A New Glycosidic Acid, Muricatic Acid D, and Resin Glycosides, Muricatins X and XI, from the Crude Resin Glycoside Fraction of the Seeds of Ipomoea muricata

Abstract

Alkaline hydrolysis of crude resin glycoside fraction of the seeds of Ipomoea muricata (L.) Jacq. (Convolvulaceae) yielded a new glycosidic acid, muricatic acid D; three known glycosidic acids, namely, muricatic acids A, B, and C; and three known organic acids, namely, isobutyric, 2S-methylbutyric, and 2S-methyl-3S-hydroxybutyric acid. Two new genuine resin glycosides with macrolactone structures (jalapins), muricatins X and XI, were also isolated from the fraction. Their structures were determined using spectroscopic data and chemical evidence.

Introduction

The seeds of Ipomoea muricata (L.) Jacq. (Convolvulaceae) are used as a laxative and carminative folk medicine in India.¹⁾ Noda et al. reported that alkaline hydrolysis of the crude resin glycoside fraction of the seeds of I. muricata yielded three organic acids, namely, isobutyric, 2S-methylbutyric, and 2R-methyl-3R-hydroxybutyric (2R,3R-nilic) acid; and a glycosidic acid fraction composed of L-rhamnose, D-fucose, D-quinovose, and 11R-hydroxyhexadecanoic (jalapinolic) acid,¹⁾ whose absolute configuration was later corrected as S.²⁾ They also discussed the isolation and structural elucidation of three glycosidic acids, namely, muricatic acids A–C.^1,3) Eight genuine resin glycosides having macrolactone structures (jalapins),⁴⁾ namely, muricatins I–VIII were also reported.^3,5) In a previous paper, we reported the isolation and structural elucidation of a new type of resin glycoside, namely, muricatin IX, from the seeds of I. muricata, in which an organic acid connects the sugar moiety and the aglycone moiety to form a macrocyclic ester ring.⁶⁾ Recently, Wang et al. reported the isolation and structural elucidation of nine jalapins, namely, calonyctins B–J, from the seeds of I. muricata, four of which were structurally similar to muricatin IX.⁷⁾ The present study, which is part of our ongoing research on resin glycosides from the seeds of I. muricata, reinvestigates glycosidic acid and organic acid components generated by alkaline hydrolysis of the crude resin glycoside fraction of the seeds. The isolation and structural elucidation of two new jalapins from the crude resin glycoside fraction were also reported.

Results and Discussion

Silica gel column chromatography was used to separate a part of the EtOAc-soluble fraction (crude resin glycoside fraction)⁶⁾ of the seeds of I. muricata, followed by HPLC using octadecyl silica (ODS) and naphthylethyl group bonded silica (πNAP) columns, to obtain compounds 1 and 2.

The ¹H-NMR spectrum of 1 showed that it likely composed a new glycosidic acid with glucopyranosyl residue. Therefore, we re-examined the components in the crude resin glycoside fraction, including the glycosidic acids. The alkaline hydrolysis products of a part of this fraction were separated into organic acid and glycosidic acid fractions.

The organic acid fraction was acylated with p-bromophenacyl bromide, followed by chromatographic separation to obtain p-bromophenacyl isobutyrate (3), p-bromophenacyl 2-methylbutyrate (4), and p-bromophenacyl nilate (5)⁸⁾ (Fig. 1). The absolute configurations of 4 and 5 were defined as S and 2S,3S, respectively, by comparing the results of specific rotation and chiral HPLC analysis (Fig. 1). The absolute configurations at C-2 and C-3 of 5 were different from those reported previously.¹⁾

Fig. 1. Structures of 1–10 and 6a–11a

Acidic hydrolysis of a part of the glycosidic acid fraction yielded aglycone and monosaccharide fractions. Methylation of the former with trimethylsilyldiazomethane–hexane yielded methyl 11S-jalapinolate (6), which was identified by the ¹³C-NMR spectral data of 6 and the ¹H-NMR spectral data of its (+)-α-methoxy-α-trifluoromethylphenylacetic acid ester (6a)²⁾ (Fig. 1). The monosaccharide fraction was converted into thiocarbamoyl–thiazolidine derivatives and then analyzed using HPLC, according to a procedure reported by Tanaka et al.⁹⁾ Derivatives of D-glucose, D-fucose, D-quinovose, and L-rhamnose were detected.

The remaining glycosidic acid fraction was treated with trimethylsilyldiazomethane–hexane and then subjected to ODS column chromatography and HPLC using πNAP and ODS columns to yield four methyl esters (7–10) of glycosidic acids, among which 9 was identified as muricatic acid C methyl ester, based on its reported physical and ¹H- and ¹³C-NMR spectral data.³⁾ Upon alkaline hydrolysis, 7–10 gave the corresponding free glycosidic acids (7a–10a). Compounds 7a and 8a were identified as muricatic acids A and B, respectively, based on their NMR spectral data¹⁾ (Fig. 1). The ¹H- and ¹³C-NMR data of 9a have not been reported in literature.

