An ursadienedioic acid glycoside from Crossopteryx febrifuga

003 I 9422/9 I $3.00 + 0.00 Q 1991 PergamonPressplc

Phyrochemistry, Vol. 30, No. 9, pp. 3069 3072, 1991 Printedin Great Britain.

AN URSADIENEDIOIC ACID GLYCOSIDE CROS~OP~~R YX FEBRlFUGA

FROM

BABADY-BILA, TSHIAMUENE NGALAMULUME,AMURI KILONDA, SUZANNE TOPPET.* FRANS COMPERNOLLE* and GEORGES HOORNAERT+t Laboratoire de Chimie des Substances Naturelles, Wpartement de Chimie, Universite de Kinshasa, 8. P. 137, Kinshasa XI, Zaire; lDepartement Scheikunde, Laboratorium voor Organische Synthese, KULeuven, Celestijnenlaan 200 F, 3030 Heverlee, Belgium (Received in revised&m 22 January 199f)

Key Word Index-Crossopteryx a-r-rhamnopyranosyl

febrijiiu; Rubiaceae; 38-hydroxyurs-12,20(3O)diene-27,28aioic ether; @glucopyranosyl ester.

acid glycoside;

the root bark of Crossopreryx febr~~a a triterpene saponin characterized by a novel ursadienedioic acid aglycone part was isolated. The structure 3~-(a-L-rhamnopyranosyioxy)-28-0-(8-D-giucopyranosyl)urs12,20(30tdiene-27,28-dioic acid was established on the basis of ‘H and 13CNMR, and mass spectrometry of various derivatives prepared before and after acidic cleavage of the 28-O-glucosyl ester. Total acidic hydrolysis led to hydration of the exocyclic 20(30)-double bond and further transformations of the aglycone group. Abstract-From

INTRODUffiON Crossopteryx febrgiga

Benth is a plant widely used in African folk medicine against fevers and sundry diseases [ 1,2]. Previous work reports that this plant contains the triterpenoid /?-quinovin, the glycoside shanzhizide methyl ester, a cytotoxic triterpenoid and flavonoids 133, crossoptines A and B 143. Recently, the isolation and structure elucidation of two saponins from this plant were reported [S]. We now describe the structure elucidation of a new saponin from the root bark of Crossopteryx febrifuga Benth. RESULTS AND

lb IC

DISCUSSION

The

HPLC fractionation of the butanolic extract of the root bark of C. febrijiiga yielded la as a powder, mp 174-176”, [ali + 85” (MeOH; c 0.4). The molecular formula C42H640 14 was deduced from DEPT ’ 3C NMR data (Table 1). These data and those obtained from ‘H NMR spectroscopy (Table 2) indicated the presence of two sugar units, linked to different sites of the aglycone. The presence of a glucopyranosyl unit linked to a carboxyl group of the aglycone was revealed by the ‘HNMR anomeric signal at 65.43 in methanol-d4 (or 6.21 in pyridine-d,). The 13C-‘H correlated spectrum showed that this proton is attached to the anomeric carbon observed at 695.7 in methanol-d, (or 95.8 in pyridine-d,. The /?-configuration of this glucopyranosyl unit was derived from the coupling constant values (J H.1H.2=8 Hz and J,.,_,,,= 163 Hz) and from the cheiical shift values [6, 73. The ‘H and the 13C NMR data of the second sugar unit suggested the presence of the ~-rhamnopyranosyl unit linked to the aglycone by an ether bond. The

tAuthor to whom correspondence should be addressed.

