- Email: [email protected]
Saponins from Crossopteryx febrifuga
Phytochemistry, Vol. 29, No. 8, pp. 2629-2635, 1990. Printedin Great Britain.
0
SAPONINS FROM CROSSOPTERYX PIERLUIGI GARIBOLDI,
LUISELLA VERorrA*t
0031-9422/90 $3.00+0.00 1990 PergamonPress plc
FEBRIFUGA
and
BRUNO GABETTA $
Dipartimento
di Scienze Chimiche, Universitl di Camerino, Via S. Agostino 1,62032 Camerino, Italy; TDipartimento di Chimica Organica e Industriale, Universita di Milano, Via Venezian 21,20133 Milano, Italy; $ Laboratori Ricerca e Sviluppo, Invemi della Beffa, Via Ripamonti 99, 20141 Milano, Italy (Received 11 December
1989)
Key Word Index-Crossopteryx febrijiiga; Rubiaceae; roots; bisdesmosidic saponins; 28,3/?,6/3,16a,23_pentahydroxyolean-12-en-28-oic acid glycosides; 2D NMR experiments; antiedemic, analgesic and mucolitic activities.
Abstract-Two bisdesmosidic saponins from the roots of Crossopteryx febrijiiga were isolated by means of reversed phase HPLC. They were characterized on the basis of chemical and spectral data as 3-O-/&D-glucopyranosyl2/3,3fi,6/?,16u,23-pentahydroxyolean-12-en-28-oic acid 28-O-[a-L-rhamnopyranosyl( 1+3)] [b-D-XylOpyranOsyl(1 -t4)] [a-L-rhamnopyranosyl( l-+2)$-L-arabinopyranoside and 3-0-[/?-D-apiofuranosyl( 1+3)] B-D-glucopyranosyl-2B,3B,6B,16cr,23-pentahydroxyolean-l2-en-28-oic acid 28-O-[cc+rhamnopyranosy1(1+3)] [j?-D-XylOpyranOsyl(l-43 [a-L-rhamnopyranosyl( 1+2)]a-L-arabinopyranoside. 2D NMR experiments were useful in providing complete information on the structure and geometry of the new sapogenin present.
INTRODUCTION Crossopteryx febrifuga (Rubiaceae) is a shrub, occurring in dry woodland on sandy soils of Southern Africa. Parts of the tree provide a remedy for fever, the specific name referring to this use [l]. In this paper we wish to report the isolation and the structure of two bisdesmosidic saponins from the plant. In laboratory testings they showed antiedemic, analgesic and mucolitic activities [Z]. RESULTS AND DISCUSSION
A methanolic extract of the roots was purified by complexation with cholesterol, followed by column chromatography on silica gel. A single fraction was subjected to reversed phase HPLC affording two products, 1 and 2. Compound 1 showed a molecular ion at m/z 1238 [Mlin the FAB mass spectrum, and fragments at m/z 1092 [M -146]-, 1076 [M-162]-, 681 [M-1-556]and 519 [681- 162]-. Treatment of 1 with sodium methoxide and subsequent purification on silica gel afforded a product (3) showing molecular ion at m/z 695 [M -H] - and a fragment at m/z 533 [M-H162]-, indicating the presence of a hexose unit in the molecule. Compound 2 showed a molecular ion at m/z 1370 [Ml-, fragments at m/z 1238 [M-132]-, 1224 [M -146]-, 1076 [1238- 162]-, 814 [M-556]-, 681 [814 -132-l]-, 519 [681-162-J-. Treatment of 2 with sodium methoxide afforded a product (4) showing molecular ion at m/z 827 [M - l] - and a fragment at m/z 695 [827 - 132]-. The enzymatic hydrolysis with /3-glucosidase from Helix pomatia [3] of the original mixture of 1 and 2 gave, together with unreacted starting material, a compound 5, showing a molecular ion at m/z 1076 [Mland a fragment at m/z 5 19 [M - 556 - l] -. The unreacted product showed the same composition as the starting
mixture 1 and 2. From these considerations 1 and 2 are bidesmosidic saponins, carrying the same ester glycosidic moiety; 2 weighs 132 mass units more than 1, that is a pentose unit. These data are confirmed by the corresponding ‘HNMR spectra (500 MHz, pyridine-d,) 1 possesses five anomeric signals (66.43, d, J = 3 Hz; 6.15, d, J=ZHz; 5.62, d, J-2Hz; 5.12, d, J=IHz; 5.06, d, J =8 Hz) while 2 shows six anomeric signals (66.43, d, J =3 Hz; 6.22, d, J=4 Hz; 6.16, d, J=2Hz; 5.64, d, J=2 Hz; 5.10, d, J=8 Hz; 5.06, d, J=8 Hz). The treatment of the original mixture (1 + 2) with sodium methoxide gave two products (3+4) in the ratio 1: 1. The treatment of the latter mixture with HCl-methanol,
*Author to whom correspondence should be addressed. 2629
bH bH
1
R’=
2
R’=
H
d
= CHIOH
R’ = CH,OH
P. GARIBoLDI et al.
2630
HO
ti&2Me”o,-d: OH HO
HO
OH
OH
4
3 R = CHIOH
0 R2 = CH20H
R' = OH 6;)'; on OH
HO R = CH,OH
6 R = CH>OH 5
OAc
Me0 OMe
CH,OAc
C,,Hd’4
Meo@$ Me
CI~HIOO
00 OMe
Me0
OMe
-3H,O
7
followed by silylation [4], permitted the identification of D-ghCOSC and D-apiose in the ratio 2: 1 by capillary GC and comparison with pure standards obtained by methanalysis, followed by silylation, of apiin. This fact confirmed the hypothesis of bidesmosidic saponins, 1 carrying an 0-D-glucosyl unit, and 2 with 0-~glucosyl, O-Dapiosyl units. The same mixture (3 + 4) was submitted to enzymatic hydrolysis with /?-glucosidase from H. pomatia, affording, as main compound, the aglycone methyl ester (6), of M, 534 (EIMS at 20 eV), suggesting a molecular formula C3rHS007. The two fragments at m/z 260 and 201 are diagnostic for a RDA fragmentation, followed by loss of water, leaving four hydroxyl groups in A and B rings and one hydroxyl group in D or E rings. The acid hydrolysis of (3+4) gave a mixture of three products, among which the aglycone methyl ester (6) and its dehydrated product can be recognized.
1 1
Cl 1H1601 -H*O
C,,HzaOz m/z1260
m/z 201
The structure of (6) was determined as follows. Molecular weight and mass fragmentations suggested a pentacyclic triterpenoid with a A I2 double bond and five hydroxyl groups. The ‘H decoupled 13CNMR together with DEPT experiments (Tables 1 and 3) confirmed such a hypothesis and established the presence of four secondary alcohols, one primary alcohol, one carbomethoxy and six methyl groups. Analysis of the ‘HNMR spectrum (300 MHz; pyridine-d,) (Table 3) displayed six singlets for the methyl groups and allowed the assignment and relative stereochemistry of the two hydroxyl groups in position 2 and 3, while the primary alcohol should be connected to a quaternary carbon (AB system). A double quantum filtered ‘H-‘H 2D correlation spectrum (DQFCOSY) [S] provided assignments for all ‘H chemical shifts while most of the coupling constants were extracted from the high resolution 1D spectrum. Some
Saponins from Crossopteryx febrijiga
2631
Table 1. 13C NMR chemical shifts of aglycone moieties in pyridine-d, C
1
2
3
4
6
5
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 21 28 29 30
41.0
47.4 70.4 83.4 44.2 49.0 67.6 41.7 39.8 48.5 37.1 24.5 *
47.2 70.8 83.4 44.1 48.9 61.6 41.3 39.6 48.3 37.1 24.3 123.4 144.1 42.8 36.1 74.5 49.3 41.6 46.6 31.1 36.0 32.8 65.3 17.0 19.2 18.7 26.6 178.2 33.4 24.8 52.1
46.8 10.7 83.0 43.8 48.5 67.3 41.0 39.2 48.0 36.8 23.9 *
47.3 71.7 12.9 43.4 49.0 67.5 41.0 39.2 48.0 37.0 24.0 123.1 143.8 42.5 35.8 14.2 49.0 41.2 46.9 30.8 35.8 32.4 66.9 16.0 18.9 18.4 21.2 177.8 33.1 24.5 51.7
47.6 71.9 73.4 43.5 49.3 67.9 41.4 39.6 48.3 31.2 24.2 *
COOMe
71.0 83.1 44.0 48.8 67.6 41.2 39.5 48.2 31.0 24.0 123.0 143.5 42.1 36.0 74.0 49.8 41.2 46.6 31.1 35.9 32.0 65.3 17.8 19.1 18.9 21.2 178.0 33.2 25.0
143.8 43.0 36.4 74.4 50.0 41.7 46.9 31.2 36.3 32.3 65.4 l?.l 19.3 18.9 21.1 176.4 33.6 25.2
143.8 42.4 35.8 74.5 48.9 41.2 46.3 30.8 35.1 32.4 65.0 16.7 18.8 18.3 21.2 177.8 33.1 24.5 51.7
143.8 42.8 36.2 14.1 49.1 41.4 41.2 31.0 35.9 32.1 61.3 18.4 19.6 19.2 21.4 176.1 33.3 24.9
*These signals are hidden by pyridine-d, resonances.
