
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Table 1 13 C-NMR data of compounds 1, 3, and 4 (C5D5N) Carbon
VIa
1
3
4
Carbon
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Glc(1) 1 2 3
37.32 31.74 71.73 42.40 140.91 121.33 31.86 31.66 50.03 36.71 21.94 38.99 44.60 56.61 32.16 81.64 71.73 14.63 19.56 208.22 31.74
37.62 32.48 71.17 43.34 141.84 120.89 31.96 31.49 50.19 36.79 20.88 38.86 44.86 54.48 33.73 80.78 72.10 14.65 19.47 208.27 32.93
37.62 32.46 71.22 43.34 141.74 120.86 31.90 31.43 50.17 36.74 20.89 38.67 44.77 54.41 33.56 80.53 71.89 14.59 19.44 207.85 32.46
37.58 32.49 71.27 43.31 141.86 120.75 31.90 31.38 50.12 36.74 20.79 38.66 44.85 54.39 33.40 80.58 71.92 14.54 19.41 208.41 32.49
104.88 75.11 78.31
104.67 76.35 78.23
104.70 76.38 78.32
4 5 6 glc(2) 1 2 3 4 5 6 glc(3) 1 2 3 4 5 6 glc(4) 1 2 3 4 5 6
a
1
3
4
70.48 76.28 69.02
70.92 76.53 69.57
70.89 76.84 69.09
102.62 84.82 78.07 70.58 78.31 69.37
102.75 85.01 78.00 70.81 78.12 62.40
104.94 75.04 78.22 71.09 76.84 69.73
105.43 75.39 78.36 71.60 78.36 62.71
106.55 76.38 78.12 71.22 78.86 62.24
105.41 75.04 78.15 71.47 78.32 62.57
106.64 76.54 78.25 71.28 78.82 62.34
VI ⫽ 16 ␣-methyoxy-pregnenolone.
vent, the residues were suspended in H2O and extracted sequentially with petroleum ether, ethyl ether, and n-BuOH. The BuOH extracts (370 g) were subjected to silica gel column chromatography and eluted with CHCl3/MeOH in order of increasing MeOH concentration (100:0– 60:40) to give 520 fractions. Fractions 266–520 were separated on a D101 macroporous resin column and eluted sequentially with 100% water, 30% EtOH, 50% EtOH, 70% EtOH, and 95% EtOH. The fraction from the 30% EtOH elution was subjected to column chromatography on Silica gel using CHCl3/MeOH (4:1–1:1) as a gradient eluent to afford 145 fractions. Fractions 41– 62 were isolated using a RP C18 and Sephadex LH-20 column and finally, semipreparative HPLC (50% MeOH, 2.5 ml/min) to give 3 (24 mg). Fractions 63– 80 were subjected to RP C18 and Sephadex LH-20 column chromatography and finally, semipreparative HPLC (50% MeOH, 2.5 ml/min) to yield 4 (15 mg). Fractions 81– 89 were chromatographed on a RP C18 and Sephadex LH-20 column to afford 2 (80 mg). Fractions 90–104 were subjected to RP C18 and Sephadex LH-20 column chromatography to yield 1 (400 mg).
using CHCl3/CH3OH/H2O/HOAc (30:12:4:6) as the development system. 2.5. Stelmatocryptonoside A (1) 25 White powder, mp 193⬃195°C; [␣]D ⫽ ⫺31.6 (H2O); FAB-MS (posit.) m/z: 1003.2 [M ⫹ Na]⫹; HRSI-MS (posit.) m/z: 981.4457 [M ⫹ H]⫹ (calcd. for C45H73O23: 981.4537), 819.4000 [M ⫺ glc]⫹, 657.3572 [M ⫺ 2glc]⫹, 495.2934 [M ⫺ 3glc]⫹, 333.2428 [M ⫺ 4glc]⫹; For the 1H NMR (500 MHz) and 13C NMR (125 MHz) data, see Table 1 and Table 2.