Compound 10a, named muricatic acid D, was obtained as an amorphous powder and showed an [M–H]⁻ ion peak at m/z 871, along with fragment ion peaks at m/z 725 [871–146 (6-deoxyhexosyl unit)]⁻, 579 [725–146]⁻, 417 [579–162 (hexosyl unit)]⁻, and 271 [417–146]⁻ in negative-ion FAB-MS (Fig. 2). The molecular formula of 10a was determined to be C₄₀H₇₂O₂₀ using high-resolution (HR)-positive-ion FAB-MS. The ¹H-NMR spectrum of 10 showed signals arising from the four anomeric protons [δ 6.27 (1H, s), 5.84 (1H, d, J = 6.5 Hz), 5.15 (1H, d, J = 8.0 Hz), 4.83 (1H, d, J = 7.5 Hz)] and the three secondary methyl groups [δ 1.90 (3H, d, J = 6.5 Hz), 1.53 (3H, d, J = 6.5 Hz), 1.46 (3H, d, J = 6.5 Hz)] assignable to H₃-6 of 6-deoxyhexosyl residues. The spectrum also showed signals attributed to one methoxy group [δ 3.63 (3H, s)], one equivalent methylene group [δ 2.32 (2H, t, J = 7.5 Hz)] adjacent to a carbonyl group, and one primary methyl group [δ 0.87 (3H, t, J = 6.5 Hz)], ascribable to the jalapinoyl residue (Jla). The ¹³C-NMR spectrum of 10 showed signals attributed to four anomeric carbons (δ 106.1, 102.4, 102.0, 101.9) and one carboxyl carbon (δ 174.0). These NMR signals were assigned based on ¹H–¹H correlation spectroscopy (COSY) and heteronuclear multiple quantum coherence (HMQC) spectral data (Tables 1, 2). The assigned data suggested that 10 was composed of 1 mol each of methyl 11S-jalapinolate, L-rhamnose, D-quinovose, D-fucose, and D-glucose. The coupling constants of the signals due to anomeric and methine protons and the chemical shifts of ¹³C-NMR signals¹⁰⁾ corresponding to the sugar moiety indicated that all the monosaccharide units were in the pyranose form. Furthermore, it was observed that the glycosidic linkage of the rhamnosyl residue (Rha) was in the α mode in ¹C₄ conformation, while those of the quinovosyl (Qui), fucosyl (Fuc), and glucosyl (Glc) residues were in the β mode in ⁴C₁ conformation. The ¹³C-NMR signals due to the sugar moiety of 10 were compared with those of methyl pyranosides, as reported in the literature.^10,11) Glycosylation shifts^12,13) were observed at C-2 (+3.4 ppm) of Qui, C-2 (+4.4 ppm) of Glc, and C-4 (+10.7 ppm) of Rha. In addition, the ¹³C-NMR signal assignable to C-11 of Jla showed a downfield shift of 9.6 ppm, compared with that of methyl 11S-jalapinolate.¹⁴⁾ This data suggests that the sugar linkages of 10 were located at OH-2 of Qui, OH-2 of Glc, OH-4 of Rha, and OH-11 of Jla. The arrangement of sugar linkages was determined using the heteronuclear multiple bond correlation (HMBC) spectrum of 10, i.e., by the observation of key cross peaks between H-1 of Qui and C-11 of Jla, H-1 of Glc and C-2 of Qui, H-1 of Rha and C-2 of Glc, and H-1 of Fuc and C-4 of Rha (Fig. 3). Accordingly, the structure of 10a was determined as 11S-jalapinolic acid 11-O-β-D-fucopyranosyl-(1→4)-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→2)-β-D-quinovopyranoside (Fig. 1), which was previously reported as the structure of albinosinic acid G.¹⁵⁾ However, the ¹H- and ¹³C-NMR spectral data of 10a were not consistent with those of albinosinic acid G. The differences (Δδ = δ10a–δalbinosinic acid G) in chemical shifts at H-4 (Δδ = −0.15) and H-5 (Δδ = 0.17) of Qui, H-3 (Δδ = −0.22) and H-5 (Δδ = 0.18) of Rha, H-5 (Δδ = −0.21) of Fuc, C-2 (Δδ = 9.7) and C-3 (Δδ = 4.8) of Qui, C-3 (Δδ = 4.0), C-4 (Δδ = 11.2), and C-5 (Δδ = 3.9) of Rha, C-4 (Δδ = −3.9) of Glc, and C-2 (Δδ = −6.9), C-3 (Δδ = −3.8), and C-4 (Δδ = −3.2) of Fuc were especially large.

Fig. 2. Fragment Ions Observed in the Negative-Ion FAB-MS of 1 and 10a

Fig. 3. ¹H–¹³C Long-Range Correlations Observed for 1 and 10 in the HMBC Spectra (in Pyridine-d₅, 500 MHz)

