Phytochemistry, Vol. 30,No. 10,pp. 3395-3400,199l Printedin GreatBritain.

OO31-9422/91 $3.00 + 0.00 0 1991PcrgamonPressplc

TRITERPENE SAPONINS FROM VERBASCUM

SONGARICUM

KARLHEE SEIFERT, ALFRED PREI~S, SIEGFRIED JOHNE, JORGEN SCHMIDT, NGUYEN T. LIEN,* CATHERINE LAVAUD and GEORGES MASSIOT~

Institute for Plant Biochemistry, Halle 4050 HallefSaale Weinberg 3, Germany; *Institute for Chemistry, National Centre for

Scientific Research of Vietnam, Hanoi, Vietnam; tFaculty of Pharmacy, 51096 Reims CeciexRue Cognacq-jay 51, France (Received in revised form 1 February 1991)

Key Word Index-verbascum songaricum, Scrophuiariaceae; triterpenoid saponins; songarosaponin A, B and C.

Abstract-Songarosaponin A, B and C isolated from the aerial parts of Y~~~QSCWII sung&cum were shown to be 3-U{[ol-~-rhamnopyranosyl-(l-+4)-/?-D-glucopyranosyl-( 1-+3)]-~~-D-glucopyranosyl-(1+2)]-#&D-fucopyranosyl~-olea11,13-diene-3@23,28-triol, 3-0-([n-L-rhamnopyranosyl-( l-+~)-~-D-~~UCO~~~~~OS~~-(~-+3)]-[~-D-glucopyranosyl(1+2)]-fi-D-fucopyranosyI)-olea-1 l-ene-3/$13,23,28-tetrol and 3-O-(@-D-glucopyranosyl-(~ -+4)]-[B-D-glucopyranosyl-( 1~3)]-[~-D-glUCOpy~UOSyl-( 1~2~]-~-~fucopyranosyl~-l3~,28~poxyolea-l l-ene3/?,23-diol.

IN’IlKODUCTlON Verbuscum

sozrguricum

Schrenk is a perennial plant occurring wild in many parts of Central Asia. We report on the isolation and structural elucidation of three new triterpenoid saponins [ 13. RESUI,Ts AND DiXXJ!%ION

The methanolic

extract of the dried aerial parts of on repeated chromatographic purification on a silica gel column and prep. TLC yielded songarosaponin A (I), B (2) and C (3). Songarosaponin A (1) shows the characteristic tJV-maxima of an olea-11,13diene (243, 252 and 262 am) [2]. Songarosaponin B (2) can be converted to 1 by treatment with 1 M sulphuric acid. Methanolysis of l-3 gave triterpene B tolea-12,18 (17)diene-3/&23-dial] f2, 33. Hydrolysis of the methyl glyco sides obtained after methanolysis of 1 and 2 yielded glucose, fucose and rhamnose and of 3 glucose and fucose (GC-MS of the peracetylated monosaccharides in comparison with reference compounds). Complete methylation of 1 and 2, and hydrolysis of the permethyl products, led to 2,3,4,6-tetra-O-methyl-glucopyranose, 2,3,4-tri-0-methyl-rhamnopyranose (TLC compared with reference compounds), 2,3,6-tri-O-methylglucopyranose and 4-~-methyl-fucopyrano~ (characterized by GC-MS and ‘HNMR spectroscopy of the acetates). The partially methylated monosaccharides were transformed to alditols by sodium borohydride and acetylated. The alditol acetates were investigated by means of GC and GC-MS [4,5] and identified as 1,5-d& Q-acetyl-2,3,4-tri-O-methybrhamnitol, 1,5-di-O-acetyl2,3,4,6-tetra-O-methyl-glucitol, 1,23,5-tetra-O-acetyl4-O-methyl-ftitol and 1,4,5-tri-0-aeetyl-2,3,6-tri-Omethyl-glucitol. Hydrolysis of ~~ethylsongarosa~~in C gave 2,3,4,6-tetra-O-methyl-~ucopyrano~, 2,3,6-u-i-Omethyl-glucopyranose and 4-O-methyl-fucopyranose and after reduction and acetylation the appropriate alditol acetates. Verbascm

