1980 Chem. Pharm. Bull. Vol.28, 2753-2762

by Hikaru Okabe, Yumi Miyahara, Tatsuo Yamauchi, Kazumoto Miyahara, Toshio Kawasaki
The fruit of Momordica charantia L.…

Studies on the Constituents of Momordica charantia L. I. Isolation and Characterization of Momordicosides A and B, Glycosides of a Pentahydroxyl-Cucurbitane Triterpene

Author: Hikaru Okabe, Yumi Miyahara, Tatsuo Yamauchi, Kazumoto Miyahara, Toshio Kawasaki

Type of Publication: Pre-Clinical

Date of Publication: 1980

Publication: Chem. Pharm. Bull. Vol.28, 2753-2762, 1980

Organization: Fukuoka University and Kyushi University

Abstract: Two triterpene glycosides, momodicosides A and B, were isolated from the seeds of Momordica charantia L. (Cucurbitaceae). Their stucture were determined on the basis of spectral and chemical evidence and by X-ray analysis as the 3-O--gentiobioside and 3-O--D-xylopyranosyal (1-4)-[ -D-glucopyranosyl(1-6)] --D-glucopyranoside, respectively, of cucurbit-5-ene-3-, 22(S), 23(R), 24(R), 25-pentaol

The fruit of Momordica charantia L. (cucurbitaceae) (Niga-uri or Tsuru-reishi in Japanese) has been used as bitter stomachic southern Japan, and as a laxative and an anthelmintic for children in India.³⁾ An antidiabetic effect of its alcohol extract was also reported.⁴⁾ As a first in chemical studies of the bitter principles of the fruit, the seed was investigated. This paper deals with the isolation and stucture elucidation of two kinds of oligoglycosides of a cucurbitane derivative, named momordicosides A (I) and B (II).

The defatted methanol extractive of the seeds was partitioned between butanol and water and the latter layer, containing vicine (III),⁵⁾ was discarded. The butanol layer was subjected to successive column chromatographies and two compounds, I, C₄₂H₇₂O₁₅2H₂O_, mp 181-187 (dec.), []_D + 1.05⁰, and II, C₄₇H₈₀O₁₉. 3/2H₂O, mp 238-242⁰ (dec.), []_D + 6.15⁰, were isolated.

On mild methanolysis, I gave methyl glucoside and an aglycone (IV), while II furnished a product identical with I, methyl xyloside and glucoside, and IV, indicating II to be a xyloside of I. IV was assumed to retain its stucture in glucoside I and II, sine IV was also obtained on enzymatic hydrolysis of I, and the carbon –13 nuclear magnetic resonance (CMR) spectrum of IV was almost identical with those of I and II, except for the signals due to sugar moieties and those of the aglycone carbons affected by the glycoside linkage.

IV was formulated as C₃₀H₅₂O₅ based on the elementary analysis results and the molecular ion peak (M+) observed at m/z 492 in the field desorption mass spectrum (FD-MS). The proton nuclear magnetic resonance (PMR) of IV exhibited signals of seven tertiary (0.96 (x2), 0.94, 1.12, 1.40, 1.67 and 1.75 ppm) and one secondary (1:43 ppm) methyl groups along with four methines bearing hydroxyl groups (J= ca 4 Hz). The presence of four secondary and one tertiary hydroxyl groups was seen in the CMR spectrum of IV as five peaks between 70 and 76 ppm, four of which were transformed to doublets by off-resonance decoupling measurement, while one remained as a singlet. In the rgion from 34.8 to 49.2 ppm, four quarternary carbons signals were observed, and olfinic carbons were found at 143.0 (singlet) and 119.1 ppm (doublet).

Its molecular formula and the presence of one double bond, seven tertiary methyl group and four C-C-bonded and one hydroxylated quaternary carbons, as described above, suggested IV to be a tetracyclic triterpenoid. The PMR and CMR patterns due to a proton and carbon-of a double bond, respectively, were similar to those of cucurbit-5-ene derivatives,⁶ and the EI-MS spectrum of IV showed peaks at m/z 152 and 340 which could be considered to be provided by the typical retro-Diels-Adler fragmentation of ring B of a cucurbit-5-ene derivative having hydroxyl group in ring A.^6a,7)

IV was acetylated with Ac₂O and pyridine at room temperature to give a tetraacetate (V) and at 90⁰ to give a pentaacetate (VI). The hudroxyl group acylable only on heating was tentatively located at C-25 of the cucurbitane nucleus, and the two singlets at 1.67 and 1.75 ppm in the PMR spectrum of IV were assigned to the 26-and-27-methyl groups.

