Agaveside C, a steroidal glycoside from Agave cantala

1336

Short Reports

nor-3/I-methoxyserrat-l4-en-2l-onc Council (Republic

and lo the National

Science

of Chma) for financial support.

5. Tsuda, Y.. Sano, T.. Kawaguchi. Tetrahdron I~tters 1279. 6. Inubusht, Lctrtw

Li, H. L. (cd.) (1975) Flora (f

Vol.

Tetrahedron

9. Y. S. (1988)

Chem. Sot. (Tuipei)

M&n,

W..

Woodward,

and Djerassi. C.

Phyto-

IO. Harada.

N.

Spectroscopy-

B. A. and Rowe, J. W. (1984)

40. 42 17.

Chap.

E. H. T.

Teerrahedron

and Fang, J. M. (1975) J. Chin.

Il.

f.etfrrs

Am. Chem.

and Nakanishi. Exciron

SW.

K. (1983)

Coupling

W., Klyne, W. at,

4013. Dichroic

Circular

rn Oryonic Sfereochrmisfr,v.

lOand 11. University Science Books. Mill Valley, CA.

Conner. A. J. Drg.

22, 341,

R. B.. Moscowitz.

(1961) J.

H., Haromy,

Chem.

T. P. and Sundar~iln~am.

M. (lY81)

46. 2987.

P~~?o~~em~scr~. Vol. 30, No. 4. pp. 1336 1339, 199 1 Printed in Great Btltain

AGAVESIDE

T~frahedrnn

2745.

1. pp. 518-525.

Epoch, Taiwan.

4. Cheng, Y. S.. Chen.

Y. (1964)

x. Rowe, J. W. and Bower. C. 1.. (1965)

T&van

2. Fang, J. M., Su, W. C. and Cheng, chemistry 27, 1395. 3. Conner. A. H., Nagasampagi,

Y., Sano. 1. and Tsuda.

1303.

7. Kutncy, J. P. and Elgendorf, G. (1969) Tetrahedron 25.37.53.

REFERENCES 1.

K. and Inubushi. Y (1964)

01131 9422.91 $3.~+B.~ 922 cm- ‘) revealed 25R stereochemistry which was consistent with the j3C shielding data. The 500 MHz *H NMR exhibited two singlets at SO.64 and 0.76 and four doublets (J = 6.0 Hz) at 60.75, 1.03,1.06 and 1.07 corresponding to two tertiary methyl groups at C-18 and C-19, and two secondary methyls at C-21, C-27 and C-6 of the rhamnopyranosyl units. The anomeric proton signals were observed at 64.78, 4.80, 5.05, 5.08, 5.17 and 5.44 and were correlated with ‘% resonances at 6 102.91. 104.67, 104.81, 105.45, 104.81 and 104.97 respectively in a one bond CH correlation experiment, thus

