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14,15-Secopregnane-Type Glycosides with 5α:9α-Peroxy and Δ6,8(14)-diene Linkages from the Roots of Cynanchum stauntonii

Three new 14,15-secopregnane-type glycosides, stauntosides UA, UA1, and UA2, were isolated from the roots of Cynanchum stauntonii. The three compounds share the first reported and same basic structural features of 3β-hydroxy-14:16,15:20,18:20-triepoxy-5α:9α-peroxy-14,15-secopregnane-6,8(14)-diene named as stauntogenin G as the aglycones. The structures of the new compounds were characterized on the basis of extensive spectroscopic analyses, mainly 1D and 2D NMR and MS methods and chemical analysis. The isolation and identification of the new compounds graced the structural diversity of pregnane-type steroids from C. stauntonii.


Results and Discussion
The roots of C. stauntonii were extracted with 95% EtOH. The 95% EtOH extract was concentrated and partitioned using petroleum ether and EtOAc. The EtOAc-soluble fraction was separated using multiple column chromatographies and preparative HPLC. As a result, three new compounds reported herein were yielded, all as white amorphous powders. All three compounds showed positive Libermann-Burchard and Keller-Kiliani reactions, suggesting their glycosidated steroids or triterpenoids categories with 2-deoxysugars in their sugar moieties [2,15]. The category of steroidal glycosides was determined according to their shared common features in the NMR spectra. The three or four distinguishable anomeric proton signals of sugars in the 1 H-NMR spectra indicated the presence of corresponding sugar moieties (Table 1). When putting off the carbon signals of the three or four hexose units, all three compounds left twenty-one carbons from the C21-pregnane moieties (Tables 1 and 2). All three compounds showed two singlets of methyl groups in their 1 H-NMR spectra.
The δ values of the relatively lower-field singlet of methyl groups in each compound, i.e., δH 1.54 for all three compounds, along with the carbon signals at δC 118.4, 119.6, and 118.2 in the 13 C-NMR spectra for compounds 1-3, respectively, suggested that they belonged to the unusual 14,15-seco-(or 13,14:14,15-diseco-) C21-pregnane-type steroids. These singlets of methyl groups in the 1 H-NMR spectra were born of Me-19 and 21 of the C21-pregnane skeleton, respectively. These carbon signals are the typical features of a dioxygenated secondary carbon-20 structure and they were confirmed by the correlations from Me-21 to C-17 and 20 in the respective HMBC spectrum of the three compounds. No carbonyl carbon was present in the 13 C-NMR spectra of all three compounds, assigning them to be the 14,15-seco-C21-pregnane-type steroids [2,3,7].

Results and Discussion
The roots of C. stauntonii were extracted with 95% EtOH. The 95% EtOH extract was concentrated and partitioned using petroleum ether and EtOAc. The EtOAc-soluble fraction was separated using multiple column chromatographies and preparative HPLC. As a result, three new compounds reported herein were yielded, all as white amorphous powders. All three compounds showed positive Libermann-Burchard and Keller-Kiliani reactions, suggesting their glycosidated steroids or triterpenoids categories with 2-deoxysugars in their sugar moieties [2,15]. The category of steroidal glycosides was determined according to their shared common features in the NMR spectra. The three or four distinguishable anomeric proton signals of sugars in the 1 H-NMR spectra indicated the presence of corresponding sugar moieties (Table 1). When putting off the carbon signals of the three or four hexose units, all three compounds left twenty-one carbons from the C 21 -pregnane moieties (Tables 1 and 2). All three compounds showed two singlets of methyl groups in their 1 H-NMR spectra.
