P~yt~~emistry, Vol. 31, No. 4, pp. 1427-1428, 1992
0031~9422/92 95.00+0.00 Q 1992Pergamon Press plc
Printed in Great Britain.
TRITERPENOID
SAPONINS
FROM
SIJDHER K. UNIYAL Department of ~he~stry,
ROOTS
OF CLEMATIS
GRATA
and 0. P. SATI*
H.N.B. Garhwal University, Srinagar (UP.), 246 174, India (Received 25 June 1991)
Key Word Index-Clematis
grata;
Ranunculaceae; roots; triterpenoid saponins.
Abstract-Two triterpenoid saponins obtained from the roots of Clematis grnta were characterized on the basis of chemical and spectral evidence as [a-L-rhamnopyranosyl( 1-t4)+Dglucopyranosyl(l-+ 3)]-3,23/I-di-hydroxy-olean12-en-28-oic acid and [fl-D-giucopyranosyl( 1-+4)-~-D-xylopyranosyl(l+3)-a-r,-rhamnopyranosyl( 1+ 3)]-3& hydroxy-olean-I2~n-28-oic acid. INTRODUCMON In the previous paper [l] we reported the isolation and structure elucidation of some triterpenoid saponins which have been isolated from the ethanolic extract of the roots of Clematis grata Wall. In continuation of our study the saponin mixture from the ethanol extract of the roots was further fractionated. We now deal with the isolation and characterization of two new triterpenoid saponins, 1 and 2. RESCllfS AND DISCUSSION The concentrated aqueous ethanolic extract of the roots on solvent partitions and repeated column chromatography afforded two compounds, 1 and 2, which gave characteristic tests for triterpenoids. On acidic hydrolysis, 1 gave hederagenin and two monosaccharides, rhamnose and glucose, in the ratio 1: 1, while 2 afforded oleanolic acid and three monosaccharides, glucose, xylose and rhamnose in the ratio 1: 1: 1. The identity of oleanolic acid and hederagenin were confirmed by direct comparison with authentic samples (co-TLC, mmp and co-IR). The sugars were identified by paper chromatography. On alkaline hydrolysis both 1 and 2 remained unchanged. The molecular mass and sugars sequence were established by FAB mass spectrometry in the negative ion mode. The FAB mass spectrum of 1 showed peaks at m/z 779 [M-H]-, 633 [(M-H)--(146)]-, 471 [(M-H)-(146+ 162)]- corresponding to the sequential loss of a deoxy-hexosyl and hexosyl units. The permethylate of 1 prepared by the method of ref. [2], on acidic hydrolysis afforded 2,3,4-tri-0-methyl+rhamnose and 2,3,6-to-O-methyl-D-~ucose. The identities of permethylated sugars were confirmed by comparison with available authentic samples Cl, 31. The partial hydrolysis of 1 afforded la in addition to hederagenin. Interglycosidic linkages were determined by the acidic hydrolysis of the permethyl ether of la. On acidic hydrolysis, la-permethylate gave 2,3,4,6-tetra-O-methyl-D-glucase. The type of linkage at the glycosidic points and the position of linkage of sugars and aglycone were further confirmed by ‘H and 13CNMR spectra. The C-3 of ‘Author to whom correspondence
should be addressed.
