C₂₁ steroid derivatives from the Dai herbal medicine Dai-Bai-Jie, the dried roots of Marsdenia tenacissima, and their screening for anti-HIV activity

Abstract

Twenty-three new C₂₁ steroidal glycosides, marstenacissides C1–C10 (1–10), D1–D7 (11–17) and E1–E6 (18–23), and four new C₂₁ steroids, 11α,12β-O-ditigloyl-tenacigenin C (24), 11α-O-benzoyl-12β-O-tigloyl-tenacigenin C (25), 11α-O-tigloyl-12β-O-benzoyl-tenacigenin C (26) and 11α-O-tigloyl-12β-O-benzoyl-marsdenin (27), were isolated from the Dai herbal medicine Dai-Bai-Jie, derived from the roots of Marsdenia tenacissima. The chemical structures of all compounds were established by spectroscopic techniques, including high-resolution mass spectrometry and NMR spectroscopy, as well as by comparison with reported spectral data. The anti-HIV activities of these compounds were screened, and the compounds obtained displayed inhibitory effects against HIV-1 with inhibition rates of 36.4–81.3% at 30 μM.

Introduction

Dai-Bai-Jie is a Dai herbal medicine widely used by Dai people living in Laos, Burma and the Yunnan province of China for the purposes of detoxification, decreasing swelling, alleviating pain etc. It is not only used as a Dai herbal medicine, but also as the main medicinal material for preparations such as Ya-jie tablets and Bai-jie capsules. Originally Dai-Bai-Jie was mistakenly thought to be the root of Dregea sinensis Hemsl. (Asclepiadaceae), but Dregea sinensis has significant differences in distribution, morphology and anatomy, pharmacognosy, molecular biology and chemical composition. It was finally proved that the original plant of Dai-Bai-Jie matches the characteristics of Marsdenia tenacissima (Roxb.) Moon (Asclepiadaceae) in the Flora of China recently published in English [1]. This plant is a perennial climber extensively distributed in the south of China, especially in Yunnan province.

As an important Dai herbal medicine, Dai-Bai-Jie derived from the dried roots of M. tenacissima has seen a lack of systematic phytochemical research to date. Previously, a series of polyoxypregnane glycosides form M. tenacissima were reported, where the part of the plant studied was the stem [2,3,4,5,6,7,8,9,10,11,12,13]. Aiming to investigate the constituents of Dai-Bai-Jie, our phytochemical study was carried out on the dried roots of M. tenacissima. Previously, we reported the work which led to the isolation of thirty-one C₂₁ steroidal glycosides [14, 15]. In this continued work, twenty-seven C₂₁ steroid derivatives were obtained, comprising twenty-three new C₂₁ steroidal glycosides (1–23) and four new C₂₁ steroids (24–27) (Fig. 1). The structures of all compounds were established by spectroscopic techniques, including mass spectrometry and NMR spectroscopy, as well as by comparison with reported spectral data. Herein, we mainly present the isolation and structure elucidation of these compounds, as well as their preliminary anti-HIV testing.

Fig. 1

Structures of 1–27

Results and discussion

Twenty-three new C₂₁ steroidal glycosides (1–23) and four new C₂₁ steroids (24–27) were obtained from the 95% alcoholic extract of Dai herbal medicine Dai-Bai-Jie, the dried roots of M. tenacissima, by silica gel and MCI resin column chromatography, and repeated semi-preparative RP-HPLC with different solvent systems.

Marstenacisside C1 (1) displayed a molecular formula of C₄₇H₇₂O₁₉ according to the [M–H]⁻ ion at m/z 939.4587 in the HRESIMS spectrum. In its ¹H-NMR spectrum, three methyl singlet signals at δ 1.35 (3H, s, CH₃-18), 1.22 (3H, s, CH₃-19) and 2.17 (3H, s, CH₃-21), and three methine protons indicative of secondary alcoholic functions at δ 3.76 (1H, m, H-3), 5.72 (1H, t, J = 10.2 Hz, H-11) and 5.24 (1H, d, J = 10.2 Hz, H-12) were observed. Combination of the ¹H- and ¹³C-NMR data indicated a C₂₁ steroidal skeleton for 1. Comparing the NMR data of the C₂₁ steroidal skeleton of 1 with those of volubiloside C isolated from Dregea volubilis [16], the C₂₁ steroid skeleton could be identified as (8β, 9α,17α)-5-ene-20-one-3β,11α,12β,14β-tetradroxypregnane (drevogenin P). The proton signals of δ 1.93 (3H, s, Ac-H-2), 7.18 (1H, qq, J = 7.1, 1.3 Hz, Tig-H-3), 1.68 (3H, d, J = 7.1, Tig-H-4) and 1.97 (3H, s, Tig-H-5) and the carbon signals of δ 170.2 (Ac-C-1), 21.4 (Ac-C-2) and 167.9 (Tig-C-1), 128.5 (Tig-C-2), 139.1 (Tig-C-3), 14.4 (Tig-C-4) and 12.2 (Tig-C-5) indicated the existence of an acetyl group and a tigloyl group in the molecule. The acetyl and tigloyl group were determined be located at C-11and C-12, respectively, due to the HMBC correlations between δ 5.72 (H-11) and 170.2 (Ac-C-1), and between δ 5.24 (H-12) and 167.9 (Tig-C-1) (Fig. 2). Accordingly, the aglycone structure of 1 was identified as 11-O-acetyl-12-O-tigloyl-drevogenin P, and all its proton and carbon signals were assigned by combined use of ¹H–¹H COSY, HSQC and HMBC experiments (Table 1). The relative configurations of the aglycone were further confirmed by correlations of H-1/H-3, H-1/H-9, H-9/H-12, H-12/H-17, H-11/H-19, H-11/H-18, H-8/H-11 and H-18/H-21 in the ROESY spectrum (Fig. 3). In the ¹H- and ¹³C-NMR spectra of 1, three anomeric protons at δ 4.84 (1H, dd, J = 9.7, 1.4 Hz), 5.08 (1H, d, J = 8.1 Hz) and 4.94 (1H, d, J = 7.7 Hz) and three anomeric carbon signals at δ 97.7, 101.9 and 106.6 were found, which suggested the existence of three sugar units in the molecule. By NMR spectroscopic data analysis as well as by comparison with the reported NMR data [5, 17], the sugar units were identified as canarose, 6-deoxy-3-O-methly-allose and glucose. Using ¹H–¹H COSY, HSQC and HMBC spectra, the proton spin systems and the carbon resonances of each sugar were fully assigned (Table 2). The β configuration of each sugar was determined by the large coupling constants (³ J _1,2 > 7 Hz). The connectivity of sugar units was established by the HMBC correlations between δ 5.08 (Allo-H-1) and 88.4 (Can-C-4), and δ 4.94 (Glc-H-1) and 82.7 (Allo-C-4), and the HMBC correlation between δ 4.84 (Can-H-1) and 77.4 (C-3) suggested the glycosidation site at C-3 (Fig. 2). Consequently, the structure of 1 was elucidated as 3-O-β-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α-O-acetyl-12β-O-tigloyl-drevogenin P.

Fig. 2

Key HMBC and ¹H–¹H COSY correlations for 1

Table 1 ¹³C-NMR data for aglycones of 1–27 (150 MHz for 1–4, 6, 8, 9 and 11–28, and 125 MHz for 5, 7 and 10, pyridine-d ₅)

Fig. 3

Key ROESY correlations for the aglycone of 1

Table 2 ¹³C-NMR data for sugar moieties of 1–23 (150 MHz for 1–4, 6, 8, 9 and 11–23, and 125 MHz for 5, 7 and 10, pyridine-d ₅)

The ¹³C-NMR data due to the sugar moieties of 2–10 agreed well with those of 1, which provided the evidence that 2–10 had the same sugar moiety as 1, and the identical glycosidation shifts observed in 2–10 suggested their moieties were all located at the C-3 hydroxyl group.

Marstenacisside C2 (2) had a molecular formula of C₅₀H₇₆O₁₉ as determined by HRESIMS [M−H]⁻ ion at m/z 979.4924. The NMR data of 2 showed a pattern analogous to 1, except for an ester group at C-11. In the ¹³C-NMR spectrum of 2, the carbon signals of 167.8, 167.1, 138.7, 138.5, 129.0, 128.5, 14.3, 14.3, 12.1 and 12.0 suggested two tigloyl groups in the molecule. For this reason, the ester group at C-11 was deduced to be a tigloyl group. Therefore, the structure of 2 was elucidated to be 3-O-β-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α,12β-di-O-tigloyl-drevogenin P.

Marstenacisside C3 (3) presented a [M–H]⁻ ion at m/z 941.4778 in its HRESIMS spectrum, and its molecular formula was deduced to be C₄₇H₇₄O₁₉. 3 had two mass units more than that of 1. Comparison of the NMR data of 3 with those of 1 revealed that they had almost identical structures except the differences in the A ring portion. No characteristic olefinic carbon signals of C-5 and C-6 were observed in the ¹³C-NMR spectrum of 3. By comparing the NMR data with those of condurangosides A and B isolated from condurango cortex [18], the C₂₁ steroidal skeleton of 3 was identified as (5β,8β,9α,17α)-20-one-3β,11α,12β,14β-tetradroxypregnane (deacylcondurangogenin A). Thus, the structure of 3 was elucidated as 3-O-β-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α-O-acetyl-12β-O-tigloyl-decaylconduragogenin A.

