Phytochemistry,Vol. 30, No. 11, pp. 3703 3707, 1991 Printed in Great Britain.
00314422/91 $3.00+0.00 © 1991PergamonPress plc
TRITERPENES FROM M U C U N A BIRD WOODIANA YI DING, JUNEI KINJO, CHONG-REN YANG* and TOSHIHIRO NOHARA Faculty of Pharmaceutical Sciences, Kumamoto University, Oe-honmachi, Kumamoto 862, Japan; *Laboratory of Phytochemistry, Kunming Institute of Botany, Chinese Academy of Science, China
(Received in revisedform 1 May 1991) Key Word Iodex--Mucuna birdwoodiana; Leguminosae; triterpenoid glycoside;methyl maslinate; methyl asiatate; lfl,2a,3fl,23-tetrahydroxyolean-12-en 28-oate; lfl,2~t,3fl,23-tetrhydroxyurs-12-en28-oate; mucunagenin a; mucunagenin b.
Abstraet--Methanolic extracts of the stalks of Mucuna birdwoodiana on acid hydrolysis and subsequent methylation with diazomethane provided four triterpene sapogenols. On the other hand, investigation of glycosides after methylation of the same extract led to the isolation of four triterpene glycosides. On the basis of chemical and spectral evidence, their structures were characterized as methyl asiatate, methyl maslinate, two new sapogenols, methyl lfl,2ct,3fl,23-tetrahydroxyolean-12-en 28-oate (mucunagenin a), its urs-12-en isomer (mucunagenin b), 3-0-(6-0methyl-fl-D-glucuronopyranosyl) methyl asiatate 3-O-[~-L-arabinopyranosyl(l~2)]-6-O-methyl-fl-D-glucuronopyranosyl methyl maslinate, 3-O-[ct-L-arabinopyranosyl(1--*2)]-6-O-methyl-fl-D-glucuronopyranosylmethyl asiatate and 3-O-(6-O-methyl-fl-D-glucuronopyranosyl) asiatic acid 28-O-fl-D-glucopyranoside.
INTRODUCTION
Mucuna birdwoodiana (Leguminosae) grows in southern China. It has been recognized as being effective in promoting blood circulation or to relieve stasis. It has been widely used to treat pain or numbness of the wrists, knees or other joints and irregular menstruation [1]. Moreover, it is known that seeds of this plant contain LD O P A [21 and stalks include phenolic compounds [3]. However, no study on the triterpenoid compounds of this drug has so far been undertaken. We deal with the isolation and structure elucidation of triterpenoid components.
RESULTS
AND DISCUSSION
Dried stalks were extracted with methanol and then partitioned between n-BuOH and water. The solvent from the organic layer was removed to give a residue which was firstly hydrolysed and methylated with diazomethane in order to survey component sapogenols prior to the investigation of their glycosides; four sapogenols (1~1) were obtained. Sapogenols 1 anal 2 were identified as methyl 2~,3fl,23-trihydroxyurs-12-en-28oate (methyl asiatate) [-4] and methyl 2ct,3fldihydroxyolean-12-en-28-oate (methyl maslinate) [-4-6] on the basis of their physical data, EI mass and tH and 3C NMR spectra. Sapogenols 3 and 4 were recognized as new triterpenes. Sapogenol 3, needles, showed a [M] ÷ at m/z 518 and fragment peaks at m/z 262 [-C17H2602] +, 203 [-C15H23] +, 189 [-CI4H21] + [7] and 133 [-CloH13] + derived from D, E ring via retro-Diels-Alder fission in the EI mass spectrum (Fig. 1), indicating that 3 possesses four hydroxyl groups in the A, B-ring on the olean-12-en skeleton. The t3CNMR spectrum of 3 revealed the presence of signals due to four oxygenated carbon signals
[666.0 (t), 72.9 (d), 74.1 (d) and 83.0 (d)], one trisubstituted double bond (6123.0 and 142.1), and one carbomethoxy group (6 51.0 and 178.7) (Table 1). Moreover, 13C N M R signals due to C-18-C-22 (640.7, 45.3, 30.1, 33.3 and 31.6) suggested that 3 is an olean-12-en derivative. The acetate (3') of 3 showed signals due to three methine protons at 64.93 (d, J=9.9Hz), 5.16 (d, J=9.9Hz), 5.25 (t, J =9.9Hz) adjacent to the acetoxyl group and one acetoxymethine protons at 63.56 and 3.82 (AB q, J = 11.7 Hz) together with signals due to the typical H-18 at 62.84 (brd, J = 10.3 Hz) of an olean-12-ene type of triterpene and four acetyl groups at 31.95, 1.96, 1.99 and 2.11 in its t H N M R spectrum. The 1H-tH COSY of 3 disclosed that three signals at 64.93, 5.25 and 5.17 could be assigned to H-I, H-2 and H-3, respectively. The J values of these signals indicated that all protons were oriented axially, so that the structure of 3 could be characterized as methyl lfl,2ct,3/~,23-tetrahydroxyolean12-en-28-oate, named mucunagenin a. Sapogenol 4, needles, showed similar E1 mass and 13C N M R spectra to those of 3. The ~H N M R spectrum of the corresponding tetraacetate (4') also showed the presence of lfl,2~,3fl,23-tetraacetoxyl groups at 65.16, (d, J = 10.2 Hz), 5.27 (t, J = 10.2 Hz), 4.93 (d, J = 10.2 Hz) and 3.55, 3.82 (ABq, J = 12.4 Hz). However, on the basis of the ~HNMR spectrum of 4' and the t3CNMR spectrum (Table 1) of 4, compound 4 could be distinguished from 3 by showing characteristic urs- 12-en type signals: H- 18 at 62.19 (d, J = 12.8 Hz); C-18~C-22 at 3 52.6, 39.0, 38.8, 30.6 and 36.6. Consequently the structure of 4 could be represented as methyl lfl,2~,3fl,23-tetrahydroxyolean- 12en-28-oate, named mucunagenin b. On the other hand, separation and purification of the n-BuOH layer using Bondapak Ct8, silica gel, MCI gel, CHP 20P column chromatographies yielded the triterpene glycosides upon methylation with diazomethane providing four compounds (5-8).
3703
YI DING et al.