Short Reports

1337

1 R=Olc 3 R=H

confirming the hexaglycosidic nature of compound 1. Acid hydrolysis of 1 afforded an aglycone (2), [M] T m/r 432, C2,H4.,04, identified as 22,3/Sdihydroxy-25R-5aspirostane (gitogenin) from its mass spectral data and by ‘H and ‘%NMR spectroscopy [2, 43. The sugars obtained from the saponin hydrolysates were glucose, rhamnose and xylose. The relative ratio of monosaccharides was determined (HPLC) as 3 : 2: 1, thus, 1 is considered as gitogenin hexaglycoside. Positive ionization fast atom bombardment mass spectrometry [S] of compound 1, in agreement with its molecular formula C,,H,,,O,,, gave rise to a peak at m/z 1365 corresponding to the quasi-molecular ion [M +Na]‘. The presence of other ions corresponding to various cleavages of the molecular ion allowed the gross saccharide composition to be deduced. Fragments of approximately equal abundance were observed at m/z 1233 [(M+Na)-132]‘, 1219 [(M +Na)146]- and 1203 [(M + Na)- 162]+ inferring the presence of three terminal sugar moieties. These correspond to the loss of a pentosyl (xylosyl, 6-deoxyhexosyl (rhamnosyl) and hexosyl (glucosyl) moiety respectively. This was complemented by the presence of an ion at m/z 925 [(M + Na) -440]+ due to the loss of all of the three terminal sugar residues. A fragment ion at m/z 1087 [(M + Na) - 2781 L formed by loss of a disaccharide moiety consisting of one xylosyl and one rhamnosyl units, and a signal at m/z 895 [(M + Na)-4701’ corresponding to the loss of a trisaccharide moiety consisting of two glucosyl and one rhamnosy1 units confirmed the substitution of these saccharide moieties to the internal glucosyl unit. The other fragment ions at m/z 763 [(M + Na)- 6021’ and 617 [(M +Na) -748]+ were attributed to the loss of one xylosyl + two glucosyl+ one or two rhamnosyl moieties respectively. The interglycosidic linkages as well as the position of attachment of the sugar chain to the aglycone were The assignment established by ‘%Z NMR spectroscopy. of the carbon resonances due to the sugar moieties of 1 was carried out by a comparison with those reported for the saccharide chain of related glycosides [2,6,7] and the chemical shifts of methyl-0-glycosides [S] as well as by considering the glycosidation effects [2, 63. The appearance of anomeric carbon resonances at 6 105.45, 104.97, 104.81 x 2, 104.67 and 102.91 clearly confirmed its hexa-

glycosidic nature and demonstrated that all the monosaccharide units are substituted on C-l. Thus, the carbon chemical shifts of the terminal xylose, rhamnose and glucose could be attributed. The C-2 and C-3 of both the inner glucose were observed at ca 681 and 86 indicating their involvement in glycosidic linkage formation [l, 93. One of the glucose moieties is substituted to rhamnose at C-4 [lo], as the C-4 resonance was observed at 680.42. The rhamnose moieties are substituted at C-2 of the glucose moieties as C-2 resonated at ca 681. The inter glycosidic linkage between the inner glucose is (l-3) from the close resemblance to chemical shifts reported for other glycosides of A. cantala [ 1, 1 I]. The proposed interglycosidic linkages were consistent with the finding that methylation of 1 with NaH-Me1 in hexamethyl phosphoric triamide (HM PA) followed by acid hydrolysis liberated 4,6-di-O-methyl-D-glucose (2 mol), 2,3,4,6-tetra-O-methyl-D-glucose (1 mol), 2,3-di-Omethyl-L-rhamnose (1 mol), 2,3,4-tri-O-methyl-t.-rham nose (1 mol) and 2,3,4-tri-O-methyl-D-xylose (1 mol). The presence of xylose, rhamnose and glucose as the terminal sugar moieties was confirmed by the detection of these monosaccharides, on partial hydrolysis of compound 1 on TLC in a HCl atmosphere [12]. Enzymatic hydrolysis of 1 with /I-glucosidase afforded a prosapogenin (3) which had five anomeric resonances at 6 102.95, 103.98, 104.90, 104.75 and 105.02 in its r3C NMR spectrum, thus indicating it to be a pentaglycoside. The absence of a resonance at ca 688 together with the appearance of a resonance at 681.82 supported a (1+3) interglucosidic linkage, as well as rhamnosyl substitution at C-2 of the second glucosyl moiety. The + ve FAB mass spectrum showed a quasi molecular ion, [M +Na]‘, at m/z 1203 followed by fragments at m/z 1071 and 1057 due to the loss of terminal xylosyl and rhamnosy1 unit respectively. The genesis of fragments at m:z 925 and 895 corresponding to the loss of disaccharide moieties consisting of xylose and rhamnose, and rhamnose and glucose respectively were consistent with the attachment of xylorhamnosyl to the inner glucose. Other fragments in the FAB mass spectrum as well as its “C NMR shielding data were in full agreement with the proposed structure. The hexaglycoside moiety in 1 is linked at C-3 of the

