2017 Volume 65 Issue 8 Pages 754-761
From the leaves of Zanthoxylum ailanthoides, 4′-O-p-E-coumaric acid esters of 2-propanol β-D-glucopyranoside, megastigmane and megastigmane glucosides were isolated. Their structures were elucidated by spectroscopic evidence. The absolute configurations of the megastigmane and aglycone of megastigmane glucosides were determined by the octant rule and modified Mosher’s method after protection of carboxylic acids by p-bromophenacyl esters and primary alcohols by pivaloyl esters.
The genus Zanthoxylum belonging to the family Rutaceae comprises about 200 species that are found in tropical and moderate areas of both hemispheres. Z. ailanthoides SIEBOLD et ZUCCARINI is a tall, deciduous tree that grows in Japan (Honshu and Kyushu), Okinawa, Taiwan, China and the Philippines.1) Some less polar compounds such as amides and benzenoids, nor-lignan and phenyl propanoids, and pheophorbides with antiproliferative and apoptosis-inducing activities against human leukemia cells, HL-60 and WEHI-3 have been isolated from the stem bark, stem wood and leaves of Z. ailanthoides, respectively.2–4) In the course of continuing investigation, Z. ailanthoides was chosen for phytochemical study. The leaves of Z. ailanthoides are known to be used for a carminative and the fruits for a stomachic.5) This paper deals with the isolation of polar compounds from the leaves of Z. ailanthoides.
Air-dried leaves of Z. ailanthoides were extracted with MeOH three times and the concentrated MeOH extract was partitioned with solvents of increasing polarity. The 1-BuOH-soluble fraction was separated by means of various chromatographic procedures including column chromatography (CC) on a highly porous synthetic resin (Diaion HP-20), normal silica gel and reversed-phase octadecyl silica gel (ODS) CC, droplet counter-current chromatography (DCCC), and HPLC to afford 2-propanol β-D-glucopyranoside 4′-O-p-E-coumaric acid ester (1), a new megastigmane and five megastigmane glucosides, named zanthoionic acid (2) and zanthoionosides A–E (3–7), respectively. These novel compounds were isolated along with the known compounds dendranthemoside B (8),6) kiwiionoside (9),7) icariside B1 (10),8) citroside A (11),9) linalool 3-O-α-L-arabinopyranosyl-(1″→6′)-β-D-glucopyranoside (12)10) and sibiricose A5 (13)11) (Fig. 1).
Compound 1 was isolated as an amorphous powder and its elemental composition was determined as C18H24O8 by high-resolution (HR)-electrospray ionization (ESI)-MS. The IR spectrum showed absorption bands ascribable to hydroxy groups (3307 cm−1), an ester carbonyl functional group (1701 cm−1) and an aromatic ring (1512 cm−1). The UV absorption band also suggested the presence of an aromatic ring. In the 1H-NMR spectrum, two non-equivalent methyl signals (δH 1.23 and 1.27) were observed and these methyl groups were connected through an oxymethine signal (δH 4.07) in 1H–1H correlation spectrometry (COSY). The NMR spectra exhibited anomeric proton (δH 4.41) and carbon (δC 102.6) signals (Table 1), and HPLC analysis of the hydrolyzate of 1 revealed the presence of D-glucose as a sugar unit. Thus, the core unit of 1 was established as 2-propanol D-glucopyranoside and the mode of sugar linkage was inferred as β from the coupling constant (J=7.7 Hz) of the anomeric proton. The remaining seven 13C signals accounted for nine carbons, due to a symmetrically substituted benzene ring (Table 1), along with proton signals of a trans double bond [δH 7.68 (1H, d, J=16.0 Hz, H-7″) and 6.39 (1H, d, J=16.0 Hz, H-8″)], and they were assigned as those of a p-E-coumaroyl group. COSY connectivity from H-1′ to H-4′ and heteronuclear multiple-bond correlation (HMBC) between deshielded H-4′ (δH 4.84) and C-9″ (δC 168.6) substantiated the structure of 1 as 2-propanol β-D-glucopyranoside 4′-O-p-E-coumarate, as shown in Fig. 2. Non-equivalent upfield shifts of methyl groups of 2-propanol on β-D-glucopyranosylation are well discussed in the literature.12)
| C | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
|---|---|---|---|---|---|---|---|
| 1 | 22.1 | 39.6 | 34.1 | 34.4 | 33.6 | 34.1 | 34.0 |
| 2 | 72.8 | 56.8 | 46.1 | 46.4 | 45.2 | 45.7 | 46.1 |
| 3 | 23.8 | 214.2 | 71.4 | 75.5 | 70.3 | 82.6 | 71.3 |
| 4 | 50.1 | 78.3 | 39.5 | 88.7 | 78.4 | 78.3 | |
| 5 | 35.0 | 33.9 | 27.3 | 32.8 | 32.9 | 33.8 | |
| 6 | 58.0 | 58.0 | 59.9 | 58.2 | 58.2 | 58.2 | |
| 7 | 131.0 | 133.2 | 134.7 | 133.6 | 133.8 | 134.3 | |
| 8 | 134.5 | 136.0 | 132.8 | 133.3 | 133.3 | 132.1 | |
| 9 | 74.0 | 78.0 | 74.9 | 74.3 | 74.4 | 72.8 | |
| 10 | 178.7 | 24.1 | 67.8 | 67.6 | 67.7 | 75.5 | |
| 11 | 21.4 | 24.1 | 23.9 | 32.3 | 32.3 | 32.5 | |
| 12 | 31.0 | 32.8 | 32.8 | 23.8 | 23.8 | 24.0 | |
| 13 | 21.8 | 17.2 | 21.8 | 17.2 | 17.2 | 17.2 | |
| 1′ | 102.6 | 102.3 | 102.8 | 105.7 | 107.0 | 104.9 | |
| 2′ | 75.3 | 75.4 | 75.4 | 75.2 | 76.0 | 75.3 | |
| 3′ | 75.9 | 78.2 | 78.5 | 77.9 | 78.3 | 78.0 | |
| 4′ | 72.7 | 71.4 | 71.9 | 71.2 | 71.8 | 71.6 | |
| 5′ | 76.1 | 77.9 | 77.9 | 77.6 | 78.0 | 77.9 | |
| 6′ | 62.6 | 62.5 | 63.0 | 62.3 | 63.0 | 62.7 | |
| 1″ | 127.2 | ||||||
| 2″,6″ | 131.3 | ||||||
| 3″,5″ | 116.9 | ||||||
| 4″ | 161.4 | ||||||
| 7″ | 147.2 | ||||||
| 8″ | 114.9 | ||||||
| 9″ | 168.6 |
Zanthoionic acid (2) was isolated as an amorphous powder and its elemental composition was determined to be C13H20O4 by HR-ESI-MS. The IR spectrum exhibited absorptions for hydroxy groups, two types of carbonyl groups (1701 and 1713 cm−1) and a double bond. In the 1H-NMR spectrum, signals for two singlet methyl, one doublet methyl and one trans double bond were observed and the presence of two methylene, one oxymethine, two methine, carbonyl, carboxyl and quaternary carbons was confirmed by 13C-NMR and from distortionless enhancement by polarization transfer spectra. Two 1H–1H COSY correlations from H2-4 through H-9, and H-2 eq and H-4 eq by long-range coupling, as well as HMBC correlations between H3-11 and H3-12 and C-2 and C-6, H2-2 and H2-4 and C-3, H-6 and C-1, H-8 and C-10, and other diagnostic correlations as shown in Fig. 3 established the scaffold of 2 to have a megastigmane skeleton with a carboxylic acid at the end of the side chain. The coupling constant of H-6 (J=10.7 Hz) confirmed that H-5 and H-6 were in axial positions and by application of the octant rule,13) a positive Cotton effect at 298 nm enabled the assignment of the absolute configuration of C-5 as R and thus C-6 also as R. The absolute configuration at the 9-position was determined to be R by the modified Mosher’s method, using 2 p-bromophenacyl ester (2a)14) (Fig. 3). Therefore, the structure of 2 was elucidated as 5R,6R,7E,9R-megastigman-3-on-7-en-9-ol-10-oic acid as shown in Fig. 2.