The finding that IV afforded mono-(VII) and di- (VIII) acetonides, and the coupling patterns ( 3.83 (d, J = 9 Hz),  4.10 (dd, J = 7, 9 Hz) and  4.36 (d, J = 7 hz) of three protons on three carbinyl carbons of VIII, suggested that two glycol system were adjacent to each other. When the monoacetonide (VII) was subjected to periodate oxidation, an aldehyde IX) was obtained. An FD-MS spectrum of IX showed the (M + H)^ ion peak at m/z 472, indicating that bond fission in VII took place between C-24 and 25. On the other hand, when IV was treated with periodic acid in methanol, the bond between C-22 and –23 was cleaved ti give mixture of an aldehyde (X) and its dimethyl acetal (IX) which retained one secondary hydroxyl group. Therefore, four hydroxyl groups were thought to be located at C-22, -23, 24 and –25.

The remaining one hydroxyl group was assumed to be at C-3, and this was confirmed by converting XI to an oxo derivative (XII), and further to the corresponding 2-hydroxy-1-n-3-one (XIII); the PMR spectrum of the latter showed the proton at C-1 at 6.12 ppm as a doublet (J = 3 Hz), also supporting the presence of a proton at C-10.

Consequently, IV was presumed to be a cucurbit-5-ene-3, 22, 23, 24, 25-pentaol. In order to confirm the cucurbitane framework and to determine the configurations of substituents, X-ray analysis of IV was carried out by the direct method. The final atomic parameters, bond lengths and bond agles for nonhydrogen atoms are given in Tables I, II and III, respectively, and an ORTEP drawing is shown in Fig.1.

The aglycone was thus identified as cucurbit-5-ene-3,22(S), 23(R), 24(R), 25(R)-pentaol (IV) or its enantiomer (IV’).

The CD spectrum of XII showed a negative Cotton curve ([0]₂₉₄ – 8030⁰) indicating the presence of a 10-hydrogen atom and a 5,6-double bond.⁸⁰

Thus, the absolute structure was established as IV.

Momordicoside A (I) showed the (M + Na)^- peak at m/z 839 in the FD-MS spectrum, and the anomeric carbon signals at 105.2 and 106.9 ppm in the CMR spectrum. The EI-MS spectrum of its acetate (XIV) showed a fragment peak (m/z 619) due to an acetylated biose moiety. These data suggest that the sugar moiety of I is composed of two moles of glucose. The signals in the CMR spectrum ascribed to the sugar carbons were quite similar to those of pregnenolone gentioside.¹⁰⁾

When I was methylatede in tetrahydrofuran with NaH and CH₃I, two methylates (XV and XVI) were obtained. Methanolysis of their XV or XVI furnished equal amounts of methyl pyranosides of 2, 3, 4, 6-tetra-O-methyl glucose and 2, 3, 4-tri-O-methyl glucose. The linkage between the two glucose units as well as that to the aglycone were regarded as  in view of the coupling constants (ca. 7 Hz) of the two anomeric protons in the PMR spectrum of XV> The aglycones provided by methanolysis of XV and XVI were 25, 26-anhydro- (XVII), 22, 25-anhydro (XVIII) and 22, 23, 24, 25-tetra-o-methyl (XIX) derivative from the former, and XVII, XVIII and 2, 23, 24,-tri-o-methyl ether (XX) from the latter. Thus, XV is the permethylate (missing text), while XVI has a free hydroxyl group at C-25.

Sugar linkage with the hydroxyl group at C-3 in I was confirmed by its CMR spectrum in which a downfield shift of the C-3 signal compared with that in IV was observed, and by the result that XIX was affordable by methanolysis of XV.

Accordingly, I was concluded to be the 3-O--gentiobioside of IV.

On comparison of the FD-MS spectra and the molecular formulae of I and II, the sugar moiety of the latter appeared to be composed of two moles of glucose and one of xylose. The EI-Ms spectrum of II-acetate (XXI) showed peaks at m/z 331 and 259, corresponding to the pyronium ions originated from the terminal acetylated glucose and xylase residues, indicating a branched chain trisaccharide structure. II-Permethylate (XXIIa) was subsequently methanolyzed and the resulting methylated monosaccharide were identified by GLC as methyl pyranosides of 2, 3,4-tri-O-methyl xylose, 2, 3, 4, 6-tetra-O-methyl glucose and 2, 3,-di-O-methyl glucose. Since I was formed by partial hydrolysis of II, the xylose unit II should be linked to the hydroxyl group at C-4 of the glucose combined with the aglycone. The mode of linkage of the xylose unit was determined as  judging from the coupling constant (6 Hz) of its anomeric proton signal in the PMR spectrum of XXIIa.

Thus, II is the 3-O--D-xylopyranosyl (1-4)-[-D-glucopyranosyl (1-6)]- -D-glucopyranoside of IV.

More than thirty cucurbitane derivatives have so far been isolated from plants, mainly of Cucurbitaceae and also of Primulaceae,¹¹⁾ Cruciferae,¹²⁾ Begoniaceae¹³⁾ and Datiscaceae.¹⁴ They are, in general, highly oxygenated and the oxygen functions are distributed in the nucleus and side chain (C-3 and C-11 being invariably oxygenated).