2.6. Stelmatocryptonoside B (2) 25 White powder, mp 184⬃186°C; [␣]D ⫽ ⫺44.1 (H2O); TOF-MS (posit.) m/z: 1003.49 [M ⫹ K]⫹, 987.49 [M ⫹ Na]⫹; HRSI-MS (posit.) m/z: 987.4755 [M ⫹ Na]⫹ (calcd. for C46H76O21Na: 987.4771); For the 1H NMR (500 MHz) and 13C NMR (125 MHz) data, see Table 3.
2.4. Acidic hydrolysis on TLC
2.7. Stelmatocryptonoside C (3)
A sample (1 mg) was dissolved in 1 ml MeOH and loaded on a TLC plate. The plate was suspended over a solution of 10 ml 6N HCl at a temperature of 60°C for 30 min. After hydrolysis, HCl absorbed by the silica gel on the plate was evaporated. Then, the plate was chromatographed
25 White powder, mp 207⬃209°C; [␣]D ⫽ ⫺33.3 (H2O); ⫹ TOF-MS (posit.) m/z: 841.4 [M ⫹ Na] ; HRSI-MS (posit.) m/z: 841.3815 [M ⫹ Na]⫹ (calcd. for C39H62O18Na: 841.3828); For the 1H NMR (500 MHz) and 13C NMR (125 MHz) data, see Table 1 and Table 2.
Q. Zhang et al. / Steroids 67 (2002) 347–351 Table 2 1 H-NMR data of compounds 1, 3, and 4 (C5D5N) Proton Aglycone 3-H 4-H 6-H 16-H 17-H 18-Me 19-Me 21-Me Glc(1) 1-H 2-H 3-H 4-H 5-H 6-H Glc(2) 1-H 2-H 3-H 4-H 5-H 6-H Glc(3) 1-H 2-H 3-H 4-H 5-H 6-H Glc(4) 1-H 2-H 3-H 4-H 5-H 6-H
1
3
4
3.78 (m) 2.57 (2H, brs) 5.26 (m) 5.19 (brt, 7.2 Hz) 2.93 (d, 6.4 Hz) 0.59 (s) 0.88 (s) 2.46 (s)
3.81 2.58 5.28 5.24 2.91 0.61 0.91 2.33
4.88 (d, 7.5 Hz) 4.03 (t, 7.5 Hz) 4.19 (t, 7.0 Hz) 4.43 (t, 7.0 Hz) 3.92 (m) 4.54, 4.20 (m)
4.91 (d, 7.5 Hz) 4.05 (m) 4.20 (m) 4.40 (m) 4.01 (m) 4.32, 4.60 (m)
4.82 (d, 7.5 Hz)
5.05 (d, 7.8 Hz) 3.91 (t, 7.8 Hz) 4.27 (m) 4.27 (m) 4.02 (m) 4.72, 4.29 (m)
5.19 (d, 8.0 Hz) 4.07 (m) 4.09 (t, 7.6 Hz) 4.22 (m) 4.01 (m) 4.40, 4.48 (m)
5.01 (d, 7.5 Hz)
4.98 (d, 7.3 Hz) 4.03 (t, 7.3 Hz) 4.20 (m) 4.20 (m) 3.89 (m) 4.49, 4.37 (m)
5.29 (d, J ⫽ 7.5 Hz) 4.07 (m) 4.16 (m) 4.20 (m) 3.81 (m) 4.34, 4.60 (m)
5.06 (d, 7.5 Hz)
(m) (2H, brs) (m) (brt, 7.0 Hz) (d, 6.5 Hz) (s) (s) (s)
3.80 2.59 5.32 5.19 2.92 0.59 0.92 2.41
(m) (2H, brs) (m) (brt, 7.5 Hz) (d, 6.5 Hz) (s) (s) (s)
5.12 (d, 7.6 Hz) 4.02 (t, 7.8 Hz) 4.10 (m) 4.10 (m) 3.89 (m) 4.57, 4.33 (m)
2.8. Stelmatocryptonoside D (4) 25 White powder, mp 216⬃220°C (dec.); [␣]D ⫽ ⫺39.1 ⫹ (H2O); TOF-MS (posit.) m/z: 841.0 [M ⫹ Na] ; HRSI-MS (posit.) m/z: 841.3820 [M ⫹ Na]⫹ (calcd. for C39H62O18Na: 841.3828); For the 1H NMR (500 MHz) and 13C NMR (125 MHz) data, see Table 1 and Table 2.
