C21 Steroidal Glycosides from the Roots of Oxypetalum caeruleum

Abstract

The MeOH extract from dried roots of Oxypetalum caeruleum (Apocynaceae) plants yielded seventeen new pregnane glycosides, some of which had the acylated-ramanone or -isoramanone type aglycone. The structures of these compounds were established using NMR, MS spectroscopic analysis and chemical evidence.

Introduction

In the course of researching phytochemicals, we have reported the isolation and structural determination of pregnane glycosides in Asclepiadaceae plants. This family was recently included in the Apocynaceae, and the genus Oxypetalum was also classified to Apocynaceae. O. caeruleum is indigenous to South America and is distributed as a garden plant in Japan. Previous research on the constituents of the aerial parts of this plant afforded a few pregnanes and their glycosides with tetracyclic triterpenoids and lignanes.¹⁾ As pregnane glycosides were characteristic of the Apocynaceaus (Asclepiadaceaus) family, we carried out an investigation of these compounds in the roots of O. caeruleum, and found seventeen new pregnane glycosides. The present paper describes their isolation and structural determination.

Results and Discussion

A MeOH extract from the dried roots of O. caeruleum plants was suspended in water, and this suspension was extracted with diethylether and partitioned into an ether-soluble fraction and a water-soluble fraction. The residue of the ether-soluble fraction was subjected to silica gel column chromatography and semi-preparative HPLC to give seventeen pregnane glycosides.

To acquire the component aglycones and sugars, the fraction containing pregnane glycosides from chromatography was subjected to acid hydrolysis, affording four new (acylated) pregnanes (2a, 3a, 6a, and 13a) and one presumed known compound, 12-O-benzoylramanone (1a).²⁾

The acylated aglycones 2a and 3a were suggested to have the molecular formula C₂₇H₄₀O₅ and C₂₈H₄₂O₅, based on high resolution (HR)-electrospray ionization (ESI)-MS [2a; m/z: 467.2766 [M + Na]⁺, 3a; m/z: 481.2924 [M + Na]⁺]. By comparing the ¹³C-NMR spectroscopic data of 2a and 3a with those of 1a, these aglycones were presumed to be 12-O-acylated-ramanones. The ¹H-NMR spectrum of the ester moiety in 2a displayed signals for two singlet methyl groups [δ_H 1.74 (3H, br s), 1.63 (3H, br s)], one olefinic proton [δ_H 5.27 (1H, br t, J = 7.0 Hz)], and two methylene protons [δ_H 2.91 (2H, br d, J = 7.0 Hz)]. The ¹³C-NMR spectrum of the ester moiety in 2a showed six carbon signals, which were classified as one carbonyl carbon (δ_C 171.2), two olefinic carbons (δ_C 135.4, 115.6), one sp³ methylene (δ_C 33.4), and two sp³ methyls (δ_C 25.7, 18.0) based on the result of the distortionless enhancement by polarization transfer (DEPT) experiment. On the other hand, the ¹H-NMR spectrum of the ester moiety in 3a exhibited signals for three methyl groups [δ_H 2.13 (3H, d, J = 1.0 Hz), 1.06 (6H, d, J = 7.0 Hz)], one olefinic proton [δ_H 5.50 (1H, br s)], and one methine proton [δ_H 2.36 (overlapping)]. The ¹³C-NMR spectrum of the ester moiety in 3a showed seven carbon signals, which included one carbonyl carbon (δ_C 165.6), two olefinic carbons (δ_C 166.5, 112.9), one sp³ methine (δ_C 38.1), and three sp³ methyls (δ_C 21.0, 20.8, 16.6) in consideration of the results of the DEPT experiment. Because these NMR spectroscopic data were in good agreement with those of the ester moieties of umbelloside IV³⁾ and cynanchogenin,⁴⁾ the ester moieties of 2a and 3a were concluded to be 4-methyl-3-pentenoyl and ikemaoyl groups, respectively. Thus, the structures of 2a and 3a were elucidated as presented in Chart 1.

Chart 1. Structures of Compounds 1–17

The molecular formula of the acylated aglycone, 13a was deduced to be C₃₀H₃₈O₅ on the basis of HR-ESI-MS. The ¹³C- and ¹H-NMR spectra of 13a were similar to those of 1a, but the C-20 carbonyl carbon signal was observed at δ_C 217.1, together with signals due to the (trans)-cinnamoyl group (δ_H 7.74, 7.57 × 2, 7.42 × 3, 6.50, and δ_C 166.8, 145.5, 134.1, 130.6, 129.0 × 2, 128.2 × 2, 117.8). This δ value of C-20 suggested that this carbonyl group existed at the 17β-side, and that the aglycone was isoramanone.^2,5) Thus, this acylated aglycone was determined to be 12-O-(E)-cinnamoylisoramanone.

The aglycone 6a had the molecular formula C₂₁H₃₄O₄, according to the results of HR-ESI-MS. In the ¹H- and ¹³C-NMR spectrum of 6a, the signals for the ester moiety disappeared, and one more carbonyl signal was observed at δ_C 211.9 instead of the one oxygenated methine carbon signal seen in 1a, 2a, 3a and 13a. Observations of the ³J_CCCHs between this carbonyl signal and H-18 (δ_H 1.19)/H-9 (δ_H 1.49) in a ¹H-detected hetero-nuclear multiple-bond connectivity (HMBC) experiment suggested that this signal was at the C-12 position (see Chart 2). In the ¹H-NMR spectrum, the H-17 signal [δ_H 4.29 (1H, dd, J = 10.0, 4.0 Hz)] was shifted down-field, affected by the C-12 carbonyl group, and its multiplicity and J value were almost consistent with those of 13a. Moreover, a rotating frame nuclear Overhauser effect correlation spectroscopy (ROESY) experiment displayed a ROE between H-18 and H-21 (δ_Η 2.30) (see Chart 2). This evidence suggested that H-17 possessed the α-orientation. Therefore, the structure of 6a was determined as shown in Chart 1, and it was named oxypetalumone.

Chart 2. Observed Key ¹H–¹H COSY, HMBC, and ROE Correlations for Compound 6a

The acquired sugar mixtures were fractionated to cymarose and oleandrose using silica gel chromatography. The absolute configurations of these sugars were believed to be D-forms on the basis of the optical rotation values.^6,7)

The sugar sequences of compounds 4–7, 9, 11–13 and 15 were identified as shown in Chart 1. They were previously determined to be pregnane glycosides, consistent with their NMR spectroscopic data in the literature.^8–13) The (acylated) aglycone of each compound except for 7 was confirmed by HPLC analysis following acid hydrolysis.