The aglycone (IV of momordicosides A (I) and B (II) is also typical cucurbitane derivative, but is unusual in that four of the five hydroxyl groups are located in the side chain and in that there is no oxygen function at C-11.

Experimental¹⁵⁾

Extraction and Isolation of I, II and III – Seeds (3.34 kg) of Momordica charantia L. were crushed and percolated successively with MeOH (201) and MeOH-H₂O (1:1) (201). The MeOH solution was concentrated under reduced pressure to 1/10 volume, repeatedly extracted with hexane to remove lipids. The MeOH solution was concentrated to dryness, suspended in water and extracted with BuOH. The BuOH solution was concentrated to dryness to give a light brown powder (55 g). This was repeatedly chromatographed on silica gel (50-100 times the weight of material) using CHCI₃-MeOH-H₂O (70:25:3) as an eluent to give (4.3 g) and II (0.3 g).

I: colorless needles from MeOH, mp 181-187⁰ (dec.), []_D²⁰ + 1.05⁰ (c=0.96, CHCI₃-MeOH (2:1), FD-MS m/z: 839 (M + Na) ^, 855 (M + K)⁺. Anal. Calcd for C₄₂H_72O15_.2H₂O: C, 59.13; H, 8.98. Found: 58.95; h, 8.85. CMr: olefinic carbons: 14.2 (s), 118.66(d) , 72.41.63(d), 71.19, 62.72(t); quaternary carbons: 49.12, 4678, 41.66, 34.70.

II: colorless needles from MeOH-CHCI₃, mp 238-242⁰ (dec.), [a] _D²⁰ + 6.15⁰ (c= 0.98, CHCI₃-MeOH 2:1). FD-MS m/z: 971 (M + Na)⁺. Anal Cald for C₄₇H₈₀O₁₉. 3/2H₂O: C, 57.83; H, 8.57. Found: C, 57.60; H, 8.43. CMR: olefinic carbons: 142.85(s), 118.48(d); oxygen-bearing carbons: 106.58(d), 104.82(d), 87.19(d), 79.98(d), 79.51(d), 76.11. 74.94, 74.71, 74.24(s), 72.25, 71.25, 71.48, 71.48, 71.07, 71.07, 70.66,68.83, 67.38, 62.64(t); quaternary carbons: 49.04, 46.70, 41.60, 34.63.

The aqueous layer from the BuOH-H₂O extraction of the defatted NeOH extractives and the MeOH H₂O extract were combined and concentrated. On standing at room temperature colorless needles (11 g) were separated out, and these were recrystallized from water to give III: colorless, mp 250-255⁰ (decrease.), [_D²⁵ – 13.5⁰ (c= 2.15, 0.1% NaOH). Anal. Cald for C₁₀H₁₆N₄.1/2H₂O: C, 38.33; H, 5.43; N, 17.89. Found: C, 38.66; H, 5.47; N, 17.95. UV _max^{0.5 NHCI} nm (): 273 (15800), _max ^H2O nm (): 275 (12700), 210 (20400), _max^{0.1 NnaOH} nm (): 268 (9700), 235 (5624), 217 98500). CMR (1_N KOH): base moiety, 170.3, 160.9, 158.8, 118.6; sugar moiety, 108.0 (1’), 78.6 (3’), 77.9 (5’), 74.7 (2’), 71.5 (4’), 62.6 (4’), 62.6 (6’). IR (KBr): superimposable on the spectrum of vicine¹⁶ isolated from faba beans beans (Vicia faba L.) according to the method at Lin et a.¹⁷

Methanolysis of I, Identification of the Component Sugar and Isolation of the Aglycone (IV) – A solution :1 (610 mg) in 1_N HCI-MeOH (20 ml) was stirred for 5 days at room temperature then neutralized with Ag₂CO_3. The precipitates were filtered off and the filtrate was evaporated to dryness under reduced pressure. The residue was chromatographed on silica gel (160 g). Elution with 3% MeOH-CHI₃ gave an aglycone 223 mg), which was crystallized from MeOH to give colorless needles (IV): mp 193-195⁰ []_D²⁰ + 48.1⁰ 0.94, CHCI₃-MeOH (2:1). FD-MS m/z 492 (M^). Anal Cald for C₃₀H₅₂O₅.1/2H₂O: C, 71.86; H, 19.58. Found: C, 72.04; H, 10.79. PMR: .90 (6H, s, C-CH₃x2), 0.94 (3H,s, C-CH₃), 1.40 (3H,s, C-CH₃), 1.43 (3H, d, J= 6Hz, >CH-CH₃), 1.67, 1.75 (3H each, s, HO-C(CH₃)₂), 3.74 (h, br, s,>CH-OH), 4.07 (H, d, J= 9 Hz, -CH (OH)-CH(OH)-), 4.37 (H, d, J= 9 Hz, - CH(OH)-CH-H)-), 4.57 (H, d, J= 4 Hz,>CH-OH), 5.61(H, br d, J= 4 Hz, >C=CH-). CMR: olefinic carbons: 143.02 s, 119.12(d); oxygen-bearing carbons: 75.94(d), 75.23(d), 74.30(s), 72.30(d), 71.13(d); quartenary carbons: 19.16, 46.82, 41.60. EI-MS m/z: 492 (M^), 474 (M^-H₂O), 340, 322, 177, 159, 163, 152, 134, 59.

