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Steroidal glycosides from Allium albopilosum and A. ostrowskianum
0031~9422/93 $6.00+0.00 0 1993 PergantonPressLtd
Ph~toc~e~stry, Vol. 34, No. 3, pp. 799-805, 1993
Printedin Great Britain.
STEROIDAL
~OS~IHIRO
MIMAKI,
GLYCOSIDES FROM ALLIUM AND A. ~~T~~~~K~A~UM
ALBOPILOSUM
KAZUHIROKAWA~HIMA,T~SHI~~RDKANMOTO and YUTAKA SASHIDA*
Tokyo College of Pharmacy, 1432-1,Horinouchi, Hachioji, Tokyo 192-03,Japan (Rec~i#e~ in r~~e~~r~
Key Word Index-Allium hydroxy-3-methylglutaric
26 February
1993)
albopilosum; A. ostrowskianum; Liliaceae; bulbs; steroidal saponins; acid ester; cholestane glycosides.
3-
Abstract-Chemical studies of the bulbs of A&urn a~~~pilo~rn and A. ostrowskianum have led to the isolation of two new steroidal saponins and four new cholestane glycosides together with several known compounds. The structures of the new compounds were ~tablished by the spectroscopic data, hydrolysis and chemical correlations as (25 R and S)5cc-spirostane-2a,3#?,6~-triol 3-~-HO-~-D-glucopyranosyl-~1 ~2)-O-[3-O-acetyl-~-R-xylopyranosyl-(l-3)]-O-& D-glucopyranosyl-~1~4)-~-D-galactopyranoside~, (25R)-2-O-[(S)-3-hydroxy-3-methylglutaroyl]-~~-spirostane2~,3~,6~-t~ol 3-~-HO-~-D-glucopyranosyi-( 1~2)-~-[~-~-xylopyranosyl-( 1~3)]-~-~-D-~ucopyr~osyl-~I -+4)-@D-galactopyranoside), (22S)-cholest-S-ene-l&3/&16/&22-tetraol l-O-or+rhamnopyranoside 16-O-{O-a-L-rhamnopyranosyl-(1+3)-B-D-glucopyranoside}, l/$3&168-trihydroxycholest-5-en-22-one l-0-a-L-rhamnopyranoside 16-O-{@ a-L-rhamnopyranosyl-(I-+3)-B-D-glucopyranoside), lfi,3~,16~-trihydroxy-Sa-cholestan-22-one 1-O-a-L-rhamnopyranoside 16-O-(O-a-t-rharnnopyranosyl-(l~3)-8-1D-glucopyranoside) and (22S~cholest-S-ene-1~,3~,16~,22-tetraol l~O-~O-~-~glucopyranosyl~l~3~~-D-glucop~a~oside~.
1NTRoDljClTC.W
The genus A&m contains ca 400 species and has a distribution in the northern hemisphere [l]. In preceding papers, we described the isolation and structural elucidation of new steroidal saponins and/or cholestane glycosides from the bulbs of A. gi~a~t~ [2,33, A. u~u~~ns@ [3] and A. sc~u~ertii f4, 51. As part of our ongoing chemical analysis of A&urn species, we have investigated the bulbs of A. albopilosum and A. ostrowskia~u~ on which no chemical work appears to have been done. This paper reports the structural elucidation of new steroidal saponins and cholestane glycosides from these two AUium species.
B~UL’E
AND DJXUSSION
A methanolic extract of the fresh bulbs of A. albo~~~osumwas partitioned between 1-butanol and water. Chromatographic fractionation of the I-butanoi phase gave l-10. Compounds l-5 were known compounds, and by spectroscopic data and direct TLC comparison with authentic samples, the respective structures were identified as alliogenin (1) [2,6], alliogenin 2-0-~-D-glucopyr” anoside (2) [23, 3-O-acetylalliogenin 2-0-/&D-glucopyranoside (3) [Z], aginoside (4) [3, 71 and 26-O-/?-Dglucopyranosyl-(25~ and S)-Sa-furostane-2u,3&6fl,22~,
*Author to whom correspondence should be addressed.
26-peutaol 3-~-~~-~-D-glucopyra~osyl-~I42)-~-[~-Dxylopyranosyl-( l-+3)]-O+D-glucopyranosyl-( l-+4)~-D-galactopyranoside~ (s) [5]. Compound 6 was obtained as an amorphous powder, [aID -52.0” (methanol). The molecular formula, Cs2Hs40z5, was estimated by the negative-ion FAB mass Saturn (m/z 1108 EM]-), the 13G NMR site and elemental analysis. The IR spectrum showed the presence of hydroxyl group(s) (3400 cm-‘) and an acetyl group 11730 cmmf), the presence of the latter also being indicated by the ‘HNMR (S1.91, 3H, s) and 13CNMR (6170.5 and 21.1) spectra. Treatment of 6 with 4% potassium hydroxide in ethanol gave a steroidai saponin, which was identified as a 3: 2 mixture of aginoside (4) and its C-25 epimer (turoside A) [S]. In the 13CNMR spectrum of 6, the signal due to the xylose C-3 was shifted to lower field by 0.6 ppm, whereas the signals due to C-2 and C-4 were moved to higher fields by 2.1 and 2.0 ppm, respectively, compared with that of 4, indicating that the acetyl group was linked to the xylose C-3 hydroxyl position. Thus, the structu~ of 6 was determined to be (25 R and S)-.5a-spirostane-2a,38,6@-triol 3-O-(0-/?-~~u~p~~yl~l~2~~-[3-~-~l-~-~xylop~anosyl( 1~3~]-0-~-D-gl~opyran~yl-( 1~4)-~-~~lactopyranoside) . The spectral data of 7 (CJ6H9,,0z8) were similar to those of 4, suggesting that it was a steroidal saponin of the same type. The presence of a 3-hydroxy-3-methylglutaroyl group in the molecule was recognized by the IR El720 err- ‘(C=O)], ‘H NMR [S 3.12 (2H, s), 3.08 (2H, s)
799
Y. MIMAKI et al.
800
27
1 2 3
R’ Ii
R2 H H AC
Glc Glc
OH
OH
(S)-HMG:
5
and 1.76 (3H, s)], and 13CNMR C6174.7 and 171.2 (C=O), 70.1 (C), 46.8 and 46.7 (CH,), and 28.1 (Me)] spectra. Alkaline methanolysis of 7 with 3% sodium methoxide in methanol gave 4 and 3-hydroxy-3methylglutaric acid monomethyl ester (7a). The above data were consistent with 7 being a 3-hydroxy-3methylglutaric acid ester of 4. The ester linkage position in the aglycone C-2 hydroxyl position of 7 was formed from the 3-hydroxy-3-methylglutaric acid as was evident in the shift due to acylation in the ’ 3C NMR spectrum; the signal due to C-2 was moved downfield by 4.5 ppm
compared with that of 4, and all other signals remained almost unaffected. The absolute configuration of the asymmetric centre of the 3-hydroxy-3-methylglutaroyl moiety of 7 was determined by the following chemical correlation. The methyl ester moiety of 7a was reduced with lithium borohydride in tetrahydrofuran at o”, and the reaction mixture, after dilution with water, was a.llowed to stand in acidic conditions for 72 hr to give (3R)mevalonolactone (7b) [9] (Scheme 1). Thus, the asymmetric configuration of the 3-hydroxy-3-methylglutaroyl moiety of 7 was confirmed to be S, and the OH
3% NaoMe 7
4
+
Homok*
y&
_
m
0
0 7a
Scheme
1.
Reactions used to determine the absolute configuration of 7.
‘% (6 0 7b
0
Saponins from Allium bulbs
full structure of 7 was characterized as (25@2-O-[(S)3-hydroxy-3-methylglutaroyl]-S~-spirostan~-2~,3~,6~trio1 3-~-~O-~-~~u~pyranosyl~l-+2)-0-~-~xylopyranosyl-(l-i3)-O-P-D-glucopyranosyl-( l-+4)-B-D-galactopyranoside). ~orn~u~~ g-10 were cholesta~e bi~esmosides and their molecular formulas were determined by negativ~ion FAB mass spectroscopy, r3CNMR and elemental anaIysis to be C,,H,,O,,, C&I&17 and %H7&& resp~tively. The IR spectrum of 8 showed the presence of hydroxyi group(s) (3430 cm-‘). The ‘HNMR spectrum showed three anomeric proton signals at 66.21 {br s), 5.63 (br s) and 4.65 (d, J=7.8 Hz), five secondary methyl proton signals at 61.69 (5x6.1 Hz), X.65 (J=5.8 Hz), 1.14 (J = 6.9 Hz), 0.95 (.I = 6.0 Hz) and 0.92 (J = 6.1 Hz) and two angular methyl proton signals at 61.23 and 1.01. The signals at S 1.69 and 1.65 were due to the methyl groups of 6-deoxyhexoses. Acid hydrolysis of 8 with 1 M hydrochloric acid (dioxane-water, I : 1) gave D-glucose, L-rhamnose and aglycone @a), which was identified as (22S)cholest-5-ene-l~,3~,l6~,ZZ-tetraol, i.e. the aglycone of schubertoside D [4]. The i3C NMR signals of the WCCharide moiety of 8 were assigned by comparing them with those of reference methyl glycosides taking into account the downfield shifts caused by ~-gly~osyiation, confirming the presence of two terminal a+rhamnopyranosyI units (697.7, 72.9, 73.0, 73.7, 70.7 and 18.7; 6102.9, 72.6, 72.8,74.2,69.8 and 18.6) and a 3-substitute /J-D-glucopyranosyl unit (6 106.7, 75.8, &Q.o, 69.7, 78.1 and 62.6) in
R2 R&l-(1-#)-Glc H Gtc GIG
801
the molecule. Furthermore, the glycosylation-induced downfield shifts were observed at the aglycone C-l and C-16 positions in 8 when the whole 13CNMR signals were compared with those of 8a, accounting for the rhamnose and rhamnosyl-(l+3)-glucose linkages to the C-l and C-16 hydroxyl positions, respectively, or C16 and C-l. Mild hydrolysis ofg with 0.2 M hydrochloric acid gave two partial hydrolysates (8b and e). The ‘H and 13C NMR spectra of Sb and c showed the presence of a & D-~u~pyranosyl unit in 8b, and a ~-D-~ucopyranosyl unit and an a-t-rhamnopyranosyi unit in &. The downtield shifts by ~-glycosylation were observed at the aglycone C-16 in 8b, and C-l and C-16 in & in comparison of the r3C signals between 8b and a, and between & and a. The structures of 86 and c were assigned as ~22S)-~holest-5-en~-l~,3~,16~,22-tetraol 16O-P-D-glueopyranoside and ~22~hol~t-5-ene-l~,3~,16~,22-tetraol l-~-~-L-rhamnopyranoside 16-0-~-D-gIu~pyra~oside, respectively. Thus, the s~u~tu~ of 8 was characte~d as (22S)-~holest-5-ene-l~,3~,16~,22-tetraol l-O-a-L-rhamnopyranoside 1~0-HO-~-L-rh~opyr~osyliI -+3)-j%. ~glucopyranoside~. The spectral properties of 9 were essentially identical to those of 8. The IR and i 3CNMR spectra of 9 showed the existence of a carbonyi group (IR: 1700 cm-‘; i3CNMR: 6214.8). The ‘HNMR signal due to the C-20 methine proton of 9 was moved to lower field by 0.71 ppm in comparison with that of 8. On treatment of9 with sodium borohydride, it was Stereos~ci~~alIy reduced to yield & Thus, the structure of 9 was assigned as 1#3,3~,16~-
9
10
tua
tA1 W-W &a-H)
GIG GIcs(1-+3)-Gle
GIG+ -+3)-Glc:
Rha-(1-+3)-Gle:
R
Rha-(1-@-Gie Rha-(l~3~Gl~ Glc
Y. MIMAKI et al.
802
spectral data of the aglycone moieties of 4, 6, 7. 8,8a, Sb, &, 9, 10, 1Oaand 12 (pyridine-d,)
Table 1. 13C NMR 6 4
(25R)
(25s)
7
8
8a
8b
&
9
47.2 70.5 84.7 32.3” 47.9 70.0 40.8 30.1 54.6 37.1 21.4 40.1 40.9 56.3 32.0” 81.3 63.1 16.6 17.2 42.0 15.0 109.2 31.8 29.3 30.6 66.9 17.3
47.1 70.5 84.6 32.3” 47.9 70.0 40.8 30. I 54.6 37.1 21.4 40.1 40.9 56.3 31.9” 81.1 63.1 16.6 17.2 42.0 15.0 109.2 31.8” 29.3 30.6 66.9 17.3
_. -
47.1 75.0 84.8 32.3’ 47.9 70.0 40.8 30.1 54.6 37.1 21.4 40.2 40.9 56.3 32.0” 81.1 63.1 16.6 17.2 42.0 15.0 109.2 31.8” 29.3 30.6 66.9 17.3
81.3 36.0 68.1 43.7 139.2 125.1 31.5 33.4 50.8 42.9 24.8 40.5 42.2 55.3 37.1 82.7 58.1 13.8 14.6 36.1 12.8 73.4 33.5 36.8 28.7 23.0” 23.1”
78.2 44.0 68.2 43.6 140.4 124.5 32.1 33.3 51.6 43.6 24.2 41.3 42.5 55.3 37.5 75.4 58.4 13.7” 13.9” 36.2 15.3 71.5 32.3 36.8 28.5 22.8” 23.0b
78.2 44.0 68.2 43.7 140.3 124.6 32.2 33.1 51.5 43.5 24.3 40.9 42.2 55.4 37.3 82.7 58.2 13.8” 13.9’ 36.0 12.6 73.2 33.8 36.8 28.9 23.0b 23.1b
81.4 36.1 68.1 43.8 139.2 125.1 31.6 33.5 50.9 42.9 24.8 40.6 42.3 55.4 37.2 82.6 58.1 13.9 14.6 36.1 12.7 73.2 33.8 36.8 28.9 23.1 23.1
81.3” 35.9 68.1 43.7 139.1 125.0 31.4 33.3 50.8 42.8 24.8 40.5 41.7 54.6 36.8 81.1” 57.1 14.0 14.6 44.2 16.6 214.8 32.9 38.6 28.0 22.6b 23.0b
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 I5 16 17 18 19 20 21 2.2 23 24 25 26 27
_“. _ .-. .._ -. -. .42.5 14.8 109.7 26.4” 26.2a 27.6 65.1 16.3
10
10a
81.8 37.3 67.6 39.5 42.9 28.9 32.0 36.3 54.9 41.6 24.4 40.7 41.8 54.5 36.8 81.1 57.3 14.1 8.1 44.2 16.6 214.9 32.9 38.6 28.0 22.5a 23.0”
81.8 37.3 67.6 39.6 42.9 29.0 32.0 36.4 55.0 41.6 24.5 40.8 41.8 54.5 37.1 81.0 57.3 14.2 8.2 44.1 16.6 214.6 33.0 38.8 28.1 22.6” 23.0”
12 78.1 43.9 68.1 43.6 140.3 124.5 32.1 33.0 51.4 43.5 24.2 40.7 42.1 55.4 37.1 82.9 58.1 13.78 13.9” 36.0 12.6 73.3 33.6 36.8 28.9 23.0b 23.2b
Assignments wtth same superscript may he reversed in each column.
trihydroxycholest-5-en-22-one side
l-O-a-L-rhamnopyrano-
16-O-{O-a-L-rharnnopyranosyl-(l--+3)-,!?-D-ghtcopyr-
anoside}. The 13C NMR spectrum of 10 showed a close simiiarity to that of 9 except for signals due to the aglycone C-5 and C-6, and their neighbouring carbons. Hydrolysis of IO with 0.2 M hydrochlo~c acid produced L-rhamnose and a partial hydrolysate (IO@. The latter was subsequently reduced with sodium borohydride to provide a cholestane bisdesmoside (lob), identified by IR and ‘H NMR spectra as (22S)-Sa-cholestane-1&3,!?,16/$22-tetraol l-O-c+ L-rhamnopyranoside 16-O-/&D-glucopyranoside, which was previously isolated from the bulbs of Ornithogalum thqprsoides[lo]. Thus, the structure of 10 was determined to be 1&3p,l6#?-trihydroxy-5cc-cholestan-22-one l-O-a-~rh~nopymno~~ 1~0-~0”~-L-rhamnopyranosyl~l --)I3)~-D-glucopyranoside~. A methanofic extract of A. ostro~skiunum bulbs was analysed in a similar manner to that described for A. albopilosum to give 4, 5, II and 12. Compound 11 was identified as F-gitonin [l l-131 from its spectral data. TheNMR spectral data of 12 (C39H66018) showed that it possessed an identical aglycone structure to 8b, but differed from it in its saccharide structure. The i3C NMR spectrum indicated the presence of a terminal p-D-ghicopyranosyl unit and a 3-substituted j?-D-glucopyfanosyl unit, leading to the structure of the sugar moiety as &D
glucopyranosyl-(1+3)-/?-D-glucopyranose. Thus, the structure of 12 was formulated as (22Qcholest-5-ene1~,3/?,16B,22-tetraol 16-0-{0-fl-D-glucopyranosyl-(l-+3)P-D-giucopyranoside}. Compounds 6-10 and 12 are new naturally occurring compounds. A&urn species are known to be rich sources of steroidal saponins [14]. The results of our continuing studies on this genus suggested that species native to Central Asia, e.g. A. schubertii [4], A. stipitafum [15], A. albopilosum and A. ostrowskianum contain cholestane glycosides as well as steroidal saponins. EXPERIMENTAL
General. ‘H and 13C NMR (ppm, f in Hz): 400 MHz (‘H NMR) and 100.6 MHz (13C NMR) with TMS as int. standard.
CC: silica gel (Fuji-Silysia
HP-20 (Mitsubishi-kasei),
Sephadex
Chemic~),
Diaion
LH-20 {Pha~acia),
ODS silica gel (Nacalai Tesque) and Ambedite IR-120B (Organo). TLC: precoated Kieselgel 60 F,,, (0.25 mm thick, Merck) and RF18 F,,, S (0.25 mm thick, Merck). Spots were visualized by spraying with 10% H,SO, soln followed by heating. HPLC: TSK-gel Silica-60 column (Tosoh, 4.6 id. x 250 mm, 5 ,um) for analytical HPLC, and Kaseisorb LC ODS-120-5 column (Tokyo-k.asei-kogyo, 10 i.d. x 250 mm, 5 pm) for prep. HPLC, UV and RI detection.
