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Steroidal glycosides and cardenolide glycosides from Asclepias fruticosa
Pergamon
0031-9422(94)E0209-B
STEROIDAL
GLYCOSIDES AND CARDENOLIDE ASCLEPIAS FRUTICOSA
Phytochemistry, Vol. 37, No. 1, pp. 217-226, 1994 Copyrioht 8 1994 Ekvier Science Ltd Pnnted in Gnat Britain All rights reserved 0031-9422,94 $7.00+0.00
GLYCOSIDES
FROM
TSU?OMU WARASHINAand TADATAKANORO Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, Yada 52-1, Shizuoka, 422, Japan (Received 7 December 1993)
Key Word Index-Asclepias
fruticosa;
Asclepiadaceae; pregnane glycoside; cardenolide glycoside.
Abstract-Asclepias fruticosa afforded, in addition to five known pregnane glycosides and 11 known cardenolide glycosides, four new pregnane glycosides and eleven new cardenolide glycosides. Structures of these compounds were elucidated by spectroscopic methods and from chemical evidence.
INTRODUCTION In connection with a study on the constituents of some plants in the Asclepiadaceae, we have investigated Asclepias fruticosa L. The isolation and structures of five pregnane glycosides from this plant have been reported [ 11. We describe now the identification of four pregnane glycosides and 11 cardenolide glycosides in addition to the known compounds 6 [2,3], 7-8 [2], 9 [2,4], 10 [2], 11 CZ 31, 12 PI, 13 [Sl, 23 C63,24 C71,25 C81. 1 H
RESULTSAND DISCUSSION The methanolic extract of the whole plant of A. frutiafforded new compounds 2-5, 14-22, 26 and 27. Compounds 2 and 3 gave 1 as the aglycone moiety on acid hydrolysis which was identified as lineolon by comparison of the 13C and ‘HNMR spectral data with the reported data [9, lo]. Compounds 4 and 5 were obtained in a small amount (2 and 1 mg, respectively) and acid hydrolysis was not possible to identify the aglycone moiety. However, the ’ 3C NMR spectra 4 and 5 suggested that the aglycone moiety in each case was isolineolon [lo]. Compound 2 showed four anomeric proton signals 64.93 (lH, dd, 5=9.5, 2.0Hz), 4.55 (lH, dd, 5=9.5, 2.0 Hz), 4.83 (lH, dd, J=9.5, 2.0 Hz) and 4.56 (lH, dd, J =9.5, 2.0 Hz) in the ‘HNMR spectrum. In the “C NMR spectrum, four anomeric carbon signals were observed at 6 95.9,100.4 x 2 and 99.2. The signal due to C3 of the glycone at 6 78.0 shifted downfield by comparison with that of lineolon. This glycosylation shift indicated that the sugar chain was attached at the C-3 position. Acid hydrolysis gave digitoxose, olivose and oleandrose as the sugar moiety and analysis by gas chromatography showed that the relative ratio of these monosaccharides was two digitoxoses to one olivose and one oleandrose (see Experimental). The FAB-mass spectrum of 2 revealed
2 digitoxoee 41 3 digitoxoee 3
olivoee 41 digitoxcse 41 deandroee deandross 41 digitoxoee 41 oleandroee
cosa
R
4 digitoxcee 41 5 digitoxoee 41
deandrase 41 deandroee 4(
digitoxoee 9 cymarose 5
deandroee deandrose
a [M+Na]+ ion peak at m/z 921, which was larger by 534 mass units than that of lineolon, and the extra mass units corresponded to the total M, of these four monosaccharides. The C-HCOSY spectrum of 2 revealed the cross peaks between the anomeric carbon signals at 695.9, 100.4, 99.2, 100.4 and the anomeric proton signals at 64.93 (lH, dd, J=9.5,2.0 Hz), 4.55 (lH, dd, 5=9.5,2.0 Hz), 4.83 (lH,dd, J=9.5,2.0 Hz), 4.56 (lH, 217
T. WARASHINA and T. NORO
218
dd, J= 9.5, 2.0 Hz), respectively. In the ‘H NMR spec-
R5
I36
Me
a-H
H
H
CHO
a-H
H
H
R’
6
R4
I?3
a-H, @OH
7
l-l
8 9
H
a-H, &OH
Cl-IO
a-H
H
H
H
a-OH. P-H
CHO
a-H
H
H
10
OH
a-H, &OH
CHO
a-H
H
H
il
H
a-H, fi-0l-i
Me
a-H
H
OH
12
H
a-OH, P-H
Me
a-H
H
OH
13
H
a-H, P-OH
CHO
a-H
H
OH
14
H
Me
a-H
H
OH
I”
, ..nS
II
N,
3
S
15
WI
a-H, P-OH
Me
a-H
H
OH
16
H
a-H, P-OH
Me
a-OH
H
H
17
H
a-H, P-OH
CHO
a-OH
H
H
18
H
CHO
P-OH
H
H
II
I
N,
,\as
19
t-i
a-H, B-OH
CHO
P-OH
H
H
20
l-l
a-H. P-OH
Me
P-OH
H
OH
21
H
a-OH. P-H
Me
P-OH
H
OH
22
H
CHO
a-H
OH
H
II
I
N,
.,S
R3
R2
H
k R’O
H
~ n
R’ 23
H
24
H
R2
R3
Me C&OH
a-H
CHO
a-H
a-H
25
p-Gdeoxyalbse
26
&8-deoxyallose CHO
p-OH
27
p-6deoxyallose
a-OH
Me
trum, the characteristic signals of H-3fl in digitoxopyranosewereseenat~4.22(IH,q,J=3.OHz)and4.2S(lH,q, J=3.0 Hz), and the signal of H-40 in digitoxopyranose were observed at S 3.21 (1H, dd, J = 9.5, 3.0 Hz) and 3.24 (lH, dd, J =9.5, 3.0 Hz). In the HMBC spectrum, 3J,,li, s were confirmed between these characteristic H38 signals and the anomeric carbon signals as follows, 64.22 (If-I, q, J=3.0Hz) and 695.9, 64.25 (fH, q, J = 3.0 Hz) and 6 99.2. From the above results, the signal at 695.9, 64.93 (IH, dd, J=9.5, 2.0Hz) and 699.2, 64.83 (lH, dd, J =9.5, 2.0 Hz) were assigned to the anomeric carbon and proton signals of digitoxopyranoses. Similarly, a 3Jo,, was observed between the methoxyl proton signals at 6 3.40 (3H, s) and the C-3 signal at 6 80.4, and 2J,,, s were seen between this C-3 signal and H-2 signals at S 1.48 (lH, dt, J=9.5, 12.0 Hz) and 2.32 (lH, ddd, J= 12.0, 4.5, 2.0 Hz). The carbon signal at 6 35.3 which had ’ J(,--, s with the above H-2 signals revealed a 2Jt,,, with the anomeric proton signal at 6 4.56 (1H, dd, J = 9.5,2.0 Hz). Thus, the anomeric signals at 6 100.4 and 64.56 (IH, dd, J=9.5. 2.0 Hz) belonged to oleandropyranose, and the remaining anomeric signals at d 100.4 and 6 4.55 (lH, dd, J = 9.5,2.0 Hz) were assigned to olivopyranose. In addition, the sugar linkage was determined by the HMBC and the difference nuclear Overhauser effect (NOE) spectra. The signal at 695.9 due to the anomeric carbon of digitoxopyranose showed a 3JtCOCHtto the signal due to the H-3 of lineolon [S 3.56 (lH, m)J, and the C-3 signal of lineolon exhibited a 3Jc,-wHj to the anomeric proton one of digitoxopyranose cS4.93 (lH, dd, J = 9.5,2.0 Hz)]. Accordingly, the sugar which was attached at the C-3 position of the aglycone was decided to be digitoxopyranose. Secondly, the C-4 and H-4 signals of the above digitoxopyranose [S 82.8 and 3.21 (lH, dd, J = 9.5, 3.0 Hz)] showed 3Jo,,,, s to the anomeric proton and carbon signals of olivopyranose [S 4.55 (lH, dd, J =9.5, 2.0 Hz) and 100.41, respectively. Thirdly, the C-4 and H-4 signals of olivopyranose [S 87.9 and 2.97 (lH, t, J =9.5 Hz)] displayed 3JtcwH, s to the anomeric proton and carbon signals of another digitoxopyranose 164.83 (lH, dd, J=9.5,2.0 Hz) and 99.21. Finally, between the C4 and H-4 signals of digitoxopyranose [S 82.2 and 3.24 (lH, dd, J =9.5, 3.0 Hz)] and the anomeric ones of oleandropyranose cS4.56 (lH, dd, J=9.5, 2.0 Hz) and 100.41, 3Jtcw,, s were observed. In the difference NOE spectrum, irradiation at the anomeric proton signal at 6 4.93 (lH, dd, J =9.5, 2.0 Hz) which was assigned to digitoxopyranose revealed an NOE to the H-3 signal of the aglycone [6 3.56 (lH, m)]. Similarly, NOES were observed between the anomeric proton signal of olivopyranose [S 4.55 (lH, dd, J = 9.5, 2.0 Hz)] and the H-4 signal of the above digitoxopyranose ES3.21 (IH, dd, J =9.5, 3.0 Hz)], the H-l signal of another digitoxopyranose [S 4.83 (lH, dd, J = 9.5,2.0 Hz)] and the H-4 signal of olivopyranose [S 2.97 (iH, t, J = 9.5 Hz)], the H-l signal of oleandropyranose [S 4.56 (IH, dd, J = 9.5,2.0 Hz)] and the H-4 signal of the above digitoxopyranose [6 3.24 (lH, dd, J=9.5, 3.0 Hz)]. Based on the above evidence, the structure of 2 was determined to be lineolon 3-O-&
Glycosides from Asclepiasfiticosa
219
OH