133X

Short Reports

aglycone as the signals of 1 and 2 were superimposable except for those of C-2, C-3 and C-4 which were somewhat different due to glycosylation shifts. The C-3 signal was found at ii8.40 lower field, while the C-2 and C-4 signals were observed at 6 1.08 and 1.02 upfield positions in the case of I relative to 2, demonstrating the monodesmosidic nature of 1 and C-3 as the site of sugar linkage [2, 73. Consequently, the structure of agaveside C was assigned as 3/Y-0-j z-L-rhamnopyranosyl-( 1+2)-/&Dglucopyrdnosyl-( I +3)-fi-D-glucopyranosyl-[fi-D-xylopyranosyl-( l-4)-r-L-rhamnopyranosyl-( I-2)]-B-D-glucopyranosyl}2r-hydroxy-25K-Sz-spirostane (1).

EXPERIMENTAL Mp: uncorr. ‘H and 13CNMR: 500 and 125 MHz respect~vcly. pyridine-d, or CDCI, with TMS as mt. standard under the conditions reported previously [13J; IR: KBr; Positive ionization FABMS (Finnigan MAT 90 high resolution spectrometer): glycerol and a Noba-Na matrix. The sample in MeOH (1 ~1) was mlxcd with 1 PI of matrix on the FAB target. Accelerating boltage 5 kV. IWO resolution. using Xe with 8 kV energy, Scans wcrc from 100 10 1500 amu at 10 scans dec’. TLC: silica gel G. The spots were visualized by spraying with 10% alcoholic H,SO, followed by heating. l:‘xtracrion and isolarion ofagcrueside C (1). The fruits (2 kg) of wild A. cantala were collected from Srinagar, Garhwal, U.P. and specimens identdied by the Forest Research Institute, Dehradun, IJ.P where a voucher specimen has been deposited. The fruits were powdered and extracted with MeOH. A part of the crude McOH extract (3 g) was subjected to VLC using CHCI,-MeOH (19: 1, 9: I. 17:3, X.2. 3: 1. 7:3, 3:2, l:l, 1:2), the eluates were collecctcd m lOOmI portions. Frs 12-13 obtained from elution with CHCl,-MeOH (3:2- 1: 1)showed two spots. Further purification of this residue (180mg) on silica gel PLC using CHCl,-McOH Hz0 (15:8: 2, organic layer) as eluent, afforded 0.06 g agaveside C (1). Aguwsidr C (1). C,,H,,,O,,. powder, mp 256260”, [z]i’ -39.4’ (MeOH: c 2.6). IR v:Zcrn-‘: 3397 (br OH), 2927, 1377, 1243, 1157, 1074 (br, C-O-C), 922,899 and 866 (899 > 920). 25R splroketal): ‘H NMR:d;0.64(s, Me),0.76(.$, Me),0.75(d, Me), 1.03 (d. Me). 1.06 (d. Me). 1.07 (d, Me), 4.78, 4.80, 5.05, 5.08, 5.17 and 5.44 (anomeric-H); “C NMR: 645.11 (C-l), 72.00 (C-2), 84.85 (C3). 34.98 (C-4), 45.1 1 (C-5). 29.72 (C-6). 32.27 (C-7). 35.06 (C-8). 55 49 (C-91. 35.89 (C-IO), 21.74 (C-l I), 40.19 (C-12). 40.61 (C-13). 56.89(C-14). 32.25 (C-15). 80.42 (C-16), 62.77 (C-17). 15.49 (C-18). 12.89 (C-19). 42.77 (C-20). 14.02 (C-21). 109.78 (C-22). 31.05 (C23). 29.32 (C-24). 29.72 (C-25). 67.34 (C-26). 17.07 (C-27). 102.91 (C-l’), 81.69 (C-2’), 86.22 (C-3’). 69.46 (C-4’), 78.93 (C-5’). 6l.l4(C6’). 104.67 (C- I”), 80.42 (C-2”), 88.72 (C-3”). 69.46 (C-4”). 78.93 (C5”), 61.14 (C-6”), 104.81 (C-l”‘), 71.13 (C-2”‘), 73.04 (C-3”‘). 73.04 (C-4”‘). 67.34 (C-5”‘). 17.78 (C-6”‘). 104.67 (C-l”“). 72.47 (C-2”“). 77.92 (C-3”“), 72 00 (C-4”“), 75.82 (C-5”“). 63.43 (C-6”“), 104.97 (C-I ““). 72.00 (C-2”“) 69.46 (C-3” “). 80.42 (C-4” “), 67.34 (C-5”“‘), 15.49 (C-6”“‘). 105.45 (C- I”‘“‘), 73.38 (C-2”““). 75.82 (C-3”““). 71.13 (C-4”““), 67.34 (C-5”““); FARMS: 1365 LM + Na] +, 1342 [M] +, 1233[M+Na)-XylJ+, 1219[(M+Na)-Rha],l203[(M+Na) -Glc] +, 1087 [(M +Na)-Rha-Xyl]‘, 1057 [(M +Na)-Rha - Glc)] ’ , 925 [(M +Na)-Xyl-Rha-Glc]-, 895 [(M+Na) -Xyl-ZxGlc-Rha]‘, 617 [(M+Na)-Xyl-2xGlc-2 x Rha]‘. 455 [(M+Na)-Xyl-3xGlc-2xRha]’ 431, 415, 399, 139, 115. Actd hydrolysis of compound 1. Compound 1 (15 mg) in HCI --dioxane (1: 1) was refluxcd for 5 hr. The aglycone was extracted with CHCI, (4mg). needles, mp 268-270” (dec.), IR