Zanthoionoside A (3) was isolated as an amorphous powder and its elemental composition was determined to be C19H34O8 by HR-ESI-MS. A strong IR absorption band at 3305 cm−1 indicated 3 was a glycosidic compound. In the 1H-NMR spectrum, signals for two singlet methyls, two doublet methyls and two olefinic protons coupled in trans geometry were observed. The 13C-NMR spectrum exhibited six signals assignable to glucopyranose and HPLC analysis of the enzymatic hydrolyzate revealed the presence of D-glucose. The remaining 13 signals were indicative that 3 was a derivative of megastigmane and comprised of four methyls, one methylene, three oxymethines, two methines, one disubstituted double bond and a quaternary carbon. The 1H–1H COSY spectrum showed a proton chain from H2-2 to H3-10 and geminal dimethyl protons were correlated with methylene carbon in the HMBC spectrum. Thus, two hydroxy groups were placed on C-3 and C-4. Considering the coupling constants, J2–3=2.9 and 3.3 Hz, suggested that H-3 was in the equatorial position, and the hydroxy group at the 3-position was in an axial position; J4–5=10.4 Hz also suggested that H-4 and H-5 were in axial positions. From the coupling constant, J5–6=10.8 Hz, it could be inferred that the side chain was equatorially linked at the 6-position. The mode of sugar linkage was β, evidenced by the coupling constant of the anomeric proton, which showed a cross peak with the C-9 carbon in the HMBC spectrum. Therefore, the structure of 3 was elucidated as megasitgam-7-ene-4,5,9-triol 9-O-β-D-glucopyranose. The absolute configurations were assigned by the application of the modified Mosher’s method to the aglycone of 3 (3a). From MS spectral data, two units of α-methoxy-α-trifluoromethylphenylacetic acid (MTPA) were introduced into 3a and the positions of esterification were deduced to be on the hydroxy groups at the 4- and 9-positions from the obvious downfield shifts (δH 4.74 and 5.54, respectively for 3b and δH 4.69 and 5.54, respectively for 3c) of H-4 and H-9 compared with the spectra of 3 and 3a. The coupling pattern and coupling constants of H-4 of 3b and 3c were the same as those of 3 and 3a, suggesting that the esterification occurred on the hydroxy group at the 4-position. From this evidence, the absolute configurations were assigned as 3S, 4R, 5S, 6S, and 9R (Fig. 4).
Zanthoionoside B (4) was isolated as an amorphous powder and its elemental composition was determined to be C19H34O8 by HR-ESI-MS. NMR spectroscopic data indicated that 4 was also a derivative of megastigmane with one primary and two secondary hydroxyl groups. From the 1H–1H COSY correlation from H2-2 to H2-10, the primary alcohol was placed at the terminal carbon, C-10. The 13C-NMR spectrum exhibited six signals assignable to those of glucopyranose, and HPLC analysis of the enzymatic hydrolyzate revealed the presence of D-glucose. The mode of sugar linkage was β based on the coupling constant of the anomeric proton, and the position of the sugar linkage was determined to be on the hydroxy group at the 3-position based on the HMBC correlation between the anomeric proton and C-3. The coupling constants of H-3 (J=3.7, 2.8, 2.8 and 2.2 Hz) indicated that the hydroxy group at the 3-position was in the axial orientation and as J5–6=10.3 Hz, the C-13 methyl group and the side chain were in the equatorial positions. Enzymatic hydrolysis of 4 gave an aglycone (4a) whose primary alcohol was protected as pivaloyl ester (4b). Application of the modified Mosher’s method to 4b revealed the absolute configuration at C-3, C-5, C-6 and C-9 to be R, R, R and S, respectively (Fig. 4). Therefore, the structure of 4 was elucidated as 3R,5R,6R,7E,9S-megastigman-7-ene-3,9,10-triol 3-O-β-D-glucopyranoside, as shown in Fig. 2.
Zanthoionoside C (5) was isolated as an amorphous powder and its elemental composition was determined as C19H34O9 by HR-ESI-MS. NMR spectroscopic data indicated that 5 was a similar compound to 4 with one additional hydroxy group. Methylene protons from C-2 through C-10 were correlated by the 1H–1H COSY spectrum and geminal dimethyl protons showed cross peaks with C-2 in the HMBC spectrum. Thus, a 3,4,9,10-tetrol structure was proposed for 5 and the position of the sugar linkage was determined to be on the hydroxy group at C-4 by HMBC. The coupling constants, J2–3=3.0 and 3.1 Hz indicated that H-3 was in the equatorial position, and J4–5=10.7 Hz and J5–6=10.9 Hz indicated substituents at C-4, C-5 and C-6 in the equatorial positions. Enzymatic hydrolysis of 5 gave an aglycone (5a) and the primary alcohol was protected as a pivaloyl ester (5b). (R)- and (S)-MTPA were prepared from the pivaloyl ester to give 4,9-diesters (5c and d) (Fig. 4). As a result, the structure of 5 was elucidated as 3S,4R,5S,6S,7E,9R-megastigman-7-ene-3,4,9,10-tetrol 4-O-β-D-glucopyranoside, as shown in Fig. 2.
Zanthoionosides D and E (6 and 7) were isolated as amorphous powders and their elemental composition was determined as C19H34O9 by HR-ESI-MS, which was the same as that of 5. NMR spectroscopic data showed strong similarity to those of 5. On enzymatic hydrolysis, 6 and 7 gave D-glucose and a common aglycone, whose physico-chemical properties were the same as those of 5a. In the HMBC spectra of 6 and 7, anomeric protons showed cross peaks with C-3 and C-10, respectively. Therefore, the structures of 6 and 7 were elucidated as 3S,4R,5S,6S,7E,9R-megastigman-7-ene-3,4,9,10-tetrols 3-O-β-D-glucopyranoside and 10-O-β-D-glucopyranoside, respectively, as shown in Fig. 2.
From the leaves of Z. ailanthoides, 2-propanol β-D-glucopyranoside 4′-O-p-E-coumarate (1), zanthoionic acid (2) and zanthoionosides A–E (3–7) were isolated. Their structures were elucidated mainly by the use of NMR spectroscopic evidence. The absolute configuration of the cyclohexanone moiety of 2 was determined using the octant rule and that of the side chain was determined using the modified Mosher’s method (Fig. 4). The absolute configurations of zanthoionosides A–E (3–7) were determined using the modified Mosher’s method. The isolation of a megastigmane with a carboxylic acid functional group like 2 was a relatively rare example from a natural source. In general, megastimanes and their glycosides do not have remarkable biological activity. However, they are known as constituents of wine flavor15,16) and a structurally related abscisic acid is a plant dormancy hormone.17) Inhibitory activity of histamine release from antigen-stimulated RBL-2H3 cells was also reported for some megastigmane glycosides.18)
Optical rotations and circular dichroism (CD) spectra were measured on JASCO P-1030 and JASCO J-720 spectropolarimeters, respectively. IR and UV spectra were measured on Horiba FT-710 and JASCO V-520 UV/Vis spectrophotometers, respectively. 1H- and 13C-NMR spectra were taken on a Bruker Avance III spectrometer at 600 and 150 MHz, respectively, with tetramethylsilane as an internal standard. Positive-ion HR-ESI-MS was performed with an Applied Biosystems QSTAR XL system ESI (Nano Spray)-TOF-MS.