1
2
R’ OH H
RZ Glc(3--cl)Rha Rha(3~1)Xy1(4+1)Glc
hederagenin appeared at 679.8 while C-4 of glucose appeared at 679.5, revealing deshielding of ca 6.1 and ca 8 ppm, respectively, for these carbon resonances in comparison to the reported values for hederganin [4] and methylpyranoside [S], as a result of glycosidation in these positions. The anomeric protons were observed at 64.34 (d, 3=7 Hz) and 5.14 (s). Thus, 1 is [tr-L-rhamnopyranosyl(l~4~~-D-glucopyranosyl(l -+ 3)]-3,23#&dihydroxy-olean-12-en-28-oic acid. The FAB mass spectrum of 2 showed peaks at m/z 895 [M-H]-, 733 [(M-H)-162]-, 601 [(M-H)(162+132)]-, and 455 [(M-H)-(162+132+146)]corresponding to the sequential loss of hexosyl, pentosyl and deoxyhexosyl units, respectively. Compound 2 was permethylated by Kuhn’s method 13-J. Completion of the reaction was checked by IR spectroscopy. Acidic hydrolysis of Zpermethylate afforded 2,3,4,6-tetra-Omethyl-D-gluco~, 2,3-di-O-methyl-~xylose and 2,4-di0-methyl-L-rhamnose. Compound 2 was partially hydrolysed to yield prosapoganins 2a and 2b. The permethyl ether of 2a on acidic hydrolysis afforded 2,3,4tri-0-methyl-D-xylose and 2,4-di-0-methyl+rhamnose, while the permethyl ether of 2b gave 2,3,4-tri-o-methylt-rhamnose. In the ’ %ZNMR spectrum of 2, the C-3 of the aglycone, C-4 of xylose and C-3 of rhamnose were observed at 583.8, 78.2 and 82.4, respectively, revealing deshielding of ca 5.8, cx 7.0 and ca 10.0 ppm for these carbon resonances in comparison to the reported values, as a result of glycosidation at these points. The anomeric
1427
1428
Short Reports
signals of sugars were observed at 64.32 (d, .J = 7.0 HZ), 4.48(d, J =7.0 Hz) and 5.08(s). Thus, 2 is [B-D-glucopyranosyl( l-+4)-p-D-xylopyranosyl( l-3)-cl-t-rhamnopyranosyl( l-+ 3)]-3fl-hydroxy-olean-l2-ett-28-oic acid.
Mps: uncorr; FAB-MS: negative ion mode. NMR spectra were recorded at 300 MHz in DMSO with TMS at int. standard; CC: silica gel (Merck, @J-120 mesh); TLC: Kieselgel 6OG (Merck). The spots on TLC were visualized by spraying with 10% H,SO,; PC: Whatman No. 1 paper using a decending mode and aniline hydrogen phthalate as developer. Extraction and fractionation of the extract. Roots of Clematis grata (2 kg) collected from Roomdhar (Tehri Garhwal), UP. and identified by Ethnomedictal Plant Identification Lab., H.N.B. Garhwal University, Srinagar, were extracted with 90% aq. ethanol (4 x 1 1).The ethanolic extract was co& to dryness and partitioned between n-BuOH and Hz0 (1: 1, 1 I). The n-BuOH layer was coned under red. pres. and dissolved in 20 ml MeOH and fractionated by CC over silica gel using CHCl,-MeOH. Repeated CC afforded 1 (4~m~ and 2 (600 mg). Compound 1. Flakes from MeOH, mp 218-220”; IR v:11:cm-‘: 3400, 1690; ‘HNMR: 60.71, 0.87, 0.92, 1.02, 1.16, 1.23 (each Me, 6 x tert. Me), 4.34 (1 H, d, 5=7.0 Hz, H-l of Glc), 5.14 (1 H. s, H-l of Rba); 13CNMR: C-l to C-30 of aglycone: 6383,275, 79.8,42.6.47.4, 17.8,32.9,39.2,47.4,37.4,23.6,121.7, 144.0. 41.7, 27.5, 23.2,46.2. 41.3,46.7, 30.4, 32.9, 36.3, 68.7, 13.0, 15.7, 17.1. 25.7. 