Sulfated Glucosides of an Aliphatic Alcohol and Monoterpenes, and Megastigmanes from the Leaves of Meliosma pinnata spp. arnottiana

Abstract

Chemical study of the leaves of Meliosma pinnata spp. arnottiana afforded five sulfated glucosides of oct-1-en-3-ol (1) and cyclic linalool derivatives (2–5), and two megastigmanes (6, 7). Their structures were elucidated by extensive investigation of one- and two-dimensional NMR spectroscopic data, and the absolute structures of the megastigmanes were determined by the modified Mosher’s method.

Meliosma pinnata (ROXB.) MAXIM. ssp. arnottiana (WALP.) BEUSEKOM is a tall deciduous tree belonging to the family Sabiaceae. However, the International Plant Names Index currently classifies Meliosma species into the Meliosmaceae,¹⁾ whereas the latest taxonomic publication observes the classical taxon Sabiaceae.²⁾ The Sabiaceae comprise about one hundred species in five major genera, and M. pinnata spp. arnottiana grows wild in the Amami Islands, the Okinawa Islands and Taiwan.³⁾ This plant is also known as a feeding plant for the larvae of a butterfly, Dichorragia nesimachus.⁴⁾ In a previous paper, the constituents of a closely related plant, M. lepidota ssp. squamulata, were investigated.⁵⁾ However, there has been no report on the constituents of the title plant and its medicinal use is also uncertain. This paper deals with isolation work on the plant.

Results and Discussion

From the leaves of Meliosma pinnata ssp. arnottiana, seven new compounds, i.e., sulfated glucosides of oct-1-en-3-ol (1) and monoterpenes (2–5), and megastigmanes (6, 7), together with four known ones, (3S,6S)-cis-linalool-3,7-oxide β-D-glucopyranoside (8),⁶⁾ (3S,5R,6S,9R)-3,6-dihydroxy-5,6-dihydro-β-ionol (9),⁷⁾ alangionoside A (10),⁸⁾ and (2R,3R,5R,6S,9R)-3-hydroxy-5,6-epoxy-β-ionol 2-O-β-D-glucopyranoside (11)⁹⁾ (Fig. 1) were isolated using various kinds of chromatographic techniques. This paper deals with structural elucidation of the new compounds.

Fig. 1. Structures of the New Compounds Isolated and a Reference Compound

Compound 1, [α]_D²¹ −28.6, was isolated as colorless needles and its elemental composition was determined to be C₁₄H₂₆O₉S by observation of a quasi-molecular ion peak at 369.1214 [M−H]⁻ in the high-resolution (HR)-electrospray ionization (ESI)-MS. The IR spectrum showed absorption bands at 3364 and 1235 cm⁻¹ assignable to hydroxy groups and a sulfonic acid moiety, respectively. The ¹H-NMR spectrum exhibited a triplet methyl signal, three olefinic signals arising from a terminal double bond [δ_H 5.09 (H-1b), 5.20 (H-1a), and 5.89 (H-2)] and an anomeric proton [δ_H 4.35 (d, J=7.9 Hz)]. The ¹³C-NMR spectrum comprised one methyl, four methylene, one oxymethine and two olefinic carbon signals [(δ_C 116.1 (t) and 141.0 (d)] together with six sugar signals (Table 1). Sugar analysis revealed the presence of D-glucose, and from the coupling constant of the anomeric proton in the ¹H-NMR spectrum, the sugar linkage mode was assigned as β. The significant correlation between H-2 and H-3 (δ_H 4.12) observed in the ¹H–¹H correlation spectrum (COSY) enabled placing of the hydroxy group at the 3-position, to which the sugar moiety was attached. Thus, the structure of 1 was expected to be oct-1-en-3-ol β-D-glucopyranoside with a sulfate functional group. The linkage position of the sulfate group was deduced to be the 4′-position of the sugar moiety from the obvious down-field shifts of the 4′-proton and carbon signals in the NMR spectra. The absolute configuration of the 3-position of matsutake alcohol (1a) (aglycone of 1) isolated from natural sources was generally found to be R.^10,11) Since application of the β-D-glucosylation-induced ¹³C chemical shift trend rule to 1 and 1a was not successful in a previous experiment,¹²⁾ 1 was catalytically reduced to give 1,2-dihydro derivative of 1 (1b), and the rule was applied to 1b and octan-3-ol (1c).¹³⁾ The upfield shift value for the 2-position was calculated to be −2.4 ppm and that for the 4-position to be −3.5 ppm (Table 1), which clearly demonstrated that the absolute configuration of the 3-position of 1b is S. Therefore, the overall structure of 1 was elucidated to be (3R)-oct-1-en-3-ol β-D-glucopyranoside 4′-O-sulfate, as shown in Fig. 1.