3704
It R1 H
-CH2COOMe73~
OR
OOR 3
~
~ 1 8 = t2 .I-
R20"
~
262
~
/
R1
R2
R3
1
H
H
CH 3
4 §
OH H
7
H
8
H
H ~-D-GIc UA-Me
CH 3
c(-L-Ara pyr(1-~2)-~-D-GIc UA-Me ~-D-GIc UA-Me
H
RI
J~-D-GIc
R3
2
H
H
CH 3
3
OH
H
CH20 H
6
H
(~-L-Ara pyr(1-~2)-~-D-Glc UA-Me
203
M M-Me M-COOMe M-AcOH M-AcOH-Me M-AcOH-COOMe M-2AcOH M-2AcOH-Me M-2AcOH-COOMe M-3AcOH M-3AcOH-Me M-3AcOH-COOMe M-4AcOH M-4AcOH-COOMe
3 -
R2
133
CH 3
OOMe
/'\..R
-C5Hlo 70
CH 3
H
R20
189
oCOOMe 59
CH 3
Glycoside 1 (5), on acid hydrolysis, yielded an aglycone which was identical with methyl asiatate (1). The negative FAB mass spectrum of 5 showed peaks due to [M + nitrobenzyl alcohol (NBA)]- at m/z 845, [ M - H ] - at m/z 691 and [M - H -glucuronic acid (UA)] - at m/z 501, indicating that it contains only one molecule of glucuronic acid. Comparison of the 13C NMR spectrum of 5 with that of 1 disclosed that the structure of 5 could be characterized as a monoglycoside, 3-O-(6-O-methyl-f-Dglucuronopyranosyl) methyl asiatate. Glycoside 2 (6), on acid hydrolysis, provided an aglycone, which was identical with methyl maslinate (2). The negative FAB mass spectrum of 6 showed peaks due to [ M + N B A ] - at m/z 961, [ M - H ] - at m/z 807 and [M -H - U A -pentose (Pen)] - at m/z 485, indicating that 6 contains one molecule of glucuronic acid and a pentose. On the basis of a detailed ~H-~H COSY study of the acetate (6') of 6, all signals due to the sugar moiety could be assigned as given in the Experimental. Thus, the structure of sugar is ~-L-arabinopyranosyl (1--,2)-6-O-
1' 628 (7) 613 (3} 569 (6) 568 (9) 553 (4) 509 (5) 508 (7) 493 (3) 449 (4) 448 (5) 433 (4) 389 (3) ---
2' 570 (11) - -
511 (6) 510 (9) 495 (4) 450 (11) 435 (7) 391 (5) -- -
-- -
---
3' 686 (10) - -
627 (48) 626 (100) -567 (35) 566 (74) -507 (48) 506 (74) -447 (23) 446 (21) 387 (11)
Fig. 1. Molecular and fragment ion peaks (re~z)in the EI mass spectra of compounds 1'-3'.
methyl-fl-D-glucuronopyranoside. Furthermore, comparison of the 13CNMR spectrum of 6 with that of 5 (Table 1) also suggested that one additional arabinopyranosyl moiety attached to 5 forming 6. The downfield shift (+7.5 ppm) at C-2 and the upfield shift ( - 1.4 ppm) at C-3 on the glucuronic acid moiety in 6 revealed that the linkage between the two sugars should be 1--,2. Therefore, 6 is 3-O-[~-L-arabinopyranosyl (1-*2)]-6-O-methylfl-D-glucuronopyranosyl methyl maslinate. Glycoside 3 (7), on acid hydrolysis, afforded the same aglycone as that obtained from 5 by comparing R I values on TLC and 1H NMR spectra. The negative FAB mass spectrum of 7 showed peaks due to [M + NBA] - at m/z 977, [ M - H I - at m/z 823, [ M - H - P e n ] at m/z 691 and [ M - H - U A - P e n ] at m/z 501, indicating that 7 contained one molecule of glucuronic acid and a pentose in the sugar residue which was identical to that of 6. On the other hand, the ~3C NMR spectrum of 7 (Table 1) also showed the same chemical shifts as those of 6 in the sugar moiety. Consequently, 7 is 3-O-[~-L-arabinopyranosyl (1-~2)]-6-O-methyl-fl-D-glucuronopyranosyl methyl asiatate. Glycoside 4 (8), exhibited two anomeric proton signals at65.27(1H, d,J=7.3 Hz)and 66.26(1H, d,J=8.1 Hz)in
Triterpenes from
Mucunabirdwoodiana
3705
Table 1. 13CNMR spectral data for sapogenols 1-4 (400 MHz, CDCI3) and glycosides 5-8 (400 MHz, pyridine-ds) C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 CO2Me G-UA 1 2 3 4 5 6 CO2Me Ara 1 2 3 4 5 Glc 1 2 3 4 5 6
1
2
3
4
5
6
7
8
47.7 68.8 78.1 42.1 c 48.0 18.4 33.1 39.4 b 47.8 38.2 23.7 125.5 138.4 42.3 o 28.5 24.8 48.2 53.4 39.4b 39.3b 31.0 37.4 66.3 13.0 17.1a 17.2a 23.8 178.4 17.4a 21.3 51.6
47.8 68.6 83.8 39.9b 56.0 18.9 33.2 39.9b 48.2 38.6 23.7 122.5 143.6 42.2 c 28.3 24.0 46.7 42.0 ~ 46.5 31.0 34.2 33.2 29.4 16.8 17.5 16.8 26.0 178.0 33.2 23.5 51.5
72.9 74.1 83.0 42.2 48.0 17.1 31.9 41.0 44.6 39.3 26.3 123.0 142.1 41.8 27.2 22.8 46.4 40.7 45.3 30.1 33.3 31.6 66.0 12.4a 12.2a 16.4 25.2 178.7 32.3 22.5 51.0
73.5 75.0 83.5 42.5 ~ 48.3 17.6 32.5 40.0 45.3 40.0 26.7 126.3 137.2 42.8 c 28.0 24.2 48.0 52.6 39.0 38.8 30.6 36.6 67.6 13.2b 13.2b 17.1a 23.7 178.1 17.1~ 21.2 51.5
47.5 66.8 88.1 42.3 47.4 18.1 32.9 39.9b 47.9 37.7 23.7 125.8 138.7 44.7 28.4 24.5 48.3 53.3 39.2 b 39.2b 30.9 37.0 63.5 14.6 17.3~ 17.3~ 23.8 177.7 17.5~ 21.2 51.5
47.5 66.3 95.2 37.7 55.5 18.3 32.8 40.8 47.7 39.5 23.3 122.7 144.0 41.8 ~ 27.9 23.7 47.2 41.6 ¢ 45.9 30.7 33.9 32.7 28.6 16.6 17.0a 17.2a 26.0 177.9 33.1 23.6 51.5
47.5 66.4 88.1 42.1 47.1 17.9 32.7 39.7b 47.8 37.5 23.5 125.7 138.5 44.7 28.2 24.4 48.1 53.1 39.1b 39.1b 30.6 36.8 63.0 13.8 17.0" 17.2~ 23.7 177.6 17.3a 21.2 51.3
47.4 66.7 87.9 42.2 47.0 18.0 32.9 40.0 47.9 37.6 23.7 125.9 138.2 44.5 28.5 24.5 48.2 53.1 39.2 38.9 30.6 36.0 63.3 14.5 17.2a 17.4a 23.6 176.1 17.6a 21.1 --
105.8 75.1 77.8 73.0 77.8 170.2 52.1
104.5 82.6 76.4 73.5 77.4 169.9 52.1
103.7 82.7 76.2 73.5 77.2 169.9 52.1
105.6 .75.0 77.6 72.8 76.6 170.1 52.0
106.6 72.6 74.3 69.2 67.2
106.5 72.4 74.2 69.1 67.1
m
m
m
m
m
m
w
m
m
95.5 73.9 78.7 71.1 79.0 62.2
"-Clnterchangeable values.