songakwn

The mass spectroscopic fragmentation behaviours of songarosaponin A (I), its peracetate (4) and permethyf ether (7) were as follows: The [M - l]- ion (m/z 1071) was found in the negative ion FAB spectrum of songarosaponin A (1). The ions m (m/z 925), II (m/z 763) and o (m/z 909) appear by splitting of the rhamnopyranosyi, the rhamnopyranosyl-(l-+4)-glucopyranosyl and the glucopyranosyl moiety. The positive ion mass spectra of 4 and 7 showed the characteristic ions i (4 m/z 273,7 m/z 189Xj (4 m/z 561,7 m/z 393), k (4 m/z 331,7 m/z 219) and I(4 m/z 523,7 m/z 467). The ions Eand k of 4 and 7 were in accord with the terminal position of rhamnopyranose and glucopyranose in the glycosidic part of sogarosaponin A (1). The disaccharide fragment j confirmed the linkage of rhamnopyranose and glucopyranose. Thus, the terminal rhamnose could be attached to either of the glucose units. Songarosaponin B (2) was transformed to songarosapomn A (1) by treatment with 1 M sulphuric acid and so the sequence of the sugar chain derived from the mass spectra of 4 and 7 is also valid for 2. Sugars and their points of attachment were determined by means of ‘HNMR measurements (difhzrence spin decoupling, NUE difference experiments, 211)COSY, 2D ROESY, 200 MHz, 300 MHz) [6-93 of peracetylsongarosaponin A (4) and peracetyl songarosaponin C (6) (Table 1). The following protons of 4 (CDCl,) were identified by spin decoupling [lo]: 6 1.16 H-6’ fucose, H-6”’ rhamnose, 63.64 H-5’ fucose, 63.63 H-5”” terminal glucose, 63.82 H-S” rhamnose, 63.51 H-5” glucose, 54.10 H-6“ A glucose, 64.76 H-6” B glucose, 63.92 H-4” glucose. NOES were especially notable at glucopyranoses and fucopyranoses between 1,3-bis axial protons. The NOE of H-5 at HICCshould be the largest due to the shortest distance. Thus, the H-l of the terminal glucose, the l&linked glucose and fucose were determined by irradiation of the appropriate H-5 protons. The ~-conflation of the terminal glucose, the 1,4-linked glucose and the fucose were deduced from NOE experiments (H-5 axial+H-1 axial) and coupling constants J,,, = 7.3-7.4 &. The

3395

Triterpene sapoains from Vmix2mi.m songaricum

3397

Table 1. Continued

2.08 2.11 2.12 2.13 2.15 2.18

s s s s (6H) s s

3

3.50 m

11

6.41

12

(dd, J 1*,12=lo.6Hz,J=&6Hz) 5.59 ,,=10.6)3[2, J=O.S ImE2) (dd, J 1%.

23A

4.26

23B

(d, JZU, 4.08

28A

(dct,J,,,, 4.20

28B

tdd,J 2s~. 3.98 (d, J 28A.

2.08 2.09 2.10 2.12 2.13 2.16 218

1.98 s 2.05 s 2.06 s 2.19 s 2.22 s 2.33 s 2.34 s 2.53 s $!6J 2A,j = 11.4 Hz, J28,3 = 4.8 Hz) 6.72 (dd, JI,. ia = 10.8 Hz, J= 28 Hz) 5.86 (d, J Il. x2= 10.8 Hz)

s s s s s s s

5.85

(d, J 11,Iz= 10.3 Hz) 5.36 (dd, J, 1, 12 = 10.3 Hz, J = 2.9 Hz}

4.98 23B=

11.6

Hz)

Cd,J23A, 4,56

238 =

11.6

Hz1

(dt J23,, 4.56

288 =

11.2

Hz)

2s~ =

11.2

Hz)

(d, J 28A, 4.47 (d, J 28A.

a)

238 =

11.7

23B=

f 1 Hz)

28B =

1 1 HZ)

28B=

lk3

Hz)

/I-D-fucopyranose 1’ 2 3 4’ 5’ 6

4.28 3.83 3.76 5.19 3.64 1.16

4.28 3.82 3.74 5.20 3.64 1.16

t, 1 2” 3” 4” 5” WA 6”B

4.72 4.84 5.11 3.92 3.51 4.10 4.76

5.09 5.52 5.61 4.27 3.46 5.10 4.62

4.69 4.91 5.07 3.87 3.48 4.01 4.72

11, 1 2 tl? I,, 3 ,,I 4 #I, 5 I,, 6

cl-L-rhamnopyranose 4.86 5.10 5.18 5.04 3.82 1.16

a-mhamnopyrmosz 5.22 5.81 5.92 5.78 4.38 1.51

/3-D-glucopyranose terminal 4.56 4.94 5.16 5.09 3.68 4.06 4.42

,rt, 1 2”” ,,,I 3 4,111 ,#,F 5 6’“‘A 6”“B

4.64 4.92 5.12 5.12 3.63 4.06 4.35

5.24 5.70 5.79 5.89 3.65 4.17 4.71

4.66 4.56 4.10 5.66 3.73 1.50 j-D-glucopyranose

l&linked

H-6”‘A H-6”‘B

4.63 4.92 5.12 5.12 3.63 4.05 4.33

Typical coupling constants in Hz for peracetylated sugars: /3-D-fucopyranosez J,, z = 7.3, J,, 3 =9.7, J,, 4 = 3.3, J,, 5 = 1.0, J,, 6 = 6.2; fl-mgfucopyranose: J,, z=7.4, J,,,=9.0, J,,,=9.5, J4, $=9.5, J,, sa=2.8, J,, 6B= 12.6; a-L-rhamnopyranose: J,,2=2.0, Ja, 3=3.2, J3,4=9,9, Jq, 5 =9.9, J,, 6=6.2.