ambiguities in ‘H assignments were left in the aliphatic part of the spectra; they were solved after analysis of a 13C-‘H 2D correlation spectrum; as all carbon resonances were well resolved, the corresponding inphase f,traces (‘H) confirmed ‘H chemical shifts and, when possible, allowed coupling constant evaluation. A further 13C-lH 2D correlation spectrum was run, but optimized for long-range couplings (‘JW and 3J& (COLOC experiment [6]) which established the connections between directly bonded carbons and hydrogens. Results of this experiment are summarized in Table 4; all quaternary carbons could be correctly assigned and located within the protonated frame of the molecule. Due to the well known problem involved in NOE measurements at medium field strengths for mediumsized molecules, a 2D NOE in the ‘rotating frame’ (ROESY) [7] was performed. The most relevant results from this experiment are listed in Table 5. Assignment of ‘H and 13C resonances due to the six methyl groups could be reconfirmed together with their relative stereochemistry as well as -CH,OH equatorial geometry at C4. Most of the stereochemistry and conformation of the molecule were straightforwardly deducible by the coupling patterns of the ‘H resonances resulting in a regular all-trans chair conformation for rings A-D. The strong deshielding effect induced by the carbomethoxy group on
H-18 (6 3.43) is diagnostic for a cis fusion between rings D and E, while a strong NOE between C-30 methyl and H18 suggests a chair conformation to be predominant also for ring E. All the NMR data collected for compound 6 are self-consistent and show that it has a Al2 oleanane skeleton with the depicted new pentahydroxy derivatization. The ‘H and ’ 3C NMR spectra of 3, supported by 2DCOSY and DEPT experiments, fully define its structure and the position of linkage of the glucose unit. The anomeric proton resonance (doublet at 65.24 with J = 8 Hz) reflects a p-glucosidic linkage, being the 3-OH of compound 6 involved in the linkage as confirmed by the 13C NMR glycosylation shifts. The ‘H and 13CNMR spectra of 4, supported by DEPT experiments, and 13C--lH 2D correlation spectrum, confirmed the presence of two anomeric protons (6 5.15, d, J = 8.0 Hz, corresponding to carbon at 6 105.4 and 6.20, d, J= 3.0 Hz, corresponding to carbon at S 111.2). Comparison of ’ 3C NMR signals of 4 with those of 3 and literature data for linked D-apiose [S] suggested the presence of B-D-apiOfUranOSyl(I--+ 3)-fl-D-glucopyranosyl moiety. Once the 3-0-glycosyl substituents had been defined, it remained to investigate the ester bonded glycosidic moiety. Compound 5 was treated with HCl-methanol, followed by silylation [4], identifying L-
Saponins from Crossopteryx febrijuga Table 4. Cross-peaks from the COLOC experiment C
C-H connectivities
1 4* 5 7 8* 9 10 11 14* 15 17* 18 19 20* 21 22 24 25 26 27 29 30
H-25 H-2, H-5, H-23, H-24 H-7Et, H-25 H-26 H-6, H-9, H-26, H-27 H-lAt, H-7E, H-12, H-25, H-26 H-2, H-5, H-6, H-25 H-9, H-12 H-9, H-12, H-16, H-18, H-26, H-27 H-27 H-15E, H-21A, H-21E, H-22A, H-22E H-12, H-16, H-19A, H-22A H-18, H-29, H-30 H-19A, H-22A, H-29, H-30 H-29, H-30 H-21A H-3, H-5 H-lA, H-lE, H-5 H-7A, H-7E, H-9 H-15A, H-15E H-19A, H-30 H-19A, H-21A, H-29
Table 5. Cross-peaks from the ROESY experiment Protons connected by NOE
1At
H-2Et H-lA, H-3A H-2E, H-5A H-3A, H-6E, H-7A, H-9A H-5A, H-23’ (upfield) H-5A, H-27 H-5A, H-27 H-25 H-18E, (H-19A) H-26 H-27 H-22E H-12, H-19E, H-30 H-27 H-18E H-16E H-6E H-24 H-23” (downfield), H-25 H-l lA, H-24, H-26 H-l 5A, H-25 H-7A, H-9A, H-15E, H-19A H-30 H-18A, H-29
2E 3A 5A 6E IA 9A 11A 12 15A 15E 16E 18E 19A 19E 22E 23’ (upfield) 23” (downfield) 24 25 26 27 29 30
[666 -terminal permethylrhamnose] +, 420 [580-dimethylhexose unit] +, 246 [dimethyl hexalditol diacetate -HI+ and 189 [C,H, TO,]+. Methanolysis of 7 confirmed the presence of methyl 2,3,4-tri-O-methyl-L-rhamnopyranoside, methyl 2,4-di-0-methyl-D-xylopyranoside and methyl 2,3-di-0-methyl+rhamnopyranoside thus unambiguously showing that the two saponins have structures 1 and 2. EXPERIMENTAL
* Quaternary carbons. tA = axial, E = equatorial.
H
2633
l
*NOE’s due to geminal protons have been omitted. Only cross-peaks with intensity either comparable or stronger than those displayed by geminal protons have been listed. tA = axial, E = equatorial.
Plant material was collected in Mozambique in 1986 and authenticated by Dr U. Boni (Inverni della Beffa). A voucher specimen is available at the Department of Pharmacognosy of Inverni della Befla (Milan). Mps: uncorr. Precoated Kieselgel60 F 254and RP 18 (Merck) were used for TLC. Spots were detected by spraying with H,S04-MeOH (1:9) followed by heating. Kieselgel 60 (7&230 mesh, Merck) was employed for CC. Reversed phase HPLC was carried out on a Varian 5000 liquid chromatograph, equipped with a Varian RI-3 refractive index. Analytical separation was obtained on a PBondapack C,, column (1Om 4.6 x 150 mm, Waters), eluting with MeCN-H,O (1:3) at a flow rate of 1 mlmin-I. Preparative separation was carried out on a Viosfer ODS column (10 pm, 1 x 25 cm, Violet), eluting with MeCN-H,O (1: 3), at a flow rate of 2.2 ml min-‘. A Hibar pre-column (4 x 30 mm, Merck), filled with Perisorb RP 8 (3w pm, Merck), was used. Before injection the samples, dissolved in H,O, were filtered on Millex-HV filters (0.45 pm, Millipore). MPLC was carried out with a Buchi 681 pump (Buchi) and a Buchi B685 column (2.6 x 30 cm) filled with LiChroprep RP 18 (4&63pm, Merck). NMR spectra were recorded on 200, 500 and 300 MHz spectrometers; 2D experiments were run at 300 MHz for ‘H and 75.4 MHz for 13C. DQFCOSY and ROESY spectra were accumulated and processed in the phase-sensitive mode. 1K data memory was used in the F2 dimension, and 256 t, increments, zero-filled to 1024 before Fourier transformation. The mixing time for ROESY was 0.8 sec. Two 2D heterocorrelated spectra via l.Ic_” were run; one for oxygenated carbons (60-80 ppm) and one for aliphatic carbons (15-55 ppm). 1K and 2K data memory were used in the F2 dimension and 128 (zero-filled to 256) and 512 (zero-filled to 1024) t, increments respectively. The COLOC experiment utilized 2K in the F2 dimension and 128 t, increments (zero-filled to 512);only the aliphatic carbons were observed (15-55 ppm) while all the ‘H spectrum was excited for polarization transfer. Delays were set to optimize polarization transfers due to a ‘J, 8 Hz, approximately the maximum expected value for couplings involving CH and CH, protons. Samples were dissolved in CDCl, or pyridine-d, and TMS was used as int. standard. Negative FABMS spectra were obtained on a VG 7070 mass spectrometer; samples were dissolved in a glycerol matrix and placed on a steel target prior to bombardment with Ar atoms of energy 7-8 kV. Sugars (silylated and methylated) were analysed on a WCOT CP SilS-CB fused silica capillary column (Chrompack, 25 m x 0.32 mm i.d., film thickness 0.11 pm, carrier H, 0.45 kgcmm2. Inj. temp. 290, det. temp. 300, prog. 150-280 at 4” min- ’ (silyl derivatives) and 8&250 at 4” min- ’ (methyl derivatives). Extraction and isolation. Thinly minced dried roots (2 kg) were exhaustively extd by percolation with hot MeOH (30 1).The ext was coned to 0.8 1, taken up with 50% MeOH (2.7 1) and left overnight at room temp. After filtration, the soln was added with cholesterol (270 g), refluxed for 5 hr with stirring, then left overnight at room temp. The solid material was filtered in uacuo, washed with 50% MeOH (0.5 I), dissolved in CHCl,-MeOH 1: 1 (4.7 I) and H,O added (1.7 1).The organic phase was sepd and the
2634
P.