Glc

K’ H

ti

fi

Me H

H Me

K’

la

Id

anomeric proton was observed at 64.77 (br s) in methanol-d, (or 5.14 in pyridine-d,) and the anomeric C-atom at 6104.4 in methanol-d, (or 104.2 in pyridine-d,). The r-configuration of the rhamnopyranosyl unit was deduced from the coupling constant values J,.., HSG2 = 1.5 Hz and J,.., H.,1= 168.6 Hz. This configuration’was confirmed by thk good agreement of the C’i and C’Gvalues for the rhamnopyranosyl unit in 13C NMR (672.9 and 69.8, respectively, in pyridine-d,) with those described [S] for methyl ~-L-rhamnopyranoside (672.5 and 69.4, respectively, in pyridine-d,); the values for the /_Ganomer are 675.3 and 73.4, respectively. The structure of the aglycone part was inferred as 20(30)-ene quinovic acid by comparison of the NMR data with those of an authentic sample of 3-O-acetylated quinovic acid (2) isolated from Heinsiu crinata [9], and with those published [ 10, 11J. The main differences were the disappearance of signals due to the CH-20 and Me-30 of quinovic acid, and the appearance of signals characteristic of a terminal double bond. In the ‘H NMR spectrum

3069

3070

BABADY-BILA et al. Table

I. ‘%I NMR spectral

la and 2 (in CD,OD*

data of compounds

or pyridine-&t

at 30”)

C

la*

lat

2t

DEPT

C

la*

W

2t

DEPT

1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16

40.8 26.6 90.3 40.0 57.5 19.4 37.9 40.8 48.0 36.6 24.0 130.9 133.5 57.7 26.7 26.0

38.9 26.4 88.3 39.1 55.6 18.7 37.4 40.2 47. I 37.0 23.6 129.7 133.4 56.9 25.9 25.5

38.6 26.4 80.8 37.9 55.6 18.7 37.4 40.0 47.2 37.2 23.4 129.0 134.3 56.8 24.0 25.6

CHz CH2

17 18 19 20 21 22 23 24 25 26 27 28 29 30 MeCO MeCO

49.9 56.5 36.6 154.0 32.9 39.8 28.8 16.9 16.7 19.2 179.5 177.3 17.0 106.6

49.1 56.9 36.1 153.1 32.2 38.7 28.1 16.8 16.5 19.1 178.4 175.9 16.5 106.1

48.8 55.0 39.4 37.8 30.7 37.2 28.1 17.1 16.5 18.9 178.1 180.2 18.3 21.4 170.6 21.1

C

Rhamnose 1 2 3 4 5 6

at CJ 104.4 72.7 72.8 73.9 69.9 17.8

Table

CH C CH CHz CHz C CH C CHz CH C C (332 CH2

Glucose 104.2 72.4 72.9 74. I 69.8 18.5

CH CH CH CH CH CH,

2. ‘H NMR spectral

data of compound

1 2 3 4 5 6

at 28-COO 95.7 74.1 78.3 71.5 78.6 62.6

la (in CD,OD*

6 (ppm)

CH CH CjCH CH, (342 CH, CH, CH3 CH3 coo coo CH, CH2/CH,

C CH,

95.8 74.1 78.9 71.3 79.2 62.5

at 25’ or pyridine-d,t

6 (wm) t

CH CH CH CH CH CHz

at 90”)

Group

l

t

M; J (Hz)

Group

*

3x-H 12-H

3.12

3.11

dd; 5, 12

5.67

5.97

m (0; 3

29-Me 30 CH2(H, and Ha

1.1 4.70 4.72

1.38 4.73 4.76

d;6 hr s hr s

18-H 23-Me 24-Me 25-Me 26-Me

2.42 0.85 0.86 0.98 1.04

2.82 0.86 0.87 1.01 1.23

d; 10 s s s s

6’-Me 1‘-H I “-H

1.29 4.77 5.43

1.61 5.14 6.21

d;6 br s (d); 1.5 d; 8

(pyridine-d,) two broad singlets at 64.73 (1H) and 4.76 (1H) were observed, whereas the 13C NMR spectrum showed the vinylic methylene carbon at 6106.1 and the vinylic quaternary carbon at 6 153.1. Most of the 13C NMR absorptions, except for some carbons, were comparable to those of the 3-O-acetate of quinovic acid (2). This report is the first to describe a natural 20(30)-ene quinovic acid derivative. The assignment of the rhamnopyranosyl unit as a 3O-ether derivative is supported by the 13C signal observed for C-3 (690.3 in methanol-d,or 688.3 in pyridined,). The site of attachment of the glucosyl unit (C-28) was derived by comparing the 13C NMR shifts for C-27 and C-28 with those of quinovic acid 3-O-acetate (2) and commercial ursolic acid (3) in pyridine-ds. For diacid 2 the upfield signal at 6 178.1 was attributed to C02H-27 and the downfield absorption at 6180.2 to C02H-28. According to 13C NMR data reported for model car-

boxylic acids for C-27 on

and

esters

M; J(Hz)