Compound 1, named muricatin X, was obtained as an amorphous powder. The molecular weight of 1 was determined to be 938, by using FAB-MS in both positive- and negative-ion modes. Furthermore, the molecular formula of 1 was established as C₄₅H₇₇O₂₀ by HR-negative-ion FAB-MS. Alkaline hydrolysis of 1 yielded an organic acid fraction and a glycosidic acid. The GC analysis of the organic acid fraction revealed the presence of 2-methylbutyric acid. The glycosidic acid was identified as 10a by ¹H-NMR spectroscopy. The ¹H-NMR spectrum of 1 showed signals corresponding to two primary methyl groups, four secondary methyl groups, one H-2 of 2-methylbutyryl residue (Mba), one non-equivalent H₂-2 of Jla, and four anomeric protons (Table 1). The ¹³C-NMR spectrum of 1 showed signals for 45 carbons, including four anomeric carbons and two carboxyl carbons (Table 2). This data suggests that 1 is a jalapin composed of 1 mol each of 2-methylbutyric acid and 10a.^4,16) The NMR signals were assigned with two-dimensional NMR techniques, similar to the method used for 10. Comparison of the ¹H-NMR signals of the sugar moiety between 1 and 10 revealed the acylation shifts (Δδ = δ1–δ10) of the signals due to H-3 (Δδ = 1.01) of Glc and H-2 (Δδ = 1.43) of Rha, indicating the location of the ester linkages at OH-3 of Glc and OH-2 of Rha. The sites of the ester linkage of Mba and Jla were determined to be at OH-2 of Rha and OH-3 of Glc, respectively, using the HMBC spectrum of 1, with key cross-peaks observed between H-3 of Glc and C-1 of Jla and between H-2 of Rha and C-1 of Mba (Fig. 3). The locations of these linkages were supported by the fragment ion peaks observed in the negative-ion FAB-MS of 1, in which the fragment ion peaks at m/z 271 [jalapinolic acid–H]⁻ and 417 [271 + 146 (6-deoxyhexosyl unit)]⁻ were the same as those of 10a. However, 1 showed a peak at m/z 561 [417 + 144 (hexosyl unit−18 (H₂O))]⁻ instead of at m/z 579, which was observed in the spectrum of 10a (Fig. 2). The difference of 18 mass units suggests that the ester linkage of Jla is located at OH-3 of Glc.¹⁶⁾ Consequently, the structure of 1 was determined to be 11S-jalapinolic acid 11-O-β-D-fuucopyranosyl-(1→4)-O-(2-O-2S-methylbutyryl)-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)-β-D-quinovopyranoside, intramolecular 1,3″-ester (Fig. 1).

Table 1. ¹H-NMR Spectral Data for 1, 2, and 10 (in Pyridine-d₅, 500 MHz)

Position	1	2	10
Qui-1	4.78 d (8.0)	4.79 d (8.0)	4.83 d (7.5)
2	4.52 dd (8.0, 9.0)	4.50 dd (8.0, 9.0)	4.30 dd (7.5, 9.0)
3	4.28 dd (9.0, 9.0)	4.29a)	4.42a)
4	3.54 dd (9.0, 9.0)	3.53 dd (9.0, 9.0)	3.57 dd (9.0, 9.0)
5	3.68 dq (9.0, 6.0)	3.67 dq (9.0, 6.0)	3.69a)
6	1.59 d (6.0)a)	1.57 d (6.0)	1.53 d (6.5)
Qui′-1		5.29 d (8.0)
2		4.01 dd (8.0, 9.0)
3		4.09 dd (8.0, 9.0)
4		3.69a)
5		3.71a)
6		1.60 d (6.0)
Rha-1	5.50 d (1.0)	5.53 d (1.0)	6.27 s
2	5.80a)	5.78 dd (1.0, 3.5)	4.73 br s
3	4.64 br d (9.5)	4.69 dd (3.5, 9.5)	4.80 br d (9.5)
4	4.25 dd (9.5, 9.5)	4.29 dd (9.5, 9.5)	4.43 dd (9.5, 9.5)
5	4.77a)	4.81 dq (9.5, 6.5)	5.00 dq (9.5, 6.5)
6	1.91 d (6.5)	1.93 d (6.5)	1.90 d (6.5)
Glc-1	5.95 d (7.0)	5.96 d (7.5)	5.84 d (6.5)
2	4.16 dd (7.0, 9.0)	4.17 dd (7.5, 9.0)	4.19a)
3	5.80 dd (9.0, 9.0)	5.80 dd (9.0, 9.0)	4.20a)
4	4.37a)	4.37 dd (9.0, 9.0)	4.06a)
5	3.83 ddd (3.5, 3.5, 9.0)	3.83 ddd (3.5, 3.5, 9.0)	3.85 m
6	4.30a)	4.29a)	4.42a)
6	4.30a)	4.29a)	4.25 dd (5.5, 11.0)
Fuc-1	5.17 d (8.0)		5.15 d (8.0)
2	4.37a)		4.36 dd (8.0, 9.0)
3	4.06 dd (3.0, 9.0)		4.05a)
4	3.99 d (3.0)		3.95 br s
5	3.79 q (6.5)		3.68a)
6	1.52 d (6.5)		1.46 d (6.5)
Jla-2	2.89 ddd (2.0, 8.0, 17.0)	2.86 ddd (2.0, 8.0, 17.0)	2.32 t (7.5)
2	2.43 ddd (1.5, 10.5, 17.0)	2.44 ddd (2.0, 11.0, 17.0)
11	3.88 m	3.87 m	3.88 m
16	0.86 t (7.0)	0.86 t (7.0)	0.87 t (6.5)
OCH3			3.63 s
Mba-2	2.56 ddq (7.0, 7.0, 7.0)	2.54 ddq (7.0, 7.0, 7.0)
3	1.80 m	1.78 m
3	1.52a)	1.51 m
4	0.96 dd (7.5, 7.5)	0.93 dd (7.5, 7.5)
5	1.24 d (7.0)	1.23 d (7.0)

δ in ppm from TMS. Coupling constants (J) in Hz are given in parentheses. a) Signals were overlapped with other signals.