GARIBALDI
aq. phase extd with CHCI, (2 x 1.7 1). The aq. phase was coned in uacuo to yield 22.5 g saponins. This residue was chromatographed on silica gel (1.3 kg), previously conditioned with CHCI,-MeOH-H,O (14: 6: 1) and eluted with the same mixture at a flow rate of 1 lhr-‘. Fractions (500 ml) were collected according to their composition, taken to dryness and afforded 12.8 g of a mixture showing a single spot on TLC (silica gel and RP 18), but consisting of two substances in the same ratio as shown by HPLC analysis. Compound l(86.5 mg) was obtained by prep. HPLC and pptd by EtOAc as crystals, mp 213-214” (dec), [a];’ -48.0” (MeOH; c 1.05). FABMS m/z (C58H94028): 1238 [M] -, 1092 [M - 146]-, 1076 CM-162]-, 681 [M-l-556]-, 519 [681-162]-. ‘H NMR (500 MHz, pyridine-d,): 66.43 (d, J =3 Hz, H-l Ara), 6.15 (d, J=2Hz, H-l Rha), 5.68 (t, J=3 Hz, H-12), 5.62 (d, J =2Hz,H-1 Rha),5.19(m,H-6),5.14(m,H-16),5.12(d,5=8Hz, H-l Glc), 5.06 (d, J=8 Hz, H-l Xyl), 4.90 (m, H-2), 4.72 (d, J =4Hz, H-3), 3.60(dd, J=14, 4Hz, H-18), 2.72 (t, J=!3 Hz, H19A), 2.20, 1.96, 1.73, 1.64, 1.14, 0.98 (s, -Me), 1.64, 1.63 (d, J = 7 Hz, -Me); ‘%Z NMR (75.4 MHz, pyridine-d,): see Tables 1 and 2. Compound 2 (57.7 mg) was obtained by prep. HPLC and pptd by EtOAc as needles, mp 221-222” (dec), [a];* -56.3” (MeOH; ~0.67). FABMS m/z (C,,H,,,O,,): 1370 [Ml-, 1238 [M -132]1224 [M-146]-, 1076 [1238-!62]-, 814 [M -556]-, 681 [814-132-l]-, 519 [681-162]-. ‘HNMR (500 MHz, pyridine-d,): 66.43 (d, J = 3 Hz, H-l Ara), 6.22 (d, J =4 Hz,H-I Api), 6.16(d, J=2 Hz, H-l Rha), 5.67(t, J=3 Hz, H12), 5.64(d, J=2 Hz, H-l Rha), 5.18(m, H-6), 5.13(m, H-16), 5.10 (d, J=8 HZ, H-l Glc), 5.06 (d, J=8 Hz, H-l Xyl), 4.90 (m, H-2), 4.72 (d, J=4 Hz, H-3), 3.60 (dd, J= 14, 4 Hz, H-18). 2.73 (t, J = 13 Hz, H-19A), 2.20, 1.95, 1.75, 1.63, 1.15, 0.97 (s, -Me), 1.64, 1.63 (d, J = 7 Hz, -Me); 13C NMR (52.4 MHz, pyridine-d,): see Tables 1 and 2. Transesterijcation of 1. To a solution of NaOMe-MeOH (SO mg Na were dissolved in 4 ml dry MeOH) 56 mg 1 in 2 ml dry MeOH were added. The solution was refluxed for 2 hr. then was neutralized HOAc, diluted with H,O, and extracted with nBuOH (2 x 15 ml). The crude residue was purified by CC on silica gel eluting with CHCl,-MeOH (3: 1) and afforded 27 mg of 3. Mp 21@212” (iso-Pr),O-EtOH (99:1), [ZIP NO (MeOH; c 1.07). FABMS (C,,H,,O,,): m/z 695 [M-H]-, 533 [M - 162 -HI-. ‘H NMR (300 MHz, pyridine-d,): 65.62 (t. J=3 Hz, H12), 5.24 (d, J=8 Hz, H-l Glc), 5.18 (m, H-6), 5.01 (m, H-16), 4.92 (m, H-2), 4.59 (d, J = 11 Hz, H-23’), 4.38 (d, J =4 Hz, H-3). 4.03 (d, J=ll Hz,H-23”),3.43(dd,J=14,4Hz,H-18),2.76(t,J=13Hz, H-19A), 2.27. 2.03, 1.77, 1.50, 1.07, 0.98 (s, -Me); 13CNMR (75.4 MHz, pyridine-d,): see Tables 1 and 2. Transesterifcation of 2. Compound 2 (43 mg) was treated with NaOMe-MeOH, in the same manner as 1. The crude residue was purified by CC on silica gel eluting with CHCl,-MeOH (3: 1) and afforded 15 mg of 4. Mp 225-230” (iso-Pr),O-EtOAc (3 : 21,Cali’ -21.6” (MeOH; ~0.66). FABMS (C42H68016): m/z 827[M-HI-,695 [M-132-H]-,‘HNMR(3OOMHz,pyridine-d,): 66.20(d, J=3 Hz, H-l Api), 5.60@, J= 3 Hz, H-12), 5.16 (m, H-6), 5.15 (d, J=8 Hz, H-l G!c),4.98(m, H-16),4.87(m, H-2), 4.52 (d, J=ll Hz, H-23’), 4.33 (d, J=4Hz, H-3), 4.02 (d, J = 11 Hz, H-23”), 3.42 (dd, J= 14, 4 Hz, H-18), 2.78 (t, J= 13 Hz. H-19A), 2.25 2.03, 1.78, 1.50, 1.08, 0.98 (s, -Me); 13CNMR (75.4 MHz, pyridine-d,): see Tables 1 and 2. Enzymatic hydrolysis of 1 and 2. The original mixture of 1 and 2 (3.0047 g) was hydrolysed with the hepatopancreatic juice of 40 snails (Helix pomatia) dil. with H,O (200 ml) and filtered. The soln was stirred at 35” for 20 hr; the mixt. was then extracted with n-BuOH (2 x 200 ml) to afford a residue which was purified by RP-MPLC on a C,, stationary phase, eluting with MeCN-H,O
et al. (1:3) (400 ml), MeCN-H,O (3:7) (600 ml) and MeCN-H,O (2: 3) (800 ml). Fractions were collected according to their composition and afforded 593 mg of 5 and 809 mg of unreacted sample (1+2) showing the same composition of the starting mixture (HPLC analyses). Compound 5 showed mp 231-232” (EtOAc), [E];’ -49.7’ (MeOH; (’ 1.02). FABMS (C,2H,,0,,) m/z: 1076 [Ml-, 519 [M-556-H]-. ‘HNMR (3GOMHz, pyridine-d,): 66.47 (d, J = 3 Hz, H-l Ara), 6.14 (d, J= 2 HZ, H-l Rha), 5.64(d, J=2 Hz, H-l RhaJ.5.61 (t, J=3 Hz,H-!2), 5.22(m, H-6), 5.15(m, H-16), 5.06(d, J=8 Hz, H-l Xyl),4.95(m,H-2),4.78 (d, J=4 Hz, H-3), 3.62 (dd, J= !4,4 Hz, H-18), 2.75 (t. J= 13 Hz, H-19A), 2.28, 2.02, 1.76, 1.66, 1.15, 0.97 (s, -Me), 1.67, 1.65 (d, J = 7 Hz, -Me). “C NMR (75.4 MHz. pyridine-d,): see Tables 1 and 2. Transesterification of the original mixture 1+2. The mixture 1+2 (2 g) was dissolved in 10 ml MeOH and added to a NaOMe-MeOH soln (1.3g Na in 100 ml MeOH). The mixture was refluxed with stirring for 1.30 hr, neutralized with HOAc dild with H,O and extd with n-BuOH (2 x 100 ml). The crude residue was purified by CC on silica gel eluting with CHCl,-MeOH (7:3) (500 ml) and afforded 583 mg of 3+4. Methanolysis and siIylation of 3 +4. The mixture 3+4 (30 mg) was treated with 0.5 M HCI-MeOH (2 ml) and refluxed under N, for 7 hr. The solvent was removed with a llow of N,. a soln of BSTFA-pyridine (1: 1)added (0.5 ml) and the mixture warmed