-Absttract-Soyasapmiin III has been characterized and the structure of a new triterpenoid saponin, hispidacin, has been elucidated as soyasapogenol ~-3-U-~-~-rham~opyranopyranosyl(l42~~-~-glu~opyran~syl(l -*2)$-D-&k ~uro~opyrano~de by a combination of fast~atom ~mbardment mass s~trurnet~~ lsCNMR spectroscopy, and some chemical t~a~sf~~atio~s. Mechanism uf transforma~on of soyasapogenol B to soyasap~gen~ls D, and F has also been rationalized.

INTRODUCTION

Gaertn (syn M. ~~~~~~~~~~~ Willd) is an annual semi-erect or prustrate herb occurring in many parts of India. The plant is used as green fodder and its leaves are rich in ascorbic acid and /?-carotene [I]. Isolation of a saponin, hederin and its sapogenin hederegenin, has been reported by WalterE21. In continuation of our chemical investigation on the ph~a~uti~lly important naturally uccurring saponins [3-91 we report the isolation and structure elucidation of a new triterpenoid trisaccharide from the aerial part of this plant along with ~h~a~t~ri~tiun of soyasaponin III,

~~~~~~u

~~s~~~

RESULTS AND DISCWON

The rr-butanul soluble fraction of the metha~oli~ extract of the leaves and twigs of M. ~~~~~~~cm purifrcation by repeated column chromatography and preparative TLC led to the isolation of two triterpenoid saponins, as judged by Liebermann-Burchard and Molish tests, as well as by the furmatiun of stable froth when shaken with water. The more polar sapunin, designated as hispidacin (I), was found to be homogeneous by TLC, PC and HPLC. However, on hydrolysis with aqueous methanolic hydrachloric acid it yielded a mixture of aglycones (2-4) and sugar constituents identifiied by PC as cr-gfucuroni~ acid, ct-glucuse, and L-rhamnose by direct comparison with authentic samples. The aglycone (2) was eventually characterized as soyasapogenof B by cumparison of its mp, [at&,, ‘H and 13C NMR and mass spectral data with those of an authentic sample [lo]. The aglycone (3) did not show any olefinic proton signal in its ‘H NMR spectrum, although the presence of unsaturatio~ was revealed by the tetranitr~methane test and molecular formula, Moreover, the “H NMR spectrum showed a methoxy signal at 53.32. The mass spectrum exhibited a molecular ion peak at nr/z 472 and the discernible fragment ion peaks were reminiscent of A’3’f81pentacyclic triterpenes f I 11. Compound 4 was present in the hydrolysate only in traces and, although it could not be isolated in the pure state, its

presence in contamination with soyasapogenol B (2) was revealed by the mass spectrum which showed in addition to the retru~Diels-Alar ion (rr) at mfi 234+an ion (b) at m/z 221 and a related ion at m/z 203 (after loss uf water) consistent with the presence of a 13: 18 double bond. As the i%Z NMR spectrum of hispidacin (Table 1) did not display signals ascribable to a tetrasubstituted double bond, the cumpounds 3 and 4 were assumed to be artefaets of 2 formed during acid hydrolysis of the saponin. This presumption was proved to be correct by transformation uf aglycone 2 to the products 3 and 4 by similar acid treatment, Iurzysta Cl23 and Price & ial. [t3] have reoently reported that compounds 3 (soyasapogenol D) and 4 {suyasapogen~l F) are artefacts of soyasapog enol B which arise during acid hydrolysis, However, the mechanism of formation of these products which apparently seems to be rather unusual, does not appear to have been proposed as yet. It is noteworthy that longispinogenin or its glycosides corchorusins A and D, containing 168 (axial) hydroxy group [43 did not yield any A*3(1*‘-aglycone on similar treatment with aqueous rnethan~~~ hydru~hlo~~ acid. It was, therefore indicated that the 22s (axial~hydr~xy group present in soyasapogenol B (2) or its glycosides might have some role in inducing this transfo~atio~~ Assuming all-chair conformatiun uf 2 having D/E cisfusion, inspection of its Dreidiag model disclosed that the 22#&axial hydroxyl experienws strong steric interaction with the 20/I-axial methyl, in addition to that with the 17methyl group. Migration of the double bond from the 12: 13 to 13: 18 position transforms the 22@-hydroxyl from axial tu equatorial orientation, thereby releasing the 1,3-diaxial interaction. Substitutiuu of the 22-hydroxy group in 4 with 1xmethoxy group may then occur ~1s shown in Scheme 1 via (a) its protunation, (b) elimination uf the protonated group through pa~i~pa~uu of the 13 : 18 double bond which is favuurably disposed to form a protonated cyclopropane as intermediate and (c) attack by a molecule of methanol on the intermediate carbocatiun, to generate a methoxy derivative with retention of ~un~guration. That the 2Zmethoxy group in soyasapogenol D (3) has the equatorial (@) disposition is

3389

S.

