Oleanane glycosides from Glycyrrhiza yunnanensis roots

0031-9422/‘92 $5.00+0.00 C) 1992PergamonPressplc

Vol. 31,No. 5, pp. 17471752,1992 Phytochemistry, Printedin GreatBritain.

OLEANANE

GLYCOSIDES

KAZUHIRO OHTANI, KAWJKI

Institute

of Pharmaceutical

FROM

GLYCYRRHZZA

YUNNANENSIS

OGAWA, RYOJI KASAI,? CHONG-REN YANG,~ JUN ZHOU~ and OSAMU TANAKA

ROOTS*

KAZUO YAMASAKI,

Sciences, Hiroshima University School of Medicine, Kasumi Minami-ku, $.Kunming Institute of Botany, Academia Sinica, Kunming, Yunnan, China

Hiroshima

734, Japan;

(Received 9 September 1991)

Key Word Index--Glycyrrhiza yunnunensis; Leguminosae; liquorice; roots; oleanane-type triterpene glycosides; yunganosides A,, B,, C,, D,, E,, F,; yunganogenins C, D, E, F.

Abstract-From the roots of Glycyrrhiza yunnanensis, collected in Yunnan, Chin-a, six new oleanane-type triterpene glycosides named yunganosides A,, B,, C 1, D,, E2 and F, were isolated together with hypaphorine. The structures of these glycosides were established by spectroscopic and chemical means.

INTUODUCTION Glycyrrhizae

Radix (liquorice,

roots and stolons

of

Glycyrrhiza spp.) is a very important herbal medicine. It is

used not only in the prescriptions of traditional oriental medicine, but also as a source of glycyrrhizin, a wellknown sweetener and medicine. A number of species and varieties of Glycyrrhiza have been found, and of these, G. glabra L., G. urarensis Fisch. et DC. and G. injlata Batalin are chiefly used as source plants of this herbal medicine. Glycyrrhiza yunnanensis Cheng f. et L. K. Tai grows in the northern region of Yunnan, China. The roots of this plant, called ‘yunnan gan cao’ in Chinese, have always been used in this district as a medicine in place of the plants described above. Recently, Zeng et al. reported the isolation of macedonic acid and two new triterpenoids named glyyunnansapogenins C and E from the hydrolysate of a crude glycoside fraction obtained from this plant [l]. This paper describes the isolation and structural elucidation of six new glycosides named yunganosides A, (I), B, (2), C, (3), D, (4), E, (5) and F, (6) from the same plant. BFSULTSANDDISCUSSION A methanolic extract of the roots of G. yunnanensis was chromatographed on a column of highly porous polymer resin with water, 10% MeOH, 40% MeOH, 60% MeOH and MeOH, successively. Crystallization of the 10% MeOH fraction gave a crystalline compound, which was identified as a-carboxy-N,N,N-trimethyl-lH-indole-3ethanaminium hydroxide inner salt ( = hypaphorine, the betaine of tryptophane) by comparison of physical and spectral data with those of ref. [2]. The 60% MeOH eluate, a crude glycoside fraction, was chromatographed

*Part 1 in the series ‘Studies on the constituents of Glycyrrhiza yunnanensis’. tAuthor to whom correspondence should be addressed.