at 60’ for 3 hr. This solution was directly analysed by capillary CC identifying glucose and apiose in the ratio 2: 1 by co-injection with appropriate standards. Enzymatic hydrolysis of3+4. The mixture 3+4 (87 mg) was hydrolysed with the hepatopancreatic juice of six snails (Helix pomatia) dild with H,O (10 ml) and filtered. The solution was stirred at 35” for 40 hr, the mixture was then extd with n-BuOH (2 x 10 ml) to afford a residue which was purified by CC on silica gel eluting with CHCl,-MeOH (9:l) (lOOmI) and CHCl,-MeOH (4: 1) (100 ml). Fractions were collected according to their composition and afforded 38 mg 6 and 14 mg of unreacted sample (3 +4). Compound 6 showed mp 255-256” (isoPr),O, [a];’ + 19.4’ (MeOH; c 1.00). ‘H and 13C NMR see Tables 1,3-5.EIMS(20eV)(C,,H,,O,~m/~534[M]’.260,201. (Found: C, 69.6; H, 9.2: 0, 21.2. C,,H,,O, requires: C. 69.7; H, 9.3; 0, 21.0%). Acid hydrolysis of 3+4. A soln of 3 +4 (90 mg) in 15 ml 2% H,SO, was refluxed for 10 hr. The soln was extd with EtOAc (3 x 20 ml) affording 68 mg of a mixture which was purified by CC on silica gel with increasing amounts of MeOH in CHCI,. Fractions were collected according to their composition and allowed to isolate 14 mg of dehydrated 6. 12 mg 6 and 27 mg of dehydrated 3 + 4. Methanolysis and silylation of 5. Compound 5 (14 mg) was treated with 0.5 M HCI-MeOH and refluxed under N, for 5 hr. The solvent was removed with a flow of N,, a solution of BSTFA-pyridine (1: 1) was added and the mixture was warmed at 60” for 3 hr. This soln was directly analysed by capillary CC identifying arabinose, xylose and rhamnose in the ratio 1 : I:2 by co-injection with appropriate standards. Permethylation and reduction of5 Compound 5 (107 mg) was dissolved in 3 ml DMSO, 350 mg t-NaOBu (Merck), 70 mg dry powdered NaOH and then 0.5 ml Me1 were added with stirring at room temp. After 3 hr the mixt. was poured into ice and extd with CHCI, (3 x 3 ml). The organic phase was washed with H,O and evapd to dryness. The crude residue (Sa) was directly treated with LiAlH, in THF (5 ml) and refluxed for 2 hr. A solution of NH,Cl was added dropwise; the mixture was filtered on celite in uacuo and the residue washed with H,O. The aq. phase was extd with Et,0 (1 x 5 ml) and CHCI, (3 x 5 ml). The CHCI, ext. (117 mg) was purified by CC on silica gel, eluting with
Saponins from Crossopteryx febrijiiga CHzClz-iso-PrOH (19: 1) (80 ml), CH,Cl,-iso-PrOH (93:7) (50 ml), CH,Cl,-iso-PrOH (9: 1) (50 ml). Fractions were collected according to their composition and afforded 58 mg 7a. Hydrolysis of7a. Compound 7a (4 mg) was treated with 0.5 M HCI-MeOH under N,, at reflux for 2 hr. Methyl 2,3,4-&i-Omethyl+rhamnopyranoside, methyl 2,4-di-O-methyl-D-xylopyranoside, methyl 2,3-di-O-methyl-L-rhamnopyranoside were identified by capillary co-GC with appropriate standards [13]. Acetylation of 7~. Compound 7~ (34 mg) was acetylated (A@-pyridine 1: 1)at room temp. overnight. The crude residue was purified by CC on silica gel eluting with petrol-EtOAc (3: 7) (40 ml) and afforded 23 mg 7. Oil, [a]ks - 57.2” (CHCI,; c 1.01). EIMS (20 eV) m/z 666 [M -2 x HOAc] +, 580, 420, 189
CW%@J+.
Hydrolysis of5a. Compound 5a (5 mg) was treated with 0.5 M HCI-MeOH under N,, reflux for 2 hr. Methyl 2,3,4-tri-O-methyl+rhamnopyranoside, methyl 2,4-di-O-methyl-D-xylopyranoside, methyl 2,3-di-O-methyl-L-rhamnopyranoside, methyl 3,4di-0-methyl+arabinopyranoside were identified by capillary co-GC with appropriate standards [13].
Acknowledgements-We are grateful to Istituto di Ricerche Farmacologiche M. Negri (Bergamo-Italy) for FABMS analyses. Part of this work was supported by Tecnofarmaci SpA. Financial assistance from the Minister0 della Pubblica Istruzione of Italy (40% and 60%) is gratefully acknowledged. REFERENCES 1. Palgrave, K. C. (1988) Trees of Southern Africa 5th Edn, p, 844. Struik, Cape Town.
2635
2. Foresta, P., Ghirardi, O., Gabetta, B. and Cristoni, A. (1986) (to Sigma Tau- Invemi della Beffa), Ital. Pat. 48208 A/86, l/7/1986. Chem. Abs. 109, (26): 236992~. 3. Canon& L., Orsini, F. and Pelizzoni, F. (1976) Gazz. Chim. Ital. 106, 889. 4. Bombardelli, E., Conti, M., Magistretti, M. J. and Martinelli, E. M. (1983) J. Chromatogr. 297, 593. 5. Piantini, U., Sorenson, 0. W. and Ernst, R. R. (1982) J. Am. Chem. Sot. 104,680. 6. Kessler, H., Griesinger, C., Zarbock, J. and Loosli, H. R. (1984) J. Magn. Reson. 57, 331.
7. Kessler, H., Griesinger, C., Kerssebaum, R., Wagner, K. and Ernst, R. R. (1987) J. Am. Chem. Sot. 109,607. 8. Ishii, H., Kitagawa, I., Matsushita, K, Shirakawa, K., Tori, K., Tozyo, T., Yoshikawa, M. and Yoshimura, Y. (1981) Tetrahedron Letters 22, 1529. 9. Ishii, H., Tori, K., Tozyo, T. and Yoshimura, Y. (1984) J.
Chem. Sot. Perkin Trans. 661. 10. Ishii, H., Tori, K., Tozyo, T. and Yoshimura, Y. (1978) Chem. Letters 719.
11. Kasai, R., Miyatoshi, M., Nie, R., Zhou, J., Matsumoto, K., Morita, T., Nishi, M., Miyahara, K. and Tanaka, 0. (1988) Phytochemistry 27, 1439. 12. Ciucanu, I. and Kerek, F. (1984) Carbohydr. Res. 131, 209. 13. Carpani, G., Orsini, F., Sisti, M. and Verotta, L. (1989) Gazz. Chim. Ital. 119, 463. 14. De Marco, A., Gariboldi, P., Molinari, H. and Verotta, L. (1990) Carbohydr. Res. (in press).