180.2, 32.9, 23.0; carbons of sugar moiety: glucosyl: 104.6 (C-i), 74.9 (C-2), 79.8 (C-3), 79.3 (C-4) 79.8 (C-5), 64.0 (C-6); rhamnosyl: 102.4 (C-l), 71.3 (C-2), 72.2 (C-3), 74.9 (C-4) 68.8 (C-S), 17.8(C-S); FAB-MS (negative ion) m/z: 779,633, 471, 423, 297, 248. Acidic hydrolysis of compound 1. Compound I (20 mg) was refluxed with 2 M HCl-MeOH (1: 1, 10 ml) at loo” for 2 hr to afford the aglycone, identified as hederagenin, needles (MeOH), mp 310-312”, identity confirmed by co-TLC, mmp and co-IR with an authentic sample. The neutralized (AgsCOs) and coned hydrolysate showed the presence of glucose and rhamnose on PC (RI values 0.18 and 0.37, respectively, solvent rtBuOH-HOAc-HtO, 4: 1: 5). Alkalzne hydrolyses ofcompound1. Compound 1 (10 mg) was refluxed at loo” for 2 hr with KOH in MeOH-H,O (1: 1) and recovered unchanged. Partial hydrolysis of compound 1. Compound 1 (SOmg) was heated with 1 M HCl-n-BuOH (1: 1, 15ml) at 70” for 1 hr. The n-BuOH layer was washed with 5% NaHCO, and then with Hz0 and coned in uacuo to afford a residue which was purified by CC to give hederagenin (10 mg) and partial product la
t-rhamnose and 53,6-tri-O-methyl-D-glucose (Ra 0.84 and 1.01, respectively, PC, solvent, n-BuOH-EtOH-H,O, 4: 1: 5),whereas la permethylate hydrolysate gave 2,3,4,6-tetra-O-methyl-oglucose (Re 1.0). Compound 2. Crystallized from MeOH, mp 228230”: FABMS (negative ion) m/z: 895,733,601,455,439,394,325,297,208; ‘HNMR: 60.70, 0.78, 0.84, 0.87, 0.92, 1.12, 1.22 (each Me, 7 x tert. Me), 4.32 (1 H, d, J ~7.0 HZ, H-i of Glc), 4.48 (1 H, d, 3=7.0 Hz, H-1 of Xyl), 5.08 (1 H, s, H-l of Rha), 5.22 (1 H, brs, H-12); i3C NMR, C-l to C-30 of aglycone: 639.4,26.3,83.8,39.1, S&8,19.2, 34.2,40.2,48.8,37.6,24.2,122.1,144.8,42.6,28.6,24.1, 46.4,41.8,46.4, 31.6, 34.8, 33.1, 287, 16.9, 15.8, 17.6,27.2, 178.8, 33.5, 24.2; sugar moiety: glucosyl: 104.3, (C-l), 75.6 (C-2), 78.4 (C-3), 71.4 (C-4), 78.1 (C-5), 61.9 (C-6); xylosyl: 106.3 (C-l), 74.8 (C-2), 75.9 (C-3), 78.2 (C-4), 65.3 (C-S), rhamnosyk 101.2 (C-l), 71.6 (C-2), 82.4 (C-3), 72.2 (C-4), 70.2 (C-5), 19.1 (C-6). Acidic hydrolysis of compound 2. A solution of 2 (20 mg) in 2 M Hal-MeOH (1: 1, 10ml) was heated under retlux for 2 hr and worked-up in the same way as 1 to give the aglycone (6 mg), glucose, xylose and rbamnose. The aglycone, prisms, mp 306-308”, was identified as oleanolic acid by direct comparison with as authentic sample co-TLC, mmp and co-IR. Alkaline ~ydrofysjs ~fcompound 2. Com~und 2 (10 mg) was hydrolysed with KOH in MeOH-H,O (1: I) as above and recovered unchanged. Partial hydrolysis of compound 2. Compound 2 (100 mg) was heated with 1 M HCl-n-BuOH (1: 1, 30 ml) at 70” for 2 hr and worked-up in the same way as 1. The product was chromatographed on silica gel (50 g) with a gradient of tHC&-MeGH (MeOH, O-10%) to give oleanolic acid (20mg), and partial products 2a (30 mg) and 2b (30 mg). Permethylation of campounds 2, Zp and 2b. Compounds 2 (20 mg), 2a (1.5mg) and 2b (15 mg) were separately permethylated with Me1 (6 ml), Ag,O (100 mg) in DMF (2 ml) and worked-up in the same way as 1, The product was chromatographed on silica gel (10 g) with CsH,-MeZCO (17:3) to give permethylates of 2 (15 mg) 2a (10 mg) and 2b (10mg). Acidic hydrolysis of permethylates of compounds 2, 2a and 2b. Permethylates of 2, Za and 2b (8 mg each) were separately
refluxed with 2 M HCI-MeOH (1: 1, 5 ml) for 2 hr. The neutralized and coned hydrolysate of the ~~ethylate of 2 gave 2,3,4,6-tetra-O-methyl-~-gluc~, 2,3~i-O-methyl-D-xylo~ and 2,4-di-0,methyl-r-rhamnose, permethylate of 2s gave 2,3,4-t& O-methyl-o-xylose and 2,4-di-0-methyl-L-rhamnose, whereas 2Bpermethylate afforded 2,3,4-tri-O-methyl-L-rhamnose. Acknowledgement-We are grateful to Prof. T. Kawasaki (Setsunan, Japan) for FAB-MS; RSIC, CDRI Lucknow for NMR spectra and CSIR. New Delhi for financial assistance.