Table 2. ¹³C-NMR Spectral Data for 1, 2, and 10 (in Pyridine-d₅, 125 MHz)

Position	1	2	10		1	2	10
Qui-1	102.6	102.7	102.4	Jla-1	172.5	172.4	174.0
2	76.4	76.6	80.0	2	34.5	34.5	34.2
3	78.9	78.9	79.2	11	80.9	80.9	80.6
4	77.1	77.1	77.1	16	14.2	14.2	14.3
5	72.6	72.7	72.6	OCH3			51.2
6	18.5	18.5	18.5	CH2	35.1	35.1	35.3
Qui′-1		105.9		CH2	35.0	35.1	34.9
2		76.2		CH2	32.1	32.1	32.3
3		78.1		CH2	31.6	31.6	30.6
4		76.6		CH2	29.5	29.6	30.1
5		73.0		CH2	28.3	28.3	29.9
6		18.5		CH2	28.0	28.0	29.6
Rha-1	99.5	99.2	102.0	CH2	27.0	27.0	29.4
2	72.9	73.2	72.0	CH2	25.6	25.6	25.3
3	69.7	69.8	72.4	CH2	24.8	24.9	25.3
4	84.7	84.4	84.3	CH2	23.7	23.7	25.3
5	68.0	68.1	67.9	CH2	22.9	22.9	23.0
6	18.6	18.8	18.8	Mba-1	176.0	176.0
Glc-1	99.8	99.9	101.9	2	41.3	41.3
2	81.0	80.7	79.2	3	27.1	27.1
3	79.3	79.3	78.9	4	11.7	11.7
4	69.9	69.9	72.4	5	17.0	17.0
5	76.9	76.9	77.6
6	61.8	61.8	63.2
Fuc-1	106.5		106.1
2	73.2		73.3
3	75.4		75.4
4	72.7		72.7
5	71.7		71.6
6	17.1		17.2

δ in ppm from TMS.

Compound 2, named muricatin XI, was also obtained as an amorphous powder. The molecular formula of 2 was the same as that of 1, as determined by HR-negative-ion FAB-MS. The alkaline hydrolysis of 2 yielded 2-methylbutyric acid and scammonic acid A (11a).¹⁷⁾ The ¹H- and ¹³C-NMR spectra of 2 were superimposable on those of 1, except for the appearance of signals due to the terminal β-D-quinovopyranosyl residue and the absence of the signals due to the terminal β-D-fucopyranosyl residue (Tables 1, 2). The HMBC spectrum of 2 also exhibited key correlations between H-3 of Glc and C-1 of Jla and between H-2 of Rha and C-1 of Mba. The negative-ion FAB-MS of 2 also showed the same fragment ion peaks as those observed for 1, i.e., at m/z 853, 791, 707, 561, 417, and 271. Consequently, 2 was determined to be an epimer of 1, in which the β-D-fucopyranosyl residue in 1 was replaced by the β-D-quinovopyranosyl residue, as shown in Fig. 1.

The study of the seeds of I. muricata resulted in the isolation and structural elucidation of one new glycosidic acid, namely, muricatic acid D, and two new genuine resin glycosides, namely, muricatins X and XI. In addition, the absolute configuration of the nilic acid component of the crude resin glycoside fraction of the seeds of I. muricata was corrected from 2R,3R to 2S,3S. Consequently, the absolute configuration of nilic acid residues in muricatins VII and IX was also corrected from 2R,3R to 2S,3S.

Experimental

The instruments and materials used were as cited in the preceding report⁶⁾ unless otherwise specified.

Isolation of 1 and 2

A part (32.0 g) of the EtOAc-soluble fraction (fr.) (42.0 g, crude resin glycoside fr.) was chromatographed on silica gel [support, Art.1.07734, Merck, Darmstadt, Germany; solvent, CHCl₃–MeOH–H₂O (1 : 0 : 0, 30 : 1 : 0, 10 : 1 : 0, 10 : 2 : 0.1)] to furnish fractions (frs.) 1–24. HPLC [column, COSMOSIL 5C₁₈-AR-II, Nacalai Tesque, Inc. (Kyoto, Japan), 20 mm i.d. × 250 mm (column 1); solvent, 90% MeOH] of fr. 22 (389 mg) yielded frs. 22-1–22-13. Fraction 22-10 (34 mg) was subjected to HPLC [column, COSMOSIL πNAP, Nacalai Tesque, Inc., 20 mm i.d. × 250 mm (column 2); solvent, 90% MeOH] to afford 2 (10 mg) and 1 (18 mg).

Muricatin X (1): Amorphous powder, [α]_D²⁰−34.2° (c = 2.3, MeOH). Positive-ion FAB-MS m/z: 961 [M + Na]⁺. Negative-ion FAB-MS m/z: 937 [M–H]⁻, 853 [937–84 (2-methylbutyryl unit)]⁻, 835 [937–102 (2-methylbutyric acid)]⁻, 791 [937–146]⁻, 707 [791–84]⁻, 561 [707–146]⁻, 417 [561–144]⁻, 271 [417–146]⁻. HR-negative-ion FAB-MS m/z: 937.5002 (Calcd for C₄₅H₇₇O₂₀⁻, 937.5014). ¹H-NMR spectral data: see Table 1. ¹³C-NMR spectral data: see Table 2.