0
SAPONINS FROM CROSSOPTERYX PIERLUIGI GARIBOLDI,
LUISELLA VERorrA*t
0031-9422/90 $3.00+0.00 1990 PergamonPress plc
FEBRIFUGA
and
BRUNO GABETTA $
Dipartimento
di Scienze Chimiche, Universitl di Camerino, Via S. Agostino 1,62032 Camerino, Italy; TDipartimento di Chimica Organica e Industriale, Universita di Milano, Via Venezian 21,20133 Milano, Italy; $ Laboratori Ricerca e Sviluppo, Invemi della Beffa, Via Ripamonti 99, 20141 Milano, Italy (Received 11 December
1989)
Key Word Index-Crossopteryx febrijiiga; Rubiaceae; roots; bisdesmosidic saponins; 28,3/?,6/3,16a,23_pentahydroxyolean-12-en-28-oic acid glycosides; 2D NMR experiments; antiedemic, analgesic and mucolitic activities.
Abstract-Two bisdesmosidic saponins from the roots of Crossopteryx febrijiiga were isolated by means of reversed phase HPLC. They were characterized on the basis of chemical and spectral data as 3-O-/&D-glucopyranosyl2/3,3fi,6/?,16u,23-pentahydroxyolean-12-en-28-oic acid 28-O-[a-L-rhamnopyranosyl( 1+3)] [b-D-XylOpyranOsyl(1 -t4)] [a-L-rhamnopyranosyl( l-+2)$-L-arabinopyranoside and 3-0-[/?-D-apiofuranosyl( 1+3)] B-D-glucopyranosyl-2B,3B,6B,16cr,23-pentahydroxyolean-l2-en-28-oic acid 28-O-[cc+rhamnopyranosy1(1+3)] [j?-D-XylOpyranOsyl(l-43 [a-L-rhamnopyranosyl( 1+2)]a-L-arabinopyranoside. 2D NMR experiments were useful in providing complete information on the structure and geometry of the new sapogenin present.
INTRODUCTION Crossopteryx febrifuga (Rubiaceae) is a shrub, occurring in dry woodland on sandy soils of Southern Africa. Parts of the tree provide a remedy for fever, the specific name referring to this use [l]. In this paper we wish to report the isolation and the structure of two bisdesmosidic saponins from the plant. In laboratory testings they showed antiedemic, analgesic and mucolitic activities [Z]. RESULTS AND DISCUSSION
A methanolic extract of the roots was purified by complexation with cholesterol, followed by column chromatography on silica gel. A single fraction was subjected to reversed phase HPLC affording two products, 1 and 2. Compound 1 showed a molecular ion at m/z 1238 [Mlin the FAB mass spectrum, and fragments at m/z 1092 [M -146]-, 1076 [M-162]-, 681 [M-1-556]and 519 [681- 162]-. Treatment of 1 with sodium methoxide and subsequent purification on silica gel afforded a product (3) showing molecular ion at m/z 695 [M -H] - and a fragment at m/z 533 [M-H162]-, indicating the presence of a hexose unit in the molecule. Compound 2 showed a molecular ion at m/z 1370 [Ml-, fragments at m/z 1238 [M-132]-, 1224 [M -146]-, 1076 [1238- 162]-, 814 [M-556]-, 681 [814 -132-l]-, 519 [681-162-J-. Treatment of 2 with sodium methoxide afforded a product (4) showing molecular ion at m/z 827 [M - l] - and a fragment at m/z 695 [827 - 132]-. The enzymatic hydrolysis with /3-glucosidase from Helix pomatia [3] of the original mixture of 1 and 2 gave, together with unreacted starting material, a compound 5, showing a molecular ion at m/z 1076 [Mland a fragment at m/z 5 19 [M - 556 - l] -. The unreacted product showed the same composition as the starting
mixture 1 and 2. From these considerations 1 and 2 are bidesmosidic saponins, carrying the same ester glycosidic moiety; 2 weighs 132 mass units more than 1, that is a pentose unit. These data are confirmed by the corresponding ‘HNMR spectra (500 MHz, pyridine-d,) 1 possesses five anomeric signals (66.43, d, J = 3 Hz; 6.15, d, J=ZHz; 5.62, d, J-2Hz; 5.12, d, J=IHz; 5.06, d, J =8 Hz) while 2 shows six anomeric signals (66.43, d, J =3 Hz; 6.22, d, J=4 Hz; 6.16, d, J=2Hz; 5.64, d, J=2 Hz; 5.10, d, J=8 Hz; 5.06, d, J=8 Hz). The treatment of the original mixture (1 + 2) with sodium methoxide gave two products (3+4) in the ratio 1: 1. The treatment of the latter mixture with HCl-methanol,
*Author to whom correspondence should be addressed. 2629
bH bH
1
R’=
2
R’=
H
d
= CHIOH
R’ = CH,OH
P. GARIBoLDI et al.
2630
HO
ti&2Me”o,-d: OH HO
HO
OH
OH
4
3 R = CHIOH
0 R2 = CH20H
R' = OH 6;)'; on OH
HO R = CH,OH
6 R = CH>OH 5
OAc
Me0 OMe
CH,OAc
C,,Hd’4
Meo@$ Me
CI~HIOO
00 OMe
Me0
OMe
-3H,O
7
followed by silylation [4], permitted the identification of D-ghCOSC and D-apiose in the ratio 2: 1 by capillary GC and comparison with pure standards obtained by methanalysis, followed by silylation, of apiin. This fact confirmed the hypothesis of bidesmosidic saponins, 1 carrying an 0-D-glucosyl unit, and 2 with 0-~glucosyl, O-Dapiosyl units. The same mixture (3 + 4) was submitted to enzymatic hydrolysis with /?-glucosidase from H. pomatia, affording, as main compound, the aglycone methyl ester (6), of M, 534 (EIMS at 20 eV), suggesting a molecular formula C3rHS007. The two fragments at m/z 260 and 201 are diagnostic for a RDA fragmentation, followed by loss of water, leaving four hydroxyl groups in A and B rings and one hydroxyl group in D or E rings. The acid hydrolysis of (3+4) gave a mixture of three products, among which the aglycone methyl ester (6) and its dehydrated product can be recognized.
1 1
Cl 1H1601 -H*O
C,,HzaOz m/z1260
m/z 201
The structure of (6) was determined as follows. Molecular weight and mass fragmentations suggested a pentacyclic triterpenoid with a A I2 double bond and five hydroxyl groups. The ‘H decoupled 13CNMR together with DEPT experiments (Tables 1 and 3) confirmed such a hypothesis and established the presence of four secondary alcohols, one primary alcohol, one carbomethoxy and six methyl groups. Analysis of the ‘HNMR spectrum (300 MHz; pyridine-d,) (Table 3) displayed six singlets for the methyl groups and allowed the assignment and relative stereochemistry of the two hydroxyl groups in position 2 and 3, while the primary alcohol should be connected to a quaternary carbon (AB system). A double quantum filtered ‘H-‘H 2D correlation spectrum (DQFCOSY) [S] provided assignments for all ‘H chemical shifts while most of the coupling constants were extracted from the high resolution 1D spectrum. Some
Saponins from Crossopteryx febrijiga
2631
Table 1. 13C NMR chemical shifts of aglycone moieties in pyridine-d, C
1
2
3
4
6
5
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 21 28 29 30
41.0
47.4 70.4 83.4 44.2 49.0 67.6 41.7 39.8 48.5 37.1 24.5 *
47.2 70.8 83.4 44.1 48.9 61.6 41.3 39.6 48.3 37.1 24.3 123.4 144.1 42.8 36.1 74.5 49.3 41.6 46.6 31.1 36.0 32.8 65.3 17.0 19.2 18.7 26.6 178.2 33.4 24.8 52.1
46.8 10.7 83.0 43.8 48.5 67.3 41.0 39.2 48.0 36.8 23.9 *
47.3 71.7 12.9 43.4 49.0 67.5 41.0 39.2 48.0 37.0 24.0 123.1 143.8 42.5 35.8 14.2 49.0 41.2 46.9 30.8 35.8 32.4 66.9 16.0 18.9 18.4 21.2 177.8 33.1 24.5 51.7
47.6 71.9 73.4 43.5 49.3 67.9 41.4 39.6 48.3 31.2 24.2 *
COOMe
71.0 83.1 44.0 48.8 67.6 41.2 39.5 48.2 31.0 24.0 123.0 143.5 42.1 36.0 74.0 49.8 41.2 46.6 31.1 35.9 32.0 65.3 17.8 19.1 18.9 21.2 178.0 33.2 25.0
143.8 43.0 36.4 74.4 50.0 41.7 46.9 31.2 36.3 32.3 65.4 l?.l 19.3 18.9 21.1 176.4 33.6 25.2
143.8 42.4 35.8 74.5 48.9 41.2 46.3 30.8 35.1 32.4 65.0 16.7 18.8 18.3 21.2 177.8 33.1 24.5 51.7
143.8 42.8 36.2 14.1 49.1 41.4 41.2 31.0 35.9 32.1 61.3 18.4 19.6 19.2 21.4 176.1 33.3 24.9
*These signals are hidden by pyridine-d, resonances.