K. SEIFERTet al.

coupling constant Jr,2 = 2.0 Hz indicated the a-configuration of the L-rhamnopyranose. The expected value for the /I-configuration is Jr,, = 1.1 Hz [1X]. A further condition for the dissemination of alternative sugar chain sequences in 1 was the identi~cation of the fucose protons H-2’ and H-3’. This was possible by difference decoupling of the anomeric fucose proton and by analysis of a high temperature spectrum (333 K) which is better resolved. The fucose signals H-2’ and H-3’ were detected due to a small shift of the H-5”’ rhamnose. In two further experiments the NOES between H-l”” terminal glucose and H-2’ fucose and H-l” 1,4-linked glucose and H-3’ fucose were found. According to the data the sequence of sugar chain in 1 is probable, but model considerations show that conformations can exist in which H-l”” terminal glucose is spatially near H-3’ fucose and H-l” 1,4-linked glucose spatially near H-2‘ fucose. Thus, further experiments should be carried out for the detection of long-range interactions between H-2’, H-3’ fucose and H- 1”” terminal glucose, H- 1” 1,4-linked glucose. The assignment of sugar protons was also possible using 20 COSY and 2D ROESY. 2D ROESY (300 MHz, CsDb) of peracetylsongarosaponin A (4) showed the following transfers of magnetization: H- 1’ fucose-H-3 aglycone, H-2’ fucose-H- 1”” terminal glucose, H-3’ fucose-H- 1” 1,4-linked glucose, H-4” 1,4-linked glucoseH-l”’ rhamnose. Therefore, the sequence of sugar chain of songarosaponin A and B, is as in structures 1 and 2. The lH NMR spectrum of peracetylsongarosaponin C (6) (CDCI,, 300 MHz) showed the signals of a terminal glucose instead of the rhamnose protons of peracetylsongarosaponin A (4). The chemical shifts and coupling constants of the protons of fucose, 1,4-linked glucose and terminal glucose of compounds 4 and 6 were in accord. The following Overhauser effects were determined by means of 2D ROESY (300 MHz): H-l’ fucose-H-3 aglycone, H-2’ fucose-H-l“” terminal glucose, H-3’ fucose-H1” 1,4-linked glucose, H-4” 1,4-linked glucose-H-l”’ terminal glucose. The ’ 3C NMR spectral data of the aglycones of songarosaponin A (1) and songarosaponin C (3) were in good agreement with the 13C NMR data of triterpene A (olea11,13-diene-3#I,23,2&triol) [33 and l&dehydrosaikogenin G (13fl,28epoxyolea- 11-ene-3/3,23-diol) [ 121 (Table 2). The glycosylation in position C-3 of compounds 1,2,3 and 4 leads to a C-3 downfield shift in comparison with triterpene A [33 and 16-dehydrosaikog~in G [16-J. The chemical shift of S 87.3 for C-13 of songarosaponin B (2) confirmed the presence of a hydroxyl group in this position [7]_ The shift value of C-28 of songarosaponin C (S 77.0) was shifted downfield in comparison with C-28 of songarosaponin B (663.0) and was in good agreement with C-28 of l&dehydrosaikogenin G (S 77.1) [12]. The 13C NMR chemical shifts of songarosaponin B (2) were assigned by comparison with the 13C NMR data of verbascosaponin (3-O-( r~-L-rhamnopyranosyl-( l-+4)-@D-glucopyranosyl-( l-+4)]-[P-D-glucopyranosyl-f l--+2)3/3-r>-fucopyranosylj-olea-1 I-ene-3&13,23,28-tetrol) f33, which is different from 2 in the points of attachment at fucose (1,24-linked), The 13C NMR data of tridecaacetylsongarosaponin A (4) were assigned by means of a SFORD spectrum and comparison with the r3C NMR chemical shifts of triterpene A [3] and the appropriate acetylated sugars [13, 14-j. The reduced coupling con-

Table 2. t 3C NMR spectral data of the triterpenoid moieties of compounds l-4 1

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

(CD,OD) 39.6 26.9 85.0 44.9 * 17.4 32.4 41.4 56.2 36.5 127.7 126.9 138.1 43.9 33.3 25.3 41.9 136.5 37.8 33.5 34.2 30.2 64.2 13.2 18.4 19.4 19.4 65.1 34.2 24.5