oleandropyranosyl-(l+4)-/Ldigitoxopyranosyl-( l-+4)+ olivopyranosyl-( 1+4)-#?-digitoxopyranoside. Compound 3, with the molecular formula C4.,H,601, based on the FAB-mass spectrum, showed the signals of lineolon and four monosaccharides the same as 2, but signals for one more methoxyl were observed at 6 56.7 and 3.41 (3H, s) in the 13C and ‘HNMR spectra, respectively. Acid hydrolysis, followed by NaBH, reduction, revealed that digitoxopyrano~ and oleandropyranose existed in a ratio of one to one in this compound. These restdts suggested that the four monosaccharides were two digitoxopyranoses and two oleandropyranoses, and the new methoxyl signals belonged to oleandropyranose. In the HMBC spectrum, the anomeric carbon signals at 6 95.8 and 98.5 showed a 3Jtcccw, to the signals at 64.22 (lH, q, 3=3.0Hz) and 4.23 (lH, q, f = 3.0 Hz), respectively, which are the chara~te~sti~ H-34 signals of digitoxopyranose. By consideration of this argument and the result of the C-HCOSY spectrum, these anomeric carbon signals and proton signals at 64.93 (lH, dd, J=9.5, 2.0 Hz) and 5.00 (lH, dd, J=9.5, 2.0 Hz) were assigned to digitoxopyranoses and the remaining two anomeric signals [S 100.2, 4.50 (lH, dd, J =9.5, 2.0 Hz) and 6 100.3, 4.55 (lH, dd, J=9.5, 2.0 Hz)] were assigned to oleandropyranoses. About the sugar linkage, 3J~cW.j s were observed in the HMBC spectrum as follows, 6 78.0 [the C-3 of aglycone] and 6 4.93 (lH, dd, J=9.5,2.0 Hz) [the H-l of digitoxopyranose], S 82.7 [the C-4 of digitoxopyranose] and 6 4.50 (lH, dd, f =9.5, 2.0 Hz) [the H-l of ol~ndropyranose], 6 82.3 [the C-4 of oleandropyranose] and 6 5.00 (lH, dd, f=9.5, 2.0 Hz) [the H-l of another digitoxopyranose], 6 82.7 [the C-4 of digitoxopyranose] and 6 4.55 (lH, dd, J = 9.5,2.0 Hz) [the H-l of terminal oleandropyranose]. Moreover, in the difference NOE spectra, irradiating at each anomeric proton signal, NOES were observed between the H-l of ~gitoxopyranose [S 4.93 (lH, dd, J = 9.5,2.0 Hz)] and the H-3 of the aglycone [&3.56 (lH, m)], H-l of oleandropyranose [S 4.50 (lH, dd, J = 9.5,2.0 Hz)] and the H-4 of the above digitoxopyranose [S 3.20 (lH, dd, J = 9.5, 3.0 Hz)], the H-l of another digitoxopyranose [S 5.00 (lH, dd, J = 9.5,2.0 Hz)], and the H-4 of the above oleandropyranose cS3.19 (lH, t, J-9.5 Hz)], the H-l of terminal oleandropyranose [S 4.55 (lH, dd, J = 9.5, 2.0 Hz)] and the H-4 of digitoxopyranose cS3.21 (lH, dd, J =9.5,3.0 Hz)]. Based on the above evidence, the structure of 3 was determined to be lineolon 3-O-j-oleandro-
OH
pyranosyl-( 1 + 4)+digitoxopyranosyl-( 1 + 4)+oleandropyranosyl-(1+4)+digitoxopyranoside. As the FAB-mass spectrum of 4 revealed a [M +Na]’ ion peak at m/z 935 which was the same as that of 3, the molecular formula was deduced to be C.,,H,60,,. Acid hydrolysis followed by NaBH, reduction suggested that digitoxopyranose and oleandropyranose existed in a ratio of one to one in this compound. The ‘HNMR spectrum of 4 exhibited four anomeric proton signals E64.94 (lH, dd, J=9.5, 2.OHz), 4.50 (lH, dd, 3=9.5, 2.0 Hz), 5.01 (lH, dd, J=9.5, 2.0Hz) and 4.55 (lH, dd, J =9.5, 2.0 Hz)]. From these results, 4 had two digitoxopyranoses and two oleandropyranoses as the sugar moiety. As to the chemical shifts of this sugar moiety in the ‘H and 13C NMR spectra, they almost corresponded to those of 3. Thus, the sugar linkage was deduced to be the same as that of 3. In the difference NOE spectra, irradiation at the anomeric proton signal at 6 4.94 (1H, dd, J = 9.5,2.0 Hz) which was assigned to digitoxopyranose brought an NOE to the H-3 signal of the aglycone [S 3.56 (lH, m)]. Similarly, NOES were observed between the H-l of oleandropyranose [S 4.50 (lH, dd, J=9.5, 2.0 Hz)] and the H-4 of the above di~toxopyrano~ cS3.20 (lH, dd, J=9.5, 3.0Hz)], the H-l of another digiitoxopyranose [s 5.01 (lH, dd, J = 9.5,2.0 Hz)] and the H-4 of the above oleandropyranose [S 3.19 (lH, c, J = 9.0 Hz)], the H-l of terminal oleandropyranose [S 4.55 (1 H, dd, J = 9.5,2.0 Hz)] and the H-4 of digitoxopyranose [S 3.22 (lH, dd, 1=9.5,3.0 Hz)]. Thus, the structure of 4 was deduced to be isolineolon 3-O-~-oleandropyranosyl(1~4)-~-~~toxopyranosyl~l+4)-/?-oleandropyranosyl(l-+4)-@digitoxopyranoside. Compound 5 showed the signals of isolineolon and four monosaccharides the same as 4 in the ‘H and 13CNMR spectra. The monosaccharides were determined to be one di~toxopyr~o~, two oleandropyranose and one cym~opyrano~ by acid hydrolysis followed by NaBH, reduction. The ‘H and 13CNMR spectra of the sugar moiety in this compound almost corresponded to those of lineolon 3-U+oleandropyranosyl-(l+4)-bcymaropyranosyl-( 1 + 4)+oleandropyransoyl-( 1 --, 4)-j?digitoxopyranoside El]. Moreover, in the NOE spectra, NOES were observed between the H-l of digitoxopyranose [S 4.93 (IH, dd, 3=9.5, 2.0 Hz)] and the H-3 of the aglycone [S 3.56 (lH, m)], the H-l of oleandropyranose [S 4.50 (lH, dd, J =9.5,2.0 Hz)] and the H-4 of the above digitoxopyranose [S 3.20 (lH, dd, J =9.5,3.0 Hz)], the H-
T.
222
WARASMNA and T. NOKO
Table 3. ‘H NMR spectral data of 2-5 (in CDCl, solution at 35”, at 500 MHz) H
2
Aglycone moiety 3 6 12 17 18 19 21 Sugar moiety
3.56 IH, 5.33 lH, 3.70 lH, 3.38 lH, 1.27 3H, 1.13 3H, 2.24 3H,
1 ;?a 2b 3 4 5 6 1 2a 2b 3 4 5 6 -0Me 1 2a 2b 3 4 5 6 -0Me 1 2a 2b 3 4 5 6 -0Mo
digito. 4.93 1H, 1.70% 2.09 lH, 4.22 lH, 3.21 lH, 3.80 IH, 1.24 3H, olv. 4.55 lH, 1.60 iH, 2.24*
m br s br d (I LO) t (10.0) s s s
dd (9.5, 2.0)
3
4
S
3.56 lH, M 5.33 iH, br s 3.70 lH, br d (11.0) 3.37 1H. t (10.0) 1.27 3H, s 1.14 3H, s 2.23 3H, s
3.56 lH, m 5.38 IH, Sr s
3.56 lH, m 5.38 IH, br s
1.18 3H, s 1.08 3H, s 2.27 3H, s
1.19 3H, s 1.09 3H, s 2.27 3H, s
dig&o,
4.93 1W, dd (9.5,Z.0)
4.94 lH, dd (9.5,2.0)
1.70* 1.70* ddd (14.0, 3.0, 2.0) 2.08 lH, ddd (14.0, 3.0, 2.0) 2.10 lH, 4.21 IH, 4.22 lH, 4 (3.0) q (3.0) dd (9.5, 3.0)
3.20 1H, dd (9.5,3.0)
3.20 1H,
dq (9.5, 6.5) d (6.5)
3.80 1H, dq (9.5, 6.5) 1.24 3H, d (6.5) ole. 4.50 1H, dd (9.5, 2.0) 1.55 lH, dt (10.0, 12.5) 2.27 IH, ddd (12.5,4.5, 2.0)
3.80 lH, 1.23 3H, ole. 4.50 lH, 1.55 lH,
dd (9.5, 2.0) dt (10.0, 12.5)
3.53’ 2.97 lH, t (9.5) 3.32 IH, dq (9.5, 6.5) 1.26 3H, d (6.5)
digito. 4.93 lH, 1.70: ddd (14.0, 3.0, 2.0) 2.10 IH, 4.22 lH, g (3.0) 3.20 lH, dd (9.5,3.0) 3.79 lH, dq (9.5,6.5) 1.23 3H, d (6.5) ale. 4.50 lH, dd (9.5,2.0) 1.55’ dt (10.0, 12.0)
dig&o.
3.32*
2.27* 3.32*
dd (9.5, 2.0) ddd (14.44.0,
2.0)
4 (3.0) dd (9.5, 3.0) dq (9.5,6.5) d (6.5)
dd (9.5, 2.0)
2.2v 3.3@
3.17 IH, t (9.0) 3.19 lH, t (9.5) 3.19 lH, t (9.0) 3.33 lH, dq (9.5, 6.5) 3.32 lH, dq (9.5, 6.5) 3.32 lH, dq (9.5, 6.5) 1.28 3H, d (6.5) 1.28 3H, d (6.5) 1.29 3H, d (6.5) 3.40 3H, s 3.41 3H, s 3.41 3H, s dig&o. dig&o. digito. 4.83 1H, dd (9.5,2.0) :g lH, dd (9.5 2.0) 5.00 iH, dd (9S, 2.0) 5.01 lH, dd (9.5, 2.0) 1.73* 1.67* f .67* 1.55* 2.18 lH, ddd (14.0, 3.0, 2.0) 2.11 lH, ddd (14.0, 3.0, 2.0) 2.12 lH, ddd (14.0, 3.5, 2.0) 2.14 lH, ddd (14.0, 4.0, 2.0) 4.25 lH, q (3.0) 3.81 lH, q (3.0) 4.23 lH, q (3.0) 4.23 lH, q (3.0) 3.23 1H, dd (9.5, 3.0) 3.24 lH, dd (9.5, 3.0) 3.21 lN, dd (9.5, 3.0) 3.22 IH, dd (9.5 3.0) 3.90 lH, dq (9.5, 6.5) 3.93 lH, dq (9.5, 6.5) 3.83 IN, dq (9.5, 6.5) 3.83 lH, dq (9.5,&S) 1.28 3H, d (6.5) 1.26 3H, d (6.5) 1.25 3H, d (6.5) 1.26 3H, d (6.5) 3.45 3H, s ole. ole. ObZ. ale. 4.56 lH, dd (9.5, 2.0) 4.50 lH, dd (9.5,2.0) 4.55 IH, dd (9.5, 2.0) 4.55 lH, dd (9.5,2.0) 1.48 lH, dt (9.5, 12.0) 1.51 lH, dt (9.5, 12.5) 1.48 lH, dt (9.5, 125) 1.48 Hi, di (10.0, 12.0) 2.32 IH, ddd (12.44.5, 2.0) 2.32 fH, ddd (12.5,4.5, 2.0) 2.32 IH, ddd (12.0, 4.5, 2.0) 2.33 lH, ddd (12.5,4.5,2.0) 3.17* 3.17s 3.15* 3.W 3.12 lH, t (9.0) 3.12 lH, t (9.0) 3.13 lH, t (9.0) 3.13 lH, t (9.0) 3.33 1H, dq (9.0, 6.5) 3.33 IH, dq (9.0, 6.5) 3.29 lH, dq (9.5.6.5) 3.33 lH, dq (9.5, 6.5) 1.32 3H, d (6.5) 1.31 3H, d 6.5) 1.32 3H, d (6.5) 1.31 3H, d (6.5) 3.39 3H, s 3.40 3H, s 3.40 3H, s 3.40 3H, s
Signal assignments were done based on the consequence of 2D-NMR (C-H COSY and HMBC) spectra and/or the decoupling experiments. *Overlapped with other signals.