Table 1. ¹³C-NMR Spectroscopic Data for 1–5, 8, 1b and c (150 MHz, CD₃OD)

	1	1b	1cb)	2	8	3	4	5
1	116.1	10.0	10.4	111.5	111.5	111.1 (110.3)c)	112.0	112.2 (111.5)c)
2	141.0	28.7 (−2.4)a)	31.1	147.5	147.5	148.4 (148.0)	145.5	144.9 (144.4)
3	83.1	82.1 (+8.3)a)	73.9	74.9	74.9	75.2 (73.5)	84.5	84.9 (83.5)
4	35.7	34.5 (−3.5)a)	38.0	33.7	33.8	29.2 (28.8)	38.7	38.1 (37.3)
5	25.7	25.8	26.6	25.7	25.8	21.1 (20.9)	28.1	28.1 (26.9)
6	33.0	33.3	33.2	85.9	85.8	77.5 (77.1)	85.3	86.9 (84.8)
7	23.7	23.7	23.8	77.2	77.2	76.6 (74.9)	80.3	80.8 (79.2)
8	14.4	14.4	14.5	22.0	22.1	28.4 (28.4)	23.5	23.7 (22.6)
9				30.1	30.1	27.0 (26.9)	22.8	21.1 (23.2)
10				32.3	32.3	30.6 (30.2)	25.9	26.9 (26.8)
1′	103.0	103.3		106.0	106.3	101.2 (101.7)	98.2	98.6 (98.4)
2′	75.4	75.3		75.4	75.4	75.1 (75.0)	75.4	75.1 (75.0)
3′	77.0	76.9		76.9	78.2	77.0 (76.3)	76.8	76.7 (76.3)
4′	77.8	78.0		77.8	71.7	78.1 (77.2)	77.8	78.0 (76.9)
5′	76.2	76.1		76.1	77.8	76.3 (76.3)	76.1	76.1 (75.8)
6′	62.5	62.7		62.6	62.9	62.8 (62.0)	62.6	62.6 (61.8)

a) Δ_δ1b–δ1c. b) Data from ref. 12. c) Data for pyridine-d₅.

Compound 2, [α]_D²⁸ +19.7, was isolated as an amorphous powder and its elemental composition was determined to be C₁₆H₂₈O₁₀S by HR-ESI-MS. The IR spectrum showed absorption bands at 3393 and 1267 cm⁻¹ assignable to hydroxy groups and a sulfonic acid moiety, respectively. In the ¹H-NMR, signals assignable to three singlet methyls, a terminal double bond (δ_H 4.95, 5.03 and 5.96), and an anomeric proton (δ_H 4.36) were observed. The ¹³C-NMR spectrum comprised the signals of three methyls, two methylenes, one oxygenated methine, two oxygenated tertiary carbons and a terminal mono-substituted double bond [δ_C 111.5 (t) and 147.5 (d)] together with six sugar carbon signals. The three degrees of unsaturation required a mono-cyclic structure, probably due to an oxyran ring between two oxygenated tertiary carbons. From this evidence, 2 was expected to be a sulfated analogue of (3S,6S)-cis-linalool-3,7-oxide β-D-glucopyranoside (8) isolated from Zanthoxylum piperitum,⁶⁾ which co-occurred in this plant. The sulfated position was deduced be the same as that in 1 and 8 from the sugar ring signals in the ¹³C-NMR spectrum superimposable with those of 1 (Table 1). Therefore, the structure of 2 was elucidated to be the 4′-O-sulfate of 8, as shown in Fig. 1.

Compound 3, [α]_D²⁵ −24.2, was isolated as an amorphous powder and its elemental composition was the same as that of 2. NMR spectroscopic data indicated that 3 was an analogous compound to 2, and H-6 appeared at δ_H 3.59 with coupling constants of J=2.6 and 5.7 Hz, indicating that H-6 was in an equatorial orientation. Since the β-axial proton at the 4-position (δ_H 2.06 in pyridine-d₅) showed a significant cross peak with 10-methyl protons (δ_H 1.15 in pyridine-d₅) in the phase-sensitive (PS) nuclear Overhauser enhancement spectroscopy (NOESY) spectrum, the vinyl substituent at the 3-position and the glucopyranoxy group at the 6-position are in the trans relationship. Based on the assumption that the biosynthetic precursors of 2 and 3, linalool has the same stereochemistry at the 3-position, i.e., S and the significant difference of the ¹³C-NMR chemical shifts of the C-6 (2: δ_C 85.9 and 3: δ_C 77.5), the structure of 3 was tentatively assigned as an epimeric compound of 2, namely (3S*,6R*)-trans-linalool-3,7-oxide β-D-glucopyranoside 4′-O-sulfate. However, the possibility remains that the aglycones of 2 and 3 are enantiomers of each other.

Compound 4, [α]_D²⁰ −12.8, was isolated as an amorphous powder and its elemental composition was determined to be C₁₆H₂₈O₁₀S. The IR spectrum also showed absorption bands for hydroxy groups and a sulfonic acid moiety. The functionalities indicated by the NMR spectra were the same as those of 2 and 3, and the NMR spectra of the aglycone were essentially the same as those of (3R,6S)-cis-linalool-3,6-oxide β-D-glucopyranoside isolated from Z. piperitum.⁶⁾ Therefore the structure was elucidated to be (3S,6S)-cis-linalool-3,6-oxide β-D-glucopyranoside 4′-O-sulfate, as shown in Fig. 1.