the t H N M R s p e c t r u m . O n acid hydrolysis it afforded a n aglycone (la), w h o s e m e t h y l ester was identical to 1 o n T L C . T h e E1 m a s s s p e c t r u m of l a s h o w e d a [ M ] ÷ at m/z 488 a n d f r a g m e n t i o n p e a k s a t m/z 203 a n d 248, which were 14 m u lower t h a n t h o s e of 1, s u g g e s t i n g t h a t the 28-
c a r b o x y l g r o u p exists in the free form. T h e negative F A B m a s s s p e c t r u m o f g s h o w e d p e a k s d u e to [ M + N B A ] - at m/z 993, [ M - H ] - at m/z 839, [ M - H - h e x o s e ( H e x ) ] at m/z 677 a n d [ M - H - U A - H e x ] at m/z 487. O n alkaline hydrolysis, 8 afforded a p r o s a p o g e n i n w h i c h was
YI DING et al.
3706
identical with 5 upon methylation with diazomethane. A comparative study o f the 13C N M R spectrum of 8 with that of 1 (Table 1) indicated the glycosylation shifts (I-8, 91 at C-3 and C-28 in the aglycone moiety. An anomeric carbon signal due to the ester type of the glucosyl linkage appeared at 395.5, indicating that 8 is a 3,28-btsdesmoside. Thus, the structure of 8 was established as 3-0-(6-0methyl-fl-D-glucuronopyranosyl) asiatic acid 28-O-fl-Dglucopyranoside. Finding two new sapogenols possessing lfl,2~t,3fl,23tetrahydroxyl groups in the A-ring of olean- or urstriterpene is worthy of note. Moreover, asiatic acid and maslinic acid have so far been isolated from several plants [4, 10"1, but they have rarely been obtained as glycosides. It has been reported that maslinic triglycoside occurs in Swertia cincta [6!, but the sugar moiety is different from that found in M. birdwoodiana.
EXPERIMENTAL
1H and 13CNMR spectra were measured on a 400 MHz instrument and chemical shifts are given on a 6 (ppm) scale with TMS as int. standard. CC was carried out with MCI gel CHP 20P (75-100 mm, Mitsubishi) and Kieselgel 60 (70-230 and 230-400 mesh, Merck). TLC was conducted on precoated Kieselgel 60 F254 plates (Merck), and detection was achieved by spraying with 10% H2SO4 followed by heating. Extraction and separation. Dried stalks (6 kg) ofM. birdwoodiana (Tutcher) were extracted with MeOH and the extract (298.3 g) partitioned between n-BuOH and H20. The n-BuOH layer was evapd under red. pres. to give a residue (216.2 g), a part (3.36 g) of which was refluxed in 1 M HCI-MeOH at 100° for 2 hr and then neutralized with 3% KOH-MeOH. Removal of MeOH followed by partition between H20 and EtOAc provided the EtOAc extract which was methylated with excess CH2N2-Et20 and then subjected to a silica gel CC with CHC13-MeOH-H20 (200:20:1) to afford frs 6-25 (450mg), which were further chromatographed on silica gel with nhexane-Me2CO (1 : 1) to give frs 32-50 which were recrystallized from MeOH, to give 2 (18.5 mg). Frs 181-209 were also recrystallized from MeOH to give 1. Frs 210-270 were repeatedly subjected to silica gel CC with CHC13-MeOH-H20 (100:10: 1) and CHCla-Me2CO (1 : 1), and then recrystallized from MeOH, giving 3 (22.5 mg) and 4 (26.7 mg). The remaining n-BuOH portion (208.9 g) was refluxed with nhexane. The n-hexane insol, portion (161.4 g) was subjected to Sephadex LH-20 CC using MeOH to give frs 1-7. Frs 3-5 were collected and chromatographed on a silica gel column with CHCI3-MeOH-H20 (40:10:1--,14:6:1). Frs 64-66, 67-72 and the MeOH eluate were respectively treated with excess CH2N2-Et20 followed by CC over silica gel using CHCI3-MeOH-H20 (90: 10:1--,40: 10: 1) and Bondapak with 40---,70% MeOH to provide glycosides 1 (5) (94.5 mg), 2 (6) (54.2 mg), 3 (7), (86.3 mg) and 4 (8) (135.6 mg). Sapogenol 1 (1). Plates, mp 228-230 ° (MeOH). [~]22+45.1° (MeOH; c 0.50). 13CNMR (Table 1). 1-Acetate (1'). A soln of 1 (10 mg) in Ac20-pyridine (1 : 1) was heated at 80° for 2 hr. The reaction mixt. was evapd to dryness and then chromatographed on silica gel using n-hexane-EtOAc (2: 1), giving the peracetate (1') as a powder. 1H NMR (CDCI3) 6: 0.75-1.10 (m, 6xMe), 1.98, 2.02, 2.09 (3 x OAc), 2.23 (1H, d, J = l l . 4 H z , H-18), 3.57 (1H, d, J=12.4Hz, H-23), 3.60 (s, CO2Me ), 3.85 (1H, d, J = 12.4 Hz, H-23'), 5.07 (1H, d, J = 10.3 Hz, H-3), 5.16 (m, H-2), 5.25 (1H, t-like, H-12). EIMS (Fig. 1). Sapogenol 2 (2). Needles, mp 223-224 ° (MeOH). [ct]23 +48.1 ° (MeOH; c 0.16). ~3CNMR (Table 1).