Muricatin XI (2): Amorphous powder, [α]_D¹⁸−34.1° (c = 1.3, MeOH). Positive-ion FAB-MS m/z: 961 [M + Na]⁺. Negative-ion FAB-MS m/z: 937, 853, 835, 791, 707, 561, 417, 271. HR-negative-ion FAB-MS m/z: 937.5034 (Calcd for C₄₅H₇₇O₂₀⁻, 937.5014). ¹H-NMR spectral data: see Table 1. ¹³C-NMR spectral data: see Table 2.

Alkaline Hydrolysis of 1 and 2

Solutions of 1 (5 mg) and 2 (5 mg) in 1 M KOH–1,4-dioxane (1 : 1, 1 mL) were each heated at 95 °C for 1 h. The reaction mixture was adjusted to pH 4 with 1 M HCl and then diluted with H₂O (5 mL) and extracted with ether (5 mL × 3). The ether layer was dried over MgSO₄ and evaporated to furnish an organic acid fr., which was analyzed by using GC [column, Unisole 30T (5%) glass column, GL Sciences, Tokyo, Japan, 3.2 mm i.d. × 2.0 m (column 3); carrier gas, N₂ (1.0 kg/cm²); column temperature, 120 °C; retention time (t_R) (min): 4.75 (2-methylbutyric acid) for 1 and 2].

The aqueous layer was desalted by MCI gel CHP 20 column chromatography (support, Mitsubishi Chemical Industries Co., Ltd., Tokyo, Japan; solvent, H₂O, acetone) to give a glycosidic acid as an amorphous powder (2 mg from 1, 2 mg from 2). The glycosidic acids derived from 1 and 2 were identical with 10a and scammonic acid A (11a), respectively, by comparison of ¹H-NMR data with those of authentic sample or those already reported.¹⁷⁾

Alkaline Hydrolysis of the Crude Resin Glycoside Fraction

A part (2.03 g) of the crude resin glycoside fr.⁶⁾ was dissolved in 1 M KOH–1,4-dioxane (9 : 1, 10 mL) and heated at 95 °C for 1 h. After cooling, the reaction mixture was adjusted to pH 4 with 1 M HCl and shaken with ether (10 mL × 5). The ether layer was dried over MgSO₄ and evaporated in vacuo to give an oil (270 mg, organic acid fr.). The H₂O layer was chromatographed over MCI gel CHP20 (solvent, H₂O, MeOH). The MeOH eluate was evaporated to dryness to afford an amorphous powder (1205 mg, glycosidic acid fr.).

Identification of Organic Acids from the Crude Resin Glycoside Fraction

An aliquot of the organic acid fr. was analyzed by GC [column, column 3; column temperature, 120 °C; carrier gas, N₂ (1.0 kg/cm²); t_R (min): 2.96 (isobutyric acid), 4.74 (2-methylbutyric acid)]. An aliquot of the organic acid fr. was methylated with diazomethane–ether and then the reaction mixture was analyzed by GC [column, column 3; column temperature, 90 °C; carrier gas, N₂ (1.2 kg/cm²); t_R (min): 6.30 (methyl nilate)]. Organic acid fr. (268 mg) in acetone (10 mL) was neutralized with triethylamine and then the solvent was removed. A mixture of the residue and p-bromophenacyl bromide (750 mg) in acetone (20 mL) was left to stand at room temperature for 1 h and then concentrated in vacuo to give a residue. The residue was subjected to a silica gel column [support, Art.1.09385, Merck; solvent, hexane–acetone (50 : 1, 3 : 1, 1 : 1)] to afford frs. 25–39. Fractions 26 (80 mg) and 37 (70 mg) were each subjected to HPLC [column, COSMOSIL 5SL-II, Nacalai Tesque, Inc., 20 mm i.d. × 250 mm; solvent, hexane–EtOAc (50 : 1) for fr. 26, hexane–EtOAc (3 : 1) for fr. 37] to yield p-bromophenacyl 2S-methylbutyrate (4, 25 mg) and p-bromophenacyl isobutyrate (3, 15 mg) from fr. 26 and p-bromophenacyl 2S,3S-nilate (5, 2 mg) from fr. 37.

3: Amorphous powder. ¹H-NMR spectral data (in CDCl₃, 500 MHz) δ: 1.26 (6H, d, J = 7.0 Hz, H₃-3 and H₃-4), 2.74 (1H, sept, J = 7.0 Hz, H-2), 5.28 (2H, s, OCH₂CO), 7.64 (2H, d-like, J = 8.5 Hz, aromatic H), 7.78 (2H, d-like, J = 8.5 Hz, aromatic H).

4: Amorphous powder, [α]_D²⁵ + 9.9° (c = 3.1, CHCl₃). ¹H-NMR spectral data (in CDCl₃, 500 MHz) δ: 0.98 (3H, dd, J = 7.5, 7.5 Hz, H₃-4), 1.24 (3H, d, J = 7.0 Hz, H₃-5), 1.56 (1H, ddq, J = 14.0, 7.0, 7.5 Hz, Ha-3), 1.79 (1H, ddq, J = 14.0, 7.0, 7.5 Hz, Hb-3), 2.56 (1H, ddq, J = 7.0, 7.0, 7.0 Hz, H-2), 5.29 (2H, s, OCH₂CO), 7.63 (2H, d-like, J = 8.5 Hz, aromatic H), 7.78 (2H, d-like, J = 8.5 Hz, aromatic H).