Marstenacisside C4 (4) had a molecular formula of C₅₀H₇₈O₁₉ as determined by HRESIMS [M−H]⁻ ion at m/z 981.5067. The NMR data of 4 showed a pattern analogous to 3, except for the difference of the ester group at C-11. In the ¹³C-NMR spectrum of 4, the carbon signals of 167.8, 167.1, 138.7, 138.5, 129.0, 128.5, 14.3, 14.3, 12.1 and 12.0 suggested two tigloyl groups in the molecule. The tigloyl group was therefore determined to be located at C-11. Accordingly, the structure of 4 was elucidated to be 3-O-β-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α,12β-di-O-tigloyl-decaylconduragogenin A.

Marstenacisside C5 (5) had a molecular formula of C₅₄H₇₂O₂₀ determined by HRESIMS ion [M−H]⁻ at m/z 1039.4574. In the ¹³C-NMR spectrum of 5, the carbon signals at δ 166.8, 166.0, 133.5, 133.2, 130.5, 130.0 (×3), 129.9 (×2), 128.7 (×2) and 128.6 (×2) suggested that double benzoyl groups existed in the molecule. The molecular formula of 5 suggested that it had one more oxygen atom in the C₂₁ steroid skeleton, and by comparing the ¹³C-NMR data of the C₂₁ steroid skeleton between 5 and 1, the significant chemical shifts at C-7 (δ 35.6, +7.4 ppm) and C-8 (δ 76.2, +37.8 ppm) of 5 suggested it had one more hydroxyl group at C-8 than 1. Thus, the C₂₁ steroid skeleton of 5 was deduced to be (9α,17α)-5-ene-20-one-3β,8β,11α,12β,14β-pentadroxypregnane, coinciding with marsdenin [18, 19]. Confirmed by combined use of ¹H–¹H COSY, HSQC and HMBC experiments, the structure of 5 was elucidated as 3-O-β-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α,12β-di-O-benzoyl-marsdenin.

Marstenacisside C6 (6) had a molecular formula of C₅₀H₇₆O₂₀ determined by HRESIMS ion [M–H]^– at m/z 995.4868. The NMR data of 6 presented a pattern analogous to 5 except for the difference in diester groups. In the ¹³C-NMR spectrum of 6, the carbon signals at δ 168.0, 166.9, 138.6, 138.4, 129.2, 128.6, 14.3 (×2) and 12.1 (×2) showed the existence of two tigloyl groups in the molecule. Therefore, the structure of 6 was elucidated as 3-O-β-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α,12β-di-O-tigloyl-marsdenin.

Marstenacisside C7 (7) displayed a molecular formula of C₅₂H₇₄O₂₀ as determined by HRESIMS ion [M−H]⁻ at m/z 1017.4708. The NMR data of 7 showed a pattern analogous to 5 and 6 except for the difference of diester groups. Comparison of NMR data of 7 with those of 5 and 6 showed that it had the same C-11 tigloyl group as 6 and the same C-12 benzoyl group as 5. Thus, the structure of 7 was identified as 3-O-β-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α-O-tigloyl-12β-O-benzoyl-marsdenin.

Marstenacisside C8 (8) had a molecular formula of C₅₂H₇₆O₂₀ as determined by HRESIMS ion [M–H]^– at m/z 1019.4888. The NMR data of 8 suggested it had almost the same structure as 7 except for the obvious difference of C-4 to C-6, and the chemical shift of C-4 (35.7), C-5 (45.8) and C-6 (25.4) of 8 suggested the sp³ hybridization of these carbon atoms. Further comparing the NMR data with those reported in the literature [5, 7, 18], the C₂₁ steroid skeleton of 8 was deduced to be (5α,9α,17α)-20-one-3β,8β,11α,12β,14β-pentadroxypregnane (tenacigenin C). Accordingly, the structure of 8 was elucidated as 3-O-β-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α-O-tigloyl-12β-O-benzoyl-tenacigenin C.

Marstenacisside C9 (9), with a molecular formula of C₅₀H₇₈O₂₀ (HRESIMS [M−H]⁻ ion at m/z 997.5027), had an almost identical structure to 8 except for the ester group at C-12 by comparison of their NMR data. In the ¹³C-NMR spectrum of 9, the carbon signals of two tigloyl groups at δ 168.0, 167.0, 138.4, 138.3, 129.3, 128.7, 14.3, 14.3, 12.0 and 12.0 were observed, suggesting that the ester group at C-12 was a tigloyl group. Therefore, the structure of 9 was identified as 3-O-β-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α,12β-O-ditigloyl-tenacigenin C.

Marstenacisside C10 (10) had a molecular formula of C₅₂H₇₄O₁₉ as determined by HRESIMS ion [M−H]⁻ at m/z 1001.4797. 10 had two hydrogen atoms and one carbon atom less than 8. By comparing their NMR data, it was suggested 10 had almost identical carbon signals to 8 except for obvious differences at C-8 (66.3, −10 ppm) and C-14 (71.7, −14 ppm), suggesting an epoxy structure between C-8 and C-12. Further comparing the NMR data with those reported in the literature [5, 10], the C₂₁ steroid skeleton of 10 was identified as (5α,9α,17α)-8β,14β-epoxy-20-one-3β,11α,12β-tridroxypregnane (17β-tenacigenin B). Thus, the structure of 10 was elucidated as 3-O-β-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α-O-tigloyl-12β-O-benzoyl-17β-tenacigenin B.

Marstenacisside D1 (11) displayed a molecular formula of C₄₁H₆₂O₁₄ as determined by HRESIMS ion [M+HCOO]⁻ at m/z 823.4079. 11 had 162 mass units less than 1, and the NMR data suggested that 11 had an almost identical structure to 1 except for a terminal glucose less than 1. In the anomeric regions of the ¹H- and ¹³C-NMR spectra of 11, two anomeric protons at δ 4.86 (1H, d, J = 10.4 Hz) and 5.12 (1H, d, J = 7.2 Hz), and two anomeric carbon signals at δ 103.3 and 98.2 were observed. By analysis of NMR spectroscopic data, it was deduced that the sugar moiety consisted of a canarose and a 6-deoxy-3-O-methyl-allose. Using ¹H–¹H COSY, HSQC and HMBC experiments, the proton spin systems and the carbon resonances of each sugar could be fully assigned (Table 2). The β configurations of the sugars were all determined by the large coupling constants (³ J _1,2 > 7 Hz). The long correlations between δ 4.86 (Can-H-1) and 77.4 (C-3), and between δ 5.12 (Allo-H-1) and 88.6 (Can-C-4) in the HMBC spectrum deduced the glycosidation site and the sequence of the sugar chain. Therefore, the sugar moiety was deduced as 3-O-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranoside. Accordingly, 11 was elucidated as 3-O-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α-O-acetyl-12β-O-tigloyl-drevogenin P.

Compounds 12–17 presented the same ¹³C-NMR data on sugar moieties as 11, showing that 12–17 contained the same sugar moiety as 1. Moreover, the identical glycosidation shifts observed in 2–10 suggested their moieties were all located at the C-3 hydroxyl group.

Marstenacisside D2 (12) had a molecular formula of C₄₄H₆₆O₁₄ as determined by HRESIMS [M+HCOO]⁻ ion at m/z 863.4423. The NMR data of 12 showed a pattern analogous to 11, except for the ester group at C-11. In the ¹³C-NMR spectrum of 12, two groups of tigloyl carbon signals at δ 167.8, 167.1, 138.7, 138.5, 129.0, 128.5, 14.3, 14.3, 12.1 and 12.0 showed the ester group at C-11 to be tigloyl. Therefore, the structure of 12 was elucidated as 3-O- 6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α,12β-O-ditigloyl-drevogenin P.

Marstenacisside D3 (13) had a molecular formula of C₄₄H₆₆O₁₅ as determined by HRESIMS [M+HCOO]⁻ ion at m/z 879.4347. 13 had 162 mass units less than 6. Comparison of the NMR data of 13 with 6 suggested that 13 had the same aglycone structure as 6. Thus, the structure of 13 could be elucidated as 3-O-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α,12β-O-ditigloyl-marsdenin.

Marstenacisside D4 (14) had a molecular formula of C₄₃H₆₀O₁₅ as determined by HRESIMS [M+HCOO]⁻ ion at m/z 861.3910. The NMR data of 14 showed that it had an almost identical aglycone structure to 5 except the difference of an ester group at C-11. In the ¹³C-NMR spectrum of 14, carbon signals at δ 170.0 and 21.3 were observed, suggesting that the ester group at C-11 was acetyl. As a result, the structure of 14 was elucidated as 3-O-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α-acetyl-12β-di-O-tigloyl-marsdenin.

Marstenacisside D5 (15) had a molecular formula of C₄₆H₆₄O₁₅ as determined by HRESIMS [M+HCOO]⁻ ion at m/z 901.4215. The NMR data of 15 showed a pattern analogous to 14, except for an ester group at C-11. The NMR data of 15 also suggested that it had an almost identical aglycone structure to 13 except for the ester group at C-12. Thus, the diester groups linking to C-11 and C-12 of 15 could be determined to be tigloyl and benzoyl, respectively. Therefore, the structure of 15 was elucidated as 3-O-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α-O-tigloyl-12β-O-benzoyl-marsdenin.