Elution with 25% MeOH-CHI₃ gave resinous glycoside (160 mg) ([] _D²⁵ + 93.1⁰ (c= 1.23, MeOh)). Its PMR spectrum exhibited two methoxyl proton signals at 3.46 and 3.61 in ration of 2:1 and anomeric proton signals at 4.73 (d, J= 7 Hz) and 5.17 ppm (d, J= 4 Hz) (methoxyl proton signals: methyl -_D-glucoside, 3.46: -anomer, 3.61 ppm. Anomeric proton signals: methyl -_D-glucopyranoside, 5.17 (d, J = 4 Hz) ; -anomer, 4.73 ppm (d, J= & Hz)). The methyl glycoside was acetylated in the usual manner and examined by GLC.¹⁸⁾ It gave two peaks with retention times (4.6 and 5.2 min) identical with those of methyl 2,3,4,6tetra-O-acetyl--_D-glucopyranoside and its

- anomer.

Enzymatic Hydrolysis of I- I (80 mg) was suspended in water (8 ml) and cellulose (Type I, Sigma Chemical Co.) ( 80 mg) was added. The mixture was shaken for a week at 38⁰. The reaction mixture was extracted with CHCI₃ and then with BuOH. Both extracts were combined and chromatorgraph on silica gel (15 g). The fraction eluted witg 3% MeOH-CHCI₃ gave the aglycone (33 mg) as colorless needles (crystallized from MeOH), identical with IV (IR and mixed mp).

Methanolysis of II, Identification of the Component Sugars and isolation of Momordicoside A (I) and the Aglycone (IV) – II (660 mg) was dissolved in 1 N HCI-MeOH (20 ml) and stirred fo 5 hour at room temperature. The reaction mixture was neutralized with Ag₂CO₃ and worked up in the same way as for I. The product was treated with 15% acetone- H₂O and the insoluble material (550 mg) was chromatographed on silica gel (70 g), using CHCI₃-MeOH-H₂O (70:25:3) as an eluent to give Fr. 1 (143 mg), Fr.2 (170 mg) and Fr. (170 mg). Fr. 1 was further chromatographed on silica gel (30 g) (eluent, 3% MeOH-CHCI₃) to provide a thin- layer-chromatographically homogeneous compound, which was crystallized from MeOH to give colorless needles (46 mg): mp 192-195⁰. The IR spectrum was superimposable on that of IV and the melting point was not depressed on admixture with IV. Fr. 2 was crystallized from MeOH-H₂O to give colorless needles (94 mg): mp 180-188⁰ (dec.). This product gave the same IR and CMR spectral as I. Fr. 3 gave II on recystallization. The 15% acetone-soluble substance 9122 mg) showed two spots on TLC and their Rf values were identical with those of methyl -_D-xylopyranoside (Rf: 0.46, CHCI₃-MeOH-H₂O (70:35:5) and methyl -_D-glucopyranoside (Rf: 0.32)_. This mixture was subjected to column chromatography (silica gel, 11 g; CHCI₃-MeOH-H₂O (70:25:3) to give two methyl glycoside. The less polar one (27 mg) showed [] _D²⁵⁰ + 127.6⁰ (c= 1.34, MeOH)¹⁹⁾ and its PMR spectrum exhibited methoxyl proton signals at 3.49 and 3.58 ppm (ration 3:1). It was acetylated and subjected to GLC, ¹⁸⁾ giving two peaks with t_R 1.60 and 1.80 min (methyl -_D-xylopyranoside triacetate, t_R 1.55; -anomer, 1.80). In the same way, the polar methyl glycoside was identified as a mixture of methyl -_D-glycopyranoside and its -anomer.