803
Saponins from AIlium buibs Table 2. 13CNMR spectral data of the saccharide moieties of 4, 6, 7, 8, C 1’ 2 3 4 5 6 1” 2” 3” 4” 5” 6” 1”’ 2”‘ 3” 4”’ 5” 6”’ 1”” ll,, 2 3”” 4”’ 5”” 1”“’ 2”“’ 3”” 4”“’ 5”“’ 6”‘*’ AC
4
6
7
8
8b
&
103.2 72.5 X5.6* 79.4 75.8” 60.6 104.8b 81.1 87.1 70.6’ 17.6 63.0 lOLOb 76.1’ 78.2d 71.4 78.F 42.8 104.7” 75.1 78.5d 70.sc 67.3 -_
103.1 72.5 75.6’ 79.4 75.8@ 60.7 104.8 8i.1 87.3 70.6 77.6 62.8 104.8 75.F 78.2 71.5 78.2 62.8 104.3 73.0 79.1 68.8 66.9 -
103.2 72.5 75.6’ 79.1 75.8’ 60.7 104.7b 81.1 86.6 70.5’ 77.6 63.0 105.0b 75.T 78.2@ 71.5 78.F 62.8 104.7& 75.2 78.1d 70.6’ 67.3 171.2 46.7e 70.1 46.V 174.7 28.1 -
97.7 72.9’ 73.0” 73.7 70.7 18.7b 106.7 75.8 84.0 69.7’ 78.1 62.6 102.9 72.6’ 72.8” 74.2 69.8’ 18.6b
107.0 75.7 78.2* 71.8 78.8’ 63.0 -. -
97.8 72.9= 73.t-P 73.7 70.7 18.7 107.0 75.7 78.2b 71.8 78.8b 63.0 -_ -
^ ___ -
170.5 21.1
-
-
Sb, &, 9, 10, 1Oa and 12 (pyridine-d,) 9
97.7 72.99 73.W 73.7 70.7 18.7b 105.3 76.2 83.8 69.9 18.2 62.6 102.9 72.6’ 72.8” 74.2 69.9 lZ1k6~ “--
10 98.5 72.98 73.28 73.8 71.0 18.7” 105.4 76.2 83.7 69.8 78.2 62.6 102.9 72.6’ 72.8= 74.2 69.8 t8.6b --_._ ..-
10a 98.5 72.9’ 73.2” 73.8 71.1 18.8 105.7 75.9 78.7 71.8 78.3 62.9 -_ .--. -_-
12 106.1” 74.3 89.3 69.7 77.9 62.4b 106.5” 75.5 78.7’ 71.6 78.3’ 62.5b
.-
-
Assignments with the same superscript may be reversed in each column.
Extra&ti~~ and ~o~at~~~. Fresh bulbs of A. al~pi~os~ (5.6 kg) purchased from Heiwaen, Japan, were extracted with hot MeOH. The extract was coned to almost dryness under red. pres. and the residue, after dilution with H,O, was extracted with n-BuOH. The n-BuOH phase was fractionated on a silica gel column with a gradient mixt. of CHCl,-MeOH (19:1, 9: 1 and 4: 1) and finally with MeOH to give 5 frs. Fr. 2 was sepd by silica gel CC with EtOAc-MeOH (9: 1) and ODS silica gel CC with MeOH-H,O (4: 1) to yield l(289 mg) and 3 (17 mg). Fr. 3 was further fractionated by silica gel CC with EtOAc-MeOH (4: 1) into 3 frs (3a-c). Fr. 3b was subjected to ODS silica gel CC with MeOH-H,O (7: 3 and 3 : 2) to yield 2 (102 mg), 8 (44 mg), 9 (7 mg) and 10 (42 mg). Fr. 3c was chromatographed on silica gel with CHCl,-MeOH (4:l) and ODS silica gel with MeOH-H,O (7:3) to yield 4 (136 mg), 6 (18 mg) and 7 (1 g). Fr. 5 was chromatographed on silica gel with EtOAc-MeOH (4:1) and ODS silica gel with MeOH-H,O (7 : 3) to yield 5 (309 mg). Fresh bulbs of A. ostrowskianum (1.1 kg) were treated in a similar manner to those of A. al~opilos~~, The n-BuOH phase was chromato~aphed on silica gel with a gradient mixt. of CHCl,-MeOH (9:1, 4:l and 2:l) and finally
with MeOH to give 8 frs. Fr. 5 was sepd by ODS silica gel CC with MeOH-H,O (7:3) and finally purified by HPLC with MeOH-Hz0 (7: 3) to yield 12 (14 mg). Fr. 8 was further fractionated by ODS silica gel CC with MeOH -H,O (7:3) into 5 frs @a-e). Fr. 8b was purified by HPLC with MeOH-H,O (7: 3) to yield 5 (1 g) and fr. 8e by HPLC with MeOH-H,O (9: 1) to yield 4 (1 g) and 11 (18 mg). Compound
6. Amorphous powder. [u]i4 -52.0” (MeOH; ~0.10). Found: C, 52.04; H, 7.40. Calcd for CJ2Ha40z5. 5H,O: C, 52.08; H, 7.90%. Neg. FABMS m\Xcm-‘: 3400 (OH), 2925 (CH), m/z 1108 [MlIR vKar 1730 (C=O), 1370, 1260, 1050, 1034 980,960, 920, 895, 800. “H NMR (pyridine-d,): 65.63 (lH, t, J =9.2 Hz, H3”“),5.47(1H,d,J=7.8Hz,H-l”‘),5.30(1H,d,J=7.8Hz, H-l”“), 5.23 (lH, d, f=7.8 Hz, H-l”), 4.96 (lH, d, J =7,8 Hz, H-l’), 1.91(3H,s,Ac), 1.29(3H,s, H-19), l.l4(d, J=6.9 Hz, H-21 of(25S)-isomer), 1.13 (d, 5=7.8 Hz, H-21 of (25R)-isomer), 1.07 (d, 5=7.1 Hz, H-27 of (25S)isomer), 0.85 (s, H-18 of (25R)-isomer), 0.84 (s, H-18 of (2X?)-isomer), 0.68 (s, J = 5.7 Hz, H-27 of (25R)-isomer). Alkaline ~ydro~ys~ of 6. Compound 6 (5 mg) was treated with 4% KOH in EtOH at room temp. for 30 min. The reaction mixt. was passed through an Amberlite IR-
804
Y. MIMAKI
120B column and subjected to ODS silica gel CC with MeOH-H,O (7: 3) to yield 3.9 mg of a 3: 2 mixt. of 4 and turoside A. Compound 7. Amorphous powder. [a];” -64.0” (pyridine; ~0.10). Found C, 54.79; H, 7.68. Calcd for C,,H,,O,,.H,O: C, 54.71; H, 7.68%. Neg. FABMS m/z 1210 [Ml-. IR vzf; cm-‘: 3400 (OH), 2950 (CH), 1720 (C=O), 975, 955, 915, 895. ‘HNMR (pyridine-d,): 65.56 (lH, d, J=7.7Hz, H-l”‘), 5.35 (IH, ddd, J=9.7, 9.7, 5.6Hz,H-2),5.20(1H,d,J=7.7Hz,H-l””),5.19(1H,d,J =7.5 Hz, H-l”), 4.95 (lH, d, J=7.7 Hz, H-l’), 3.12 (2H, s, H-2”“’ or H-4”“‘), 3.08 (2H, s, H-2”“’ or H-4”“‘), 1.76 (3H, s, H-6”“‘), 1.29 (3H, s, H-19), 1.13 (3H, d, J=6.8 Hz, H-21), 0.85 (3H, s, H-18), 0.68 (3H, d, J=5.7 Hz, H-27). Alkaline hydrolysis of 7. Compound 7 (300 mg) was treated with 3% NaOMe in MeOH at room temp. for 1.5 hr. The reaction mixt. was passed through an Amberlite TR-120B column and subjected to Sephadex LH-20 CC with MeOH to yield 4 (200 mg) and 7a (30 mg). Compound 7a, viscous syrup. [a]:’ +6.0” (CHCl,; ~0.10). IR ~2:’ cm- ‘. 3452 (OH), 2954, 2925 and 2854 (CH), 1718 (C=O), 1439, 1377, 1349, 1260, 1204, 1116, 1093,1015,979,895,802. ‘H NMR (chloroform-d,): 63.73 (3H, s, OMe), 2.72 and 2.65 (each lH, ABq, J= 15.6 Hz, H-2 or H-4), 2.71 and 2.63 (each lH, ABq, J = 15.7 Hz, H2 or H-4), 1.40 (3H, s, H-6). Preparation of(3R)-m@valonoluctone (7b)from 7. A mixt. of 7a (25 mg) and LiBH, (10 mg) in dry THF was stirred at 0” for 3 hr. After acidification with 2 M HCI, the reaction mixt. was allowed to stand for 72 hr and chromatographed on silica gel using CHCl,-MeOH (19: 1) to give 7b (7.2 mg). Compound 7h, viscous syrup. [u];’ -6.9” (CHCl,; ~0.72). IR vg:t cm-‘: 3400 (OH), 2950 and 2900 (CH), 1720 (C=O), 1403,1308,1265,1230,1132, 1072, 1026. ‘H NMR (chloroform-d,): 64.61 (lH, ddd, J =11.3, 9.7, 5.0Hz, H-5a), 4.35 (lH, ddd, J=11.3, 5.1, 4.1 Hz, H-5b), 2.67 (lH, dd, J=17.4, 1.8 Hz, H-2a), 2.54 (lH, d, J= 17.4 Hz, H-2b), 1.94 (2H, M, H-4), 1.41 (3H, s, H-6). Compound 8. Amorphous powder. [a]i4 -40.0” (MeOH; ~0.10). Found: C, 59.52; H, 8.51. Calcd for C,,H,,O,,.H,O: C, 59.58; H, 8.67%. Neg. FABMS m/z 887 [M-H]-, 741 [M-Rha]-. IR vff; cm-‘: 3430 (OH), 2960 and 2925 (CH), 1450, 1370, 1260, 1090, 1025, 980, 800. ‘H NMR (pyridine-d,): 66.21 (lH, br s, H-l”‘), 5.63 (lH, br s, H-l’), 5.55 (lH, br d, J=5.4 Hz, H-6), 4.65 (lH,d, J=7.8 Hz,H-1”). 3.86(1H, m, H-3), 3.80(1H,dd, J = 11.5, 3.5 Hz, H-l), 2.52 (lH, m, H-20), 1.69 (3H, d, J = 6.1 Hz, H-6”‘), 1.65 (3H, d, J = 5.8 Hz, H-6’), 1.23 (3H, s, H-19), 1.14 (3H, d, J=6.9 Hz, H-21), 1.01 (3H, s, H-18), 0.95 (3H, d, J= 6.0 Hz, H-26 or H-27), 0.92 (3H, d, J =6.1 Hz, H-26 or H-27). Acid hydrolysis of& Compound 8 (10.2 mg) was dissolved in 1 M HCl in dioxane-H,O (1: 1) and refluxed for 1 hr. After cooling, the reaction mixt. was neutralized with 1 M NaOH and chromatographed on a silica gel column using a gradient mixt. of CHCl,-MeOH (19: 1 and 4: 1) to give 8s (2.3 mg) and a sugar fr. Compound 8a, amorphous powder. [a];” - 58.0” (CHCl,; c 0.10). EIMS m/z (rel. int.): 416 [M-H,O]+ (lOO), 398 [M-2H,O]’ (25), 318
et al.