Marstenacisside D6 (16) displayed a molecular formula of C₄₄H₆₄O₁₅ as determined by HRESIMS [M+HCOO]⁻ ion at m/z 901.4191, which was same as 15. Comparison of the ¹³C-NMR data of 16 and 15 suggested they had almost identical carbon signals. However, the carbon chemical shifts of 16 at δ 72.6 (C-11) and 78.3 (C-12) were obviously different to those of 15 at δ 71.6 (C-11) and 79.4 (C-12), proving that 16 had the same diester groups as 15 but with different esterification positions. Thus, the diester groups linking to C-11 and C-12 of 16 could be determined to be benzoyl and tigloyl, respectively. Consequently, the structure of 16 was elucidated as 3-O-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α-benzoyl-12β-O-tigloyl-marsdenin.

Marstenacisside D7 (17) displayed a molecular formula of C₄₆H₆₆O₁₅ as determined by HRESIMS [M+HCOO]⁻ ion at m/z 903.4367. The NMR data of 17 presented a pattern analogous to 16 except for the obviously different chemical shift of C-4 to C-6. Based on comparison of the ¹³C-NMR spectra, 17 was proved to have the same C₂₁ steroid skeleton as 8 and 9. Thus, the structure of 17 was elucidated as 3-O-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α-benzoyl-12β-O-tigloyl-tenacigenin C.

Marstenacisside E1 (18) displayed a molecular formula of C₄₅H₆₈O₁₄ as determined by HRESIMS ion [M−H]⁻ at m/z 831.4525. In the anomeric regions of the ¹H- and ¹³C-NMR spectra of 18, two anomeric protons at δ 4.75 (1H, dd, J = 7.7, 2.0 Hz) and 5.29 (1H, d, J = 8.0 Hz), and two anomeric carbon signals at δ 98.0 and 102.0 were observed, suggesting two sugar units in the molecule. By comparing the NMR data of 18 and 12, it was found that they had identical structures except for the difference in internal sugars. The proton signal δ 3.50 (3H, s) and carbon signal 57.1 in the ¹H- and ¹³C-NMR spectra suggested a –OCH₃ group in the initial sugar unit. By comparison with previously reported values, the initial sugar unit was identified as oleandrose [4], and the sugar moiety of 18 was further identified to be 6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-oleandropyranoside. Consequently, the structure of 18 was elucidated as 3-O-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-oleandropyranosyl-11α,12β-O-ditigloyl-drevogenin P.

The NMR data indicated that 19–23 had the same sugar moiety, 3-O-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-oleandropyranoside, as 18, but differed in their aglycone moieties.

Marstenacisside E2 (19) displayed a molecular formula of C₄₅H₆₈O₁₅ (HRESIMS ion [M−H]⁻ at m/z 847.4548). The aglycone NMR data of 19 showed a pattern analogous to 6 and 13, and by comparing their NMR data, it was deduced that 19 had the same C₂₁ steroidal skeleton (marsdenin) and diester groups (11α-O-tigloyl and 12β-O-tigloyl) as 6 and 13. Therefore, the structure of 19 was elucidated as 3-O-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α,12β-O-ditigloyl-marsdenin.

Marstenacisside E3 (20) displayed a molecular formula of C₄₇H₆₆O₁₅ (HRESIMS ion [M−H]⁻ at m/z 869.4307). The NMR data of 20 showed a pattern analogous to 7 and 15, and comparison of their NMR data indicated that they had the same C₂₁ steroidal skeleton (marsdenin) and the same diester groups (11α-O-tigloyl and 12β-O-benzoyl). Therefore, the structure of 20 was elucidated as 3-O-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α-O-tigloyl-12β-O-benzoyl-marsdenin.

Marstenacisside E4 (21) displayed a molecular formula of C₄₇H₆₆O₁₅ (HRESIMS ion [M−H]⁻ at m/z 869.4407). The NMR data of 21 showed a pattern analogous to 16, indicating by comparison of their NMR data that they had the C₂₁ steroidal skeleton of marsdenin and the diester groups of 11α-O-benzoyl and 12β-O-tigloyl. Therefore, the structure of 20 was elucidated as 3-O-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α-O-benzoyl-12β-O-tigloyl-marsdenin.

Marstenacisside E5 (22) displayed a molecular formula of C₄₅H₇₀O₁₅ (HRESIMS ion [M−H]⁻ at m/z 849.4713). The NMR data of 22 showed a pattern analogous to 9, indicating by comparison of their NMR data that 22 had the same C₂₁ steroidal skeleton of tenacigenin C and the diester groups of 11α-O-benzoyl and 12β-O-tigloyl as 9. Therefore, the structure of 22 was elucidated as 3-O-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α,12β-O-ditigloyl-tenacigenin C.

Marstenacisside E6 (23) displayed a molecular formula of C₄₇H₆₆O₁₄ (HRESIMS ion [M−H]⁻ at m/z 853.4401). The NMR data of 23 showed a pattern analogous to 10, indicating by comparison of their NMR data that 23 had the same C₂₁ steroidal skeleton of 17β-tenacigenin B and the diester groups of 11α-O-tigloyl and 12β-O-benzoyl as 10. Therefore, the structure of 23 was elucidated as 3-O-6-deoxy-3-O-methyl-β-allopyranosyl-(1→4)-β-canaropyranosyl-11α-O-tigloyl-12β-O-benzoyl-tenacigenin C.

In the anomeric regions of the ¹H- and ¹³C-NMR spectra of 24–27, no characteristic anomeric protons and anomeric carbon signals were observed, suggesting no sugar moiety in the molecules of these compounds. Based on analyses of their ¹H- and ¹³C-NMR data, 24–27 were identified to be C₂₁ steroid diester derivatives.

11α,12β-O-Ditigloyl-tenacigenin C (24) had a molecular formula of C₃₁H₄₆O₈ (HRESIMS [M−H]⁻ ion at m/z 545.3143). The NMR data suggested that 24 had the same C₂₁ steroidal skeleton of tenacigenin C and 11α,12β-O-ditigloyl groups as 9. Therefore, the structure of 24 was elucidated to be 11α,12β-O-ditigloyl-tenacigenin C.

11α-O-Benzoyl-12β-O-tigloyl-tenacigenin C (25) had a molecular formula of C₃₃H₄₄O₈ (HRESIMS [M−H]⁻ ion at m/z 567.2981). The NMR data suggested that 25 had the same C₂₁ steroidal skeleton of tenacigenin C and ester groups of 11α-O-benzoyl and 12β-O-ditigloyl as 17. Therefore, the structure of 25 was elucidated to be 11α-O-benzoyl-12β-O-tigloyl-tenacigenin C.

11α-O-Tigloyl-12β-O-benzoyl-tenacigenin C (26) had a molecular formula of C₃₃H₄₄O₈ (HRESIMS [M−H]⁻ ion at m/z 567.2987). The NMR data suggested that 26 had the same C₂₁ steroidal skeleton of tenacigenin C and ester groups of 11α-O-tigloyl-12β-O-benzoyl as 8. Therefore, the structure of 26 was elucidated to be 11α-O-tigloyl-12β-O-benzoyl-tenacigenin C.

11α-O-Tigloyl-12β-O-benzoyl-marsdenin (27) had a molecular formula of C₃₃H₄₂O₈ (HRESIMS [M−H]⁻ ion at m/z 565.6849). The NMR data suggested that 27 had the same C₂₁ steroidal skeleton of marsdenin and ester groups of 11α-O-acetyl and 12β-O-benzoyl as 7 and 15. Therefore, the structure of 27 was elucidated to be 11α-O-acetyl-12β-O-benzoyl-marsdenin.

The relative structures of twenty-seven C₂₁ steroidal glycosides were elucidated and presented in this work. Due to lack of reference sugars, the absolute configurations of the monosaccharides of canarose, 6-deoxy-3-O-methyl-allose and glucose involved in this paper were not determined. According to the literature reporting C₂₁ steroidal glycosides isolated from this plant [4, 5, 7, 10], it was supposed that the configurations of 6-deoxy-3-O-methyl-allose and glucose should be D configurations, and the canarose was also supposed to have the D configuration by comparing the spectroscopic data with those of canarose reported in compounds from plants of the Asclepiadaceae family [20, 21].

Compounds 1–27 were screened for anti-HIV activites. As shown in Table 3, 17, 23, 26 and 27, at 30 μM, had minor inhibitory effects on HIV-1 with the inhibition rates ($\overline{x}$, n = 3) of 81.28, 80.9, 79.8 and 79.5%, respectively. 5–8, 10–15, 18–20, 24 and 25 had slight inhibitory effects on HIV-1 with inhibition rates of 59.0–72.8%, and the other compounds exhibited negligible effects with low inhibition rates of 19.4–49.5%. The above preliminary results suggested that the C₂₁ steroid and the C₂₁ steroidal diglycoside displayed relative stronger activities than the C₂₁ steroidal triglycoside and C₂₁ steroidal tetrglycoside.

Table 3 HIV-1 inhibition rate of 1–27 (each 30 μM) and efavirenz (0.01 μM)

This paper presents a further systematic study on the isolation and structure elucidation of new polyoxypregnane glycosides from the Dai medicine Dai-Bai-Jie, the roots of M. tenacissima, and their anti-HIV activites, as an aid to understanding the constituents of this ethnic medicine, which will benefit the development of this medicine as well as its preparation. The phytochemical work on Dai-Bai-Jie showed that the C₂₁ steroid derivatives of Dai-Bai-Jie had differences from those obtained from Dregea sinensis in aglycone, sugar moiety and ester group, but were more consistent with those of Marsdenia plants, providing the evidence to finally identify the original plant of Dai-Bai-Jie as Marsdenia tenacissima (Roxb.) Moon in terms of chemotaxonomy.