2-Acetate (2'). Compound 2 was acetylated as described above to yield 2'. 1HNMR (CDC13) 6:0.72-1.25 (m, 7 × Me), 1.98, 2.05 (2 × OAt), 2.82 (1H, dd, J=4.3, 11.9 Hz, H-18), 3.60 (s, CO2Me), 4.74 (1H, d, J = 10.3 Hz, H-3), 5.27 (m, H-2), 5.36 (1H, tlike, H-12). EIMS (Fig. 1). Sapogenol 3 (3). Needles, mp 235-236 ° (MeOH). [~t]24 + 35.3° (MeOH; c 0.22). EIMS m/z (rel. int.): 518 [M] +, (14), 500 [M - H 2 0 ] + (35), 482 [ M - 2 H 2 0 ] + (21), 262 [C17H2602] + (72) (D/E ring fragment of RDA), 203 [262-CO2Me] + (100), 189 [262-CH2CO2Me] + (37), 133 [203-C5Hlo] + (17). 13CNMR (Table 1). 3-Acetate (3'). Compound 3 was acetylated as described above to yield 3'. 1H NMR (CDCI3) 6:0.70-1.22 (m, 6 x Me), 1.95, 1.96, 1.99, 2.11 (4 x OAc), 2.84 (1H, br d, J = 10.3 Hz, H- 18), 3.56 (1H, d, J = 11.7 Hz, H-23), 3.61 (s, CO2Me), 3.82 (1H, d, J = 11.7 Hz, H23'), 4.93 (1H, d, J=9.9 Hz, H-3), 5.17 (1H, d, J=9.9 Hz, H-I), 5.19 (1H, t-like, H-12), 5.25 (1H, t, J = 9.9 Hz, H-2). EIMS (Fig. 1). Sapooenol 4 (4). Needles, mp 165-166 ° (MeOH). [0t]24 + 38.9 (MeOH; c 0.40). EIMS m/z (rel. int.): 518 [M] ÷ (9), 500 [M --H20"] + (26), 482 ['M--2H20"] + (14), 262 [C17H2602] + (57) (D/E ring fragment of RDA), 203 [262-CO2Me] + (100), 189 [262-CH2CO2Me] + (31), 133 [203-CsH~o] + (19). 13CNMR (Table 1). 4-Acetate (4'). Compound 4 was acetylated as described for 1 to yield 4'. tH NMR (CDCIa) ~3:0.72-1.23 (m, 6 x Me), 1.96, 1.99, 2.00, 2.10 (4 x OAt), 2.19 (1H, d, J = 12.8 Hz, H-18), 3.55 (1H, d, J = 12.4 Hz, H-23), 3.59 (s, CO2Me), 3.82 (1H, d, J = 12.4 Hz, H23'), 4.93 (1H, d, J = 10.2 Hz, H-3), 5.14 (1H, t-like, H-12), 5.16 (1H, d, J = 10.2 Hz, H-I), 5.27 (1H, t, J = 10.2 Hz, H-2). Glycoside 1 (5). Powder. [ct]~3 + 8.05° (MeOH; c 0.36). Negative FAB-MS (m/z): 845 [ M + N B A ] - , 691 [ M - H I - , 677 [ M - M e ] - , 501 [ M - H - U A ] - . ~HNMR (pyridine-ds) 6:0.85-1.08 (s, 6 x Me), 2.39 (1H, d, J = 11.4 Hz, H-18), 5.33 (1H, t-like, H-12). laCNMR (Table 1). Acid hydrolysis of compound 5. A soln of 5 (5 mg) in 1 M HC1-MeOH was refluxed at 100° for 2 hr and then neutralized with 3% KOH-MeOH. Removal of MeOH followed by partition between H20 and EtOAc provided the EtOAc extract which was column chromatographed over silica gel to give sapogenol 1. Glycoside 2 (6). Powder. [a]~3+22.3 ° (MeOH; c 0.35). 1HNMR (pyridine-ds) 6:0.81-1.32 (m, 7 x Me), 2.24 (1H, dd, J =4.8, 12.8 Hz, H-18), 5.35 (1H, t-like, H-12). Negative FAB-MS (m/z): 961 [ M + N B A ] - , 807 [ M - H I - , 485 [ M - H - P e n -UA]-. 1aC NMR (Table 1). Acid hydrolysis of 6 gave 2. 6-Acetate (6'). Compound 6 was acetylated to give the peracetate (6'). ~H NMR (CDCI3) 6: 0.71, 0.90, 0.92, 0.96, 1.02, 1.10, 1.11 (each 3H, s, 7 x Me), 1.97, 1.98, 2.00, 2.04, 2.09, 2.11 (each 3H, s, 6 x OAc), 2.83 (1H, dd, J=3.9, 12.1 Hz, H-18), 3.21 (1H, d, J = 9.9 Hz, H-3), 3.55 (1H, d, J = 12.8 Hz, Ara H-5), 3.62, 3.70 (each 3H, s, 2 x CO2Me), 3.84 (1H, dd, J=7.7, 9.0 Hz, UA H-2), 3.96 (1H, d, J = 9.1 Hz, UA H-5), 4.01 (1H, dd, J = 2.0, 12.9 Hz, Ara H5'), 4.53 (1H, d, J=7.7 Hz, UA H-l), 4.55 (1H, d, J=7.7 Hz, Ara H-I), 4.93 (1H, dd, J = 3.7, 9.9 Hz, Ara H-3), 5.09 (1H, dd, J = 3.7, 9.9 Hz, Ara H-3), 5.09 (1H, t, J =9.3 Hz, UA H°4), 5.10 (1H, dd, J = 7.8, 9.5 Hz, Ara H-2), 5.13 (1H, m, H-2), 5.21 (1H, m, Ara H-4), 5.24 (1H, t, J=9.3 Hz, UA H-3), 5.27 (1H, t-like, H-12). Glycoside 3 (7). Powder, I-~t]~4+19.2° (MeOH; c 0.26). ~HNMR (pyridine-ds) 6:0.84-1.15 (m, 6 x Me), 2.39 (1H, d, J = l l . 4 H z , H-18), 5.33 (IH, t-like, H-12). Negative FAB-MS (m/z): 977 [ M + N B A ] - , 823 [ M - H I - , 691 [ M - H - P e n ] - , 501 [ M - H - P e n - U A ] - . Acid hydrolysis of 7 gave 1. Glycoside 4 (8). Powder. [~t]~'*+l.5 ° (MeOH; c 0.47). 1HNMR (pyridine-ds) 6:0.87-1.15 (m, 6 x Me)., 2.50 (1H, d, J = 11.4 Hz, H-18), 5.39 (1H, t-like, H-12). Negative FABMS (m/z): 993 [M +NBA] -, 839 [M - H I -, 677 [ M - H - Hex] -, 487 [M - H - Hex - UA] -.
Triterpenes from Mucuna birdwoodiana
Alkaline hydrolysis of compound 8. A soln of 8 in 3% KOH-MeOH was refluxed for 30 min. The reaction mixt. was neutralized with 1 M HCI-MeOH and methylated with excess CH2N2-Et20 to yield a product identical with 1 (TLC, 1H NMR). Acknowledgements--We are grateful to Dr S. Yahara, Mr K. Takeda and Mr T. Iriguchi of this Faculty for measurements of NMR and MS and for their valuable suggestions.
REFERENCES
1. Chiang Su New Medical College (ed.) (1977) Chinese Drug Dictionary Vol. I, p. 2071. Shanghai Science and Technology Publishing.