2.30; S, 5.14, FAR-MS m/z: 612 [M+Na]+, ‘H and 13C NMR: Tables 4 and 5. Compound I5 . Prisms from MeOH, mp 222-226”. f=]g2 + 60.8” (M&H: c 0.2). Calcd for C&H&,, *H,O: C, 61.25; H, 7.80; found: C, 61.33; H, 7-87. FAB-MS m/z: 551 [M+H] ‘. ‘H and 13CNMR: Tables 4 and 5. Cornp~u~ 16. Amorphous powder. [@]A2 + 12.8” (MeOH; ~0.6). Calcd for Cz&,,O, .9/4H,O: C, 6OSS; H, 8.15; found: C, 60.50; H, 7.86. FAB-MS m/z: 535 /M -I-H] +. ‘H and 13CNMR: Tables 4 and 5.
Compound 17. Amorphous powder. [a];* +445” (MeOH; ~0.3). Ca.i& for C29H,,010* 5/4H@: C, 60.99; H, 7.50; found: C, 61.08; H, 7.57. FAB-MS m/z: 549 [M +N_7+. ‘H and 13CNMR: Tables 4 and 5. Compou~ 18. Amorphous powder. [E];’ -I-11.4” (MeOH; ~0.7). Calcd for C31H41N0$*7/2Hz0: C, 55.84; H, 7.26; N, 2.1@ S, 4.81; found: C, 55.78; H, 7.04, N, 2.17; S, 4.72. FAB-MS m/z: 604 [M+H]+. ‘H and 13CNMR: Tables 4 and 5. Compound 19. Amorphous powder. [a];* +67.4”
Glycosides from
223
Asclepiasfiuticosa
Table 4. r3C NMR spectral data of 14-22, 26 and 27 (at 125.65 MHz)
Aglycone moiety 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Sugar moiety 1’ 2 3 4 5 6 Other 1” 2”
42.2 69.8 72.2 32.0 44.9 27.7 26.7 40.6 49.2 38.1 21.1 38.5 48.8 81.7 73.3’ 37.1 48.8 16.5 13.9 173.6 73.4= 118.2 174.4
43.0 69.4 73.0 33.0 45.0 28.4 26.9 41.0 49.4 38.1 21.6 38.7 49.0 81.8 73.0 38.0 49.4 16.9 14.0 175.2 73.8 118.1 174.4
42.9 69.6 73.0 33.0 45.1 28.3 27.2 41.9 50.1 37.9 21.7 32.8 53.6 85.0 31.8 38.7 85.9 15.6 13.9 181.1 74.1 117.5 174.7
36.6 69.8 72.3 32.7 43.5 28.0 21.2 43.5 48.9 53.0 22.3 34.0 53.4 84.6 31.2 38.6 85.8 15.3 208.1 180.8 74.0 117.6 174.7
36.1 69.7 71.3 33.0 43.3 27.2 26.5 42.4 48.5 52.4 21.0 33.3 51.5 87.9 30.2 36.7 86.2 12.4 206.8 171.3 72.9 116.5 173.6
36.6 69.8 72.2 34.0 43.4 27.8 26.9 42.4 48.5 52.9 21.5 33.0 51.7 87.0 30.9 37.0 86.4 12.8 208.0 172.8 73.2 116.8 173.9
42.8 69.5 72.9 33.0 44.9 28.3 26.8 40.7 49.3 38.0 20.9 33.1 51.7 85.7 70.8 48.7 85.1 13.6 13.8 172.5 73.3 116.9 173.9
42.6 69.1 73.1 33.0 45.0 28.2 26.8 40.7 49.3 38.0 20.9 33.0 51.7 85.9 70.8 48.6 85.1 13.6 13.8 172.6 73.3 116.9 173.9
36.6 70.3 72.0 33.8 43.6b 28.1’ 28.W 42.7* 48.8e 53.0 22.4 40.3 49.3 84.4 42.5* 76.5 62.2 16.1 207.8 174.3 74.4 118.1 174.3
31.5’ 31.2’ 76.5 36.3 43.0s 28.6 26.9 42.7’ 48.5 51.7 21.4 33.1 51.6 87.1 30.8 37.1 86.5 12.9 208.7 172.9 73.3 116.7 173.9
31.4 30.1h 77.2 34.9 44.4 29.1h 27.3 42.5 50.1 36.0 21.5 39.2 53.6 85.1 31.8 38.7 85.9 15.6 12.3 181.2 74.1 117.4 174.6
95.1 91.5 99.6 47.1 68.2 20.9
95.5 93.8 75.4 72.9 70.2 18.8
95.6 91.9 71.8 38.3 66.5 21.6
95.5 91.9 71.7 38.3 66.6 21.6
94.9 91.5 99.5 47.0 68.2 20.9
95.5 92.0 71.7 38.3 66.6 21.6
95.7 91.9 71.8 38.3 66.5 21.6
97.5 92.8 74.0 39.9 68.6 21.6
96.8 93.1 100.1 48.6” 68.5 21.4
99.6 72.5 72.9 74.4 70.4 18.8
99.5 72.6 72.9 74.5 70.4 18.9
-
-
-
-
-
-
160.6 43.5b
-
-
160.0 42.6
160.1 42.7
“‘Assignments may be interchanged within each column. *Measured in CDCl, solution at 35”. tMeasured in pyridine-ds solution at 35 ‘.
(MeOH; ~0.7). Calcd for C,,H,,O,, - 7/4H,O: C, 60.04; H, 7.56; found: C, 60.02; H, 7.65. FAB-MS nrjz: 549 [M +Hl +. ‘H and “CNMR: Tables 4 and 5. Compound 20. Amorphous powder. [cl]p i-66.9” (MeOH; c 0.8). Calcd for C,sH,,O,, - 7/4H,O: C, 59.83; H, 7.88; found: C, 59.96; H, 7.86. FAB-MS m/r 551 [M +Hl +. ‘H and 13CNMR: Tables 4 and 5. Co~pou~ 21. Amorphous powder. [x]gz +41.3 (MeOH; c 0.8). Calcd for C2sH,20,, .9/4H,O: C, 58.92; H, 7.93; found: C, 58.99; H, 7.76. FAB-MS m/z: 551 [M +HI +. ‘H and r3CNMR: Tables 4 and 5. Compound 22. Amorphous powder. [u]i2 -3.3” (MeOH, ~0.4). FAB-MS m/z: 604 [M+H]*. ‘H and r3CNMR: Tables 4 and 5. Compound 26. Amorphous powder. [a]$r +5.2” (MeOH; c 0.7). Calcd for CzsH~,OIO~ 5/4H,O: C, 60.77; H, 7.83; found: C, 60.75; H, 7.84. FAB-MS m/z: 551 [M +Hl ’ ‘H and 13CNMR: Tables 4 and 5. PHYTO 37:1-P
Com~u~ 27. Amorphous powder. [u]i2 -38.2” (MeOH; c 1.4). Calcd for C29H,,0,. H,O: C, 62.80; H, 8.36; found: C, 62.55; H, 8.33. FAB-MS mfr 537 [M +H]+. ‘H and r3CNMR: Tables 4 and 5. As 4, 5 and 22 could be acquired only by a small amount, the elemental analyses of these two compounds were not performed. Deg~~ution of the crude pregnane g~ycoside. The MeOH eluate on Diaion HP-20 column (see Experimental) was chromatographed on a silica gel column and the fr. eluted with CHCl,-MeOH (93 : 7) on this silica gel column was rechromatographed on semi-prep. HPLC (ODS: MeCN-H,O gradient system). The crude fr. was eluted with 2931% MeCN on this semi-prep. HPLC. This crude pregnane glycoside fr. (10 mg) was dissolved in dioxane (1 ml) and 0.2 M H,SO, (5 drops) and this soln was heated at 60” for 90 min. After addition of H,O (1 ml) to this reaction mixt., dioxane was removed by blowing
Aglycone moiety 2 3 4ax 8 9 15 16a 16b 17 18 19 21a 21b 22 Sugar moiety 1’ 2 3 4’a 4’b 5 6 Other 1” 2”a 2”b
H
* 3H, 3H, lH, lH, lH, br d (18.0) br s
br d (18.0)
s s
t (7.0)
cd (12.0, 3.0)
rd (12.0, 3.0)
q (12.0)
lH, lH, lH, 3H,
dd (12.5, 11.5) dd (12.5, 1.5) m d (6.5)
7.55 lH, br s 3.89 lH, br d (17.0) 3.87 lH, br d (17.0)
2.25 1.72 4.28 1.23 4.46 lH, m 1.72 3H, d (6.5)
q (11.5) cd (11.5, 3.0) td (11.5, 3.0)
td (11.5, 4.5)
3H, s 3H, s lH, dd (18.5, 1.5) lH,dd (18.5, 1.5) lH, t (1.5)
* lH, lH, lH, lH,
-
4.25 2.02 2.20 4.55 1.38
lH, lH, lH, lH, 3H, d (6.5)
m
br t (12.5)
br d (12.5)
br s
5.44 lH, s
1.16 0.72 5.19 5.64 6.67
4.55 4.35 1.49 1.67 1.02
16$
-
4.27 2.03 2.19 4.54 1.38
lH, lH, lH, lH, 3H,
d (6.5)
m
br t (12.5)
br d (12.5)
br s
5.44 lH, s
1.09 3H, s 10.07 lH, s 5.18 lH, br d (18.5) 5.61 lH, br d (18.5) 6.66 lH, br s
4.49 lH, ddd (12.0, 11.0, 4.5) 4.37 lH, td (12.0, 4.5) 1.56 lH, q (12.0)
171
’ H NMR spectral data of 14-22, 26 and 27 (at 500 MHz)
ddd (12.0, 10.0, 4.5) cd (12.0, 4.5)
4.46 lH, d (3.0) 4.23 lH, dd (9.5, 3.0)
2.64 1.00 0.70 5.00 5.29 6.10
lH, lH, lH, lH, lH, lH,
-
s s dd (18.0, 1.5) dd (18.0, 1.5) t (1.5)
4.60 4.31 1.50 1.68 1.09 4.79
5.49 lH, s
* 3H, 3H, lH, lH, lH,
2.66 0.92 0.87 4.81 5.05 5.86
f (8.0)
cd (12.0, 4.5)
cd (12.0, 3.5)
q (11.5)
ddd (11.5, 10.0, 4.5) td (11.5, 4.5)
1%
5.08 lH, s
lH, lH, lH, lH, lH, lH,
4.15 4.06 1.47 1.60 1.11 4.56
w
Table 5.