Oxypetalumoside A₁ (1) was considered to have the molecular formula C₄₂H₆₀O₁₁, based on HR-ESI-MS. The ¹H- and ¹³C-NMR spectra showed two anomeric proton and carbon signals at δ_H 4.86, 4.50 and δ_C 95.9, 101.4, with signals due to the acylated aglycone identified as 1a by acid hydrolysis. A comparison of the ¹³C-NMR spectroscopic data of 1 with those of 1a established glycosylation shifts at the C-2, C-3, and C-4 positions [C-2 (−1.9 ppm), C-3 (+6.1 ppm), C-4 (−3.2 ppm)].¹⁴⁾ Thus, 1 was glycosylated at C-3, and deduced to be 12-O-benzoylramanone 3-O-diglycoside. Acid hydrolysis of 1 afforded cymarose and oleandrose as the component sugars, and the J values of each anomeric proton (J = 9.5, 2.0 Hz) in the ¹H-NMR spectrum indicated that these sugars had one β-D-cymaropyranosyl group and one β-D-oleandropyranosyl group. The ¹³C- and ¹H-NMR signal assignments for the sugar moiety (see Table 3 and Experimental) were based on the results of two-dimensional (2D)-NMR [¹H–¹H shift correlation spectroscopy (COSY) and ¹H-detected heteronuclear single-quantum correlation spectroscopy (HSQC)] measurement. The sugar sequence was determined from the ROESY measurement. ROEs were observed between δ_H 4.86 (H-1′ of β-D-cymaropyranose) and 3.55 (H-3 of the aglycone) and δ_H 4.50 (H-1″ of β-D-oleandropyranose) and 3.24 (H-4′ of β-D-cymaropyranose). Moreover, in the HMBC experiment, ³J_COCHs were observed between δ_C 77.4 (C-3 of the aglycone) and δ_H 4.86 (H-1′ of β-D-cymaropyranose), δ_C 95.9 (C-1′ of β-D-cymaropyranose) and δ_H 3.55 (H-3 of the aglycone), δ_C 82.7 (C-4′ of β-D-cymaropyranose) and δ_H 4.50 (H-1″ of β-D-oleandropyranose), and δ_C 101.4 (C-1″ of β-D-oleandropyranose) and δ_H 3.24 (H-4′ of β-D-cymaropyranose). Thus, 1 was established to be 12-O-benzoylramanone 3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranoside. Compounds 2–17 were also glycosylated at the C-3 position of each aglycone on the basis of glycosylation shifts in their ¹³C-NMR spectra.

Table 1. ¹³C-NMR Spectroscopic Data for the Aglycone and Ester Moieties of Compounds 1–3, 6, 7 and 13

Carbon No.	1	2	3	6	7	13
Aglycone moiety
C-1	37.1	37.0	37.1	37.0	37.0	37.2
-2	29.4	29.4	29.4	29.4	29.4	29.4
-3	77.4	77.4	77.4	77.3	77.4	77.5
-4	38.6	38.6	38.6	38.7	38.6	38.6
-5	139.7	139.7	139.7	138.7	139.5	138.8
-6	121.1	121.0	121.0	121.9	121.2	122.1
-7	26.4	26.4	26.4	27.6	26.2	27.3
-8	37.0	37.0	37.0	35.4	36.7	35.6
-9	42.8	42.8	42.9	44.7	42.7	43.0
-10	37.1	37.0	37.0	37.3	37.0	37.1
-11	26.4	26.1	26.4	36.4	27.1	26.2
-12	72.2	71.9	70.7	211.9	72.3	75.9
-13	54.2	53.7	53.8	62.7	51.1	53.7
-14	87.4	87.4	87.5	86.3	87.1	85.7
-15	31.5	31.3	31.4	33.6	30.5	34.4
-16	20.7	20.3	20.4	24.2	22.8	24.4
-17	59.6	59.6	59.9	52.0	51.7	57.1
-18	13.8	13.6	13.8	15.3	13.7	9.8
-19	19.4	19.3	19.4	19.1	19.4	19.3
-20	209.4	209.4	209.0	218.1	72.3	217.2
-21	31.7	31.9	31.5	33.4	26.0	33.2
Ester moiety
C-α	165.0	—	—	—	165.9	166.7
-β	—	—	—	—	—	117.7
-γ	—	—	—	—	—	145.5
-1′	130.1	171.1	165.5	—	131.0	134.1
-2′	129.5	33.3	112.9	—	129.4	128.2
-3′	128.3	115.6	166.4	—	128.2	129.0
-4′	132.9	135.3	38.1	—	132.9	130.6
-5′	128.3	25.7	20.9a)	—	128.2	129.0
-6′	129.5	18.0	20.8a)	—	129.4	128.2
-7′	—	—	16.6	—	—	—
-1″	—	—	—	—	169.5	—
-2″	—	—	—	—	21.4	—

Measured in CDCl₃ at 175 MHz. a) Interchangeable in each column.

Table 2. ¹H-NMR Spectroscopic Data for the Aglycone and Ester Moieties of Compounds 1–3, 6, 7 and 13

Proton No.	1	2	3	6	7	13
Aglycone moiety
H-1	1.81 (1H, dt, 13.5, 4.0)	1.80*	1.82 (1H, dt, 13.5, 4.0)	1.72 (1H, dt, 13.5, 4.0)	1.79 (1H, dt, 13.5, 4.0)	1.84*
	1.12 (1H, td, 13.5, 4.0)	1.08 (1H, td, 13.5, 4.0)	1.09 (1H, td, 13.5, 4.0)	1.06 (1H, td, 13.5, 4.0)	1.08 (1H, td, 13.5, 4.0)	1.10 (1H, td, 13.5, 4.0)
-2	1.94*	1.93*	1.93*	1.94 (1H, m)	1.90*	1.93*
	1.54*	1.54*	1.53*	1.54*	1.52*	1.55*
-3	3.55 (1H, m)	3.53 (1H, m)	3.55 (1H, m)	3.51 (1H, m)	3.54 (1H, m)	3.54 (1H, m)
-4	2.39*	2.38 (1H, m)	2.36*	2.38*	2.37 (1H, m)	2.34*
	2.21*	2.18*a)	2.19*	2.20 (1H, m)	2.20*	2.21*
-6	5.43 (1H, br s)	5.40 (1H, br s)	5.41 (1H, br s)	5.43 (1H, br s)	5.42 (1H, br s)	5.42 (1H, br s)
-7	2.22*	2.19*a)	2.19*	2.40*	2.22*	2.33*
	1.86 (1H, m)	1.82*	1.84*	1.88*	1.83*	1.81*
-8	1.74 (1H, td, 11.5, 5.5)	1.68 (1H, td, 11.0, 5.0)	1.69*	2.16 (1H, ddd, 12.5, 10.5, 5.0)	1.70 (1H, ddd, 12.5, 10.5, 5.0)	1.81*
-9	1.31*	1.26*	1.25 (1H, td, 12.0, 3.5)	1.45 (1H, td, 12.5, 5.0)	1.27 (1H, td, 12.5, 5.0)	1.29*
-11	1.94*	1.82*	1.84*	2.57 (1H, t, 12.5)	1.84*	1.81*
	1.44 (1H, q, 12.0)	1.27*	1.30*	2.30*	1.50*	1.55*
-12	4.91 (1H, dd, 12.0, 4.5)	4.58 (1H, dd, 11.0, 4.0)	4.59 (1H, dd, 11.5, 4.5)	—	5.05 (1H, dd, 11.5, 4.5)	4.74 (1H, dd, 12.0, 4.5)
-15	2.01 (1H, m)	1.93*	1.92*	1.77*	1.88*	2.01*
	1.63 (1H, m)	1.57*	1.57*	1.39*	1.62*	1.88*
-16	2.36*	2.32*	2.36*	1.85*	1.87*	2.03*
	1.77*	1.71*	1.72*	1.77*	1.48*	1.98*
-17	3.25 (1H, t, 9.0)	3.21 (1H, t, 9.0)	3.17 (1H, t, 9.0)	4.27 (1H, dd, 10.0, 4.0)	2.49 (1H, q, 9.5)	3.17*
-18	1.51 (3H, s)	1.36 (3H, s)	1.39 (3H, s)	1.16 (3H, s)	1.36 (3H, s)	1.09 (3H, s)
-19	0.98 (3H, s)	0.97 (3H, s)	0.97 (3H, s)	1.04 (3H, s)	0.98 (3H, s)	0.99 (3H, s)
-20	—	—	—	—	4.65 (1H, br s)	—
-21	2.04 (3H, s)	2.21 (3H, s)	2.16 (3H, s)	2.27 (3H, s)	1.11 (3H, br d, 6.0)	2.21 (3H, s)
Ester moiety
-β	—	—	—	—	—	6.48 (1H, d, 16.0)
-γ	—	—	—	—	—	7.73 (1H, d, 16.0)
-2′	7.96 (1H, br d, 8.0)	2.90 (2H, br d, 7.5)	5.49 (1H, br s)	—	7.97 (1H, br d, 8.0)	7.56 (1H, m)
-3′	7.43 (1H, br t, 8.0)	5.26 (1H, br t, 7.5)	—	—	7.43 (1H, br t, 8.0)	7.42*
-4′	7.55 (1H, br t, 8.0)	—	2.35*	—	7.56 (1H, br t, 8.0)	7.42*
-5′	7.43 (1H, br t, 8.0)	1.74 (3H, br s)	1.06 (3H, d, 7.0)	—	7.43 (1H, br t, 8.0)	7.42*
-6′	7.96 (1H, br d, 8.0)	1.63 (3H, br s)	1.06 (3H, d, 7.0)	—	7.97 (1H, br d, 8.0)	7.56 (1H, m)
-7′	—	—	2.12 (3H, d, 1.0)	—	—	—
-2″	—	—	—	—	1.74 (3H, s)	—