[ 123, an upfield

shift is expected

the basis of its allylic position and the larger number of y-substituents (6 for C-27 and 4 for C-28). The assignment of C02H-28 for diacid 2 (6180.2) was confirmed by the value, 6 179.9, found for 28-monoacid 3. On the basis of these results, the signal at 6178.4 in the spectrum of acid ester la was attributed to a free CO*H27 group. The upfield absorption at 6 175.9 was assigned to the 28-ester group, in agreement with the value (6 175.9) reported for a 30-norolean-12,20(29)-dien-28-0p-D-glucopyranosyl] ester [13] and the value (6 176.2) for a quinovic acid 28-O-[P-D-glucopyranosyl] ester c141.

To confirm the site of attachment of glucose, the free and glucosyl esterified carboxy groups were differentiated through methyl esterification and transesterification, respectively. Heating la with t-BuOK in methanol afforded the C-28 methyl ester lc and the saponified

Ursadienedioic acid glycoside from

2 3

R’ AC H

Crossopteryx

R’ CO,H Me

4

3071

febrijiga

R’ Me OH

R’ 011 “I Me

Table 3. Comparison of EI mass spectra* of trimethylsilyl derivatives of methyl esters lc and Id, diacid lb and saponin la+ 1C

ld

lb

w

WI+

932 (6)

932 (8)

990(11)

[M -Me] [M -CO#MeJ+ [M - RhamnO]+

917 (1.5) 815 (5) 553 (50)

917 (1.6) 815 (2) 553 (30)

975 (8) 873 (10) 611 (36)

[M - RhamnO - Me,SiOH] + [M - RhamnO - HCO,SiMe,]’ [Rhamn] +

463 (47) 435 (45) 363 (100)

463 (3) 435 (10) 363 (100)

521 (57) 493 (38) 363 (100)

990 (4)$ 918 (12)5 903 (6) 801 (15) 611 (50)$ 539 (4O)g 521 (60) 493 (60) 363 (100)

*Intensities relative lo m/z 363; other ions due lo the rhamnopyranosyl unit were detected at m/z 73,204 and 217. tlnitial loss of glucosyl with $ transfer of MeSSi or 8 transfer of H to the acid moiety.

product lb, which were isolated by TLC. Esterification of la was accomplished by using MezNCH(OMe)z as a methylating reagent. Subsequent hydrolysis of the glucosyl ester group with 2 M aqueous HCl at room temperature gave the regioisomeric C-27 methyl ester Id. A comparison of the EI mass spectra (Table 3) of the pentasilylated diacid lb ([Ml’ at m/z 990) and the tetrasilylated methyl esters lc and Id ([Ml’ at m/z 932) showed the expected mass shift for most fragment ions, e.g. the ions corresponding to loss of the rhamnosyloxy group (m/z 61 l-553). Characteristic fragmentations in the spectrum of the C-27 silyl esters derived from lb and lc were due to cleavage of the allylic 27-COzSiMe, group (m/z 873 and 815) and to combined losses of the rhamnosyloxy group and either Me,SiOH (m/z 521 and 463) or HC0,SiMe3 (m/z 493 and 435). For the non allylic C-28 silyl ester derived from ld the ions at m/z 815, 463 and 435 were suppressed to a large extent. The spectrum (Table 3) of the silylated saponin la was similar to that for lb, since fragmentation was initiated by loss of the glucosyl group with transfer of either a H-atom (m/z 918) or, to a lesser extent, of a Me$i group (m/z 990) to the acid moiety. Acid hydrolysis of saponin la with 2 M HCI in methanol-water at room temperature afforded diacid lb as the main product, identical with the product derived from treatment with base (TLC and MS analysis). Further acid cleavage of the 3-0-rhamnosyl group was effected by heating at 50”. Two genin fractions were isolated

