Four new resin glycosides, calyhedins VII–X, from the rhizomes of Calystegia hederacea

Abstract Four new resin glycosides with macrolactone structures (jalapins), namely, calyhedins VII (1)–X (4), were isolated from the rhizomes of Calystegia hederacea Wall. (Convolvulaceae). The structures of 1–4 were determined based on spectroscopic data. They were classified into three ring types: a 27-membered ring (1), a 22-membered ring (2, 3), and a 23-membered ring (4). Their sugar moieties were partially acylated using five organic acids, including (E)-2-methylbut-2-enoic acid, 2S-methylbutyric acid, and 2 R-methyl-3R-hydroxybutyric acid. Compound 4 was the first genuine resin glycoside with calyhedic acid F as the glycosidic acid component. Additionally, the cytotoxic activities of 1, 2, and 4 towards HL-60 human promyelocytic leukaemia cells were evaluated. All compounds demonstrated almost the same activity as the positive control, cisplatin. Graphical Abstract


Introduction
Calystegia hederacea Wall. (Convolvulaceae) is a perennial herbaceous vine that grows widely throughout India and East Asia. All parts of this plant are used for the treatment of menoxenia and gonorrhoea (Hotta et al. 1989). In a previous study, we reported four monosaccharides (D-glucose, D-fucose, D-quinovose, and L-rhamnose), three organic acids [(E)-2-methylbut-2-enoic (tiglic), 2S-methylbutyric, and 2 R-methyl-3R-hydroxybutyric (2 R,3R-nilic) acids], and two hydroxyl fatty acids [11S-hydroxyhexadecanoic (jalapinolic) and 12S-hydroxyhexadecanoic acids] as components of the crude resin glycoside fraction of the whole plants of C. hederacea (Ono et al. 2020a). The absolute configurations of the monosaccharides were determined by high-performance liquid chromatography (HPLC) analysis of their thiocarbamoyl-thiazolidine derivatives and those of 2-methylbutyric acid and nilic acid were defined by comparing the specific rotations of their p-bromophenacyl esters and the 1 H NMR spectral data for the (-)-a-methoxy-a-trifluoromethylphenylacetic acid (MTPA) ester of p-bromophenacyl nilate with those of authentic samples. The absolute configurations of the two hydroxyl fatty acids were determined by comparing the 1 H NMR spectral data for the (þ)-MTPA ester of methyl jalapiniolate with those of an authentic sample and applying Mosher's method to methyl 12-hydroxyhexadecanoate (Ono et al. 2020a). We also isolated six new glycosidic acids, calyhedic acids A-F, and two known glycosidic acids, calysolic acids A and B as the components of the crude resin glycoside fraction (Ono et al. 2020a(Ono et al. , 2020b. We also reported the isolation and structural elucidation of six new genuine macrolactone-type resin glycosides (jalapins) (Ono et al. 2021) using 11Sjalapinolic acid or 12S-hydroxyhexadecanoic acid as the aglycone. These glycosides were named calyhedins I-VI, obtained from the rhizomes of C. hederacea. It was found that either of the aglycones could be distinguished via methylene carbon signals in the 13 C NMR spectrum, particularly the signal due to C-14. If the chemical shift of the signal due to C-14 was approximately d 32, the aglycone was 11S-jalapinolic acid, whereas if it was approximately d 28, the aglycone was 12S-hydroxyhexadecanoic acid (Ono et al. 2021). As part of an ongoing study of resin glycosides obtained from this plant, the current report investigates the isolation and elucidation of the structures of four new genuine resin glycosides from the rhizomes of C. hederacea. Additionally, the cytotoxic activity of three isolated resin glycosides towards HL-60 human promyelocytic leukaemia cells was evaluated.