Measured in CDCl₃ at 700 MHz.*: Overlapping with other signals. a) Interchangeable in each column.

Table 3. ¹³C-NMR Spectroscopic Data for the Sugar Moieties of Compounds 1, 4, 6, 8–12, 14–16 and 17

Carbon No.	1	4	6	8	9	10	11	12	14	15	16	17
	Cym	Cym	Cym	Cym	Cym	Cym	Cym	Ole	Ole	Ole	Ole	Ole
C-1′	95.9	95.9	95.9	95.9	95.9	95.9	95.9	97.7	97.6	97.6	97.7	97.5
-2′	35.6	35.5a)	35.5a)	35.4a)	35.6	35.6	35.7	36.4a)	36.5a)	36.5	36.5a)	36.5a)
-3′	77.0	77.0b)	77.0b)	77.0b)	77.0	76.9	77.0	78.9	79.3b)	79.3a)	79.3	79.3b)
-4′	82.7	82.5c)	82.5c)	82.4	82.7	82.6	82.7	82.2	82.6c)	82.5b)	82.4	82.5c)
-5′	68.4	68.5	68.5	68.5	68.4	68.4	68.4	71.1	71.1d)	70.9	70.9	70.9
-6′	18.2	18.2d)	18.2	18.2	18.2	18.2	18.2	18.4b)	18.4	18.4	18.4	18.4
	Ole	Cym	Cym	Cym	Ole	Ole	Ole	Cym	Ole	Ole	Ole	Ole
C-1″	101.4	99.7	99.6	99.6	101.3	101.3	101.3	98.4	100.2	100.2	100.1	100.1
-2″	35.3	35.4a)	35.4a)	35.5a)	36.2	36.4	36.4a)	35.4	36.4a)	36.5	36.6a)	36.4a)
-3″	80.6	76.9b)	76.9b)	76.9b)	79.0	79.1	78.9b)	77.0	79.2b)	79.2a)	79.3	79.2b)
-4″	75.3	82.6c)	82.4c)	82.6	82.3a)	82.5	82.3c)	82.6	82.5c)	82.6b)	82.7	82.6c)
-5″	71.5	68.3	68.2	68.3	70.9b)	71.0	70.9	68.7	71.0d)	71.1c)	71.1	71.1d)
-6″	18.0	18.3d)	18.2	18.2	18.4	18.3	18.4d)	18.2	18.4	18.4	18.4	18.4
	—	Ole	Ole	Can	Ole	Can	Ole	Ole	Ole	Ole	Can	Ole
C-1‴	—	101.4	101.3	101.4	100.1	100.4	100.2	101.3	100.2	100.2	100.4	100.2
-2‴	—	35.3a)	36.2	38.3	36.4	38.5	36.3a)	36.2a)	36.4a)	36.5	38.1	36.4a)
-3‴	—	80.6	79.1	69.4	79.2	69.5	79.1b)	79.1	79.2b)	78.9	69.5	78.9
-4‴	—	75.3	82.2	88.3	82.5a)	88.4	82.4c)	82.2	82.5c)	82.3	88.1	82.3
-5‴	—	71.5	70.9	70.2	71.1b)	70.4	71.2	70.9	70.9d)	71.3c)	70.6	71.2d)
-6‴	—	18.0	18.4	17.9	18.4	17.9	18.4d)	18.3b)	18.4	18.3	17.9	18.4
	—	—	Ole	Ole	Ole	Ole	Cym	Ole	Ole	Cym	Cym	Cym
C-1′‴	—	—	100.2	100.9	100.3	101.0	98.3	100.2	100.3	98.5	99.3	98.5
-2′‴	—	—	35.3a)	35.1	35.4	35.1	33.8	35.3	35.4	35.3	35.3	35.4
-3′‴	—	—	80.6	80.3	80.7	80.3	77.5	80.7	80.7	76.9	76.7	77.0
-4′‴	—	—	75.4	75.0	75.4	75.0	72.4	75.4	75.4	82.6b)	82.2	82.6c)
-5′‴	—	—	71.6	72.0	71.6	72.0	71.0	71.6	71.6	68.7	69.0	68.6
-6′‴	—	—	18.0	17.6	18.0	17.6	18.3d)	18.0	18.0	18.3	17.9	18.2
	—	—	—	—	—	—	—	—	—	Ole	Ole	Ole
C-1″‴	—	—	—	—	—	—	—	—	—	101.5	101.5	101.3
-2″‴	—	—	—	—	—	—	—	—	—	35.2	35.2	36.2
-3″‴	—	—	—	—	—	—	—	—	—	80.6	80.5	79.1
-4″‴	—	—	—	—	—	—	—	—	—	75.4	75.3	82.2
-5″‴	—	—	—	—	—	—	—	—	—	71.5	71.6	70.9
-6″‴	—	—	—	—	—	—	—	—	—	18.0	18.0	18.3
	—	—	—	—	—	—	—	—	—	—	—	Ole
C-1‴‴	—	—	—	—	—	—	—	—	—	—	—	100.2
-2‴‴	—	—	—	—	—	—	—	—	—	—	—	35.3
-3‴‴	—	—	—	—	—	—	—	—	—	—	—	80.7
-4‴‴	—	—	—	—	—	—	—	—	—	—	—	75.4
-5‴‴	—	—	—	—	—	—	—	—	—	—	—	71.6
-6‴‴	—	—	—	—	—	—	—	—	—	—	—	18.0
OMes	58.1	58.1	58.1	58.2	58.1	58.0	58.1	57.9	56.7 × 2	57.9	58.4	57.9
	56.3	57.9	57.9	57.9	56.7	56.8	57.1	56.6	56.5	56.8	57.0	56.8
		56.3	56.6	56.5	56.6	56.5	56.7	56.4 × 2	56.4	56.6	56.5	56.6 × 2
			56.3		56.4		56.6			56.5	56.3	56.5
										56.3		56.3

Cym: D-cymaropyranose, Ole: D-oleandropyranose, Can: D-canaropyranose Measured in CDCl₃ at 175 MHz. a)–d) Interchangeable in each column.