3H, lH, lH, lH, lH,
s s dd (18.5, 2.0) dd (18.5, 2.0) t (2.0)
lH, ddd (12.0, 9.5, 4.5) lH, td (12.0, 4.5) lH, q (12.0) lH, td (11.5, 3.0)
lH, lH, lH, 3H,
dd (13.5, 1.5) dd (13.5, 11.5) m d (6.5)
3.87 lH, dd (17.5, 1.5) 3.89 lH, dd (17.5, 1.5)
7.52 lH, br s
1.72 2.24 4.28 1.23
5.07 lH, s
1.01 9.98 4.81 4.99 5.85
-
3.96 4.09 1.47 1.59
1st
lH, ddd (12.0, 9.5, 4.5) lH, td (12.0, 4.5) lH, q (12.0) lH, td (11.5, 3.0) *
-
4.26 2.02 2.18 4.55 1.37
lH, lH, lH, lH, 3H,
d (6.5)
m
br t (12.0)
br d (12.0)
br s
5.43 lH, s
1.11 3H, s 10.06 lH, s 5.02 lH, br d (18.5) 5.15 lH, br d (18.5) 6.19 lH, br s
4.49 4.39 1.57 1.88 1.27
19$
4.25 lH, 2.00 lH, 2.18 lH, 4.53 lH, 1.36 3H,
d (6.5)
m
dt (13.5, 25) td (13.5, 2.5)
t (2.5)
5.03 lH, s
ddd (11.0, 10.0,4.0) td (11.0, 4.0) q (11.0) td (120, 3.0)
2.00 lH, 2.27 lH, 4.59 lH, 1.39 3H, 7.61 lH, br s 3.86 lH, br d (16.5) 3.92 lH, br d (16.5)
-
dd (12.0, 1.5) t (12.0) m d (6.5)
5.58 lH, s
5.05 lH, td (8.0,4.0) 3.04 lH, d (4.0) 0.94 3H s 9.89 H-i, s 5.03 lH, br d (17.5) 5.17 lH, br d (17.5) 6.23 H-I, br s
4.47 lH, 4.43 lH, 1.55 lH, 1.78 lH,
4.11 lH, dd (12.0,4.5) 2.00 lH, ddd (12.0, 4.5, 1.5) 2.10 lH, q (12.0) 3.74 lH, m 1.34 3H, d (6.5)
s s dd (18.0, 1.5) dd (18.0, 1.5) t (1.5)
dd (15.5, 9.5)
4.79 lH, dd (9.5, 3.0) 244 lH, dd (15.5, 3.0)
5.44 lH, s
ddd (120, 10.0,4.5) td (12.0, 4.5) q (12.0) td (11.5, 3.0)
3.09 lH, 1.24 3H, 0.70 3H, 5.03 lH, 5.16 lH, 6.21 lH,
4.54 lH, 4.29 lH, 1.52 lH, 1.80 lH,
4.55 lH, ddd (11.5, 9.5,4.5) 4.32 lH, td (11.5, 4.5) 1.48 lH, q (11.5) 1.79 lH, td (11.5, 3.0) 1.06 * 4.87 lH, dd (9.5, 3.0) 2.43 lH, dd (15.5, 3.0) 3.09 lH, dd (15.5, 9.5) 1.23 3H, s 0.72 3H, s 5.02 lH, dd (18.0, 1.5) 5.15 lH, dd (18.0, 1.5) 6.20 lH, br s
4.35 lH, dq (9.5, 6.5) 1.64 3H, d (6.5)
4.36 lH, dq (9.5, 6.5) 1.64 3H, d (6.5)
dd (9.5, 3.0)
t (3.0)
d (8.0) dd (8.0, 3.0)
5.39 lH, 3.94 lH, 4.68 lH, 3.69 lH,
5.37 lH, d (8.0) 3.92 lH, dd (8.0, 3.0) 4.68 lH, t (3.0) 3.69 lH, dd (9.5, 3.0)
s s dd (18.0, 1.5) dd (18.0, 1.5) t (1.5)
1.19 3H, 0.69 3H, 5.21 lH, 5.66 lH, 6.68 lH,
-
3.96 lH, m
1.14 3H, s 10.02 lH, s 5.03 lH, br d (18.5) 5.17 lH, br d (18.5) 6.21 lH, br s
-
4.00 lH, m
Signal assignments were done based on the consequence of ZD-NMR (C-H COSY, NMBC and H-H COSY) spectra. *Overlapping with other signals. tMeasured in CDCI, solution at 35”. SMeasured in pyridine-d, solution at 35”.
8 9 15 16a 16b 17 18 19 21a 21b 22 Sugar moiety 1’ 2 3 4’a 4’b 5 6 Other 1” 2” a 2”b
4tiX
Aglycone moiety 2 3
Table 5. Continued
226
T. WARASHINAand
air on a hot water bath for a short time. The soln was then heated at 60” for 90 min once more. After hydrolysis, the soln was passed through a Mitsubishi Diaion HP-20 column and the absorbed material was eluted with MeOH. The MeOH eluate was coned to dryness and the residue was recrystallized from MeOH to give pure lineolon (1) (1 mg), mp 237-244”, [a];’ + 12.7” (MeOH; ~0.38) (lit. mp 233-239”, [I]:: + 13”; MeOH) [13]. ‘H NMR [(CD,),SO]: 6 1.07 (3H, s, H-19), 1.28 (3H, s, H18), 2,10(3H, s, H-21), 3.34(1H, m, H-3), 3.30 (overlapped, H-12 or 17) 5.19 (lH, br s, H-6). 13CNMR [(CD,),SO]: S 13.8 (C-18), 17.8 (C-19), 20.8 (C-16) 27.9 (C-11), 30.9 (C2), 31.4 (C-21), 33.4 (C-15) 34.3 (C-7), 36.3 (C-lo), 38.2 (Cl), 42.1 (C-4), 43.7 (C-9), 56.4 (C-13) 60.0 (C-17), 67.8 (C12), 70.3 (C-3), 73.3 (C-8), 86.4 (C-14), 117.9 (C-6), 139.2 (C-5), 209.2 (C-20) [9, lo]. However, isolineolon was not acquired. Acid hydrolysis of 2-5,26 and 21. Compounds 2 and 3 (ca 1 mg) dissolved in dioxane (4 drops) and 0.2 N H,SO, (1 drop) was heated at 60” for 90 min. After addition of H,O (3 drops) to this reaction mixt., dioxane was removed as before and then the soln was heated at 60” for 90 min. After hydrolysis, the soln was passed through a Mitsubishi Diaion HP-20 column and eluted with MeOH. The MeOH eluate was coned to dryness and the residual aglycone was identified by HPLC with an authentic sample [Conditions: column; YMC ODS, flow rate; 1.0 ml min- r, 40% MeOH in water, R, (min); lineolon 9.41. Hydrolysis of 4 and 5 were not carried out, because of the unavailability of an authentic sample of aglycone and small amounts available of 4 and 5. Thus, the aglycone moieties of 4 and 5 were identified as isolineolon on the basis of a comparison of the l3 C NMR spectral data with that of isolineolon [lo]. Acid hydrolysis, to confirm the monosaccharides in the sugar chain of 2-5, were done with a small amount (ca 0.1 mg) in the same way as above. Compounds 26 and 27 (ca 0.1 mg) were dissolved in dioxane and 10% H,SO, (each one drop) and this soln was heated at loo” for 1 hr. After hydrolysis, the soln was passed through an Amberlite IRA-60E column and the eluate was coned to give a residue, which was reduced with NaBH, (ca 1 mg) for 1 hr at room temp. The reaction mixt. was passed through an Amberlite IR-120B column and the eluate was coned to dryness. Boric acid was removed by codistillation with MeOH and the residue was acetylated with Ac,O and pyridine (each one drop) at loo” for 1 hr. The reagents were evapd in t’acuo. From each glycoside, cymaritol acetate, oleandritol acetate, digitoxitol acetate, olivitol acetate and 6-deoxyallitol acetate were detected by GC [Conditions: column; Supelco SP-2380 capillary column (0.25 mm x 30 m), column temp.; 200”, carrier
T. NORO
gas; N,, R, (min); cymaritol acetate 6.6, oleandritol acetate 7.5, digitoxitol acetate 9.4, olivitol acetate 10.5 and 6deoxyalhtol acetate 13.41. The relative ratio of each monosaccharide in 2-5 was determined based on the peak area. Synthesis of 17~hydroxydigitoxigenin. The synthesis of 17a-hydroxydigitoxigenin from digitoxigenin (40 mg) was carried out as reported in ref. [ 1I]. 17a-Hydroxydigitoxigenin (3 mg), mp 213-218” from MeOH. [aliz - 17.5” (MeOH; ~0.1) (lit. mp 215-219”, [x]k6 -22.8”; MeOH) [ll]. r3C NMR [C,D,N] : 6 15.7 (C-18), 21.4, 21.7 (C-7 and C-l I), 24.2 (C-19) 27.4, 28.8, 30.4 (C-l, C-2 and C-6), 31.9 (C-15), 33.2 (C-12) 34.4 (C-4), 35.8, 35.9 (C-9 and Clo), 36.8 (C-5), 38.7 (C-16), 42.9 (C-8), 53.7 (C-13), 66.0 (C3) 74.1 (C-21), 85.3 (C-14), 86.0 (C-17), 117.4 (C-22), 174.7 (C-23), 181.3 (C-20). Acknowledgement-We
thank the staff of the Central Analytical Laboratory of this school for measurement of mass spectra and the element analyses.
REFERENCES 1.
Warashina, T. and Noro, T. (1994) Chem. Pharm. Bull.
42, 322. 2. Cheung, H. T. A., Chiu, F. C. K., Watson, T. R. and Wells, R. J. (1983) J. Chem. Sot. Perkin Trans. I 2827. 3. Cheung, H. T. A. and Watson, T. R. (1980) J. Chem. Sot., Perkin Trans. I 2162. 4. Lee, S. M. and Seiber, J. N. (1983) Phytochemistry 22, 923. 5. Rodriges-Hahn, L. and Fonseca, G. (1991) Phytochemistry 30, 3941. 6. Rangaswami, S. and Reichstein, T. (1949) Helu. Chim. Acta 32, 939. 7. Abe, F., Mori, Y. and Yamauchi, T. (1991) Chem. Pharm. Bull. 39, 2709. 8. Cheung, H. T. A., Nelson, C. J. and Watson, T. R. (1988) J. Chem. Sot., Perkin Trans. I 1851.