(12), 83 (47), 69 (48). JR vk,ft: cm- ‘: 3450 (OH), 2950 (CH), 1260, 1090, 1010, 800. ‘H NMR (pyridine-d,): 6 5.65 (lH, br d, J=5.7 Hz, H-6), 4.16 (lH, br d, J=8.1 Hz, H-22), 3.97 (lH, m, H-3), 3.83 (lH, br d, J= 11.9 Hz, H-l), 1.38 (3H,s,H-19), 1.27(3H,s,H-18), 1.20(3H,d, J=7.0 Hz,H21), 0.90 (3H x 2, d, J = 6.6 Hz, H-26 and H-27). The sugar fr. was treated with a large excess of (-)-a-methylbenzylamine and Na[BH,CN] for 3 hr at 40”, followed by acetylation with Ac,O in pyridine. The l-[(S)-Nacetyl-r-methylbenzylaminol-I-deoxyalditol acetate derivatives of the monosaccharides were analysed by HPLC under the following conditions: column, a TSK-gel Silica60 (4.6 mm i.d. x 250 mm, 5 pm); solvent, hexane-EtOH l., detection, UV 230 nm. The (19:1);flowrate,1.2mlmin derivatives of D-glucose and L-rhamnose were detected. R, (min): D-glucose, 29.7; L-rhamnose, 19.2. Partial hydrolysis of 8. A soln of 8 (30 mg) in 0.2 M HCl (dioxane-H,O, 1: 1,2 ml) was heated for 30 min at loo”.
The reaction mixt. was coned to dryness and passed through a Diaion HP-20 column to give L-rhamnose and a glycoside fr. The glycoside fr. was subjected to ODS silica gel CC with MeOH-H,O (7:3) to give 8b (1 mg) and & (4.3 mg). Compound 8b, amorphous powder. [a]; -8.0” (MeOH; ~0.10). IR vale cm-‘: 3400 (OH), 2930 (CH), 2850, 1260, 1080, 1030. ‘H NMR (pyridine-d,): 65.56(1H, brd, J=5.9Hz, H-6),4.75(1H,d, J=7.7Hz, H-l’), 3.95 (lH, m, H-3), 3.79 (lH, m, H-l), 1.34 (3H, s, H19), 1.20 (3H, d, J= 7.0 Hz, H-21), 1.09 (3H, s, H-18), 0.94 (3H x 2, d, J= 6.1 Hz, H-26 and H-27). Compound 8c, amorphous powder. [ol]; - 36.0’ (MeOH; c 0.10). Neg. FABMS m/z 741 [M-H]-. IR vE:i cm-‘: 3420 (OH),
2920 (CH), 1370, 1070, 1030,980. ‘H NMR (pyridine-d,): 65.63(1H,brs,H-1’),5.54(1H,brd, J=5.6Hz,H-6),4.75 (lH,d, J=7.7 Hz, H-l”), 3.96(1H, m, H-3), 3.80(1H,dd, J = 12.0, 3.9 Hz, H-l), 1.65 (3H, d, J=5.8 Hz, H-6’), 1.22 (3H, s, H-19), 1.16 (3H, d, J = 7.0 Hz, H-21), 1.06 (3H, s, H18), 0.94 (3H, d, J=6.2 Hz, H-26 or H-27), 0.93 (3H, d, J =6.3 Hz, H-26 or H-27). Compound 9. Amorphous powder. [a]k4 -40.0 (MeOH; ~0.10). Found: C, 60.57; H, 8.48. Calcd for C45H74017: C, 60.93; H, 8.41%. Neg. FABMS m/z 885 [M-H]-, 740 [M-Rha]-. IR vg: cm-r: 3430 (OH), 2930 (CH), 1700 (C=O), 1380,1080,1040,985. ‘H NMR (pyridine-d,): b 6.17 (lH, br d, J= 1.1 Hz, H-l”‘), 5.64 (lH, br s, H-l’), 5.56 (lH, br d, J=5.5 Hz, H-6), 4.48 (lH, d, J =7.9 Hz, H-l”), 3.88 (lH, m, H-3), 3.80 (lH, dd, J= 11.6, 3.7 Hz, H-l), 3.23 (lH, qd, J= 10.8, 7.3 Hz, H-20), 1.69 (3H, d, J=6.2 Hz, H-6”‘), 1.68 (3H, d, J=5.9 Hz, H-6’), 1.22 (3H, s, H-19), 1.08 (3H, d, J=7.3 Hz, H-21), 0.92 (3H, d, J- 6.5 Hz, H-26 or H-27), 0.90 (3H, s, H-18), 0.88 (3H, d, J=6.4 Hz, H-26 or H-27). Reduction of9. A mixt. of 9 (5 mg) and NaBH, (5 mg) in MeOH was stirred for 1 hr. The reaction mixt. was subjected to Sephadex LH-20 CC with MeOH and prep. HPLC with MeOH-H,O (7: 3) to yield 8 (1 mg). Compound 10. Amorphous powder. [a]h4 -30.0“ (MeOH; ~0.10). Found: C, 59.02; H, 8.59. Calcd for C,,H,,O,, . H,O: C, 59.58; H, 8.67%. Neg. FABMS m/z 887 [M-H]-, 741 [M-Rha]-. IR v$,t; cm-‘: 3450 (OH), 2960 (CH), 1700 (C=O), 1380,1130,1080,1045,985.
805
Saponins from AIlium bulbs ‘H NMR (pyridine-d,): 66.19 (lH, br s, H-l”‘), 6.00 (IH, brs,H-l’),4.47(1H,d,J=7.7Hz,H-l”),3.89(1H,m,H-3), 3.72 (lH, dd, J=11.7, 4.2Hz, H-l), 3.23 (lH, m, H-20), 1.69 (3H x 2, d, J= 6.1 Hz, H&and H-6”‘), 1.08 (3H, d, J =7.2 Hz, H-21),0.96 (3H, s, H-19),0.92(3H,d,J=6.4 Hz, H-26 or H-27), 0.87 (3H, d, J = 6.4 Hz, H-24 or H-27), 0.83 (3H, s, H-18). Paths hydrolysis of 10. A soln of 10 (30 mg) in 0.2 M HCI (dioxane-H,O, 1: 1,2 ml) was heated for 30 min at 100”. The reaction mixt. was coned to dryness and passed through a Diaion HP-20 column to give L-rhamnose and a glycosides fr. The glycoside fr. was subjected to ODS silica gel CC with MeOH-H,O (7: 3) to give 1Oa (4 mg). Compound lOa, amorphous powder. [ali -28.0” (Mew, ~0.10). Neg. FABMS m/z 742 [Ml-; IR VE cm-‘: 3360 (OH), 2930 (CH), 1700 (C=O), 1375, 1080. “H NMR (pyridine-d,): 6 5.60 (lH, br s, H-l’), 4.58 (lH, d, J=7.7 Hz,H-l”), 3.89(1H,m,H-3), 3.73(1H,dd, J= 11.0, 3.9H~H-l),2.8l(lH,~H-20),1.70(3H,d,J=6.OH~H63, 1.10 (3H, d, J=7.3 Hz, H-21), 0.97 (3H, s, H-19), 0.89 (3H x 2, d, J = 6.3 Hz, H-26 and H-27). Reduction of 1Oa. A mixt. of 1Oa (4 mg) and NaBH, (4 mg) in MeOH was stirred for 1 hr at room temp. The reaction mixt. was subjected to Sephadex LH-20 CC with MeOH and prep. HPLC with MeOH-H,O (7: 3) to yield lob (1 mg). Compound 12. Amorphous powder. [ali -16.0” (MeOH; ~0.10). Found: C, 61.72; H, 8.32. Calcd for C&,H,,O,,*3H,O: C, 57.62; H, 8.93%. Neg. FABMS m/z 757 [M-H]-, IR vz cm-‘: 3370 (OH), 2950 (CH), 1370, 1260, 1160, 1080, 1040. ‘H NMR (pyridine-d,): 65.56 (lH, br d, J=5.3 Hz, H-6), 5.26 (lH, d, J=7.9 Hz, H-1”),4.73(1H,d,J=7.7 Hz,H-1’),3.%(lH,m,H-3),3.80 (lH, H-l, overlapping with H-5’), 1.34 (3H, s, H-19), 1.20 (3H, d, J=7.0 Hz, H-21), 1.08 (3H, s, H-18), 0.98 (3H, d, J = 6.2 Hz, H-26 or H-27), 0.94 (3H, d, J=6.3 Hz, H-26 or H-27). Ac~nowledgeme~t~We are grateful to Dr Y. Shida and Mrs Y. Katoh of the Central Analytical Centre of our
College for the measurement of mass spectra, and Mr H. Fukaya for elemental analyses. REFERENCES
1. Hotta, M. (1989) in Useful Plants of the World, p. 63. Heibonsha, Tokyo. 2. Sashida, Y., Kawashima, K. and Mimaki, Y. (1991) Gem. Pharm. Buff. 39, 698. 3. Kawashima, K., Mimaki, Y. and Sashida, Y. (1991) Phytochemistry 30, 3063. 4. Kawashima, K., Mimaki, Y. and Sashida, Y. (1991) Chem. Phurm. Bull. 39,276l. 5. Kawashima, K., Mimaki, Y. and Sashida, Y. (1993) Phytochemistry 32, 1267. 6. Khristulas, F. S., Gorovits, M. B., Luchnskqa, V. N. and Abubakirov, N. K. (1970) Khim. Prir. Soedin. 489. 7. Ke~~nbaev, A. N., Gorovits, M. B., Gorovits, T. T. and Abubakirov, N. K. (1976) Khim. Prir. Soedin. 480. 8. Pirtskhalava, G. V., Gorovits, M. B., Gorovits, T. T. and Abubakirov, N. K. (1978) Khim. Prir. Soedin. 355. 9. Eberle, E. and Arigoni, D. (1960) Helu. Chim. Acta. 1508.
