Ent-Abietane Diterpenoids from Euphorbia fischeriana and Their Cytotoxic Activities

The roots of Euphorbia fischeriana have been used as a traditional Chinese medicine for the treatment of tuberculosis and ringworm. In the current study, diterpenoids from the ethyl acetate extract of the roots E. fischeriana and their cytotoxic effects against five cancer lines were investigated. Two new ent-abietane diterpenoids, euphonoids H and I (1–2), as well as their two analogues (3–4) were first isolated from this source. The structures of the two new compounds were elucidated on the basis of spectroscopic data and quantum chemical calculation. Their absolute configurations were assigned via ECD spectrum calculation. The isolated compounds were evaluated for their antiproliferative activities against five cancer cell lines. Compounds 1 and 2 exhibited significant inhibitory effects against human prostate cancers C4-2B and C4-2B/ENZR cell lines with IC50 values ranging from 4.16 ± 0.42 to 5.74 ± 0.45 μM.


Introduction
Natural products are promising sources for the discovery of novel agents/active templates for the development of effective agents against a variety of human diseases [1]. Due to their great structural diversity and wide range of bioactivities [2], diterpenoids have been a constant focus of drug discovery. Among the naturally occurring cyclic diterpenoids (e.g., abietane, labdane and clerodane diterpenoids), tricyclic abietane diterpenoids are of particular significance. These diterpenoids were reported to be present in species of Lamiaceae, Cupressaceae, Pinaceae and Euphorbiaceae as well as in several higher plants [3,4] and were shown to possess antitumor, antioxidant, antibacterial and anti-inflammatory effects [5]. Sugiol, an abietane diterpenoid previously isolated from Metasequoia glyptostroboides (Cupressaceae), has been developed as an advanced multimodal anti-inflammatory disease targeting tool [6]; Euphelionolides A, D, I and L, four ent-abietane diterpenoids isolated from Euphorbia helioscopia (Euphorbiaceae), were demonstrated to be effective free-radical scavengers acting via various reaction pathways [7]; 6-hydroxy-5,6-dehydrosugiol, a derivative of sugiol isolated from the stem bark of Cryptomeria japonica, was shown to be a potent androgen receptor antagonist in PCa cells [8].
The roots of Euphorbia fischeriana Steud have been used as a traditional Chinese medicine for the treatment of lymphoid tuberculosis and ringworm [9]. Previous phytochemical studies showed that polycyclic diterpenoids including ent-abietanes, ent-atisanes, ent-kauranes, ent-isopimaranes and ent-pimaranes possessing a common 6/6/6-tricyclic ring are the major constituents of E. fischeriana [10,11]. In our earlier study, a series of entabietane diterpenoids with significant cytotoxicity have been isolated from this plant [12]. As part of our continuing efforts toward novel antitumor diterpenoids, the chemical constituents of the roots of E. fischeriana were reinvestigated. As a result, two previously undescribed ent-abietanes and two known analogues were isolated from the roots of E. fischeriana. The new compounds showed significant cytotoxicity against human prostate