A highly porous synthetic resin (Diaion HP-20) was purchased from Mitsubishi Kagaku (Tokyo, Japan). Silica gel CC and reversed-phase (ODS) open CC were performed on silica gel 60 (Merck, Darmstadt, Germany) and Cosmosil 75C18-OPN (Nacalai Tesque, Kyoto, Japan) [Φ=50 mm, L=20 cm, linear gradient: MeOH–H2O (1 : 9, 1 L)→(1 : 1, 1 L) and then (1 : 1 500 mL)→(3 : 7, 500 mL), fractions of 10 g being collected], respectively. DCCC (Tokyo Rikakikai, Tokyo, Japan) was equipped with 500 glass columns (Φ=2 mm, L=40 cm), the lower and upper layers of a solvent mixture of CHCl3–MeOH–H2O–n-PrOH (9 : 12 : 8 : 2) being used as the stationary and mobile phases, respectively. Five-gram fractions were collected and numbered according to their order of elution with the mobile phase. HPLC was performed on an ODS-3 column (Inertsil; GL Science, Tokyo, Japan; Φ=6 mm, L=25 cm), and the eluate was monitored with a UV detector at 254 nm and a refractive index monitor.
(R)- and (S)-MTPA, and β-glucosidase (Almond, 10000 U/270.3 mg) were products of Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Plant MaterialLeaves of Z. ailanthoides were collected in Kunigami-son, Kunigami-gun, Okinawa in July 2008 and a voucher specimen was deposited in the Herbarium of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Hiroshima University (08-ZA-Okinawa-0708).
Isolation and ExtractionLeaves of Z. ailanthoides (10.6 kg) were extracted three times with MeOH (45 L×3) at room temperature for one week and then concentrated to 6 L in vacuo. The concentrated extract was washed with n-hexane (6 L, 161 g), and then the MeOH layer was concentrated to a gummy mass. The latter was suspended in water (6 L) and then extracted with EtOAc (6 L) to give 177 g of an EtOAc-soluble fraction. The aqueous layer was extracted with 1-BuOH (6 L) to give a 1-BuOH-soluble fraction (134 g), and the remaining water layer was concentrated to furnish 577 g of a water-soluble fraction.
The 1-BuOH-soluble fraction (133 g) was subjected to Diaion HP-20 CC (Φ=50 mm, L=50 cm), using H2O–MeOH (4 : 1, 3 L), (3 : 2, 3 L), (2 : 3, 3 L), and (1 : 4, 3 L), and MeOH (3 L), 1 L-fractions being collected. The residue (23.3 g) in fractions 4–8 was subjected to silica gel (500 g) CC with increasing amounts of MeOH in CHCl3 [CHCl3 (3 L), and CHCl3–MeOH (49 : 1, 3 L), (24 : 1, 3 L), (23 : 2, 3 L), (9 : 1, 3 L), (7 : 1, 3 L), (17 : 3, 3 L), (4 : 1, 3 L), (3 : 1, 3 L), (7 : 3, 3 L) and (3 : 2, 3 L)], 500-mL fractions being collected. The residue (4.25 g) in fractions 26–33 was subjected to open ODS CC to give a residue (179 mg) in fractions 73–82 which was purified by HPLC (H2O–MeOH, 3 : 1) to give 6.9 mg of 9 and 10.0 mg of 8 from the peaks at 8 and 12 min, respectively. The residue (1.67 g) in fractions 44–49 was subjected to open ODS CC. The residue (303 mg) in fractions 78–92 was applied to DCCC to give a residue (155 mg) in fractions 31–38, which was then purified by HPLC (H2O–MeOH, 1 : 3) to give 18.0 mg of 13 from the peak at 28 min. The residue (151 mg) in fractions 93–100 was again subjected to silica gel CC (70 g) with increasing amounts of MeOH in CHCl3 [CHCl3 (400 mL), and CHCl3–MeOH (19 : 1, 400 mL), (9 : 1, 400 mL), (17 : 3, 400 mL), (4 : 1, 400 mL), (3 : 1, 400 mL), (7 : 3, 400 mL), (13 : 7, 400 mL), (3 : 2, 400 mL) and (1 : 1, 400 mL)], 10-g fractions being collected to afford 7.5 mg of 10, 14.3 mg of 7, and 84.7 mg of 5 in fractions 182–190, 211–223, and 224–260, respectively. The residue (148 mg) in fractions 101–107 was again subjected to silica gel CC (50 g) with the same solvent system as above to yield 21.1 mg of 11 and 78.1 mg of 6 in fractions 185–206 and 207–257, respectively.
The residue (30.5 g) in fractions 9–15 obtained on Diaion HP-20 CC was subjected to silica gel (700 g) CC with increasing amounts of MeOH in CHCl3 [CHCl3 (3 L), and CHCl3–MeOH (49 : 1, 3 L), (24 : 1, 3 L), (23 : 2, 3 L), (9 : 1, 3 L), (7 : 1, 3 L), (17 : 3, 3 L), (33 : 7, 3 L), (4 : 1, 3 L), (3 : 1, 3 L), (7 : 3, 3 L) and (3 : 2, 3 L)], 500-mL fractions being collected. The residue (1.83 g out of 7.05 g) in fractions 33–41 was subjected to open ODS CC. The residue (253 mg) in fractions 117–127 was purified by DCCC to give 16.1 mg of 1 in fractions 63–68. The residue (2.06 out of 5.04 g) in fractions 42–47 was subjected ODS CC. The residue (112 mg) in fractions 91–99 was purified by DCCC to give 11.2 mg of 3 in fractions 39–41. The residue (239 mg) in fractions 116–125 was subjected to DCCC and the residue (54.4 mg) in fractions 29–38 was purified by HPLC (H2O–MeOH, 13 : 7) to give 10.8 mg of 4 from the peak at 18 min.
The residue (16.5 g) in fractions 30–38 obtained on HP-20 CC was subjected to silica gel CC (400 g) with increasing amounts of MeOH in CHCl3 [CHCl3 (2 L), and CHCl3–MeOH (49 : 1, 2 L), (24 : 1, 2 L), (47 : 3, 2 L), (23 : 2, 2 L), (9 : 1, 2 L), (7 : 1, 2 L), (17 : 3, 2 L), (33 : 7, 2 L), (4 : 1, 2 L), (3 : 1, 2 L) and (7 : 3, 2 L)], 300-mL fractions being collected. The residue (2.12 g) in fractions 46–52 was separated by open ODS CC to give 409 mg of 2 in fractions 52–64. The residue (238 mg) in fractions 221–233 was purified by DCCC to give 134 mg of 12 in fractions 82–111.
2-Propanol β-D-Glucopyranoside 4′-O-E-Coumaric Acid Ester (1) Colorless amorphous powder, [α]D25–18.5 (c=1.07, MeOH); IR νmax (film) cm−1: 3307, 1701, 1512, 1161, 1028; UV λmax (MeOH) nm (log ε): 306 (3.62); 1H-NMR (CD3OD, 600 MHz) δ: 7.68 (1H, d, J=16.0 Hz, H-7″), 7.47 (2H, d, J=8.2 Hz, H-2″ and 6″), 6.81 (2H, d, J=8.2 Hz, H-3″ and 5″), 6.39 (1H, d, J=16.0 Hz, H-8″), 4.84 (1H, overlapped by DHO signal, H-4′), 4.41 (1H, d, J=7.7 Hz, H-1′), 4.07 (1H, septet, J=6.2 Hz, H-2), 3.64 (1H, dd, J=9.3, 9.2 Hz, H-3′), 3.62 (1H, dd, J=12.2, 2.0 Hz, H-6′a), 3.43 (1H, ddd, J=8.3, 5.9, 2.0 Hz, H-5′), 3.54 (1H, dd, J=12.2, 5.9 Hz, H-6′b), 3.26 (1H, dd, J=9.3, 7.7 Hz, H-2′), 1.27 (3H, d, J=6.2 Hz, H3-3, pro-R), 1.23 (3H, d, J=6.2 Hz, H3-1, pro-S); 13C-NMR (CD3OD, 150 MHz): Table 1; HR-ESI-MS (positive-ion mode): m/z: 391.1368 [M+Na]+ (Calcd C18H24O8Na: 391.1363).