2 (pyridine-d,)

3 (pyridine-d,)

4 (CDCI,)

38.6 25.8 84.7 43.7 47.8 17.6 31.5 41.6 53.6 36.2 131.9 132.6 84.8 44.0 25.8 25.8 41.9 51.4 37.3 31.6 35.0 31.0 64.5 12.6 18.6 19.5 19.7 63.0 33.5 23.5

38.6 25.6 84.7 43.8 47.8 17.6 31.5 41-6 53.7 36.2 132.0 131.6 84.8 44.1 25.8 26.0 41.9 51.4 37.3 31.7 35.0 31.0 64.5 12.7 18.6 19s 19.8 77.0 33.6 23.5

38.2a 25.2b 83.6 42.0’ 47.7 18.0 32.2 40.4 54.4 36.6 126.6 125.4 137.1 38.0 33.1 24.3b 42.2” 133.2 37.8” 32.9 35.0 30.1 65.Sd 12.5 18.3 16.7 65.9d 32.2 24.3

The assignment of the methylene carbons of l-3 was done by means of reference compounds [3, 12-j. “-dAssignments may be reversed. * Superimposed with solvent. jSuperimposed with acetyl moieties.

slants (‘J L.)c,n) of the SFORD spectrum are correlated with the ‘H NMR chemical shifts of the corresponding sugar protons.

General. Mps: corr. ‘H NMR spectra were recorded at 80,200 and 300 MHz. 13C NMR were obtained at 50 and 75 MHz. The positive (10-16 eV) and negative (2-4 eV) ion mass spectra were recorded on an electron attachment mass spectrograph from the Research Institute ‘Manfred von Ardenne’, Dresden (unheated ion source, inlet temperature 90-120”, electron current 10 mA, accelerating voltage 40 kV). The matnx for the FAB-MS was glycerin, A gas chromatograph with FID was used for GC. CC was carried out on silica gel 40 (0.063-0.2 mm). TLC on silica gel sheets (0.3 mm, HF,,) and prep. TLC on silica gcelsheets (1 mm, PF,,,). TLC colour reactions “triterpcne reagent’ (1% soln of vanillin in 50% H,PO,) and ‘sugar reagent’ (4% ethanolic aniline_4%ethanolic diphenylamine-H,P0,, 5 : 5 : 1). isolation c;rfsaponins. Pfants of V. songaricum Schrenk were cultivated from seeds in the garden of the fnstitute for Plant Biochemistry, Halle (a voucher specimen of V. songaricum is deposited in the herbarium of the Institute). Dried powdered

3399

3400

K. SEIFERT et al.

2, Breton, J. L. and Gonzalez, A. G. (1963) J, Chem. Sec. 1401. 3. Tschesche, R., Sepulveda, S. and Braun, T. M. (1980) Gem. Ber. 113, 1754. 4, Bjiimdal, H., Hellerqvist, C, C., Lindberg, B. and Svenssan, S. (1970) Angew. Chem. 82,646. 5. Lanngren, J. and Pilotti, A. (1971) Aetu Chem. Scad. W, 1144,

6. Massiot, G., Lavaud, C., Le Men-Olivier, L., Van Binst, G., Miller, S. P. F. and Fales, II. M. (1988) J, Gem. Sot. Perkin Z-FansI 3071. 7. Massiot, G., Lavaud, C., Guillaume, D., Le Men-Olivier, L. and Van Binst, G. (1986) J. Chem, Sot., Gem. Commun. 1485.

8. Dabrowski, J, Dabrowski, U, Hanfland, P., Kordowiez, M. and Hall, W. E. (1986) Magn. Reson. Chem. 24,59. 9. Waltho, J. P., Williams, D. H., Mahato, S. B., Pal, B. C. and

Bama, C. J. (1986) J. Gem. Sot. Perkin Trans I 1527. 10. Sanders, J. K. M. and Mersch, J. D. (1986) Progress in Nuclear Magnetic Resonance Spectroscopy Vol. 15 (Emsley, J. W., Feeney, J. and SutclifSe, L. H,, eds), p. 353. Pergamon Press, Oxford. 11. Late, C, Nguyen Phuoc Du, A. M., Winternitz, F., Wylde, R. and Pratviel-Sosa, F. (1978) Carboh. Res. 67,91, 12. Tori, K., Yoshimura, Y., Seo, S., Sakurawi, K., Tomita, Y. and Ishii, H. (1976) Tetrahedron Letters 46, 4163. 13. Bock, K., Pedersen, C. and Pedersen, H. (1984) A& Carbohr. Chem. 42,193.

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