Experimental

General experimental procedures

Optical rotations were measured with a JASCO J-810 polarimeter (JASCO Corporation, Japan). HRESIMS were recorded on a Synapt MS (Waters Corporation, USA). NMR spectra were recorded on a Varian ^UNITYINOVA 600 spectrometer (600 MHz for ¹H-NMR and 150 MHz for ¹³C-NMR, Palo Alto, CA, USA) and a Bruker DRX-500 spectrometer (500 MHz for ¹H-NMR and 125 MHz for ¹³C-NMR, Karlsruhe, Germany), which were measured in pyridine-d ₅ and the chemical shifts are given in δ (ppm). The HPLC analyses were performed on an Agilent 1100 system (Agilent Technologies, USA) equipped with a Venusil XBP C18 column (4.6 mm × 250 mm, ODS, 5 μm, Bonna-Agela, China) and an Alltech 2000 evaporative light scattering detector (temp: 110 °C, gas: 2.4 L/min, Alltech Corporation, USA). The preparative HPLC separations were performed on a NP7000 module (Hanbon Co. Ltd, China) equipped with a Shodex RID 102 detector (Showa Denko Group, Japan) and a Venusil XBP C18 column (8.0 mm × 250 mm, ODS, 5 μm, Bonna-Agela, China). TLC was performed on silica gel GF254 plates (Qingdao Marine Chemical, China). Silica gel H (Qingdao Marine Chemical, China), MCI resin (50 μm, Mitsubishi Chemicals, Japan) and UniPS 40–300 polymer (50 μm, Nano-Mircro Co. Ltd, China) were used for column chromatography.

Plant material

The roots of Marsdenia tenacissima were collected from Zhenyuan, Simao, Yunnan province of China in October 2011 and were identified by Prof. Li-Xia Zhang. A voucher specimen (NO.111010) was deposited in the herbarium of the Beijing Institute of Radiation Medicine, Beijing, China.

Extraction and isolation

The dried roots of M. tenacissima (3 kg) were crushed and extracted with 95% EtOH (24 L) under reflux three times, then the extract was further partitioned between EtOAc and H₂O. The residue (78 g) of the EtOAc-soluble fraction was fractionated on a silica gel column eluted with CH₃Cl₃–MeOH (50:1→7:1) to afford 374 fractions (Fr.₁ 1–374).

Fr.₁ 220–250 was subjected to a MCI resin column eluted with MeOH–H₂O (v/v, 70:30, 80:20) to afford 14 fractions (Fr.₂ 1–14). By repeated HPLC separation with CH₃OH–H₂O and CH₃CN–H₂O, 1 (51.3 mg), 3 (27.3 mg) and 6 (83.1 mg) were obtained from Fr.₂ 7–9; 2 (9.8 mg), 4 (13.1 mg), 6 (40.3 mg) and 9 (54.1 mg) were obtained from Fr.₂ 10; 2 (66.4 mg) and 7 (57.4 mg) were obtained from Fr.₂ 11; 5 (31.3 mg), 8 (19.4 mg) and 10 (67.0 mg) were obtained from Fr.₂ 12.

Fr.₁ 39–98 was subjected to a UniPS 40–300 polymer column eluted with MeOH–H₂O (v/v, 70:30, 75:25) to afford 34 fractions (Fr.₃ 1–34). By repeated HPLC separation with CH₃OH–H₂O and CH₃CN–H₂O, 11 (35.6 mg) and 13 (73.4 mg) were isolated from Fr₃ 9–14; 14 (10.1 mg) was isolated from Fr₃ 15–16; 12 (7.1 mg), 15 (55.6 mg), 16 (17.4 mg), 17 (6.7 mg) and 27 (7.4 mg) were isolated from Fr.₃ 26–31.

Fr.₁ 33–38 was subjected to a MCI resin column eluted with MeOH–H₂O (v/v, 75:25, 100:0) to afford 10 fractions (Fr.₄ 1–10). Fr.₄ 4–8 was further subjected to a MCI resin column eluted with MeOH–H₂O (v/v, 70:30) to afford 42 fractions (Fr.₅ 1–42). By repeated HPLC separation with CH₃OH–H₂O, CH₃CN–H₂O and (CH₃)CO–H₂O, 24 (6.5 mg) was obtained from Fr.₅ 11–13; 18 (27.8 mg), 19 (7.1 mg), 21 (28.7 mg), 22 (39.8 mg), 25 (7.8 mg), 26 (7.5 mg) were obtained from Fr.₅ 16–23; 18 (16.2 mg), 20 (55.0 mg), 23 (12.6 mg) were obtained from Fr.₅ 24–42. The details of the isolation procedure are available as the supporting information.