Zanthoionic Acid (2) Colorless amorphous powder, [α]D25–3.4 (c=0.50, MeOH); IR νmax (film) cm−1: 3332, 2959, 2931, 1713, 1701, 1650; 1H-NMR (CD3OD, 600 MHz) δ: 5.74 (1H, dd, J=15.2, 5.4 Hz, H-8), 5.56 (1H, dd, J=15.2, 9.8 Hz, H-7), 4.43 (1H, d, J=5.4 Hz, H-9), 2.42 (1H, d, J=13.2 Hz, H-2ax), 2.30 (1H, ddd, J=13.7, 4.4, 2.3 Hz, H-4 eq), 2.15 (1H, dd, J=13.7, 11.9 Hz, H-4ax), 1.96 (1H, dd, J=10.7, 9.8 Hz, H-6), 1.90 (1H, overlapped, H-5), 2.02 (1H, dd, J=13.2, 2.3 Hz, H-2 eq), 0.99 (3H, s, H3-12), 0.94 (3H, d, J=6.2 Hz, H3-13), 0.82 (3H, s, H3-11); 13C-NMR (CD3OD, 150 MHz): Table 1; CD [θ] (nm): +307 (298) (c=4.17×10−5 M, MeOH); HR-ESI-MS (positive-ion mode): m/z: 263.1254 [M+Na]+ (Calcd C13H20O4Na: 263.1254).
Zanthoionoside A (3) Colorless amorphous powder, [α]D25–0.54 (c=0.75); IR νmax (film) cm−1: 3305, 2931, 2880, 1457, 1368, 1267, 1228, 1070, 1032; 1H-NMR (CD3OD, 600 MHz) δ: 5.50 (1H, dd, J=15.6, 6.4 Hz, H-8), 5.40 (1H, dd, J=15.6, 9.9 Hz, H-7), 4.36 (1H, quintet, J=6.4 Hz, H-9), 4.35 (1H, d, J=7.7 Hz, H-1′), 3.91 (1H, m, H-3), 3.84 (1H, overlapped, H-6′a), 3.68 (1H, dd, J=12.1, 4.8 Hz, H-6′b), 3.17 (1H, overlapped, H-2′), 3.35–3.23 (3H, overlapped, H-3′, 4′ and 5′), 3.06 (1H, dd, J=10.4, 3.3 Hz, H-4), 1.77 (1H, m, H-5), 1.75 (1H, dd, J=14.6, 3.3 Hz, H-2 eq), 1.47 (1H, dd, J=10.8, 9.9 Hz, H-6), 1.41 (1H, dd, J=14.6, 2.9 Hz, H-2ax), 1.29 (3H, d, J=6.4 Hz, H3-10), 1.04 (3H, s, H3-11), 0.89 (3H, d, J=6.4 Hz, H3-13), 0.83 (3H, s, H3-12); 13C-NMR (CD3OD, 150 MHz): Table 1; HR-ESI-MS (positive-ion mode): m/z: 413.2153 [M+Na]+ (Calcd C19H34O8Na: 413.2146).
Zanthoionoside B (4) Colorless amorphous powder, [α]D21–31.4 (c=0.69, MeOH); IR νmax (film) cm−1: 3362, 2943, 1558, 1456, 1368, 1076, 1030; 1H-NMR (CD3OD, 600 MHz) δ: 5.52 (1H, ddd, J=15.4, 9.9, 0.7 Hz, H-7), 5.40 (1H, dd, J=15.4, 6.4 Hz, H-8), 4.30 (1H, d, J=7.9 Hz, H-1′), 4.11 (1H, dddd, J=7.3, 6.4, 4.5, 0.7 Hz, H-9), 4.04 (1H, dddd, J=3.7, 2.8, 2.8, 2.2 Hz, H-3), 3.85 (1H, dd, J=11.8, 2.4 Hz, H-6′a), 3.66 (1H, dd, J=11.8, 5.3 Hz, H-6′b), 3.50 (1H, dd, J=11.1, 4.5 Hz, H-10a), 3.43 (1H, dd, J=11.1, 7.3 Hz, H-10b), 3.36 (1H, dd, J=8.8, 8.8 Hz, H-3′), 3.26 (1H, overlapped, H-4′), 3.24 (1H, overlapped, H-5′), 3.16 (1H, dd, J=8.8, 7.9 Hz, H-2′), 2.00 (1H, dddd, J=14.7, 2.8, 2.8, 2.4 Hz, H-4 eq), 1.89 (1H, m, H-5), 1.82 (1H, ddd, J=14.5, 2.4, 2.2 Hz, H-2 eq), 1.39 (1H, dd, J=10.3, 9.9 Hz, H-6), 1.37 (1H, dd, J=14.5, 3.7 Hz, H-2ax), 1.05 (1H, m, H-4ax), 1.03 (3H, s, H3-11), 0.84 (3H, s, H3-12), 0.79 (3H, d, J=6.6 Hz, H3-13); 13C-NMR (CD3OD, 150 MHz): Table 1; HR-ESI-MS (positive-ion mode): m/z: 413.2142 [M+Na]+ (Calcd C19H34O8Na: 413.2146).
Zanthoionoside C (5) Colorless amorphous powder, [α]D21–3.08 (c=3.41, MeOH); IR νmax (film) cm−1: 3381, 2967, 2936, 2878, 1652, 1508, 1456, 1072, 1011; 1H-NMR (CD3OD, 600 MHz) δ: 5.47 (1H, dd, J=15.6, 9.5 Hz, H-7), 5.40 (1H, dd, J=15.6, 6.2 Hz, H-8), 4.36 (1H, d, J=7.8 Hz, H-1′), 4.17 (1H, ddd, J=3.1, 3.1, 3.0 Hz, H-3), 4.12 (1H, ddd, J=7.5, 6.2, 4.3 Hz, H-9), 3.81 (1H, dd, J=12.0, 2.0 Hz, H-6′a), 3.69 (1H, dd, J=12.0, 5.0 Hz, H-6′b), 3.51 (1H, dd, J=11.1, 4.3 Hz, H-10a), 3.44 (1H, dd, J=11.1, 7.5 Hz, H-10b), 3.26 (1H, dd, J=8.8, 7.8 Hz, H-2′), 3.37–3.35 (3H, overlapped, H-3′, 4′ and 5′), 3.18 (1H, dd, J=10.7, 3.1 Hz, H-4), 1.93 (1H, m, H-5), 1.74 (1H, dd, J=14.7, 3.1 Hz, H-2 eq), 1.51 (1H, dd, J=10.9, 9.5 Hz, H-6), 1.43 (1H, dd, J=14.7, 3.0 Hz, H-2ax), 1.04 (3H, s, H3-12), 0.97 (3H, d, J=6.4 Hz, H3-13), 0.82 (3H, s, H3-11); 13C-NMR (CD3OD, 150 MHz): Table 1; HR-ESI-MS (positive-ion mode): m/z: 429.2093 [M+Na]+ (Calcd C19H34O9Na: 429.2095).