10. Kubo, S., Mimaki, Y., Sashida, Y., Nikaido, T. and Ohmoto, T. (1992) Bull. Chem. Sot. Jpn. 65, 1120. 11. Kawasaki, T. and Nishioka, I. (1964) Chem. Pharm. Bull. 12, 1311. 12. Kawasaki, T., Nishioka, I., Komori, T., Yamauchi, T. and Miyahara, K. (1965) Tetrahedron 21,299. 13. Nakano, K., Matsuda, E., Tsurumi, K., Yamasaki, T., Murakami, K., Takaishi, Y. and Tomimatsu, T. (1988) Phybochemistry 27, 3235. 14. Kravets, S. D., Vollerner, Yu. S., Gorovits, M. B. and Abubakirov, N. K. (1990) Khim. Prir. Soedin. 429. 15. Vollerner, Yu. S., Kravets, S. D., Shashkov, A. S., Tashkhodzhaev, B., Gorovits, M. B., Yagudaev, M. R. and Abubakirov, N. K. (1991) Khim. Prir. Soedin. 23 1.
Ph~toc~e~stry, Vol. 34, No. 3, pp. 799-805, 1993
Printedin Great Britain.
STEROIDAL
~OS~IHIRO
MIMAKI,
GLYCOSIDES FROM ALLIUM AND A. ~~T~~~~K~A~UM
ALBOPILOSUM
KAZUHIROKAWA~HIMA,T~SHI~~RDKANMOTO and YUTAKA SASHIDA*
Tokyo College of Pharmacy, 1432-1,Horinouchi, Hachioji, Tokyo 192-03,Japan (Rec~i#e~ in r~~e~~r~
Key Word Index-Allium hydroxy-3-methylglutaric
26 February
1993)
albopilosum; A. ostrowskianum; Liliaceae; bulbs; steroidal saponins; acid ester; cholestane glycosides.
3-
Abstract-Chemical studies of the bulbs of A&urn a~~~pilo~rn and A. ostrowskianum have led to the isolation of two new steroidal saponins and four new cholestane glycosides together with several known compounds. The structures of the new compounds were ~tablished by the spectroscopic data, hydrolysis and chemical correlations as (25 R and S)5cc-spirostane-2a,3#?,6~-triol 3-~-HO-~-D-glucopyranosyl-~1 ~2)-O-[3-O-acetyl-~-R-xylopyranosyl-(l-3)]-O-& D-glucopyranosyl-~1~4)-~-D-galactopyranoside~, (25R)-2-O-[(S)-3-hydroxy-3-methylglutaroyl]-~~-spirostane2~,3~,6~-t~ol 3-~-HO-~-D-glucopyranosyi-( 1~2)-~-[~-~-xylopyranosyl-( 1~3)]-~-~-D-~ucopyr~osyl-~I -+4)-@D-galactopyranoside), (22S)-cholest-S-ene-l&3/&16/&22-tetraol l-O-or+rhamnopyranoside 16-O-{O-a-L-rhamnopyranosyl-(1+3)-B-D-glucopyranoside}, l/$3&168-trihydroxycholest-5-en-22-one l-0-a-L-rhamnopyranoside 16-O-{@ a-L-rhamnopyranosyl-(I-+3)-B-D-glucopyranoside), lfi,3~,16~-trihydroxy-Sa-cholestan-22-one 1-O-a-L-rhamnopyranoside 16-O-(O-a-t-rharnnopyranosyl-(l~3)-8-1D-glucopyranoside) and (22S~cholest-S-ene-1~,3~,16~,22-tetraol l~O-~O-~-~glucopyranosyl~l~3~~-D-glucop~a~oside~.
1NTRoDljClTC.W
The genus A&m contains ca 400 species and has a distribution in the northern hemisphere [l]. In preceding papers, we described the isolation and structural elucidation of new steroidal saponins and/or cholestane glycosides from the bulbs of A. gi~a~t~ [2,33, A. u~u~~ns@ [3] and A. sc~u~ertii f4, 51. As part of our ongoing chemical analysis of A&urn species, we have investigated the bulbs of A. albopilosum and A. ostrowskia~u~ on which no chemical work appears to have been done. This paper reports the structural elucidation of new steroidal saponins and cholestane glycosides from these two AUium species.
B~UL’E
AND DJXUSSION
A methanolic extract of the fresh bulbs of A. albo~~~osumwas partitioned between 1-butanol and water. Chromatographic fractionation of the I-butanoi phase gave l-10. Compounds l-5 were known compounds, and by spectroscopic data and direct TLC comparison with authentic samples, the respective structures were identified as alliogenin (1) [2,6], alliogenin 2-0-~-D-glucopyr” anoside (2) [23, 3-O-acetylalliogenin 2-0-/&D-glucopyranoside (3) [Z], aginoside (4) [3, 71 and 26-O-/?-Dglucopyranosyl-(25~ and S)-Sa-furostane-2u,3&6fl,22~,
*Author to whom correspondence should be addressed.
26-peutaol 3-~-~~-~-D-glucopyra~osyl-~I42)-~-[~-Dxylopyranosyl-( l-+3)]-O+D-glucopyranosyl-( l-+4)~-D-galactopyranoside~ (s) [5]. Compound 6 was obtained as an amorphous powder, [aID -52.0” (methanol). The molecular formula, Cs2Hs40z5, was estimated by the negative-ion FAB mass Saturn (m/z 1108 EM]-), the 13G NMR site and elemental analysis. The IR spectrum showed the presence of hydroxyl group(s) (3400 cm-‘) and an acetyl group 11730 cmmf), the presence of the latter also being indicated by the ‘HNMR (S1.91, 3H, s) and 13CNMR (6170.5 and 21.1) spectra. Treatment of 6 with 4% potassium hydroxide in ethanol gave a steroidai saponin, which was identified as a 3: 2 mixture of aginoside (4) and its C-25 epimer (turoside A) [S]. In the 13CNMR spectrum of 6, the signal due to the xylose C-3 was shifted to lower field by 0.6 ppm, whereas the signals due to C-2 and C-4 were moved to higher fields by 2.1 and 2.0 ppm, respectively, compared with that of 4, indicating that the acetyl group was linked to the xylose C-3 hydroxyl position. Thus, the structu~ of 6 was determined to be (25 R and S)-.5a-spirostane-2a,38,6@-triol 3-O-(0-/?-~~u~p~~yl~l~2~~-[3-~-~l-~-~xylop~anosyl( 1~3~]-0-~-D-gl~opyran~yl-( 1~4)-~-~~lactopyranoside) . The spectral data of 7 (CJ6H9,,0z8) were similar to those of 4, suggesting that it was a steroidal saponin of the same type. The presence of a 3-hydroxy-3-methylglutaroyl group in the molecule was recognized by the IR El720 err- ‘(C=O)], ‘H NMR [S 3.12 (2H, s), 3.08 (2H, s)
799
Y. MIMAKI et al.
800
27
1 2 3
R’ Ii
R2 H H AC
Glc Glc
OH
OH
(S)-HMG:
5
and 1.76 (3H, s)], and 13CNMR C6174.7 and 171.2 (C=O), 70.1 (C), 46.8 and 46.7 (CH,), and 28.1 (Me)] spectra. Alkaline methanolysis of 7 with 3% sodium methoxide in methanol gave 4 and 3-hydroxy-3methylglutaric acid monomethyl ester (7a). The above data were consistent with 7 being a 3-hydroxy-3methylglutaric acid ester of 4. The ester linkage position in the aglycone C-2 hydroxyl position of 7 was formed from the 3-hydroxy-3-methylglutaric acid as was evident in the shift due to acylation in the ’ 3C NMR spectrum; the signal due to C-2 was moved downfield by 4.5 ppm
compared with that of 4, and all other signals remained almost unaffected. The absolute configuration of the asymmetric centre of the 3-hydroxy-3-methylglutaroyl moiety of 7 was determined by the following chemical correlation. The methyl ester moiety of 7a was reduced with lithium borohydride in tetrahydrofuran at o”, and the reaction mixture, after dilution with water, was a.llowed to stand in acidic conditions for 72 hr to give (3R)mevalonolactone (7b) [9] (Scheme 1). Thus, the asymmetric configuration of the 3-hydroxy-3-methylglutaroyl moiety of 7 was confirmed to be S, and the OH
3% NaoMe 7
4
+
Homok*
y&
_
m
0
0 7a
Scheme
1.
Reactions used to determine the absolute configuration of 7.
