Highly oxygenated ent-atisane and podocarpane diterpenoids from Excoecaria agallocha

Abstract A new highly oxygenated ent-atisane diterpenoid, namely excagallonoid A (1), together with five known analogues (2 − 6) were isolated from the leaves and twigs of Excoecaria agallocha. Their structures were elucidated on the basis of extensive spectroscopic analyses (HRESIMS, UV, IR, 1 D and 2 D NMR), and the absolute configurations of 1 and 5 were determined by single-crystal X-ray diffraction. Compound 1 represents the first example of an ent-atisane diterpenoid featuring a vicinal 2,3-diol moiety. Compounds 1 and 4 exhibited weak cytotoxicities in vitro against RKO colon cancer cells with IC50 values of 28.7 ± 1.98 and 32.6 ± 2.81 µM, respectively. Graphical Abstract


Introduction
ent-Atisanes are a relatively small group of diterpenoids characterised by the 6/6/6/6carbocyclic carbon framework. To date, more than 150 ent-atisane diterpenoids have been isolated, and most of them were isolated from the species of the family Euphorbiaceae, Lamiaceae, and Rosaceae (Drummond et al. 2020). The structural diversity of this class of diterpenes arises from a variety of oxidation and functional group patterns on the basic scaffold. Over the past few decades, their fascinating chemical structures and important biological activities have attracted great interest from both synthetic and pharmacological communities Girotra 1963, 1964;Taguchi et al. 2003;Huang et al. 2007;Trana et al 2019).
Excoecaria agallocha L. (Euphorbiaceae) is a mangrove plant widely distributed in Guangdong and Guangxi Provinces of China and several countries of Southeast Asia (Editor Committee for Flora of China 1997). Its leaves and latex are used as fish poison in some Asian countries, while the barks are used in Thailand folk medicine for the treatment of flatulence (Chan et al. 2018). Previous phytochemical studies of this plant have led to the identification of numerous diterpenoids (Anjaneyulu et al. 2003;Wang et al. 2007;Annam et al. 2015;Jiang et al. 2019Jiang et al. , 2020, triterpenoids (Zou et al. 2006), polyphenols (Li et al. 2012), linear alkane hydrocarbons (Satyan et al. 2012), and flavonoids (Rifai et al. 2011). Some of these compounds showed diverse biological properties, including anti-cancer, antiviral, and anti-inflammatory activities (Li et al. 2010(Li et al. , 2012Rifai et al. 2011). In this study, a new ent-atisane diterpenoid, excagallonoid A (1), with a vicinal 2,3-diol moiety, together with five known diterpenoids (2 À 6) were isolated from the leaves and twigs of E. agallocha. Their structures were characterised on the basis of comprehensive spectroscopic data analyses, and the absolute configurations of 1 and 5 were established by single-crystal X-ray diffraction experiments. Compounds 1 and 4 showed weak cytotoxicities against RKO colon cancer cells. Herein, the isolation and structural elucidation, and cytotoxic activity of these compounds are described.