Compound 5, [α]_D²⁵ +3.23, was isolated as an amorphous powder and its elemental composition was the same as the aforementioned linalool derivatives. NMR spectra of 5 were similar to those of 4, and judging from the PS-NOESY correlation between H-2 (δ_H 5.90 in pyridine-d₅) and H-6 (δ_H 4.05 in pyridine-d₅), the vinyl and propan-2-ol substituents are in the trans relationship. Assuming as for 3 that the biosynthetic precursor is the same, 3S-linalool, the structure of 5 was tentatively elucidated to be (3S*,6R*)-trans-linalool-3,6-oxide β-D-glucopyranoside 4′-O-sulfate, as shown in Fig. 1. However, the possibility remains that the aglycones of 4 and 5 are enantiomers of each other.

Compound 6, [α]_D²¹ −10.9, was isolated as a syrup and its elemental composition was determined to be C₁₃H₂₄O₄ by HR-ESI-MS. The IR spectrum showed absorption bands assignable to hydroxy groups (3383 cm⁻¹) and a double bond (1456 cm⁻¹). In the ¹H-NMR spectrum, signals for two singlet and two doublet methyls, a trans double bond and three oxygenated methines were observed (Table 2). The ¹³C-NMR spectrum exhibited four methyl, one methylene, one methine, three oxygenated methines, one oxygenated tertiary, one double bond and one quaternary carbon signals. Thus, the structure of 6 was expected to be that of a megastigmane with four hydroxy substituents. From two COSY sequences, C-2 to C-13 and C-7 to C-10, together with the heteronuclear multiple-bond correlation spectrum (HMBC) shown in Fig. 2, the planar structure of 6 was assigned as 2,3,6,9-tetrahydroxymegastigm-7-ene. From the coupling constant of H-2 (J=9.7 Hz), the two hydroxy groups at the 2- and 3-positions placed in equatorial orientations and the methyl group at the 5-position was also placed equatorially from the coupling constant between H-4 and H-5 (J=13.1 Hz). The PS-NOESY correlations of H-7 and H₃-11 (axial), and H-3 (axial) and H₃-11 allowed placing the side chain in the equatorial position. Finally, the modified Mosher’s method¹⁴⁾ was applied to 6 to give 3,9-α-methoxy-α-trifluoromethylphenylacetic acid (MTPA) diesters (Fig. 3), and then the structure of 6 was elucidated to be (2S,3S,5R,6S,7E,9R)-2,3,6,9-tetrahydroxymegastigm-7-ene, as shown in Fig. 1.

Table 2. NMR Spectroscopic Data for 6 and 7 (C: 150 MHz; H: 600 MHz, CD₃OD)

	6		7
	C	H	C	H
1	44.9	—	34.6	—
2	79.0	3.47 d 9.7 Hz	48.6	1.73 dd 12.9, 4.3 Hz
				1.32 dd 12.9, 11.7 Hz
3	71.7	3.58 ddd 11.5, 9.7, 5.1 Hz	69.7	3.52 ddd 11.7, 9.8, 4.3 Hz
4	37.8	1.70 ddd 12.6, 5.1, 4.0 Hz	84.4	3.16 d 9.8 Hz
		1.50 ddd 13.1, 12.6, 11.5 Hz
5	34.7	1.98 dqd 13.1, 6.8, 4.0 Hz	76.7	—
6	80.0	—	60.8	1.87 d 9.8 Hz
7	133.8	5.58 dd 15.9, 1.1 Hz	125.9	5.66 ddd 15.1, 9.8, 0.9 Hz
8	135.6	5.71 dd 15.9, 5.9 Hz	141.2	5.60 dd 15.1, 5.3 Hz
9	69.2	4.30 qdd 6.4, 5.9, 1.1 Hz	69.0	4.26 qdd 6.4, 5.3, 0.9 Hz
10	24.1	1.25 3H, d 6.4 Hz	23.7	1.25 3H d 6.4 Hz
11	17.8	0.87 3H s	17.8	0.96 3H s
12	21.5	0.96 3H s	21.5	0.89 3H s
13	16.1	0.79 3H, d 6.8 Hz	16.1	1.15 3H s

Fig. 2. ¹H–¹H COSY and HMBC Correlations of 6

Fig. 3. Results of Modified Mosher’s Method for 6 (a) and 7 (b)

Compound 7, [α]_D²⁰ −12.7, was isolated as a syrup and its elemental composition was the same as that of 6. One-dimensional NMR spectra indicated the same functionalities as those of 6, and two-dimensional NMR spectra revealed the planar structure of 7 to be 3,4,5,9-tetrahydroxymegastigm-7-ene. The two hydroxy groups at the 3- and 4-positions were placed in equatorial orientations from the axial proton at the 3-position (J=9.8 Hz). The PS-NOESY correlations H-7 and H₃-11 (axial), H-3 and H₃-11, H-4 (axial) and H-6, and H-3 and H₃-13 placed the hydroxy group at the 5-position and the side chain were placed in equatorial orientations. The modified Mosher’s method was also applied to 7 to give 4,9-MTPA diesters (Fig. 3), and thus the structure of 7 was established to be (3R,4S,5S,6R,7E,9R)-3,4,5,9-tetrahydroxymegastigm-7-ene, as shown in Fig. 1.