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2. Cai, J. and Zhu, Z. (1990) Zhongcaoyao 21, 103. 3. Goda, Y., Shibuya, M. and Sankawa, U. (1987) Chem. Pharm. Bull. 35, 2657. 4. Furuya, T., Orihara, Y. and Hayashi, C. (1987) Phytochemistry 26, 715. 5. Seo, S., Tomita, Y. and Toil, K. (1975) Tetrahedron Letters 1, 7. 6. Zhang, J. and Mao, Q. (1984) Yaoxue Xuebao 19, 819. 7. Cong, P. (1987) The Application of Mass Spectra in Natural Organic Chemistry p. 686, The Science Publishing Co. 8. Kasai, R., Suzuo, M., Asakawa, J. and Tanaka, O. (1977) Tetrahedron Letters 175. 9. Toil, K., Seo, S., Yoshimura, Y., Arita, H. and Tomita, Y. (1977) Tetrahedron Letters 179. 10. Yaguchi, Y. and Sakurai, N. (1988) Chem. Pharm. Bull. 36, 1419.
00314422/91 $3.00+0.00 © 1991PergamonPress plc
TRITERPENES FROM M U C U N A BIRD WOODIANA YI DING, JUNEI KINJO, CHONG-REN YANG* and TOSHIHIRO NOHARA Faculty of Pharmaceutical Sciences, Kumamoto University, Oe-honmachi, Kumamoto 862, Japan; *Laboratory of Phytochemistry, Kunming Institute of Botany, Chinese Academy of Science, China
(Received in revisedform 1 May 1991) Key Word Iodex--Mucuna birdwoodiana; Leguminosae; triterpenoid glycoside;methyl maslinate; methyl asiatate; lfl,2a,3fl,23-tetrahydroxyolean-12-en 28-oate; lfl,2~t,3fl,23-tetrhydroxyurs-12-en28-oate; mucunagenin a; mucunagenin b.
Abstraet--Methanolic extracts of the stalks of Mucuna birdwoodiana on acid hydrolysis and subsequent methylation with diazomethane provided four triterpene sapogenols. On the other hand, investigation of glycosides after methylation of the same extract led to the isolation of four triterpene glycosides. On the basis of chemical and spectral evidence, their structures were characterized as methyl asiatate, methyl maslinate, two new sapogenols, methyl lfl,2ct,3fl,23-tetrahydroxyolean-12-en 28-oate (mucunagenin a), its urs-12-en isomer (mucunagenin b), 3-0-(6-0methyl-fl-D-glucuronopyranosyl) methyl asiatate 3-O-[~-L-arabinopyranosyl(l~2)]-6-O-methyl-fl-D-glucuronopyranosyl methyl maslinate, 3-O-[ct-L-arabinopyranosyl(1--*2)]-6-O-methyl-fl-D-glucuronopyranosylmethyl asiatate and 3-O-(6-O-methyl-fl-D-glucuronopyranosyl) asiatic acid 28-O-fl-D-glucopyranoside.
INTRODUCTION
Mucuna birdwoodiana (Leguminosae) grows in southern China. It has been recognized as being effective in promoting blood circulation or to relieve stasis. It has been widely used to treat pain or numbness of the wrists, knees or other joints and irregular menstruation [1]. Moreover, it is known that seeds of this plant contain LD O P A [21 and stalks include phenolic compounds [3]. However, no study on the triterpenoid compounds of this drug has so far been undertaken. We deal with the isolation and structure elucidation of triterpenoid components.
RESULTS
AND DISCUSSION
Dried stalks were extracted with methanol and then partitioned between n-BuOH and water. The solvent from the organic layer was removed to give a residue which was firstly hydrolysed and methylated with diazomethane in order to survey component sapogenols prior to the investigation of their glycosides; four sapogenols (1~1) were obtained. Sapogenols 1 anal 2 were identified as methyl 2~,3fl,23-trihydroxyurs-12-en-28oate (methyl asiatate) [-4] and methyl 2ct,3fldihydroxyolean-12-en-28-oate (methyl maslinate) [-4-6] on the basis of their physical data, EI mass and tH and 3C NMR spectra. Sapogenols 3 and 4 were recognized as new triterpenes. Sapogenol 3, needles, showed a [M] ÷ at m/z 518 and fragment peaks at m/z 262 [-C17H2602] +, 203 [-C15H23] +, 189 [-CI4H21] + [7] and 133 [-CloH13] + derived from D, E ring via retro-Diels-Alder fission in the EI mass spectrum (Fig. 1), indicating that 3 possesses four hydroxyl groups in the A, B-ring on the olean-12-en skeleton. The t3CNMR spectrum of 3 revealed the presence of signals due to four oxygenated carbon signals
[666.0 (t), 72.9 (d), 74.1 (d) and 83.0 (d)], one trisubstituted double bond (6123.0 and 142.1), and one carbomethoxy group (6 51.0 and 178.7) (Table 1). Moreover, 13C N M R signals due to C-18-C-22 (640.7, 45.3, 30.1, 33.3 and 31.6) suggested that 3 is an olean-12-en derivative. The acetate (3') of 3 showed signals due to three methine protons at 64.93 (d, J=9.9Hz), 5.16 (d, J=9.9Hz), 5.25 (t, J =9.9Hz) adjacent to the acetoxyl group and one acetoxymethine protons at 63.56 and 3.82 (AB q, J = 11.7 Hz) together with signals due to the typical H-18 at 62.84 (brd, J = 10.3 Hz) of an olean-12-ene type of triterpene and four acetyl groups at 31.95, 1.96, 1.99 and 2.11 in its t H N M R spectrum. The 1H-tH COSY of 3 disclosed that three signals at 64.93, 5.25 and 5.17 could be assigned to H-I, H-2 and H-3, respectively. The J values of these signals indicated that all protons were oriented axially, so that the structure of 3 could be characterized as methyl lfl,2ct,3/~,23-tetrahydroxyolean12-en-28-oate, named mucunagenin a. Sapogenol 4, needles, showed similar E1 mass and 13C N M R spectra to those of 3. The ~H N M R spectrum of the corresponding tetraacetate (4') also showed the presence of lfl,2~,3fl,23-tetraacetoxyl groups at 65.16, (d, J = 10.2 Hz), 5.27 (t, J = 10.2 Hz), 4.93 (d, J = 10.2 Hz) and 3.55, 3.82 (ABq, J = 12.4 Hz). However, on the basis of the ~HNMR spectrum of 4' and the t3CNMR spectrum (Table 1) of 4, compound 4 could be distinguished from 3 by showing characteristic urs- 12-en type signals: H- 18 at 62.19 (d, J = 12.8 Hz); C-18~C-22 at 3 52.6, 39.0, 38.8, 30.6 and 36.6. Consequently the structure of 4 could be represented as methyl lfl,2~,3fl,23-tetrahydroxyolean- 12en-28-oate, named mucunagenin b. On the other hand, separation and purification of the n-BuOH layer using Bondapak Ct8, silica gel, MCI gel, CHP 20P column chromatographies yielded the triterpene glycosides upon methylation with diazomethane providing four compounds (5-8).
3703
YI DING et al.