Results and discussion
The dried leaves and twigs of E. agallocha were powdered and extracted with 95% EtOH. The crude extract was suspended in H 2 O and then partitioned sequentially with petroleum ether (PE) and EtOAc (each 2 L Â 3). The EtOAc fraction was separated by repeated column chromatography over silica gel, C 18 reversed phase (RP-C 18 ), and finally HPLC to give ent-atisane and podocarpane diterpenoids (1 À 6) ( Figure 1 The 13 C NMR spectrum of 1 showed 20 carbon resonances that were classified by DEPT and HSQC spectra as a ketone carbonyl carbon (d C 216.9), a exocyclic double bond (d C 147.4 and 106.8), three methyls, six sp 3 methylenes, five sp 3 methines (including two oxygenated at d C 78.3 and 70.7), and three quaternary carbons. One ketocarbonyl and one double bond accounted for two out of the six degrees of unsaturation, suggesting a tetracyclic ring system for 1. The aforementioned spectroscopic information was similar to that of ent-atisanes previously isolated from the genus Excoecaria (Kang et al. 2005;Jiang et al. 2020), suggesting that compound 1 was an ent-atisane diterpenoid.
The relative configuration of 1 was partially established via the NOESY data (supporting information Figure S2) and 1 HÀ 1 H coupling constants. The strong NOE correlations of H-1b/H-3, H-3/H-5, and H-5/H-9 suggested that they were on the axial positions and were randomly designated as b-orientations. The equatorial and b-orientation of H-2 was indicated by a small coupling constant (J ¼ 3.7 Hz) between H-2 and H-3. Consequently, the NOE correlation of H-1a/Me-20 assigned Me-20 to be a-oriented, while the b-orientations of C8 À C15 and C12 À C16 bonds were suggested by the NOE correlations of H-9/H-15b, H-15b/H-17b, and H-17a/H-12. Finally, the relative configuration of 1 was further secured by a successful X-ray diffraction experiment [Flack parameter ¼ 0.1 (9)], which also established the absolute configuration of 1 (supporting information Figure S3) as 2 R,3S,5S,8S,9S,10R,12R. Thus, the structure of 1 was established and named as excagallonoid A.
It is worth noting that the oxidized positions of the ent-atisane scaffold at C-16, C-17, and C-3 (96%, 82% and 59% occurrence, respectively) are common in nature, but both C-2 and C-3-oxidated ent-atisanes are relatively rare (Drummond et al. 2020). Compound 1 is the first ent-atisane diterpenoid containing a vicinal 2,3-diol moiety.   Figure S1) of 5 was determined as the same as the known diterpenoid 6,12-dihydroxy-13-methylpodocarpa-5,8,11,13-tetraene-3,7-dione previously isolated from the Drypetes gossweileri (Ngouela et al. 2003), based on the detailed 2 D NMR analyses (HSQC, HMBC, and 1 HÀ 1 H COSY) (supporting information Figure S1). The relative configuration of the only chiral center (C-10) was tentatively assigned as R Ã in previous report (Ngouela et al. 2003). In the current study, the absolute configuration of 5 (10S) was determined for the first time by single crystal X-ray diffraction analysis with Cu Ka radiation [Flack parameter ¼ 0.005 (6)] (supporting information Figure S4). In addition, the optical rotation, UV, and IR of 5 were also reported for the first time in this study.
Since numerous these diterpenoids have been reported to possess cytotoxicity (Drummond et al. 2020), all isolates (1 À 6) were evaluated for their cytotoxicity against RKO colon cancer cell line. Results revealed that compounds 1 and 4 showed weak cytotoxicities with IC 50 values of 28.7 ± 1.98 and 32.6 ± 2.81 mM, respectively, while other compounds were inactive (IC 50 > 100 mM). 5-FU was used as a positive control (IC 50 ¼ 9.6 ± 1.2 mM).

General experimental procedures
X-ray data were collected using an Agilent Xcalibur Nova X-ray diffractometer. UV spectra were obtained using Shimadzu UV-2450 spectrophotometry. Optical rotations were measured on a Rudolph Autopol I automatic polarimeter. Melting points were measured on an X-4 melting instrument. IR spectra were obtained using a Bruker Tensor 27 spectrometer. 1 D and 2 D NMR spectra were measured on a Bruker AM-400 spectrometer. HRESIMS were performed on a Waters Micromass Q-TOF spectrometer. Semipreparative HPLC separations were carried out by photodiode array (PDA) analysis using a Wufeng LC-100 apparatus with a Kromasil 100-5 C 18 column (250 Â 10 mm, 5 mm). TLC analysis was carried out on silica gel plates (Marine Chemical Ltd., Qingdao, China). Silica gel (300 À 400 mesh, Qingdao Haiyang Chemical Co., Ltd.) and reversedphase C 18 (RP-C 18 ) silica gel (12 nm, S-50 mm, YMC Co., Ltd.) were used for column chromatography (CC).

Plant material
The leaves and twigs of E. agallocha were collected from Guangdong Province, China, in June 2019. The identification of the plant was done by one of the author (X. Li). A voucher specimen (201906-EA) was deposited at The Second Affiliated Hospital of Guangzhou University of Chinese Medicine.

Cell viability assay
In vitro cytotoxic activity against the RKO colon cancer cells were tested by using the MTT method (Alley et al. 1988), and with 5-FU as the positive control.

Conclusion
In summary, excagallonoid A (1), the first example of ent-atisane-2,3-diol diterpenoid, together with five known diterpenoids (2 À 6) were obtained from the leaves and twigs of E. agallocha. Their structures were elucidated by detailed spectroscopic analysis, and the absolute configurations of 1 and 5 were confirmed by combined singlecrystal X-ray diffraction. Compounds 5 and 6 were isolated from this genus for the first time. In bioassay, compounds 1 and 4 showed weak cytotoxicity against RKO colon cancer cell line. The current study expanded the structural diversity of polycyclic diterpenoids from the Excoecaria species.