on silica gel, and then purified by HPLC to give compounds l-6. On methylation with CH,N,, compounds l-6 afforded the methyl esters lb-6b, respectively. On hydrolysis with glycyrrhizin hydrolase [3,4], compound 1 yielded an aglycone la. On inspection of the spectral and physical data, compound la was found to be identical with kuzusapogenol C, previously isolated from the roots of Pueraria lobata as a sapogenin [S]. Rhamnose and glucuronic acid were identified in the methanolysate of 1. The ‘H and i3CNMR spectra of lb showed that this compound is a dimethyl ester and has three monosaccharide units. Therefore, 1 has two glucuronide moieties. Compound 1 was methylated by Hakomori’s procedure [6] followed by esterification with CH,N, to give a permethylated compound, which was subjected to sequence analysis of the sugar moiety by CC-MS [7,8] as follows. LiAlD, reduction of the permethylated compound of 1 converted the CO,Me groups of the glucuronide moieties to CD,OH. The product was then hydrolysed with HCl in aqueous MeOH. The resulting mixture of methylated monosaccharides was subjected to reduction with NaBH, followed by acetylation. GC-MS analysis [7,8] of the product showed the presence of terminal rhamnopyranose and 2-linked glucuronopyranose, the latter of which was detected as 6-&1,2,5,6-tetra-Oacetyl-3,4-di-0-methylhexitol. The anomeric configuration of each sugar unit was determined as c(for rhamnose and /l for two glucuronic acids from the coupling constants of anomeric proton signals as well as the chemical shifts of carbon signals of the sugar moiety of lb. These indicated that the sugar moiety of compound 1 can be formulated as a-Rha(l+2)-/?-GlcA( 1-+2)+GlcA. This was supported by comparison of the carbon signals due to the sugar moiety of lb with the reference data of the methyl ester of liquorice-saponin D, which was isolated from G. urarensis and has the same sugar moiety [9]. In comparing the 13CNMR spectra of lb and la, glycosylation shifts [lo] were observed for the signals due to C-2, C-3 and C-24, indicating that 1 is the 3-0glycoside of la. Based on these results, the structure of 1 was established as shown.

1747

K. OHTANI et 01.

1748

1,

K’ i

B-OH

R’=H

S:R=O

2 R’=H

R’=OH

6’ R = $-OH,

3 R’ = a-OH

R’ = H

la-6a.

respe‘ave

aglycone

of

o-H

l-6

Rhamnose and glucuronic acid were identified in the methanolysate of 2. On enzymatic hydrolysis of 2 with glycyrrhizin hydrolase, 2a was obtained as the aglycone. Compound 2a was identified as soyasapogenol B, previously isolated from soy-beans as a sapogenin [l 11, by comparison of the physical and r3CNMR data with those of refs [S and 111. The carbon signals of the sugar moieties of the dimethyl ester (2b) were essentially superimposable on those of 1b and the glycosylation shifts were observed for the signals due to C-2, C-3 and C-24. These results led to the formulation of 2 as shown. On enzymatic hydrolysis with glycyrrhizin hydrolase, 3 afforded a new aglycone named yunganogenin C (3a). The negative FAB-MS of 3a indicated a peak due to [M-H]-at m/z 457. The r3C NMR spectrum of 3a showed the presence of a trisubstituted double bond, two secondary hydroxyl groups and a primary hydroxyl group. Furthermore, the carbon signals of 3a appeared at almost the same positions as those of la and 2a except for the signals attributable to the E ring. The EI-MS of 3a showed a characteristic fragment peak at m/z 233 due to retro Diels -Alder cleavage as shown. These suggested that 3a is an isomer of la and 2a with respect to the position or configuration of a secondary hydroxyl group located in the E ring. Assignment of proton signals due to the 29- and 30gem-dimethyl groups of 3a was substantiated by ‘H-‘H and rH-13C correlation spectroscopy (COSY) experiments. In the correlation via long-range coupling (COLOC) spectrum of 3a, C-H long-range correlations were observed between the carbinyl carbon (674.5) and both the 29- and 30-methyl protons as shown in Table 1. Further, the carbinyl proton signal at 63.70 was observed as double triplets (.I=08 and 3.7 Hz). These suggested that the hydroxyl group on the E ring of 3a must be located at 21a (axial). Therefore, the structure of 3a is formulated as 3/?,21c(,24-trihydroxy-olean-12-ene. The ‘H and 13C NMR spectra ofthe dimethyl ester (3b) revealed that the sugar moiety is identical with those of lb and 2b, and the glycosylation shifts around C-3 disclosed that 3 is a 3-0-glycoside of 3s. These observations led to the formulation of 3 as shown. On enzymatic hydrolysis with glycyrrhizin hydrolase, compound 4 afforded a new aglycone named yunganogenin D (4a). The negative FAB-MS of 4a showed a peak