5: Amorphous powder. [α]_D²⁵ + 15.4° (c = 0.2, CHCl₃). ¹H-NMR spectral data (in CDCl₃, 500 MHz) δ: 1.25 (3H, d, J = 7.0 Hz, H₃-5), 1.30 (3H, d, J = 6.5 Hz, H₃-4), 2.63 (1H, dq, J = 6.5, 7.0 Hz, H-2), 3.97 (1H, m, H-3), 5.34 (1H, d, J = 16.0 Hz, OCH₂CO), 5.44 (1H, d, J = 16.0 Hz, OCH₂CO), 7.66 (2H, d-like, J = 8.5 Hz, aromatic H), 7.79 (2H, d-like, J = 8.5 Hz, aromatic H).

Compound 5 was analyzed by HPLC [column, COSMOSIL CHiRAL 5B, Nacalai Tesque, Inc., 4.6 mm i.d. × 250 mm; solvent, hexane–2-propanol (9 : 1); flow rate, 0.8 mL/min; column temperature, 25 °C; detector, Shimadzu SPD-10A UV (250 nm)]. The t_R of 5 (24.54 min) was in better agreement with that (24.61 min) of p-bromophenacyl 2S,3S-nilate⁸⁾ rather than that (26.01 min) of p-bromophenacyl 2R,3R-nilate.¹⁸⁾

Acidic Hydrolysis of the Glycosidic Acid Fraction from the Crude Resin Glycoside Fraction

A part (99 mg) of the glycosidic acid fr. in 1 M HCl (2 mL) was heated at 95 °C for 1 h. The reaction mixture was diluted with H₂O (5 mL) and then extracted with ether (5 mL × 6). The ether extract was dried over MgSO₄ and concentrated in vacuo to give a residue (26 mg). Treatment of the residue with trimethylsilyldiazomethane–hexane, followed by evaporation, gave a powder, which was chromatographed over a silica gel column [support, Art. 1.09385; solvent, hexane–acetone (20 : 1, 10 : 1, 7 : 1, 0 : 1)] to afford methyl jalapinolate (6, 5 mg).

6: Amorphous powder, [α]_D²⁴ +0.7° (c = 0.7, CHCl₃). ¹H-NMR spectral data (in CDCl₃, 500 MHz) δ: 0.89 (3H, t, J = 7.0 Hz, H₃-16), 2.30 (2H, t, J = 7.5 Hz, H₂-2), 3.58 (1H, m, H-11), 3.67 (3H, s, OCH₃). ¹³C-NMR data (in CDCl₃, 125 MHz) δ: 14.1 (C-16), 22.7, 25.0, 25.4, 25.6, 29.1, 29.2, 29.4, 29.5, 29.7, 31.9, 34.1, 37.5 (×2), 51.5 (OCH₃), 72.1 (C-11), 174.4 (C-1).

The aqueous layer was neutralized with Amberlite MB-3 (Organo Co., Tokyo, Japan) and then evaporated in vacuo to give a monosaccharide fr. (48 mg). A part (1 mg) of the monosaccharide fr. was dissolved in pyridine (0.2 mL) containing L-cysteine methyl ester hydrochloride (1 mg) and heated at 60 °C for 1 h. A solution (0.01 mL) of o-tolylisothiocyanate (0.1 mL) in pyridine (1.0 mL) was added to the mixture, which was heated at 60 °C for 1 h. The reaction mixture was analyzed by HPLC [column, COSMOSIL 5C₁₈-AR-II, Nacalai Tesque, Inc., 4.6 mm i.d. × 250 mm; solvent, 25% CH₃CN in 50 mM H₃PO₄; flow rate, 0.8 mL/min; column temperature, 35 °C; detector, Shimadzu SPD-10A UV detector (250 nm); t_R (min): 16.75 (D-glucose deriv.), 22.76 (D-fucose deriv.), 26.27 (D-quinovose deriv.), 27.88 (L-rhamnose deriv.)].

(+)-α-Methoxy-α-trifluoromethylphenylacetyl chloride (15 mg) was added to solution of 6 (2 mg) in pyridine (0.5 mL) and CCl₄ (5 drops) and left to stand at room temperature overnight. The solvent was removed under an N₂ stream to give a residue. The residue was purified by chromatography over a silica gel column [support, Art. 1.09385; solvent, hexane–EtOAc (30 : 1, 20 : 1)] to give 6a (2 mg).

6a: ¹H-NMR spectral data (in CDCl₃, 500 MHz) δ: 0.877 (3H, t, J = 7.0 Hz, H₃-16), 2.302 (2H, t, J = 7.5 Hz, H₂-2), 3.560 (3H, d-like, J = 1.0 Hz, OCH₃), 3.668 (3H, s, OCH₃), 5.084 (1H, m, H-11), approx. 7.389 (3H), approx. 7.545 (2H).