‘% (6 0 7b
0
Saponins from Allium bulbs
full structure of 7 was characterized as (25@2-O-[(S)3-hydroxy-3-methylglutaroyl]-S~-spirostan~-2~,3~,6~trio1 3-~-~O-~-~~u~pyranosyl~l-+2)-0-~-~xylopyranosyl-(l-i3)-O-P-D-glucopyranosyl-( l-+4)-B-D-galactopyranoside). ~orn~u~~ g-10 were cholesta~e bi~esmosides and their molecular formulas were determined by negativ~ion FAB mass spectroscopy, r3CNMR and elemental anaIysis to be C,,H,,O,,, C&I&17 and %H7&& resp~tively. The IR spectrum of 8 showed the presence of hydroxyi group(s) (3430 cm-‘). The ‘HNMR spectrum showed three anomeric proton signals at 66.21 {br s), 5.63 (br s) and 4.65 (d, J=7.8 Hz), five secondary methyl proton signals at 61.69 (5x6.1 Hz), X.65 (J=5.8 Hz), 1.14 (J = 6.9 Hz), 0.95 (.I = 6.0 Hz) and 0.92 (J = 6.1 Hz) and two angular methyl proton signals at 61.23 and 1.01. The signals at S 1.69 and 1.65 were due to the methyl groups of 6-deoxyhexoses. Acid hydrolysis of 8 with 1 M hydrochloric acid (dioxane-water, I : 1) gave D-glucose, L-rhamnose and aglycone @a), which was identified as (22S)cholest-5-ene-l~,3~,l6~,ZZ-tetraol, i.e. the aglycone of schubertoside D [4]. The i3C NMR signals of the WCCharide moiety of 8 were assigned by comparing them with those of reference methyl glycosides taking into account the downfield shifts caused by ~-gly~osyiation, confirming the presence of two terminal a+rhamnopyranosyI units (697.7, 72.9, 73.0, 73.7, 70.7 and 18.7; 6102.9, 72.6, 72.8,74.2,69.8 and 18.6) and a 3-substitute /J-D-glucopyranosyl unit (6 106.7, 75.8, &Q.o, 69.7, 78.1 and 62.6) in
R2 R&l-(1-#)-Glc H Gtc GIG
801
the molecule. Furthermore, the glycosylation-induced downfield shifts were observed at the aglycone C-l and C-16 positions in 8 when the whole 13CNMR signals were compared with those of 8a, accounting for the rhamnose and rhamnosyl-(l+3)-glucose linkages to the C-l and C-16 hydroxyl positions, respectively, or C16 and C-l. Mild hydrolysis ofg with 0.2 M hydrochloric acid gave two partial hydrolysates (8b and e). The ‘H and 13C NMR spectra of Sb and c showed the presence of a & D-~u~pyranosyl unit in 8b, and a ~-D-~ucopyranosyl unit and an a-t-rhamnopyranosyi unit in &. The downtield shifts by ~-glycosylation were observed at the aglycone C-16 in 8b, and C-l and C-16 in & in comparison of the r3C signals between 8b and a, and between & and a. The structures of 86 and c were assigned as ~22S)-~holest-5-en~-l~,3~,16~,22-tetraol 16O-P-D-glueopyranoside and ~22~hol~t-5-ene-l~,3~,16~,22-tetraol l-~-~-L-rhamnopyranoside 16-0-~-D-gIu~pyra~oside, respectively. Thus, the s~u~tu~ of 8 was characte~d as (22S)-~holest-5-ene-l~,3~,16~,22-tetraol l-O-a-L-rhamnopyranoside 1~0-HO-~-L-rh~opyr~osyliI -+3)-j%. ~glucopyranoside~. The spectral properties of 9 were essentially identical to those of 8. The IR and i 3CNMR spectra of 9 showed the existence of a carbonyi group (IR: 1700 cm-‘; i3CNMR: 6214.8). The ‘HNMR signal due to the C-20 methine proton of 9 was moved to lower field by 0.71 ppm in comparison with that of 8. On treatment of9 with sodium borohydride, it was Stereos~ci~~alIy reduced to yield & Thus, the structure of 9 was assigned as 1#3,3~,16~-
9
10
tua
tA1 W-W &a-H)
GIG GIcs(1-+3)-Gle
GIG+ -+3)-Glc:
Rha-(1-+3)-Gle:
R
Rha-(1-@-Gie Rha-(l~3~Gl~ Glc
Y. MIMAKI et al.
802
spectral data of the aglycone moieties of 4, 6, 7. 8,8a, Sb, &, 9, 10, 1Oaand 12 (pyridine-d,)
Table 1. 13C NMR 6 4
(25R)
(25s)
7
8
8a
8b
&
9
47.2 70.5 84.7 32.3” 47.9 70.0 40.8 30.1 54.6 37.1 21.4 40.1 40.9 56.3 32.0” 81.3 63.1 16.6 17.2 42.0 15.0 109.2 31.8 29.3 30.6 66.9 17.3
47.1 70.5 84.6 32.3” 47.9 70.0 40.8 30. I 54.6 37.1 21.4 40.1 40.9 56.3 31.9” 81.1 63.1 16.6 17.2 42.0 15.0 109.2 31.8” 29.3 30.6 66.9 17.3
_. -
47.1 75.0 84.8 32.3’ 47.9 70.0 40.8 30.1 54.6 37.1 21.4 40.2 40.9 56.3 32.0” 81.1 63.1 16.6 17.2 42.0 15.0 109.2 31.8” 29.3 30.6 66.9 17.3
81.3 36.0 68.1 43.7 139.2 125.1 31.5 33.4 50.8 42.9 24.8 40.5 42.2 55.3 37.1 82.7 58.1 13.8 14.6 36.1 12.8 73.4 33.5 36.8 28.7 23.0” 23.1”
78.2 44.0 68.2 43.6 140.4 124.5 32.1 33.3 51.6 43.6 24.2 41.3 42.5 55.3 37.5 75.4 58.4 13.7” 13.9” 36.2 15.3 71.5 32.3 36.8 28.5 22.8” 23.0b
78.2 44.0 68.2 43.7 140.3 124.6 32.2 33.1 51.5 43.5 24.3 40.9 42.2 55.4 37.3 82.7 58.2 13.8” 13.9’ 36.0 12.6 73.2 33.8 36.8 28.9 23.0b 23.1b
81.4 36.1 68.1 43.8 139.2 125.1 31.6 33.5 50.9 42.9 24.8 40.6 42.3 55.4 37.2 82.6 58.1 13.9 14.6 36.1 12.7 73.2 33.8 36.8 28.9 23.1 23.1
81.3” 35.9 68.1 43.7 139.1 125.0 31.4 33.3 50.8 42.8 24.8 40.5 41.7 54.6 36.8 81.1” 57.1 14.0 14.6 44.2 16.6 214.8 32.9 38.6 28.0 22.6b 23.0b
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 I5 16 17 18 19 20 21 2.2 23 24 25 26 27
_“. _ .-. .._ -. -. .42.5 14.8 109.7 26.4” 26.2a 27.6 65.1 16.3
10
10a
81.8 37.3 67.6 39.5 42.9 28.9 32.0 36.3 54.9 41.6 24.4 40.7 41.8 54.5 36.8 81.1 57.3 14.1 8.1 44.2 16.6 214.9 32.9 38.6 28.0 22.5a 23.0”
81.8 37.3 67.6 39.6 42.9 29.0 32.0 36.4 55.0 41.6 24.5 40.8 41.8 54.5 37.1 81.0 57.3 14.2 8.2 44.1 16.6 214.6 33.0 38.8 28.1 22.6” 23.0”
12 78.1 43.9 68.1 43.6 140.3 124.5 32.1 33.0 51.4 43.5 24.2 40.7 42.1 55.4 37.1 82.9 58.1 13.78 13.9” 36.0 12.6 73.3 33.6 36.8 28.9 23.0b 23.2b
Assignments wtth same superscript may he reversed in each column.
trihydroxycholest-5-en-22-one side
l-O-a-L-rhamnopyrano-
16-O-{O-a-L-rharnnopyranosyl-(l--+3)-,!?-D-ghtcopyr-
anoside}. The 13C NMR spectrum of 10 showed a close simiiarity to that of 9 except for signals due to the aglycone C-5 and C-6, and their neighbouring carbons. Hydrolysis of IO with 0.2 M hydrochlo~c acid produced L-rhamnose and a partial hydrolysate (IO@. The latter was subsequently reduced with sodium borohydride to provide a cholestane bisdesmoside (lob), identified by IR and ‘H NMR spectra as (22S)-Sa-cholestane-1&3,!?,16/$22-tetraol l-O-c+ L-rhamnopyranoside 16-O-/&D-glucopyranoside, which was previously isolated from the bulbs of Ornithogalum thqprsoides[lo]. Thus, the structure of 10 was determined to be 1&3p,l6#?-trihydroxy-5cc-cholestan-22-one l-O-a-~rh~nopymno~~ 1~0-~0”~-L-rhamnopyranosyl~l --)I3)~-D-glucopyranoside~. A methanofic extract of A. ostro~skiunum bulbs was analysed in a similar manner to that described for A. albopilosum to give 4, 5, II and 12. Compound 11 was identified as F-gitonin [l l-131 from its spectral data. TheNMR spectral data of 12 (C39H66018) showed that it possessed an identical aglycone structure to 8b, but differed from it in its saccharide structure. The i3C NMR spectrum indicated the presence of a terminal p-D-ghicopyranosyl unit and a 3-substituted j?-D-glucopyfanosyl unit, leading to the structure of the sugar moiety as &D
glucopyranosyl-(1+3)-/?-D-glucopyranose. Thus, the structure of 12 was formulated as (22Qcholest-5-ene1~,3/?,16B,22-tetraol 16-0-{0-fl-D-glucopyranosyl-(l-+3)P-D-giucopyranoside}. Compounds 6-10 and 12 are new naturally occurring compounds. A&urn species are known to be rich sources of steroidal saponins [14]. The results of our continuing studies on this genus suggested that species native to Central Asia, e.g. A. schubertii [4], A. stipitafum [15], A. albopilosum and A. ostrowskianum contain cholestane glycosides as well as steroidal saponins. EXPERIMENTAL
General. ‘H and 13C NMR (ppm, f in Hz): 400 MHz (‘H NMR) and 100.6 MHz (13C NMR) with TMS as int. standard.
CC: silica gel (Fuji-Silysia
HP-20 (Mitsubishi-kasei),
Sephadex
Chemic~),
Diaion
LH-20 {Pha~acia),
ODS silica gel (Nacalai Tesque) and Ambedite IR-120B (Organo). TLC: precoated Kieselgel 60 F,,, (0.25 mm thick, Merck) and RF18 F,,, S (0.25 mm thick, Merck). Spots were visualized by spraying with 10% H,SO, soln followed by heating. HPLC: TSK-gel Silica-60 column (Tosoh, 4.6 id. x 250 mm, 5 ,um) for analytical HPLC, and Kaseisorb LC ODS-120-5 column (Tokyo-k.asei-kogyo, 10 i.d. x 250 mm, 5 pm) for prep. HPLC, UV and RI detection.