990, 960, 930, 905, 870 (Intensity 905. 930, (25R)vi::cm-‘: spiroketal): MS m/z 432 [M] *, 417, 318, 289, 139 [C,H,,O]+; “CNMR (C-l-C-27): 45.08, 73.08, 76.36, 36.00, 44.85, 27.78, 32.06, 34.48. 54.32. 37.55, 21.18, 40.12, 40.69. 56.19. 31.72. 80.70, 62.21, 16.52, 12.48.41.71. 14.47, 109.32, 31.42.2X.X1, 30.30.66.76, 17.05. Identified as gitogenin by direct comparison with an authentic sample. The aq. portion revealed the presence of glucose, rhamnose and xylose on PC (BuOH-HOAc-H,O, 4: 1: 5). The molar ralio(3:2: 1) was determined by HPLC (30cm x 3.9mm l.d., carbohydrate analysis column. MeCN--H,O 73: 27 as solvent). Permethylution oj’compound 1 and hydrolysis o/‘rhe producr. A soln of compound I (20mg) In HMPA (5ml) was treated with NaH (300mg) and Mel (5 ml) at room temp. for 3 hr. The reaction was worked-up and the residue purified by prep. TLC [petrol (bp 60.80‘)-EtOAc, 1: I]. Hydrolysis of the permethylate of 1 was perlormed by refluxing with 1 M HCI - MeOH (I : 1.5 ml). PC (n-BuOH-EtOH-H,O, 5: I :4) of the neutralized and concentrated hydrolysate showed the presence of 2,3,4.6-lelraO-methyl-D-glucose, 2,3,4-tri-O-methyl-L.-rhamnose, 2.3.4-trl-0methyl-D-xylose, 2.3-di-O-methyl-L-rhamnose and 4.6-di-Omethyl-D-glucose. Purriul hydrolysis on TLC of compound 1. Compound 1 was applied on silica gel TLC and left in an HCI atoms at room temp. (30”) for 1 hr. HCI vapour was eliminated under hot ventilation and then authentic samples of the sugars were applied lo the chromatoplate. The chromatoplate was developed with the solvent system CHCl,- MeOH--Me,CO -H,O (3: 3 : 3: 1) and spots detected by spraying with 10% MeOH --H,SO, followed by heating. Glucose, rhamnose and xylose were identdied. Enzymatic hydrolysis ofcompound 1. A mlxt. of I (30 mg) and /l-glucosidase (from almond) m a soln of HOAc NaOAc buffer (pH 4.5) was incubated at 3X for 48 hr. The products were purified by silica gel CC to afford compound 3 (18mg), amorphous powder, IR v:1):crn- ‘. 3395 (br) 2925, 1380, 1245. 1160, 1075 (br), 920, 895 and 866 (895) 920, 25R spiroketal): “C NMR (C-IC-27): 645.15, 72.02, 84.86, 35.00,45.12. 29.75, 32.30, 35.08, 55.52, 35.92, 21.74, 40.20, 40.65, 56.92, 32.25, 80.43, 62.81, 15.52. 12.89, 42.79, 14.00. 109.81. 31.06. 29.32, 29.72. 67.34. 17.07. glc (C,-C,); 102.95, 81.72, 86.32. 69.51. 78.90. 61.21; 103.98, 81.X2, 78.72, 71.52, 78.93, 61.20. Rha (C,C,): 104.75, 71.15. 73.00. 73.02, 67.30, 17.80. 104.90, 72.11, 69.52, 80.42, 67.34, 16.50; Xyl ((Z-C,): 105.02, 73.45; 75.82, 71.11, 67.40, FABMS: 1203 [M +Na]‘, 1071 [(M+Na)-Xyl]+, 1057[(M+Na)-Rha]‘,925 [(M+Na)-Xyl-Rha]‘, 895 [(M+Na)-Rham-Glc]‘; 779 [(M+Na)-Xyl-2xRha]‘;763L(M+Na)-Xyl-Rha-Glc]’, 617 (M+Na)-Xyl-Glc-ZxRhaJ’, 455 [(M+Na)-Xyl2xGlc-2xRha]‘,431,415, 399. 139. 115.