Zanthoionoside D (6) Colorless amorphous powder, [α]D21+11.7 (c=4.84, MeOH); IR νmax (film) cm−1: 3364, 2957, 2924, 2873, 1604, 1508, 1456, 1074, 1029; 1H-NMR (CD3OD, 600 MHz) δ: 5.47 (1H, ddd, J=15.5, 9.7, 0.8 Hz, H-7), 5.39 (1H, dd, J=15.5, 6.2 Hz, H-8), 4.39 (1H, d, J=7.8 Hz, H-1′), 4.11 (1H, m, H-9), 3.91 (1H, m, H-3), 3.87 (1H, dd, J=11.8, 1.5 Hz, H-6′a), 3.68 (1H, dd, J=11.8, 4.5 H-6′b), 3.50 (1H, dd, J=11.1, 4.5 Hz, H-10a), 3.43 (1H, dd, J=11.1, 7.4 Hz, H-10b), 3.27 (3H, overlapped, H-3′, 4′ and 5′), 3.23 (1H, dd, J=9.2, 7.8 Hz, H-2′), 3.06 (1H, dd, J=10.7, 3.3 Hz, H-4), 2.06 (1H, dd, J=14.5, 3.3 Hz, H-2 eq), 1.80 (1H, m, H-5), 1.46 (1H, dd, J=10.8, 9.7 Hz, H-6), 1.39 (1H, dd, J=14.5, 2.8 Hz, H-2ax), 1.01 (3H, s, H3-12), 0.91 (3H, d, J=6.4 Hz, H3-13), 0.82 (3H, s, H3-11); 13C-NMR (CD3OD, 150 MHz): Table 1; HR-ESI-MS (positive-ion mode): m/z: 429.2093 [M+Na]+ (Calcd C19H34O9Na: 429.2095).
Zanthoionoside E (7) Colorless amorphous powder, [α]D21–15.2 (c=0.65, MeOH); IR νmax (film) cm−1: 3335, 2964, 2932, 2873, 1684, 1508, 1457, 1075, 1033; 1H-NMR (CD3OD, 600 MHz) δ: 5.51 (1H, ddd, J=15.5, 10.1, 1.0 Hz, H-7), 5.38 (1H, dd, J=15.5, 6.3 Hz, H-8), 4.32 (1H, m, H-9), 4.30 (1H, d, J=7.7 Hz, H-1′), 3.93 (1H, dd, J=10.3, 3.1 Hz, H-10a), 3.91 (1H, ddd, J=3.3, 3.1, 3.1 Hz, H-3), 3.86 (1H, dd, J=11.9, 1.7 Hz, H-6′a), 3.66 (1H, dd, J=11.9, 5.6 Hz, H-6′b), 3.41 (1H, dd, J=10.3, 8.8 Hz, H-10b), 3.39–3.28 (3H, overlapped, H-3′, 4′ and 5′), 3.22 (1H, dd, J=9.3, 7.7 Hz, H-2′), 3.06 (1H, dd, J=10.4, 3.3 Hz, H-4), 1.78 (1H, m, H-5), 1.74 (1H, dd, J=14.7, 3.1 Hz, H-2 eq), 1.47 (1H, dd, J=10.8, 10.1 Hz, H-6), 1.40 (1H, dd, J=14.7, 3.1 Hz, H-2ax), 1.04 (3H, s, H3-12), 0.90 (3H, d, J=6.6 Hz, H3-13), 0.82 (3H, s, H3-11); 13C-NMR (CD3OD, 150 MHz): Table 1; HR-ESI-MS (positive-ion mode): m/z: 429.2094 [M+Na]+ (Calcd C19H34O9Na: 429.2095).
p-Bromophenacyl Ester (2a) of Zanthoionic Acid (2)A solution of 2 (3.0 mg) in 1 mL of EtOH was neutralized by the addition of 0.1 M NaOH using phenolphthalein as an indicator. To the neutralized solution was added zanthoionic acid (2) (0.3 mg) to make it slightly acidic. p-Bromophenacylbromide (6 mg) in 1 mL of EtOH was added and the reaction mixture was kept at 60°C for 12 h. The ethanol was evaporated and the resulting residue was purified by prep. TLC (silica gel, CHCl3) to give 1.2 mg of zanthoionic acid p-bromophenacyl ester (2a). Zanthoionic acid p-bromophenacyl ester (2a): Colorless amorphous powder, [α]D26–26.3 (c=0.08, CHCl3); IR νmax (film) cm−1: 3380, 2958, 2871, 1760, 1707, 1067, 669; 1H-NMR (CDCl3, 600 MHz) δ: 7.77 (2H, d, J=8.4 Hz, H-3′ and 5′), 7.65 (2H, d, J=8.4 Hz, H-2′ and 6′), 5.75 (1H, overlapped, H-8), 5.73 (1H, overlapped, H-7), 5.43 (1H, d, J=16.2 Hz, H-8′a), 5.38 (1H, d, J=16.2 Hz, H-8′b), 4.90 (1H, d, J=9.3 Hz, H-9), 2.40 (1H, ddd, J=13.7, 3.2, 2.5 Hz, H-4a), 2.29 (1H, d, J=13.4 Hz, H-2a), 2.12 (1H, dd, J=13.4, 2.5 Hz, H-2b), 2.04 (1H, dd, J=13.7, 12.7 Hz, H-4b), 1.97 (2H, overlapped, H-5 and 6), 0.98 (3H, s, H3-12), 0.97 (3H, d, J=5.8 Hz, H3-13), 0.85 (3H, s, H3-11); 13C-NMR (CDCl3, 150 MHz): 210.7 (C-3), 190.0 (C-7′), 173.1 (C-10), 133.8 (C-8), 132.6 (C-4′), 132.4 (C-2′ and 6′), 129.5 (C-7 and 1′), 129.2 (C-3′ and 5′), 71.3 (C-9), 66.7 (C-8′), 57.0 (C-6), 56.0 (C-2), 49.3 (C-4), 38.5 (C-1), 33.4 (C-5), 30.5 (C-12), 21.3 (C-11), 21.1 (C-13); HR-ESI-MS (positive-ion mode): m/z: 459.0774 [M+Na]+ (Calcd C21H25O579BrNa: 459.0778).
Preparation of (R)- and (S)-MTPA Esters (2b and c) of Xanthoionic Acid p-Bromophenacyl Ester (2a)A solution of 2a (0.6 mg) in 1 mL of dehydrated CH2Cl2 was reacted with (R)-MTPA (36 mg) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (39 mg) and 4,4-dimethylaminipyridine (DMAP) (20 mg), and then the mixture was occasionally stirred at 25°C 12 h. After the addition of 1 mL of CH2Cl2, the solution was washed with H2O (1 mL), 4 N HCl (1 mL), NaHCO3-saturated H2O, and then brine (1 mL), successively. The organic layer was dried over Na2SO4 and then evaporated under reduced pressure. The residue was purified by prep. TLC [silica gel, CHCl3–(CH3)2CO (20 : 1)] to furnish an (R)-MTPA ester, 2b (0.3 mg). Through a similar procedure, the (S)-MTPA ester (2c) (0.2 mg) was prepared from 2a (0.6 mg) using (S)-MTPA (44 mg), EDC (36 mg) and 4-DMAP (20 mg). Xanthoionic acid p-bromophenacyl ester (R)-MTPA ester (2b): Colorless amorphous powder; 1H-NMR (CDCl3, 600 MHz) δ: 7.76 (2H, d, J=8.4 Hz, H-3′ and 5′), 7.65 (2H, d, J=8.4 Hz, H-2′ and 6′), 7.41–7.39 (5H, overlapped, aromatic protons), 5.78 (2H, overlapped, H-7 and 8), 5.65 (1H, d, J=6.3 Hz, H-9), 5.50 (1H, d, J=16.2 Hz, H-8′a), 5.30 (1H, d, J=16.2 Hz, H-8′b), 3.57 (3H, s, –OCH3), 2.40 (1H, ddd, J=13.7, 3.1, 2.5 Hz, H-4a), 2.29 (1H, d, J=13.5 Hz, H-2a), 2.11 (1H, dd, J=13.5, 2.5 Hz, H-2b), 2.04 (1H, overlapped, H-4b), 1.94 (1H, m, H-5), 1.93 (1H, m, H-6), 0.96 (3H, s, H3-12), 0.88 (3H, d, J=5.9 Hz, H3-13), 0.79 (3H, s, H3-11); HR-ESI-MS (positive-ion mode): m/z: 675.1172 [M+Na]+ (Calcd C31H32O779BrF3Na: 675.1176). Xanthoionic acid p-bromophenacyl ester (S)-MTPA ester (2c): Colorless amorphous powder; 1H-NMR (CDCl3, 600 MHz) δ: 7.74 (2H, d, J=8.6 Hz, H-3′ and 5′), 7.64 (2H, d, J=8.6 Hz, H-2′ and 6′), 7.43–7.38 (5H, overlapped, aromatic protons), 5.86 (2H, overlapped, H-7 and -8), 5.68 (1H, d, J=6.8 Hz, H-9), 5.44 (1H, d, J=16.1 Hz, H-8′a), 5.28 (1H, d, J=16.1 Hz, H-8′b), 3.57 (3H, s, –OCH3), 2.41 (1H, ddd, J=13.7, 3.6, 2.4 Hz, H-4a), 2.29 (1H, d, J=13.5 Hz, H-2a), 2.13 (1H, dd, J=13.5, 2.4 Hz, H-2b), 2.04 (1H, overlapped, H-4b), 1.98 (2H, m, H-5 and -6), 0.98 (3H, s, H3-12), 0.93 (3H, d, J=5.8 Hz, H3-13), 0.83 (3H, s, H3-11); HR-ESI-MS (positive-ion mode): m/z: 675.1171 [M+Na]+ (Calcd C31H32O779BrF3Na: 675.1176).