3390

B.MAHATC)

OR'

2 8 9

b

i)R' 1 7

?a

R’ H H Ac

RZ H Me Me

R' R2 COzH H COzMe Me Me CH20Me

+

H,k

OR HO

Ii ~ c

b

R = HoxMe

revealed by its lH NMR spectrum as well as that of its acetate (5) which show two double doublets {J = 6,X 1 Hz) centred at 62.80 attributable to 22-axial hydrogen arising out of its axial-axial and axial-equatorial interactions with the adjacent Hz-21. The isomeric methoxy compound (a) with a 12 : 13 double bond and axial (B) methoxy group displayed signals assigned to H-22 at S 2.81 (dd, J = 2.9, 6.5 Hz) as reported by Kitagawa et at. t141. The positive-ion FAB spectrum of hispidacin fl) in a ~ycerol-thiogly~rol matrix showed a protonated ion at m/z 943 ascribable to [M + H] +. A cationized ion at m/z 965 was also discernible. Thus, the M, of the glycoside (1) was determined to be 942. The major fragment ions (Scheme 2) are in full accord with the sequence of the monosaccharide units in the assigned structure for hispidacin (1). The r3C NMR spectra of soyasapogenol B (2) and hispidacin (1) were useful in the determination of the points of attachment of the trisaccharide moiety to the aglycone as well as the intersugar linkages. The signal assignments were made by single-frequency off-resonance decoupling @FORD) taking into consideration the r3C NMR chemical shifts of soyasapogenol B, p-D-&curonopyranoside [ 151, 8-D-glucoside, a-r.-rhamnopyranoside [ 163 and glycosylation shift values [17-193. The attachment of the trisaccharide unit in saponin 1 to the C-3 hydroxy group of aglycone 2 was evident from

3

R = Me

4

K=H

R = I-1or Me

the shift of carbon atoms C-2, C-3 and C-4 of compound 1 by - 1.2, 9.1 and - 0.4, respectively, whereas l-+2 linkages between L-rhamnose, D-glucose, and D-gh&Xironic acid were deduced from the Glc-2 and Glut-2 resonances which experienced downfield glycosylation shifts of 5.7 and 4.3 ppm, respectively. Permethylation of 1 by Hakomori’s method [ZO] afforded the permethylate (7) which on LiAlH, reduction followed by acid hydrolysis liberated 3,4,6-tri-o-methylD-giUCOSe, 2,3,4-tri-O-methyl-t-rhamnose and 3,4-d&0methyl-D-glucose identified by GLC and the partially methylated aglycone (8). This compound (8) yielded an acetate (9) which showed in its ‘H NMR spectrum a signal at 64.60 (lH, &I, J = 10.5 and 6 Hz} assignable to the axial proton at C-3 bearing the equatorial acetoxy group. The results demonstrated that the terminal rhamnose is linked through its anomeric hydroxyl to the 2position of glucose which in turn is connected to giucuronic acid by (l-*2) linkage and the latter being connected to the C-3 hydroxyl of the aglycone. The /I-configurations (4CI conformation) for the glucuronopyranosyl and glucopyranosyl units and an a-configuration (‘Cd conformation) for the rhamnopyranoside were inferred from the J values of the respective anomeric protons in the ‘H NMR spectrum of the permethylate 7 (see Experimental). On the basis of the above, the structure of hispidacin is soyasapogenol B-3-O-cr+rhamnopyranosyl

