•Nine 14,15-secopregnane-type C21-steriosides were isolated from Cynanchum stauntonii.•An unusual 14,15-secopregnane with 5:9-peroxy bridge moiety was characterized.•Two sugar moieties composed of deoxysugars were characterized.•Active compounds selectively inhibited production of nitric oxide.•Bioactive C21-steriosides underlie the traditional application of C. stauntonii.Nine 14,15-secopregnane-type C21-steriosides, stauntosides U, V, V1-V3, W and C1-C3, as well as two known C21-steriosides, were isolated from the roots of Cynanchum stauntonii. Stauntosides U, V and V1-V3 share the same basic structural features of 8α:14α,14:16,15:20,18:20-tetraepoxy-14,15-secopregn-6-ene-3β,5α,9α-triol, with the numbering system following that of C21-pregnanes. The aglycones of stauntosides U, V and V1-V3 are classified into two subcategories, the 5,9-dihydroxy groups and 5α:9α-peroxy bridge, according to the oxidative states of the two hydroxy groups at the C-5 and C-9 positions. The anti-inflammatory activity of the major compounds was assessed in an in vitro inflammatory model of mouse peritoneal macrophages using IC50 values of the inhibition of nitric oxide (NO) production as an indicator. Stauntosides V1 and V3 exhibited target activity with IC50 values of 9.3 μM and 12.4 μM, respectively, compared with dexamethasone, which was used as a positive control.IC50 of anti-inflammatory activies: stauntoside V1 12.4 μM; stauntoside V3 9.3 μM.Download high-res image (141KB)Download full-size image

•More information on the chemical constituents of Ephedra sinica are described.•Five new acetamide glycosides were characterized.•The N,N-bissubstituted acetamides showed obvious rotamerism in solution.Five new N-mono-/bis-substituted acetamide glycosides, N-{4-O-[3-O-(4-O-α-l-rhamnopyranosyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (1), N-methyl-N-{4-O-[3-O-(4-O-α-l-rhamnopyranosyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (2), N-methyl-N-{4-O-[3-O-(6-O-benzoyl-4-O-α-l-rhamnopyranosyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (3), N-methyl-N-{4-O-[3-O-(6-O-benzoyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (4), and N-methyl-N-{4-O-[3-O-(6-O-trans-cinnamoyl-4-O-α-l-rhamnopyranosyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (5), along with one known acetamide derivative, N-methyl-N-(4-hydroxyphenethyl)-acetamide, the shared aglycone of 2–5, were isolated from the ethanol extract of the stems of Ephedra sinica. The structures of these new compounds were elucidated on the basis of extensive spectroscopic examination, mainly including multiple 1D and 2D NMR and HRESIMS examinations, and qualitative chemical tests. All N,N-bissubstituted acetamide glycosides were found to show the obvious rotamerism, as in the case of the isolated known N-methyl-N-(4-hydroxyphenethyl)-acetamide, under the experimental NMR conditions, with the ratios of integrated intensities between anti- and syn-rotamers always being found to be about 4 to 3.Five new N-mono-/N,N-bis-substituted acetamide glycosides were isolated from the stems of Ephedra sinica. The structures of these new compounds were elucidated on the basis of extensive spectroscopic analyses.

•Twenty five 13-substituted quaternary coptisine derivatives were synthesized.•Introduction of alkyls into 13-position led to significant increases of cytotoxicity.•Extending the alkyl side chain increased the cytotoxic activity.•Aromaric methyl and analogs of arylmethyls causes cytotoxicity on IEC-6.•Introducing other arylmethyls into 13-position showed no effect on tested activity.Twenty five 13-substituted quaternary coptisine derivatives were synthesized to test their cytotoxicities against several cancer cell-lines and on intestinal epithelial cell-6 (IEC-6) in vitro to evaluate structure–activity relationship (SAR). Introduction of the alkyl groups into the C-13 position of quaternary coptisine (1) led to significant increase of the cytotoxic activity, while the substitution of arylmethyl groups and others at the same position showed no effect on improving cytotoxicities against the same cancer cell-lines. The cytotoxicities of quaternary 13-alkylcoptisines was significantly reinforced as the length of the aliphatic chain increased, with quaternary 13-n-undecylcoptisine (4l) showing 7, 23, 12, and 9 times, respectively, more active than quaternary coptisine (1) against HCT, A549, Bel7402, and C33A, and being 4, 11, 2, and 3 times, respectively, more active than the positive control, fluorouracil (5-FU), against the same cell-lines, by IC50 values. In comparison to quaternary 13-n-undecylcoptisine (4l) and the above references, quaternary 13-n-dodecylcoptisine (4m) almost showed the same cytotoxicities. In contrast with the n-alkyl chains, the arylmethyl substituents at C-13 displayed low cytotoxicity, except for naphthyl rings or phenyl rings with CF3 or methyl substituents. However, their low cytotoxicity could make them useful as drug candidates for other diseases (bowel, etc).Twenty five 13-substituted quaternary coptisine derivatives were synthesized and tested for their cytotoxicity to evaluate the structure–activity relationships, with explicit influencing factor on improving activity being obtained.

Nine new steroidal glycosides, named as stauntosides C–K (2, 5, 7–10, 13, 14, and 16), along with seven known compounds (1, 3, 4, 6, 11, 12, and 15) were isolated from the 95% ethanol extract of the roots of Cynanchum stauntonii. The structures of these new compounds were elucidated on the basis of extensive spectroscopic analyses, mainly 1D and 2D NMR, and HRESI-MS, and qualitative chemical methods. Their significance in terms of the chemotaxonomy of C. stauntonii is discussed.Graphical abstractNine new steroidal glycosides were isolated from the roots of Cynanchum stauntonii. The structures of these new compounds were elucidated on the basis of extensive spectroscopic analyses and chemical methods.Download full-size imageHighlights► More information about the chemical constituents of Cynanchum stauntonii were given. ►·Sixteen compounds were identified, including nine new steroidal glycosides. ►·Two new 14,15-secopregnane-type skeleton aglycones were reported for the first time. ► Several new oligosaccharide chains were found for the first time.