Results and Discussion
The dry roots of E. fischeriana were repeatedly extracted with 95% EtOH at room temperature and the extract was successively partitioned with petroleum ether, ethyl acetate (EtOAc) and n-butanol. The EtOAc fraction was subjected repeatedly to column chromatography over silica gel, ODS gel, Sephadex LH-20 and semipreparative HPLC to yield two previously undescribed ent-abietane diterpenoids (1)(2). In addition to the above new compounds, two known diterpenoids ( Figure 1) were also obtained: raserranes A (3) and B (4). The structures of all compounds were well-characterized by NMR analysis and quantum chemical calculation.
Molecules 2022, 27, x FOR PEER REVIEW 4 of 9 establish the relative configuration, the chemical shifts of four conformers were predicted at the B3LYP/6-311+G (d, p) level in chloroform (Figure 4). The results showed that the calculated chemical shifts of conformer 1b was in the best agreement with the experimental values among those predicted for 1a, 1b, 1c and 1d. Further DP4+ analyses verified that conformer 1b was assigned with a 99.99% probability among all the conformers (Figure 4). These results suggested that compound 1 had the structure of conformer 1b with the relative stereochemistry of 5R*, 8R*, 9R*, 10R*, 11R*, 15S*.   The absolute configuration of 1 was established by comparing its experimental ECD spectrum with those calculated at the CAM-B3LYP/6-31+G(d) level in acetonitrile. As shown in Figure 5, the experimental ECD curve of 1 showed first negative and second positive Cotton effects around 250 and 213 nm, respectively, which matched well the calculated ECD spectrum of 5R, 8R, 9R, 10R, 11R, 15S-1 (Figure 5a). Thus, compound 1, as an ent-abietane diterpenoid, was established as depicted and named euphonoid H. establish the relative configuration, the chemical shifts of four conformers were predicted at the B3LYP/6-311+G (d, p) level in chloroform (Figure 4). The results showed that the calculated chemical shifts of conformer 1b was in the best agreement with the experimental values among those predicted for 1a, 1b, 1c and 1d. Further DP4+ analyses verified that conformer 1b was assigned with a 99.99% probability among all the conformers (Figure 4). These results suggested that compound 1 had the structure of conformer 1b with the relative stereochemistry of 5R*, 8R*, 9R*, 10R*, 11R*, 15S*.   The absolute configuration of 1 was established by comparing its experimental ECD spectrum with those calculated at the CAM-B3LYP/6-31+G(d) level in acetonitrile. As shown in Figure 5, the experimental ECD curve of 1 showed first negative and second positive Cotton effects around 250 and 213 nm, respectively, which matched well the calculated ECD spectrum of 5R, 8R, 9R, 10R, 11R, 15S-1 (Figure 5a). Thus, compound 1, as an ent-abietane diterpenoid, was established as depicted and named euphonoid H. establish the relative configuration, the chemical shifts of four conformers were predict at the B3LYP/6-311+G (d, p) level in chloroform (Figure 4). The results showed that t calculated chemical shifts of conformer 1b was in the best agreement with the expe mental values among those predicted for 1a, 1b, 1c and 1d. Further DP4+ analyses verifi that conformer 1b was assigned with a 99.99% probability among all the conformers (F ure 4). These results suggested that compound 1 had the structure of conformer 1b w the relative stereochemistry of 5R*, 8R*, 9R*, 10R*, 11R*, 15S*.   The absolute configuration of 1 was established by comparing its experimental EC spectrum with those calculated at the CAM-B3LYP/6-31+G(d) level in acetonitrile. shown in Figure 5, the experimental ECD curve of 1 showed first negative and seco positive Cotton effects around 250 and 213 nm, respectively, which matched well the c culated ECD spectrum of 5R, 8R, 9R, 10R, 11R, 15S-1 (Figure 5a). Thus, compound 1, as ent-abietane diterpenoid, was established as depicted and named euphonoid H. The absolute configuration of 1 was established by comparing its experimental ECD spectrum with those calculated at the CAM-B3LYP/6-31+G(d) level in acetonitrile. As shown in Figure 5, the experimental ECD curve of 1 showed first negative and second positive Cotton effects around 250 and 213 nm, respectively, which matched well the calculated ECD spectrum of 5R, 8R, 9R, 10R, 11R, 15S-1 (Figure 5a). Thus, compound 1, as an ent-abietane diterpenoid, was established as depicted and named euphonoid H.
The molecular formula of 2 was determined to be C 20 H-17)]. The 13 C NMR (Table 1) and HSQC data of 2 revealed the presence of three methyls, five methylenes, five methines (including two oxygenated ones at δ C 64.8 and 55.3 and seven quaternary carbons (including two olefinic ones at δ C 166.2 and 127.5). The above NMR characteristic features of 2 resembled those of jolkinolide B [14], the major differences being the replacement of the 17-CH 3 group in jolkinolide B by an aldehyde group (δ C 185.0) in 2. HMBC correlations from H-17 (δ H 9.97, s) to C-13 (δ C 166.2) and C-15 (δ C 127.5) further confirmed the above deduction. The NOESY correlation H-5/H-9 suggested that H-5 and H-9 were β-oriented, whereas the NOESY correlations H 3 -20/H-11 and H 3 -20/H-14 indicated that H-11, H-14 and CH 3 -20 were α-oriented. Subsequently, quantum chemical calculation of NMR chemical shifts was run on the proposed structure of 2. As indicated by R 2 ( 13 C: 0.9979), CMAD ( 13 C: 1.88 ppm) and CLAD ( 13 C: 4.79 ppm) good consistency was observed between the theoretically predicted and experimental chemical shifts, which validated the proposed structure for 2 ( Figure 6). Subsequently, ECD calculation (Figure 5b) of the two enantiomers of 2 enabled the establishment of the absolute configuration of 2 to be 5S, 8S, 9R, 10R, 11R, 12R, 14R. The structure of 2 was therefore established as depicted and named euphonoid I.  (Table 1) and HSQC data of 2 revealed t presence of three methyls, five methylenes, five methines (including two oxygenated on at δC 64.8 and 55.3 and seven quaternary carbons (including two olefinic ones at δC 166 and 127.5). The above NMR characteristic features of 2 resembled those of jolkinolide [14], the major differences being the replacement of the 17-CH3 group in jolkinolide B an aldehyde group (δC 185.0) in 2. HMBC correlations from H-17 (δH 9.97, s) to C-13 ( 166.2) and C-15 (δC 127.5) further confirmed the above deduction. The NOESY correlati H-5/H-9 suggested that H-5 and H-9 were β-oriented, whereas the NOESY correlatio H3-20/H-11 and H3-20/H-14 indicated that H-11, H-14 and CH3-20 were α-oriented. Su sequently, quantum chemical calculation of NMR chemical shifts was run on the propos structure of 2. As indicated by R 2 ( 13 C: 0.9979), CMAD ( 13 C: 1.88 ppm) and CLAD ( 13 C: 4. ppm) good consistency was observed between the theoretically predicted and expe mental chemical shifts, which validated the proposed structure for 2 ( Figure 6). Sub quently, ECD calculation (Figure 5b) of the two enantiomers of 2 enabled the establis ment of the absolute configuration of 2 to be 5S, 8S, 9R, 10R, 11R, 12R, 14R. The structu of 2 was therefore established as depicted and named euphonoid I.  (Table 1) and HSQC data of 2 revealed the presence of three methyls, five methylenes, five methines (including two oxygenated ones at δC 64.8 and 55.3 and seven quaternary carbons (including two olefinic ones at δC 166.2 and 127.5). The above NMR characteristic features of 2 resembled those of jolkinolide B [14], the major differences being the replacement of the 17-CH3 group in jolkinolide B by an aldehyde group (δC 185.0) in 2. HMBC correlations from H-17 (δH 9.97, s) to C-13 (δC 166.2) and C-15 (δC 127.5) further confirmed the above deduction. The NOESY correlation H-5/H-9 suggested that H-5 and H-9 were β-oriented, whereas the NOESY correlations H3-20/H-11 and H3-20/H-14 indicated that H-11, H-14 and CH3-20 were α-oriented. Subsequently, quantum chemical calculation of NMR chemical shifts was run on the proposed structure of 2. As indicated by R 2 ( 13 C: 0.9979), CMAD ( 13 C: 1.88 ppm) and CLAD ( 13 C: 4.79 ppm) good consistency was observed between the theoretically predicted and experimental chemical shifts, which validated the proposed structure for 2 ( Figure 6). Subsequently, ECD calculation (Figure 5b) of the two enantiomers of 2 enabled the establishment of the absolute configuration of 2 to be 5S, 8S, 9R, 10R, 11R, 12R, 14R. The structure of 2 was therefore established as depicted and named euphonoid I.  The other two known diterpenoids (3)(4) were identified to be ent-abietane diterpenoids raserranes A (3) and B (4) by comparison of their NMR data with those reported in the literature, these four diterpenoids were discovered for the first time from this species [15].