Triterpenoid saponins from Medicago hispida

3391

Table 1. 13C NMR spectral data [S, (If:O.l)} of compounds 1 and 2 1

C

(DMSO-I,)

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

38.6 27.3’ 89.5 43.0 55.6 19.8 32.1 39.8 46.3 36.6 24.8 122.0 143.6 42.3 27.2’ 29.6 38.0 45.5 46.3 30.2 42.4 75.2 24.3 67.4 15.6b 16.2b 25.1 28.0 33.4 21.7

2 (pyridine-d,)

.“Me C

39.2

Glut 1

28.5a 80.4 43.4 56.5 19.3 33.8 40.3 48.4 37.3 24.2 122.6 144.7 42.5 26.6 28.8” 38.0 45.6 47.0 30.8 42.7 75.8 23.6 64.6 16.2b 17.0b 25.8 28.8 33.2 21.0

Glut 2 Glut 3 Glut 4 Glut 5 Glut 6 Glc 1 Gb 2 Glc 3 Glc 4 Glc 5 Glc 6 Rha 1 Rha 2 Rha 3 Rha4 Rha.5 Rha 6

I 103.4 81.4 76.4 73.s 77.6’ 175.1 99.4c 79.2 77.6b 71.7 77*4b 61.8 99.7” 71.9 71.8 74.0* 70.0 17.3

I

H@

tie

“-“Assignments within a column may be interchanged. Glut: Glucuronic acid.

OMe ( 1+ 2)-b-D-@ucopyranosyI aside {I).

( f 4 2)-/?-D-glucuronopyran-

The less polar saponin (10)on acid hydrolysis afforded soyasapogenoi B (2), D-ghzuronic acid and D-galaCtOSe. The acid rearranged products 3 and 4 were also found to have been formed as minor products along with the aglycone, soyasapogenol B. Permethylation of saponin 10, followed by LiAlH4 reduction and subsequent acid hydrolysis, furnished 22,24-d&O-methyl soyasapogenol B (8), 2,3,4,6-tetra-0-methyl+galactose, and 3,4-di-Omethyl-D-glucose. These results, as well as the mp, [a]n and ‘H NMR spectrum of saponin 10, were in full agreement with those reported for soyasaponin III [213. This is the first report of soyasaponin III from 1M.kispida. EXPJSRIMENTAL

The plant material was identified at the Indian Botanic Garden, Howrah and a voucher specimen is deposited at the herbarium of IICB. Mpsz are uncorr. Prep. TLC was performed on silica gel G plates deveioped with CHCIS-Ha0 with McOH added until the soln became clear. Paper chr~to~phy was carried out on Whatman paper No. 1 using the solvent system

i

Me

3 Scheme 1.

BuOH-pyridine-Hz0 (6:4:3); a satd soln of aniline oxalate in H,O was used as staining agent. GLC was carried out on a Hewlett-Packard model 5730A instrument with the following columns; (i) ECNSS-M, 3% on Gas Chrome Q at 190” for alditol acetates and (ii) OV-225 on Gas Chrome Q for partially methylated alditol acetates. ‘H and 13C NMR spectra were recorded at 99.6 MHz and 25.05 MHz in pyridine-d, or DMSO-d, solns, respectively, with TMS as int. standard. FAB-MS: 8 kV. A mixt. of gly~~l/thio~y~ol was used as a matrix and DMSO as solvent. The sample was ionized by bombardment with Xe atoms produced by a saddle field ion source from Ion Tech. E&MS: 70 eV.