The molecular formulae of oxypetalumosides B₁ (2) and C₁ (3) were considered to be C₄₁H₆₄O₁₁ and C₄₂H₆₆O₁₁ based on HR-ESI-MS. Acid hydrolysis afforded 2a from 2 and 3a from 3 with cymarose and oleandrose. Because the ¹H- and ¹³C-NMR spectroscopic data of their sugar moieties were consistent with those of 1, they had the same sugar sequence as 1. Thus, 2 and 3 were determined to be 12-O-(4-methyl-3-pentenoyl)ramanone 3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranoside and 12-O-ikemaoylramanone 3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranoside, respectively.

HR-ESI-MS showed the molecular formula of oxypetalumoside E₃ (7) to be C₅₈H₈₈O₁₈, and it was presumed to be diacylated-pregnane 3-O-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside. Based on the ¹H- and ¹³C-NMR spectroscopic data of the aglycone moiety of 7, the oxygenated methine proton and carbon signals were observed at δ_H 4.65 and δ_C 72.3, instead of the carbonyl carbon signal, together with the signals due to the benzoyl (δ_H 7.97 × 2, 7.43 × 2, 7.56 and δ_C 165.9, 132.9, 131.0, 129.4 × 2, 128.2 × 2) and acetyl groups (δ_H 1.74 and δ_C 169.5, 21.4). Because the H-21 signal was shown at δ_H 1.11 as the doublet methyl signal (J = 6.0 Hz), these oxygenated methine proton and carbon signals were assigned at the H-20 and C-20 positions, which was supported by the ¹H–¹H COSY and HMBC experiments (see Chart 3). In the ROESY measurement, ROEs were shown between H-17(δ_H 2.49) and H-18 (δ_H 1.36), H-12 (δ_H 5.05) and H-20, and H-21 and H-16 (δ_H 1.87) (see Chart 3). These results suggested that H-17 existed at the β-side and C-20 possessed the S-configuration. The linked position of the ester moieties was confirmed by the results of the HMBC experiment [a ³J_COCH between C-α (δ_C 165.9) and H-12 (δ_H 5.05)] and/or the acylation shifts for the H-12 and H-20 signals (see Chart 3 and Table 2). Therefore, this di-acylated aglycone was elucidated as 12-O-benzoyl-20-O-acetyl-17-epi-baucerigenin,³⁾ and the structure of 7 is presented in Chart 1.

Chart 3. Observed Key ¹H–¹H COSY, HMBC, and ROE Correlations for the Aglycone and Ester Moieties of Compound 7

Based on HR-ESI-MS, the molecular formulae of oxypetalumosides D₄ (8) and D₆ (10) were considered to be C₄₈H₇₆O₁₆, being smaller by one CH₂ unit than those of 6 and 9. The NMR spectra and acid hydrolysis suggested that both compounds were oxypetalumone 3-O-tetraglycosides. The sugar moieties of 8 consisted of two β-D-cymaropyranosyl groups, one β-D-canaropyranosyl group, and a terminal β-D-oleandropyranosyl group, while those of 10 consisted of one β-D-cymaropyranosyl group, one inner β-D-oleandropyranosyl group, one β-D-canaropyranosyl group, and a terminal β-D-oleandropyranosyl group. Comparing the δ values for the anomeric carbon and proton signals of the terminal β-D-oleandropyranosyl (δ_C 100.9 and δ_H 4.48 in 8; δ_C 101.0 and δ_H 4.49 in 10) and the β-D-canaropyranosyl groups (δ_C 101.4 and δ_H 4.51 in 8; δ_C 100.4 and δ_H 4.71 in 10) with those of curassaviosides J₁ and K₁,¹⁵⁾ the terminal β-D-oleandropyranosyl group was attached at the C-4 position of the β-D-canaropyranosyl group in both compounds. This canaropyranosyl group was linked at the C-4 position of the β-D-cymaropyranosyl group in 8, and at the C-4 position of the β-D-oleandropyranosyl group in 10. Thus, the sugar sequences of 8 and 10 were deduced to be 3-O-β-D-oleandropyranosyl-(1→4)-β-D-canaropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside and 3-O-β-D-oleandropyranosyl-(1→4)-β-D-canaropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranoside. These sugar linkages were confirmed by observations of ROEs between the anomeric proton signal of each monosaccharide and the H-4 signals of neighboring sugars in the ROESY experiment and ³J_COCHs in the HMBC measurement. The structures of 8 and 10 are presented in Chart 1.

Oxypetalumoside F₉ (14) was proposed to be 12-O-(E)-cinnamoylisoramanone 3-O-tetraglycoside, with the molecular formula C₅₈H₈₆O₁₇ based on the NMR spectroscopic data and HR-ESI-MS. Because acid hydrolysis of 14 only afforded oleandrose, its sugar moiety consisted of four β-D-oleandropyranosyl groups, in consideration of the J value of each anomeric proton signal. Additionally, as 14 was a monodesmoside of 12-O-(E)-cinnamoylisoramanone from the NMR spectroscopic data, its sugar sequence was deduced to be 3-O-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranoside, and this was confirmed by the results of the HMBC and ROESY experiments. ROEs were observed between δ_H 3.54 (H-3 of the aglycone) and 4.53 (H-1′ of β-D-oleandropyranose), δ_H 4.66 (H-1″ of β-D-oleandropyranose) and 3.16 (H-4′of β-D-oleandropyranose), δ_H 4.66 (H-1‴ of β-D-oleandropyranose) and 3.17 (H-4″ of β-D-oleandropyranose), and δ_H 4.72 (H-1″″ of β-D-oleandropyranose) and 3.17 (H-4‴ of β-D-oleandropyranose) in the ROESY experiment. The HMBC experiment revealed the ³J_COCHs between the H-1/C-4 signals, and the H-4/C-1 signals of the neighboring sugars. The structure of 14 is presented in Chart 1.

The molecular formula of oxypetalumoside D₁₁ (16) was determined to be C₅₅H₈₈O₁₉, on the basis of HR-ESI-MS. The NMR spectroscopic data and acid hydrolysis suggested that 16 was oxypetalumone 3-O-pentaglycoside, with sugar moiety consisting of two inner β-D-oleandropyranosyl groups, one β-D-cymaropyranosyl group, one β-D-canaropyranosyl group, and a terminal β-D-oleandropyranosyl group. Assignments of the anomeric carbon and proton signals of this terminal β-D-oleandropyranosyl group at δ_C 101.5 and δ_H 4.51 indicated that it was attached at the C-4 position of the β-D-cymaropyranosyl group, as with 15. Moreover, the anomeric carbon and proton signals of this β-D-cymaropyranosyl group were assigned at δ_C 99.3 and δ_H 4.74, and this proton signal revealed a ROE to the characteristic H-4 (δ_H 2.97) signal of the β-D-canaropyranosyl group. Accordingly, the terminal β-D-oleandropyranpsyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-canaropyranosyl array and inner β-D-oleandropyranopsyl-(1→4)-β-D-oleandropyranosyl array seemed to be presented in its sugar sequence, based on a comparison of the ¹H- and ¹³C-NMR spectroscopic data of 16 with those of 14 and 15. Anomeric carbon and proton signals for the β-D-canaropyranosyl group at δ_C 100.4 and δ_H 4.70 indicated that this β-D-canaropyranosyl group was linked at the C-4 position of the inner β-D-oleandropyranosyl group, the same as for 10 and currasavioside J₁.¹⁵⁾ Thus, the sugar linkage of 16 was established as shown in Chart 1, which was further supported by the results of the ROESY and HMBC experiments. The structure of 16 is presented in Chart 1.