Marstenacisside C1 (1): C₄₇H₇₂O₁₉; white amorphous powder; $[\alpha ]_{\varvec{D}}^{25}$  = +15.6 (c = 0.0875, CH₃OH). HRESIMS (negative): m/z 939.4587 [M−H]⁻ (calcd. for C₄₇H₇₁O₁₉, 939.4590). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.76 (1H, m, H-3), 5.48 (1H, br d, J = 5.6 Hz, H-6), 5.72 (1H, t, J = 10.2 Hz, H-11), 5.24 (1H, d, J = 10.2 Hz, H-12), 3.14 (1H, dd, J = 9.1, 4.8 Hz, H-17), 1.35 (3H, s, 18-CH₃), 1.22 (3H, s, 19-CH₃), 2.17 (3H, s, 21-CH3), 1.93 (3H, s, Ac-H-2), 7.18 (1H, qq, J = 7.1, 1.3 Hz, Tig-H-3), 1.68 (3H, d, J = 7.1 Hz, Tig-H-4), 1.97 (3H, s, Tig-H-5), 4.82 (1H, dd, J = 10.0, 1.6 Hz, Can-H-1), 1.93 (1H, m, Can-H-2a), 2.48 (1H, m, Can-H-2b), 3.96 (1H, m, Can-H-3), 3.28 (1H, t, J = 8.8 Hz, Can-H-4), 3.55 (H, qd, J = 8.8, 6.2 Hz, Can-H-5), 1.49 (3H, d, J = 6.1 Hz, Can-H-6), 5.07 (1H, d, J = 8.0 Hz, Allo-H-1), 3.87 (1H, J = 8.0, 2.5 Hz, Allo-H-2), 4.45 (1H, br t, J = 2.5 Hz, Allo-H-3), 3.72 (1H, dd, J = 9.7, 2.5 Hz, Allo-H-4), 4.29 (1H, dq, J = 9.7, 6.2 Hz, Allo-H-5), 1.56 (3H, d, J = 6.2, Allo-H-6), 3.79 (1H, s, Allo-3-OCH₃), 4.93 (1H, d, J = 7.7 Hz, Glc-H-1), 3.99 (1H, m, Glc-H-2), 4.23 (1H, dd, J = 8.8, 8.7 Hz, Glc-H-3), 4.19 (1H, dd, J = 9.2, 8.8 Hz, Glc-H-4), 3.97 (1H, m, Glc-H-5), 4.35 (1H, dd, J = 11.6, 5.4 Hz, Glc-H-6a), 4.53 (1H, dd, J = 11.6, 2.4 Hz, Glc-H-6b). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside C2 (2): C₅₀H₇₆O₁₉; white amorphous powder; $[\alpha ]_{\varvec{D}}^{25}$ = +31.8 (c = 0.0592, CH₃OH). HRESIMS (negative): m/z 979.4924 [M−H]⁻ (calcd. for C₅₀H₇₅O₁₉, 979.4903). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.76 (1H, m, H-3), 5.50 (1H, br d, J = 5.7 Hz, H-6), 5.88 (1H, t, J = 10.4 Hz, H-11), 5.24 (1H, d, J = 10.0 Hz, H-12), 3.18 (1H, dd, J = 9.3, 4.8 Hz, H-17), 1.38 (3H, s, 18-CH₃), 1.30 (3H, s, 19-CH₃), 2.16 (3H, s, 21-CH₃), 6.91 (1H, qq, J = 7.1, 1.4 Hz, Tig₁-H-3), 1.57 (3H, d, J = 7.1 Hz, Tig₁-H-4), 1.78 (3H, s, Tig₁-H-5), 7.06 (1H, qq, J = 7.1, 1.3 Hz, Tig₂-H-3), 1.64 (3H, d, J = 7.1 Hz, Tig₂-H-4), 1.89 (3H, s, Tig₂-H-5), 4.82 (1H, dd, J = 10.0, 1.6 Hz, Can-H-1), 1.49 (1H, d, J = 6.1 Hz, Can-H-6), 5.07 (1H, d, J = 8.0 Hz, Allo-H-1), 1.56 (3H, d, J = 6.2, Allo-H-6), 3.79 (1H, s, Allo-3-OCH₃), 4.93 (1H, d, J = 7.7 Hz, Glc-H-1). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside C3 (3): C₄₇H₇₄O₁₉; white amorphous powder; $[\alpha ]_{\varvec{D}}^{25}$ = +9.6 (c = 0.0583, CH₃OH). HRESIMS (negative): m/z 941.4778 [M−H]⁻ (calcd. for C₄₇H₇₃O₁₉, 939.4746). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.80 (1H, m, H-3), 5.56 (1H, t, J = 10.2 Hz, H-11), 5.24 (1H, d, J = 9.8 Hz, H-12), 3.14 (1H, dd, J = 8.8, 5.0 Hz, H-17), 1.32 (3H, s, 18-CH₃), 0.95 (3H, s, 19-CH₃), 2.16 (3H, s, 21-CH₃), 1.92 (3H, s, Ac-H-2), 7.15 (1H, qq, J = 7.1, 1.4 Hz, Tig-H-3), 1.67 (3H, d, J = 7.2 Hz, Tig-H-4), 1.96 (3H, s, Tig-H-5), 4.85 (1H, dd, J = 9.7, 1.4 Hz, Can-H-1), 1.57 (1H, d, J = 6.2 Hz, Can-H-6), 5.09 (1H, d, J = 8.1 Hz, Allo-H-1), 1.58 (3H, d, J = 6.2, Allo-H-6), 3.80 (1H, s, Allo-3-OCH₃), 4.94 (1H, d, J = 7.7 Hz, Glc-H-1). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside C4 (4): C₅₀H₇₈O₁₉; white amorphous powder; $[\alpha ]_{\varvec{D}}^{25}$ = + 24.2 (c = 0.0492, CH₃OH). HRESIMS (negative): m/z 981.5067 [M−H]⁻ (calcd. for C₅₀H₇₇O₁₉, 981.5059). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.81 (1H, m, H-3), 5.72 (1H, t, J = 10.5 Hz, H-11), 5.23 (1H, d, J = 9.9 Hz, H-12), 3.17 (1H, dd, J = 9.3, 5.1 Hz, H-17), 1.35 (3H, s, 18-CH₃), 1.02 (3H, s, 19-CH₃), 2.16 (3H, s, 21-CH₃), 6.91 (1H, qq, J = 7.2, 1.0 Hz, Tig₁-H-3), 1.59 (3H, d, J = 7.1 Hz, Tig₁-H-4), 1.78 (3H, s, Tig₁-H-5), 7.04 (1H, qq, J = 7.1, 1.1 Hz, Tig₂-H-3), 1.63 (3H, d, J = 7.1 Hz, Tig₂-H-4), 1.88 (3H, s, Tig₂-H-5), 4.82 (1H, dd, J = 9.7, 1.4 Hz, Can-H-1), 1.52 (1H, d, J = 6.2 Hz, Can-H-6), 5.08 (1H, d, J = 8.1 Hz, Allo-H-1), 1.57 (3H, d, J = 6.2, Allo-H-6), 3.79 (1H, s, Allo-3-OCH₃), 4.93 (1H, d, J = 7.7 Hz, Glc-H-1). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside C5 (5): C₅₄H₇₂O₂₀; white amorphous powder; $[\alpha ]_{\varvec{D}}^{20}$ = +71.8 (c = 0.0311, CH₃OH). HRESIMS (negative): m/z 1039.4574 [M−H]⁻ (calcd. for C₅₄H₇₁O₂₀, 1039.4539). ¹H-NMR data (500 MHz, pyridine-d ₅): δ 3.85 (1H, m, H-3), 5.44 (1H, d, J = 4.6 Hz, H-6), 6.75 (1H, t, J = 10.7 Hz, H-11), 5.79 (1H, d, J = 10.0 Hz, H-12), 3.31 (1H, overlap, H-17), 1.80 (3H, s, 18-CH₃), 1.74 (3H, s, 19-CH₃), 2.10 (3H, s, 21-CH₃), 8.04 (2H, qq, J = 7.1, 1.4 Hz, Bz₁-H-3, 7), 7.20 (2H, overlap, Bz₁-H-4, 6), 7.31 (3H, t, J = 7.4 Hz, Bz₁-H-5), 8.12 (2H, d, J = 7.9 Hz, Bz₂-H-3, 7), 7.20 (2H, dd, J = 7.8, 7.4 Hz, Bz₂-H-4, 6), 7.39 (3H, t, J = 7.4 Hz, Bz₂-H-5), 4.87 (1H, d, J = 9.6 Hz, Can-H-1), 1.50 (1H, d, J = 6.0 Hz, Can-H-6), 5.08 (1H, d, J = 8.0 Hz, Allo-H-1), 1.58 (3H, d, J = 6.2, Allo-H-6), 3.80 (1H, s, Allo-3-OCH₃), 4.95 (1H, d, J = 7.8 Hz, Glc-H-1). ¹³C-NMR data (125 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside C6 (6): C₅₀H₇₆O₂₀; white amorphous powder; $[\alpha ]_{\varvec{D}}^{25}$ = +26.2 (c = 0.0725, CH₃OH). HRESIMS (negative): m/z 995.4868 [M−H]⁻ (calcd. for C₅₀H₇₅O₂₀, 995.4852). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.85 (1H, m, H-3), 6.42 (1H, t, J = 10.5 Hz, H-11), 5.46 (1H, d, J = 10.2 Hz, H-12), 3.19 (1H, dd, J = 9.0, 5.4 Hz H-17), 1.65 (3H, s, 18-CH₃), 1.58 (3H, s, 19-CH₃), 2.18 (3H, s, 21-CH₃), 6.96 (1H, dq, J = 6.6, 1.2 Hz, Tig₁-H-3), 1.58 (3H, d, J = 8.4 Hz, Tig₁-H-4), 1.81 (3H, s, Tig₁-H-5), 7.07 (1H, dq, J = 7.2, 1.8 Hz, Tig₂-H-3), 1.63 (3H, d, J = 7.2 Hz, Tig₂-H-4), 1.89 (3H, s, Tig₂-H-5), 4.88 (1H, dd, J = 9.6, 1.8 Hz, Can-H-1), 1.52 (1H, d, J = 6.0 Hz, Can-H-6), 5.08 (1H, d, J = 8.4 Hz, Allo-H-1), 1.57 (3H, d, J = 6.0, Allo-H-6), 3.83 (1H, s, Allo-3-OCH₃), 4.93 (1H, d, J = 7.8 Hz, Glc-H-1). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside C7 (7): C₅₂H₇₄O₂₀; white amorphous powder; $[\alpha ]_{\varvec{D}}^{25}$ = +45.1 (c = 0.0592, CH₃OH). HRESIMS (negative): m/z 1017.4708 [M−H]⁻ (calcd. for C₅₂H₇₃O₂₀, 1017.4695). ¹H-NMR data (500 MHz, pyridine-d ₅): δ 3.87 (1H, m, H-3), 5.43 (1H, brs, H-6), 6.57 (1H, t, J = 10.7 Hz, H-11), 5.66 (1H, d, J = 10.1 Hz, H-12), 3.32 (1H, overlap, H-17), 1.75 (3H, s, 18-CH₃), 1.71 (3H, s, 19-CH₃), 2.09 (3H, s, 21-CH₃), 6.77 (1H, q, J = 7.0 Hz, Tig-H-3), 1.38 (3H, d, J = 7.0 Hz, Tig-H-4), 1.55 (3H, s, Tig-H-5), 8.29 (2H, d, J = 7.8 Hz, Bz-H-3, 7), 7.43 (2H, dd, J = 7.8, 7.3 Hz, Bz-H-4, 6), 7.52 (1H, t, J = 7.3 Hz, Bz-H-5), 4.86 (1H, br d, J = 9.6 Hz, Can-H-1), 1.53 (1H, overlap, Can-H-6), 5.09 (1H, d, J = 7.9 Hz, Allo-H-1), 1.59 (3H, d, J = 6.0, Allo-H-6), 3.81 (1H, s, Allo-3-OCH₃), 4.96 (1H, d, J = 7.8 Hz, Glc-H-1). ¹³C-NMR data (125 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside C8 (8): C₅₆H₇₆O₂₀; white amorphous powder; $[\alpha ]_{\varvec{D}}^{25}$ = +43.0 (c = 0.0558, CH₃OH). HRESIMS (negative): m/z 1019.4888 [M−H]⁻ (calcd. for C₅₂H₇₅O₂₀, 1019.4852). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.88 (1H, m, H-3), 6.58 (1H, t, J = 10.8 Hz, H-11), 5.79 (1H, d, J = 10.2 Hz, H-12), 3.32 (1H, overlap, H-17), 1.70 (3H, s, 18-CH₃), 1.50 (3H, s, 19-CH₃), 2.03 (3H, s, 21-CH₃), 6.80 (1H, dq, J = 7.1, 1.4 Hz, Tig-H-3), 1.35 (3H, d, J = 7.1 Hz, Tig-H-4), 1.51 (3H, s, Tig-H-5), 8.24 (2H, d, J = 7.8 Hz, Bz-H-3, 7), 7.40 (2H, dd, J = 7.8, 7.4 Hz, Bz-H-4, 6), 7.49 (3H, t, J = 7.4 Hz, Bz₂-H-5), 4.88 (1H, d, J = 9.6 Hz, Can-H-1), 1.55 (1H, d, J = 6.2 Hz, Can-H-6), 5.08 (1H, d, J = 8.2 Hz, Allo-H-1), 1.57 (3H, d, J = 6.2, Allo-H-6), 3.80 (1H, s, Allo-3-OCH₃), 4.93 (1H, d, J = 7.8 Hz, Glc-H-1). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside C9 (9): C₅₀H₇₈O₂₀; white amorphous powder; $[\alpha ]_{\varvec{D}}^{25}$ = + 25.9 (c = 0.0625, CH₃OH). HRESIMS (negative): m/z 997.5027 [M−H]⁻ (calcd. for C₅₀H₇₇O₂₀, 997.5008). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.89 (1H, m, H-3), 6.45 (1H, t, J = 10.5 Hz, H-11), 5.39 (1H, d, J = 10.2 Hz, H-12), 3.24 (1H, dd, J = 9.2, 5.7 Hz, H-17), 1.61 (3H, s, 18-CH₃), 1.48 (3H, s, 19-CH₃), 2.13 (3H, s, 21-CH₃), 6.94 (1H, qq, J = 7.1, 1.4 Hz, Tig₁-H-3), 1.59 (3H, d, J = 7.1 Hz, Tig₁-H-4), 1.79 (3H, s, Tig₁-H-5), 7.02 (1H, qq, J = 7.1, 1.4 Hz, Tig₂-H-3), 1.60 (3H, d, J = 7.1 Hz, Tig₂-H-4), 1.86 (3H, s, Tig₂-H-5), 4.88 (1H, dd, J = 9.6, 1.8 Hz, Can-H-1), 1.55 (1H, d, J = 6.2 Hz, Can-H-6), 5.08 (1H, d, J = 8.2 Hz, Allo-H-1), 1.57 (3H, d, J = 6.2, Allo-H-6), 3.80 (1H, s, Allo-3-OCH₃), 4.93 (1H, d, J = 7.8 Hz, Glc-H-1). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside C10 (10); C₅₂H₇₄O₁₉; white amorphous powder; $[\alpha ]_{\varvec{D}}^{25}$ = + 18.4 (c = 0.0550, CH₃OH). HRESIMS (negative): m/z 1001.4797 [M−H]⁻ (calcd. for C₅₂H₇₃O₁₉, 1001.4746). ¹H-NMR data (500 MHz, pyridine-d ₅): δ 3.85 (1H, m, H-3), 5.86 (1H, t, J = 10.1 Hz, H-11), 5.53 (1H, d, J = 10.1 Hz, H-12), 3.08 (1H, dd, J = 11.6, 6.4 Hz, H-17), 1.56 (3H, s, 18-CH₃), 1.27 (3H, s, 19-CH₃), 2.02 (3H, s, 21-CH₃), 6.76 (1H, q, J = 7.1 Hz, Tig-H-3), 1.35 (3H, d, J = 7.0 Hz, Tig-H-4), 1.50 (3H, s, Tig-H-5), 8.16 (2H, d, J = 7.9 Hz, Bz-H-3, 7), 7.37 (2H, dd, J = 7.9, 7.4 Hz, Bz-H-4, 6), 7.46 (3H, t, J = 7.4 Hz, Bz₂-H-5), 4.87 (1H, br d, J = 9.7 Hz, Can-H-1), 1.60 (1H, d, J = 6.2 Hz, Can-H-6), 5.11 (1H, d, J = 8.0 Hz, Allo-H-1), 1.55 (3H, overlap, Allo-H-6), 3.81 (1H, s, Allo-3-OCH₃), 4.96 (1H, d, J = 7.9 Hz, Glc-H-1). ¹³C-NMR data (125 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside D1 (11): C₄₁H₆₂O₁₄; white amorphous powder; $[\alpha ]_{\varvec{D}}^{20}$ = +26.2 (c = 0.0311, CH₃OH). HRESIMS (negative): m/z 823.4079 [M+HCOO]⁻ (calcd. for C₄₂H₆₃O₁₆, 823.4116). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.78 (1H, m, H-3), 5.49 (1H, br d, J = 6.0 Hz, H-6), 5.74 (1H, t, J = 10.2 Hz, H-11), 5.25 (1H, d, J = 10.2 Hz, H-12), 3.15 (1H, dd, J = 9.0, 4.8 Hz, H-17), 1.35 (3H, s, 18-CH₃), 1.23 (3H, s, 19-CH₃), 2.17 (3H, s, 21-CH3), 1.93 (3H, s, Ac-H-2), 7.16 (1H, q, J = 7.2 Hz, Tig-H-3), 1.68 (3H, d, J = 6.6 Hz, Tig-H-4), 1.97 (3H, s, Tig-H-5), 4.86 (1H, d, J = 10.4 Hz, Can-H-1), 1.93 (1H, m, Can-H-2a), 2.43 (1H, m, Can-H-2b), 3.95 (1H, m, Can-H-3), 3.37 (1H, t, J = 9.0 Hz, Can-H-4), 3.62 (H, m, Can-H-5), 1.48 (3H, d, J = 6.1 Hz, Can-H-6), 5.12 (1H, d, J = 7.2 Hz, Allo-H-1), 3.94 (1H, m, Allo-H-2), 4.06 (1H, t-like, Allo-H-3), 3.61 (1H, m, Allo-H-4), 4.20 (1H, m, Allo-H-5), 1.46 (3H, d, J = 6.0, Allo-H-6), 3.81 (1H, s, Allo-3-OCH₃). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside D2 (12): C₄₄H₆₆O₁₄; white amorphous powder; $[\alpha ]_{\varvec{D}}^{20}$ = + 58.8 (c = 0.0289, CH₃OH). HRESIMS (negative): m/z 863.4423 [M+HCOO]⁻ (calcd. for C₄₅H₆₇O₁₆, 863.4429). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.78 (1H, m, H-3), 5.51 (1H, br d, J = 6.0 Hz, H-6), 5.88 (1H, t, J = 10.4 Hz, H-11), 5.30 (1H, d, J = 9.6 Hz, H-12), 3.17 (1H, dd, J = 9.6, 4.8 Hz, H-17), 1.39 (3H, s, 18-CH₃), 1.30 (3H, s, 19-CH₃), 2.17 (3H, s, 21-CH₃), 6.92 (1H, qq, J = 6.0, 1.2 Hz, Tig₁-H-3), 1.57 (3H, d, J = 7.2 Hz, Tig₁-H-4), 1.78 (3H, s, Tig₁-H-5), 7.06 (1H, qq, J = 6.6, 1.2 Hz, Tig₂-H-3), 1.64 (3H, d, J = 7.2 Hz, Tig₂-H-4), 1.89 (3H, s, Tig₂-H-5), 4.82 (1H, dd, J = 9.6, 1.8 Hz, Can-H-1), 1.48 (1H, d, J = 6.0 Hz, Can-H-6), 5.10 (1H, d, J = 7.8 Hz, Allo-H-1), 1.57 (3H, d, J = 6.0, Allo-H-6), 3.81 (1H, s, Allo-3-OCH₃). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside D3 (13): C₄₄H₆₆O₁₅; white amorphous powder; $[\alpha ]_{\varvec{D}}^{20}$ = +49.8 (c = 0.0389, CH₃OH). HRESIMS (negative): m/z 879.4347 [M+HCOO]⁻ (calcd. for C₄₅H₆₇O₁₇, 879.4378). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.85 (1H, m, H-3), 6.51 (1H, t, J = 8.4 Hz, H-11), 5.45 (1H, d, J = 10.4 Hz, H-12), 3.19 (1H, dd, J = 8.4, 4.8 Hz H-17), 1.65 (3H, s, 18-CH₃), 1.57 (3H, s, 19-CH₃), 2.17 (3H, s, 21-CH₃), 6.97 (1H, dq, J = 7.2, 1.2 Hz, Tig₁-H-3), 1.58 (3H, d, J = 8.4 Hz, Tig₁-H-4), 1.80 (3H, s, Tig₁-H-5), 7.07 (1H, dq, J = 7.2, 1.2 Hz, Tig₂-H-3), 1.62 (3H, d, J = 7.2 Hz, Tig₂-H-4), 1.90 (3H, s, Tig₂-H-5), 4.86 (1H, dd, J = 9.6, 1.8 Hz, Can-H-1), 1.46 (1H, d, J = 6.0 Hz, Can-H-6), 5.10 (1H, d, J = 7.4 Hz, Allo-H-1), 1.57 (3H, d, J = 6.6, Allo-H-6), 3.81 (1H, s, Allo-3-OCH₃). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside D4 (14): C₄₃H₆₀O₁₅; white amorphous powder; $[\alpha ]_{\varvec{D}}^{20}$ = +59.4 (c = 0.0211, CH₃OH). HRESIMS (negative): m/z 861.3910 [M+HCOO]⁻ (calcd. for C₄₄H₆₁O₁₇, 861.3909). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.87 (1H, m, H-3), 5.39 (1H, br d, J = 6.0 Hz, H-6), 6.39 (1H, t, J = 10.4 Hz, H-11), 5.60 (1H, d, J = 9.6 Hz, H-12), 3.26 (1H, dd, J = 9.0, 4.8 Hz, H-17), 1.74 (3H, s, 18-CH₃), 1.71 (3H, s, 19-CH₃), 2.07 (3H, s, 21-CH₃), 1.60 (3H, s, Ac-H-2), 8.37 (2H, d, J = 6.6 Hz, Bz-H-3, 7), 7.48 (2H, t, J = 7.8 Hz, Bz-H-4, 6), 7.54 (3H, overlap, Bz₂-H-5), 4.88 (1H, dd, J = 9.6, 3.0 Hz, Can-H-1), 1.47 (1H, d, J = 7.2 Hz, Can-H-6), 5.10 (1H, d, J = 7.4 Hz, Allo-H-1), 1.60 (3H, d, J = 7.2, Allo-H-6), 3.81 (1H, s, Allo-3-OCH₃). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside D5 (15): C₄₆H₆₄O₁₅; white amorphous powder; $[\alpha ]_{\varvec{D}}^{20}$ = + 67.0 (c = 0.0556, CH₃OH). HRESIMS (negative): m/z 901.4215 [M−H]⁻ (calcd. for C₄₇H₆₅O₁₇, 901.4222). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.87 (1H, m, H-3), 6.55 (1H, t, J = 10.4 Hz, H-11), 5.64 (1H, d, J = 9.6 Hz, H-12), 3.27 (1H, 1H, dd, J = 8.4, 4.8 Hz, H-17), 1.73 (3H, s, 18-CH₃), 1.69 (3H, s, 19-CH₃), 2.08 (3H, s, 21-CH₃), 6.81 (1H, dq, J = 7.2, 1.2 Hz, Tig-H-3), 1.37 (3H, d, J = 6.0 Hz, Tig-H-4), 1.54 (3H, s, Tig-H-5), 8.28 (2H, d, J = 7.2 Hz, Bz-H-3, 7), 7.42 (2H, t, J = 8.4 Hz, Bz-H-4, 6), 7.51 (3H, t, J = 7.2 Hz, Bz-H-5), 4.87 (1H, dd, J = 9.6, 1.8 Hz, Can-H-1), 1.46 (1H, d, J = 6.0 Hz, Can-H-6), 5.11 (1H, d, J = 8.4 Hz, Allo-H-1), 1.57 (3H, d, J = 6.0, Allo-H-6), 3.81 (1H, s, Allo-3-OCH₃). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside D6 (16): C₄₆H₆₄O₁₅; white amorphous powder; $[\alpha ]_{\varvec{D}}^{20}$ = +58.8 (c = 0.0278, CH₃OH). HRESIMS (negative): m/z 901.4191 [M+HCOO]⁻ (calcd. for C₄₇H₆₅O₁₇, 901.4222). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.84 (1H, m, H-3), 6.61 (1H, t, J = 10.2 Hz, H-11), 5.58 (1H, d, J = 10.4 Hz, H-12), 3.22 (1H, dd, J = 7.2, 3.0 Hz, H-17), 1.71 (3H, s, 18-CH₃), 1.69 (3H, s, 19-CH₃), 2.18 (3H, s, 21-CH₃), 6.90 (1H, dq, J = 7.2, 1.2 Hz, Tig-H-3), 1.42 (3H, d, J = 8.4 Hz, Tig-H-4), 1.61 (3H, s, Tig-H-5), 8.18 (2H, d, J = 6.6 Hz, Bz-H-3, 7), 7.38 (2H, t, J = 7.8 Hz, Bz-H-4, 6), 7.45 (3H, t, J = 6.0 Hz, Bz-H-5), 4.83 (1H, dd, J = 9.6, 1.8 Hz, Can-H-1), 1.45 (1H, d, J = 6.0 Hz, Can-H-6), 5.09 (1H, d, J = 7.8 Hz, Allo-H-1), 1.53 (3H, d, J = 6.0, Allo-H-6), 3.81 (1H, s, Allo-3-OCH₃). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside D7 (17): C₄₆H₆₆O₁₅; white amorphous powder; $[\alpha ]_{\varvec{D}}^{20}$ = +97.5 (c = 0.0139, CH₃OH). HRESIMS (negative): m/z 903.4367 [M+HCOO]⁻ (calcd. for C₄₇H₆₇O₁₇, 903.4378). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.86 (1H, m, H-3), 6.64 (1H, t, J = 10.4 Hz, H-11), 5.52 (1H, d, J = 10.2 Hz, H-12), 3.27 (1H, dd, J = 8.4, 4.8 Hz, H-17), 1.66 (3H, s, 18-CH₃), 1.52 (3H, s, 19-CH₃), 2.14 (3H, s, 21-CH₃), 6.86 (1H, dq, J = 7.2, 1.2 Hz, Tig-H-3), 1.38 (3H, d, J = 7.2 Hz, Tig-H-4), 1.52 (3H, s, Tig-H-5), 8.17 (2H, d, J = 7.2 Hz, Bz-H-3, 7), 7.39 (2H, t, J = 7.8 Hz, Bz-H-4, 6), 7.45 (3H, t, J = 7.2 Hz, Bz₂-H-5), 4.87 (1H, dd, J = 9.6, 1.8 Hz, Can-H-1), 1.46 (1H, d, J = 6.0 Hz, Can-H-6), 5.10 (1H, d, J = 7.8 Hz, Allo-H-1), 1.52 (3H, d, J = 6.0, Allo-H-6), 3.80 (1H, s, Allo-3-OCH₃). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside E1 (18): C₄₅H₆₈O₁₄; $[\alpha ]_{\varvec{D}}^{20}$ = + 32.9 (c = 0.035, CH₃OH). HRESIMS (negative): m/z 831.4525 [M−H]⁻ (calcd. for C₄₅H₆₇O₁₄, 831.4531). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.81 (1H, m, H-3), 5.30 (1H, m, H-6), 5.90 (1H, m, H-11), 5.52 (1H, d-like, H-12), 3.19 (1H, m, H-17), 1.39 (3H, s, 18-CH₃), 1.31 (3H, s, 19-CH₃), 2.16 (3H, s, 21-CH₃), 6.92 (1H, m, Tig₁-H-3), 1.64 (3H, overlap, Tig₁-H-4), 1.78 (3H, s, Tig₁-H-5), 7.07 (1H, m, Tig₂-H-3), 1.64 (3H, overlap, Tig₂-H-4), 1.89 (3H, s, Tig₂-H-5), 4.75 (1H, dd, J = 7.7, 2.0 Hz, Ole-H-1), 1.53 (1H, overlap, Ole-H-6), 3.50 (1H, s, Ole-3-OCH₃), 5.29 (1H, d, J = 8.0 Hz, Allo-H-1), 1.61 (3H, d, J = 6.0, Allo-H-6), 3.81 (1H, s, Allo-3-OCH₃). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside E2 (19): C₄₅H₆₈O₁₅; $[\alpha ]_{\varvec{D}}^{20}$ = + 59.2 (c = 0.0245, CH₃OH). HRESIMS (negative): m/z 847.4548 [M−H]⁻ (calcd. for C₄₅H₆₇O₁₅, 847.4480). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.86 (1H, m, H-3), 5.42 (1H, d, J = 5.9 Hz, H-6), 6.43 (1H, t, J = 8.4 Hz, H-11), 5.46 (1H, d, J = 10.2 Hz, H-12), 3.19 (1H, dd, J = 8.7, 4.6 Hz, H-17), 1.68 (3H, s, 18-CH₃), 1.65 (3H, s, 19-CH₃), 2.17 (3H, s, 21-CH₃), 6.96 (1H, dq, J = 7.1, 1.3 Hz, Tig₁-H-3), 1.58 (3H, d, J = 7.3 Hz, Tig₁-H-4), 1.81 (3H, s, Tig₁-H-5), 7.06 (1H, m, Tig₂-H-3), 1.62 (3H, overlap, Tig₂-H-4), 1.88 (3H, s, Tig₂-H-5), 4.77 (1H, dd, J = 9.7, 2.0 Hz, Ole-H-1), 1.53 (1H, J = 6.2 Hz, Ole-H-6), 3.49 (1H, s, Ole-3-OCH₃), 5.29 (1H, d, J = 8.1 Hz, Allo-H-1), 1.61 (3H, overlap, H-6), 3.81 (1H, s, Allo-3-OCH₃). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside E3 (20): C₄₇H₆₆O₁₅; $[\alpha ]_{\varvec{D}}^{20}$ = + 63.3 (c = 0.019, CH₃OH). HRESIMS (negative): m/z 869.4307 [M−H]⁻ (calcd. for C₄₇H₆₅O₁₅, 869.4323). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.87 (1H, m, H-3), 5.42 (1H, d, J = 5.9 Hz, H-6), 6.56 (1H, t, J = 10.4 Hz, H-11), 5.65 (1H, d, J = 9.6 Hz, H-12), 3.28 (1H, 1H, dd, J = 9.9, 4.9 Hz, H-17), 1.73 (3H, s, 18-CH₃), 1.69 (3H, s, 19-CH₃), 2.07 (3H, s, 21-CH₃), 6.82 (1H, dq, J = 7.2, 1.2 Hz, Tig-H-3), 1.37 (3H, d, J = 6.0 Hz, Tig-H-4), 1.53 (3H, s, Tig-H-5), 8.28 (2H, d, J = 7.2 Hz, Bz-H-3, 7), 7.41 (2H, t, J = 8.4 Hz, Bz-H-4, 6), 7.50 (3H, t, J = 7.1 Hz, Bz-H-5), 4.77 (1H, dd, J = 9.7, 2.0 Hz, Ole-H-1), 1.53 (1H, J = 6.2 Hz, Ole-H-6), 3.49 (1H, s, Ole-3-OCH₃), 5.29 (1H, d, J = 8.0 Hz, Allo-H-1), 1.62 (3H, overlap, H-6), 3.81 (1H, s, Allo-3-OCH₃). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside E4 (21): C₄₇H₆₆O₁₅; $[\alpha ]_{\varvec{D}}^{20}$ = +48.6 (c = 0.0494, CH₃OH). HRESIMS (negative): m/z 869.4407 [M−H]⁻ (calcd. for C₄₇H₆₅O₁₅, 869.4323). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.86 (1H, m, H-3), 6.61 (1H, t, J = 10.6 Hz, H-11), 5.59 (1H, d, J = 10.4 Hz, H-12), 3.22 (1H, dd, J = 9.2, 4.6 Hz, H-17), 1.71 (3H, s, 18-CH₃), 1.69 (3H, s, 19-CH₃), 2.18 (3H, s, 21-CH₃), 6.99 (1H, dq, J = 7.1, 1.2 Hz, Tig-H-3), 1.42 (3H, d, J = 7.1 Hz, Tig-H-4), 1.61 (3H, s, Tig-H-5), 8.19 (2H, d, J = 7.1 Hz, Bz-H-3, 7), 7.37 (2H, t, J = 7.6 Hz, Bz-H-4, 6), 7.44 (3H, t, J = 7.5 Hz, Bz-H-5), 4.74 (1H, dd, J = 9.7, 2.0 Hz, Ole-H-1), 1.52 (1H, J = 6.2 Hz, Ole-H-6), 3.49 (1H, s, Ole-3-OCH₃), 5.28 (1H, overlap, Allo-H-1), 1.60 (3H, overlap, H-6), 3.81 (1H, s, Allo-3-OCH₃). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside E5 (22): C₄₅H₇₀O₁₅; $[\alpha ]_{\varvec{D}}^{20}$ = +44.3 (c = 0.0305, CH₃OH). HRESIMS (negative): m/z 849.4713 [M−H]⁻ (calcd. for C₄₅H₆₉O₁₅, 849.4636). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.88 (1H, m, H-3), 6.45 (1H, t, J = 11.0 Hz, H-11), 5.40 (1H, d, J = 10.1 Hz, H-12), 3.25 (1H, dd, J = 9.3, 5.4 Hz, H-17), 1.62 (3H, s, 18-CH₃), 1.50 (3H, s, 19-CH₃), 2.13 (3H, s, 21-CH₃), 6.94 (1H, qq, J = 6.8, 1.4 Hz, Tig₁-H-3), 1.59 (3H, overlap, Tig₁-H-4), 1.79 (3H, s, Tig₁-H-5), 7.03 (1H, qq, J = 6.9, 1.4 Hz, Tig₂-H-3), 1.60 (3H, overlap, Tig₂-H-4), 1.86 (3H, s, Tig₂-H-5), 4.81 (1H, dd, J = 9.7, 2.0 Hz, Ole-H-1), 1.53 (1H, J = 6.0 Hz, Ole-H-6), 3.50 (1H, s, Ole-3-OCH₃), 5.30 (1H, overlap, Allo-H-1), 1.61 (3H, overlap, H-6), 3.82 (1H, s, Allo-3-OCH₃). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