GO md.
Fer~er~y~u~~onufcomp~unds 1 and la. Compounds I(15 mg) and la (10 mg) were separately ~rmethylat~ with Me1 (5 ml), Ag,O (1OOmg) in DMF (I ml), at room temp. on stirring for 24 hr. Reaction mixtures were filtered, filtrates diluted with HzO, extracted wtth CHCl3 and washed with water. Complete methylation was checked by IR. The syrups obtained were purified by CC to yield the permethylate of 1 (10 mg) and of la (6 mg). Acidic hydrolysis of the permethylates of 1 and la. Permethylates of 1 and la (5 mg each) were separately refluxed with 2 M HCI-MeOH (1: 1,3 ml) for 2 hr. The neutralized and coned hydrolysate from 1 permethylate contained 2,3,4-t&O-methyl-
1. Sati, 0. P., Uniyal, S. K, Bahuguna, S. and Kikucbi, T. (19aO) Phytochemistry 29, 3676. 2. Kuhn, R., Egge, H., Brossmer, R., Gauche, A., Prischmann,
H., Klesse, P., Lechinger, W., Robm, E. and Tschampal, D. (1960) Angew Chem. 72,805. 3. Sati, 0. P., Bahuguna, S., Uniyal, S. and Bhakuni, D. S. (1989) Phytochemistry 28, 575. 4. Tori, K., Seo, S., Shimaoka, A. and Tomita, Y. (1974) Tetrahedron Letters 4227. 5. Seo, S., Tom&a, Y., Tori, K. and Yoshimura, Y. (1978) .i. Am. Chem. Sac. 100, 1331.
0031~9422/92 95.00+0.00 Q 1992Pergamon Press plc
Printed in Great Britain.
TRITERPENOID
SAPONINS
FROM
SIJDHER K. UNIYAL Department of ~he~stry,
ROOTS
OF CLEMATIS
GRATA
and 0. P. SATI*
H.N.B. Garhwal University, Srinagar (UP.), 246 174, India (Received 25 June 1991)
Key Word Index-Clematis
grata;
Ranunculaceae; roots; triterpenoid saponins.
Abstract-Two triterpenoid saponins obtained from the roots of Clematis grnta were characterized on the basis of chemical and spectral evidence as [a-L-rhamnopyranosyl( 1-t4)+Dglucopyranosyl(l-+ 3)]-3,23/I-di-hydroxy-olean12-en-28-oic acid and [fl-D-giucopyranosyl( 1-+4)-~-D-xylopyranosyl(l+3)-a-r,-rhamnopyranosyl( 1+ 3)]-3& hydroxy-olean-I2~n-28-oic acid. INTRODUCMON In the previous paper [l] we reported the isolation and structure elucidation of some triterpenoid saponins which have been isolated from the ethanolic extract of the roots of Clematis grata Wall. In continuation of our study the saponin mixture from the ethanol extract of the roots was further fractionated. We now deal with the isolation and characterization of two new triterpenoid saponins, 1 and 2. RESCllfS AND DISCUSSION The concentrated aqueous ethanolic extract of the roots on solvent partitions and repeated column chromatography afforded two compounds, 1 and 2, which gave characteristic tests for triterpenoids. On acidic hydrolysis, 1 gave hederagenin and two monosaccharides, rhamnose and glucose, in the ratio 1: 1, while 2 afforded oleanolic acid and three monosaccharides, glucose, xylose and rhamnose in the ratio 1: 1: 1. The identity of oleanolic acid and hederagenin were confirmed by direct comparison with authentic samples (co-TLC, mmp and co-IR). The sugars were identified by paper chromatography. On alkaline hydrolysis both 1 and 2 remained unchanged. The molecular mass and sugars sequence were established by FAB mass spectrometry in the negative ion mode. The FAB mass spectrum of 1 showed peaks at m/z 779 [M-H]-, 633 [(M-H)--(146)]-, 471 [(M-H)-(146+ 162)]- corresponding to the sequential loss of a deoxy-hexosyl and hexosyl units. The permethylate of 1 prepared by the method of ref. [2], on acidic hydrolysis afforded 2,3,4-tri-0-methyl+rhamnose and 2,3,6-to-O-methyl-D-~ucose. The identities of permethylated sugars were confirmed by comparison with available authentic samples Cl, 31. The partial hydrolysis of 1 afforded la in addition to hederagenin. Interglycosidic linkages were determined by the acidic hydrolysis of the permethyl ether of la. On acidic hydrolysis, la-permethylate gave 2,3,4,6-tetra-O-methyl-D-glucase. The type of linkage at the glycosidic points and the position of linkage of sugars and aglycone were further confirmed by ‘H and 13CNMR spectra. The C-3 of ‘Author to whom correspondence
should be addressed.