3. Results and discussion Stelmatocryptonoside A (1) was isolated as a white powder. The FAB-MS spectrum gave a quasi-molecular ion peak at m/z 1003.2 [M ⫹ Na]⫹, and the HRSI-MS exhibited a quasi-molecular ion peak at m/z 981.4557 [M ⫹ H]⫹ (calcd. for C45H73O23: 981.4537), from which the molecular formula C45H72O23 was deduced. Compound 1 showed positive Liebermann–Burchard and Molish reactions. The 1H NMR spectrum of 1 showed signals for methyl groups at ␦
349
0.59 (3H, s), 0.88 (3H, s), and 2.46 (3H, s), one trisubstituted olefinic proton at ␦ 5.26 (1H, m), and four anomeric protons at ␦ 5.12 (1H, d, J ⫽ 7.8 Hz), 5.05 (1H, d, J ⫽ 7.8 Hz), 4.98 (1H, d, J ⫽ 7.3 Hz), and 4.88 (1H, d, J ⫽ 7.8 Hz). The 13C NMR spectrum revealed the presence of a pair of characteristic olefinic carbons at ␦ 120.89 and 141.84, one carbonyl carbon at ␦ 208.27, and four anomeric carbons at ␦ 106.64, 102.62, 105.43, and 104.88. All of the above data, together with the biogenesis relationship, suggested that 1 was a steroidal glycoside with the skeleton of pregn-5-en-20-one. The signals at ␦ 5.19 (1H, d, J ⫽ 7.2 Hz) and 3.78 (1H, m) in the 1H NMR spectrum and the corresponding signals at ␦ 80.78 and 71.17 in the 13C NMR spectrum indicated the presence of two oxygenated groups in the aglycone. In the 13 C NMR spectrum, the signals for the aglycone moiety of 1 were in good agreement with those of 16␣-methyoxypregnenolone (VI) (3-hydroxy-16 ␣ -methoxy-pregn-5-en20-one) [4] (Table 1), except for the absence of the signal for the methoxy group, indicating that the aglycone of 1 was 3, 16␣-dihydroxy-pregn-5-en-20-one. This was ultimately confirmed by the results of one- and two-dimensional NMR spectra. The stereochemistry of 16␣-OH was further determined to be ␣ configuration by the NOE correlation between H-16 (␦ 5.19) and H-18 (␦ 0.59). Acid hydrolysis of 1 on TLC produced glucose, which was identical to an authentic sample. The signals of anomeric protons and carbons in the 1H and 13C NMR spectra showed that 1 was a tetraglycoside consisting of four glucose residues with -anomeric configuration. One and two-dimensional NMR techniques (COSY, DEPT, HMQC, TOCSY, TOCSY-1D, and HMBC) permitted assignments of all the 1H and 13C NMR signals for the aglycone and sugar moieties of 1 (Table 1 and Table 2). In the HMBC spectrum, significant correlations were observed between ␦ 4.88 (glc(1) H-1) and ␦ 80.78 (aglycone C-16), ␦ 5.05 (glc(2) H-1) and ␦ 69.02 (glc(1) C-6), ␦ 4.98 (glc(3) H-1) and ␦ 69.37 (glc(2) C-6), and ␦ 5.15 (glc(4) H-1) and ␦ 84.82 (glc(2) C-2), suggesting that the positions of the glycosidic linkages