3704
It R1 H
-CH2COOMe73~
OR
OOR 3
~
~ 1 8 = t2 .I-
R20"
~
262
~
/
R1
R2
R3
1
H
H
CH 3
4 §
OH H
7
H
8
H
H ~-D-GIc UA-Me
CH 3
c(-L-Ara pyr(1-~2)-~-D-GIc UA-Me ~-D-GIc UA-Me
H
RI
J~-D-GIc
R3
2
H
H
CH 3
3
OH
H
CH20 H
6
H
(~-L-Ara pyr(1-~2)-~-D-Glc UA-Me
203
M M-Me M-COOMe M-AcOH M-AcOH-Me M-AcOH-COOMe M-2AcOH M-2AcOH-Me M-2AcOH-COOMe M-3AcOH M-3AcOH-Me M-3AcOH-COOMe M-4AcOH M-4AcOH-COOMe
3 -
R2
133
CH 3
OOMe
/'\..R
-C5Hlo 70
CH 3
H
R20
189
oCOOMe 59
CH 3
Glycoside 1 (5), on acid hydrolysis, yielded an aglycone which was identical with methyl asiatate (1). The negative FAB mass spectrum of 5 showed peaks due to [M + nitrobenzyl alcohol (NBA)]- at m/z 845, [ M - H ] - at m/z 691 and [M - H -glucuronic acid (UA)] - at m/z 501, indicating that it contains only one molecule of glucuronic acid. Comparison of the 13C NMR spectrum of 5 with that of 1 disclosed that the structure of 5 could be characterized as a monoglycoside, 3-O-(6-O-methyl-f-Dglucuronopyranosyl) methyl asiatate. Glycoside 2 (6), on acid hydrolysis, provided an aglycone, which was identical with methyl maslinate (2). The negative FAB mass spectrum of 6 showed peaks due to [ M + N B A ] - at m/z 961, [ M - H ] - at m/z 807 and [M -H - U A -pentose (Pen)] - at m/z 485, indicating that 6 contains one molecule of glucuronic acid and a pentose. On the basis of a detailed ~H-~H COSY study of the acetate (6') of 6, all signals due to the sugar moiety could be assigned as given in the Experimental. Thus, the structure of sugar is ~-L-arabinopyranosyl (1--,2)-6-O-
1' 628 (7) 613 (3} 569 (6) 568 (9) 553 (4) 509 (5) 508 (7) 493 (3) 449 (4) 448 (5) 433 (4) 389 (3) ---
2' 570 (11) - -
511 (6) 510 (9) 495 (4) 450 (11) 435 (7) 391 (5) -- -
-- -
---
3' 686 (10) - -
627 (48) 626 (100) -567 (35) 566 (74) -507 (48) 506 (74) -447 (23) 446 (21) 387 (11)
Fig. 1. Molecular and fragment ion peaks (re~z)in the EI mass spectra of compounds 1'-3'.
methyl-fl-D-glucuronopyranoside. Furthermore, comparison of the 13CNMR spectrum of 6 with that of 5 (Table 1) also suggested that one additional arabinopyranosyl moiety attached to 5 forming 6. The downfield shift (+7.5 ppm) at C-2 and the upfield shift ( - 1.4 ppm) at C-3 on the glucuronic acid moiety in 6 revealed that the linkage between the two sugars should be 1--,2. Therefore, 6 is 3-O-[~-L-arabinopyranosyl (1-*2)]-6-O-methylfl-D-glucuronopyranosyl methyl maslinate. Glycoside 3 (7), on acid hydrolysis, afforded the same aglycone as that obtained from 5 by comparing R I values on TLC and 1H NMR spectra. The negative FAB mass spectrum of 7 showed peaks due to [M + NBA] - at m/z 977, [ M - H I - at m/z 823, [ M - H - P e n ] at m/z 691 and [ M - H - U A - P e n ] at m/z 501, indicating that 7 contained one molecule of glucuronic acid and a pentose in the sugar residue which was identical to that of 6. On the other hand, the ~3C NMR spectrum of 7 (Table 1) also showed the same chemical shifts as those of 6 in the sugar moiety. Consequently, 7 is 3-O-[~-L-arabinopyranosyl (1-~2)]-6-O-methyl-fl-D-glucuronopyranosyl methyl asiatate. Glycoside 4 (8), exhibited two anomeric proton signals at65.27(1H, d,J=7.3 Hz)and 66.26(1H, d,J=8.1 Hz)in
Triterpenes from
Mucunabirdwoodiana
3705
Table 1. 13CNMR spectral data for sapogenols 1-4 (400 MHz, CDCI3) and glycosides 5-8 (400 MHz, pyridine-ds) C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 CO2Me G-UA 1 2 3 4 5 6 CO2Me Ara 1 2 3 4 5 Glc 1 2 3 4 5 6
1
2
3
4
5
6
7
8
47.7 68.8 78.1 42.1 c 48.0 18.4 33.1 39.4 b 47.8 38.2 23.7 125.5 138.4 42.3 o 28.5 24.8 48.2 53.4 39.4b 39.3b 31.0 37.4 66.3 13.0 17.1a 17.2a 23.8 178.4 17.4a 21.3 51.6
47.8 68.6 83.8 39.9b 56.0 18.9 33.2 39.9b 48.2 38.6 23.7 122.5 143.6 42.2 c 28.3 24.0 46.7 42.0 ~ 46.5 31.0 34.2 33.2 29.4 16.8 17.5 16.8 26.0 178.0 33.2 23.5 51.5
72.9 74.1 83.0 42.2 48.0 17.1 31.9 41.0 44.6 39.3 26.3 123.0 142.1 41.8 27.2 22.8 46.4 40.7 45.3 30.1 33.3 31.6 66.0 12.4a 12.2a 16.4 25.2 178.7 32.3 22.5 51.0
73.5 75.0 83.5 42.5 ~ 48.3 17.6 32.5 40.0 45.3 40.0 26.7 126.3 137.2 42.8 c 28.0 24.2 48.0 52.6 39.0 38.8 30.6 36.6 67.6 13.2b 13.2b 17.1a 23.7 178.1 17.1~ 21.2 51.5
47.5 66.8 88.1 42.3 47.4 18.1 32.9 39.9b 47.9 37.7 23.7 125.8 138.7 44.7 28.4 24.5 48.3 53.3 39.2 b 39.2b 30.9 37.0 63.5 14.6 17.3~ 17.3~ 23.8 177.7 17.5~ 21.2 51.5
47.5 66.3 95.2 37.7 55.5 18.3 32.8 40.8 47.7 39.5 23.3 122.7 144.0 41.8 ~ 27.9 23.7 47.2 41.6 ¢ 45.9 30.7 33.9 32.7 28.6 16.6 17.0a 17.2a 26.0 177.9 33.1 23.6 51.5
47.5 66.4 88.1 42.1 47.1 17.9 32.7 39.7b 47.8 37.5 23.5 125.7 138.5 44.7 28.2 24.4 48.1 53.1 39.1b 39.1b 30.6 36.8 63.0 13.8 17.0" 17.2~ 23.7 177.6 17.3a 21.2 51.3
47.4 66.7 87.9 42.2 47.0 18.0 32.9 40.0 47.9 37.6 23.7 125.9 138.2 44.5 28.5 24.5 48.2 53.1 39.2 38.9 30.6 36.0 63.3 14.5 17.2a 17.4a 23.6 176.1 17.6a 21.1 --
105.8 75.1 77.8 73.0 77.8 170.2 52.1
104.5 82.6 76.4 73.5 77.4 169.9 52.1
103.7 82.7 76.2 73.5 77.2 169.9 52.1
105.6 .75.0 77.6 72.8 76.6 170.1 52.0
106.6 72.6 74.3 69.2 67.2
106.5 72.4 74.2 69.1 67.1
m
m
m
m
m
m
w
m
m
95.5 73.9 78.7 71.1 79.0 62.2
"-Clnterchangeable values.