9. Yamagishi, T., Hayashi, K., Mitsuhashi, H., Imanari, M. and Matsushita, K. (1973) Tetrahedron Letters 3527.
10. Yamagishi, T., Hayashi, K., Mitsuhashi, H., Imanari, M. and Matsushita, K. (1973) Tetrahedron Letters 3531.
11. Saito, Y., Kanemasa, Y. and Okada, M. (1970) Chem. Pharm. Bull. 18, 629. 12. Abe, F., Mori, Y. and Yamauchi, T. (1992) Chem. Pharm. Bull. 40, 2917. 13. Deepak, D., Khare, A. and Khare, M. P. (1989) Phytochemistry 28, 3255.
0031-9422(94)E0209-B
STEROIDAL
GLYCOSIDES AND CARDENOLIDE ASCLEPIAS FRUTICOSA
Phytochemistry, Vol. 37, No. 1, pp. 217-226, 1994 Copyrioht 8 1994 Ekvier Science Ltd Pnnted in Gnat Britain All rights reserved 0031-9422,94 $7.00+0.00
GLYCOSIDES
FROM
TSU?OMU WARASHINAand TADATAKANORO Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, Yada 52-1, Shizuoka, 422, Japan (Received 7 December 1993)
Key Word Index-Asclepias
fruticosa;
Asclepiadaceae; pregnane glycoside; cardenolide glycoside.
Abstract-Asclepias fruticosa afforded, in addition to five known pregnane glycosides and 11 known cardenolide glycosides, four new pregnane glycosides and eleven new cardenolide glycosides. Structures of these compounds were elucidated by spectroscopic methods and from chemical evidence.
INTRODUCTION In connection with a study on the constituents of some plants in the Asclepiadaceae, we have investigated Asclepias fruticosa L. The isolation and structures of five pregnane glycosides from this plant have been reported [ 11. We describe now the identification of four pregnane glycosides and 11 cardenolide glycosides in addition to the known compounds 6 [2,3], 7-8 [2], 9 [2,4], 10 [2], 11 CZ 31, 12 PI, 13 [Sl, 23 C63,24 C71,25 C81. 1 H
RESULTSAND DISCUSSION The methanolic extract of the whole plant of A. frutiafforded new compounds 2-5, 14-22, 26 and 27. Compounds 2 and 3 gave 1 as the aglycone moiety on acid hydrolysis which was identified as lineolon by comparison of the 13C and ‘HNMR spectral data with the reported data [9, lo]. Compounds 4 and 5 were obtained in a small amount (2 and 1 mg, respectively) and acid hydrolysis was not possible to identify the aglycone moiety. However, the ’ 3C NMR spectra 4 and 5 suggested that the aglycone moiety in each case was isolineolon [lo]. Compound 2 showed four anomeric proton signals 64.93 (lH, dd, 5=9.5, 2.0Hz), 4.55 (lH, dd, 5=9.5, 2.0 Hz), 4.83 (lH, dd, J=9.5, 2.0 Hz) and 4.56 (lH, dd, J =9.5, 2.0 Hz) in the ‘HNMR spectrum. In the “C NMR spectrum, four anomeric carbon signals were observed at 6 95.9,100.4 x 2 and 99.2. The signal due to C3 of the glycone at 6 78.0 shifted downfield by comparison with that of lineolon. This glycosylation shift indicated that the sugar chain was attached at the C-3 position. Acid hydrolysis gave digitoxose, olivose and oleandrose as the sugar moiety and analysis by gas chromatography showed that the relative ratio of these monosaccharides was two digitoxoses to one olivose and one oleandrose (see Experimental). The FAB-mass spectrum of 2 revealed
2 digitoxoee 41 3 digitoxoee 3
olivoee 41 digitoxcse 41 deandroee deandross 41 digitoxoee 41 oleandroee
cosa
R
4 digitoxcee 41 5 digitoxoee 41
deandrase 41 deandroee 4(
digitoxoee 9 cymarose 5
deandroee deandrose
a [M+Na]+ ion peak at m/z 921, which was larger by 534 mass units than that of lineolon, and the extra mass units corresponded to the total M, of these four monosaccharides. The C-HCOSY spectrum of 2 revealed the cross peaks between the anomeric carbon signals at 695.9, 100.4, 99.2, 100.4 and the anomeric proton signals at 64.93 (lH, dd, J=9.5,2.0 Hz), 4.55 (lH, dd, 5=9.5,2.0 Hz), 4.83 (lH,dd, J=9.5,2.0 Hz), 4.56 (lH, 217
T. WARASHINA and T. NORO
218
dd, J= 9.5, 2.0 Hz), respectively. In the ‘H NMR spec-
R5
I36
Me
a-H
H
H
CHO
a-H
H
H
R’
6
R4
I?3
a-H, @OH
7
l-l
8 9
H
a-H, &OH
Cl-IO
a-H
H
H
H
a-OH. P-H
CHO
a-H
H
H
10
OH
a-H, &OH
CHO
a-H
H
H
il
H
a-H, fi-0l-i
Me
a-H
H
OH
12
H
a-OH, P-H
Me
a-H
H
OH
13
H
a-H, P-OH
CHO
a-H
H
OH
14
H
Me
a-H
H
OH
I”
, ..nS
II
N,
3
S
15
WI
a-H, P-OH
Me
a-H
H
OH
16
H
a-H, P-OH
Me
a-OH
H
H
17
H
a-H, P-OH
CHO
a-OH
H
H
18
H
CHO
P-OH
H
H
II
I
N,
,\as
19
t-i
a-H, B-OH
CHO
P-OH
H
H
20
l-l
a-H. P-OH
Me
P-OH
H
OH
21
H
a-OH. P-H
Me
P-OH
H
OH
22
H
CHO
a-H
OH
H
II
I
N,
.,S
R3
R2
H
k R’O
H
~ n
R’ 23
H
24
H
R2
R3
Me C&OH
a-H
CHO
a-H
a-H
25
p-Gdeoxyalbse
26
&8-deoxyallose CHO
p-OH
27
p-6deoxyallose
a-OH
Me
trum, the characteristic signals of H-3fl in digitoxopyranosewereseenat~4.22(IH,q,J=3.OHz)and4.2S(lH,q, J=3.0 Hz), and the signal of H-40 in digitoxopyranose were observed at S 3.21 (1H, dd, J = 9.5, 3.0 Hz) and 3.24 (lH, dd, J =9.5, 3.0 Hz). In the HMBC spectrum, 3J,,li, s were confirmed between these characteristic H38 signals and the anomeric carbon signals as follows, 64.22 (If-I, q, J=3.0Hz) and 695.9, 64.25 (fH, q, J = 3.0 Hz) and 6 99.2. From the above results, the signal at 695.9, 64.93 (IH, dd, J=9.5, 2.0Hz) and 699.2, 64.83 (lH, dd, J =9.5, 2.0 Hz) were assigned to the anomeric carbon and proton signals of digitoxopyranoses. Similarly, a 3Jo,, was observed between the methoxyl proton signals at 6 3.40 (3H, s) and the C-3 signal at 6 80.4, and 2J,,, s were seen between this C-3 signal and H-2 signals at S 1.48 (lH, dt, J=9.5, 12.0 Hz) and 2.32 (lH, ddd, J= 12.0, 4.5, 2.0 Hz). The carbon signal at 6 35.3 which had ’ J(,--, s with the above H-2 signals revealed a 2Jt,,, with the anomeric proton signal at 6 4.56 (1H, dd, J = 9.5,2.0 Hz). Thus, the anomeric signals at 6 100.4 and 64.56 (IH, dd, J=9.5. 2.0 Hz) belonged to oleandropyranose, and the remaining anomeric signals at d 100.4 and 6 4.55 (lH, dd, J = 9.5,2.0 Hz) were assigned to olivopyranose. In addition, the sugar linkage was determined by the HMBC and the difference nuclear Overhauser effect (NOE) spectra. The signal at 695.9 due to the anomeric carbon of digitoxopyranose showed a 3JtCOCHtto the signal due to the H-3 of lineolon [S 3.56 (lH, m)J, and the C-3 signal of lineolon exhibited a 3Jc,-wHj to the anomeric proton one of digitoxopyranose cS4.93 (lH, dd, J = 9.5,2.0 Hz)]. Accordingly, the sugar which was attached at the C-3 position of the aglycone was decided to be digitoxopyranose. Secondly, the C-4 and H-4 signals of the above digitoxopyranose [S 82.8 and 3.21 (lH, dd, J = 9.5, 3.0 Hz)] showed 3Jo,,,, s to the anomeric proton and carbon signals of olivopyranose [S 4.55 (lH, dd, J =9.5, 2.0 Hz) and 100.41, respectively. Thirdly, the C-4 and H-4 signals of olivopyranose [S 87.9 and 2.97 (lH, t, J =9.5 Hz)] displayed 3JtcwH, s to the anomeric proton and carbon signals of another digitoxopyranose 164.83 (lH, dd, J=9.5,2.0 Hz) and 99.21. Finally, between the C4 and H-4 signals of digitoxopyranose [S 82.2 and 3.24 (lH, dd, J =9.5, 3.0 Hz)] and the anomeric ones of oleandropyranose cS4.56 (lH, dd, J=9.5, 2.0 Hz) and 100.41, 3Jtcw,, s were observed. In the difference NOE spectrum, irradiation at the anomeric proton signal at 6 4.93 (lH, dd, J =9.5, 2.0 Hz) which was assigned to digitoxopyranose revealed an NOE to the H-3 signal of the aglycone [6 3.56 (lH, m)]. Similarly, NOES were observed between the anomeric proton signal of olivopyranose [S 4.55 (lH, dd, J = 9.5, 2.0 Hz)] and the H-4 signal of the above digitoxopyranose ES3.21 (IH, dd, J =9.5, 3.0 Hz)], the H-l signal of another digitoxopyranose [S 4.83 (lH, dd, J = 9.5,2.0 Hz)] and the H-4 signal of olivopyranose [S 2.97 (iH, t, J = 9.5 Hz)], the H-l signal of oleandropyranose [S 4.56 (IH, dd, J = 9.5,2.0 Hz)] and the H-4 signal of the above digitoxopyranose [6 3.24 (lH, dd, J=9.5, 3.0 Hz)]. Based on the above evidence, the structure of 2 was determined to be lineolon 3-O-&
Glycosides from Asclepiasfiticosa
219
OH