Experimental

General

Melting point (mp) was measured with a Yanagimoto micro melting point apparatus and is uncorr. Optical rotations were measured on a JASCO P-1030 polarimeter and IR spectra on a Horiba FT-710 spectrophotometer. ¹H- and ¹³C-NMR spectra were taken on Brucker Avance III 600 spectrometers at 600 MHz and 150 MHz, respectively, with tetramethylsilane as an internal standard. Positive- and negative-ion HR-MS was performed with an Applied Biosystem QSTAR XL system ESI (Nano Spray)-MS.

A highly-porous synthetic resin (Diaion HP-20) was purchased from Mitsubishi Kagaku (Tokyo, Japan). Silica gel column chromatography (CC) and reversed-phase [octadecylsilanized silica gel (ODS)] open CC were performed on silica gel 60 (Merck, Darmstadt, Germany) and Cosmosil 75C₁₈-OPN (Nacalai Tesque, Kyoto, Japan), respectively. The droplet counter-current chromatograph (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 column (Inertsil; GL Science, Tokyo, Japan), and the eluate was monitored with a UV detector at 254 nm and a refractive index monitor. (R)- and (S)-MTPAs were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Plant Material

The leaves of Meliosma pinnata spp. arnottiana were collected in Kunigami-gun, Okinawa, Japan, in July 1993, and the plant was identified by Dr. Takakazu Shinzato of the Subtropical Field Research Center, Faculty of Agriculture, University of the Ryukyus. A voucher specimen was deposited in the Herbarium of the Faculty of Pharmaceutical Sciences, Hiroshima University (93-Okinawa-MPA-0727).

Extraction and Isolation

Leaves of M. pinnata spp. arnottiana (2.57 kg) were extracted three times with MeOH (4.5 L×3) at room temperature for one week and then concentrated to 3 L in vacuo. The concentrated extract was washed with n-hexane (3 L, 61.7 g), and then the MeOH layer was concentrated to a gummy mass. The latter was suspended in water (3 L) and then extracted with EtOAc (3 L) to give 22.2 g of an EtOAc-soluble fraction. The aqueous layer was extracted with 1-BuOH (3 L) to give a 1-BuOH-soluble fraction (21.7 g), and the remaining water layer was concentrated to furnish 52.2 g of a water-soluble fraction.

The 1-BuOH-soluble fraction (21.7 g) was subjected to Diaion HP-20 CC (Φ=35 mm, L=40 cm), using H₂O–MeOH (4 : 1, 500 mL), (3 : 2, 500 mL), (2 : 3, 500 mL), and (1 : 4, 500 mL), and MeOH (500 mL), 100 mL-fractions being collected. The residue (903 mg) in fractions 13–16 was subjected to silica gel (250 g) CC with a linear gradient solvent system [CHCl₃–MeOH (9 : 1), 2 L→CHCl₃–MeOH (1 : 9), 2 L], 10-g fractions being collected. The residue (119 mg) in fractions 16–27 was subjected to DCCC and the residue (10.4 mg) in fractions 23–25 was purified by HPLC [Inertsil Phenyl, GL Science, 6.0 mm×250 mm, H₂O–MeOH (39 : 1), 1.6 mL/min] to give 2.3 mg of 3 from the peak at 11 min. The residue (84.3 mg) in fractions 28–33 was subjected to DCCC and the residue (16.8 mg) in fractions was purified by HPLC [Inertsil Phenyl, GL Science, 6.0 mm×250 mm, H₂O–MeOH (19 : 1), 1.6 mL/min] to give 6.9 mg of 2, 6.3 mg of 4, and 4.6 mg of 5 from the peaks at 16 min, 17 min, and 18 min, respectively. The residue (178 mg) in fractions 42–57 was subjected to DCCC to give 44.4 mg of 1 in fractions 20–22, and the residue (7.1 mg) in fractions 31–34 to HPLC [Inertsil ODS, GL Science, 10.0 mm×250 mm, H₂O–MeOH (7 : 3), 2.8 mL/min] to give 1.3 mg of 11 from the peak at 21 min. The residue (76.5 mg) in fractions 58–67 was subjected to DCCC, and that (19.6 mg) in fractions 61–82 was purified by HPLC [Inertsil ODS, 6.0 mm×250 mm, H₂O–MeOH (13 : 7), 1.6 mL/min] to give 1.3 mg of 6 and 1.1 mg of 7 from the peaks at 19 min and 20 min, respectively. The residue (88.4 mg) in fractions 68–81 was subjected to DCCC to give 8.0 mg of 10 in fractions 26–29. The residue (69.5 mg) in fractions 82–99 was subjected to DCCC to give 40.7 mg of 9 in fractions 71–88.

The residue (2.02 g) in fractions 17–21 obtained on Diaion HP-20 CC was separated by reversed-phase open CC with a linear gradient solvent system [H₂O–MeOH (9 : 1), 2 L→H₂O–MeOH (1 : 9), 2 L, 50 mm×25 cm], 10 g-fractions being collected. The residue (180 mg) in fractions 94–110 was subjected to DCCC to give the residue (26.3 mg) in fractions 57–75 which was purified by HPLC [Inertsil ODS, 6.0 mm×250 mm, H₂O–MeOH (3 : 2), 1.6 mL/min] to give 2.4 mg of 8 from the peak at 17 min.