were as shown in Fig. 1. The glucoses linked together at C-2, C-6, C-6, and the sugar chain bound to C-16 of the genin. The linkages were further proved by the results of the NOESY-1D spectrum (Fig. 1). Based on these observations, the structure of 1 was unambiguously established as 3,16␣-dihydroxy-pregn-5-en-20-one-16-O-D-glucopyranosyl-(132)-[-D-glucopyranosyl-(136)]-D-glucopyranosyl-(136)--D-glucopyranoside Stelmatocrytonoside B (2) was obtained as a white powder. The TOF-MS spectrum showed quasi-molecular ion peaks at m/z 1003.49 [M ⫹ K]⫹, 987.49 [M ⫹ Na]⫹, and the HRSI-MS gave a quasi-molecular ion peak at m/z 987.4755 [M ⫹ Na]⫹ (calcd. for C46H76O21Na: 987.4771), from which the molecular formula C46H76O21 was deduced. Compound 2 showed positive Liebermann–Burchard and Molish reactions. In the 1H NMR spectrum, 2 showed signals for three methyl groups at ␦ 0.61 (3H, s), 0.97 (3H, s) and 1.44 (3H, d, J ⫽ 5.5 Hz), a trisubstituted olefinic
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Table 3 NMR spectral data of compound 2 (C5D5N) Position
7a
2
␦H 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Digi 2 3 4 5 6 ⫺OCH3 Glc(1) 1 2 3 4 5 6 Glc(2) 1 2 3 4 5 6 Glc(3) 1 2 3 4 5 6 a
5.40 (m)
0.67 1.02 3.75 1.61 4.69 4.86
(s) (s) (brq, 6.2 Hz) (d, 6.2 Hz) (d, 7.8 Hz) (dd, 7.8, 9.7 Hz)
1.49 (d, 6.4 Hz) 3.51 (s) 5.26 (d, 7.6 Hz)
5.37 (d, 7.8 Hz)
␦C
␦C
DEPT
37.76 32.61 71.23 43.47 141.77 121.31 32.17 31.92 50.42 36.90 21.05 39.07 41.52 58.06 26.86 24.45 56.63 12.62 17.40 81.72 23.19 104.19 76.21 85.38 68.36 72.19 17.32 56.67 104.40 75.23 77.77 71.23 77.28 69.95 105.17 75.45 78.19 71.90 78.19 62.94
37.74 32.53 71.32 43.39 141.68 121.37 32.54 31.87 50.32 36.83 21.09 39.13 41.51 58.07 26.88 24.44 56.58 12.60 19.59 81.62 23.24 104.29 76.14 85.26 68.22 70.65 17.32 56.58 104.29 75.14 78.08 71.32 77.19 69.85 105.13 75.45 77.04 71.96 76.68 69.96 105.52 75.14 78.21 71.41 78.34 62.54
CH2 CH2 CH CH2 C CH CH2 CH CH C CH2 CH2 C CH CH2 CH2 CH CH3 CH3 CH CH3 CH CH CH CH CH CH3 CH3 CH CH CH CH CH CH2 CH CH CH CH CH CH2 CH CH CH CH CH CH2
␦H
3.80 (m) 2.57 (2H, m) 5.36 (m)
0.78 (m)
1.09 (m) 1.62 (m)
0.98 (m) 0.61 (s) 0.97 (s) 3.71 (brq, 6.5 Hz) 1.54 (d, 5.5 Hz) 4.66 (d, 7.5 Hz) 4.76 (dd, 7.5, 10.0 Hz) 3.57 (dd, 3.1, 7.5 Hz) 3.94 (m) 3.62 (brq, 7.0 Hz) 1.44 (d, 7.0 Hz) 3.46 (s) 5.21 (d, 7.5 Hz) 3.91 (t, 7.5 Hz) 4.32 (m) 4.22 (m) 3.88 (m) 4.35, 4.80 (m) 5.25 (d, 8.0 Hz) 3.98 (m) 4.10 (m) 4.12 (m) 3.83 (m) 4.35, 4.69 (m) 5.01 (d, 8.0 Hz) 4.00 (t, 8.0 Hz) 4.17 (m) 4.20 (m) 3.86 (m) 4.32, 4.45 (m)
7 ⫽ 3,20-dihydroxy-pregn-5-en-20--D-glucopyranosyl-(136)--D-glucopyranosyl-(132)--D-digitalopyranoside.