oleandropyranosyl-(l+4)-/Ldigitoxopyranosyl-( l-+4)+ olivopyranosyl-( 1+4)-#?-digitoxopyranoside. Compound 3, with the molecular formula C4.,H,601, based on the FAB-mass spectrum, showed the signals of lineolon and four monosaccharides the same as 2, but signals for one more methoxyl were observed at 6 56.7 and 3.41 (3H, s) in the 13C and ‘HNMR spectra, respectively. Acid hydrolysis, followed by NaBH, reduction, revealed that digitoxopyrano~ and oleandropyranose existed in a ratio of one to one in this compound. These restdts suggested that the four monosaccharides were two digitoxopyranoses and two oleandropyranoses, and the new methoxyl signals belonged to oleandropyranose. In the HMBC spectrum, the anomeric carbon signals at 6 95.8 and 98.5 showed a 3Jtcccw, to the signals at 64.22 (lH, q, 3=3.0Hz) and 4.23 (lH, q, f = 3.0 Hz), respectively, which are the chara~te~sti~ H-34 signals of digitoxopyranose. By consideration of this argument and the result of the C-HCOSY spectrum, these anomeric carbon signals and proton signals at 64.93 (lH, dd, J=9.5, 2.0 Hz) and 5.00 (lH, dd, J=9.5, 2.0 Hz) were assigned to digitoxopyranoses and the remaining two anomeric signals [S 100.2, 4.50 (lH, dd, J =9.5, 2.0 Hz) and 6 100.3, 4.55 (lH, dd, J=9.5, 2.0 Hz)] were assigned to oleandropyranoses. About the sugar linkage, 3J~cW.j s were observed in the HMBC spectrum as follows, 6 78.0 [the C-3 of aglycone] and 6 4.93 (lH, dd, J=9.5,2.0 Hz) [the H-l of digitoxopyranose], S 82.7 [the C-4 of digitoxopyranose] and 6 4.50 (lH, dd, f =9.5, 2.0 Hz) [the H-l of ol~ndropyranose], 6 82.3 [the C-4 of oleandropyranose] and 6 5.00 (lH, dd, f=9.5, 2.0 Hz) [the H-l of another digitoxopyranose], 6 82.7 [the C-4 of digitoxopyranose] and 6 4.55 (lH, dd, J = 9.5,2.0 Hz) [the H-l of terminal oleandropyranose]. Moreover, in the difference NOE spectra, irradiating at each anomeric proton signal, NOES were observed between the H-l of ~gitoxopyranose [S 4.93 (lH, dd, J = 9.5,2.0 Hz)] and the H-3 of the aglycone [&3.56 (lH, m)], H-l of oleandropyranose [S 4.50 (lH, dd, J = 9.5,2.0 Hz)] and the H-4 of the above digitoxopyranose [S 3.20 (lH, dd, J = 9.5, 3.0 Hz)], the H-l of another digitoxopyranose [S 5.00 (lH, dd, J = 9.5,2.0 Hz)], and the H-4 of the above oleandropyranose cS3.19 (lH, t, J-9.5 Hz)], the H-l of terminal oleandropyranose [S 4.55 (lH, dd, J = 9.5, 2.0 Hz)] and the H-4 of digitoxopyranose cS3.21 (lH, dd, J =9.5,3.0 Hz)]. Based on the above evidence, the structure of 3 was determined to be lineolon 3-O-j-oleandro-
OH
pyranosyl-( 1 + 4)+digitoxopyranosyl-( 1 + 4)+oleandropyranosyl-(1+4)+digitoxopyranoside. As the FAB-mass spectrum of 4 revealed a [M +Na]’ ion peak at m/z 935 which was the same as that of 3, the molecular formula was deduced to be C.,,H,60,,. Acid hydrolysis followed by NaBH, reduction suggested that digitoxopyranose and oleandropyranose existed in a ratio of one to one in this compound. The ‘HNMR spectrum of 4 exhibited four anomeric proton signals E64.94 (lH, dd, J=9.5, 2.OHz), 4.50 (lH, dd, 3=9.5, 2.0 Hz), 5.01 (lH, dd, J=9.5, 2.0Hz) and 4.55 (lH, dd, J =9.5, 2.0 Hz)]. From these results, 4 had two digitoxopyranoses and two oleandropyranoses as the sugar moiety. As to the chemical shifts of this sugar moiety in the ‘H and 13C NMR spectra, they almost corresponded to those of 3. Thus, the sugar linkage was deduced to be the same as that of 3. In the difference NOE spectra, irradiation at the anomeric proton signal at 6 4.94 (1H, dd, J = 9.5,2.0 Hz) which was assigned to digitoxopyranose brought an NOE to the H-3 signal of the aglycone [S 3.56 (lH, m)]. Similarly, NOES were observed between the H-l of oleandropyranose [S 4.50 (lH, dd, J=9.5, 2.0 Hz)] and the H-4 of the above di~toxopyrano~ cS3.20 (lH, dd, J=9.5, 3.0Hz)], the H-l of another digiitoxopyranose [s 5.01 (lH, dd, J = 9.5,2.0 Hz)] and the H-4 of the above oleandropyranose [S 3.19 (lH, c, J = 9.0 Hz)], the H-l of terminal oleandropyranose [S 4.55 (1 H, dd, J = 9.5,2.0 Hz)] and the H-4 of digitoxopyranose [S 3.22 (lH, dd, 1=9.5,3.0 Hz)]. Thus, the structure of 4 was deduced to be isolineolon 3-O-~-oleandropyranosyl(1~4)-~-~~toxopyranosyl~l+4)-/?-oleandropyranosyl(l-+4)-@digitoxopyranoside. Compound 5 showed the signals of isolineolon and four monosaccharides the same as 4 in the ‘H and 13CNMR spectra. The monosaccharides were determined to be one di~toxopyr~o~, two oleandropyranose and one cym~opyrano~ by acid hydrolysis followed by NaBH, reduction. The ‘H and 13CNMR spectra of the sugar moiety in this compound almost corresponded to those of lineolon 3-U+oleandropyranosyl-(l+4)-bcymaropyranosyl-( 1 + 4)+oleandropyransoyl-( 1 --, 4)-j?digitoxopyranoside El]. Moreover, in the NOE spectra, NOES were observed between the H-l of digitoxopyranose [S 4.93 (IH, dd, 3=9.5, 2.0 Hz)] and the H-3 of the aglycone [S 3.56 (lH, m)], the H-l of oleandropyranose [S 4.50 (lH, dd, J =9.5,2.0 Hz)] and the H-4 of the above digitoxopyranose [S 3.20 (lH, dd, J =9.5,3.0 Hz)], the H-
T.
222
WARASMNA and T. NOKO
Table 3. ‘H NMR spectral data of 2-5 (in CDCl, solution at 35”, at 500 MHz) H
2
Aglycone moiety 3 6 12 17 18 19 21 Sugar moiety
3.56 IH, 5.33 lH, 3.70 lH, 3.38 lH, 1.27 3H, 1.13 3H, 2.24 3H,
1 ;?a 2b 3 4 5 6 1 2a 2b 3 4 5 6 -0Me 1 2a 2b 3 4 5 6 -0Me 1 2a 2b 3 4 5 6 -0Mo
digito. 4.93 1H, 1.70% 2.09 lH, 4.22 lH, 3.21 lH, 3.80 IH, 1.24 3H, olv. 4.55 lH, 1.60 iH, 2.24*
m br s br d (I LO) t (10.0) s s s
dd (9.5, 2.0)
3
4
S
3.56 lH, M 5.33 iH, br s 3.70 lH, br d (11.0) 3.37 1H. t (10.0) 1.27 3H, s 1.14 3H, s 2.23 3H, s
3.56 lH, m 5.38 IH, Sr s
3.56 lH, m 5.38 IH, br s
1.18 3H, s 1.08 3H, s 2.27 3H, s
1.19 3H, s 1.09 3H, s 2.27 3H, s
dig&o,
4.93 1W, dd (9.5,Z.0)
4.94 lH, dd (9.5,2.0)
1.70* 1.70* ddd (14.0, 3.0, 2.0) 2.08 lH, ddd (14.0, 3.0, 2.0) 2.10 lH, 4.21 IH, 4.22 lH, 4 (3.0) q (3.0) dd (9.5, 3.0)
3.20 1H, dd (9.5,3.0)
3.20 1H,
dq (9.5, 6.5) d (6.5)
3.80 1H, dq (9.5, 6.5) 1.24 3H, d (6.5) ole. 4.50 1H, dd (9.5, 2.0) 1.55 lH, dt (10.0, 12.5) 2.27 IH, ddd (12.5,4.5, 2.0)
3.80 lH, 1.23 3H, ole. 4.50 lH, 1.55 lH,
dd (9.5, 2.0) dt (10.0, 12.5)
3.53’ 2.97 lH, t (9.5) 3.32 IH, dq (9.5, 6.5) 1.26 3H, d (6.5)
digito. 4.93 lH, 1.70: ddd (14.0, 3.0, 2.0) 2.10 IH, 4.22 lH, g (3.0) 3.20 lH, dd (9.5,3.0) 3.79 lH, dq (9.5,6.5) 1.23 3H, d (6.5) ale. 4.50 lH, dd (9.5,2.0) 1.55’ dt (10.0, 12.0)
dig&o.
3.32*
2.27* 3.32*
dd (9.5, 2.0) ddd (14.44.0,
2.0)
4 (3.0) dd (9.5, 3.0) dq (9.5,6.5) d (6.5)
dd (9.5, 2.0)
2.2v 3.3@
3.17 IH, t (9.0) 3.19 lH, t (9.5) 3.19 lH, t (9.0) 3.33 lH, dq (9.5, 6.5) 3.32 lH, dq (9.5, 6.5) 3.32 lH, dq (9.5, 6.5) 1.28 3H, d (6.5) 1.28 3H, d (6.5) 1.29 3H, d (6.5) 3.40 3H, s 3.41 3H, s 3.41 3H, s dig&o. dig&o. digito. 4.83 1H, dd (9.5,2.0) :g lH, dd (9.5 2.0) 5.00 iH, dd (9S, 2.0) 5.01 lH, dd (9.5, 2.0) 1.73* 1.67* f .67* 1.55* 2.18 lH, ddd (14.0, 3.0, 2.0) 2.11 lH, ddd (14.0, 3.0, 2.0) 2.12 lH, ddd (14.0, 3.5, 2.0) 2.14 lH, ddd (14.0, 4.0, 2.0) 4.25 lH, q (3.0) 3.81 lH, q (3.0) 4.23 lH, q (3.0) 4.23 lH, q (3.0) 3.23 1H, dd (9.5, 3.0) 3.24 lH, dd (9.5, 3.0) 3.21 lN, dd (9.5, 3.0) 3.22 IH, dd (9.5 3.0) 3.90 lH, dq (9.5, 6.5) 3.93 lH, dq (9.5, 6.5) 3.83 IN, dq (9.5, 6.5) 3.83 lH, dq (9.5,&S) 1.28 3H, d (6.5) 1.26 3H, d (6.5) 1.25 3H, d (6.5) 1.26 3H, d (6.5) 3.45 3H, s ole. ole. ObZ. ale. 4.56 lH, dd (9.5, 2.0) 4.50 lH, dd (9.5,2.0) 4.55 IH, dd (9.5, 2.0) 4.55 lH, dd (9.5,2.0) 1.48 lH, dt (9.5, 12.0) 1.51 lH, dt (9.5, 12.5) 1.48 lH, dt (9.5, 125) 1.48 Hi, di (10.0, 12.0) 2.32 IH, ddd (12.44.5, 2.0) 2.32 fH, ddd (12.5,4.5, 2.0) 2.32 IH, ddd (12.0, 4.5, 2.0) 2.33 lH, ddd (12.5,4.5,2.0) 3.17* 3.17s 3.15* 3.W 3.12 lH, t (9.0) 3.12 lH, t (9.0) 3.13 lH, t (9.0) 3.13 lH, t (9.0) 3.33 1H, dq (9.0, 6.5) 3.33 IH, dq (9.0, 6.5) 3.29 lH, dq (9.5.6.5) 3.33 lH, dq (9.5, 6.5) 1.32 3H, d (6.5) 1.31 3H, d 6.5) 1.32 3H, d (6.5) 1.31 3H, d (6.5) 3.39 3H, s 3.40 3H, s 3.40 3H, s 3.40 3H, s
Signal assignments were done based on the consequence of 2D-NMR (C-H COSY and HMBC) spectra and/or the decoupling experiments. *Overlapped with other signals.