Results and discussion
Air-dried rhizomes of C. hederacea were extracted using methanol (MeOH). The extract was treated with Diaion HP20, silica gel, Sephadex LH-20, and ODS column chromatography as well as HPLC on ODS and naphthylethyl group bonded silica (p NAP) to yield four compounds (1-4).
Compound 3 (calyhedin IX) was obtained as an amorphous powder. The negativeion FAB-MS and positive-ion FAB-MS indicated that the molecular weight of 3 was 1806, which was 2 mass units smaller than that of 2, and the molecular formula was C 83 H 138 O 42 , determined via HR-positive-ion FAB-MS. The 1 H and 13 C NMR spectra of 3 were similar to those of 2, except for the appearance of signals due to 1 mol of tigloyl residue (Tig) and the absence of signals due to 1 mol of 2-methylbutyryl residue (Tables S1, S2). In addition, acylation shifts were observed for the same signals as those observed for 2. In the HMBC spectrum of 3, key correlations were observed between H-6b [  6.05 (1H, d, J ¼ 3.5 Hz)] of dd,J ¼ 10.0,10.0 Hz)] of Rha and C-1 (d 175.6) of Nla',dd,J ¼ 8.0,9.5 Hz)] of dd,J ¼ 9.5,9.5 Hz)] of Glc' and C-1 (d 166.5) of Tig,dd,J ¼ 9.5,9.5 Hz)] of Glc' and C-1 (d 175.5) of Mba" ( Figure S1). Furthermore, the negative-ion FAB-MS of 3 yielded fragment ion peaks at m/z 1313 and 1171 ( Figure S2). These data indicate that Nla, Nla', Mba, Mba", Tig, and Agl were linked with C-6 of Glc, C-4 of Rha, C-2 of Rha, C-4 of Glc', C-3 of Glc', and C-2 of Glc', respectively. Accordingly, 3 was proposed to be an analogue of 2, in which the Mba' of 2 was replaced by Tig (Figure 1).
Compound 4 (calyhedin X) was obtained as an amorphous powder, and its molecular weight was determined to be 1662 by both positive-and negative-ion FAB-MS. HRpositive-ion FAB-MS showed the molecular formula of 4 to be C 77 H 130 O 38 . The 1 H and 13 C NMR spectra of 4 were similar to those of calyhedin VI (8) (Ono et al. 2021), except for the additional signals due to 2 mol of niloyl residues and the disappearance of the signals due to 1 mol of b-glucopyranosyl and 2 mol of 2-methylbutyryl residues (Tables S1, S2). In particular, the 13 C NMR data of Agl (C-14, d 27.7) were almost the same as those of 8, indicating that the aglycone of 4 was 12S-hydroxyhexadecanoic acid. Meanwhile, a comparison of the 13 C NMR signals arising from the sugar moiety of 4 with those of 8 revealed upfield shift (5.6 ppm) and downfield shift (2.1 ppm) of the signals corresponding to C-3 and C-4, respectively, of Rha. Furthermore, the HMBC spectrum of 4 showed correlations between H-1 [  Figure S1). These data indicate that 4 comprised 1 mol of calyhedilic acid F (9) (Ono et al. 2020b), 2 mol of 2methylbutyric acid, and 3 mol of nilic acid. By comparing the chemical shifts of the 1 H NMR signals between 4 and 9, downfield shifts due to acylation were observed for the same signals as those of 2. In addition, the HMBC spectrum of 4 showed key crosspeaks between H-6b [ Compounds 1, 2, and 4 were evaluated for their cytotoxic activity towards HL-60 human promyelocytic leukaemia cells because it had been previously reported that the calyhedins II (10) and III (11), which have similar macrolactone structures to those of 1, 2, and 4, exhibited cytotoxicity towards HL-60 cells (Ono et al., 2021). All the tested compounds demonstrated cytotoxic activities (1, IC 50 7.50 ± 0.72 mM; 2, IC 50 8.53 ± 0.39 mM; 4, IC 50 8.43 ± 0.88 mM) similar to that of the positive control, cisplatin (IC 50 13.0 ± 2.44 mM). The activity of 3 could not be determined because there was insufficient material left after completion of the structural elucidation procedures. Although the cytotoxicity of many resin glycosides has been reported, further studies are needed to clarify the structure-activity relationship of resin glycosides to cytotoxicity due to their structural diversity (Pereda-Miranda et al., 2005;Yin et al., 2009;Fan et al., 2014Fan et al., , 2015Bautista et al., 2016;Corona-Castañeda B et al., 2016;Sura et al., 2019;Ono et al., 2019Ono et al., , 2020aOno et al., , 2021Xu et al., 2020;Lu et al., 2021).

Plant material
The rhizomes of C. hederacea were collected in Fukuoka Prefecture, Japan, in October 2012, and identified by one of authors (Professor Okawa M.). A voucher specimen (CHRFU2012) has been deposited at the laboratory of Natural Products Chemistry, School of Agriculture, Tokai University.

Cell
The HL-60 human promyelocytic leukaemia cells (JCRB0085) was obtained from Japanese Collection of Research Bioresources (Tokyo, Japan).

Conclusion
All the compounds isolated from the rhizomes of Calystegia hederacea, namely calyhedins VII (1)-X (4), had intramolecular cyclic ester structures. Therefore, they were classified as jalapin-type resin glycosides (Ono, 2017). Furthermore, they were classified into three ring types: a 27-membered ring with an ester linkage of the carboxyl group of 11S-hydroxyhexadecanoic acid residue at OH-2 of the fourth glucosyl residue (1), a 22membered ring with an intramolecular ester group between the carboxyl group of 11S-hydroxyhexadecanoic acid residue and OH-2 of the second glucosyl residue (2 and 3), and a 23-membered ring with an ester linkage between the carboxyl group of the 12S-hydroxyhexadecanoic acid residue and OH-2 of the second glucosyl residue (4). Compound 4 was the first genuine resin glycoside with calyhedic acid F as the glycosidic acid component. Compounds 1, 2, and 4 showed almost the same cytotoxic activity towards HL-60 cells as that of cisplatin.