S. B. MAHATO

3392

-H*O #-+ mk!44f--+

w

m/z 423 .-,

+

H

HO

HO

mlz 797 1

m/z965 [H+ Na]” m/z 819 [M + Na-Rha]+ m/z943 fM+H]+ m/r899 [M+H-COzl+

m/z925 [M+H-H,O]+ Scheme 2.

fsoladion ofsuponins. The air-dried powdered aerial part of M. hispi& (1.25 kg) was successively extracted with petrol (60-80”), CHCI, and MeGH. The methanotic extract, on removal of the solvent under red. pres. yielded a viscous dark brown mass which was partitioned between n-BuGH and Hz0 The organic layer was coned to dryness under red. pres, to give a residue (45 g) which was chromatographed on silica gel (95Og). Graded elution was effected with CHCI, followed by CHCI,-MeOH mixt. Frs (250 ml each) were monitored by TLC. The partially sepd more polar and less polar fr. were further purified by rechromatography on silica gel column follwed by prep. TLC for some of the frs. Thus, the more polar fr. (O&g) and the less polar fr. (0.26g) were isolated. Hispidacin (1). The more polar fr. on crystallization from aq. MeOH afforded microneedles, mp 245-247”; [a],-22.5” (MeOH; ~0.42); ‘H NMR {DMSQ-d,): 60.70 (3H, s), 0.75 (3H, s) 0.79 (3H, s), 0.81(3H, s), 0.83 (3H, s), 0.92 (3H, s), 1.02 (3H, s), 1.04 (3H, d, 3=7 Hz), 1.12 (3H, s) (Found: C, 58.84; H, 8.50; C4sH7s01s -2H,O requires 58.88; H, 8.44%). HydroZysis of hispiducin (1). Compound l(O.25 g) was hydrolysed with 2 M HCl in aq. MeOH (60 ml) under reflux for 1Ohr. The usual work-up followed by chromatographic purification on a silica gel column afforded soyasapogenol B (2) as the major product 180 mg) which contained traces of soyasapogenol F (4), and soyasapogenol D (3) (16 mg) as the minor aglycone. Soyasapogenol B (2) was purified by repeated crystallizations from MeGH to give needles, mp 257-259” (lit. [Zl], mp 258-260’). Its MS, ‘H and 13C NMR spectra were comparable to those of authentic sample. The acid rearranged minor sapogenol, soyasapogenol D (3) was crystallized from MeOH as needles, mp 242-W; ‘H NMR (CDC1,):50.78,0.82,0.98,l.16,1.24(alls,7x Me),280(1H,dd,J =6, 11 Hz, HZ?,,), 3.35 (3H, s, OMe), 4.18 (lH, d, J- 10 Hz, H-

24), MS m/z (rel. int.): 472 [M]” (8), 440 [M -MeOH]+ (4), 248 [ion cl* (17), 235 [’ion hJ+ (19), 216 [c-MeOH]+, (37), 203 @ - MeGH] +, (lOO),175 (43), 99 (74). Acetylation of compound 3 with A@--pyridine on a waterbath for 1 hr followed by usual work-up and crystallization from MeOH furnished needles of acetate 5, mp 182-184. ‘HNMR (CDCl,): 60.73, 0.84, 0.89, 0.96, 1.00, 1.15, 1.51 (a11s, 7x Me), 2.01, 2.02 (each s, 2 x OAc), 2.80 (1H, dd, .I = 6, Xl Hz, H-22,,), 4.09 (1H, d, J = 12 Hz, H-24), (4.60, dd, J = 6,10 Hz, H-3); MS m/z 556 CM]’ 524 [M-MeOH)‘, 496 [M-HOAcJ’. The filtrate from the hydrolysate was neutralized with Ag,CO,, filtered and a portion of the filtrate coned under red. pres. and tested for carbohydrates by paper chromoatography with the solvent system n-BuOH-pyridine-H,O (6:4:3). Spots corresponding to D-glucuronic acid, b-glucose and t-rhamnose were detected by comparison with authentic samples, The other portion of the coned filtrate was reduced with NaBH, and worked-up as usual. The residue was acetyiated with Ac,O-pyridine (1: 1)at water-bath temp. for 1 hr, dried in oacuo, puriCed by chromatography over silica gel, and subjected to GLC analysis using column (i). Two peaks corresponding to glucitol