ambiguities in ‘H assignments were left in the aliphatic part of the spectra; they were solved after analysis of a 13C-‘H 2D correlation spectrum; as all carbon resonances were well resolved, the corresponding inphase f,traces (‘H) confirmed ‘H chemical shifts and, when possible, allowed coupling constant evaluation. A further 13C-lH 2D correlation spectrum was run, but optimized for long-range couplings (‘JW and 3J& (COLOC experiment [6]) which established the connections between directly bonded carbons and hydrogens. Results of this experiment are summarized in Table 4; all quaternary carbons could be correctly assigned and located within the protonated frame of the molecule. Due to the well known problem involved in NOE measurements at medium field strengths for mediumsized molecules, a 2D NOE in the ‘rotating frame’ (ROESY) [7] was performed. The most relevant results from this experiment are listed in Table 5. Assignment of ‘H and 13C resonances due to the six methyl groups could be reconfirmed together with their relative stereochemistry as well as -CH,OH equatorial geometry at C4. Most of the stereochemistry and conformation of the molecule were straightforwardly deducible by the coupling patterns of the ‘H resonances resulting in a regular all-trans chair conformation for rings A-D. The strong deshielding effect induced by the carbomethoxy group on
H-18 (6 3.43) is diagnostic for a cis fusion between rings D and E, while a strong NOE between C-30 methyl and H18 suggests a chair conformation to be predominant also for ring E. All the NMR data collected for compound 6 are self-consistent and show that it has a Al2 oleanane skeleton with the depicted new pentahydroxy derivatization. The ‘H and ’ 3C NMR spectra of 3, supported by 2DCOSY and DEPT experiments, fully define its structure and the position of linkage of the glucose unit. The anomeric proton resonance (doublet at 65.24 with J = 8 Hz) reflects a p-glucosidic linkage, being the 3-OH of compound 6 involved in the linkage as confirmed by the 13C NMR glycosylation shifts. The ‘H and 13CNMR spectra of 4, supported by DEPT experiments, and 13C--lH 2D correlation spectrum, confirmed the presence of two anomeric protons (6 5.15, d, J = 8.0 Hz, corresponding to carbon at 6 105.4 and 6.20, d, J= 3.0 Hz, corresponding to carbon at S 111.2). Comparison of ’ 3C NMR signals of 4 with those of 3 and literature data for linked D-apiose [S] suggested the presence of B-D-apiOfUranOSyl(I--+ 3)-fl-D-glucopyranosyl moiety. Once the 3-0-glycosyl substituents had been defined, it remained to investigate the ester bonded glycosidic moiety. Compound 5 was treated with HCl-methanol, followed by silylation [4], identifying L-
Saponins from Crossopteryx febrijuga Table 4. Cross-peaks from the COLOC experiment C
C-H connectivities
1 4* 5 7 8* 9 10 11 14* 15 17* 18 19 20* 21 22 24 25 26 27 29 30
H-25 H-2, H-5, H-23, H-24 H-7Et, H-25 H-26 H-6, H-9, H-26, H-27 H-lAt, H-7E, H-12, H-25, H-26 H-2, H-5, H-6, H-25 H-9, H-12 H-9, H-12, H-16, H-18, H-26, H-27 H-27 H-15E, H-21A, H-21E, H-22A, H-22E H-12, H-16, H-19A, H-22A H-18, H-29, H-30 H-19A, H-22A, H-29, H-30 H-29, H-30 H-21A H-3, H-5 H-lA, H-lE, H-5 H-7A, H-7E, H-9 H-15A, H-15E H-19A, H-30 H-19A, H-21A, H-29
Table 5. Cross-peaks from the ROESY experiment Protons connected by NOE
1At
H-2Et H-lA, H-3A H-2E, H-5A H-3A, H-6E, H-7A, H-9A H-5A, H-23’ (upfield) H-5A, H-27 H-5A, H-27 H-25 H-18E, (H-19A) H-26 H-27 H-22E H-12, H-19E, H-30 H-27 H-18E H-16E H-6E H-24 H-23” (downfield), H-25 H-l lA, H-24, H-26 H-l 5A, H-25 H-7A, H-9A, H-15E, H-19A H-30 H-18A, H-29
2E 3A 5A 6E IA 9A 11A 12 15A 15E 16E 18E 19A 19E 22E 23’ (upfield) 23” (downfield) 24 25 26 27 29 30
[666 -terminal permethylrhamnose] +, 420 [580-dimethylhexose unit] +, 246 [dimethyl hexalditol diacetate -HI+ and 189 [C,H, TO,]+. Methanolysis of 7 confirmed the presence of methyl 2,3,4-tri-O-methyl-L-rhamnopyranoside, methyl 2,4-di-0-methyl-D-xylopyranoside and methyl 2,3-di-0-methyl+rhamnopyranoside thus unambiguously showing that the two saponins have structures 1 and 2. EXPERIMENTAL
* Quaternary carbons. tA = axial, E = equatorial.
H
2633
l
*NOE’s due to geminal protons have been omitted. Only cross-peaks with intensity either comparable or stronger than those displayed by geminal protons have been listed. tA = axial, E = equatorial.
Plant material was collected in Mozambique in 1986 and authenticated by Dr U. Boni (Inverni della Beffa). A voucher specimen is available at the Department of Pharmacognosy of Inverni della Befla (Milan). Mps: uncorr. Precoated Kieselgel60 F 254and RP 18 (Merck) were used for TLC. Spots were detected by spraying with H,S04-MeOH (1:9) followed by heating. Kieselgel 60 (7&230 mesh, Merck) was employed for CC. Reversed phase HPLC was carried out on a Varian 5000 liquid chromatograph, equipped with a Varian RI-3 refractive index. Analytical separation was obtained on a PBondapack C,, column (1Om 4.6 x 150 mm, Waters), eluting with MeCN-H,O (1:3) at a flow rate of 1 mlmin-I. Preparative separation was carried out on a Viosfer ODS column (10 pm, 1 x 25 cm, Violet), eluting with MeCN-H,O (1: 3), at a flow rate of 2.2 ml min-‘. A Hibar pre-column (4 x 30 mm, Merck), filled with Perisorb RP 8 (3w pm, Merck), was used. Before injection the samples, dissolved in H,O, were filtered on Millex-HV filters (0.45 pm, Millipore). MPLC was carried out with a Buchi 681 pump (Buchi) and a Buchi B685 column (2.6 x 30 cm) filled with LiChroprep RP 18 (4&63pm, Merck). NMR spectra were recorded on 200, 500 and 300 MHz spectrometers; 2D experiments were run at 300 MHz for ‘H and 75.4 MHz for 13C. DQFCOSY and ROESY spectra were accumulated and processed in the phase-sensitive mode. 1K data memory was used in the F2 dimension, and 256 t, increments, zero-filled to 1024 before Fourier transformation. The mixing time for ROESY was 0.8 sec. Two 2D heterocorrelated spectra via l.Ic_” were run; one for oxygenated carbons (60-80 ppm) and one for aliphatic carbons (15-55 ppm). 1K and 2K data memory were used in the F2 dimension and 128 (zero-filled to 256) and 512 (zero-filled to 1024) t, increments respectively. The COLOC experiment utilized 2K in the F2 dimension and 128 t, increments (zero-filled to 512);only the aliphatic carbons were observed (15-55 ppm) while all the ‘H spectrum was excited for polarization transfer. Delays were set to optimize polarization transfers due to a ‘J, 8 Hz, approximately the maximum expected value for couplings involving CH and CH, protons. Samples were dissolved in CDCl, or pyridine-d, and TMS was used as int. standard. Negative FABMS spectra were obtained on a VG 7070 mass spectrometer; samples were dissolved in a glycerol matrix and placed on a steel target prior to bombardment with Ar atoms of energy 7-8 kV. Sugars (silylated and methylated) were analysed on a WCOT CP SilS-CB fused silica capillary column (Chrompack, 25 m x 0.32 mm i.d., film thickness 0.11 pm, carrier H, 0.45 kgcmm2. Inj. temp. 290, det. temp. 300, prog. 150-280 at 4” min- ’ (silyl derivatives) and 8&250 at 4” min- ’ (methyl derivatives). Extraction and isolation. Thinly minced dried roots (2 kg) were exhaustively extd by percolation with hot MeOH (30 1).The ext was coned to 0.8 1, taken up with 50% MeOH (2.7 1) and left overnight at room temp. After filtration, the soln was added with cholesterol (270 g), refluxed for 5 hr with stirring, then left overnight at room temp. The solid material was filtered in uacuo, washed with 50% MeOH (0.5 I), dissolved in CHCl,-MeOH 1: 1 (4.7 I) and H,O added (1.7 1).The organic phase was sepd and the
2634
P.