due to [M -H] _ at m/z 455. The ’ 3C NMR spectrum of 4a (Table 2) revealed the presence of two double bonds, two secondary and one primary hydroxyl groups as well as seven quaternary methyl groups. Furthermore, the carbon signals assignable to the A ring of 4a were in good agreement with those of la-3a. The UV spectrum of 4a showed characteristic absorption bands at 242, 250 and 258 nm due to an olean-11,13-diene-type triterpene [12]. These results suggested that 4a is 3,24-dihydroxy-olean11,13-diene with an additional secondary hydroxyl group at the E ring. In the COLOC spectrum of 4a, C--H correlations were observed as shown in Table 1. On the basis of this observation as well as the NOE experiment shown in Fig. 1 and the coupling pattern of the carbinyl proton (63.81, lH, dd. J= 10.8, 4.0 Hz), the hydroxyl group on the E ring of 4a must be located at 228 (equatorial). Therefore, compound 4a can be formulated as 3/?,22/?,24-trihydroxy-olean-11,13-diene. Comparison of the ‘3C NMR spectrum of the dimethyl ester (4b) with that of 4a showed that 4 is the 3-0glycoside of 4a having three monosaccharide units. The carbon signals due to the sugar moiety of 4b appeared essentially at the same positions as those of lC3b. These observations led to the formulation of 4 as shown. On enzymatic hydrolysis with glycyrrhizin hydrolase, compound 6 yielded a new aglycone named yunganogat enin F (6a) which showed a peak due to [M-H] m/z 453 in the negative FAB-MS. The r3CNMR spectrum of 6a revealed that the presence of two double bonds, two secondary hydroxyl groups and a carboxyl group. The UV spectrum of 6a showed almost the same absorption band pattern as that of 4a. The carbon signals due to the A-C ring of 6a were in good agreement with those of saikogenin C [12]. The COLOC (Table 1) and NOESY (Fig. 1) spectra of 6a disclosed the presence of a hydroxyl group and a carboxyl group at C-22 and C20, respectively. Furthermore, the carbinyl proton signal of H-22 showed double doublets (J = 10.7, 4.0 Hz), suggesting that the secondary hydroxyl group at C-22 must be oriented /3 (equatorial). Based on these results, compound 6a can be formulated as 3p,22/?-dihydrowy-olean11,13-dien-30-oic acid. Glucuronic acid was identified in the methanolysate of 6. The structure of the sugar moiety of 6 was determined as b-GlcA( l-+2)-/?-GlcA from the results of methylation

Glycosides

Table

1. rH-‘%

Compound

long-range

correlation

H

from Glycyrrhiza

by COLOC of 3a-6a correlations) Correlated

(two and

three

bond

C

2.20 2.47 1.07 3.70 0.98 1.19 1.04

(H-18) (H-19) (H-19) (H-21) (H-28) (H-29) (H-30)

145.4 (C-13)/42.4 (C-14)/33.3 (C-17)/42.8 (C-19)/36.0 33.3 (C-17)/47.6 (C-18)/36.0 (C-20) 145.4 (C-13)/33.3 (C-17)/47.6 (C-18)/36.0 (C-20) 36.0 (C-20)/44.6 (C-22) 31.0 (C-16)/33.3 (C-17)/47.6 (C-18)/44.6 (C-22) 42.8 (C-19)/36.0 (C-20)/74.5 (C-21)/17.7 (C-30) 42.8 (C-19)/36.0 (C-20)/74.5 (C-21)/29.9 (C-29)

4a

2.57 1.90 3.81 0.98 1.24 1.04

(H-19) (H-19) (H-22) (H-28) (H-29) (H-30)

138.3 134.9 42.0 35.6 38.4 38.4

(C-13)/42.0 (C-18)/43.2 (C-17)/43.3 (C-16)/42.0 (C-19)/43.2 (C-19)/43.2

(C-17)/134.9 (C-18)/43.2 (C-20)/43.3 (C-20) (C-21) (C-17)/134.9 (C-18)/75.6 (C-22) (C-20)/43.3 (C-21)/29.1 (C-30) (C-20)/43.3 (C-21)/25.0 (C-29)

