Clerodane-type Diterpene Glycosides from Dicranopteris pedata

Graphic Abstract Three new clerodane-type diterpene glycosides, (5R,6S,8R,9S,10R)-6-O-[β-d-glucopyranosyl-(1 → 4)-α-l-rhamnopyranosyl]cleroda-3,13(16),14-diene (1), (5R,6S,8R,9S,10R,13S)-6-O-[β-d-glucopyranosyl-(1 → 4)-α-l-rhamnopyranosyl]-2-ox-oneocleroda-3,13-dien-15-ol (2), (5R,6S,8R,9S,10R)-6-O-[β-d-glucopyranosyl-(1 → 4)-α-l-rhamnopyranosyl]-(13E)-2-oxoneocleroda-3,14-dien-13-ol (3), together with two known compounds 4 and 5 were isolated from Dicranopteris pedata. The structures of these compounds were elucidated by detailed spectroscopic analysis, and the absolute configuration of compound 2 was determined by ECD calculations. In addition, compound 1 exhibited weak inhibitory activities against SMMC-7721, MCF-7 and SW480. Supplementary Information The online version contains supplementary material available at 10.1007/s13659-021-00315-y.


Introduction
The genus Dicranopteris is consists of about 10 species, distributed mainly in Asia and widely in Japan, India, Vietnam and China. There are six species in China and they mainly distribute in the south of the Yangtze River such as Yunnan, Sichuan and Guizhou provinces [1]. The whole plant of D. pedata is commonly used as a folk medicine in ancient China for the treatment of hemorrhage, dysentery and empyrosis [2,3]. Previous researches revealed a diversity of pharmacological properties of the extract of D. pedata, such as antioxidant, antibacterial, antinociceptive, anti-inflammatory and antipyretic activities [4][5][6][7][8]. Up to date, various secondary metabolites including flavonoids [9,10], phenols [11], proanthocyanidins [12] and clerodane-type diterpene glycosides [10,13] have been reported from D. pedata. Our previous investigation on this species revealed the presence of two oxygenated phenolic derivatives [14], dichotomains A and B and some clerodane-type diterpene glycosides, in which dichotomain A has exhibited weak anti-HIV activity [15,16]. As part of our ongoing search for new bioactive metabolites from fern plants, a chemical investigation on the whole plant of D. pedata led to the discovery of three previously undescribed compounds (1-3) (Fig. 1) and two known analogues (compounds 4 and 5) from the acetone extract [15,16]. This paper describes the details of isolation, structure identification, and cytotoxicity of compounds 1-3.