The δ values of the relatively lower-field singlet of methyl groups in each compound, i.e., δ H 1.54 for all three compounds, along with the carbon signals at δ C 118.4, 119.6, and 118.2 in the 13 C-NMR spectra for compounds 1-3, respectively, suggested that they belonged to the unusual 14,15-seco-(or 13,14:14,15-diseco-) C 21 -pregnane-type steroids. These singlets of methyl groups in the 1 H-NMR spectra were born of Me-19 and 21 of the C 21 -pregnane skeleton, respectively. These carbon signals are the typical features of a dioxygenated secondary carbon-20 structure and they were confirmed by the correlations from Me-21 to C-17 and 20 in the respective HMBC spectrum of the three compounds. No carbonyl carbon was present in the 13 C-NMR spectra of all three compounds, assigning them to be the 14,15-seco-C 21 -pregnane-type steroids [2,3,7].   Compound 1 possessed a molecular formula of C 40 H 58 O 15 according to its 13 C-NMR spectroscopic data and the HRESIMS (positive ion mode) data for the protonated molecular ion at m/z 779.3861 and sodium adduct molecular ion at m/z 801.3694, indicating a hydrogen deficiency index of twelve. This molecular formula has one fewer oxygen atom than that of stauntoside V 3 , a 14,15-secopregnane-type glycoside with the aglycone of stauntogenin F (3β-hydroxy-8α:14α,14:16,15:20,18:20-tetraepoxy-5α:9αperoxy-14,15-secopregn-6-ene) previously isolated by our group from C. stauntonii [3]. Its IR spectrum displayed absorption bands for hydroxy (3440 cm −1 ) and olefinic (1681 cm −1 ) functionalities, among others. Acid hydrolysis, along with derivatization and GC analysis, indicated the presence of D-canaropyranose, D-digitoxopyranose, and L-cymaropyranose in a 1:1:1 ratio. The entire 1 H and 13 C-NMR spectroscopic data for 1 are given in Tables 1 and 2, respectively. A detailed comparison of NMR data between 1 and stauntoside V 3 showed that they were very similar. In the 1 H-NMR spectrum, all the signals for 1 were nearly superimposable on their counterparts in stauntoside V 3 , the coupling constants of the three anomeric protons provided evidence that two monosaccharides shared β-glucosidic bonds and one possessed an α-glucosidic bond. In the 13 C-NMR spectrum, the primary difference was in the replacement of the signals for an oxygenated tertiary carbon at δ C 70.8 (s, C-8) and an dioxygenated secondary carbon at δ C 98.9 (s, C-14) in the known compound by the signals of an olefinic quaternary carbon at δ C 110.4 (C-8) and an oxygenated olefinic tertiary carbon at δ C 156.8 (s, C-14) in 1. The rest of the carbons of compound 1 showed full accordance with categories of carbon types with their respective counterparts in stauntoside V 3 . The conjugated highfield shifts of C-6 and C-7 by ∆ δ -10.9 and −2.7, respectively, in compound 1 compared with stauntoside V 3 were evident, which proposed a double bond linkage between C-8 and C-14. A highfield shift of ∆ δ -6.2 for C-10 compared with stauntoside V 3 was also observed, which was mainly due to the impact of the change of magnetic anisotropy from the 8:14-epoxy linkage (oxirane) in stauntoside V 3 to the ∆ 8 (14) structure in 1. For the rest of carbon atoms, except for C-12 and C-18 which, because of the same causes as C-10, showed up lowfield shifts of ∆ δ +1.4 and +1.82, respectively, the numerical range of the absolute values of ∆ δ compared with the corresponding carbons in stauntoside V 3 were less than 1.0, including those of the sugar moieties. These consistencies and differences of functional groups and chemical shifts between 1 and stauntoside V 3 , especially the molecular formula with a hydrogen deficiency index of twelve, indicated the presence of the peroxo bridge structure between C-5 and C-9, just as in stauntoside V 3 . Further, this identification was confirmed by the finding that the peroxylated downfield chemical shifts of ∆ δ +11.8 and +11.1 were observed for C-5 and C-9, respectively, in the 13 C-NMR spectrum compared with stauntoside U, another 14,15-secopregnane-type glycoside with the aglycone of stauntogenin E (8α:14α,14:16,15:20,18:20-tetraepoxy-14,15-secopregn-6-ene-3β,5α,9α-triol) previously isolated by our group from C. stauntonii [3]. The HMBC spectrum showed the same picture as those in stauntoside V 3 , with the following correlations being well-marked: H-6 to C-4, 5, 8, and 10; H-7 to C-5, 9, and 14; H-15a to C-16, 17, and 20; H-17 to C-12, 13, 14, 18, and 20; H-18a to C-12 and 14; H-18b to C-12, 14, 17, and 20; Me-19 to C-1, 5, 9, and 10; Me-21 to C-17 and 20; H-1 of α-L-cymaropyranose to C-4 of β-D-digitoxopyranose; H-1 of β-D-digitoxopyranose to C-4 of β-D-canaropyranose; and H-1 of β-D-canaropyranose to C-3, among others ( Figure 2). The typical relative configurations of 14,15-secopregnane-type steroids of compound 1, i.e., both CH 2 -18 and Me-19 in β-orientation and H-16, H-17, and Me-21 all in α-orientation, which was also the same as that of stauntoside V 3 , were affirmed in the NOESY spectrum by the same picture as those of stauntoside in the 13 C-NMR spectrum compared with stauntoside U, another 14,15-secopregnane-type glycoside with the aglycone of stauntogenin E (8α:14α,14:16,15:20,18:20-tetraepoxy-14,15-secopregn-6-ene-3β,5α,9α-triol) previously isolated by our group from C. stauntonii [3]. The HMBC spectrum showed the same picture as those in stauntoside V3, with the following correlations being well-marked: H-6 to C-4  Compound 2 possessed a molecular formula of C49H74O19 according to its 13 C-NMR data and the HRESIMS (positive ion mode) of the sodium adduct molecular ion at m/z 989.4729, indicating a hydrogen deficiency index of thirteen. Its IR spectrum displayed absorption bands for hydroxy (3393 cm −1 ) and olefinic (1646 cm −1 ) functionalities. Acid hydrolysis of 2, along with derivatization and GC analysis, indicated the presence of D-thevetopyranose, D-cymaropyranose, and L-diginopyranose in a 1:2:1 ratio. A detailed comparison of the 1 H-and 13 C-NMR data between 1 and 2 showed that 2 was nearly entirely identical to 1, with respect to their aglycone moieties (Tables 1 and 2), the numerical range of Δδ for all the 13 C-NMR signals of the aglycone of 2 compared with the corresponding carbons in 1 was between +1.1 and +1.6, which were obviously systematic errors, suggesting the same aglycone for 2 and 1. This determination was confirmed with a combined interpretation of the 2D NMR spectra of 2, including the 1 H, 1 H-COSY, HSQC, and HMBC correlations (data not shown). In addition to the resonances of the aglycone moiety, the 1 H and 13 C-NMR data (Tables 1 and 2) and the 2D NMR spectroscopic features, including the 1 H, 1 H-COSY and HMBC correlations, among others, of the sugar moiety of 2 were consistent with stauntoside V1, a 14,15-secopregnane-type glycoside previously isolated by our group from C. stauntonii [3]. Thus, the structure of 2 is 14:16,15:20,18:20-triepoxy- in the 13 C-NMR spectrum compared with stauntoside U, another 14,15-secopregnane-type glycoside with the aglycone of stauntogenin E (8α:14α,14:16,15:20,18:20-tetraepoxy-14,15-secopregn-6-ene-3β,5α,9α-triol) previously isolated by our group from C. stauntonii [3]. The HMBC spectrum showed the same picture as those in stauntoside V3, with the following correlations being well-marked: H-6 to C-4, 5, 8, and 10; H-7 to C-5, 9, and 14; H-15a to C-16, 17, and 20; H-17 to C-12, 13, 14, 18, and 20;  H-18a to C-12 and 14; H-18b to C-12  Compound 2 possessed a molecular formula of C49H74O19 according to its 13 C-NMR data and the HRESIMS (positive ion mode) of the sodium adduct molecular ion at m/z 989.4729, indicating a hydrogen deficiency index of thirteen. Its IR spectrum displayed absorption bands for hydroxy (3393 cm −1 ) and olefinic (1646 cm −1 ) functionalities. Acid hydrolysis of 2, along with derivatization and GC analysis, indicated the presence of D-thevetopyranose, D-cymaropyranose, and L-diginopyranose in a 1:2:1 ratio. A detailed comparison of the 1 H-and 13 C-NMR data between 1 and 2 showed that 2 was nearly entirely identical to 1, with respect to their aglycone moieties (Tables 1 and 2), the numerical range of Δδ for all the 13 C-NMR signals of the aglycone of 2 compared with the corresponding carbons in 1 was between +1.1 and +1.6, which were obviously systematic errors, suggesting the same aglycone for 2 and 1. This determination was confirmed with a combined