Acetylation of IV- i) IV (20 mg) was dissolved in 0.6 ml of Ac₂O-pyridine (1:1) and the solution was stirred for 15 hour at room temperature. The solution was then evaporated to dryness and the residue was chromatographed on silica gel (6 g) (4% acetone-benzene) to give a tetraacetate (V) (7 mg): fine needles (from acetone- benzene), mp 178-182⁰. PMR: 1.48 (6H, s, HO-C (CH₃)₂), 2.00 (3H, s, -Oac), 2.12 (6H, s, -OAc x2), 2.18 (3H, s, -OAc), 4.91 (H, perturbed t, >CH-OAc), 5.46 (H, d, J= 7 Hz, > CH-OAc), 5.49 (H, m, >C=CH-), 5.71 (H, br s, >CH-OAc), 6.12 (H, d, J= 7 Hz, >CH-OAc). CMR: olefinic carbons: 141.95(s), 119.44(d); oxygen-bearing carbons: 78.85(d), 76.65(d), 73.29(d), 71.58(s), 69.15(d); quaternary carbons: 49.12, 46.93, 39.91, 34.74; acetyl carbonyl carbons: 170.65, 170.65, 170.56, 170.36, 170.17. EI-MS m/z: 660 (M^), 600 (M^-AcOH), 466, 406, 194, 163, 134. ii) IV (50 mg) in 1 ml of Ac₂O-pyridine (1:1) was heated at 90⁰ for 6 hour. The solvent was removed and the residue was chromatograhed on silica gel 920 g) (2.5% acetone-benzene) to give a pentaacetate (VI) (31 mg): colorless needles (from MEOH), mp 104-107⁰. Anal. Calcd for C₄₀H₆₂O₁₀.H₂O: C, 66.62; H, 8.95. Found: C, 66.50; H, 8.73. PMR: 1.65, 1.69 (3H each, s, AcO-C(CH₃)₂), 2.04 (6H, s, -OAc x2 ), 2.16 (3H, s, -OAc), 2.18 (3H, s, -OAc), 2.21 (3H, s, -OAc), 4.92 (br s, >CH-OAc, partially overlapping with an H₂O signal),5.54 (2H, br s, >C-CH-and>CH-OAc), 5.81 (H, d, J= 8 Hz,> CH-OAc), 6.01 (H, d, J= 8 Hz,>CH_OAc). CMR: olefinic carbons 141.68(s), 119.18(d); oxygen-bearing carbons: 81.97(s), 78.63(d), 73.42(d), 72.719d), 68.61(d); quartenary carbons: 49.04, 46.87, 39.79, 34.69; acetyl carbonyl carbons: 170.16, 169.98, 169.51. EI-MS m/z: 702 (M^), 641 (M^), 642 (M^-AcOH), 508, 448, 163, 134.

Formation of Mono- (VII) and Diacetonides (VIII)- A mixture of IV (60 mg) and anhydrous CuSO₄ (160 mg) in anhydrous acetone (2.3 ml) was stirred at room temperature for 19 hour. The mixture diluted with CHCI₃ and filtered, and the filtrate was concentrated. The residue (53 mg) was repeatedly chromatographed on silica gel (200 times the weight of material), using 1% MeOH-CHCI₃ as an eluent, to give diacetonide (VIII) (7 mg) and monoacetonide (VII) (41 mg). VIII: colorless needles (from MeOH), mp 208-209⁰. PMR: 3.75 (H, br s, >CH-OH), 3.83 (h, d, J= 9 Hz), 4.10 (H, dd, J= 7, 9 Hz), 4.38 (H, d, J = 7Hz), 5.69 (br d, J= 5 Hz)>C=CH-). EI-MS m/z: 572 (M^), 557 (M^-CH₃), 554 (M^-H₂O), 443, 420 (M^-152), 405 (M^-CH₃-152), 163, 152, 134. VII: colorless needles (acetone-benzene), mp 176-178⁰. Anal. Calcd fro C₃₃H₅₆O₅.3/2H₂O: C, 70.80; H, 10.62. Found: C, 70.68; H, 10.25. PMR: 3.74 (H, br s. >CH-OH), 3.83 (H,d, j= 9 Hz), 4.32 (H, dd, J= 6,9 Hz), 4.61(H, d, J= 6 Hz), 5.61(H, d, J=4 Hz). CMR: olefinic carbons: 142.93(s), 119.00(d); oxygen-bearing carbons: 107.62(s), 84.77(d), 79.03(d), 77.47(d), 75.82(d), 73.05(s); quartenary carbons: 49.22, 46.69, 41.53, 34.72. EI-MS m/z: 532 (M^), 517 (M^-CH₃), 443, 380 (M^-152), 362 (380-H₂O), 163, 152, 134.

Periodate Oxidation of VII- VII (1.8 mg) was dissolved in o.1 ml of MeOH, and 10% NaIO₄ aqueous solution (10 l) was added. The mixture was stirred in the dark for 40 hour, then diluted with water. The precipitate were collected by centrifugation and recrystallized from MeOH to give colorless needles (IX: mp 109-115⁰. FD-MS m/z: 473 (M+H)^. Calcd for C₃₀H₄₈O₄: 472.