Enzymatic Hydrolysis of Zanthoionoside A (3)Zanthoionoside A (3) (5.6 mg) was dissolved in 1 mL of 20 mM acetate buffer and then 15 mg of β-glucosidase was added. The mixture was incubated for 18 h at 37°C. The reaction mixture was concentrated and then subjected to silica gel (50 g, Φ=2 cm, L=36 cm) CC with CHCl3 (100 mL), CHCl3–MeOH [19 : 1 (100 mL), 9 : 1 (100 mL), 17 : 3 (100 mL), 4 : 1 (100 mL), 3 : 1 (100 mL), 7 : 3 (100 mL), 13 : 7 (100 mL) and 3 : 2 (100 mL)] and MeOH (100 mL), fractions of 5 g being collected. The aglycone (3a) (1.9 mg) was obtained in fractions 116–127 and glucose was recovered in fractions 181–200. Aglycone (3a): Colorless syrup, [α]D24+33.1 (c=0.13, CHCl3); IR νmax (film) cm−1: 3309, 2928, 2861, 1512, 1456, 1272, 1228; 1H-NMR (CDCl3, 600 MHz) δ: 5.48 (1H, dd, J=15.4, 6.5 Hz, H-8), 5.37 (1H, ddd, J=15.4, 9.8, 0.8 Hz, H-7), 4.32 (1H, quintet, J=6.5 Hz, H-9), 4.00 (1H, ddd, J=3.4, 3.2, 3.2 Hz, H-3), 3.15 (1H, dd, J=10.9, 9.8 Hz, H-6), 1.81 (1H, dd, J=15.0, 3.2 Hz, H-2a), 1.76 (1H, m, H-5), 1.44 (1H, dd, J=10.9, 9.8 Hz, H-6), 1.28 (3H, d, J=6.4 Hz, H3-10), 1.04 (3H, s, H3-11), 0.91 (3H, s, H3-13), 0.82 (3H, s, H3-12); 13C-NMR (CDCl3, 150 MHz) δ: 137.2 (C-8), 130.1 (C-7), 77.3 (C-4), 70.1 (C-3), 68.9 (C-9), 56.3 (C-6), 44.8 (C-2), 34.3 (C-1), 33.0 (C-5), 31.8 (C-12), 23.7 (C-10), 23.4 (C-12), 16.5 (C-13); HR-ESI-MS (positive-ion mode): m/z: 251.1619 [M+Na]+ (Calcd C13H24O3Na: 251.1618).
Preparation of (R)- and (S)-MTPA Diesters (3b and c) of 3aIn a similar manner to that used for the preparation of 2b and c, 3b (0.1 mg) and c (0.4 mg) were prepared from 3a (0.8 mg each) using respective amounts of (R)- and (S)-MTPA (28 and 26 mg), EDC (30 and 28 mg) and 4,4-DMAP (19 and 22 mg). (R)-MTPA diester (3b): Amorphous powder, 1H-NMR (CDCl3, 600 MHz) δ: 7.43–7.33 (10 H, overlapped, aromatic protons), 5.54 (1H, quintet, J=6.5 Hz, H-9), 5.50 (2H, overlapped, H-7 and 8), 4.74 (1H, dd, J=11.4, 2.8 Hz, H-4), 4.14 (1H, br s, H-3), 3.57 (6H, s, –OCH3×2), 2.09 (1H, m, H-5), 1.81 (1H, dd, J=15.1, 2.8 Hz, H-2a), 1.52 (1H, overlapped, H-6), 1.49 (1H, overlapped, H-2b), 1.35 (3H, d, J=6.5 Hz, H3-10), 1.02 (3H, s, H3-11), 0.79 (3H, s, H3-12), 0.67 (3H, d, J=6.5 Hz, H3-13); HR-ESI-MS (positive-ion mode): m/z: 683.2414 [M+Na]+ (Calcd C33H38O7F6Na: 683.2414). (S)-MTPA diester (3c): Amorphous powder, 1H-NMR (CDCl3, 600 MHz) δ: 7.44–7.39 (10H, overlapped, aromatic protons), 5.54 (1H, quintet, J=6.5 Hz, H-9), 5.41 (2H, overlapped, H-7 and -8), 4.69 (1H, dd, J=11.2, 3.0 Hz, H-4), 4.04 (1H, br s, H-3), 3.57 (6H, s, –OCH3×2), 2.05 (1H, m, H-5), 1.78 (1H, dd, J=14.9, 3.3 Hz, H-2a), 1.56 (1H, overlapped, H-6), 1.44 (1H, overlapped, H-2b), 1.39 (3H, d, J=6.5 Hz, H3-10), 0.95 (3H, s, H3-11), 0.75 (3H, d, J=6.5 Hz, H3-13), 0.74 (3H, s, H3-12); HR-ESI-MS (positive-ion mode): m/z: 683.2416 [M+Na]+ (Calcd C33H38O7F6Na: 683.2414).
Enzymatic Hydrolysis of Zanthoionoside B (4)Zanthoionoside B (4) (6.4 mg) was hydrolyzed in a similar manner to zanthoionoside A (3) and the hydrolyzate was purified by silica gel CC (silica gel 70 g, Φ=3 cm, L=20 cm). Aglycone (4a) (3.0 mg) was recovered in fractions 84–91 and glucose was recovered in fractions 121–140. Aglycone (4a): colorless syrup, [α]D21–11.0 (c=0.28, MeOH); IR νmax (film) cm−1: 3360, 2948, 1622, 1557, 1455, 1365, 1073; 1H-NMR (CDCl3, 600 MHz) δ: 5.51 (1H, dd, J=15.5, 9.6 Hz, H-7), 5.40 (1H, dd, J=15.5, 6.3 Hz, H-8), 4.11 (1H, m, H-9), 4.03 (1H, dddd, J=3.1, 3.1, 3.0, 2.8 Hz, H-3), 3.49 (1H, dd, J=11.1, 4.6 Hz, H-10a), 3.43 (1H, dd, J=11.1, 7.3 Hz, H-10b), 1.82 (1H, m, H-5), 1.81 (1H, m, H-2a), 1.65 (1H, dt, J=14.0, 2.8 Hz, H-2b), 1.38 (1H, dd, J=10.4, 9.6 Hz, H-6), 1.35 (1H, dd, J=14.6, 3.1 Hz, H-4a), 1.19 (1H, ddd, J=14.0, 11.9, 3.1 Hz, H-4b), 1.05 (3H, s, H3-11), 0.84 (3H, s, H3-12), 0.79 (3H, d, J=6.5 Hz, H3-13); 13C-NMR (CDCl3, 150 MHz) δ: 134.5 (C-7), 132.9 (C-8), 74.5 (C-9), 68.2 (C-3), 67.7 (C-10), 59.7 (C-6), 47.4 (C-2), 42.8 (C-4), 34.3 (C-1), 32.8 (C-12), 27.2 (C-6), 23.9 (C-11), 21.7 (C-13); HR-ESI-MS (positive-ion mode): m/z: 251.1619 [M+Na]+ (Calcd C13H24O3Na: 251.1618).