Oxypetalumoside F₁₂ (17) was considered to be 12-O-(E)-cinnamoylisoramanone 3-O-hexaglycoside, with molecular formula C₇₂H₁₁₀O₂₃, on the basis of HR-ESI-MS. The sugar sequence of 17 consisted of a terminal β-D-oleandropyranosyl group and a 3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl unit, as revealed by comparison of the ¹H- and ¹³C-NMR spectroscopic data of 17 with those of 15 and 12. Additionally, the δ value of the anomeric proton and carbon signals due to this terminal β-D-oleandropyranosyl group (δ_H 4.71 and δ_C 100.2) and observation of a ROE between this anomeric proton and H-4 of the fifth β-D-oleandropyranosyl group (δ_H 3.16) in the above unit indicated that the terminal β-D-oleandropyranosyl group was connected to the C-4 position of the fifth β-D-oleandropyranosyl group, as in 12 and 13. The structure of 17 is presented in Chart 1.

In this study, we obtained pregnane glycosides plant roots, which is characteristic of the Apocynaceae. These glycosides mainly had 12-O-acylated-ramanone, -isoramanone, or an oxo-derivative at the C-12 position of isoramanone (oxypetalumone) as each aglycone. Because the previous detailed study on the constituents of the aerial parts of O. caeruleum yielded a pregnane 21-O-glycoside, differences in the structures and contents of pregnane glycosides between the aerial parts and roots were observed.

The pregnane glycosides are known to have anti-tumor, anti-microbial, anti-fungal, anti-parasitic and anti-epileptic activities.^16–24) The appetite suppressing compound P57AS3, from Hoodia gordonii, is also a pregnane glycoside. Its aglycone is hoodiogenin A (12-O-tigloylisoramanone).^25,26) Because there are few reports on plants containing 12-O-acylated-isoramanone-type glycosides, the fact that 12-O-cinnamoylisoramanone glycosides were obtained from the roots of this plant is very interesting.

Experimental

A part of this section (general procedures, plant materials, extraction and isolation, and acid hydrolysis) is described in the supplemental materials.

Oxypetalumoside A₁ (1) Amorphous powder. [α]_D¹⁸ −62 (c = 0.93, MeOH). HR-ESI-MS: m/z 763.4034 [M + Na]⁺ (Calcd for C₄₂H₆₀O₁₁Na: 763.4028). UV λ_max(MeOH) nm (log ε): 230 (4.09). ¹³C- and ¹H-NMR data of the aglycone and ester moieties (measured in CDCl₃): see Tables 1 and 2. ¹³C-NMR data of the sugar moiety (measured in CDCl₃): see Table 3. ¹H-NMR data of the sugar moiety (measured in CDCl₃) δ: 4.86 (1H, dd, 9.5, 2.0, H-1′), 4.50 (1H, dd, 9.5, 2.0, H-1″), 3.88 (1H, dq, 9.5, 6.0, H-5′), 3.82 (1H, q, 3.0, H-3′), 3.46 (3H, s, -OMe′), 3.39 (3H, s, -OMe″), 3.30 (1H, dq, 9.0, 6.0, H-5″), 3.24 (1H, dd, 9.5, 3.0, H-4′), 3.18 (1H, m, H-3″), 3.13 (1H, t, 9.0, H-4″), 1.32 (3H, d, 6.0, H-6″), 1.24 (3H, d, 6.0, H-6′).

Oxypetalumoside B₁ (2) Amorphous powder. [α]_D²² − 55 (c = 0.62, MeOH). HR-ESI-MS: m/z 755.4372 [M + Na]⁺ (Calcd for C₄₁H₆₄O₁₁Na: 755.4341). ¹³C- and ¹H-NMR data of the aglycone and ester moieties (measured in CDCl₃): see Tables 1 and 2. ¹³C- and ¹H-NMR data of the sugar moiety were in good agreement of those of 1.

Oxypetalumoside C₁ (3) Amorphous powder. [α]_D²² − 52 (c = 0.48, MeOH). HR-ESI-MS: m/z 769.4521 [M + Na]⁺ (Calcd for C₄₂H₆₆O₁₁Na: 769.4497). ¹³C- and ¹H-NHR data of the aglycone and ester moieties (measured in CDCl₃): see Tables 1 and 2. ¹³C- and ¹H-NMR data of the sugar moiety were in good agreement of those of 1.

Oxypetalumoside B₂ (4) Amorphous powder. [α]_D²² − 33 (c = 1.01, MeOH). HR-ESI-MS: m/z 899.5133 [M + Na]⁺ (Calcd for C₄₈H₇₆O₁₄Na: 899.5127). ¹³C- and ¹H-NMR data of the acylated aglycone moiety were almost consistent with those of 2. ¹³C-NMR data of the sugar moiety (measured in CDCl₃): see Table 3. ¹H-NMR data of the sugar moiety (measured in CDCl₃) δ: 4.84 (1H, dd, 9.5, 2.0, H-1′), 4.76 (1H, dd, 9.5, 2.0, H-1″), 4.50 (1H, dd, 9.5, 2.0, H-1‴), 3.87 (1H, dq, 9.5, 6.0, H-5″), 3.84 (1H, dq, 9.5, 6.0, H-5′), 3.81 (2H, q, 3.0, H-3′, -3″), 3.45 (3H, s, -OMe′), 3.45 (3H, s, -OMe″), 3.39 (3H, s, -OMe‴), 3.29 (1H, dq, 9.0, 6.0, H-5‴), 3.22 (1H, dd, 9.5, 3.0, H-4′), 3.23 (1H, dd, 9.5, 3.0, H-4″), 3.18 (1H, m, H-3‴), 3.13 (1H, t, 9.0, H-4‴), 1.32 (3H, d, 6.0, H-6‴), 1.23 (3H, d, 6.0, H-6″), 1.22 (3H, d, 6.0, H-6′).

Oxypetalumoside C₂ (5) Amorphous powder. [α]_D¹⁸ − 41 (c = 0.65, MeOH). HR-ESI-MS: m/z 913.5289 [M + Na]⁺ (Calcd for C₄₇H₇₈O₁₄Na: 913.5284). ¹³C- and ¹H-NMR data of the acylated aglycone moiety were almost consistent with those of 3. ¹³C- and ¹H-NMR data of the sugar moiety were in good agreement of those of 4.