Compound 1

Colorless needles, mp 216–217°C (MeOH), [α]_D²¹ −28.6 (c=0.27, MeOH); IR ν_max (film) cm⁻¹: 3364, 2931, 2865, 1650, 1457, 1235, 1076, 1029, 983, 831; ¹H-NMR (600 MHz, CD₃OD) δ: 5.89 (1H, ddd, J=17.3, 10.5, 7.1 Hz, H-2), 5.20 (1H, ddd, J=17.3, 1.8, 1.1 Hz, H-1a), 5.09 (1H, ddd, J=10.5, 1.8, 1.1 Hz, H-1b), 4.35 (1H, d, J=7.9 Hz, H-1′), 4.13 (1H, dd, J=9.9, 8.9 Hz, H-4′), 4.12 (1H, dt, J=7.1, 6.0 Hz, H-3), 3.83 (1H, dd, J=12.5, 2.4 Hz, H-6′a), 3.73 (1H, dd, J=12.5, 5.1 Hz, H-6′b), 3.64 (1H, dd, J=9.1, 8.9 Hz, H-3′), 3.34 (1H, ddd, J=9.9, 5.1, 2.4 Hz, H-5′), 3.29 (1H, dd, J=9.1, 7.9 Hz, H-2′), 1.69 (1H, m, H-7a), 1.52 (1H, m, H-7b), 1.26–1.41 (6H, m, H₂-4, 5, 6), 0.90 (3H, t, J=7.0 Hz, H₃-8); ¹³C-NMR (150 MHz, CD₃OD): Table 1; HR-ESI-MS (negative-ion mode) m/z: 369.1214 [M−H]⁻ (Calcd for C₁₄H₂₅O₉S: 369.1214).

Compound 2

Amorphous powder, [α]_D³² +19.7 (c=0.15, MeOH); IR ν_max (film) cm⁻¹: 3393, 2928, 1604, 1364, 1267, 1237, 1073, 1029, 985, 829; ¹H-NMR (600 MHz, CD₃OD) δ: 5.96 (1H, ddd, J=18.1, 11.3, 1.1 Hz, H-2), 5.03 (1H, dd, J=18.1, 0.8 Hz, H-1a), 4.95 (1H, dd, J=11.3, 0.8 Hz, H-1b), 4.36 (1H, d, J=7.9 Hz, H-1′), 4.09 (1H, dd, J=9.8, 8.9 Hz, H-4′), 3.88 (1H, dd, J=12.5, 2.2 Hz, H-6′a), 3.74 (1H, dd, J=12.5, 5.7 Hz, H-6′b), 3.63 (1H, dd, J=9.1, 8.9 Hz, H-3′), 3.41 (1H, dd, J=11.3, 4.2 Hz, H-6), 3.40 (1H, ddd, J=9.8, 5.7, 2.2 Hz, H-5′), 3.24 (1H, dd, J=9.1, 7.9 Hz, H-2′), 2.16 (1H, ddd, J=13.9, 4.0, 3.6 Hz, H-4a), 1.98 (1H, dddd, J=11.6, 4.2, 4.0, 1.1 Hz, H-5a), 1.80 (1H, dddd, J=13.8, 13.2, 11.3, 4.0 Hz, H-5b), 1.58 (1H, dddd, J=13.9, 13.8, 4.0, 1.1 Hz, H-4b), 1.25 (3H, s, H₃-9), 1.20 (3H, s, H₃-8), 1.11 (3H, s, H₃-10); ¹³C-NMR (150 MHz, CD₃OD): Table 1; HR-ESI-MS (negative-ion mode) m/z: 411.1323 [M−H]⁻ (Calcd for C₁₆H₂₈O₁₀S: 411.1319).