Acknowledgements-We wish lo thank Dr. D. C. Jain of this Institute for providing an authentic sample of gitogemn and Finnigan MAT, U.K. for recording FAB mass spectra. The facilities provided by the 500 MHz FT NMR National facility supported by the Department of Science and Technology at T.I.F.R., Bombay, India are gratefully acknowledged.

REFERENCES I. Uniyal, G. C., Agrawal, P. K., Thakur, R. S. and Sati, 0. P. (1990) Phytochemistry 29, 937. 2. Agrawal, P. K., Jain. D. C.. Gupta, R. K. and Thakur, R. S. (1985) Phyrochemistry 24, 2479. 3. Agrawal, P. K.. Mahmood. U. and Thakur, R. S. (1989) Heterocycles 28, 1895.

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Reports

8. Bock, K. and Pederson, C. (1983) Adv. Carbohydr. Chem. B&hem. 44, 27. 9. Nakano, K., Matsuda, E., Tsurumi, K., Yamasaki, T., Murakami, K. Takaishi, Y. and Tomimatsu, T. (1988) Phytochemistry 2l, 3235. 10. Dutton, G. G. S., Merrifield, E. H., Laffite, C., Pratviel Sosa, F. and Wylde, R. (1982) Org. Mogn. Reson. 20, 154. 11. Jain, D. C. (1987) Phytochemistry 26, 1789. 12. Amoros, M. and Girre, R. L. (1987) Phytochemistry 26,787. 13. Agrawal, P. K., Thakur, R. S., Frahm, A. W. and Schneider, M. (1990) Magn. Reson. Chem. 28, 93 1.