Results and Discussion
Compound 1 was isolated as pale yellow powder. Its molecular formula was deduced as C 32 H 52 O 10 by HRESIMS analysis (found m/z 619.3454 [M + Na] + , calcd for 619.3458), suggesting seven degrees of unsaturation. The 13 C and 1 H NMR spectra displayed two set of signals ascribed to a diterpene and two hexoses. Five methines (δ C 103.2, 72.6, 72.7, 83.5, 68.8) and a methyl (δ C 17.9) revealed the presence of rhamnose. In addition, the spectra displayed signals for a glucopyranosyl unit (five methines at δ C 105.7, 76.1, 78.1, 71.5, 78.2 and a methylene at δ C 62.7). Assignment of each glycosidic proton system was achieved by analysis of 1 H-1 H COSY, HMBC and HSQC spectra (Fig. 2). The 1 H NMR spectrum of the diterpene displayed the presence of four methyls at δ H 0.73 (s), 1.08 (s), 0.84 (d, J = 6.7 Hz) and 1.73 (d, J = 1.1 Hz, an olefinic methyl), and six olefinic protons at δ H 5.24 (br s), 6.37 (dd, J = 17.6, 10.9 Hz), 5.20 (d, J = 17.6 Hz), 5.04 (d, J = 10.9 Hz), and 4.97 (2H, d, J = 4.4 Hz). The signal at δ H 3.43 (dd, J = 11.2, 4.7 Hz) could be attributed to a proton on the oxygenated carbon. Twenty carbons of the diterpene could be classified to four methyls, seven methylenes (two olefinic at δ C 113.3, 116.2), five methines (one oxygenated at δ C 87.7; two olefinic at δ C 123.6, 140.1) and four quaternary carbons (two olefinic at δ C 144.6, 148.9). The structure of 1 was identified as a clerodane-type diterpenoid glycoside with two sugar moieties. Aforementioned 1D NMR resonances of 1 showed good agreement with the known compound 5 [16], except for the presence of one olefinic methylene and one olefinic quaternary carbon, together with the absence of a methyl and hydroxyl. The difference of compounds 1 and 5 lie in C-13, C-14, C-15 and C-16. The HMBC correlations (Fig. 2)  Thus the planar structure of compound 1 was determined.
The relative configurations of α-rhamnopyranosyl and β-glucopyranosyl moiety were identified by the coupling constants of their anomeric protons of the rhamnopyranosyl (H-1ʹ, δ H 4.79, J = 1.5 Hz) and glucopyranosyl (H-1ʺ δ H 4.59, J = 7.8 Hz), respectively. The location of the sugar chain was determined at C-6 of the aglycone by HMBC correlations ( Fig. 2) from the anomeric proton (H-1ʹ, δ H 4.79) of the rhamnopyranosyl unit to C-6 (δ C 87.7). HMBC correlation from H-1ʺ (δ H 4.59, J = 7.8 Hz) to C-4ʹ (δ C 83.5) defined a glucopyranosyl (1 → 4) rhamnosyl linkage. The absolute configurations of sugar moieties were determined by the experiments of acid hydrolysis, together with sugar derivatization and analysis with HPLC [16]. The retention times of the product in the test sample are consistent with that of standard sugar derivatives in HPLC (Rha derivative: t R = 6.68 min; Glc derivative: t R = 12.78 min), which gave d-glucose and l-rhamnose.
The presence of α-l-rhamnose and β-d-glucose were identified by the same method as compound 1. By the HMBC correlations of C-6 (δ C 85)/H-1ʹ (δ H 4.86) and C-4ʹ (δ C 83.3)/H-1ʺ (δ H 4.86, d, J = 7.8) (Fig. 2), the linkage positions of the sugar moieties were confirmed. The relative configurations of the diterpene were determined the same for their similar ROESY correlations as 1: H-6/H-10, H-6/H-8, and CH 3 -19/CH 3 -20 (Fig. 3) absolute configuration of 2 was determined by electronic circular dichroism (ECD) experiment which fitted well with the calculated ECD (Fig. 4). Finally, the structure of 2 was determined as (5R,6S,8R,9S,10R,13S) Compound 3 was obtained as pale yellow powder. It showed a molecular ion peak at m/z 651.3356 [M + Na] + (calcd for 651.3351), corresponding to the molecular formula of C 32 H 52 O 12 , which indicating seven degrees of unsaturation. The 1D NMR spectra demonstrated two hexoses and a diterpene. The spectroscopic data of the sugar moieties were consistent with 1 and 2, suggesting the presence of α-l-rhamnopyranosyl moiety and β-dglucopyranosyl moieties. The diterpene part involved five methyls (one at δ C 23.8), five methylenes (one oxygenated at δ C 59.1), five methane (one olefinic at δ C 125.5) and five quaternary carbons (one olefinic at δ C 140.2). Those spectra suggested that the structure of 3 was highly similar to that of 2. In comparison with 2, the chemical shifts of C-14, C-15 and C-16 decreased from δ C 146.1, 112.5 and 27.7 to δ C 125.5, 59.1 and 23.8 respectively, while C-13 increased from δ C 73.8 to 140.2, which testified the position of the double bond of 3 at C-13 and C-14. This was demonstrated again by the HMBC correlations of H 2 -15/C-13, C-14 and CH 3 -16/C-13, C-14. In the meantime, C-15 was determined to be a terminal oxymethylene by the chemical shift (δ C 59.1) and 1 H-1 H COSY correlation of H 2 -15/H-14. Besides, the linkage of the sugar moieties was determined by the identical way with 1 and 2. Thus the plannar structure of compound 3 was determined.

Conclusion
In conclusion, this research led to the isolation of five compounds including three new and two known clerodanetype diterpene glycosides from D. pedata.

General Experimental Procedures
Optical rotations were carried out on Autopol VI automatoc polarimeter. UV spectra were obtained using a Shimadzu UV-2401 PC spectrophotometer. A Thermo Nicolet iS10 spectrometer was used for measuring IR spectroscopy, which used KBr pellets. 1D and 2D NMR spectra were recorded on Bruker DRX-600 and spectrometers with SiMe 4 (TMS) as an internal standard. Chemical shifts (δ) are expressed in ppm with reference to the solvent signals. ESI and HRESIMS were performed on an UPLC-IT-TOF spectrometer.

Extraction and Isolation of Compounds 1-5
The dry fronds of Dicranopteris pedata (9 kg) were powdered and extracted with acetone (

Acid Hydrolysis and Derivatization
Compounds 1-3 were hydrolyzed with trifluoroacetic acid (TFA) by the procedure of the previous reported [17], with minor modifications. Compound 1 (2 mg, 3.35 μmol) was dissolved in 2 M TFA (2 mL), after the reaction mixture was heated to 120 °C and stirred for 2 h before cooling to room temperature. Then, extracted with CHCl 3 (3 × 1 mL) and

Cytotoxicity Assay
MTT assays [18] were used to evaluating the cytotoxicities of compounds  if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.

Conflict of interest All authors declare no conflict of interest.
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