2.30; S, 5.14, FAR-MS m/z: 612 [M+Na]+, ‘H and 13C NMR: Tables 4 and 5. Compound I5 . Prisms from MeOH, mp 222-226”. f=]g2 + 60.8” (M&H: c 0.2). Calcd for C&H&,, *H,O: C, 61.25; H, 7.80; found: C, 61.33; H, 7-87. FAB-MS m/z: 551 [M+H] ‘. ‘H and 13CNMR: Tables 4 and 5. Cornp~u~ 16. Amorphous powder. [@]A2 + 12.8” (MeOH; ~0.6). Calcd for Cz&,,O, .9/4H,O: C, 6OSS; H, 8.15; found: C, 60.50; H, 7.86. FAB-MS m/z: 535 /M -I-H] +. ‘H and 13CNMR: Tables 4 and 5.
Compound 17. Amorphous powder. [a];* +445” (MeOH; ~0.3). Ca.i& for C29H,,010* 5/4H@: C, 60.99; H, 7.50; found: C, 61.08; H, 7.57. FAB-MS m/z: 549 [M +N_7+. ‘H and 13CNMR: Tables 4 and 5. Compou~ 18. Amorphous powder. [E];’ -I-11.4” (MeOH; ~0.7). Calcd for C31H41N0$*7/2Hz0: C, 55.84; H, 7.26; N, 2.1@ S, 4.81; found: C, 55.78; H, 7.04, N, 2.17; S, 4.72. FAB-MS m/z: 604 [M+H]+. ‘H and 13CNMR: Tables 4 and 5. Compound 19. Amorphous powder. [a];* +67.4”
Glycosides from
223
Asclepiasfiuticosa
Table 4. r3C NMR spectral data of 14-22, 26 and 27 (at 125.65 MHz)
Aglycone moiety 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Sugar moiety 1’ 2 3 4 5 6 Other 1” 2”
42.2 69.8 72.2 32.0 44.9 27.7 26.7 40.6 49.2 38.1 21.1 38.5 48.8 81.7 73.3’ 37.1 48.8 16.5 13.9 173.6 73.4= 118.2 174.4
43.0 69.4 73.0 33.0 45.0 28.4 26.9 41.0 49.4 38.1 21.6 38.7 49.0 81.8 73.0 38.0 49.4 16.9 14.0 175.2 73.8 118.1 174.4
42.9 69.6 73.0 33.0 45.1 28.3 27.2 41.9 50.1 37.9 21.7 32.8 53.6 85.0 31.8 38.7 85.9 15.6 13.9 181.1 74.1 117.5 174.7
36.6 69.8 72.3 32.7 43.5 28.0 21.2 43.5 48.9 53.0 22.3 34.0 53.4 84.6 31.2 38.6 85.8 15.3 208.1 180.8 74.0 117.6 174.7
36.1 69.7 71.3 33.0 43.3 27.2 26.5 42.4 48.5 52.4 21.0 33.3 51.5 87.9 30.2 36.7 86.2 12.4 206.8 171.3 72.9 116.5 173.6
36.6 69.8 72.2 34.0 43.4 27.8 26.9 42.4 48.5 52.9 21.5 33.0 51.7 87.0 30.9 37.0 86.4 12.8 208.0 172.8 73.2 116.8 173.9
42.8 69.5 72.9 33.0 44.9 28.3 26.8 40.7 49.3 38.0 20.9 33.1 51.7 85.7 70.8 48.7 85.1 13.6 13.8 172.5 73.3 116.9 173.9
42.6 69.1 73.1 33.0 45.0 28.2 26.8 40.7 49.3 38.0 20.9 33.0 51.7 85.9 70.8 48.6 85.1 13.6 13.8 172.6 73.3 116.9 173.9
36.6 70.3 72.0 33.8 43.6b 28.1’ 28.W 42.7* 48.8e 53.0 22.4 40.3 49.3 84.4 42.5* 76.5 62.2 16.1 207.8 174.3 74.4 118.1 174.3
31.5’ 31.2’ 76.5 36.3 43.0s 28.6 26.9 42.7’ 48.5 51.7 21.4 33.1 51.6 87.1 30.8 37.1 86.5 12.9 208.7 172.9 73.3 116.7 173.9
31.4 30.1h 77.2 34.9 44.4 29.1h 27.3 42.5 50.1 36.0 21.5 39.2 53.6 85.1 31.8 38.7 85.9 15.6 12.3 181.2 74.1 117.4 174.6
95.1 91.5 99.6 47.1 68.2 20.9
95.5 93.8 75.4 72.9 70.2 18.8
95.6 91.9 71.8 38.3 66.5 21.6
95.5 91.9 71.7 38.3 66.6 21.6
94.9 91.5 99.5 47.0 68.2 20.9
95.5 92.0 71.7 38.3 66.6 21.6
95.7 91.9 71.8 38.3 66.5 21.6
97.5 92.8 74.0 39.9 68.6 21.6
96.8 93.1 100.1 48.6” 68.5 21.4
99.6 72.5 72.9 74.4 70.4 18.8
99.5 72.6 72.9 74.5 70.4 18.9
-
-
-
-
-
-
160.6 43.5b
-
-
160.0 42.6
160.1 42.7
“‘Assignments may be interchanged within each column. *Measured in CDCl, solution at 35”. tMeasured in pyridine-ds solution at 35 ‘.
(MeOH; ~0.7). Calcd for C,,H,,O,, - 7/4H,O: C, 60.04; H, 7.56; found: C, 60.02; H, 7.65. FAB-MS nrjz: 549 [M +Hl +. ‘H and “CNMR: Tables 4 and 5. Compound 20. Amorphous powder. [cl]p i-66.9” (MeOH; c 0.8). Calcd for C,sH,,O,, - 7/4H,O: C, 59.83; H, 7.88; found: C, 59.96; H, 7.86. FAB-MS m/r 551 [M +Hl +. ‘H and 13CNMR: Tables 4 and 5. Co~pou~ 21. Amorphous powder. [x]gz +41.3 (MeOH; c 0.8). Calcd for C2sH,20,, .9/4H,O: C, 58.92; H, 7.93; found: C, 58.99; H, 7.76. FAB-MS m/z: 551 [M +HI +. ‘H and r3CNMR: Tables 4 and 5. Compound 22. Amorphous powder. [u]i2 -3.3” (MeOH, ~0.4). FAB-MS m/z: 604 [M+H]*. ‘H and r3CNMR: Tables 4 and 5. Compound 26. Amorphous powder. [a]$r +5.2” (MeOH; c 0.7). Calcd for CzsH~,OIO~ 5/4H,O: C, 60.77; H, 7.83; found: C, 60.75; H, 7.84. FAB-MS m/z: 551 [M +Hl ’ ‘H and 13CNMR: Tables 4 and 5. PHYTO 37:1-P
Com~u~ 27. Amorphous powder. [u]i2 -38.2” (MeOH; c 1.4). Calcd for C29H,,0,. H,O: C, 62.80; H, 8.36; found: C, 62.55; H, 8.33. FAB-MS mfr 537 [M +H]+. ‘H and r3CNMR: Tables 4 and 5. As 4, 5 and 22 could be acquired only by a small amount, the elemental analyses of these two compounds were not performed. Deg~~ution of the crude pregnane g~ycoside. The MeOH eluate on Diaion HP-20 column (see Experimental) was chromatographed on a silica gel column and the fr. eluted with CHCl,-MeOH (93 : 7) on this silica gel column was rechromatographed on semi-prep. HPLC (ODS: MeCN-H,O gradient system). The crude fr. was eluted with 2931% MeCN on this semi-prep. HPLC. This crude pregnane glycoside fr. (10 mg) was dissolved in dioxane (1 ml) and 0.2 M H,SO, (5 drops) and this soln was heated at 60” for 90 min. After addition of H,O (1 ml) to this reaction mixt., dioxane was removed by blowing
Aglycone moiety 2 3 4ax 8 9 15 16a 16b 17 18 19 21a 21b 22 Sugar moiety 1’ 2 3 4’a 4’b 5 6 Other 1” 2”a 2”b
H
* 3H, 3H, lH, lH, lH, br d (18.0) br s
br d (18.0)
s s
t (7.0)
cd (12.0, 3.0)
rd (12.0, 3.0)
q (12.0)
lH, lH, lH, 3H,
dd (12.5, 11.5) dd (12.5, 1.5) m d (6.5)
7.55 lH, br s 3.89 lH, br d (17.0) 3.87 lH, br d (17.0)
2.25 1.72 4.28 1.23 4.46 lH, m 1.72 3H, d (6.5)
q (11.5) cd (11.5, 3.0) td (11.5, 3.0)
td (11.5, 4.5)
3H, s 3H, s lH, dd (18.5, 1.5) lH,dd (18.5, 1.5) lH, t (1.5)
* lH, lH, lH, lH,
-
4.25 2.02 2.20 4.55 1.38
lH, lH, lH, lH, 3H, d (6.5)
m
br t (12.5)
br d (12.5)
br s
5.44 lH, s
1.16 0.72 5.19 5.64 6.67
4.55 4.35 1.49 1.67 1.02
16$
-
4.27 2.03 2.19 4.54 1.38
lH, lH, lH, lH, 3H,
d (6.5)
m
br t (12.5)
br d (12.5)
br s
5.44 lH, s
1.09 3H, s 10.07 lH, s 5.18 lH, br d (18.5) 5.61 lH, br d (18.5) 6.66 lH, br s
4.49 lH, ddd (12.0, 11.0, 4.5) 4.37 lH, td (12.0, 4.5) 1.56 lH, q (12.0)
171
’ H NMR spectral data of 14-22, 26 and 27 (at 500 MHz)
ddd (12.0, 10.0, 4.5) cd (12.0, 4.5)
4.46 lH, d (3.0) 4.23 lH, dd (9.5, 3.0)
2.64 1.00 0.70 5.00 5.29 6.10
lH, lH, lH, lH, lH, lH,
-
s s dd (18.0, 1.5) dd (18.0, 1.5) t (1.5)
4.60 4.31 1.50 1.68 1.09 4.79
5.49 lH, s
* 3H, 3H, lH, lH, lH,
2.66 0.92 0.87 4.81 5.05 5.86
f (8.0)
cd (12.0, 4.5)
cd (12.0, 3.5)
q (11.5)
ddd (11.5, 10.0, 4.5) td (11.5, 4.5)
1%
5.08 lH, s
lH, lH, lH, lH, lH, lH,
4.15 4.06 1.47 1.60 1.11 4.56
w
Table 5.