Isolation of Glycosidic Acid Methyl Esters (7–10)

A part (1105 mg) of the glycosidic acid fr. in MeOH (30 mL) was methylated with trimethylsilyldiazomethane–hexane. The concentrated reaction mixture was subjected to Chromatorex ODS column chromatography (support, Fuji Silysia Chemical, Ltd., Aichi, Japan; solvent, 70% MeOH, 75% MeOH, 80% MeOH, 85% MeOH, 90% MeOH, MeOH) to furnish frs. 40–48. Each HPLC (column, column 2; solvent, 85% MeOH) of fr. 41 (207 mg) and fr. 42 (794 mg) gave 7 (116 mg) and 10 (18 mg) from fr. 41 and 7 (450 mg) and fr. 42-1 (27 mg) from fr. 42. Fractions 42-1 was subjected to silica gel chromatography [support, Art.1.09385; solvent, CHCl₃–MeOH–H₂O (10 : 2 : 0.1, 8 : 2 : 0.2, 7 : 3 : 0.5, 6 : 4 : 1)] to afford frs. 42-1–42-3. Fractions 42-2 (11 mg) and 46 (65 mg) were each subjected to HPLC (column, colimn 1; solvent, 82% MeOH for fr. 42-2, 80% MeOH for fr. 46) to afford 8 (5 mg) from fr. 42-2 and 9 (14 mg) from fr. 46.

10: Amorphous powder, [α]_D²⁷−44.0° (c = 2.7, MeOH). Positive-ion FAB-MS m/z: 909 [M + Na]⁺. Negative-ion FAB-MS m/z: 885 [M–H]⁻, 739 [885–146]⁻, 593 [739–146]⁻. HR-positive-ion FAB-MS m/z: 909.4672 (Calcd for C₄₁H₇₄O₂₀Na⁺, 909.4666). ¹H- and ¹³C-NMR spectral data: see Tables 1, 2

Preparation of 7a–10a

Compounds 7 (10 mg), 8 (5 mg), 9 (14 mg), and 10 (16 mg) were each heated with 1 M KOH (1 mL) at 95 °C for 1 h. After cooling, the mixture was adjusted to pH 4 with 1 M HCl and desalted by using chromatography over MCI gel CHP 20 (solvent, H₂O, acetone) to give 7a (8 mg) from 7, 8a (3 mg) from 8, 9a (9 mg) from 9, and 10a (11 mg) from 10, respectively.

9a: Amorphous powder. [α]_D²⁴−39.5° (c = 0.7, MeOH). Positive-ion FAB-MS m/z: 879 [M + Na]⁺. Negative-ion FAB-MS m/z: 855 [M–H]⁻, 709 [855–146]⁻, 563 [709–146]⁻, 417 [579–162]⁻, 271 [417–146]⁻. HR-positive-ion FAB-MS m/z: 879.4571 (Calcd for C₄₀H₇₂O₁₉Na⁺, 879.4560). ¹H-NMR (in pyridine-d₅, 500 MHz) δ: 6.29 (1H, s, H-1 of Rha), 5.74 (1H, d, J = 8.0 Hz, H-1 of Qui′), 4.98 (1H, dq, J = 9.0, 6.0 Hz, H-5 of Rha), 4.88 (1H, d, J = 7.5 Hz, H-1 of Qui), 4.83 (1H, d, J = 7.5 Hz, H-1 of Qui″), 4.81 (1H, d, J = 3.5 Hz, H-2 of Rha), 4.61 (1H, dd, J = 3.5, 9.0 Hz, H-3 of Rha), 4.45 (1H, dd, J = 9.0, 9.0 Hz, H-3 of Qui), 4.30 (1H, dd, J = 7.5, 9.0 Hz, H-2 of Qui), 4.28 (1H, dd, J = 9.0, 9.0 Hz, H-4 of Rha), 4.16 (1H, dd, J = 8.0, 8.5 Hz, H-2 of Qui′), 4.03 (1H, dd, J = 9.0, 9.0 Hz, H-3 of Qui″), 3.97 (1H, dd, J = 7.5, 9.0 Hz, H-2 of Qui″), 3.91 (1H, m, H-11 of Jla), 3.87 dd(1H, dd, J = 8.5, 8.5 Hz, H-3 of Qui′), 3.77 (1H, dq, J = 9.0, 6.0 Hz, H-5 of Qui″), 3.73 (1H, dq, J = 9.0, 6.0 Hz, H-5 of Qui), 3.64 (1H, dd, J = 9.0, 9.0 Hz, H-4 of Qui), 3.64 (1H, dd, J = 9.0, 9.0, H-4 of Qui″), 3.59 (1H, m, H-5 of Qui′), 3.55 (1H, dd, J = 8.5, 8.5 Hz, H-4 of Qui′), 2.51 (2H, t, J = 7.5 Hz, H₂-2 of Jla), 1.80 (3H, J = 6.0 Hz, H₃-6 of Rha), 1.58 (3H, J = 6.0 Hz, H₃-6 of Qui), 1.55 (3H, d, J = 6.0 Hz, H₃-6 of Qui′), 1.54 (3H, d, J = 6.0 Hz, H₃-6 of Qui″), 0.85 (3H, t, J = 7.0 Hz, H₃-16 of Jla). ¹³C-NMR (in pyridine-d₅, 125 MHz) δ: 176.4 (C-1 of Jla), 104.7 (C-1 of Qui″), 102.3 (C-1 of Rha), 102.2 (C-1 of Qui), 101.5 (C-1 of Qui′), 88.7 (C-3 of Qui′), 80.4 (C-11 of Jla), 79.4 (C-2 of Qui), 79.2 (C-3 of Qui), 78.2 (C-3 of Qui″), 78.1 (C-2 of Qui′), 77.2 (C-4 of Qui″), 76.2 (C-4 of Qui), 75.0 (C-2 of Qui″), 74.9 (C-4 of Qui′), 74.3 (C-4 of Rha), 73.4 (C-5 of Qui″), 72.7 (C-3 of Rha), 72.5 (C-5 of Qui), 72.3 (C-2 of Rha), 72.2 (C-5 of Qui′), 69.6 (C-5 of Rha), 35.2 (C-2 of Jla), 19.0 (C-6 of Rha), 18.6 (C-6 of Qui), 18.5 (C-6 of Qui′), 18.1 (C-6 of Qui″), 14.3 (C-16 of Jla).