the t H N M R s p e c t r u m . O n acid hydrolysis it afforded a n aglycone (la), w h o s e m e t h y l ester was identical to 1 o n T L C . T h e E1 m a s s s p e c t r u m of l a s h o w e d a [ M ] ÷ at m/z 488 a n d f r a g m e n t i o n p e a k s a t m/z 203 a n d 248, which were 14 m u lower t h a n t h o s e of 1, s u g g e s t i n g t h a t the 28-
c a r b o x y l g r o u p exists in the free form. T h e negative F A B m a s s s p e c t r u m o f g s h o w e d p e a k s d u e to [ M + N B A ] - at m/z 993, [ M - H ] - at m/z 839, [ M - H - h e x o s e ( H e x ) ] at m/z 677 a n d [ M - H - U A - H e x ] at m/z 487. O n alkaline hydrolysis, 8 afforded a p r o s a p o g e n i n w h i c h was
YI DING et al.
3706
identical with 5 upon methylation with diazomethane. A comparative study o f the 13C N M R spectrum of 8 with that of 1 (Table 1) indicated the glycosylation shifts (I-8, 91 at C-3 and C-28 in the aglycone moiety. An anomeric carbon signal due to the ester type of the glucosyl linkage appeared at 395.5, indicating that 8 is a 3,28-btsdesmoside. Thus, the structure of 8 was established as 3-0-(6-0methyl-fl-D-glucuronopyranosyl) asiatic acid 28-O-fl-Dglucopyranoside. Finding two new sapogenols possessing lfl,2~t,3fl,23tetrahydroxyl groups in the A-ring of olean- or urstriterpene is worthy of note. Moreover, asiatic acid and maslinic acid have so far been isolated from several plants [4, 10"1, but they have rarely been obtained as glycosides. It has been reported that maslinic triglycoside occurs in Swertia cincta [6!, but the sugar moiety is different from that found in M. birdwoodiana.
EXPERIMENTAL
1H and 13CNMR spectra were measured on a 400 MHz instrument and chemical shifts are given on a 6 (ppm) scale with TMS as int. standard. CC was carried out with MCI gel CHP 20P (75-100 mm, Mitsubishi) and Kieselgel 60 (70-230 and 230-400 mesh, Merck). TLC was conducted on precoated Kieselgel 60 F254 plates (Merck), and detection was achieved by spraying with 10% H2SO4 followed by heating. Extraction and separation. Dried stalks (6 kg) ofM. birdwoodiana (Tutcher) were extracted with MeOH and the extract (298.3 g) partitioned between n-BuOH and H20. The n-BuOH layer was evapd under red. pres. to give a residue (216.2 g), a part (3.36 g) of which was refluxed in 1 M HCI-MeOH at 100° for 2 hr and then neutralized with 3% KOH-MeOH. Removal of MeOH followed by partition between H20 and EtOAc provided the EtOAc extract which was methylated with excess CH2N2-Et20 and then subjected to a silica gel CC with CHC13-MeOH-H20 (200:20:1) to afford frs 6-25 (450mg), which were further chromatographed on silica gel with nhexane-Me2CO (1 : 1) to give frs 32-50 which were recrystallized from MeOH, to give 2 (18.5 mg). Frs 181-209 were also recrystallized from MeOH to give 1. Frs 210-270 were repeatedly subjected to silica gel CC with CHC13-MeOH-H20 (100:10: 1) and CHCla-Me2CO (1 : 1), and then recrystallized from MeOH, giving 3 (22.5 mg) and 4 (26.7 mg). The remaining n-BuOH portion (208.9 g) was refluxed with nhexane. The n-hexane insol, portion (161.4 g) was subjected to Sephadex LH-20 CC using MeOH to give frs 1-7. Frs 3-5 were collected and chromatographed on a silica gel column with CHCI3-MeOH-H20 (40:10:1--,14:6:1). Frs 64-66, 67-72 and the MeOH eluate were respectively treated with excess CH2N2-Et20 followed by CC over silica gel using CHCI3-MeOH-H20 (90: 10:1--,40: 10: 1) and Bondapak with 40---,70% MeOH to provide glycosides 1 (5) (94.5 mg), 2 (6) (54.2 mg), 3 (7), (86.3 mg) and 4 (8) (135.6 mg). Sapogenol 1 (1). Plates, mp 228-230 ° (MeOH). [~]22+45.1° (MeOH; c 0.50). 13CNMR (Table 1). 1-Acetate (1'). A soln of 1 (10 mg) in Ac20-pyridine (1 : 1) was heated at 80° for 2 hr. The reaction mixt. was evapd to dryness and then chromatographed on silica gel using n-hexane-EtOAc (2: 1), giving the peracetate (1') as a powder. 1H NMR (CDCI3) 6: 0.75-1.10 (m, 6xMe), 1.98, 2.02, 2.09 (3 x OAc), 2.23 (1H, d, J = l l . 4 H z , H-18), 3.57 (1H, d, J=12.4Hz, H-23), 3.60 (s, CO2Me ), 3.85 (1H, d, J = 12.4 Hz, H-23'), 5.07 (1H, d, J = 10.3 Hz, H-3), 5.16 (m, H-2), 5.25 (1H, t-like, H-12). EIMS (Fig. 1). Sapogenol 2 (2). Needles, mp 223-224 ° (MeOH). [ct]23 +48.1 ° (MeOH; c 0.16). ~3CNMR (Table 1).