Marstenacisside E6 (23): C₄₇H₆₆O₁₄; $[\alpha ]_{\varvec{D}}^{20}$ = +53.5 (c = 0.0194, CH₃OH). HRESIMS (negative): m/z 853.4401 [M−H]⁻ (calcd. for C₄₇H₆₅O₁₄, 851.4374). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.88 (1H, m, H-3), 5.85 (1H, t, J = 10.1 Hz, H-11), 5.52 (1H, d, J = 10.1 Hz, H-12), 3.07 (1H, dd, J = 11.5, 6.1 Hz, H-17), 1.53 (3H, s, 18-CH₃), 1.27 (3H, s, 19-CH₃), 2.00 (3H, s, 21-CH₃), 6.75 (1H, q, J = 7.1 Hz, Tig-H-3), 1.34 (3H, d, J = 7.1 Hz, Tig-H-4), 1.49 (3H, s, Tig-H-5), 8.15 (2H, dd, J = 6.8, 1.2 Hz, Bz-H-3, 7), 7.35 (2H, t, J = 8.1 Hz, Bz-H-4, 6), 7.44 (3H, t, J = 7.4 Hz, Bz₂-H-5), 4.79 (1H, dd, J = 9.7, 1.6 Hz, Ole-H-1), 1.53 (1H, overlap, Ole-H-6), 3.51 (1H, s, Ole-3-OCH₃), 5.31 (1H, d, J = 8.0 Hz, Allo-H-1), 1.65 (3H, overlap, H-6), 3.82 (1H, s, Allo-3-OCH₃). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Tables 1 and 2.