803
Saponins from AIlium buibs Table 2. 13CNMR spectral data of the saccharide moieties of 4, 6, 7, 8, C 1’ 2 3 4 5 6 1” 2” 3” 4” 5” 6” 1”’ 2”‘ 3” 4”’ 5” 6”’ 1”” ll,, 2 3”” 4”’ 5”” 1”“’ 2”“’ 3”” 4”“’ 5”“’ 6”‘*’ AC
4
6
7
8
8b
&
103.2 72.5 X5.6* 79.4 75.8” 60.6 104.8b 81.1 87.1 70.6’ 17.6 63.0 lOLOb 76.1’ 78.2d 71.4 78.F 42.8 104.7” 75.1 78.5d 70.sc 67.3 -_
103.1 72.5 75.6’ 79.4 75.8@ 60.7 104.8 8i.1 87.3 70.6 77.6 62.8 104.8 75.F 78.2 71.5 78.2 62.8 104.3 73.0 79.1 68.8 66.9 -
103.2 72.5 75.6’ 79.1 75.8’ 60.7 104.7b 81.1 86.6 70.5’ 77.6 63.0 105.0b 75.T 78.2@ 71.5 78.F 62.8 104.7& 75.2 78.1d 70.6’ 67.3 171.2 46.7e 70.1 46.V 174.7 28.1 -
97.7 72.9’ 73.0” 73.7 70.7 18.7b 106.7 75.8 84.0 69.7’ 78.1 62.6 102.9 72.6’ 72.8” 74.2 69.8’ 18.6b
107.0 75.7 78.2* 71.8 78.8’ 63.0 -. -
97.8 72.9= 73.t-P 73.7 70.7 18.7 107.0 75.7 78.2b 71.8 78.8b 63.0 -_ -
^ ___ -
170.5 21.1
-
-
Sb, &, 9, 10, 1Oa and 12 (pyridine-d,) 9
97.7 72.99 73.W 73.7 70.7 18.7b 105.3 76.2 83.8 69.9 18.2 62.6 102.9 72.6’ 72.8” 74.2 69.9 lZ1k6~ “--
10 98.5 72.98 73.28 73.8 71.0 18.7” 105.4 76.2 83.7 69.8 78.2 62.6 102.9 72.6’ 72.8= 74.2 69.8 t8.6b --_._ ..-
10a 98.5 72.9’ 73.2” 73.8 71.1 18.8 105.7 75.9 78.7 71.8 78.3 62.9 -_ .--. -_-
12 106.1” 74.3 89.3 69.7 77.9 62.4b 106.5” 75.5 78.7’ 71.6 78.3’ 62.5b
.-
-
Assignments with the same superscript may be reversed in each column.
Extra&ti~~ and ~o~at~~~. Fresh bulbs of A. al~pi~os~ (5.6 kg) purchased from Heiwaen, Japan, were extracted with hot MeOH. The extract was coned to almost dryness under red. pres. and the residue, after dilution with H,O, was extracted with n-BuOH. The n-BuOH phase was fractionated on a silica gel column with a gradient mixt. of CHCl,-MeOH (19:1, 9: 1 and 4: 1) and finally with MeOH to give 5 frs. Fr. 2 was sepd by silica gel CC with EtOAc-MeOH (9: 1) and ODS silica gel CC with MeOH-H,O (4: 1) to yield l(289 mg) and 3 (17 mg). Fr. 3 was further fractionated by silica gel CC with EtOAc-MeOH (4: 1) into 3 frs (3a-c). Fr. 3b was subjected to ODS silica gel CC with MeOH-H,O (7: 3 and 3 : 2) to yield 2 (102 mg), 8 (44 mg), 9 (7 mg) and 10 (42 mg). Fr. 3c was chromatographed on silica gel with CHCl,-MeOH (4:l) and ODS silica gel with MeOH-H,O (7:3) to yield 4 (136 mg), 6 (18 mg) and 7 (1 g). Fr. 5 was chromatographed on silica gel with EtOAc-MeOH (4:1) and ODS silica gel with MeOH-H,O (7 : 3) to yield 5 (309 mg). Fresh bulbs of A. ostrowskianum (1.1 kg) were treated in a similar manner to those of A. al~opilos~~, The n-BuOH phase was chromato~aphed on silica gel with a gradient mixt. of CHCl,-MeOH (9:1, 4:l and 2:l) and finally
with MeOH to give 8 frs. Fr. 5 was sepd by ODS silica gel CC with MeOH-H,O (7:3) and finally purified by HPLC with MeOH-Hz0 (7: 3) to yield 12 (14 mg). Fr. 8 was further fractionated by ODS silica gel CC with MeOH -H,O (7:3) into 5 frs @a-e). Fr. 8b was purified by HPLC with MeOH-H,O (7: 3) to yield 5 (1 g) and fr. 8e by HPLC with MeOH-H,O (9: 1) to yield 4 (1 g) and 11 (18 mg). Compound
6. Amorphous powder. [u]i4 -52.0” (MeOH; ~0.10). Found: C, 52.04; H, 7.40. Calcd for CJ2Ha40z5. 5H,O: C, 52.08; H, 7.90%. Neg. FABMS m\Xcm-‘: 3400 (OH), 2925 (CH), m/z 1108 [MlIR vKar 1730 (C=O), 1370, 1260, 1050, 1034 980,960, 920, 895, 800. “H NMR (pyridine-d,): 65.63 (lH, t, J =9.2 Hz, H3”“),5.47(1H,d,J=7.8Hz,H-l”‘),5.30(1H,d,J=7.8Hz, H-l”“), 5.23 (lH, d, f=7.8 Hz, H-l”), 4.96 (lH, d, J =7,8 Hz, H-l’), 1.91(3H,s,Ac), 1.29(3H,s, H-19), l.l4(d, J=6.9 Hz, H-21 of(25S)-isomer), 1.13 (d, 5=7.8 Hz, H-21 of (25R)-isomer), 1.07 (d, 5=7.1 Hz, H-27 of (25S)isomer), 0.85 (s, H-18 of (25R)-isomer), 0.84 (s, H-18 of (2X?)-isomer), 0.68 (s, J = 5.7 Hz, H-27 of (25R)-isomer). Alkaline ~ydro~ys~ of 6. Compound 6 (5 mg) was treated with 4% KOH in EtOH at room temp. for 30 min. The reaction mixt. was passed through an Amberlite IR-
804
Y. MIMAKI
120B column and subjected to ODS silica gel CC with MeOH-H,O (7: 3) to yield 3.9 mg of a 3: 2 mixt. of 4 and turoside A. Compound 7. Amorphous powder. [a];” -64.0” (pyridine; ~0.10). Found C, 54.79; H, 7.68. Calcd for C,,H,,O,,.H,O: C, 54.71; H, 7.68%. Neg. FABMS m/z 1210 [Ml-. IR vzf; cm-‘: 3400 (OH), 2950 (CH), 1720 (C=O), 975, 955, 915, 895. ‘HNMR (pyridine-d,): 65.56 (lH, d, J=7.7Hz, H-l”‘), 5.35 (IH, ddd, J=9.7, 9.7, 5.6Hz,H-2),5.20(1H,d,J=7.7Hz,H-l””),5.19(1H,d,J =7.5 Hz, H-l”), 4.95 (lH, d, J=7.7 Hz, H-l’), 3.12 (2H, s, H-2”“’ or H-4”“‘), 3.08 (2H, s, H-2”“’ or H-4”“‘), 1.76 (3H, s, H-6”“‘), 1.29 (3H, s, H-19), 1.13 (3H, d, J=6.8 Hz, H-21), 0.85 (3H, s, H-18), 0.68 (3H, d, J=5.7 Hz, H-27). Alkaline hydrolysis of 7. Compound 7 (300 mg) was treated with 3% NaOMe in MeOH at room temp. for 1.5 hr. The reaction mixt. was passed through an Amberlite TR-120B column and subjected to Sephadex LH-20 CC with MeOH to yield 4 (200 mg) and 7a (30 mg). Compound 7a, viscous syrup. [a]:’ +6.0” (CHCl,; ~0.10). IR ~2:’ cm- ‘. 3452 (OH), 2954, 2925 and 2854 (CH), 1718 (C=O), 1439, 1377, 1349, 1260, 1204, 1116, 1093,1015,979,895,802. ‘H NMR (chloroform-d,): 63.73 (3H, s, OMe), 2.72 and 2.65 (each lH, ABq, J= 15.6 Hz, H-2 or H-4), 2.71 and 2.63 (each lH, ABq, J = 15.7 Hz, H2 or H-4), 1.40 (3H, s, H-6). Preparation of(3R)-m@valonoluctone (7b)from 7. A mixt. of 7a (25 mg) and LiBH, (10 mg) in dry THF was stirred at 0” for 3 hr. After acidification with 2 M HCI, the reaction mixt. was allowed to stand for 72 hr and chromatographed on silica gel using CHCl,-MeOH (19: 1) to give 7b (7.2 mg). Compound 7h, viscous syrup. [u];’ -6.9” (CHCl,; ~0.72). IR vg:t cm-‘: 3400 (OH), 2950 and 2900 (CH), 1720 (C=O), 1403,1308,1265,1230,1132, 1072, 1026. ‘H NMR (chloroform-d,): 64.61 (lH, ddd, J =11.3, 9.7, 5.0Hz, H-5a), 4.35 (lH, ddd, J=11.3, 5.1, 4.1 Hz, H-5b), 2.67 (lH, dd, J=17.4, 1.8 Hz, H-2a), 2.54 (lH, d, J= 17.4 Hz, H-2b), 1.94 (2H, M, H-4), 1.41 (3H, s, H-6). Compound 8. Amorphous powder. [a]i4 -40.0” (MeOH; ~0.10). Found: C, 59.52; H, 8.51. Calcd for C,,H,,O,,.H,O: C, 59.58; H, 8.67%. Neg. FABMS m/z 887 [M-H]-, 741 [M-Rha]-. IR vff; cm-‘: 3430 (OH), 2960 and 2925 (CH), 1450, 1370, 1260, 1090, 1025, 980, 800. ‘H NMR (pyridine-d,): 66.21 (lH, br s, H-l”‘), 5.63 (lH, br s, H-l’), 5.55 (lH, br d, J=5.4 Hz, H-6), 4.65 (lH,d, J=7.8 Hz,H-1”). 3.86(1H, m, H-3), 3.80(1H,dd, J = 11.5, 3.5 Hz, H-l), 2.52 (lH, m, H-20), 1.69 (3H, d, J = 6.1 Hz, H-6”‘), 1.65 (3H, d, J = 5.8 Hz, H-6’), 1.23 (3H, s, H-19), 1.14 (3H, d, J=6.9 Hz, H-21), 1.01 (3H, s, H-18), 0.95 (3H, d, J= 6.0 Hz, H-26 or H-27), 0.92 (3H, d, J =6.1 Hz, H-26 or H-27). Acid hydrolysis of& Compound 8 (10.2 mg) was dissolved in 1 M HCl in dioxane-H,O (1: 1) and refluxed for 1 hr. After cooling, the reaction mixt. was neutralized with 1 M NaOH and chromatographed on a silica gel column using a gradient mixt. of CHCl,-MeOH (19: 1 and 4: 1) to give 8s (2.3 mg) and a sugar fr. Compound 8a, amorphous powder. [a];” - 58.0” (CHCl,; c 0.10). EIMS m/z (rel. int.): 416 [M-H,O]+ (lOO), 398 [M-2H,O]’ (25), 318
et al.