Compound 3

Amorphous powder, [α]_D²⁵ −24.2 (c=0.10, MeOH); IR ν_max (film) cm⁻¹: 3307, 2973, 1650, 1367, 1260, 1231, 1081, 1029, 984, 826; ¹H-NMR (600 MHz, CD₃OD) δ: 5.94 (1H, dd, J=18.0, 11.1 Hz, H-2), 5.07 (1H, dd, J=17.9, 1.1 Hz, H-1a), 4.95 (1H, dd, J=10.8, 1.1 Hz, H-1b), 4.35 (1H, d, J=7.9 Hz, H-1′), 4.11 (1H, dd, J=9.9, 9.0 Hz, H-4′), 3.89 (1H, dd, J=12.2, 2.2 Hz, H-6′a), 3.72 (1H, dd, J=12.2, 5.7 Hz, H-6′b), 3.67 (1H, dd, J=9.0, 9.0 Hz, H-3′), 3.59 (1H, dd, J=5.7, 2.6 Hz, H-6), 3.38 (1H, ddd, J=9.9, 5.7, 2.2 Hz, H-5′), 3.31 (1H, m, H-2′), 1.91 (2H, m, H-4a, 5a), 1.85 (1H, m, H-5b), 1.71 (1H, m, H-4b), 1.25 (3H, s, H₃-8), 1.24 (3H, s, H₃-9), 1.20 (3H, s, H₃-10), (600 MHz, pyridine-d₅) δ: 6.00 (1H, dd, J=17.8, 10.8 Hz, H-2), 5.24 (1H, dd, J=9.3, 9.3 Hz, H-3′), 5.06 (1H, m, H-1a), 4.94 (1H, m, H-1b), 4.78 (1H, d, J=7.8 Hz, H-1′), 4.51 (1H, dd, J=12.7, 3.5 Hz, H-6′a), 4.43 (1H, dd, J=9.5, 9.3 Hz, H-4′), 4.36 (1H, dd, J=12.7, 1.9 Hz, H-6′b), 3.96 (1H, dd, J=9.3, 7.8 Hz, H-2′), 3.80 (1H, ddd, J=9.5, 3.5, 1.9 Hz, H-5′), 3.71 (1H, dd, J=5.3, 3.8 Hz, H-6), 2.06 (1H, ddd, J=14.0, 9.6, 4.5 Hz, H-4a), 1.96 (2H, m, H₂-5), 1.68 (1H, m, H-4b), 1.37 (3H, s, H₃-9), 1.28 (3H, s, H₃-8), 1.15 (3H, s, H₃-10); ¹³C-NMR (150 MHz, CD₃OD and pyridine-d₅): Table 1; HR-ESI-MS (negative-ion mode) m/z: 411.1322 [M−H]⁻ (Calcd for C₁₆H₂₇O₁₀S: 411.1319).

Compound 4

Amorphous powder, [α]_D²⁰ −12.8 (c=0.40, MeOH); IR ν_max (film) cm⁻¹: 3303, 2976, 2942, 1650, 1457, 1370, 1231, 1082, 1034, 984, 819; ¹H-NMR (600 MHz, CD₃OD) δ: 6.00 (1H, dd, J=17.5, 10.8 Hz, H-2), 5.21 (1H, dd, J=17.5, 1.5 Hz, H-1a), 4.98 (1H, dd, J=10.8, 1.5 Hz, H-1b), 4.61 (1H, d, J=7.8 Hz, H-1′), 4.13 (1H, dd, J=9.8, 9.0 Hz, H-4′), 4.08 (1H, t, J=7.1 Hz, H-6), 3.84 (1H, dd, J=12.5, 2.4 Hz, H-6′a), 3.73 (1H, dd, J=12.5, 5.3 Hz, H-6′b), 3.68 (1H, dd, J=9.2, 9.0 Hz, H-3′), 3.39 (1H, ddd, J=9.8, 5.3, 2.4 Hz, H-5′), 3.25 (1H, dd, J=9.2, 7.8 Hz, H-2′), 2.00 (1H, m, H-5a), 1.89 (1H, m, H-4a), 1.87 (1H, m, H-5b), 1.81 (1H, m, H-4b), 1.29 (3H, s, H₃-10), 1.26 (3H, s, H₃-8), 1.23 (3H, s, H₃-9); ¹³C-NMR (150 MHz, CD₃OD): Table 1; HR-ESI-MS (negative-ion mode) m/z: 411.1327 [M–H]^– (Calcd for C₁₆H₂₇O₁₀S: 411.1319).

Compound 5

Amorphous powder, [α]_D²⁶ +3.23 (c=0.31, MeOH); IR ν_max (film) cm⁻¹: 3404, 2975, 1650, 1456, 1370, 1256, 1234, 1078, 1028, 985, 817; ¹H-NMR (600 MHz, CD₃OD) δ: 5.94 (1H, dd, J=17.3, 10.8 Hz, H-2), 5.22 (1H, dd, J=17.3, 1.5 Hz, H-1a), 4.95 (1H, dd, J=10.8, 1.5 Hz, H-1b), 4.55 (1H, d, J=7.9 Hz, H-1′), 4.13 (1H, dd, J=9.9, 9.0 Hz, H-4′), 4.01 (1H, t, J=7.0 Hz, H-6), 3.84 (1H, dd, J=12.2, 2.2 Hz, H-6′a), 3.73 (1H, dd, J=12.2, 5.3 Hz, H-6′b), 3.68 (1H, dd, J=9.2, 9.0 Hz, H-3′), 3.39 (1H, ddd, J=9.9, 5.3, 2.2 Hz, H-5′), 3.25 (1H, dd, J=9.2, 7.9 Hz, H-2′), 1.92 (1H, m, H-5a), 1.91 (1H, m, H-4a), 1.84 (1H, m, H-5b), 1.74 (1H, m, H-4b), 1.33 (3H, s, H₃-10), 1.25 (3H, s, H₃-9), 1.22 (3H, s, H₃-8), (600 MHz, pyridine-d₅) δ: 5.90 (1H, dd, J=17.4, 10.6 Hz, H-2), 5.28 (1H, dd, J=17.4, 1.7 Hz, H-1a), 5.18 (1H, dd, J=9.1, 8.9 Hz, H-3′), 5.00 (1H, m, H-1b), 4.98 (1H, m, H-1′), 4.47 (1H, dd, J=12.7, 2.1 Hz, H-6′a), 4.36 (1H, dd, J=9.1, 8.7 Hz, H-4′), 4.05 (1H, dd, J=7.2, 6.8 Hz, H-6), 3.74 (1H, m, H-5′), 3.73 (1H, br d, J=12.7 Hz, H-6′b), 3.98 (1H, dd, J=8.9 7.9 Hz, H-2′), 1.90 (1H, m, H-5a), 1.85 (1H, m, H-5b), 1.74 (1H, m, H-4a), 1.57 (1H, m, H-4b), 1.42 (3H, s, H₃-9), 1.30 (3H, s, H₃-10), 1.33 (3H, s, H₃-8); ¹³C-NMR (150 MHz, CD₃OD and pyridine-d₅): Table 1; HR-ESI-MS (negative-ion mode) m/z: 411.1325 [M−H]⁻ (Calcd for C₁₆H₂₇O₁₀S: 411.1319).