1
2
R’ OH H
RZ Glc(3--cl)Rha Rha(3~1)Xy1(4+1)Glc
hederagenin appeared at 679.8 while C-4 of glucose appeared at 679.5, revealing deshielding of ca 6.1 and ca 8 ppm, respectively, for these carbon resonances in comparison to the reported values for hederganin [4] and methylpyranoside [S], as a result of glycosidation in these positions. The anomeric protons were observed at 64.34 (d, 3=7 Hz) and 5.14 (s). Thus, 1 is [tr-L-rhamnopyranosyl(l~4~~-D-glucopyranosyl(l -+ 3)]-3,23#&dihydroxy-olean-12-en-28-oic acid. The FAB mass spectrum of 2 showed peaks at m/z 895 [M-H]-, 733 [(M-H)-162]-, 601 [(M-H)(162+132)]-, and 455 [(M-H)-(162+132+146)]corresponding to the sequential loss of hexosyl, pentosyl and deoxyhexosyl units, respectively. Compound 2 was permethylated by Kuhn’s method 13-J. Completion of the reaction was checked by IR spectroscopy. Acidic hydrolysis of Zpermethylate afforded 2,3,4,6-tetra-Omethyl-D-gluco~, 2,3-di-O-methyl-~xylose and 2,4-di0-methyl-L-rhamnose. Compound 2 was partially hydrolysed to yield prosapoganins 2a and 2b. The permethyl ether of 2a on acidic hydrolysis afforded 2,3,4tri-0-methyl-D-xylose and 2,4-di-0-methyl+rhamnose, while the permethyl ether of 2b gave 2,3,4-tri-o-methylt-rhamnose. In the ’ %ZNMR spectrum of 2, the C-3 of the aglycone, C-4 of xylose and C-3 of rhamnose were observed at 583.8, 78.2 and 82.4, respectively, revealing deshielding of ca 5.8, cx 7.0 and ca 10.0 ppm for these carbon resonances in comparison to the reported values, as a result of glycosidation at these points. The anomeric
1427
1428
Short Reports
signals of sugars were observed at 64.32 (d, .J = 7.0 HZ), 4.48(d, J =7.0 Hz) and 5.08(s). Thus, 2 is [B-D-glucopyranosyl( l-+4)-p-D-xylopyranosyl( l-3)-cl-t-rhamnopyranosyl( l-+ 3)]-3fl-hydroxy-olean-l2-ett-28-oic acid.