4. Patcl, A. V., Blunden, G., Crabb, T. A., Sauvaire, Y. and Baceou, Y. C. (1987) Fitoterapia SE, 67. 5. Price, K. R., Mellon, F. A., Self, R., Fenwick, G. R. and Osman, S. C. (1985) Biomed. Moss. Spectrum. 12, 79. 6. Agrawal, P. K. and Bansal, M. C. (1989) in Carbon-13 NMR of Flouonoids (Agrawal, P. K., ed.), p. 283. Elsevier, Amsterdam. 7. Agrawal, P. K., Srivastava, S. K. and Gaffield, G. (1.990) in Alkaloids: Chemical and Biological Perspectives Vol. 7 (Pelletier, S. W.. ed.). Wiley, New York (in press).

Phymchemisrry, Vol. 30. No. 4, pp. 1339. 1341. 1991 Q

Printed m Great Brilam.

003 1 9422/9 1 E3.00 + 0.00 1991 Pergamon Press plc

CHLOROGENONE AND NEOCHLOROGENONE FROM THE UNRIPE FRUITS OF SOLANUM TORVUM ALFXEDO Facultad

de Farmacia,

CARABOT CUERVO, GERALD

Universidad

de Los Andes, M&da,

BLUNDEN* Venezuela;

and ASMITA V. PATEL*

*School

of Pharmacy

and Biomedical

Sciences,

Portsmouth Polytechnic, King Henry I Street, Portsmouth, PO1 2DZ, U.K. (Receiued in reuisedform

7 Sepwmber

1990)

Key Word Index-Solanum hazenii; S. meridense; S. scorpioidewq S. sycophantn; S. torvwn; Solanaceae; fruits; steroidal sapogenins; steroidal alkaloids; chlorogenone; (25RtSa-spirostane-3,6dione; neochlorogenone; (25!+Saspirostane-3,6-dione.

Abstract-Either steroidal sapogenins or steroidal alkaloids have been isolated from the unripe fruits of Solanum hazenii, S, meridense, S. scorpioideum, S. sycophanta and S. torvum. Chlorogenone {(25R)-Sa-spirostane-3,6-dione} and its 2X-epimer, neochlorogenone, were extracted from S. torvum; this is the first record of these compounds as natural products.

INTRODUCTION

The distribution of steroidal sapogenins in selected speties of the Venezuelan flora is being studied Cl, 23. In this report we record the isolation and characterization of steroidal sapogenins and alkaloids from five species of Solanum, four of which have not been investigated previously. From the unripe fruits of S. torvum Swartz chlorogenone and neochlorogenone were isolated; this is the first record of these as naturally occurring compounds. RESULTS

AND DISCUSSlON

The glycosides present in dry, powdered samples of the unripe fruits of Solanum species were acid hydrolysed. The aglycones produced were extracted and compared by TLC with suitable reference compounds before isolation by preparative TLC. The isolated compounds were identified on the basis of the their IR, ‘H and 13C NMR, and mass spectra.

Both S. hazenii B&t. and S. sycophanta Dunal et DC. yielded solasodine and solasodiene; the latter compound was probably an artefact produced during hydrolysis of the saponins. From S. scorpioideum Rusby, diosgenin (TLC evidence only), tigogenin, hecogenin, sisalagenin and chlorogenin were isolated, and from S. meridense Bitter ex Pittier diosgenin and chlorogenin. On TLC investigation of the extract of S. torvum, two major yellow spots were produced after spraying with the sulphuric acid locating reagent, which were designated A and B in order of decreasing R, value. The compounds producing the spots were separated by preparative TLC. Chlorogenin and neochlorogenin were identified in B. The compounds forming spot A did not co-chromatograph with any of the reference sapogenins available to us. The IR spectrum of A showed bands at 835,876.m. 922,952 and 981 cm- * (spiroketal), in which the intensity

of the 900 cm-’ band was greater than that at 922 cm-’ (ZSR-spirostane). No bands were observed between 3500