(C-21)

sa

2.93 2.58 3.28 2.31 0.96 1.33

(H-19) (H-19) (H-21) (H-21) (H-28) (H-29)

136.1 136.1 49.4 45.7 28.0 35.8

(C-13)/49.4 (C-13)/133.5 (C-17)/35.8 (C-20)/213.0 (C-16)/49.4 (C-19)/45.7

(C-17)/133.5 (C-18)/45.7 (C-20)/47.7 (C-18)/45.7 (C-20) (C-19)/45.7 (C-20)/213.0 (C-22) (C-22) (C-17)/133.5 (C-18)/213.0 (C-22) (C-20)/47.7 (C-21)/178.2 (C-30)

(C-21)

6a

2.67 1.85 1.48 1.53 4.20 1.00 1.23

(H-19) (H-19) (H-21) (H-21) (H-22) (H-28) (H-29)

136.1 136.1 41.2 41.2 43.6 36.2 38.3

(C-13)/48.5 (C-13)/136.0 (C-20)/74.3 (C-20)/74.3 (C-21)/48.5 (C-16)/48.5 (C-19)/41.2

(C-17)/136.0 (C-18)/41.2 (C-18)/41.2 (C-20) (C-22) (C-22) (C-17) (C-17)/136.0 (C-18)/74.3 (C-20)/43.6 (C-21)/178.2

(C-21)

(C-20)/43.6

eu

of compounds

(vide sarpra) of 6 and the coupling constants of anomeric proton signals of the trimethyl ester (6b). This was further confirmed by direct comparison of the carbon signals due to the sugar moiety of 6b with those of 6’,6”dimethyl ester of glycyrrhizin [4]. Comparison of the ’ 3C NMR spectra of 6b and 6a revealed the glycosylation shifts around C-3. From these results, the structure of 6 was established as shown. Enzymatic hydrolysis of 5 with glycyrrhizin hydrolase afforded compound Sa as a new aglycone named yunganogenin E. The UV spectrum of 5a showed the absorption bands due to the same hetero-annular diene system as in the case of 4a and 6a. The negative FAB-MS of Sa exhibited a peak at m/z 451 [M-H]which is lower than that of 6a by two mass units, and the IR spectrum of

(C-20)

(C-22) (C-30)

sa Fig. 1. NOE correlation

PHYTO31:5-u

in pyridine-d,

3a

4a

analysis

1749

yunnanensis

4a, Sa and 6a (in pyridine-d,).

Sa showed an absorption band due to a ketone. The carbon signals of Sa were observed essentially at the same positions as those of 6a except for the signals due to the E ring. Furthermore, a partial structure of Sa (Fig. 1) was proposed from results of COSY, COLOC (Table 1) and NOE (Fig. 1) experiments. These evidences led to the formulation of Sa as 38-hydroxy-22-oxo-olean-11,13dien-30-oic acid. The 1%J NMR signals of the sugar moiety and around C-3 of the trimethyl ester (Sb) appeared at almost the same positions as those of 6b. Consequently, the structure of 5 is formulated as shown. We have isolated 18 additional oleanane glycosicles from this plant, but could not obtain any glycosides of the sapogenins which have the structures proposed for gly-

K. OHTANIet al.

1750

Table 2. 13C:NMR spectral data of compounds la, Mia,

C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 OMe

lb

la

39.1 26.6 90.7 44.5 56.5 18.5 33.4 40.0 47.8 36.8 24.1 122.8 144.4 42.0 26.6 29.9 35.1 47.3 46.8 36.7 73.0 47.8 23.5 63.4 15.6 16.9 26.1 28.7 29.9 17.7

38.9 28.4 80.1 43.2 56.3 19.1 33.3 40.1 48.1 37.0 24.1 122.7 144.3 41.9 26.5 29.9 35.1 47.2 46.5 36.9 72.8 47.7 23.5 64.5 16.2 16.9 26.0 28.7 29.9 17.7