Oxypetalumoside D₃ (6) Amorphous powder. [α]_D¹⁸ + 25 (c = 0.97, MeOH). HR-ESI-MS: m/z 945.5179 [M + Na]⁺ (Calcd for C₄₉H₇₈O₁₆Na: 945.5182). ¹³C- and ¹H-NMR data of the aglycone moiety (measured in CDCl₃) see Tables 1 and 2. ¹³C-NMR data of the sugar moiety (measured in CDCl₃): see Table 3. ¹H-NMR data of the sugar moiety (measured in CDCl₃) δ: 4.84 (1H, dd, 9.5, 2.0, H-1′), 4.73 (1H, dd, 9.5, 2.0, H-1″), 4.71 (1H, dd, 9.5, 2.0, H-1″″), 4.43 (1H, dd, 9.5, 2.0, H-1‴), 3.84 (1H, dq, 9.5, 6.0, H-5″), 3.81 (1H, dq, 9.5, 6.0, H-5′), 3.79 (1H, q, 3.0, H-3′), 3.77 (1H, q, 3.0, H-3″), 3.42 (6H, s, -OMe′, -OMe″), 3.38 (3H, s, -OMe‴), 3.38 (3H, s, -OMe″″), 3.36 (1H, m, H-3‴), 3.29 (1H, dq, 9.0, 6.0, H-5‴), 3.29 (1H, dq, 9.0, 6.0, H-5″″), 3.20 (1H, dd, 9.5, 3.0, H-4″), 3.19 (1H, dd, 9.5, 3.0, H-4′), 3.15 (1H, t, 9.0, H-4‴), 3.15 (overlapping, H-3″″), 3.12 (1H, t, 9.0, H-4″″), 1.33 (3H, d, 6.0, H-6″″), 1.28 (3H, d, 6.0, H-6‴), 1.20 (3H, d, 6.0, H-6″), 1.19 (3H, d, 6.0, H-6′).

Oxypetalumoside E₃ (7) Amorphous powder. [α]_D¹⁸ − 4.4 (c = 0.34, MeOH). HR-ESI-MS: m/z 1095.5870 [M + Na]⁺ (Calcd for C₅₈H₈₈O₁₈Na: 1095.5863). UV λ_max(MeOH) nm (log ε): 231 (4.18). ¹³C- and ¹H-NMR data of the aglycone and ester moieties (measured in CDCl₃): see Tables 1 and 2. ¹³C-NMR data of the aglycone and ester moieties (measured in pyridine-d₅) δ: 169.4 (C-1″), 166.4 (C-α), 139.7 (C-5), 133.3 (C-4′), 132.1 (C-1′), 130.0 × 2 (C-2′, -6′), 128.8 × 2 (C-3′, -5′), 122.3 (C-6), 86.3 (C-14), 77.1 (C-3), 73.5 (C-12), 72.8 (C-20), 52.2 (C-17), 51.9 (C-13), 43.2 (C-9), 39.2 (C-4), 37.3 (C-10), 37.1 (C-1), 36.6 (C-8), 31.0 (C-15), 30.2 (C-2), 27.8 (C-11), 27.1 (C-7), 23.6 (C-16), 21.5 (C-2″), 20.3 (C-21), 19.4 (C-19), 14.7 (C-18). ¹H-NMR data of the aglycone and ester moieties (measured in pyridine-d₅) δ: 8.34 (2H, br d, 7.5, H-2′, -6′), 7.59 (1H, br t, 7.5, H-4′), 7.54 (2H, br t, 7.5, H-3′, -5′), 5.76 (1H, br s, C14-OH), 5.44 (1H, dd, 11.5, 4.5, H-12), 5.43 (overlapping, H-6), 5.03 (overlapping, H-20), 3.83 (1H, m, H-3), 2.93 (1H, q, 9.5, H-17), 2.11 (1H, td, 11.5, 5.5, H-8), 1.87 (3H, s, H-2″), 1.73 (3H, s, H-18), 1.13 (3H, d, 6.0, H-21), 0.98 (3H, s, H-19). ¹³C- and ¹H-NMR data of the sugar moiety were in good agreement of those of 6.

Oxypetalumoside D₄ (8) Amorphous powder. [α]_D¹² + 27 (c = 0.94, MeOH). HR-ESI-MS: m/z 931.5012 [M + Na]⁺ (Calcd for C₄₈H₇₆O₁₆Na: 931.5026). ¹³C- and ¹H-NMR data of the aglycone moiety were almost consistent with those of 6. ¹³C-NMR data of the sugar moiety (measured in CDCl₃): see Table 3. ¹H-NMR data of the sugar moiety (measured in CDCl₃) δ: 4.83 (1H, dd, 9.5, 2.0, H-1′), 4.74 (1H, dd, 9.5, 2.0, H-2″), 4.51 (1H, dd, 9.5, 2.0, H-1‴), 4.48 (1H, dd, 9.5, 2.0, H-1″″), 3.86 (1H, dq, 9.5, 6.0, H-5″), 3.82 (1H, dq, 9.5, 6.0, H-5′), 3.81 (1H, q, 3.0, H-3′), 3.78 (1H, q, 3.0, H-3″), 3.59 (1H, m, H-3‴), 3.44 (3H, s, -OMe″), 3.43 (3H, s, -OMe′), 3.41 (3H, s, -OMe″″), 3.41 (overlapping, H-5″″), 3.31 (1H, dq, 9.0, 6.0, H-5‴), 3.22 (1H, dd, 9.5, 3.0, H-4″), 3.20 (1H, dd, 9.5, 3.0, H-4′), 3.20 (overlapping, H-3″″), 3.16 (1H, t, 9.0, H-4″″), 2.98 (1H, t, 9.0, H-4‴), 1.35 (3H, d, 6.0, H-6″″), 1.28 (3H, d, 6.0, H-6‴), 1.21 (3H, d, 6.0, H-6″), 1.20 (3H, d, 6.0, H-6′).

Oxypetalumoside D₅ (9) Amorphous powder. [α]_D¹⁸ + 3.3 (c = 0.46, MeOH). HR-ESI-MS: m/z 945.5183 [M + Na]⁺ (Calcd for C₄₉H₇₈O₁₆Na: 945.5182). ¹³C- and ¹H-NMR data of the aglycone moiety were almost consistent with those of 6. ¹³C-NMR data of the sugar moiety (measured in CDCl₃): see Table 3. ¹H-NMR data of the sugar moiety (measured in CDCl₃) δ: 4.84 (1H, dd, 9.5, 2.0, H-1′), 4.71 (1H, dd, 9.5, 2.0, H-1″″), 4.65 (1H, dd, 9.0, 2.0, H-1‴), 4.43 (1H, dd, 9.0, 2.0, H-1″), 3.85 (1H, dq, 9.5, 6.0, H-5′), 3.78 (1H, q, 3.0, H-3′), 3.44 (3H, s, -OMe′), 3.40 (3H, s, -OMe‴), 3.39 (3H, s, -OMe″″), 3.38 (3H, s, -OMe″), 3.37 (overlapping, H-3‴), 3.35 (1H, m, H-3″), 3.32 (overlapping, H-5‴ or -5″″), 3.31 (overlapping, H-5″″ or -5‴), 3.20 (1H, dd, 9.5, 3.0, H-4′), 3.17 (1H, t, 9.0, H-4‴), 3.16 (overlapping, H-3″″), 3.15 (1H, t, 9.0, H-4″), 3.13 (1H, t, 9.0, H-4″″), 1.34 (3H, d, 6.0, H-6″″), 1.31 (3H, d, 6.0, H-6‴), 1.28 (3H, d, 6.0, H-6″), 1.21 (3H, d, 6.0, H-6′).

