Anemhupehins A–C, Podocarpane Diterpenoids from Anemone hupehensis

Abstract Three new podocarpane diterpenoids, namely anemhupehins A–C (1–3), together with four known analogues (4–7), have been isolated from aerial parts of Anemone hupehensis. Their structures were characterized based on extensive spectroscopic data. Compounds 1 and 4 showed certain cytotoxicities against human cancer cell lines. Graphical Abstract Electronic supplementary material The online version of this article (10.1007/s13659-017-0146-6) contains supplementary material, which is available to authorized users.

Anemone hupehensis is a flowering herbaceous perennial in the Ranunculaceae family. It is about one meter high and native in central China. In previous chemical studies on this plant, a few triterpenoids and their saponins were reported [12][13][14][15]. In our continuous searching for new and bioactive natural products, we investigated the chemical constituents in aerial parts of A. hupehensis, which resulted . These data elucidated the structure of 1 to be 6,13-dihydroxy-8,11,13-podocarpatriene. An ROESY experiment revealed the relative configuration of 1 (Fig. 2), in which the cross peak of H-19/H-20 indicated that CH 3 -19 and CH 3 -20 were in the same side (b orientation), while the cross peaks of H-18/H-5, H-5/H-6, and H-6/H-18 indicated that H-6 should be a oriented. As established by the ROESY data, the energy minimized 3D structure of 1 revealed stereoconfiguration of 1. A Newman projection of C-5 and C-6 indicated the dihedral angle between H-5 and H-6 to be close to h = 90° (Fig. 2), which resulted in the coupling constants of J 5,6 = 0 Hz, that is why H-5 presented as a singlet in the 1 H NMR spectrum. All these data elucidated the structure of 1 to be 6b,13-dihydroxy-8,11,13podocarpatriene, named anemhupehin A. Compound 2 was isolated as a colorless oil. The HRE-SIMS established the molecular formula to be C 17 H 24 O 2 on the basis of a molecular ion peak at m/z 259.1704 (calcd for C 17 H 23 O 2 : 259.1704 [M -H] -). IR absorption bands (3432, 1612, 1481 cm -1 ) also indicated the presence of hydroxy and phenyl functions. The 13 C NMR and DEPT disclosed 17 carbon resonances including three methyl, four methylene, five methine, and five quaternary carbons (Table 1). These data showed similar patterns to those of 1. rather than at C-6 in 1. Detailed analyses of 2D NMR data indicated that the other parts of 2 were the same to those of 1. In the ROESY spectrum, cross peaks of H-5/H-7 suggested that OH-7 should be b oriented. Therefore, compound 2 was elucidated as 7b,13-dihydroxy-8,11,13podocarpatriene, named anemhupehin B.
Compound 3 was initially isolated as a mixture with 2. A further isolation of this mixture only obtained pure compound 2, while the amount of compound 3 is too less to obtain the clear NMR spectra. However, careful analyses of MS data of 3 and NMR spectra of the mixture (see Supporting Information), with the aid of NMR spectrum of 2, can unambiguously elucidate the structure of 3. The Analyses of 1D and 2D NMR data suggested that compound 3 should have the same planar structure to that of 1. However, the 13 C NMR shift of C-7 at d C 68.4 in 3 was significantly different from that in 2, indicating that compound 3 might be an epimer of 2. In the ROESY spectrum of the mixture, the cross peaks of 19/H-20 and H-18/H-5 suggested that C-5 and C-10 possessed the same configurations to those in compounds 2. Therefore, compound 3 was determined to be 7-epimer of 2. The 3D structure analyses of 3 and 2 also supported that OH-7 in 3 should be a oriented. Therefore, compound 3 was elucidated as 7a,13-dihydroxy-8,11,13-podocarpatriene, named anemhupehin C. Four known analogues were identified as 13,14-dihydroxy-8,11,13-podocarpatriene (4) [16], 13-hydroxy-8,11,13-podocarpatriene (5) [17], 13,14-dihydroxy-7-oxo-8,11,13-podocarpatriene (6) [17], and 13-hydroxy-7-oxo-8,11,13-podocarpatriene (7) [18]. Compound 4 was isolated as a natural product for the first time.
Compounds 1 and 4-7 were evaluated for their cytotoxicities to five human cancer cell lines. As a result, compounds 1 and 4 showed moderate activities as shown in Table 2. The other compounds were inactive to cancer cell lines (IC 50 [ 40 lM).

General Experimental Procedures
Optical rotations were measured on a Jasco-P-1020 polarimeter. IR spectra were obtained using a Bruker Tensor 27 FT-IR spectrometer with KBr pellets. NMR spectra were acquired with a Bruker DRX-600 with tetramethylsilane (TMS) used as an internal standard. HRESIMS were recorded on an API QSTAR Pulsar spectrometer. Silica gel (200-300 mesh), Sephadex LH-20 and RP-18 gel (20-45 lm) were used for column chromatography (CC). Medium pressure liquid chromatography (MPLC) was performed on a Biotage system. Preparative high performance liquid chromatography (prep-HPLC) was performed on an Agilent 1260 liquid chromatography system equipped with Zorbax SB-C18 columns (5 lm, 9.4 mm 9 150 mm or 21.2 mm 9 150 mm) and a DAD detector. Fractions were monitored by TLC and spots were visualized by heating silica gel plates immersed in H 2 SO 4 in EtOH, in combination with the Agilent 1200 series HPLC system (Eclipse XDB-C18 column, 5 lm, 4.6 9 150 mm).

Plant Material
Aerial parts of A. hupehensis were collected from Shen-LongJia of Hubei province, central China in September 2016 and identified by Prof. Ming-Qing Pan of Wuhan University. A specimen (No. COP-HF20160912.4) was deposited at South-Central University for Nationalities.