GARIBALDI
aq. phase extd with CHCI, (2 x 1.7 1). The aq. phase was coned in uacuo to yield 22.5 g saponins. This residue was chromatographed on silica gel (1.3 kg), previously conditioned with CHCI,-MeOH-H,O (14: 6: 1) and eluted with the same mixture at a flow rate of 1 lhr-‘. Fractions (500 ml) were collected according to their composition, taken to dryness and afforded 12.8 g of a mixture showing a single spot on TLC (silica gel and RP 18), but consisting of two substances in the same ratio as shown by HPLC analysis. Compound l(86.5 mg) was obtained by prep. HPLC and pptd by EtOAc as crystals, mp 213-214” (dec), [a];’ -48.0” (MeOH; c 1.05). FABMS m/z (C58H94028): 1238 [M] -, 1092 [M - 146]-, 1076 CM-162]-, 681 [M-l-556]-, 519 [681-162]-. ‘H NMR (500 MHz, pyridine-d,): 66.43 (d, J =3 Hz, H-l Ara), 6.15 (d, J=2Hz, H-l Rha), 5.68 (t, J=3 Hz, H-12), 5.62 (d, J =2Hz,H-1 Rha),5.19(m,H-6),5.14(m,H-16),5.12(d,5=8Hz, H-l Glc), 5.06 (d, J=8 Hz, H-l Xyl), 4.90 (m, H-2), 4.72 (d, J =4Hz, H-3), 3.60(dd, J=14, 4Hz, H-18), 2.72 (t, J=!3 Hz, H19A), 2.20, 1.96, 1.73, 1.64, 1.14, 0.98 (s, -Me), 1.64, 1.63 (d, J = 7 Hz, -Me); ‘%Z NMR (75.4 MHz, pyridine-d,): see Tables 1 and 2. Compound 2 (57.7 mg) was obtained by prep. HPLC and pptd by EtOAc as needles, mp 221-222” (dec), [a];* -56.3” (MeOH; ~0.67). FABMS m/z (C,,H,,,O,,): 1370 [Ml-, 1238 [M -132]1224 [M-146]-, 1076 [1238-!62]-, 814 [M -556]-, 681 [814-132-l]-, 519 [681-162]-. ‘HNMR (500 MHz, pyridine-d,): 66.43 (d, J = 3 Hz, H-l Ara), 6.22 (d, J =4 Hz,H-I Api), 6.16(d, J=2 Hz, H-l Rha), 5.67(t, J=3 Hz, H12), 5.64(d, J=2 Hz, H-l Rha), 5.18(m, H-6), 5.13(m, H-16), 5.10 (d, J=8 HZ, H-l Glc), 5.06 (d, J=8 Hz, H-l Xyl), 4.90 (m, H-2), 4.72 (d, J=4 Hz, H-3), 3.60 (dd, J= 14, 4 Hz, H-18). 2.73 (t, J = 13 Hz, H-19A), 2.20, 1.95, 1.75, 1.63, 1.15, 0.97 (s, -Me), 1.64, 1.63 (d, J = 7 Hz, -Me); 13C NMR (52.4 MHz, pyridine-d,): see Tables 1 and 2. Transesterijcation of 1. To a solution of NaOMe-MeOH (SO mg Na were dissolved in 4 ml dry MeOH) 56 mg 1 in 2 ml dry MeOH were added. The solution was refluxed for 2 hr. then was neutralized HOAc, diluted with H,O, and extracted with nBuOH (2 x 15 ml). The crude residue was purified by CC on silica gel eluting with CHCl,-MeOH (3: 1) and afforded 27 mg of 3. Mp 21@212” (iso-Pr),O-EtOH (99:1), [ZIP NO (MeOH; c 1.07). FABMS (C,,H,,O,,): m/z 695 [M-H]-, 533 [M - 162 -HI-. ‘H NMR (300 MHz, pyridine-d,): 65.62 (t. J=3 Hz, H12), 5.24 (d, J=8 Hz, H-l Glc), 5.18 (m, H-6), 5.01 (m, H-16), 4.92 (m, H-2), 4.59 (d, J = 11 Hz, H-23’), 4.38 (d, J =4 Hz, H-3). 4.03 (d, J=ll Hz,H-23”),3.43(dd,J=14,4Hz,H-18),2.76(t,J=13Hz, H-19A), 2.27. 2.03, 1.77, 1.50, 1.07, 0.98 (s, -Me); 13CNMR (75.4 MHz, pyridine-d,): see Tables 1 and 2. Transesterifcation of 2. Compound 2 (43 mg) was treated with NaOMe-MeOH, in the same manner as 1. The crude residue was purified by CC on silica gel eluting with CHCl,-MeOH (3: 1) and afforded 15 mg of 4. Mp 225-230” (iso-Pr),O-EtOAc (3 : 21,Cali’ -21.6” (MeOH; ~0.66). FABMS (C42H68016): m/z 827[M-HI-,695 [M-132-H]-,‘HNMR(3OOMHz,pyridine-d,): 66.20(d, J=3 Hz, H-l Api), 5.60@, J= 3 Hz, H-12), 5.16 (m, H-6), 5.15 (d, J=8 Hz, H-l G!c),4.98(m, H-16),4.87(m, H-2), 4.52 (d, J=ll Hz, H-23’), 4.33 (d, J=4Hz, H-3), 4.02 (d, J = 11 Hz, H-23”), 3.42 (dd, J= 14, 4 Hz, H-18), 2.78 (t, J= 13 Hz. H-19A), 2.25 2.03, 1.78, 1.50, 1.08, 0.98 (s, -Me); 13CNMR (75.4 MHz, pyridine-d,): see Tables 1 and 2. Enzymatic hydrolysis of 1 and 2. The original mixture of 1 and 2 (3.0047 g) was hydrolysed with the hepatopancreatic juice of 40 snails (Helix pomatia) dil. with H,O (200 ml) and filtered. The soln was stirred at 35” for 20 hr; the mixt. was then extracted with n-BuOH (2 x 200 ml) to afford a residue which was purified by RP-MPLC on a C,, stationary phase, eluting with MeCN-H,O
et al. (1:3) (400 ml), MeCN-H,O (3:7) (600 ml) and MeCN-H,O (2: 3) (800 ml). Fractions were collected according to their composition and afforded 593 mg of 5 and 809 mg of unreacted sample (1+2) showing the same composition of the starting mixture (HPLC analyses). Compound 5 showed mp 231-232” (EtOAc), [E];’ -49.7’ (MeOH; (’ 1.02). FABMS (C,2H,,0,,) m/z: 1076 [Ml-, 519 [M-556-H]-. ‘HNMR (3GOMHz, pyridine-d,): 66.47 (d, J = 3 Hz, H-l Ara), 6.14 (d, J= 2 HZ, H-l Rha), 5.64(d, J=2 Hz, H-l RhaJ.5.61 (t, J=3 Hz,H-!2), 5.22(m, H-6), 5.15(m, H-16), 5.06(d, J=8 Hz, H-l Xyl),4.95(m,H-2),4.78 (d, J=4 Hz, H-3), 3.62 (dd, J= !4,4 Hz, H-18), 2.75 (t. J= 13 Hz, H-19A), 2.28, 2.02, 1.76, 1.66, 1.15, 0.97 (s, -Me), 1.67, 1.65 (d, J = 7 Hz, -Me). “C NMR (75.4 MHz. pyridine-d,): see Tables 1 and 2. Transesterification of the original mixture 1+2. The mixture 1+2 (2 g) was dissolved in 10 ml MeOH and added to a NaOMe-MeOH soln (1.3g Na in 100 ml MeOH). The mixture was refluxed with stirring for 1.30 hr, neutralized with HOAc dild with H,O and extd with n-BuOH (2 x 100 ml). The crude residue was purified by CC on silica gel eluting with CHCl,-MeOH (7:3) (500 ml) and afforded 583 mg of 3+4. Methanolysis and siIylation of 3 +4. The mixture 3+4 (30 mg) was treated with 0.5 M HCI-MeOH (2 ml) and refluxed under N, for 7 hr. The solvent was removed with a llow of N,. a soln of BSTFA-pyridine (1: 1)added (0.5 ml) and the mixture warmed at 60’ for 3 hr. This