by TLC. Silylation and EIMS analysis of the more polar compound revealed incorporation of water and introduction of only three silyl groups ([Ml’ at m/z 718, C39H7,,0LSi3). This result is consistent with hydration of the exocyclic double bond to form the tertiary alcohol 4. A similar analysis for the less polar fraction showed formation of a disilyl ([Ml’ at m/z 628, CJ6H600&) and a trisilyl derivative ([M] + at m/z 700, C39H680$i3). Likewise, esterification with HC(OMe)zNMe, produced both a mono- and a dimethyl ester ([M] + at m/z 498 and 512). Reexamination of this fraction by TLC confirmed that it was a mixture of two compounds. Presumably, these correspond to a diene diacid and an isomeric lactone monoacid structure, both of which could result from acid catalysed dehydration of the alcohol intermediate 4. Total acidic hydrolysis of la at 100” led to further degradation of the aglycone group, as shown by the isolation of seven genin fractions and by EI mass spectral analysis of the silylated compounds. For instance, in the spectrum of a less polar fraction the molecular ion at m/z 512 indicated decarboxylation of one and lactonization of the other carboxy group (C3zH5z03Si, introduction of one silyl group at the 3B-OH position). Rhamnose and glucose were identified by TLC analysis of the carbohydrate fraction. On the basis of the above, the structure of the saponin is the 28-O-B-D-glucopyranosyl ester of 3/l-[a-L-rhamacid nopyranosyloxy]-urs-12,20(30)-diene-27,2&dioic (Ia).

BABADY-BILA et al.

3072 EXPERIMENTAL

Plant material was collected on the campus of the University of Kinshasa and authentilicd by a voucher specimen H. Breyne 207 kept at the herbarium of the INERA, Faculty of Sciences. University of Kinshasa. Precoated silica gel plates Alugram Sil GjUV 254 (Macherey-Nagel) were used for analytical and prep. TLC. The plates were developed with CHCIs-MeOH-Hz0 (85: 14: 1) (solvent system A). Solvent system A was used also for recovering the compounds which were detected by spraying side strips with HzSO,-MeOH (4: 1) followed by heating. Reversed phase HPLC was carried out on a 10mm RP-Cl8 lichrosorb column (id. 2.27 x 25 cm, Chrompack). The ‘H and 13C NMR spectra of la, 2 [9] and 3 were recorded at 250.1 and 62.9 MHz, respectively, using a 5 mm ‘H-‘3C dual probe. The ‘H and ‘jC NMR chemical shifts are reported in ppm relative to TMS as an int. ref. The correlations between ‘H and 13C were obtained by selective heteronuclear decoupling i3C+‘H) for la in CD30D and by means of a 2D heteronuclear correlation experiment for la and 2 in pyridine-d,. For the rhamnose and glucose units and for the methylenic envelope of the genins, assignments were tentative only for those ‘zC signals corresponding to insufficiently resolved ‘H absorptions. The DEPT pulse sequence was applied to determine the number of protons attached to each carbon. The optical rotation was measured using a polarimeter fitted with a 5 cm cell. EIMS spectra (probe, 70 eV): the ion source temp. was 15&250” as required. Silylation of compound la (0.1 mg) and other compounds described below was carried out by heating a soln in pyridine (0.1 ml) with bis-(trimethylsilyl)trifluoroacetamide (0.05 ml) at loo” for 5 min. Extraction and isolation. The dried powdered root bark of C.febrifusa (1250 g) was macerated in 80% MeOH for 48 hr, then the mixture was refluxed for 3 hr and filtered. The filtrate was evapd to dryness. The solid residue (295 g) was dissolved in 1 I of Hz0 and the aq. soln was extracted with n-BuOH. The saponins were pptd from the BuOH soln by adding a five-fold volume of Et,O. The ppt. was filtered, washed with Et,0 and dried (yield 62 g). 1 g of this mixture was subjected to HPLC fractionation using MeOH-Hz0 (4: 1) as eluent. Five frs were obtained. One of them (fr. V: 180 mg) was recycled using MeOH-Hz0 (7: 3) as eluent to afford saponin la as a powder (22 mg), mp 174-176”. [a]k +85” (MeOH; c 0.4). Transesterification of la and isolation of compounds lb and lc. To a soln of 0.5 M r-BuOK in MeOH (1.5 ml). 5 mg of la was added. The soln was heated at loo” in a sealed tube for 3.5 hr. then acidified with HOAc, diluted with Hz0 and extracted with EtOAc. Prep. TLC using solvent system A afforded lc (R, 0.33) and lb (R, 0.29). EIMS of silyl derivatives: see Table 3. Esferification of compound la and preparation of Id. Compound la (about 0.5 mg) was dissolved in MezNCH (OMe), (0.2 ml) and the soln was heated at loo” for 10 min. The reagent was evaporated and the residue hydrolysed with 2 M aq. HCl for 30 min. Extraction with EtOAc afforded Id. EIMS of silyl derivative: see Table 3. Acid hydrolysis of compound la ar room temp. Compound la (5 mg) was added to 1 ml of 2M HCI in MeOH-Hz0 (1: 1) and the soln was kept at room temp. for 1 day. For prep. TLC this soln was applied directly to the plate, affording lb as the main product. Two unidentified products (R, 0.17 and 0.20) showing polarities intermediate between those of la (R, 0.13) and lb (R, 0.29) also were detected. EIMS of silyl derivative of lb: see Table 3. Acid hydrolysis of 3-0-rhomnosyl group at 50”. Acid hydrolysis of la (3 mg) was carried out at room temp. for 1 day as