-

2b

39.1 26.6 90.6 44.5 56.5 19.0 33.4 40.0 47.9 36.8 24.0 122.6 144.9 42.5 26.6 28.9 37.9 45.5 46.9 30.9 42.4 76.0 23.4 63.2 15.7 17.1 25.8 28.8 33.3 21.1

2a

-

39.0 28.4 80.1 43.2 56.4 19.1 33.6 40.1 48.1 37 1 24.1 122.4 144.8 42.4 26.5 28.7 38.0 45.4 46.8 30.9 42.3 75.5 23.6 64.6 15.8 17.1 25.7 28.7 33.3 21.1

b (100 MHz, pyridme-d,, &values)

3b

3a

4b

4a

39.0 26.6 90.5 44.3 56.5 18.9 33.5 40.0 47.9 36.7 24.1 1224 145.3 42.4 26.7 31.0 33.3 47.6 42.8 36.0 74.5 44.6 23.2 63.0 15.6 17.0 25.6 28.8 28.1 25.4

39.0 28.2 80.1 43.1 56.5 19.1 33.6 40.0 48.2 37.1 24.2 122.4 145.4 42.4 26.7 31.0 33.3 47.6 42.9 36.0 74.5 44.6 23.5 64.5 16.2 17.1 25.6 28.8 28.1 25.5

39.0 27.3 91.6 40.0 56.8 19.2 33.8 40.8 54.6 39.4 127.3 126.3 138.3 40.4 25.1 35.6 41.8 134.9 38.4 43.1 43.3 75.6 23.0 62.8 18.3 16.8 25.4 23.6 25.0 29.1

38.9 29.0 80.1 39.8 56.4 19.6 34.0 40.7 54.8 39.8 127.3 126.3 138.3 40.8 25.0 35.6 42.0 135.0 38.4 43.2 43.3 75.6 23.3 64.5 18.3 16.9 25.4 23.6 25.0 29.1

5b

Sa

38.2 26.6 89.5 39.7 55.3 18.4 32.5 40.5 54.4 36.5 128.2 125.4 136.2 42.2 23.9 28.1 49.4 132.6 35.6 45.7 47.4 212.0 27.6 15.9 18.3 16.8 26.8 20.2 25.0 116.0 51.7

38.6 28.2 78.2 39.5 55.4 18.8 32.7 40.5 54.6 37.1 126.1 126.0 136.1 42.3 24.3 28.0 49.5 133.5 35.8 45.4 41.1 213.0 28.5 15.9 18.3 17.0 26.9 20.2 25.8 178.2

6b

6s

38.2 26.5 89.5 39.7 55.4 18 5 32.6 40.5 54.4 36.5 127.2 126.1 137.0 42.7 24.7 36.2 48.5 134.5 38.3 41.5 43.3 74.5 29.1 15.7 18.2 17.1 26.8 20.5 25.0 176.2 51.7

38.6 28.1 78.2 39.5 55.4 18.8 32.7 40.5 54.6 37.1 127.6 126.1 136.1 42.3 24.7 36.6 48.5 136.0 38.3 41.2 43.6 14.3 29.6 15.8 18.3 17.0 26.8 20.5 25.0 178.2

Table 2. (Continued) C GlcA 1 2 3 4 5 6

lb

2b

3b

4b

!5b

6b

102.4 78.4 78.4 72.5 76.5 170.4 52.2

102.6 78.5 78.7 72.8 16.7 170.3 52.0

102.4 78.4 78.4 72.5 76.5 170.4 52.0

102.9 79.7 76.8 74.3 78.0 170.0 52.0

105.1 84.5 77.6 72.1 76.7 170.2 52.0

105.2 84.5 77.5 72.7 76.7 170.2 52.0

@Me)