Compound 6

Amorphous powder, [α]_D²¹ −10.9 (c=0.43, MeOH); IR ν_max (film) cm⁻¹: 3383, 2972, 2934, 2878, 1456, 1368, 1137, 1066, 1031, 980; ¹H-NMR (600 MHz, CD₃OD): Table 2; ¹³C-NMR (150 MHz, CD₃OD): Table 2; HR-ESI-MS (positive-ion mode) m/z: 267.1560 [M+H]⁺ (Calcd for C₁₃H₂₄O₄Na: 267.1566).

Compound 7

Amorphous powder, [α]_D²⁰ −12.7 (c=0.07, MeOH); IR ν_max (film) cm⁻¹: 3363, 2966, 1651, 1457, 1392, 1127, 1078. 1044, 980; ¹H-NMR (600 MHz, CD₃OD): Table 2; ¹³C-NMR (150 MHz, CD₃OD): Table 2; HR-ESI-MS (negative-ion mode) m/z: 267.1567 [M+H]⁺ (Calcd for C₁₃H₂₄O₄Na: 267.1566).

Sugar Analysis

About 500 µg each of compounds 1–5 was hydrolyzed with 1 M HCl (0.1 mL) at 90°C for 2 h. The reaction mixtures were partitioned with an equal amount of EtOAc (0.1 mL), and the water layers were analyzed with a chiral detector (JASCO OR-2090plus) on an amino column [Asahipak NH₂P-5 4E; CH₃CN–H₂O, 4 : 1; flow rate 1 mL/min]. Compounds 1–5 gave a peak for D-glucose at the retention times of 13.7 min with a positive optical rotation sign. The peak was identified by co-chromatography with authentic D-glucose.

Catalytic Reduction of 1 to 1b

Compound 1 (9.2 mg) was reduced with PtO₂ (1 mg) and H₂ in 1 mL of MeOH for 2 h. The catalyst was removed by filtration and the resulting residue was purified by HPLC [Cosmosil π-NAP (Nacalai Tesque, Kyoto, Japan), 10 mm×250 mm, H₂O–MeOH (3 : 2), 2.8 mL/min, RI detector] to give 2.1 mg of 1 and 2.1 mg of 1,2-dihydro derivative of 1 (=1b) from the peaks at 14 min and 16 min, respectively. 1b: Amorphous powder, [α]_D²⁷ −7.2 (c=0.14, MeOH); ¹H-NMR (600 MHz, CD₃OD) δ: 4.34 (1H, d, J=7.9 Hz, H-1′), 4.11 (1H, dd, J=9.8, 9.1 Hz, H-4′), 3.87 (1H, dd, J=12.5, 1.9 Hz, H-6′a), 3.74 (1H, dd, J=12.5, 5.7 Hz, H-6′b), 3.65 (1H, dd, J=9.3, 9.1 Hz, H-3′), 3.63 (1H, quintet-like, J=5.7 Hz, H-3), 3.37 (1H, ddd, J=9.8, 5.7, 1.9 Hz, H-5′), 3.26 (1H, dd, J=9.3, 7.9 Hz, H-2′), 1.50 (1H, m, H-4a), 1.47 (1H, m, H-4b), 1.42 (1H, m, H-5a), 1.36 (1H, m, H-5b), 1.33 (1H, m, H-7a), 1.31 (1H, m, H-7b), 1.29 (2H, m, H2-6), 0.90 (3H, t, J=7.0 Hz, H₃-8); ¹³C-NMR (150 MHz, CD₃OD): Table 1; HR-ESI-MS (negative-ion mode) m/z: 371.1375 [M−H]⁻ (Calcd for C₁₄H₂₇O₉S: 371.1370).