Mps: uncorr; FAB-MS: negative ion mode. NMR spectra were recorded at 300 MHz in DMSO with TMS at int. standard; CC: silica gel (Merck, @J-120 mesh); TLC: Kieselgel 6OG (Merck). The spots on TLC were visualized by spraying with 10% H,SO,; PC: Whatman No. 1 paper using a decending mode and aniline hydrogen phthalate as developer. Extraction and fractionation of the extract. Roots of Clematis grata (2 kg) collected from Roomdhar (Tehri Garhwal), UP. and identified by Ethnomedictal Plant Identification Lab., H.N.B. Garhwal University, Srinagar, were extracted with 90% aq. ethanol (4 x 1 1).The ethanolic extract was co& to dryness and partitioned between n-BuOH and Hz0 (1: 1, 1 I). The n-BuOH layer was coned under red. pres. and dissolved in 20 ml MeOH and fractionated by CC over silica gel using CHCl,-MeOH. Repeated CC afforded 1 (4~m~ and 2 (600 mg). Compound 1. Flakes from MeOH, mp 218-220”; IR v:11:cm-‘: 3400, 1690; ‘HNMR: 60.71, 0.87, 0.92, 1.02, 1.16, 1.23 (each Me, 6 x tert. Me), 4.34 (1 H, d, 5=7.0 Hz, H-l of Glc), 5.14 (1 H. s, H-l of Rba); 13CNMR: C-l to C-30 of aglycone: 6383,275, 79.8,42.6.47.4, 17.8,32.9,39.2,47.4,37.4,23.6,121.7, 144.0. 41.7, 27.5, 23.2,46.2. 41.3,46.7, 30.4, 32.9, 36.3, 68.7, 13.0, 15.7, 17.1. 25.7. 180.2, 32.9, 23.0; carbons of sugar moiety: glucosyl: 104.6 (C-i), 74.9 (C-2), 79.8 (C-3), 79.3 (C-4) 79.8 (C-5), 64.0 (C-6); rhamnosyl: 102.4 (C-l), 71.3 (C-2), 72.2 (C-3), 74.9 (C-4) 68.8 (C-S), 17.8(C-S); FAB-MS (negative ion) m/z: 779,633, 471, 423, 297, 248. Acidic hydrolysis of compound 1. Compound I (20 mg) was refluxed with 2 M HCl-MeOH (1: 1, 10 ml) at loo” for 2 hr to afford the aglycone, identified as hederagenin, needles (MeOH), mp 310-312”, identity confirmed by co-TLC, mmp and co-IR with an authentic sample. The neutralized (AgsCOs) and coned hydrolysate showed the presence of glucose and rhamnose on PC (RI values 0.18 and 0.37, respectively, solvent rtBuOH-HOAc-HtO, 4: 1: 5). Alkalzne hydrolyses ofcompound1. Compound 1 (10 mg) was refluxed at loo” for 2 hr with KOH in MeOH-H,O (1: 1) and recovered unchanged. Partial hydrolysis of compound 1. Compound 1 (SOmg) was heated with 1 M HCl-n-BuOH (1: 1, 15ml) at 70” for 1 hr. The n-BuOH layer was washed with 5% NaHCO, and then with Hz0 and coned in uacuo to afford a residue which was purified by CC to give hederagenin (10 mg) and partial product la
t-rhamnose and 53,6-tri-O-methyl-D-glucose (Ra 0.84 and 1.01, respectively, PC, solvent, n-BuOH-EtOH-H,O, 4: 1: 5),whereas la permethylate hydrolysate gave 2,3,4,6-tetra-O-methyl-oglucose (Re 1.0). Compound 2. Crystallized from MeOH, mp 228230”: FABMS (negative ion) m/z: 895,733,601,455,439,394,325,297,208; ‘HNMR: 60.70, 0.78, 0.84, 0.87, 0.92, 1.12, 1.22 (each Me, 7 x tert. Me), 4.32 (1 H, d, J ~7.0 HZ, H-i of Glc), 4.48 (1 H, d, 3=7.0 Hz, H-1 of Xyl), 5.08 (1 H, s, H-l of Rha), 5.22 (1 H, brs, H-12); i3C NMR, C-l to C-30 of aglycone: 639.4,26.3,83.8,39.1, S&8,19.2, 34.2,40.2,48.8,37.6,24.2,122.1,144.8,42.6,28.6,24.1, 46.4,41.8,46.4, 31.6, 34.8, 33.1, 287, 16.9, 15.8, 17.6,27.2, 178.8, 33.5, 24.2; sugar moiety: glucosyl: 104.3, (C-l), 75.6 (C-2), 78.4 (C-3), 71.4 (C-4), 78.1 (C-5), 61.9 (C-6); xylosyl: 106.3 (C-l), 74.8 (C-2), 75.9 (C-3), 78.2 (C-4), 65.3 (C-S), rhamnosyk 101.2 (C-l), 71.6 (C-2), 82.4 (C-3), 72.2 (C-4), 70.2 (C-5), 19.1 (C-6). Acidic hydrolysis of compound 2. A solution of 2 (20 mg) in 2 M Hal-MeOH (1: 1, 10ml) was heated under retlux for 2 hr and worked-up in the same way as 1 to give the aglycone (6 mg), glucose, xylose and rbamnose. The aglycone, prisms, mp 306-308”, was identified as oleanolic acid by direct comparison with as authentic sample co-TLC, mmp and co-IR. Alkaline ~ydrofysjs ~fcompound 2. Com~und 2 (10 mg) was hydrolysed with KOH in MeOH-H,O (1: I) as above and recovered unchanged. Partial hydrolysis of compound 2. Compound 2 (100 mg) was heated with 1 M HCl-n-BuOH (1: 1, 30 ml) at 70” for 2 hr and worked-up in the same way as 1. The product was chromatographed on silica gel (50 g) with a gradient of tHC&-MeGH (MeOH, O-10%) to give oleanolic acid (20mg), and partial products 2a (30 mg) and 2b (30 mg). Permethylation of campounds 2, Zp and 2b. Compounds 2 (20 mg), 2a (1.5mg) and 2b (15 mg) were separately permethylated with Me1 (6 ml), Ag,O (100 mg) in DMF (2 ml) and worked-up in the same way as 1, The product was chromatographed on silica gel (10 g) with CsH,-MeZCO (17:3) to give permethylates of 2 (15 mg) 2a (10 mg) and 2b (10mg). Acidic hydrolysis of permethylates of compounds 2, 2a and 2b. Permethylates of 2, Za and 2b (8 mg each) were separately
refluxed with 2 M HCI-MeOH (1: 1, 5 ml) for 2 hr. The neutralized and coned hydrolysate of the ~~ethylate of 2 gave 2,3,4,6-tetra-O-methyl-~-gluc~, 2,3~i-O-methyl-D-xylo~ and 2,4-di-0,methyl-r-rhamnose, permethylate of 2s gave 2,3,4-t& O-methyl-o-xylose and 2,4-di-0-methyl-L-rhamnose, whereas 2Bpermethylate afforded 2,3,4-tri-O-methyl-L-rhamnose. Acknowledgement-We are grateful to Prof. T. Kawasaki (Setsunan, Japan) for FAB-MS; RSIC, CDRI Lucknow for NMR spectra and CSIR. New Delhi for financial assistance.
GO md.
Fer~er~y~u~~onufcomp~unds 1 and la. Compounds I(15 mg) and la (10 mg) were separately ~rmethylat~ with Me1 (5 ml), Ag,O (1OOmg) in DMF (I ml), at room temp. on stirring for 24 hr. Reaction mixtures were filtered, filtrates diluted with HzO, extracted wtth CHCl3 and washed with water. Complete methylation was checked by IR. The syrups obtained were purified by CC to yield the permethylate of 1 (10 mg) and of la (6 mg). Acidic hydrolysis of the permethylates of 1 and la. Permethylates of 1 and la (5 mg each) were separately refluxed with 2 M HCI-MeOH (1: 1,3 ml) for 2 hr. The neutralized and coned hydrolysate from 1 permethylate contained 2,3,4-t&O-methyl-
1. Sati, 0. P., Uniyal, S. K, Bahuguna, S. and Kikucbi, T. (19aO) Phytochemistry 29, 3676. 2. Kuhn, R., Egge, H., Brossmer, R., Gauche, A., Prischmann,
H., Klesse, P., Lechinger, W., Robm, E. and Tschampal, D. (1960) Angew Chem. 72,805. 3. Sati, 0. P., Bahuguna, S., Uniyal, S. and Bhakuni, D. S. (1989) Phytochemistry 28, 575. 4. Tori, K., Seo, S., Shimaoka, A. and Tomita, Y. (1974) Tetrahedron Letters 4227. 5. Seo, S., Tom&a, Y., Tori, K. and Yoshimura, Y. (1978) .i. Am. Chem. Sac. 100, 1331.