104.8 78.2 77.8 73.2 76.7 169.9 52.1

104.9 78.3 78.3 73.0 76.9 169.8 52.0

104.8 78.3 77.8 73.2 76.7 169.9 52.0

105.2 78.6 77.0 73.1 78.4 170.3 52.0

106.9 76.5 77.5 73.0 71.7 170.4 52.1

106.8 76.4 77.4 72.9 77.7 170.4 52.2

Rha 1 2 3 4 5 6

102.0 72.0 72.3 74.1 69.4 18.7

102.2 72.3 72.7 74.4 69.6 18.9

102.0 72.0 72.3 74.1 69.4 18.7

102.1 72.2 72.7 73.4 69.7 18.9

@Me) GlcA’ 1 2 3 4 5 6

Glycosides from Giycyrrhiza yunnanensis

yunnan-sapogenins C (3/I-hydroxy-16-oxo-olean-11,13dien-30-ok acid) and E (3&21a,24-trihydroxy-oleanl&13-dien-29-oic acid) by Zeng et al. [l]. furthermore, no giycyrrhizin seems to be present in this species. The structure of the other glycosides will be reported in the near future.

EXPERIMENTAL General. Mps: uncorr; NMR: TMS as int. standard; MS: 70 eV; CC: silica gel (Kieselgel60,70-230 mesh, Merck), silanized silica gel (LiChroprep RP-18, 40-63ym, Merck) and highly porous polymer resin @liaion HP-20 (Mitsubishi Chem. Ind. Co. Ltd)J were used. All solvent systems for chromatography were homogeneous. HPLC: TSK-gel ODS-12OT (21.5 mm x 30 cm, Toso Co. Ltd). Enzymatic hydrolysis of glycosides: to a soln of 50 mg of sample in 50 mM ACbuffer @H 4.3,5 ml), 0.5 ml of an aq. soln of glycyrrhizin hydrolase (1 Uml-i) was added. The mixture was incubated at 45” for 6 hr. The reaction mixture was diluted with 20 ml H,O, acidified with 0.1 M HCI at pH 3.0 and then extracted with CHCl,. The CHCI, layer was dried over auhyd. Na,SO, and evapd to afford the aglycone. Methylation of glycosides: the compound in dry MeOH was treated with CH,N, at room temp. The methyl ester was obtained after usual work-up. Methanolysis of glycosides and identification of the resulting monosaccharides [S] as well as methylation analysis of glycosides by GC-MS [7, 83 were carried out as described previously. Plant material. Glycyrrhiza yunnanensis Cheng f. et L. K. Tai was collected at Zhong Dian (Yunnan), China. A voucher specimen is deposited in the Herbarium of Kunming Institute of Botany, Chinese Academy of Science. Extraction and separation. The dried and powdered roots (2 kg) were extracted with hot MeOH. After removal of the solvent by evapn, the MeOH extract (256 g) was suspended in