solution was directly analysed by capillary CC identifying glucose and apiose in the ratio 2: 1 by co-injection with appropriate standards. Enzymatic hydrolysis of3+4. The mixture 3+4 (87 mg) was hydrolysed with the hepatopancreatic juice of six snails (Helix pomatia) dild with H,O (10 ml) and filtered. The solution was stirred at 35” for 40 hr, the mixture was then extd with n-BuOH (2 x 10 ml) to afford a residue which was purified by CC on silica gel eluting with CHCl,-MeOH (9:l) (lOOmI) and CHCl,-MeOH (4: 1) (100 ml). Fractions were collected according to their composition and afforded 38 mg 6 and 14 mg of unreacted sample (3 +4). Compound 6 showed mp 255-256” (isoPr),O, [a];’ + 19.4’ (MeOH; c 1.00). ‘H and 13C NMR see Tables 1,3-5.EIMS(20eV)(C,,H,,O,~m/~534[M]’.260,201. (Found: C, 69.6; H, 9.2: 0, 21.2. C,,H,,O, requires: C. 69.7; H, 9.3; 0, 21.0%). Acid hydrolysis of 3+4. A soln of 3 +4 (90 mg) in 15 ml 2% H,SO, was refluxed for 10 hr. The soln was extd with EtOAc (3 x 20 ml) affording 68 mg of a mixture which was purified by CC on silica gel with increasing amounts of MeOH in CHCI,. Fractions were collected according to their composition and allowed to isolate 14 mg of dehydrated 6. 12 mg 6 and 27 mg of dehydrated 3 + 4. Methanolysis and silylation of 5. Compound 5 (14 mg) was treated with 0.5 M HCI-MeOH and refluxed under N, for 5 hr. The solvent was removed with a flow of N,, a solution of BSTFA-pyridine (1: 1) was added and the mixture was warmed at 60” for 3 hr. This soln was directly analysed by capillary CC identifying arabinose, xylose and rhamnose in the ratio 1 : I:2 by co-injection with appropriate standards. Permethylation and reduction of5 Compound 5 (107 mg) was dissolved in 3 ml DMSO, 350 mg t-NaOBu (Merck), 70 mg dry powdered NaOH and then 0.5 ml Me1 were added with stirring at room temp. After 3 hr the mixt. was poured into ice and extd with CHCI, (3 x 3 ml). The organic phase was washed with H,O and evapd to dryness. The crude residue (Sa) was directly treated with LiAlH, in THF (5 ml) and refluxed for 2 hr. A solution of NH,Cl was added dropwise; the mixture was filtered on celite in uacuo and the residue washed with H,O. The aq. phase was extd with Et,0 (1 x 5 ml) and CHCI, (3 x 5 ml). The CHCI, ext. (117 mg) was purified by CC on silica gel, eluting with
Saponins from Crossopteryx febrijiiga CHzClz-iso-PrOH (19: 1) (80 ml), CH,Cl,-iso-PrOH (93:7) (50 ml), CH,Cl,-iso-PrOH (9: 1) (50 ml). Fractions were collected according to their composition and afforded 58 mg 7a. Hydrolysis of7a. Compound 7a (4 mg) was treated with 0.5 M HCI-MeOH under N,, at reflux for 2 hr. Methyl 2,3,4-&i-Omethyl+rhamnopyranoside, methyl 2,4-di-O-methyl-D-xylopyranoside, methyl 2,3-di-O-methyl-L-rhamnopyranoside were identified by capillary co-GC with appropriate standards [13]. Acetylation of 7~. Compound 7~ (34 mg) was acetylated (A@-pyridine 1: 1)at room temp. overnight. The crude residue was purified by CC on silica gel eluting with petrol-EtOAc (3: 7) (40 ml) and afforded 23 mg 7. Oil, [a]ks - 57.2” (CHCI,; c 1.01). EIMS (20 eV) m/z 666 [M -2 x HOAc] +, 580, 420, 189
CW%@J+.
Hydrolysis of5a. Compound 5a (5 mg) was treated with 0.5 M HCI-MeOH under N,, reflux for 2 hr. Methyl 2,3,4-tri-O-methyl+rhamnopyranoside, methyl 2,4-di-O-methyl-D-xylopyranoside, methyl 2,3-di-O-methyl-L-rhamnopyranoside, methyl 3,4di-0-methyl+arabinopyranoside were identified by capillary co-GC with appropriate standards [13].
Acknowledgements-We are grateful to Istituto di Ricerche Farmacologiche M. Negri (Bergamo-Italy) for FABMS analyses. Part of this work was supported by Tecnofarmaci SpA. Financial assistance from the Minister0 della Pubblica Istruzione of Italy (40% and 60%) is gratefully acknowledged. REFERENCES 1. Palgrave, K. C. (1988) Trees of Southern Africa 5th Edn, p, 844. Struik, Cape Town.
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2. Foresta, P., Ghirardi, O., Gabetta, B. and Cristoni, A. (1986) (to Sigma Tau- Invemi della Beffa), Ital. Pat. 48208 A/86, l/7/1986. Chem. Abs. 109, (26): 236992~. 3. Canon& L., Orsini, F. and Pelizzoni, F. (1976) Gazz. Chim. Ital. 106, 889. 4. Bombardelli, E., Conti, M., Magistretti, M. J. and Martinelli, E. M. (1983) J. Chromatogr. 297, 593. 5. Piantini, U., Sorenson, 0. W. and Ernst, R. R. (1982) J. Am. Chem. Sot. 104,680. 6. Kessler, H., Griesinger, C., Zarbock, J. and Loosli, H. R. (1984) J. Magn. Reson. 57, 331.
7. Kessler, H., Griesinger, C., Kerssebaum, R., Wagner, K. and Ernst, R. R. (1987) J. Am. Chem. Sot. 109,607. 8. Ishii, H., Kitagawa, I., Matsushita, K, Shirakawa, K., Tori, K., Tozyo, T., Yoshikawa, M. and Yoshimura, Y. (1981) Tetrahedron Letters 22, 1529. 9. Ishii, H., Tori, K., Tozyo, T. and Yoshimura, Y. (1984) J.
Chem. Sot. Perkin Trans. 661. 10. Ishii, H., Tori, K., Tozyo, T. and Yoshimura, Y. (1978) Chem. Letters 719.
11. Kasai, R., Miyatoshi, M., Nie, R., Zhou, J., Matsumoto, K., Morita, T., Nishi, M., Miyahara, K. and Tanaka, 0. (1988) Phytochemistry 27, 1439. 12. Ciucanu, I. and Kerek, F. (1984) Carbohydr. Res. 131, 209. 13. Carpani, G., Orsini, F., Sisti, M. and Verotta, L. (1989) Gazz. Chim. Ital. 119, 463. 14. De Marco, A., Gariboldi, P., Molinari, H. and Verotta, L. (1990) Carbohydr. Res. (in press).