proton at ␦ 5.36 (1H, m). The 13C NMR spectrum of 2 showed signals for a pair of characteristic olefinic carbons at ␦ 121.37 and 141.68. All of the above evidence suggested that 2 was a steroidal glycoside with the skeleton of pregn5-ene. The 13C NMR spectrum of aglycone showed two oxygenated carbon signals at ␦ 81.62 and 71.22, which suggested the presence of two oxygenerated groups in the aglycone moiety of 2. Acidic hydrolysis of 2 on TLC give glucose and digitalose. The signals at ␦ 5.25 (1H, d, J ⫽ 8.0 Hz), 5.21 (1H, d, J ⫽ 7.5 Hz), 5.01 (1H, d, J ⫽ 8.0 Hz), and
4.66 (1H, d, J ⫽ 7.5 Hz) in the 1H NMR spectrum and those at ␦ 105.52, 105.13, and 104.29 in the 13C NMR spectrum among which the signal at ␦ 104.29 was due to two anomeric carbons based on the analysis of HMQC, indicated that 2 was a tetraglycoside with -anomeric configurations. Comparison of the 13C NMR data of 2 with those of a known pregnane triglycoside, compound 7 [5] (Table 3) showed that all of the signals due to the aglycone, the digitalose and the glucose moieties of glc(1) and glc(2), were almost identical with those of 7. The exception was the presence of a
Q. Zhang et al. / Steroids 67 (2002) 347–351
351
Fig. 1. The main correlations of HMBC and NOESY-1D of compound 1.
Fig. 3. The structures of compounds 3 and 4.
set of additional signals corresponding to a terminal glucose moiety in the spectra of 2 and the downshift of the signals due to the C-6 of the glc(2) moiety of 2. Thus, it was deduced that the aglycone of 2 was 3, 20-dihydroxy-pregn-5-en, the same as that of 7, and the sugar moieties of 2 consisted of a digitalose and three glucose residues. In the HMBC spectrum, significant correlations were observed between ␦ 4.66 (dig H-1) and ␦ 81.62 (aglycone C-20), ␦ 5.21 (glc(1) H-1) and ␦ 76.14 (dig C-2), ␦ 5.25 (glc(2) H-1) and ␦ 69.85 (glc(1) C-6), and ␦ 5.01 (glc(3) H-1) and ␦ 69.96 (glc(2) C-6), suggesting that the positions of the glycosidic linkages were as shown in Fig. 2. Consequently, the structure of 2 was established as 3, 20dihydroxy-pregn-5-en-20--D-glucopyranosyl-(136)--Dglucopyranosyl-(136)--D-glucopyranosyl-(132)--Ddigitalopyranoside. Stelmatocryptonoside C (3) was obtained as a white powder. Its molecular formula, C39H62O18, was determined from HRSI-MS data. Compound 3 showed positive Liebermann-Burchard and Molish reactions. A comparison of the 13 C NMR data of 3 with those of 1 showed that the 13C NMR data of 3 were very similar to those of 1 except for the absence of the signals for a glucose residue, and no glycosidation shift at C-6 of the glc(2) moiety was observed in the
spectra of 3 (Table 1). On the basis of these observations, the structure of 3 was determined to be 3, 16␣-dihydroxypregn-5-en-20-one-16-O--D-glucopyranosyl-(132)--Dglucopyranosyl-(136)--D-glucopyranoside (Fig. 3), which was unambiguously confirmed by 2D NMR spectra data. Stelmatocryptonoside D (4) was isolated as a white powder, and its molecular formula, C39H62O18, was deduced from HRSI-MS data. Compound 4 showed positive Liebermann–Burchard and Molish reactions. A comparison of the 13 C NMR data of the sugar moieties of 4 with those of 1 showed that the 13C NMR data of 4 were very similar to those of 1 except for the absence of the signals for a glucose residue, and no glycosidation shift at C-2 of the glc(2) moiety was observed in the spectra of 4 (Table 1). On the basis of these observations, the structure of 4 was established as 3, 16␣-dihydroxy-pregn-5-en-20-one-16-O--Dglucopyranosyl-(136)--D-glucopyranosyl-(136)--Dglucopyranoside (Fig. 3), which was unambiguously confirmed by 2D NMR spectra data.
Acknowledgement This work was supported by the National Natural Science Foundation of China.
References
Fig. 2. The main correlations of the HMBC spectrum of compound 2.
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