Periodic Acid Oxidation of IV- IV (261 mg) in MeOH (16 ml) was mixed 25% HIO₄ aqueous solution (1.6 ml) and the whole was stirred in the dark for 2 days. Precipitates were centrifuged off and the supernatant was diluted with water then extracted with CHCI₃. The precipitates and CHCI₃ extract were combined and chromatographed on a silica gel (100 g) column, eluting 12% AcOEt-hexane, to give an aldehyde (X) (32 mg) and a dimethyl acetal (XI) (150 mg). X: colorless needles (MeOH), mp 156-159⁰. PMR: 3.74 (h, br s,>CH-OH), 5.64 (H, br s, J= 6 Hz,>C=CH-), 9.70 (H, D, J= 3 Hz, >CH-CHO. CMR: an aldehyde carbon: 204.49(d); olefinic carbons: 142.81(s), 118.95(d); oxygen-bearing carbon: 75.82 (d), quartenary carbons: 48.87, 46.93, 41.45, 34.86. EI-MS m/z: 372 (M^), 357 (M^-CH₃), 354 (M^-H₂O), 339, (354-H₂O), 220 (M^-152), 205 (220-CH₃), 152, 134. XI: colorless needles (MeOH), mp 155-157⁰. Anal. Calcd fro C₂₇H₄₆O₃.H₂O: C, 74.26; H, 11.08. Found: C, 74.35; H, 11.03. PMR: 3.37, 3.45 (3H each, s, -CH (OCH₃)₂), 3.72 (H, br s,>CH-OH), 4.22 (H, d, J= 1.5 Hz,>CH-CH(OCH₃)₂), 5.64 (H, br d, J=5 Hz.>C=CH-). CMR: olefinic carbons: 143.09(s), 119.00(d); oxygen-bearing carbons: 109.34(d), 75.88(d), 56.72(q), 55.61(q); quartenary carbons: 49.10, 46.58, 41.60, 34.80. EI-MS m/z: 418 (M^), 386 (M^-MeOH), 368 (386-H₂O), 266 ( M^-152), 234 (266-MeOH), 152, 134, 75 (-CH(Ome)₂^).

Oxidation of Dimethyl Acetal (XI) with CrO₃- XI (160 mg) was added to 3% Cro₃ pyridine solution (5.6 ml) and the whole was stirred at room temperature for 20 hour. CHCI₃ (24 ml) was then added to the reaction mixture, and precipitates were collected by filtration and washed with CHCI₃. The filtrate and washing were combined and shaken with water. The CHCI₃ layer was dried with anhydrous Na₂SO₄ and concentrated. The residue was chromatographed on silica gel (90 g), using CHCI₃ as an eluent, to give a ketone 980 mg), which was crystallized from MeOH to afford colorless plates (XII): mp 158-160⁰. PMR (missing text) 3.48 (3H each, s, -CH(Ome)₂), 4.24 (H, d, J= 3 Hz, -CH(Ome)₂), 5.64 (H, d, J= 6 Hz,>C=CH-). CMR: carbonyl carbon: 212.40; olefinic carbons: 142.91(s), 119.88; dimethyl acetal carbons: 109.22(d), (missing text), 55.61(q); quartenary carbons: 50.86, 48.93, 46.46, 46.46, 35.04. EI-MS m/z: 416 (M^), 384 (M^-MeOH), (missing text) 384-OCH₃), 313, 266, 234, 219, 163, 150, 75. CD (c= 0.43 x 10^-3 g.ml, dioxane) [0]²⁰⁰ (nm): 0⁰ (326), 4530⁰ (313), -7740⁰ (303), -8030⁰ (294), -1930⁰ (265), 0⁰ (244).

Air Oxidation of XII, Formation of Diosphenol (XIII) – XII (30 mg) was dissolved in 1 _N tert-BuOK, tert-BuOH (2 ml), and oxygen gas was bubbled through the solution. The mixture was stirred at room temperature until the spot of XII disappeared on TLC. After neutralization with 1 _N HCI-MeOH, and evaporation to dryness in vacuo, the residue was treated with CHCI₃. The soluble was chromatographed on silica gel (8 g) (eluent, 10% AcOEt-hexane) to give a diosphenol (XIII) (6.5 mg), which was crystallized from hexane containing s mall amount of acetone to give colorless needles, mp 173-176⁰. CHCI₃ test in EtOH: brown. PMR (CDCI₃): 3.39, 3.44 (3H each, s, -CH (Ome)₂), 4.14 (H, d, J= 2 Hz, -CH-Ome)₂), 5.70 (H, m,>C=CH-), 5.86 (H, s, echangeable with D₂O, C₂-OH), (H, d, J= 3 Hz, C₁-H). EI-MS m/z: 430 (M^), 398 (M^-MeOH), 366 (398-MeOH), 266, 234 (266-MeOH), 164, 163, 75. UV_max^DIOXANE nm (): 212 (6290), 271 97320). CD (c=0.17 x 10^-3 g/ml, dioxane) [0]²¹⁰ (nm): 0⁰ (370), -2300⁰ (360), -8100⁰ (350), -21000⁰ (330), -25300⁰ (321), -22300⁰ (313), 0⁰ (289).

X-Ray Analysis of IV- Crystal data: C₃₀O₅.H₂O (M.W.=510.762), monoclinic (from CH₃CN-H₂O), space group P2₁, =14.697(3) A, b= 7.742(2) A, c= 13.053(3) A, =99.41(2)⁰, V= 1465.3(6) A³, Z=2, D_calcd=1.16 g/cm³.