(12), 83 (47), 69 (48). JR vk,ft: cm- ‘: 3450 (OH), 2950 (CH), 1260, 1090, 1010, 800. ‘H NMR (pyridine-d,): 6 5.65 (lH, br d, J=5.7 Hz, H-6), 4.16 (lH, br d, J=8.1 Hz, H-22), 3.97 (lH, m, H-3), 3.83 (lH, br d, J= 11.9 Hz, H-l), 1.38 (3H,s,H-19), 1.27(3H,s,H-18), 1.20(3H,d, J=7.0 Hz,H21), 0.90 (3H x 2, d, J = 6.6 Hz, H-26 and H-27). The sugar fr. was treated with a large excess of (-)-a-methylbenzylamine and Na[BH,CN] for 3 hr at 40”, followed by acetylation with Ac,O in pyridine. The l-[(S)-Nacetyl-r-methylbenzylaminol-I-deoxyalditol acetate derivatives of the monosaccharides were analysed by HPLC under the following conditions: column, a TSK-gel Silica60 (4.6 mm i.d. x 250 mm, 5 pm); solvent, hexane-EtOH l., detection, UV 230 nm. The (19:1);flowrate,1.2mlmin derivatives of D-glucose and L-rhamnose were detected. R, (min): D-glucose, 29.7; L-rhamnose, 19.2. Partial hydrolysis of 8. A soln of 8 (30 mg) in 0.2 M HCl (dioxane-H,O, 1: 1,2 ml) was heated for 30 min at loo”.
The reaction mixt. was coned to dryness and passed through a Diaion HP-20 column to give L-rhamnose and a glycoside fr. The glycoside fr. was subjected to ODS silica gel CC with MeOH-H,O (7:3) to give 8b (1 mg) and & (4.3 mg). Compound 8b, amorphous powder. [a]; -8.0” (MeOH; ~0.10). IR vale cm-‘: 3400 (OH), 2930 (CH), 2850, 1260, 1080, 1030. ‘H NMR (pyridine-d,): 65.56(1H, brd, J=5.9Hz, H-6),4.75(1H,d, J=7.7Hz, H-l’), 3.95 (lH, m, H-3), 3.79 (lH, m, H-l), 1.34 (3H, s, H19), 1.20 (3H, d, J= 7.0 Hz, H-21), 1.09 (3H, s, H-18), 0.94 (3H x 2, d, J= 6.1 Hz, H-26 and H-27). Compound 8c, amorphous powder. [ol]; - 36.0’ (MeOH; c 0.10). Neg. FABMS m/z 741 [M-H]-. IR vE:i cm-‘: 3420 (OH),
2920 (CH), 1370, 1070, 1030,980. ‘H NMR (pyridine-d,): 65.63(1H,brs,H-1’),5.54(1H,brd, J=5.6Hz,H-6),4.75 (lH,d, J=7.7 Hz, H-l”), 3.96(1H, m, H-3), 3.80(1H,dd, J = 12.0, 3.9 Hz, H-l), 1.65 (3H, d, J=5.8 Hz, H-6’), 1.22 (3H, s, H-19), 1.16 (3H, d, J = 7.0 Hz, H-21), 1.06 (3H, s, H18), 0.94 (3H, d, J=6.2 Hz, H-26 or H-27), 0.93 (3H, d, J =6.3 Hz, H-26 or H-27). Compound 9. Amorphous powder. [a]k4 -40.0 (MeOH; ~0.10). Found: C, 60.57; H, 8.48. Calcd for C45H74017: C, 60.93; H, 8.41%. Neg. FABMS m/z 885 [M-H]-, 740 [M-Rha]-. IR vg: cm-r: 3430 (OH), 2930 (CH), 1700 (C=O), 1380,1080,1040,985. ‘H NMR (pyridine-d,): b 6.17 (lH, br d, J= 1.1 Hz, H-l”‘), 5.64 (lH, br s, H-l’), 5.56 (lH, br d, J=5.5 Hz, H-6), 4.48 (lH, d, J =7.9 Hz, H-l”), 3.88 (lH, m, H-3), 3.80 (lH, dd, J= 11.6, 3.7 Hz, H-l), 3.23 (lH, qd, J= 10.8, 7.3 Hz, H-20), 1.69 (3H, d, J=6.2 Hz, H-6”‘), 1.68 (3H, d, J=5.9 Hz, H-6’), 1.22 (3H, s, H-19), 1.08 (3H, d, J=7.3 Hz, H-21), 0.92 (3H, d, J- 6.5 Hz, H-26 or H-27), 0.90 (3H, s, H-18), 0.88 (3H, d, J=6.4 Hz, H-26 or H-27). Reduction of9. A mixt. of 9 (5 mg) and NaBH, (5 mg) in MeOH was stirred for 1 hr. The reaction mixt. was subjected to Sephadex LH-20 CC with MeOH and prep. HPLC with MeOH-H,O (7: 3) to yield 8 (1 mg). Compound 10. Amorphous powder. [a]h4 -30.0“ (MeOH; ~0.10). Found: C, 59.02; H, 8.59. Calcd for C,,H,,O,, . H,O: C, 59.58; H, 8.67%. Neg. FABMS m/z 887 [M-H]-, 741 [M-Rha]-. IR v$,t; cm-‘: 3450 (OH), 2960 (CH), 1700 (C=O), 1380,1130,1080,1045,985.
805
Saponins from AIlium bulbs ‘H NMR (pyridine-d,): 66.19 (lH, br s, H-l”‘), 6.00 (IH, brs,H-l’),4.47(1H,d,J=7.7Hz,H-l”),3.89(1H,m,H-3), 3.72 (lH, dd, J=11.7, 4.2Hz, H-l), 3.23 (lH, m, H-20), 1.69 (3H x 2, d, J= 6.1 Hz, H&and H-6”‘), 1.08 (3H, d, J =7.2 Hz, H-21),0.96 (3H, s, H-19),0.92(3H,d,J=6.4 Hz, H-26 or H-27), 0.87 (3H, d, J = 6.4 Hz, H-24 or H-27), 0.83 (3H, s, H-18). Paths hydrolysis of 10. A soln of 10 (30 mg) in 0.2 M HCI (dioxane-H,O, 1: 1,2 ml) was heated for 30 min at 100”. The reaction mixt. was coned to dryness and passed through a Diaion HP-20 column to give L-rhamnose and a glycosides fr. The glycoside fr. was subjected to ODS silica gel CC with MeOH-H,O (7: 3) to give 1Oa (4 mg). Compound lOa, amorphous powder. [ali -28.0” (Mew, ~0.10). Neg. FABMS m/z 742 [Ml-; IR VE cm-‘: 3360 (OH), 2930 (CH), 1700 (C=O), 1375, 1080. “H NMR (pyridine-d,): 6 5.60 (lH, br s, H-l’), 4.58 (lH, d, J=7.7 Hz,H-l”), 3.89(1H,m,H-3), 3.73(1H,dd, J= 11.0, 3.9H~H-l),2.8l(lH,~H-20),1.70(3H,d,J=6.OH~H63, 1.10 (3H, d, J=7.3 Hz, H-21), 0.97 (3H, s, H-19), 0.89 (3H x 2, d, J = 6.3 Hz, H-26 and H-27). Reduction of 1Oa. A mixt. of 1Oa (4 mg) and NaBH, (4 mg) in MeOH was stirred for 1 hr at room temp. The reaction mixt. was subjected to Sephadex LH-20 CC with MeOH and prep. HPLC with MeOH-H,O (7: 3) to yield lob (1 mg). Compound 12. Amorphous powder. [ali -16.0” (MeOH; ~0.10). Found: C, 61.72; H, 8.32. Calcd for C&,H,,O,,*3H,O: C, 57.62; H, 8.93%. Neg. FABMS m/z 757 [M-H]-, IR vz cm-‘: 3370 (OH), 2950 (CH), 1370, 1260, 1160, 1080, 1040. ‘H NMR (pyridine-d,): 65.56 (lH, br d, J=5.3 Hz, H-6), 5.26 (lH, d, J=7.9 Hz, H-1”),4.73(1H,d,J=7.7 Hz,H-1’),3.%(lH,m,H-3),3.80 (lH, H-l, overlapping with H-5’), 1.34 (3H, s, H-19), 1.20 (3H, d, J=7.0 Hz, H-21), 1.08 (3H, s, H-18), 0.98 (3H, d, J = 6.2 Hz, H-26 or H-27), 0.94 (3H, d, J=6.3 Hz, H-26 or H-27). Ac~nowledgeme~t~We are grateful to Dr Y. Shida and Mrs Y. Katoh of the Central Analytical Centre of our
College for the measurement of mass spectra, and Mr H. Fukaya for elemental analyses. REFERENCES
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