and rhamnitol acetates were obtained. Under the conditions employed no product from the glucuronic acid portion would be expected to elute. Permt~tkylatio~ of kispiducin (1). Compound 1 (185 mg) was completely methylated by the Hakomori method using Mef-DMSG-NaH. Usual work-up followed by purification by a silica gel chromatography and elution with petrol (bp 6&800)-EtOAc (3 :2) furnished the permethylate (7) as a powder (165 mg), mp 160-163” (no hydroxy absorption in the IR spectrum); ‘H NMR (CDCl,): 63.10,3.18,3.22,3.32,3.35, 3.37,3.39, 3.44,3.46(alls, 11 xOMe),4.38,4.45(lHeach,d,J=7Hz)(H-1 of glucuronide, and H-l of glucoside, 5.62 (br s, H-l of rhamno-

Triterpenoid saponins from Medicago hispida side). (Found: C, 6440, H, 9.26, CsPH,OoO1s requires C, 64.57; H, 9.19%). Reduction ofthe permethyllate (7) with LiAlH, and hydrolysis of the product. Compound 7 (150 mg) in dry Et,0 (25 ml) was treated with a suspension of LAH (150 mg) in dry Et,0 and the reaction mixt. was stirred at room temp. (26”) for 1 hr. A few drops of aq. Et,0 were then added and acidified with aq. 20% H,SO,, Extraction with Et,0 followed by usual work-up yielded the product 7a (142 mg). Compound 7a (135 mg) was hydrolysed by heating under reflux with 2 M WC1in aq. MeOH (40 ml) for 6 hr. The reaction mixt. was cooled, evaporated to dryness under red. pres. diluted with HtO, and filtered. The filtrate was neutralized with Ag,CO, and filtered. The filtrate was coned and the product reduced with NaBH,. Usual work-up followed by acetylation with Ac,O-pyridine yielded a mixt. of alditol acetates which was subjected to GLC analysis on column (ii). Three peaks were detected corresponding to the alditol acetates of 3,4,6-tri-O-methyl-D-glucose, 2,3,4-tri-O-methyl+rhamnose and 3,4-di-0-methyl-r)-glucos& by comparison of their R,s with authentic samples. The residue of the acid hydrolysate was purified by chromatography followed by crystallization from MeOH to yield the partially methylated soyasapogenol B (8) mp 192-193” [or&, +38” (CHCl,; ~0.4) (ref. [21] mp 194-195”. It furnished an acetate (9) in the usual way, mp 176-177” [orJ,+61” (CHCl,; ~0.2) (ref. [2t] mp 178-179”). Soyusaponin 111 (10). Compound 10 (0.26g) obtained from silica gel CC was crystallized from MeOH to give needles, mp 216-217” [m],+u)” (MeOH; ~0.42) (ref. [21) mp 215-216” [a]o + 15.0”).On complete acid hydrolysis of saponin 10(60 mg) with 2 M aq. HCl for 10 hr afforded soyasapogenol B (2) as the major sapogenol(25 mg) and soyasapogenols D (3), F (4) as very minor products, and D-glucuronic acid, D-galactose as carbohydrate constituents. Permethylation of 10(75 mg) by Hakomori method as described for hispidacin (1) yielded the permethylate 11 (46 mg) which on LiAlH, reduction and acid hydrolysis liberated 2,3,4,6-tetra-O-methyl-D-galactose, and 3,4-di-0-methyI-D-glucase identified by GLC of their alditol acetates using authentic samples. Acknowledgements---We thank Dr B, Pramanik (Schering Corporation, Bloomfield, N-J., U.S.A.) for the FAB spectra and Sri R. Mahato for collection of the plant and technical assistance.

3393 REFERENCES

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ofIndia Raw Materials

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by Mass Spectru-

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