Biological Activity of Isolated Compounds
The anticancer effects of the isolates 1-4 were evaluated against human breast cancer cells MDA-MB-231, human colon cancer cells HCT-15 and RKO and human prostate cancer cells C4-2B and C4-2B/ENZR (enzalutamide-resistant C4-2B cells). The IC 50 values (Table 2) indicated that the two new compounds exhibited varying degrees of growth inhibition against the five cancer cell lines. Compound 1 showed significant inhibitory activities against C4-2B and C4-2B/ENZR cell lines with IC 50 values of 5.52 ± 0.65 µM and 4.16 ± 0.42 µM, respectively. Compound 2 exhibited marked inhibitory activity towards the five human cancer cell lines (IC 50 values ranging from 4.49 ± 0.78 to 12.45 ± 3.24 µM) and was particularly active against C4-2B and C4-2B/ENZR cell lines (IC 50 values: 4.49 ± 0.78 and 5.74 ± 0.45, respectively). Macrocyclic and polycyclic diterpenes were usually encountered in the genus of Euphorbia and macrocyclic diterpenes were characteristic components of Euphorbia plants, while polycyclic diterpenes were nonspecific in this genus. Although polycyclic diterpenes were not the characteristic components of Euphorbia plants, some polycyclic diterpenes showed great potential in the development of anticancer drugs [16][17][18]. Jolkinolide B, a typical ent-abietane diterpene first isolated from Euphorbia jolkini, induced apoptosis and sensitized bladder cancer to mTOR inhibitors [19,20]; 17-hydroxy-jolkinolide B, a potent inhibitor of JAK/STAT3 signaling, is a promising anticancer drug candidate [21]. In this study, compounds 1-2 sharing the same abietane diterpene skeleton (6/6/6 carbon ring system) were shown to be promising anti-prostate cancer candidates. Among the four compounds isolated, compound 2 that possessed an α,β-unsaturated γ-lactone ring at C-12 and C-13, was very active against almost the test cancer cells. This observation was consistent with our previous discovery that such an α,β-unsaturated γ-lactone ring was beneficial for the anticancer activity of this type of diterpenoids [12]. Despite the fact that several antitumor abietane diterpenoids were reported in recent years, the pharmacophores and structure-activity relationship of abietane diterpenoids as anticancer agents were rarely investigated. Thus, synthesis of these diterpenoids and study of their structure-activity relationship and potential molecular mechanisms were of great significance for the design and development of anticancer agents.