interpretation of the 2D NMR spectra of 2, including the 1 H, 1 H-COSY, HSQC, and HMBC correlations (data not shown). In addition to the resonances of the aglycone moiety, the 1 H and 13 C-NMR data (Tables 1 and 2) and the 2D NMR spectroscopic features, including the 1 H, 1 H-COSY and HMBC correlations, among others, of the sugar moiety of 2 were consistent with stauntoside V1, a 14,15-secopregnane-type glycoside previously isolated by our group from C. stauntonii [3]. Thus, the structure of 2 is 14:16,15:20,18:20-triepoxy- Compound 2 possessed a molecular formula of C 49 H 74 O 19 according to its 13 C-NMR data and the HRESIMS (positive ion mode) of the sodium adduct molecular ion at m/z 989.4729, indicating a hydrogen deficiency index of thirteen. Its IR spectrum displayed absorption bands for hydroxy (3393 cm −1 ) and olefinic (1646 cm −1 ) functionalities. Acid hydrolysis of 2, along with derivatization and GC analysis, indicated the presence of D-thevetopyranose, D-cymaropyranose, and L-diginopyranose in a 1:2:1 ratio. A detailed comparison of the 1 H-and 13 C-NMR data between 1 and 2 showed that 2 was nearly entirely identical to 1, with respect to their aglycone moieties (Tables 1 and 2), the numerical range of ∆ δ for all the 13 C-NMR signals of the aglycone of 2 compared with the corresponding carbons in 1 was between +1.1 and +1.6, which were obviously systematic errors, suggesting the same aglycone for 2 and 1. This determination was confirmed with a combined interpretation of the 2D NMR spectra of 2, including the 1 H, 1 H-COSY, HSQC, and HMBC correlations (data not shown). In addition to the resonances of the aglycone moiety, the 1 H and 13 C-NMR data (Tables 1 and 2) and the 2D NMR spectroscopic features, including the 1 H, 1 H-COSY and HMBC correlations, among others, of the sugar moiety of 2 were consistent with stauntoside V 1 , a 14,15-secopregnane-type glycoside previously isolated by our group from C. stauntonii [3]. Thus, the structure of 2 is 14:16,15:20,18:20-triepoxy- Compound 3 possessed a molecular formula of C 48 H 72 O 19 according to its 13 C-NMR data and the HRESIMS (positive ion mode) of the sodium adduct molecular ion at m/z 975.4567, indicating a hydrogen deficiency index of thirteen. Its IR spectrum displayed absorption bands for hydroxy (3369 cm −1 ) and olefinic (1662 cm −1 ) functionalities. Acid hydrolysis of 3, along with derivatization and GC analysis, indicated the presence of D-thevetopyranose, D-digitoxopyranose, D-cymaropyranose, and L-diginopyranose in a 1:1:1:1 ratio. A detailed comparison of the 1 H-and 13 C-NMR data of 1-3 indicated that they shared the same aglycone, compound 3 being nearly entirely identical to 1 and 2, with respect to their aglycone moieties and the numerical range of ∆ δ for all the 13 C-NMR signals of the aglycone of 3 compared with the corresponding carbons in 2 being between −1.4 and −1.6 (Tables 1 and 2). This determination was confirmed with a combined interpretation of the 2D NMR spectra of 3, including the 1 H, 1 H-COSY, HSQC, and HMBC correlations (data not shown). In addition to the resonances of the aglycone moiety, the 1 H-and 13 C-NMR data (Tables 1 and 2) and the 2D NMR spectroscopic features, including the 1 H, 1 H-COSY and HMBC correlations, among others, of the sugar moiety of 3 were consistent with stauntoside W, a 14,15-secopregnane-type glycoside previously isolated by our group from C. stauntonii [3].

General Experimental Procedures
All the instruments, solvents, reagents, and experimental conditions for the measurements of IR spectra, 1D and 2D NMR spectra, and both ESIMS and HRESIMS data and for the performing of column chromatography (CC), preparative HPLC procedure, and TLC analysis were previously described [2,3].

Plant Material
The collecting, species identifying, and depositing of the roots of C. stauntonii were previously described [2,3].

Extraction and Isolation
The extraction of the dried and pulverized roots (30 kg) of C. stauntonii and the preliminary isolation of the 95% EtOH extract and the consequent EtOAc-soluble portion to afford thirteen subfractions (Fr. 1 to Fr. 13) of silica gel CC fractionation were previously described [2,3]