11α,12β-O-Ditigloyl-tenacigenin C (24): C₃₁H₄₆O₈; $[\alpha ]_{\varvec{D}}^{20}$ = + 51.3 (c = 0.0025, CH₃OH). HRESIMS (negative): m/z 545.3143 [M−H]⁻ (calcd. for C₃₁H₄₅O₈, 545.31114). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.92 (1H, m, H-3), 6.50 (1H, t, J = 10.6 Hz, H-11), 5.43 (1H, d, J = 10.1 Hz, H-12), 3.26 (1H, dd, J = 9.5, 5.3 Hz, H-17), 1.63 (3H, s, 18-CH₃), 1.55 (3H, s, 19-CH₃), 2.14 (3H, s, 21-CH₃), 7.00 (1H, qq, J = 7.1, 1.4 Hz, Tig₁-H-3), 1.61 (3H, overlap, Tig₁-H-4), 1.84 (3H, s, Tig₁-H-5), 7.05 (1H, m, Tig₂-H-3), 1.61 (6H, overlap,Tig₂-H-4), 1.89 (3H, s, Tig₂-H-5). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Table 1.

11α-O-Benzoyl-12β-O-tigloyl-tenacigenin C (25): C₃₃H₄₄O₈; $[\alpha ]_{\varvec{D}}^{20}$ = +43.8 (c = 0.0172, CH₃OH). HRESIMS (negative): m/z 567.2981 [M−H]⁻ (calcd. for C₃₃H₄₃O₈, 567.2958). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.89 (1H, m, H-3), 6.69 (1H, t, J = 10.3 Hz, H-11), 5.56 (1H, d, J = 10.1 Hz, H-12), 3.29 (1H, dd, J = 9.4, 5.2 Hz, H-17), 1.68 (3H, s, 18-CH₃), 1.58 (3H, s, 19-CH₃), 2.14 (3H, s, 21-CH₃), 6.88 (1H, dq, J = 7.1, 1.2 Hz, Tig-H-3), 1.38 (3H, d, J = 7.2 Hz, Tig-H-4), 1.52 (3H, s, Tig-H-5), 8.22 (2H, d, J = 7.0 Hz, Bz-H-3, 7), 7.40 (2H, t, J = 7.1 Hz, Bz-H-4, 6), 7.46 (3H, t-like, Bz₂-H-5). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Table 1.

11α-O-Tigloyl-12β-O-benzoyl-tenacigenin C (26): C₃₃H₄₄O₈; $[\alpha ]_{\varvec{D}}^{20}$ = + 101.5 (c = 0.022, CH₃OH). HRESIMS (negative): m/z 567.2987 [M−H]⁻ (calcd. for C₃₃H₄₃O₈, 567.2958). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.92 (1H, m, H-3), 6.63 (1H, t, J = 11.3 Hz, H-11), 5.62 (1H, d, J = 10.1 Hz, H-12), 3.35 (1H, dd, J = 9.1, 4.9 Hz, H-17), 1.72 (3H, s, 18-CH₃), 1.57 (3H, s, 19-CH₃), 2.04 (3H, s, 21-CH₃), 6.86 (1H, dq, J = 7.2, 1.2 Hz, Tig-H-3), 1.38 (3H, d, J = 7.1 Hz, Tig-H-4), 1.57 (3H, s, Tig-H-5), 8.26 (2H, d, J = 6.9 Hz, Bz-H-3, 7), 7.41 (2H, t, J = 7.9 Hz, Bz-H-4, 6), 7.50 (3H, t, J = 7.5 Hz, Bz₂-H-5). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Table 1.

11α-O-Tigloyl-12β-O-benzoyl-marsdenin (27): C₃₃H₄₂O₈; $[\alpha ]_{\varvec{D}}^{20}$ = + 34.7 (c = 0.0235, CH₃OH). HRESIMS (negative): m/z 565.6849 [M−H]⁻ (calcd. for C₃₃H₄₁O₈, 565.6837). ¹H-NMR data (600 MHz, pyridine-d ₅): δ 3.92 (1H, m, H-3), 6.59 (1H, t, J = 10.6 Hz, H-11), 5.66 (1H, d, J = 10.2 Hz, H-12), 3.29 (1H, dd, J = 9.6, 4.8 Hz, H-17), 1.75 (3H, s, 18-CH₃), 1.74 (3H, s, 19-CH₃), 2.08 (3H, s, 21-CH₃), 6.77 (1H, dq, J = 7.1, 1.3 Hz, Tig-H-3), 1.39 (3H, d, J = 7.1 Hz, Tig-H-4), 1.58 (3H, s, Tig-H-5), 8.29 (2H, d, J = 7.0 Hz, Bz-H-3, 7), 7.42 (2H, t, J = 7.5 Hz, Bz-H-4, 6), 7.50 (1H, t, J = 7.6 Hz, Bz-H-5). ¹³C-NMR data (150 MHz, pyridine-d ₅) see Table 1.

HIV inhibition assay

HIV inhibition assays were carried out according to a reported method [22]. SupT1 cells (2 × 10⁵) were co-transfected with 0.6 mg of pNL-Luc-E and 0.4 mg of pHIT/G. The VSV-G pseudo-typed viral supernatant (HIV-1) was then harvested by filtration through a 0.45-μm filter after 48 h and the concentration of viral capsid protein was determined by p24 antigen capture ELISA (Biomerieux). SupT1 cells were exposed to VSV-G pseudo-typed HIV-1 (MOI = 1) at 37.8 °C for 48 h in the absence or presence of test compounds (with efavirenz as positive control). A Luciferase Assay System (Promega) was used to determine the inhibition rate. Purities of all tested compounds were >95% detected by HPLC-ELSD. Efavirenz was obtained from the NIH-AIDS Research and Reference Reagent Program with a purity of >98% (HPLC). The tests were independently performed three times.