Oxypetalumoside D₆ (10) Amorphous powder. [α]_D²¹ + 5.8 (c = 0.63, MeOH). HR-ESI-MS: m/z 931.5046 [M + Na]⁺ (Calcd for C₄₈H₇₆O₁₆Na: 931.5026). ¹³C- and ¹H-NMR data of the aglycone moiety were almost consistent with those of 6. ¹³C-NMR data of the sugar moiety (measured in CDCl₃): Table 3 ¹H-NMR data of the sugar moiety (measured in CDCl₃) δ: 4.84 (1H, dd, 9.5, 2.0, H-1′), 4.71 (1H, dd, 9.5, 2.0, H-1‴), 4.49 (1H, dd, 9.5, 2.0, H-1″″), 4.44 (1H, dd, 9.5, 2.0, H-1″), 3.85 (1H, dq, 9.5, 6.0, H-5′), 3.79 (1H, q, 3.0, H-3′), 3.59 (1H, m, H-3‴), 3.44 (3H, s, -OMe′), 3.42 (1H, dq, 9.0, 6.0, H-5″″), 3.41 (3H, s, -OMe″″), 3.40 (3H, s, -OMe″), 3.35 (overlapping, H-3″), 3.33 (1H, dq, 9.0, 6.0, H-5‴), 3.29 (1H, dq, 9.0, 6.0, H-5″), 3.21 (1H, dd, 9.5, 3.0, H-4′), 3.20 (1H, m, H-3″″), 3.17 (1H, t, 9.0, H-4″), 3.16 (1H, t, 9.0, H-4″″), 2.98 (1H, t, 9.0, H-4‴), 1.36 (3H, d, 6.0, H-6″″), 1.31 (3H, d, 6.0, H-6‴), 1.28 (3H, d, 6.0, H-6″), 1.21 (3H, d, 6.0, H-6′).

Oxypetalumoside A₇ (11) Amorphous powder. [α]_D²² − 33 (c = 0.57, MeOH). HR-ESI-MS: m/z 537.2751 [M + 2Na]²⁺ (Calcd for (C₅₆H₈₄O₁₇Na₂)/2: 537.2746). UV λ_max(MeOH) nm (log ε): 274 (3.27), 230 (4.09). ¹³C- and ¹H-NMR data of the acylated aglycone moiety were almost consistent with those of 1. ¹³C-NMR data of the sugar moiety (measured in CDCl₃): see Table 3. ¹H-NMR data of the sugar moiety (measured in CDCl₃) δ: 4.87 (1H, dd, 9.5, 2.0, H-1″″), 4.85 (1H, dd, 9.5, 2.0, H-1′), 4.66 (1H, dd, 9.5, 2.0, H-1‴), 4.45 (1H, dd, 9.5, 2.0, H-1″), 3.86 (1H, dq, 9.5, 6.0, H-5′), 3.79 (1H, q, 3.0, H-3′), 3.63 (1H, q, 3.0, H-3″″), 3.60 (1H, dq, 9.5, 6.0, H-5″″), 3.45 (3H, s, -OMe′), 3.43 (3H, s, -OMe″″), 3.42 (3H, s, -OMe″), 3.39 (3H, s, -OMe‴), 3.37 (1H, m, H-3‴), 3.36 (1H, m, H-3″), 3.32 (1H, dq, 9.0, 6.0, H-5‴), 3.30 (1H, dq, 9.0, 6.0, H-5″), 3.22 (2H, dd, 9.5, 3.0, H-4′, -4″″), 3.18 (1H, t, 9.0, H-4‴), 3.16 (1H, t, 9.0, H-4″), 1.32 (3H, d, 6.0, H-6‴), 1.30 (3H, d, 6.0, H-6″″), 1.29 (3H, d, 6.0, H-6″), 1.23 (3H, d, 6.0, H-6′).

Oxypetalumoside D₈ (12) Amorphous powder. [α]_D²¹ + 5.0 (c = 1.03, MeOH). HR-ESI-MS: m/z 945.5192 [M + Na]⁺ (Calcd for C₄₉H₇₈O₁₆Na: 945.5182). ¹³C- and ¹H-NMR data of the aglycone moiety were almost consistent with those of 6. ¹³C-NMR data of the sugar moiety (measured in CDCl₃): see Table 3. ¹H-NMR data of the sugar moiety (measured in CDCl₃) δ: 4.94 (1H, dd, 9.5, 2.0, H-1″), 4.72 (1H, dd, 9.5, 2.0, H-1″″), 4.52 (1H, dd, 9.5, 2.0, H-1′), 4.44 (1H, dd, 9.5, 2.0, H-1‴), 3.89 (1H, dq, 9.5, 6.0, H-5″), 3.78 (1H, q, 3.0, H-3″), 3.42 (3H, s, -OMe″), 3.39 (3H, s, -OMe′), 3.39 (3H, s, -OMe‴), 3.39 (3H, s, -OMe″″), 3.37 (2H, m, H-3′, -3‴), 3.31 (overlapping, H-5‴or -5″″), 3.30 (overlapping, H-5″″ or -5‴), 3.28 (1H, dq, 9.0, 6.0, H-5′), 3.21 (1H, dd, 9.5, 3.0, H-4″), 3.17 (1H, t, 9.0, H-4′), 3.16 (1H, t, 9.0, H-4‴), 3.16 (overlapping, H-3″″), 3.14 (1H, t, 9.0, H-4″″), 1.34 (3H, d, 6.0, H-6″″), 1.29 (3H, d, 6.0, H-6‴), 1.28 (3H, d, 6.0, H-6′), 1.23 (3H, d, 6.0, H-6″).

Oxypetalumoside F₈ (13) Amorphous powder. [α]_D¹⁸ + 7.2 (c = 0.37, MeOH). HR-ESI-MS: m/z 1077.5746 [M + Na]⁺ (Calcd for C₅₈H₈₆O₁₇Na: 1077.5757). UV λ_max(MeOH) nm (log ε): 280 (4.36), 223 (4.14), 218 (4.20). ¹³C- and ¹H-NMR data of the aglycone and ester moieties (measured in CDCl₃): see Tables 1 and 2. ¹³C- and ¹H-NMR data of the sugar moiety were in good agreement of those of 12.

Oxypetalumoside F₉ (14) Amorphous powder. [α]_D¹⁸ − 8.3 (c = 0.20, MeOH). HR-ESI-MS: m/z 1077.5757 [M + Na]⁺ (Calcd for C₅₈H₈₆O₁₇Na: 1077.5757). UV λ_max(MeOH) nm (log ε): 280 (4.45), 223 (4.22), 218 (4.28). ¹³C- and ¹H-NMR data of the acylated aglycone moiety were almost consistent with those of 13. ¹³C-NMR data of the sugar moiety (measured in CDCl₃): see Table 3. ¹H-NMR data of the sugar moiety δ: 4.72 (1H, dd, 9.5, 2.0, H-1″″), 4.66 (1H, dd, 9.5, 2.0, H-1″), 4.66 (1H, dd, 9.5, 2.0, H-1‴), 4.53 (1H, dd, 9.5, 2.0, H-1′), 3.41 (3H, s, -OMe‴, -OMe′ or -OMe″), 3.40 (3H, s, -OMe″″), 3.40 (3H, s, -OMe″, -OMe‴, or -OMe′), 3.37 (3H, s, -OMe′, -OMe″, or -OMe‴), 3.36 (overlapping, H-3′, -3″, -3‴), 3.33 (1H, dq, 9.0, 6.0, H-5″), 3.33 (1H, dq, 9.0, 6.0, H-5‴), 3.32 (1H, dq, 9.0, 6.0, H-5″″), 3.30 (1H, dq, 9.0, 6.0, H-5′), 3.17 (2H, t, 9.0, H-4″, -4‴), 3.16 (1H, t, 9.0, H-4′), 3.15 (overlapping, H-3″″), 3.14 (1H, t, 9.0, H-4″″), 1.35 (3H, d, 6.0, H-6″″), 1.32 (3H, d, 6.0, H-6″ or -6‴), 1.31 (3H, d, 6.0, H-6‴ or -6″), 1.29 (3H, d, 6.0, H-6′).