1751

H,O and then washed with n-C,H,, and EtOAc, successively. The H,O layer was chromatographed on a column of highly porous potymer resin (H20, 10, 40 and 60% aq. MeOH, successively and finally MeOH). The 10% MeGH eluate (5.5 g) was crystallized from EtOH to afford needles (0.23%, yield, mp 262.5-264” (decomp.), [a]:‘+ 112” (H,G, cO.82), which was identified as hypaphorine by physical and spectral data with those of ref. [2]. The 60% MeGH eluate (9.9 g) was separated to five frs by CC on silanized silica gel with M~H-H~~HOAc (12:7:1). Fr. 2 was chromatographed on silica gel with CHCI,-MeOH-HOAc-H,O (15:6: 1: 1) and then purified by HPLC with MeOH-0.05% aq. trifluoroacetic acid (TFA) (31: 191,affording l-4 in yields of 0.08, 0.07, 0.15 and 0.05%, respectivety. Fr. 4 was ch~mato~ph~ on silica gel with CHCl,-MeOH-HOAc-H,O (10:s: 1: 1) and then purified by HPLC with MeGH-0.05% aq. TFA (3:2) to give 5 and 6 in yields of 0.1 and 0.07%, respectively. Yktnganoside AI (1). Powder, [u]B +6” (MeGH; c 1.00). (Found: C, 56.31; H, 7.90. G+sH,6021 .2H,O requires; C, 56.24; H, 7.87.) ‘3C NMR: Table 2 Methyl ester of 1 (lb). Needles from MeOH, mp 232-234”, [aJ~3+4.J” (MeOH; c 1.00). ‘HNMR (C,D,N): 63.72, 3.74 (each 3H, each s, -CO,Me), 4.98 (lH, d, J = 7.8 Hz, anomeric H of GlcA), 5.82 (lH, d, J = 7.8 Hz, anomeric H of GlcA’), 6.24(18, d, .I= 1.6 Hz, anomeric H of Rha). (Found: C, 58.96; H, 7.81, C 50H 800 21 requires: C, 59.04, H, 7.93.) Aglycone of 1 (la = kuzusapogenoi C). LeaBets from CHCI,, mp 297-298” (lit. [SJ 295-296”), [a]g3 + 89.7” (CHCI,; c 1.02)(lit. [S] + 92.8”). ‘H and i3C NMR: Tables 2 and 3, respectively. Yunganoside B, (2). Powder, [alif” -8” (MeOH, ~0.50). (Found C, 56.98; H, 7.94. C,sH,eO,,*H,O requires: C, 57.24; H, 7.81.) ‘-‘CNMR: Table 2. Methyl ester of 2 (2b). Needles from MeGH, mp 216218”, [a];? - IO” (MeOH, c 1.00). (Found: C, 58.10; H, 8.06. CsOHsOOa, .H,O requires: C, 58.01; H, 7.98.) Aglycone of 2 (2a=soyasapogenol B): mp 261-262” (lit. [ll] 260-261”), [ali3 +92” (CHCI,; c 1.0) (lit. [ll] +W). ‘H and i3C NMR: Tables 2 and 3.

Table 3. ‘H NMR spectral data of compounds 3a-6a (400 MHz, pyridine-d,, S-values) H

3a

4a

!Ia

6a

3 11 12 16

3.61 dd (4.6, 11.4) 1.92 In@ 5.33 t (3.7) 2.85 dt (4.2, 13.9) 1.21 br d (13.9) 2.20 dd (3.9, 13.9) 2.47 dd (13.0, 13.9) 1.07 ddd (0.8, 3.9, 13.0) 3.70 dt (0.8, 3.7)

3.61 dd (4.5, 11.6) 6.54 dd (3.5, 11.0) 5.63 dd (1.3, 11.0) 1.42-1.53 m

3.42 dd (4.4, 11.6) 6.50 dd (2.9, 11.0) 5.79 dd (1.3, 11.0) 2.03 ddd (3.1, 8.1, 13.9) 1.07 br d (13.9)

3.40 dd (4.4, 11.6) 6.77 dd (2.9, 10.8)

2.57 dd (2.0, 15.0) 1.90 d (15.0) 1.66-1.72 m

2.93 dd (2.6, 15.7) 2.58 d (15.7) 3.28 dd (2.4, 15.3) 2.31 d (15.3)

1.76 ti 1.53 s 3.67 d (10.8) 4.46 d (10.8) 0.98 s 1.02 s 1.33 s 0.98 s 1.19 s 1.04 s

3.81 dd (4.0, 10.8) 1.54 s 3.70 d (10.8) 4.31 d (10.8) 0.99 s 0.83 s 1.28 s 0.98 s 1.24 s 1.04 s

1.33 s 1.14 s

2.67 dd (2.0, 14.8) 1.85 d (14.8) 1.48 m 1.53 m 4.20 dd (4.0, 10.7) 1.32 s 1.12 s

0.91 s 0.78 s 1.33 s 0.96 s 1.33 s

0.89 s 0.79 s 1.35 s 1.00 s 1.23 s

18 19 21 22 23 24 25 26 27 28 29 30

Values in parentheses am coupling constants in Hz. *Interchangeable assignments.