2-Acetate (2'). Compound 2 was acetylated as described above to yield 2'. 1HNMR (CDC13) 6:0.72-1.25 (m, 7 × Me), 1.98, 2.05 (2 × OAt), 2.82 (1H, dd, J=4.3, 11.9 Hz, H-18), 3.60 (s, CO2Me), 4.74 (1H, d, J = 10.3 Hz, H-3), 5.27 (m, H-2), 5.36 (1H, tlike, H-12). EIMS (Fig. 1). Sapogenol 3 (3). Needles, mp 235-236 ° (MeOH). [~t]24 + 35.3° (MeOH; c 0.22). EIMS m/z (rel. int.): 518 [M] +, (14), 500 [M - H 2 0 ] + (35), 482 [ M - 2 H 2 0 ] + (21), 262 [C17H2602] + (72) (D/E ring fragment of RDA), 203 [262-CO2Me] + (100), 189 [262-CH2CO2Me] + (37), 133 [203-C5Hlo] + (17). 13CNMR (Table 1). 3-Acetate (3'). Compound 3 was acetylated as described above to yield 3'. 1H NMR (CDCI3) 6:0.70-1.22 (m, 6 x Me), 1.95, 1.96, 1.99, 2.11 (4 x OAc), 2.84 (1H, br d, J = 10.3 Hz, H- 18), 3.56 (1H, d, J = 11.7 Hz, H-23), 3.61 (s, CO2Me), 3.82 (1H, d, J = 11.7 Hz, H23'), 4.93 (1H, d, J=9.9 Hz, H-3), 5.17 (1H, d, J=9.9 Hz, H-I), 5.19 (1H, t-like, H-12), 5.25 (1H, t, J = 9.9 Hz, H-2). EIMS (Fig. 1). Sapooenol 4 (4). Needles, mp 165-166 ° (MeOH). [0t]24 + 38.9 (MeOH; c 0.40). EIMS m/z (rel. int.): 518 [M] ÷ (9), 500 [M --H20"] + (26), 482 ['M--2H20"] + (14), 262 [C17H2602] + (57) (D/E ring fragment of RDA), 203 [262-CO2Me] + (100), 189 [262-CH2CO2Me] + (31), 133 [203-CsH~o] + (19). 13CNMR (Table 1). 4-Acetate (4'). Compound 4 was acetylated as described for 1 to yield 4'. tH NMR (CDCIa) ~3:0.72-1.23 (m, 6 x Me), 1.96, 1.99, 2.00, 2.10 (4 x OAt), 2.19 (1H, d, J = 12.8 Hz, H-18), 3.55 (1H, d, J = 12.4 Hz, H-23), 3.59 (s, CO2Me), 3.82 (1H, d, J = 12.4 Hz, H23'), 4.93 (1H, d, J = 10.2 Hz, H-3), 5.14 (1H, t-like, H-12), 5.16 (1H, d, J = 10.2 Hz, H-I), 5.27 (1H, t, J = 10.2 Hz, H-2). Glycoside 1 (5). Powder. [ct]~3 + 8.05° (MeOH; c 0.36). Negative FAB-MS (m/z): 845 [ M + N B A ] - , 691 [ M - H I - , 677 [ M - M e ] - , 501 [ M - H - U A ] - . ~HNMR (pyridine-ds) 6:0.85-1.08 (s, 6 x Me), 2.39 (1H, d, J = 11.4 Hz, H-18), 5.33 (1H, t-like, H-12). laCNMR (Table 1). Acid hydrolysis of compound 5. A soln of 5 (5 mg) in 1 M HC1-MeOH was refluxed at 100° for 2 hr and then neutralized with 3% KOH-MeOH. Removal of MeOH followed by partition between H20 and EtOAc provided the EtOAc extract which was column chromatographed over silica gel to give sapogenol 1. Glycoside 2 (6). Powder. [a]~3+22.3 ° (MeOH; c 0.35). 1HNMR (pyridine-ds) 6:0.81-1.32 (m, 7 x Me), 2.24 (1H, dd, J =4.8, 12.8 Hz, H-18), 5.35 (1H, t-like, H-12). Negative FAB-MS (m/z): 961 [ M + N B A ] - , 807 [ M - H I - , 485 [ M - H - P e n -UA]-. 1aC NMR (Table 1). Acid hydrolysis of 6 gave 2. 6-Acetate (6'). Compound 6 was acetylated to give the peracetate (6'). ~H NMR (CDCI3) 6: 0.71, 0.90, 0.92, 0.96, 1.02, 1.10, 1.11 (each 3H, s, 7 x Me), 1.97, 1.98, 2.00, 2.04, 2.09, 2.11 (each 3H, s, 6 x OAc), 2.83 (1H, dd, J=3.9, 12.1 Hz, H-18), 3.21 (1H, d, J = 9.9 Hz, H-3), 3.55 (1H, d, J = 12.8 Hz, Ara H-5), 3.62, 3.70 (each 3H, s, 2 x CO2Me), 3.84 (1H, dd, J=7.7, 9.0 Hz, UA H-2), 3.96 (1H, d, J = 9.1 Hz, UA H-5), 4.01 (1H, dd, J = 2.0, 12.9 Hz, Ara H5'), 4.53 (1H, d, J=7.7 Hz, UA H-l), 4.55 (1H, d, J=7.7 Hz, Ara H-I), 4.93 (1H, dd, J = 3.7, 9.9 Hz, Ara H-3), 5.09 (1H, dd, J = 3.7, 9.9 Hz, Ara H-3), 5.09 (1H, t, J =9.3 Hz, UA H°4), 5.10 (1H, dd, J = 7.8, 9.5 Hz, Ara H-2), 5.13 (1H, m, H-2), 5.21 (1H, m, Ara H-4), 5.24 (1H, t, J=9.3 Hz, UA H-3), 5.27 (1H, t-like, H-12). Glycoside 3 (7). Powder, I-~t]~4+19.2° (MeOH; c 0.26). ~HNMR (pyridine-ds) 6:0.84-1.15 (m, 6 x Me), 2.39 (1H, d, J = l l . 4 H z , H-18), 5.33 (IH, t-like, H-12). Negative FAB-MS (m/z): 977 [ M + N B A ] - , 823 [ M - H I - , 691 [ M - H - P e n ] - , 501 [ M - H - P e n - U A ] - . Acid hydrolysis of 7 gave 1. Glycoside 4 (8). Powder. [~t]~'*+l.5 ° (MeOH; c 0.47). 1HNMR (pyridine-ds) 6:0.87-1.15 (m, 6 x Me)., 2.50 (1H, d, J = 11.4 Hz, H-18), 5.39 (1H, t-like, H-12). Negative FABMS (m/z): 993 [M +NBA] -, 839 [M - H I -, 677 [ M - H - Hex] -, 487 [M - H - Hex - UA] -.
Triterpenes from Mucuna birdwoodiana
Alkaline hydrolysis of compound 8. A soln of 8 in 3% KOH-MeOH was refluxed for 30 min. The reaction mixt. was neutralized with 1 M HCI-MeOH and methylated with excess CH2N2-Et20 to yield a product identical with 1 (TLC, 1H NMR). Acknowledgements--We are grateful to Dr S. Yahara, Mr K. Takeda and Mr T. Iriguchi of this Faculty for measurements of NMR and MS and for their valuable suggestions.
REFERENCES
1. Chiang Su New Medical College (ed.) (1977) Chinese Drug Dictionary Vol. I, p. 2071. Shanghai Science and Technology Publishing.
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