A crystal with approximate dimensions of 0.1×0.2×0.35 mm was mounted on a SYNTEX P₁ fully automated four-circle diffractometer, and the lattice constants were derived by a least-squares fitting of 15 reflections. The intensities of all 3621 unique reflections having 20 ≤ 55⁰ were measured using graphite-monochromated Mo K radiation (=0.71069 A) and the 20-0 scan technique. After correction by the usual borentz and polarization factors, the intensities were converted to normalized structure factors. The phases were assigned to the 270 largest E-valuye (E 1.7) by a multisolution, weighted tangent formula approach.²⁰⁰ The E-synthesis from the phase set having the least R-value showed 33 plausible nonhydrogen atoms. The complete structure (les hydrogens and with one molecule of water) was obtained by successive D-Fourier synthesis. Hydrogen atoms other than five hydroxyl protons were located on the basis of geometrical considerations.

Block-diagonal least refinement with anisotropic temperature factors for nonhydrogen atoms and isotropic temperature factors for hydrogen reduced the R-factor to the final value of 0.059 for the 2180 observed reflections (I>2.3 _I).

An ORTEP drawing of the structure, the final atomic parameters, bond lengths, and bond angles for nonhydrogen atoms are shown in Fig.1 and Tables I, II and III, respectively.

All the calculations were performed on a FACOM M-190 computer at the Computer Center of Kyushu University using the UNICS II²¹⁾ MUNTAN²⁰⁾ programs.

Acetylation of I- A solution of I (104 mg) in Ac₂O-pyridine (1:1) (2 ml) was heated on a boiling water bath for 16 hour. The mixture was then evaporated down under an air stream , and the residue (138 mg) was chromatographed on a silica gel (40 g) column, eluting with benzene-acetone mixture (4% acetone20%), to give two compounds. The less polar one (55 mg) was crystallized from EtOH to give colorless needles XIV): mp 148-152⁰. CMR: olfinic carbons: 142.09(s), 118.83(d); oxygen-bearing carbons: 101.98(d), 191.02(d), 86.66(d), 81.97(s), 73.77, 73.42, 72.71, 72.07, 71.78, 69.67, 68.91, 68.61, 62.61(t); quaternary carbons: 49.04, 46.87, 41.19, 34.80; acetyl carbonyl carbons were omitted. EI-MS m/z: 619, 331, 271 (331-AcOH), 582, 448, 134.

Methylation of I – A mixture of I (600 mg) in freshly distilled anhydrous tetrahydrofuran (7 ml) and sodium hydride (350 mg) was sonicated for 10 min. CH₃I (7 ml) was added to the mixture amd the whole altrate and washing were combined and concentrated under reduced pressure to give a thick syrup (818 mg). Chromatography on silica gel 9125 g) using 15% acetone-benzene gave two compounds: XV (196 mg) and methyl groups at C-25 (XV, 1.30 ppm; XVI, 1.46 ppm) and an additional OCH₃ at 3.26 ppm in XV.

Methanolysis of XV and XVI, Identification of Methylated Sugars and Isolation of XVII, XVII, XIX and XX – Xv 9208 mg) was dissolved in 1 _N HCI-MeOH (3 ml) and refluxed for 5 hour. After neutralization with Ag₂CO₃, the precipitates were filtered off. The filtrate was concentrated in vacuo and the residue was chromatographed on silica gel (hexane-AcOEt (5:1)) to give two fractions (Fr. 1, 61 mg; Fr. 2, 30 mg). Further elution with AcOEt gave a mixture of methylated sugars, which was examined by GLC. The results are summarized in Table IV. Fr. 2 (XIX) showed a single spot on TLC, but could be crystallized. The PMR spectrum showed signals of a hydroxymethine group at  3.74 (C₃-H, br. s), four OCH₃ (3.27, 2.42, 3.48 and 3.58 ppm) and two groups at C-25 at 1.30 ppm (6H, s).

XVI (171 mg) was methanolyzed and worked up in the manner described above to give two fractions (Fr. 1’, 22 mg; Fr. 2’, 38 mg) and a mixture of methylated sugars, which gave a GLC chromatogram identical with that of the methylated sugars from XV. Fr. 2’ was crystallized from MeOH to give colorless nedles (XX) (15 mg): mp 164-166⁰ . The PMR spectrum exhibited signals of three OCH₃ groups (3.42, 3.58 and 3.64 ppm) and a HO-C(CH₃)₂ group at 1.49 ppm (6H, s). Fr. 1’ showed the same IR spectrum as Fr. 1, and both fractions were combined and chromatographed on silica gel, using 10% AcOEt-hexane as an eluent, to give two thin-layer-chromatographed homogeneous compounds, XVII (28 mg) and XVIII (22.7 mg). XVII was crystallized from MeOH to provide colorless prisms, mp 198-200⁰. PMR: 1.84 (3H, s, CH₃-C-CH₂), 3.26, 3.43, 3.47, (3h each, s, -OCH₃), 5.17 (2H, perturbed d, CH₃-C=CH₂). CMR: olefinic carbons: 143.56(s), 143.31(s), 119.29(d), 116.08(t); oxygen-bearing carbons: 84.41(d), 81.28(d), 80.80(d), 75.97(d), 58.57(q), 57.70(q), 55.70(q); quartenary carbons: 49.17, 47.07, 41.62, 34.84. XVIII was crystallized from MeOH to give colorless needles, mp 190-193.5⁰. PMR: 3.44, 3.58 (3H each, s, -OCH₃). CMR: olefinic carbons: 143.31(s), 119.39(d); oxygen-bearing carbons: 90.64(d), 81.72(d), 81.38(d), 79.48(s), 76.02(d), 59.94(q), 59.11(q); quaternary carbons: 48.93, 47.41, 41.71, 34.89. EI-MS m/z: 502 (M^-CH₃) 350 (M^-152), 152, 134.