Preparation of Pivaloyl Ester (4b) from 4aThe aglycone (4a) (3.0 mg) was dissolved in 1 mL of pyridine and 27.5 µL of pivaloyl chloride was added. The reaction mixture was stirred for 2 h at 25°C. To the reaction mixture, 1 mL of H2O was added, followed by extraction with 2 mL of CHCl3 three times. The residue from the dried (Na2SO4) organic layer was purified by prep. TLC [silica gel, CHCl3–MeOH (9 : 1)] to give 2.2 mg of pivaloyl ester (4b). Pivaloyl ester (4b): colorless syrup, [α]D21+5.33 (c=0.15, CHCl3); IR νmax (film) cm−1: 3397, 2961, 1714, 1650, 1554, 1457, 1364, 1162; 1H-NMR (CDCl3, 600 MHz) δ: 5.58 (1H, dd, J=15.5, 9.8 Hz, H-7), 5.42 (1H, dd, J=15.5, 6.5 Hz, H-8), 4.38 (1H, m, H-9), 4.14 (1H, dd, J=11.4, 3.8 Hz, H-10a), 4.11 (1H, m, H-3), 4.03 (1H, dd, J=11.4, 7.3 Hz, H-10b), 1.88 (1H, m, H-5), 1.82 (1H, overlapped, H-2a), 1.63 (1H, ddd, J=14.4, 2.7, 2.7 Hz, H-4a), 1.40 (1H, overlapped, -6), 1.38 (1H, overlapped, H-2b), 1.22 (9H, s, H3-3′, -4′ and 5′), 1.20 (1H, overlapped, H-4b), 1.06 (3H, s, H3-11), 0.83 (3H, s, H3-12), 0.79 (3H, d, J=6.5 Hz, H3-13); 13C-NMR (CDCl3, 150 MHz) δ: 178.7 (C-1′), 134.4 (C-7), 130.4 (C-8), 71.0 (C-9), 68.4 (C-10), 67.6 (C-3), 58.3 (C-6), 46.7 (C-2), 42.1 (C-4), 38.9 (C-2′), 33.3 (C-1), 32.2 (C-12), 27.2 (C-3′, -4′ and -5′), 25.9 (C-5), 23.6 (C-11), 21.2 (C-13); HR-ESI-MS (positive-ion mode): m/z: 335.2195 [M+Na]+ (Calcd C18H32O4Na: 335.2198).
Preparation of (R)- and (S)-MTPA Diesters (4c and d) of Pivaloyl Ester (4b)In similar preparations as those used for 2b and c, 4c (0.2 mg) and d (0.2 mg) were prepared from 4b (1.1 mg each) using respective amounts of (R)- and (S)-MTPA (20 mg and 21 mg), EDC (39 mg and 35 mg) and 4,4-DMAP (20 mg and 22 mg). (R)-MTPA diester of 4b (4c): colorless syrup; 1H-NMR (CDCl3, 600 MHz) δ: 7.43–7.33 (10H, overlapped, aromatic protons); 5.64 (1H, ddd, J=7.7, 7.7, 3.2 Hz, H-9), 5.56 (1H, dd, J=15.5, 9.5 Hz, H-7), 5.38 (1H, dd, J=15.5, 7.7 Hz, H-8), 5.33 (1H, m, H-3), 4.24 (1H, dd, J=12.1, 3.2 Hz, H-10a), 4.06 (1H, dd, J=12.1, 7.7 Hz, H-10b), 3.55 (6H, s, –OCH3×2), 1.86 (1H, dddd, J=14.0, 2.6, 2.6, 2.6 Hz, H-4a), 1.71 (1H, dd, J=15.0, 2.6 Hz, H-2a), 1.56 (1H, m, H-5), 1.36 (2H, overlapped, H-2b and -6), 1.24 (1H, overlapped, H-4b), 1.13 (9H, s, H3-3′, -4′ and -5′), 0.75 (3H, H3-11), 0.71 (3H, s, H3-12), 0.65 (3H, d, J=6.1 Hz, H3-13); HR-ESI-MS (positive-ion mode): m/z: 767.2983 [M+Na]+ (Calcd C38H46O8F6Na: 767.2989). (S)-MTPA diester of 4b (4d): colorless syrup; 1H-NMR (CDCl3, 600 MHz) δ: 7.44–7.31 (10H, overlapped, aromatic protons); 5.68 (1H, ddd, J=7.7, 7.7, 3.0 Hz, H-9), 5.48 (1H, dd, J=15.4, 9.7 Hz, H-7), 5.32 (1H, m, H-3), 5.30 (1H, dd, J=15.4, 7.7 Hz, H-8), 4.33 (1H, dd, J=12.4, 3.0 Hz, H-10a), 4.06 (1H, dd, J=12.4, 7.7 Hz, H-10b), 3.56 (6H, s, –OCH3×2), 1.93 (1H, dddd, J=14.5, 3.2, 3.2, 3.2 Hz, H-4a), 1.68 (1H, overlapped, H-2a), 1.56 (1H, m, H-5), 1.37 (2H, overlapped, H-2b and 6), 1.24 (1H, overlapped, H-4b), 1.17 (9H, s, H3-3′, -4′ and -5′), 0.72 (3H, d, J=6.5 Hz, H3-13), 0.66 (3H, s, H3-11), 0.52 (3H, s, H3-12); HR-ESI-MS (positive-ion mode): m/z: 767.2988 [M+Na]+ (Calcd C38H46O8F6Na: 767.2989).
Enzymatic Hydrolysis of Zanthoionoside C (5)Zanthoionoside C (5) (6.8 mg) was hydrolyzed in a similar manner to zanthoionoside A (3) and the hydrolyzate was purified by silica gel CC {silica gel 50 g, Φ=3 cm, L=15.3 cm with CHCl3 (300 mL) and CHCl3–MeOH [19 : 1 (300 mL), 9 : 1 (300 mL), 17 : 3 (300 mL), 4 : 1 (300 mL), 3 : 1 (300 mL) and 7 : 3 (300 mL)], fractions of 10 g being collected}. Aglycone (5a) (2.0 mg) was recovered in fractions 91–120 and glucose in fractions 151–180. Aglycone (5a): colorless syrup; [α]D22+25.4 (c=0.13, MeOH); IR νmax (film) cm−1: 3395, 2961, 2925, 1654, 1558, 1508, 1456; 1H-NMR (CDCl3, 600 MHz) δ: 5.52 (1H, ddd, J=15.5, 9.9, 0.9 Hz, H-7), 5.42 (1H, dd, J=15.5, 6.3 Hz, H-8), 4.26 (1H, m, H-9), 4.00 (1H, ddd, J=3.2, 3.1, 3.1 Hz, H-3), 3.67 (1H, dd, J=11.1, 3.2 Hz, H-10a), 3.51 (1H, dd, J=11.1, 7.6 Hz, H-10b), 3.15 (1H, dd, J=10.8, 3.1 Hz, H-4), 1.82 (1H, dd, J=14.8, 3.1 Hz, H-2a), 1.77 (1H, m, H-5), 1.47 (1H, dd, J=10.8, 9.9 Hz, H-6), 1.41 (1H, dd, J=14.8, 3.1 Hz, H-2b), 1.05 (3H, s, H3-11), 0.91 (3H, d, J=6.5 Hz, H3-13), 0.82 (3H, s, H3-12); 13C-NMR (CDCl3, 150 MHz) δ: 133.4 (C-7), 131.2 (C-8), 77.1 (C-4), 73.1 (C-9), 70.0 (C-3), 66.8 (C-10), 56.5 (C-6), 44.7 (C-2), 32.9 (C-1), 32.7 (C-5), 31.8 (C-13), 23.4 (C-11), 16.5 (C-13); HR-ESI-MS (positive-ion mode): m/z: 267.1565 [M+Na]+ (Calcd C13H24O4Na: 267.1567).