described above. The soln was then heated at 50” for 3 hr and applied to a TLC plate. Development and elution with solvent system A yielded 4 (R, 0.37) and a less polar fraction (R, 0.59) as the main products. TLC analysis of the less polar fraction using CHCIa-MeOH (47: 3) revealed the presence of a 1: 1 mixture of two products (R, 0.33 and 0.40). The methyl esters of this mixture were prepared by heating a sample with HC(OMe),NMe, (0.2 ml) at 100” for 40 mitt and evaporation of the reagent. EIMS of methyl esters of less polar fraction: m/z 512 [Ml+, 498 [Ml’, 494 [M-H,O]+, 480 [M-H,O)]‘. EIMS of silyl derivatives of less polar fraction: m/z 700 [Ml+, 610,585, 467, 347. 279. 203, 189, 73. EIMS of tris-Me,Si derivative of 4: m/z (rel. int.): 718 CM]’ (2) 703 [M-Me]+ (2), 700 [M -HzO]’ (4). 628 [M-Me,SiOH]’ (3) 610 [M-HzO-Me,SiOH]’ (3). 610 [M-HzO-Me,SiOH]+ (3) 601 [M -COISiMe,]+ (3). 583 [M-HsO-COzSiMe3]’ (7). 493 (4). 465 (5). 375 (5). 279 (5). 239 (4). 73 (100). Total acid hydrolysis of compound la. Compound la (5 mg) was dissolved in 2 ml of 2% HzSO, in EtOH-Hz0 (1: 1).This soln was heated in a sealed tube at 100” for 4 hr. The soln was partially evapd in racuo and aq. soln was extracted with EtOAc. TLC of the aq. phase using i-PrOH-EtOAc-H,O (83: 11:6) showed the presence of glucose and rhamnose. The genin fraction was separated into 7 subfractions by TLC using solvent system A. EIMS of mono-Me,Si derivative of subfraction 2: m/z 512 CM]’ (lo), 497 [M-Me]’ (4). 468 [M-COz]+ (8). 407 [M -Me-Me,SiOH]’ (9). 73 [Me,Si]’ (100). Acknowledgements-The authors are indebted to the F.K.F.O. and the “Ministerie voor Wetenschapsbeleid” for financial sup port and to the A.B.O.S. (B.B.) and K. U. Leuven (A.K.) for a fellowship. They wish to thank Mr P. Valvekens for technical assistance.

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