Two pairs of epimers and three undescribed diterpenoids from Pseudocaryopteris paniculata

Abstract A phytochemical investigation on the petroleum ether partition of the whole plant of Pseudocaryopteris paniculata, yield seven new compounds: one phytanes diterpenoid (2Z,6E,10E) 14-keto-2,6,10-trimethyl pentadeca-2,6,10-trien-1-carboxylic acid (1), five clerodane diterpenoids: paniculatins A-E (2, 3a/3b, 4a/4b), one abietane diterpenoid: ent-uncinatone (5), together with 12 known compounds. Their structures were elucidated on the basis of 1D and 2D Nuclear Magnetic Resonance （NMR_, Infrared Radiation (IR), and mass spectroscopic data. Compound 2, 5, and 11 showed weak selective cytotoxic activity of 11 human cancer cell lines. Graphical Abstract


Introduction
The genus Caryopteris (Verbenaceae) comprises approximately 15 species which are widespread in Central and East Asia (Zhao et al. 2016). Many species have been used as Chinese folk medicines due to their biological activities, such as antitumor, antimicrobial, and insect-antifeeding activity (Luo et al. 2016). In previous studies by our lab, a series of diterpenoids were isolated (Zhang et al. 2017), several of which have cytotoxic activity against several human cancer cell lines.

Results and discussion
The 70% acetone extract of P. paniculata was partitioned between Petroleum ether (PE) and EtOAc. The PE phase was subjected to column chromatography (CC), medium pressure liquid chromatography (MPLC), preparative HPLC, and preparative thin-layer chromatography (TLC), 7 new compounds ( Figure 1) with 12 known compounds were gained.
Compound 2 Table S2). These data suggested compound 2 was a tricyclic diterpenoid with a pair of stereo-isomer. The 1 H and 13 C NMR spectra of 2 with aid of DEPT and HSQC spectra were very similar to 15-hydroxy-16-oxo-15,16H-hardwickiic acid methyl ester (Krishna and Singh 1999), except for the replaced position at C-15 and C-16.  Figure S11). On the basis of the deduction, compound 2 was a pair of stereo-isomer and named as paniculatin A.
Nevertheless, the chemical shifts of 1 H and 13 C NMR of these four compounds are slightly different. Compared with 3a and 3b, the chemical displacement of C-15 and C-16 in 4a and 4b moved to the low field by about 1.5 and 1.0 ppm, respectively. In the NOESY spectra of these four compounds, the correlations between H-17 ( Figures S63 and S64) and the specific rotation of four compounds in the same condition while showed different power (3a: À83.3, 3b: À94.0, 4a: À75.0, 4b: À42.2) indicated 3a and 3b were epimers of C-15 or C-16, 4a and 4b were another pair epimers of C-15 or C-16. Therefore, compounds 3a/3b and 4a/4b were named as paniculatin B-E, respectively.
Nevertheless, even in the same solvent, inspection of the 13 C NMR spectrum of compound 5 and uncinatione also revealed significant differences (Supplementary  Table S5): the C-8 and C-13 resonances in 5 were shifted upfield by 1.0 and 7.4 ppm in CDCl 3 , respectively, and C-17 resonance was shifted downfield by 7.2 ppm in CDCl 3 . This is due to the c-gauch effect. Therefore, compound 5 was the enantiomer at C-10 and C-16 with uncinatione, and named as ent-uncinatone.

General experimental procedures
Optical rotations were obtained with an Auto pol VI polarimeter. UV spectra were recorded on a TU-1901 spectrometer IR spectra were recorded with a Perkin Elmer FT-IR spectrometer. 1D-and 2D-NMR spectra were run on a Bruker AVANCE-III instrument operating at 600/400 MHz for 1 H, 125/100 MHz for 13 C, respectively, with tetramethylsilane as internal standard. HRESI-MS spectra were obtained on a Waters UPLC Premior QTOF spectrometer. Reversed phase MPLC was performed on a GRACE system. Preparative HPLC was performed on an Agilent 1260 series instrument equipped with a Shiseido Capcellpak C 18 column (250 Â 20 mm, 5 lm, flow rate: 16 mL/min).

Plant material
The whole plant of P. paniculata (C.B.Clarke) P.D.Cantino (Lamiaceae) was collected in Xingyi of Guizhou Province, People's Republic of China, in April 2015. The identification of plant material was verified by Mr. Zhaoyi Deng (Qianxinan Institute of Karst regional development). A voucher specimen (No. ZHY2015041201) has been deposited in the Shanghai R&D Center for Standardization of Traditional Chinese Medicine, Shanghai 201203, P. R. China.

Extraction and isolation
The dried and powdered plants of P. paniculata (19.4 kg) were extracted with 70% acetone (80 L Â 3, each 10 days) at room temperature and the solvent was evaporated under reduced pressure, and partitioned with petroleum ether (PE, 4 Â 20 L) and EA (4 Â 20 L), PE extract (290 g) and EA extract (200 g) were gotten.

Cell assay
The cytotoxicity of all the compounds from P. paniculata was measured by a CCK-8 method. Compounds were dissolved with dimethylsulphoxide. The cells were seeded onto 96-well microplates at a density of 1 Â 10 4 cells per well in 100 lL of medium each. After incubation, at 37 C in a humidified incubator for 24 h, the cells were treated with various concentrations of each compound. After incubation, 10% CCK-8 (10 lL in 90 lL FBS) was added to each well of the plate after removal from the medium; the cells were incubated at 37 C for 1 h. Cell viability was calculated as a percentage of viable cells in the compound-treated group vs. the control group by following equation: Cell viability (%) ¼ [1 À OD (Compound)/OD (Blank)] Â 100%.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
Financial support from the National Nature Science Foundation of China (No. 21672152) is gratefully acknowledged.