Acetylation of II – A solution of II (19 mg) in Ac₂O-pyridine (1:1) (0.5 ml) was heated on a boiling water bath for 25 hour. The reaction mixture was poured into ice-water, then the precipitates were collected by filtration, dried, and recrystallized from EtOH to give colorless fine needles (XXI), mp 133-140⁰. EI- Ms m/z: 331, 271 (331-AcOH), 259, 199 (259-AcOH), 582, 508, 448, 134.

Methylation of II – II (320 mg) was methylated in the manner described for the methylated of I to give two methylates XXIIa (94 mg), olorless needles (MeOH), mp 178-180⁰ and XXIIb (67 mg), colorless needles (MeOH), mp 167-168.5⁰. They exhibited PMR spectra similar (differing only in the number of methoxyl signals) to those of XV and XVI, respectively.

Methanolysis of XXIIa, Identification of Methylated Sugars – A solution of XXIIa (3 mg) in 1 _N HCI-MeOH (0.1 ml) was refluxed for 1 hour. The reaction mixture was worked up as usual. The aglycone was identified as a mixture of XVII, XVIII and XIX (TLC). The methylated sugars obtained were examined by GLC, and the results are shown in Table IV.

Acknowledgements. The authors are grateful to Professor I. Ueda and Associate Professor S. Kono, College of General Education, and Associate Professor T. Komori, Faculty of Pharmaceutical Sciences, Kyushu University, for providing computer programs and helpful discussions. They are also indebted to Dr. T. Nohara of Tokushima University and Mr. K. Mihashi of Fukuoka University for valuable suggestions and discussions. Thanks are also due to Mr. Y. Tanaka and Miss M. Kawamura of Kyushu University, to Mr. M. Nishi of Fukuoka University for PMR, CMR and MS measurements, and to Mr. S. Hara of Fukuoka University for elementary analyses.

References

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8. Alnusenone, a pentacyclic triterpene having a 10 -hydrogen,  5, 6-double bond and a 3-keto group gives a negative CD curve [0]₂₉₅-3630⁰), while 4,4-dimethycholest-5-en-3-one and 17-acetoxy-4,4-dimethyl-19-norlanost-5-en-3-one show positive CD curves ([0]₂₉₄ + 5148⁰ and [0]₃₀₇ + 5752⁰, respectively).⁹⁾ These CD data were referred to in the determination of the cucurbit-5-ene nucleus of gratiogenin.^6a

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15. Instruments and materials used in this work were as follows: Yanaco micromelting point apparatus (melting points), Shimadzu UV-200S double beam spectrophotometer (UV spectra), Hitachi gratin-infrared spectrophometer, model EPI-G3 (IR spectra), JASCO DIP-4 digital polarimeter (specin.rotations), JASCO J-20 sutomatic recording spectropolarimeter (CD spectra), JEOL JNM PS-100 (100 MHz) and Hitachi R-22 (90 MHz) spectrometer (PMR spectra), JEOL JMN FX-100 (25 MHz) spectrometer (CMR spectra), Shimadzu GC-3BF gas chromatograph (GLC), JEOL JMS-01SG mass spectrometer (EI-MS spectra), JEOL D-300 FD mass spectrometer (FD-MS spectra), Kieselgel 60 (70-230 mest) (E. Mecrk) (column chromatography), precoated Kieselgel 60 F₂₅₄ plates (e. Merck) (TLC). Melting points are uncorrected. PMR and CMR spectra were measured in pyridine-d₅ unless otherwise stated and chemical shifts are expressed in the -scale using tetramethylsilane as an internal standard singlet; br, broad; d, doublet; dd, doublet; t, triplet; q, quartet; m, multiplet).

16. Reported for vicine: mp 243-244⁰ (dec.), []_D²⁶⁰ – 11.7⁰, UV _max^{0.1 NHCI} nm (): 274 (16400). Merck Index.)

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18. 5⁰ 1,4-butanediol succinate on Shimalite W; 2.1 m x 3 mm; N₂ carrier gas at 1 kg/cm²; 220⁰.

19. Reported for methyl -_D-xylopyranoside: + 153.9⁰ (H₂O) [J. Am. Chem. Soc., 47, 265 (1925)].

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