5.68 dd (1.3, 10.8) 1.45-1.56 m

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K. OHTANI et al.

Yunyanoside C, (3). Powder, [r]F -10” (MeOH; ~0.75). (Found: C, 56.17; H, 7.79. C,,H,,O,, .H,O requires: C, 56.24; H, 7.87.) i3CNMR. Table 2. Methyl ester of 3 (3b). Needles from MeOH, mp 224-226’, [r]:: -7.2” (MeOH; c 0.80). (Found: C, 78.33; H, 11.01. &H5,,03 requires: C, 78.55: H, 10.99.) Yunganogentn C (3a). Needles from CHCI,, mp > 300”. [a]P + 65” (CHCI,; c 0.50). FAB-MS (neg.) m/z 457 [M-H]-’ (Found: C, 57.96; H, 8.06 C 50H 800 21 H,O requires: C, 58.01; H, 7.98.) ‘H and i3C NMR: Tables 2 and 3. Yunganoslde D, (4). Powder, [ali + 11“ (MeOH, c 0.60). (Found: C, 58.65; H, 7.30. C,,H,,O,, requires: C, 58.40, H, 7.56.) 13CNMR: Table 2. Methyl ester of 4 (4b). Needles from CHCl,-MeOH. mp 212-214‘. [u];’ f7.5’ (MeOH, ~1.0). (Found: C, 57.90; H, 7.90. C,,H,,O,, .H,O requires: C, 58.12; H, 7.81.) Yunganogenrn D (4a). Needles from CHCl,-MeOH, mp 275-277”, [aJi3 + 57’ (MeOH; c 0.70). FAB-MS (neg.): m/z 455 [M-H] *. (Found: C, 79.01; H, 10.31. C 30H 48 0 3 requires: C. 78.89; H, 10.59.) UVn;:;’ (logs) nm: 242 (4.1 l), 250 (5.16) 258 (3.96). ‘H and 13C NMR: Tables 2 and 3. Yungnnostde E, (5). Powder. [@Ii3 -42” (MeOH; ~0.65) (Found: C, 54.58; H, 7.11. C,2H,,0,,. 3H,O requires: C, 54.65; H, 7.21.) i3C NMR: Table 2. Methyl ester of 5 (Sb). Needles from MeOH, mp 221-223”, [alA -45” (MeOH; c 1.02). (Found: C, 58.20; H, 7.33. C,,H,,O,, H,O requires: C, 58.18; H, 7.38.) Yunganogenin E (%I). Needles from CHCI,-MeOH, mp > 300”, [a];” -45.6” (MeOH; ~0.70). UV1.i::” (logs) nm: 241 (4.13), 250 (5.20), 256 (3.96): FAB-MS (neg.): m/z 451 [M-H]‘. (Found: C, 74.50; H, 9.02. C 30 H 44 0 5 requires: C, 74.34; H, 9.15.) ‘H and i3C NMR: Tables 2 and 3. Yunganoside F, (6). Powder, [ml;” - 30” (MeOH; ~0.50). (Found: C, 55.88; C, 7.21. C,,H,,O,, .2H,O requires: C, 55.62: H, 7.34.) i3C NMR: Table 2. Methyl ester of 6 (6b). Needles from MeOH), mp 20%211’. [tl];” - 28” (MeOH; c 1.00). (Found: C, 58.99; H. 7.62. C,,H,,O,, requires: C, 59.20; H, 7.51.) Yunganogemn F (6a). Needles from MeOH. mp > 300”. [ali -57” (MeOH; c 1.01). UV,l~$” (log&) nm: 240.5 (4.10), 254

(5.18), 257 (3.98); FAB-MS (neg.): m/-_ 453 LM - H] - i. (Found: C. 73.98: H, 9.66. C30H4605 requires: C, 74.03; H, 9.53.) ‘H and i3C NMR: Tables 2 and 3 Acknowfedgement-This study was financially supported by Grant-in-Aid for the Mombusho International Scientific Research Program (No. 03044103. 1991) from the Ministry of Education, Science and Culture.

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