10a: Amorphous powder. [α]_D²³−40.0° (c = 1.7, MeOH). Positive-ion FAB-MS m/z: 895 [M + Na]⁺. Negative-ion FAB-MS m/z: 871 [M–H]⁻, 725 [871–146]⁻, 579 [725–146]⁻, 417 [579–162]⁻, 271 [417–146]⁻. HR-positive-ion FAB-MS m/z: 895.4504 (Calcd for C₄₀H₇₂O₂₀Na⁺, 895.4509). ¹H-NMR (in pyridine-d₅, 500 MHz) δ: 6.28 (1H, d, J = 1.5 Hz, H-1 of Rha), 5.85 (1H, d, J = 7.0 Hz, H-1 of Glc), 5.16 (1H, d, J = 7.5 Hz, H-1 of Fuc), 5.01 (1H, dq, J = 9.5, 6.5 Hz, H-5 of Rha), 4.83 (1H, d, J = 8.0 Hz, H-1 of Qui), 4.81 (1H, dd, J = 3.0, 9.5 Hz, H-3 of Rha), 4.73 (1H, dd, J = 1.5, 3.0 Hz, H-2 of Rha), 4.44 (Ha-6 of Glc), 4.44 (H-4 of Rha), 4.43 (H-3 of Qui), 4.36 (1H, dd, J = 7.5, 9.5 Hz, H-2 of Fuc), 4.31 (1H, dd, J = 8.0, 9.0 Hz, H-2 of Qui), 4.26 (1H, dd, J = 6.0, 11.5 Hz, Hb-6 of Glc), 4.22 (1H, dd, J = 9.0, 9.0 Hz, H-3 of Glc), 4.19 (1H, dd, J = 7.0, 9.0 Hz, H-2 of Glc), 4.07 (1H, dd, J = 9.0, 9.0 Hz, H-4 of Glc), 4.05 (1H, dd, J = 3.5, 9.5 Hz, H-3 of Fuc), 3.95 (1H, d, J = 3.5 Hz, H-4 of Fuc), 3.88 (H-11 of Jla), 3.86 (H-5 of Glc), 3.71 (1H, dq, J = 9.0, 6.5 Hz, H-5 of Qui), 3.68 (1H, d, J = 6.5 Hz, H-5 of Fuc), 3.57 (1H, dd, J = 9.0, 9.0 Hz, H-4 of Qui), 2.51 (2H, t, J = 7.5 Hz, H₂-2 of Jla), 1.90 (3H, d, J = 6.5 Hz, H₃-6 of Rha), 1.53 (3H, d, J = 6.3 Hz, H₃-6 of Qui), 1.46 (3H, d, J = 6.5 Hz, H₃-6 of Fuc), 0.87 (3H, t, J = 7.0 Hz, H₃-16 of Jla).¹³C-NMR (in pyridine-d₅, 125 MHz) δ: 176.2 (C-1 of Jla), 106.1 (C-1 of Fuc), 102.4 (C-1 of Qui), 102.0 (C-1 of Rha), 101.9 (C-1 of Glc), 84.2 (C-4 of Rha), 80.7 (C-11 of Jla), 79.9 (C-2 of Qui), 79.2 (C-2 of Glc), 79.2 (C-3 of Qui), 78.9 (C-3 of Glc), 77.7 (C-5 of Glc), 77.1 (C-4 of Qui), 75.4 (C-3 of Fuc), 73.3 (C-2 of Fuc), 72.7 (C-4 of Fuc), 72.5 (C-5 of Qui), 72.4 (C-4 of Glc), 72.4 (C-3 of Rha), 72.1 (C-2 of Rha), 71.6 (C-5 of Fuc), 67.9 (C-5 of Rha), 63.2 (C-6 of Glc), 35.3 (C-2 of Jla), 18.8 (C-6 of Rha), 18.5 (C-6 of Qui), 17.2 (C-6 of Fuc), 14.3 (C-16 of Jla).

Acknowledgments

We express our appreciation to Mr. H. Harazono of Fukuoka University for his measurement of FAB-MS. This research was supported in part by a Grant-in-Aid for Scientific Research (C) (JSPS KAKENHI Grant Numbers JP16K08306 and JP20K07117) from Japan Society for the Promotion Science and by the Research and Study Program/Project of Tokai University Educational System General Research Organization (Kanagawa, Japan).

Conflict of Interest

The authors declare no conflict of interest.

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