General Experimental Procedures
Optical rotations were carried out on a Rudolph Autopol I automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). The UV spectra were measured at a Shimadzu UV-2450 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). IR spectra were determined on a Bruker Tensor 37 infrared spectrophotometer (Bruker Optics, Ettlingen, Germany) with KBr disk. ECD spectra were measured on an Applied Photophysics Chirascan spectrometer (Applied Photophysics Ltd., England). NMR spectra were measured on Bruker AM-400 spectrometer with tetramethylsilane (TMS) as the internal standard. HR-ESIMS data were determined using a Waters Micromass Q-TOF spectrometer (Waters Corporation, Milford, MA, USA). The semi-preparative HPLC was performed on an Essentia LC-16 (Shimadzu, Suzhou, China). Column chromatography (CC) was used using silica gel (200-300 mesh, Qingdao Marine Chemical Factory, Qingdao, China).

Plant Material
The roots of E. fischeriana were collected in August 2015 from Tie ling city, Liaoning Province, P. R. China and identified by Prof. Qing-De Long (Guizhou Medical University). The specimens were deposited in School of Pharmaceutical Sciences, Guizhou Medical University (specimen no. 20150805).

Quantum Chemical NMR and ECD Calculations of Compound 1-2
The random conformational searches were performed by SYBYL X 2.1.1 program using MMFF94s molecular force field. The obtained conformers were subsequently optimized by using Gaussion09 software at the B3LYP/6-31G(d) level in gas phase. The optimized stable conformers were selected for further NMR calculations at the mPW1PW91/6-311 + G(d,p) level in chloroform and ECD calculations at the CAM-B3LYP/6-31 + G(d) level in acetonitrile. The overall theoretical NMR data were analyzed by using linear regression and DP4+ probability. The overall ECD data were weighted by Boltzmann distribution and produced by SpecDis version 1.70.1 software (T. Bruhn; A. Schaumlöffel; Y. Hemberger; G. Pescitelli, Berlin, Germany).

Cell Culture
Five cancer cell lines, including human prostate cancer cells (C4-2B), enzalutamideresistant C4-2B cells (C4-2B/ENZR), human breast cancer cells (MDA-MB-231) and human colon cancer cells (HCT-15 and RKO) used in this study were purchased from the Laboratory Animal Service Centre at Sun Yat-sen University (Guangzhou, China). Cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and antibiotics (100 units/mL penicillin and 100 g/mL streptomycin). These cells were incubated at 37 • C in an atmosphere of 5% CO 2 .

Cytotoxicity Assay
The cells in logarithmic growth phase were seeded a in 96-well plates at a density of 5 × 10 3 cells/well for 24 h. Then, cells were treated with different concentrations of the compounds for an additional 48 h. Subsequently, 10 µL MTT (5 mg/mL) (Sigma, Saint Louis, MO, USA) were added to each well. After incubation in the incubator for 4 h, the suspension was discarded and the dark blue crystals were solubilized in dimethyl sulfoxide (DMSO). The absorbance of the solution was detected by a multifunction micro-plate reader (Molecular Devices, Flex Station 3, Molecular Devices, San Francisco, USA) at 450 nm. IC 50 value was used to express the cytotoxic effect on the tested compounds.

Conclusions
In summary, two new highly oxygenated ent-abietane diterpenoids euphonoids H and I (1-2), together with two known analogues raserranes A (3) and B (4) were separated and identified from the EtOAc-soluble partition of the roots of E. fischeriana. Their structures were elucidated by comprehensive spectroscopic analysis, quantum chemical calculation and ECD calculations. All the compounds were isolated from E. fischerian for the first time. The two new compounds exhibited strong antiproliferative potency against the human prostate cancer cells C4-2B and C4-2B/ENZR, with IC 50 values less than 10 µM. This study not only enriches the chemical diversity of ent-abietane diterpenoids in the Euphorbia species but also forms a basis for the discovery of bioactive natural products from Euphorbiaceae herbs.
The current results, together with others' previous discoveries, suggested that entabietane diterpenoids with certain structural motifs might possess very strong anticancer activity against prostate cancer cell lines and this type of diterpenoids provided a promising skeleton for the development of anti-cancer agents for the treatment of prostate cancers.