Preparation of Pivaloyl Ester (5b) from 5aThe aglycone (5a) (2.0 mg) was dissolved in 1 mL of pyridine and 65 µL of pivaloyl chloride was added. In a similar work up as that used for 4a, 1.6 mg of pivaloyl ester (5b) was obtained. Pivaloyl ester (5b): colorless syrup: [α]D23+19.9 (c=0.10, MeOH); IR νmax (film) cm−1: 3309, 2977, 2871, 1733, 1653, 1557, 1508, 1107; 1H-NMR (CDCl3, 600 MHz) δ: 5.55 (1H, ddd, J=15.5, 10.0, 1.1 Hz, H-7), 5.41 (1H, dd, J=15.5, 6.2 Hz, H-8), 4.39 (1H, m, H-9), 4.15 (1H, dd, J=11.4, 3.7 Hz, H-10a), 4.03 (1H, dd, J=11.4, 7.2 Hz, H-10b), 4.00 (1H, ddd, J=3.3, 3.2, 3.2 Hz, H-3), 3.15 (1H, dd, J=10.3, 3.3 Hz, H-4), 1.82 (1H, dd, J=14.9, 3.2 Hz, H-2a), 1.77 (1H, m, H-5), 1.48 (1H, dd, J=10.9, 10.0 Hz, H-6), 1.41 (1H, dd, J=14.9, 3.2 Hz, H-2b), 1.23 (9H, s, H3-3′, -4′ and -5′), 1.05 (3H, s, H3-11), 0.92 (3H, d, J=6.5 Hz, H3-13), 0.82 (3H, s, H3-12); 13C-NMR (CDCl3, 150 MHz) δ: 178.7 (C-1′), 133.4 (C-7), 130.8 (C-8), 77.2 (C-4), 70.9 (C-9), 70.0 (C-3), 68.4 (C-10), 56.5 (C-6), 44.7 (C-2), 38.9 (C-2′), 32.9 (C-1), 32.7 (C-5), 31.8 (C-12), 27.2 (C-3′, -4′ and -5′), 23.4 (C-11), 16.6 (C-13); HR-ESI-MS (positive-ion mode): m/z: 351.2144 [M+Na]+ (Calcd C18H32O5Na: 351.2141).
Preparation of (R)- and (S)-MTPA Diesters (5c and d) from Pivaloyl Ester (5b)In a similar manner to that used for the preparation of 2b and c, 5c (0.1 mg) and d (0.1 mg) were prepared from 5b (0.8 mg each) using respective amounts of (R)- and (S)-MTPA (24 mg and 36 mg), EDC (30 mg and 39 mg) and 4,4-DMAP (18 mg and 25 mg). (R)-MTPA diester of 5b (5c): colorless syrup; 1H-NMR (CDCl3, 600 MHz) δ: 7.36–7.27 (10 H, overlapped, aromatic protons), 5.59 (1H, ddd, J=7.7, 7.7, 3.5 Hz, H-9), 5.57 (1H, dd, J=15.6, 10.1 Hz, H-7), 5.36 (1H, dd, J=15.6, 7.7 Hz, H-8), 4.66 (1H, dd, J=11.1, 3.1 Hz, H-4), 4.19 (1H, dd, J=12.1, 3.5 Hz, H-10a), 4.07 (1H, m, H-3), 4.01 (1H, dd, J=12.1, 7.7 Hz, H-10b), 3.58 (6H, br s, –OCH3×2), 1.77 (1H, m, H-5), 1.74 (1H, dd, J=15.0, 3.3 Hz, H-2a), 1.53 (1H, overlapped, H-6), 1.45 (1H, overlapped, H-2b), 1.06 (9H, s, H3-3′, -4′ and -5′), 0.95 (3H, s, H3-11), 0.71 (3H, s, H3-12), 0.58 (3H, d, J=6.5 Hz, H3-13); HR-ESI-MS (positive-ion mode): m/z: 783.2945 [M+Na]+ (Calcd C38H46O9F6Na: 783.2943). (S)-MTPA diester of 5b (5d): colorless syrup; 1H-NMR (CDCl3, 600 MHz) δ: 7.36–7.21 (10H, overlapped, aromatic protons), 5.64 (1H, ddd, J=7.5, 7.5, 3.2 Hz, H-9), 5.46 (1H, dd, J=15.9, 9.8 Hz, H-7), 5.27 (1H, dd, J=15.9, 7.5 Hz, H-8), 4.61 (1H, dd, J=11.4, 3.4 Hz, H-4), 4.28 (1H, dd, J=12.2, 3.2 Hz, H-10a), 4.01 (1H, dd, J=12.2, 7.5 Hz, H-10b), 3.96 (1H, m, H-3), 3.58 (6H, br s, –OCH3×2), 1.77 (1H, m. H-5), 1.72 (1H, dd, J=14.5, 3.2 Hz, H-2a), 1.55 (1H, overlapped, H-6), 1.45 (1H, overlapped, H-2b), 1.01 (9H, s, H3-3′, -4′ and -5′), 0.87 (3H, s, H3-11), 0.66 (3H, s, H3-12), 0.66 (3H, d, J=6.5 Hz, H3-13); HR-ESI-MS (positive-ion mode): m/z: 783.2946 [M+Na]+ (Calcd C38H46O9F6Na: 783.2943).
Enzymatic Hydrolysis of Zanthoionosides D (6) and E (7)Zanthoionosides D (6) (4.2 mg) and E (7) (3.4 mg) were enzymatically hydrolyzed with 45 mg of β-glucosidase in the manner described for 5. Using a similar procedure as for zanthoionosides C (5), aglycones, 6a (1.4 mg) and 7a (1.3 mg) were obtained in fractions 99–115 and 91–111, respectively and glucose was obtained in fractions 181–210. The aglycones, 6a and 7a were identical with that (5a) of zanthoionoside C ([α]D, 1H- and 13C-NMR, and HR-ESI-MS).
Sugar Analysis of Compound 1 and Zanthoionosides A–E (3–7)Compound 1 (0.8 mg) was hydrolyzed with 1 M HCl (0.1 mL) at 80°C for 2 h. The reaction mixture was partitioned with an equal amount of EtOAc (0.1 mL), and the water layer was analyzed by HPLC. Glucose fractions obtained from enzymatic hydrolysis of zanthoionosides A–E (3–7) were also analyzed by HPLC with a chiral detector (JASCO OR-2090plus) on an amino column [Asahipak NH2P-50 4E, CH3CN–H2O (3 : 1), 1 mL/min]. The hydrolyzate and glucose in the sugar fractions gave a peak for D-glucose at 9.5 min with a positive optical rotation sign. Each peak was identified by co-chromatography with an authentic sample.
The authors are grateful for access to the superconducting NMR instrument (Brucker Avance III 600) at the Analytical Center of Molecular Medicine of the Hiroshima University Faculty of Medicine, and for access to an Applied Biosystems QSTAR XL system ESI (Nano Spray)-MS at the Analysis Center of Life Science of the Graduate School of Biomedical Sciences, Hiroshima University. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Japan Society for the Promotion of Science (Nos. 22590006, 23590130 and 15H04651). Thanks are also due to the Research Foundation for Pharmaceutical Sciences and the Takeda Science Foundation for their financial support.
The authors declare no conflict of interest.