Oxypetalumoside F₁₀ (15) Amorphous powder. [α]_D¹⁸ + 1.4 (c = 0.37, MeOH). HR-ESI-MS: m/z 1221.6548 [M + Na]⁺ (Calcd for C₆₅H₉₈O₂₀Na: 1221.6544). UV λ_max(MeOH) nm (log ε): 280 (4.38), 223 (4.14), 218 (4.20). ¹³C- and ¹H-NMR data of the acylated aglycone moiety were almost consistent with those of 13. ¹³C-NMR data of the sugar moiety (measured in CDCl₃): see Table 3. ¹H-NMR data of the sugar moiety δ: 4.94 (1H, dd, 9.5, 2.0, H-1″″), 4.67 (1H, dd, 9.5, 2.0, H-1″), 4.66 (1H, dd, 9.5, 2.0, H-1‴), 4.54 (1H, dd, 9.5, 2.0, H-1′), 4.50 (1H, dd, 9.5, 2.0, H-1‴″), 3.91 (1H, dq, 9.5, 6.0, H-5″″), 3.81 (1H, q, 3.0, H-3″″), 3.44 (3H, s, -OMe″″), 3.41 (3H, s, -OMe‴), 3.40 (3H, s, -OMe″), 3.39 (3H, s, -OMe‴″), 3.38 (3H, s, -OMe′), 3.35 (overlapping, H-3′, -3″), 3.34 (overlapping, H-3‴), 3.31 (overlapping, H-5″), 3.30 (overlapping, H-5′, -5‴), 3.29 (1H, dq, 9.0, 6.0, H-5‴″), 3.24 (1H, dd, 9.5, 3.0, H-4″″), 3.18 (1H, t, 9.0, H-4‴), 3.16 (1H, t, 9.0, H-4″ or -4′), 3.15 (1H, t, 9.0, H-4′ or -4″), 3.15 (overlapping, H-3‴″), 3.13 (1H, t, 9.0, H-4‴″), 1.32 (3H, d, 6.0, H-6‴″), 1.31 (3H, d, 6.0, H-6″), 1.31 (3H, d, 6.0, H-6‴), 1.30 (3H, d, 6.0, H-6′), 1.25 (3H, d, 6.0, H-6″″).

Oxypetalumoside D₁₁ (16) Amorphous powder. [α]_D²² ≈0 (c = 0.37, MeOH). HR-ESI-MS: m/z 1075.5822 [M + Na]⁺ (Calcd for C₅₅H₈₈O₁₉Na: 1075.5812). ¹³C- and ¹H-NMR data of the aglycone moiety were almost consistent with those of 6. ¹³C-NMR data of the sugar moiety (measured in CDCl₃): see Table 3. ¹H-NMR data of the sugar moiety δ: 4.74 (1H, dd, 9.5, 2.0, H-1″″), 4.70 (1H, dd, 9.5, 2.0, H-1‴), 4.68 (1H, dd, 9.5, 2.0, H-1″), 4.54 (1H, dd, 9.5, 2.0, H-1′), 4.51 (1H, dd, 9.5, 2.0, H-1‴″), 4.00 (1H, dq, 9.5, 6.0, H-5″″), 3.84 (1H, q, 3.0, H-3″″), 3.56 (1H, m, H-3‴), 3.46 (3H, s, -OMe″″), 3.41 (3H, s, -OMe″), 3.39 (3H, s, -OMe‴″), 3.38 (3H, s, -OMe′), 3.38 (overlapping, H-3′), 3.35 (1H, m, H-3″), 3.32 (1H, 1H, dq, 9.5, 6.0, H-5‴), 3.30 (2H, dq, 9.5, 6.0, H-5′, -5″), 3.29 (1H, dq, 9.0, 6.0, H-5‴″), 3.27 (1H, dd, 9.5, 3.0, H-4″″), 3.18 (1H, m, H-3‴″), 3.16 (1H, t, 9.0, H-4″ or -4′), 3.15 (1H, t, 9.0, H-4′ or -4″), 3.13 (1H, t, 9.0, H-4‴″), 2.97 (1H, t, 9.0, H-4‴), 1.33 (3H, d, 6.0, H-6‴″), 1.31 (3H, d, 6.0, H-6″, -6′, or -6‴), 1.29 (6H, d, 6.0, H-6′, H-6‴, and/or -6″), 1.27 (3H, d, 6.0, H-6″″).

Oxypetalumoside F₁₂ (17) Amorphous powder. [α]_D¹⁸ − 1.9 (c = 0.78, MeOH). HR-ESI-MS: m/z 1365.7341 [M + Na]⁺ (Calcd for C₇₂H₁₁₀O₂₃Na: 1365.7330). UV λ_max(MeOH) nm (log ε): 280 (4.37), 223 (4.13), 218 (4.19). ¹³C- and ¹H-NMR data of the acylated aglycone moiety were almost consistent with those of 13. ¹³C-NMR data of the sugar moiety (measured in CDCl₃): see Table 3. ¹H-NMR data of the sugar moiety δ: 4.93 (1H, dd, 9.5, 2.0, H-1″″), 4.71 (1H, dd, 9.5, 2.0, H-1‴‴), 4.66 (1H, dd, 9.5, 2.0, H-1″), 4.64 (1H, dd, 9.5, 2.0, H-1‴), 4.53 (1H, dd, 9.5, 2.0, H-1′), 4.44 (1H, dd, 9.5, 2.0, H-1‴″), 3.88 (1H, dq, 9.5, 6.0, H-5″″), 3.78 (1H, q, 3.0, H-3″″), 3.42 (3H, s, -OMe″″), 3.40 (3H, s, -OMe‴), 3.39 (3H, s, -OMe″), 3.39 (3H, s, -OMe‴″), 3.39 (3H, s, -OMe‴‴), 3.37 (3H, s, -OMe′), 3.36 (overlapping, H-3‴″), 3.35 (overlapping, H-3′, -3‴), 3.34 (overlapping, H-3″), 3.30 (overlapping, H-5′, -5″, -5‴, -5‴″), 3.29 (overlapping, H-5‴‴), 3.21 (1H, dd, 9.5, 3.0, H-4″″), 3.17 (1H, t, 9.0, H-4‴), 3.16 (1H, t, 9.0, H-4‴″), 3.15 (2H, t, 9.0, H-4′, -4″), 3.15 (overlapping, H-3‴‴), 3.13 (1H, t, 9.0, H-4‴‴), 1.34 (3H, d, 6.0, H-6‴‴), 1.30 (3H, d, 6.0, H-6″), 1.29 (3H, d, 6.0, H-6‴), 1.29 (3H, d, 6.0, H-6‴″), 1.28 (3H, d, 6.0, H-6′), 1.23 (3H, d, 6.0, H-6″″).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

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