3H, lH, lH, lH, lH,
s s dd (18.5, 2.0) dd (18.5, 2.0) t (2.0)
lH, ddd (12.0, 9.5, 4.5) lH, td (12.0, 4.5) lH, q (12.0) lH, td (11.5, 3.0)
lH, lH, lH, 3H,
dd (13.5, 1.5) dd (13.5, 11.5) m d (6.5)
3.87 lH, dd (17.5, 1.5) 3.89 lH, dd (17.5, 1.5)
7.52 lH, br s
1.72 2.24 4.28 1.23
5.07 lH, s
1.01 9.98 4.81 4.99 5.85
-
3.96 4.09 1.47 1.59
1st
lH, ddd (12.0, 9.5, 4.5) lH, td (12.0, 4.5) lH, q (12.0) lH, td (11.5, 3.0) *
-
4.26 2.02 2.18 4.55 1.37
lH, lH, lH, lH, 3H,
d (6.5)
m
br t (12.0)
br d (12.0)
br s
5.43 lH, s
1.11 3H, s 10.06 lH, s 5.02 lH, br d (18.5) 5.15 lH, br d (18.5) 6.19 lH, br s
4.49 4.39 1.57 1.88 1.27
19$
4.25 lH, 2.00 lH, 2.18 lH, 4.53 lH, 1.36 3H,
d (6.5)
m
dt (13.5, 25) td (13.5, 2.5)
t (2.5)
5.03 lH, s
ddd (11.0, 10.0,4.0) td (11.0, 4.0) q (11.0) td (120, 3.0)
2.00 lH, 2.27 lH, 4.59 lH, 1.39 3H, 7.61 lH, br s 3.86 lH, br d (16.5) 3.92 lH, br d (16.5)
-
dd (12.0, 1.5) t (12.0) m d (6.5)
5.58 lH, s
5.05 lH, td (8.0,4.0) 3.04 lH, d (4.0) 0.94 3H s 9.89 H-i, s 5.03 lH, br d (17.5) 5.17 lH, br d (17.5) 6.23 H-I, br s
4.47 lH, 4.43 lH, 1.55 lH, 1.78 lH,
4.11 lH, dd (12.0,4.5) 2.00 lH, ddd (12.0, 4.5, 1.5) 2.10 lH, q (12.0) 3.74 lH, m 1.34 3H, d (6.5)
s s dd (18.0, 1.5) dd (18.0, 1.5) t (1.5)
dd (15.5, 9.5)
4.79 lH, dd (9.5, 3.0) 244 lH, dd (15.5, 3.0)
5.44 lH, s
ddd (120, 10.0,4.5) td (12.0, 4.5) q (12.0) td (11.5, 3.0)
3.09 lH, 1.24 3H, 0.70 3H, 5.03 lH, 5.16 lH, 6.21 lH,
4.54 lH, 4.29 lH, 1.52 lH, 1.80 lH,
4.55 lH, ddd (11.5, 9.5,4.5) 4.32 lH, td (11.5, 4.5) 1.48 lH, q (11.5) 1.79 lH, td (11.5, 3.0) 1.06 * 4.87 lH, dd (9.5, 3.0) 2.43 lH, dd (15.5, 3.0) 3.09 lH, dd (15.5, 9.5) 1.23 3H, s 0.72 3H, s 5.02 lH, dd (18.0, 1.5) 5.15 lH, dd (18.0, 1.5) 6.20 lH, br s
4.35 lH, dq (9.5, 6.5) 1.64 3H, d (6.5)
4.36 lH, dq (9.5, 6.5) 1.64 3H, d (6.5)
dd (9.5, 3.0)
t (3.0)
d (8.0) dd (8.0, 3.0)
5.39 lH, 3.94 lH, 4.68 lH, 3.69 lH,
5.37 lH, d (8.0) 3.92 lH, dd (8.0, 3.0) 4.68 lH, t (3.0) 3.69 lH, dd (9.5, 3.0)
s s dd (18.0, 1.5) dd (18.0, 1.5) t (1.5)
1.19 3H, 0.69 3H, 5.21 lH, 5.66 lH, 6.68 lH,
-
3.96 lH, m
1.14 3H, s 10.02 lH, s 5.03 lH, br d (18.5) 5.17 lH, br d (18.5) 6.21 lH, br s
-
4.00 lH, m
Signal assignments were done based on the consequence of ZD-NMR (C-H COSY, NMBC and H-H COSY) spectra. *Overlapping with other signals. tMeasured in CDCI, solution at 35”. SMeasured in pyridine-d, solution at 35”.
8 9 15 16a 16b 17 18 19 21a 21b 22 Sugar moiety 1’ 2 3 4’a 4’b 5 6 Other 1” 2” a 2”b
4tiX
Aglycone moiety 2 3
Table 5. Continued
226
T. WARASHINAand
air on a hot water bath for a short time. The soln was then heated at 60” for 90 min once more. After hydrolysis, the soln was passed through a Mitsubishi Diaion HP-20 column and the absorbed material was eluted with MeOH. The MeOH eluate was coned to dryness and the residue was recrystallized from MeOH to give pure lineolon (1) (1 mg), mp 237-244”, [a];’ + 12.7” (MeOH; ~0.38) (lit. mp 233-239”, [I]:: + 13”; MeOH) [13]. ‘H NMR [(CD,),SO]: 6 1.07 (3H, s, H-19), 1.28 (3H, s, H18), 2,10(3H, s, H-21), 3.34(1H, m, H-3), 3.30 (overlapped, H-12 or 17) 5.19 (lH, br s, H-6). 13CNMR [(CD,),SO]: S 13.8 (C-18), 17.8 (C-19), 20.8 (C-16) 27.9 (C-11), 30.9 (C2), 31.4 (C-21), 33.4 (C-15) 34.3 (C-7), 36.3 (C-lo), 38.2 (Cl), 42.1 (C-4), 43.7 (C-9), 56.4 (C-13) 60.0 (C-17), 67.8 (C12), 70.3 (C-3), 73.3 (C-8), 86.4 (C-14), 117.9 (C-6), 139.2 (C-5), 209.2 (C-20) [9, lo]. However, isolineolon was not acquired. Acid hydrolysis of 2-5,26 and 21. Compounds 2 and 3 (ca 1 mg) dissolved in dioxane (4 drops) and 0.2 N H,SO, (1 drop) was heated at 60” for 90 min. After addition of H,O (3 drops) to this reaction mixt., dioxane was removed as before and then the soln was heated at 60” for 90 min. After hydrolysis, the soln was passed through a Mitsubishi Diaion HP-20 column and eluted with MeOH. The MeOH eluate was coned to dryness and the residual aglycone was identified by HPLC with an authentic sample [Conditions: column; YMC ODS, flow rate; 1.0 ml min- r, 40% MeOH in water, R, (min); lineolon 9.41. Hydrolysis of 4 and 5 were not carried out, because of the unavailability of an authentic sample of aglycone and small amounts available of 4 and 5. Thus, the aglycone moieties of 4 and 5 were identified as isolineolon on the basis of a comparison of the l3 C NMR spectral data with that of isolineolon [lo]. Acid hydrolysis, to confirm the monosaccharides in the sugar chain of 2-5, were done with a small amount (ca 0.1 mg) in the same way as above. Compounds 26 and 27 (ca 0.1 mg) were dissolved in dioxane and 10% H,SO, (each one drop) and this soln was heated at loo” for 1 hr. After hydrolysis, the soln was passed through an Amberlite IRA-60E column and the eluate was coned to give a residue, which was reduced with NaBH, (ca 1 mg) for 1 hr at room temp. The reaction mixt. was passed through an Amberlite IR-120B column and the eluate was coned to dryness. Boric acid was removed by codistillation with MeOH and the residue was acetylated with Ac,O and pyridine (each one drop) at loo” for 1 hr. The reagents were evapd in t’acuo. From each glycoside, cymaritol acetate, oleandritol acetate, digitoxitol acetate, olivitol acetate and 6-deoxyallitol acetate were detected by GC [Conditions: column; Supelco SP-2380 capillary column (0.25 mm x 30 m), column temp.; 200”, carrier
T. NORO
gas; N,, R, (min); cymaritol acetate 6.6, oleandritol acetate 7.5, digitoxitol acetate 9.4, olivitol acetate 10.5 and 6deoxyalhtol acetate 13.41. The relative ratio of each monosaccharide in 2-5 was determined based on the peak area. Synthesis of 17~hydroxydigitoxigenin. The synthesis of 17a-hydroxydigitoxigenin from digitoxigenin (40 mg) was carried out as reported in ref. [ 1I]. 17a-Hydroxydigitoxigenin (3 mg), mp 213-218” from MeOH. [aliz - 17.5” (MeOH; ~0.1) (lit. mp 215-219”, [x]k6 -22.8”; MeOH) [ll]. r3C NMR [C,D,N] : 6 15.7 (C-18), 21.4, 21.7 (C-7 and C-l I), 24.2 (C-19) 27.4, 28.8, 30.4 (C-l, C-2 and C-6), 31.9 (C-15), 33.2 (C-12) 34.4 (C-4), 35.8, 35.9 (C-9 and Clo), 36.8 (C-5), 38.7 (C-16), 42.9 (C-8), 53.7 (C-13), 66.0 (C3) 74.1 (C-21), 85.3 (C-14), 86.0 (C-17), 117.4 (C-22), 174.7 (C-23), 181.3 (C-20). Acknowledgement-We
thank the staff of the Central Analytical Laboratory of this school for measurement of mass spectra and the element analyses.
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Warashina, T. and Noro, T. (1994) Chem. Pharm. Bull.
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