Preparation of (R)- and (S)-MTPA Diesters (6a, b) from 6

A solution of 6 (0.4 mg) in 1 mL of dehydrated CH₂Cl₂ was reacted with (R)-MTPA (10.0 mg) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (10.0 mg) and N,N′-dimethyl-4-aminopyridine (4-DMAP) (10 mg), the mixture being occasionally stirred at 37°C for 12 h. After the addition of 1 mL of CH₂Cl₂, the solution was washed with H₂O (1 mL), 1 M HCl (1 mL), NaHCO₃-saturated H₂O (1 mL), and then brine (1 mL), successively. The organic layer was dried over Na₂SO₄ and then evaporated under reduced pressure. The residue was purified by preparative TLC [silica gel (0.25 mm thickness), being applied for 9 cm, developed with CHCl₃–(CH₃)₂CO (19 : 1) for 9 cm, and then eluted with CHCl₃–MeOH (5 : 1)] to furnish an ester, 6a (0.3 mg). Through a similar procedure, 6b (0.4 mg) was prepared from 6 (0.6 mg) using (S)-MTPA (10.0 mg), EDC (10.0 mg), and 4-DMAP (10.0 mg). 6a: Amorphous powder, ¹H-NMR (600 MHz, CDCl₃) δ: 7.56–7.33 (10H, aromatic protons), 5.77 (1H, m, H-7), 5.19 (1H, m, H-9), 5.06 (1H, m, H-3), 3.74 (1H, dd, J=10.2, 4.2 Hz, H-2), 3.58 (3H, s, –OCH₃), 3.52 (3H, s, –OCH₃), 1.97 (1H, m, H-4a), 1.64 (1H, m, H-4b), 1.39 (3H, d, J=6.4 Hz, H₃-10), 0.93 (3H, s, H₃-11), 0.90 (3H, s, H₃-12), 0.78 (3H, d, J=6.4 Hz, H₃-13); HR-ESI-MS (positive-ion mode) m/z: 699.2365 [M+Na]⁺ (Calcd for C₃₃H₃₈O₈F₆Na: 699.2363). 6b: Amorphous powder, ¹H-NMR (600 MHz, CDCl₃) δ: 7.51–7.31 (10H, aromatic protons), 5.62 (2H, m, H-7, 8), 5.19 (1H, m, H-9), 5.06 (1H, m, H-3), 3.77 (1H, dd, J=10.2, 5.4 Hz, H-2), 3.58 (3H, s, –OCH₃), 3.52 (3H, s, –OCH₃), 1.89 (1H, m, H-4a), 1.53 (1H, m, H-4b), 1.44 (3H, d, J=6.4 Hz, H₃-10), 0.98 (3H, s, H₃-11), 0.94 (3H, s, H₃-12), 0.72 (3H, d, J=6.7 Hz, H₃-13); HR-ESI-MS (positive-ion mode) m/z: 699.2366 [M+Na]⁺ (Calcd for C₃₃H₃₈O₈F₆Na: 699.2363).

Preparation of (R)- and (S)-MTPA Diesters (7a, b) from 7

Through a similar procedure to that used for 6, 7a and b (0.03, 0.02 mg, respectively) were prepared from 7 (0.6, 0.5 mg, respectively) using (R)- and (S)-MTPAs (20.0 mg, respectively), EDC (20.0 mg, respectively), and 4-DMAP (20.0 mg, respectively). 7a: Amorphous powder, ¹H-NMR (600 MHz, CDCl₃) δ: 7.65–7.37 (10H, aromatic protons), 5.76 (1H, dd, J=15.2, 10.2 Hz, H-7), 5.67 (1H, dd, J=15.2, 7.1 Hz, H-8), 5.58 (1H, dq, J=7.1, 6.4 Hz, H-9), 4.93 (1H, d, J=10.2 Hz, H-4), 3.78 (1H, m, H-3), 3.61 (3H, s, –OCH₃), 3.52 (3H, s, –OCH₃), 2.02 (1H, d, J=10.2 Hz, H-6), 1.92 (1H, dd, J=13.3, 4.5 Hz, H-2a), 1.45 (1H, t-like, J=12.4 Hz, H-2b); 1.39 (3H, d, J=6.4 Hz, H₃-10), 1.03 (3H, s, H₃-11), 0.94 (3H, s, H₃-13), 0.84 (3H, s, H₃-12); HR-ESI-MS (positive-ion mode) m/z: 699.2368 [M+Na]⁺ (Calcd for C₃₃H₃₈O₈F₆Na: 699.2363). 7b: Amorphous powder, ¹H-NMR (600 MHz, CDCl₃) δ: 7.64–7.29 (10H, aromatic protons), 5.71 (1H, dd, J=14.5, 9.8 Hz, H-7), 5.62 (1H, dd, J=14.5, 6.1 Hz, H-8), 5.61 (1H, m, H-9), 4.93 (1H, d, J=10.1 Hz, H-6), 3.71 (1H, m, H-3), 3.62 (3H, s, –OCH₃), 3.56 (3H, s, –OCH₃), 1.98 (1H, d, J=10.1 Hz, H-6), 1.88 (1H, dd, J=13.0, 4.7 Hz, H-2a), 1.45 (1H, m, H-2b), 1.45 (3H, d, J=6.2 Hz, H₃-10), 1.13 (3H, s, H₃-13), 0.89 (3H, s, H₃-11), 0.85 (3H, s, H₃-12); HR-ESI-MS (positive-ion mode) m/z: 699.2370 [M+Na]⁺ (Calcd for C₃₃H₃₈O₈F₆Na: 699.2363).

Acknowledgment

The authors are grateful for access to the superconducting NMR instrument (Bruker Avance-III 600) at the Analytical Center of Molecular Medicine of the Hiroshima University Faculty of Medicine, and an Applied Biosystem 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 Japan Society for the Promotion of Science (15H04651), and the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of Interest

The authors declare no conflict of interest.

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