New ingenane and ingol diterpenoids from Euphorbia royleana

Abstract Phytochemical investigation on the 95% EtOH extract of the Traditional Chinese Medicine (TCM) Euphorbia royleana (Ba-wang-bian in Chinese) led to the isolation of 11 diterpenoids (1–11) and two triterpenoids (12 and 13). Among them, compounds 1 and 2 were new ingenane and ingol diterpenoids, respectively. Their structures were elucidated by a combination of spectroscopic analyses (1 D and 2 D NMR, HRMS, ECD, UV, and IR data) and chemical methods. Compounds 12 and 13 exhibited moderate cytotoxicities in vitro against human lung cancer cell line A549 with IC50 values of 14.84 ± 0.56 and 27.11 ± 1.65 µM, respectively. Graphical Abstract


Introduction
Euphorbia diterpenoids represent the characteristic metabolites occurring in the widespread genus Euphorbia (Euohorbiaceae), with over 700 analogues and 30 structurally diverse cyclic system including jatrophanes, lathyranes, ingenanes, and so forth (Vasas and Hohmann 2014). Some of them, particularly polycyclic and macrocyclic diterpenes, have been found to exert a broad spectrum of biological activities, including a-glucosidase inhibitory (Tran et al. 2020), multidrug resistance reversal (Shaker et al. 2020), anti-inflammatory properties , and cytotoxicities (Shadi et al. 2015). Notably, their fascinating structures coupled with intriguing bioactivities have been the focus of natural product chemists and pharmacologists over the past few years. For instance, euphorkanlide A, a highly modified ingenane diterpenoid featuring a hexahydroisobenzofuran-fused 19-membered macrocyclic bis-lactone ring system, exhibited significant cytotoxicities against a panel of cancer cell lines (Yan et al. 2019).
Euphorbia royleana Biosisier (Euphorbiaceae), known as 'Ba-wang-bian', has been used medicinally to relieve inflammation and rheumatic pain in Traditional Chinese Medicines (Song 1999;Ma and Michael 2008). Previous phytochemical investigations regarding this species revealed that triterpenoids and structurally diverse diterpenoids were the main metabolites, and some of them exhibited significant antiangiogenic activities (Bhat et al. 1982;Li et al. 2009). Recently, we reported the isolation of eight new diterpenoids and 22 known analogues with significant multidrug resistance reversal activity from the petroleum ether (PE) fraction of the ethanolic extract of E. royleana (Shaker et al. 2020). As part of our continuous endeavor for isolating new bioactive diterpenoids from Euphorbiaceae plants (Yan et al. 2019;Shaker et al. 2020;Zhang et al. 2020;Weng et al. 2021), the EtOAc fraction of E. royleana showed a certain cytotoxic activity against human cancer cell line A549. Subsequent chemical investigation resulted in the isolation of two new diterpenoids and 11 known compounds. Their structures were elucidated by a combination of spectroscopic analysis and chemical methods. All of the isolates were examined for their cytotoxic activities against A549 cancer cells. Compounds 12 and 13 exhibited moderate cytotoxicities against A549 cancer cells with IC 50 values of 14.8 and 27.1 mM, respectively. Herein, the isolation, structural elucidation, and cytotoxicities of these compounds were described.

Results and discussion
The air-dried powder of the whole plants of E. royleana was extracted with 95% EtOH at room temperature (rt) to give a crude extract, which was suspended in H 2 O and successively partitioned with PE, EtOAc, and n-BuOH. Various column chromatographic separations of the EtOAc fraction afforded compounds 1-13 ( Figure 1).
Compound 1, colorless oil, was assigned the molecular formula C 35 H 56 O 6 with eight indices of hydrogen deficiency (IHDs), based on the HRESIMS ion peak at m/z 595.3961 [M þ Na þ ] (calcd 595.3969). The IR spectrum exhibited absorption bands for hydroxyl (3359 cm À1 ), carbonyl (1726 cm À1 ), and double bond (1467 cm À1 ) functionalities. The 1 H NMR data ( , and a series of aliphatic methylene multiplets. The 13 C NMR and DEPT spectra resolved carbon signals for a ketone (d C 206.8), an ester carbonyl (d C 174.1), two trisubstituted double bonds (d C 138.9, 137.0, 130.3, and 128.7), five methyls, a series of sp 3 methylenes (including an oxymethylene at d C 66.5), six sp 3 methines (including two oxymethines at d C 80.9 and 73.9), an oxygenated sp 3 tertiary carbon (d C 84.5), and two quaternary carbons. Since four of eight IHDs were taken into account by a ketone, an ester carbonyl, and two double bonds, the remaining IHDs required that 1 was tetracyclic. The aforementioned data were very similar to those of coisolated compound ingenol-20-palmitate (4) (Itokawa et al. 1989), with the major difference being a different long aliphatic chain at OH-20. This was confirmed by comparison of their ESIMS data (1 showed 14 mass units less than that of 4, corresponding to the depletion of a methylene fragment) ( Figure S9 in Supplemental Material). To further determine the identity of the aliphatic chain ester, 1 was hydrolysed with excess of K 2 CO 3 to yield the fatty acid, which was subsequently esterified by the solvent methanol to give the methyl ester. The mixture was subjected to GC-MS analysis. The retention time (t R ¼ 10.96 min) and MS peak (m/z 256) of the product were identical with the methyl npentadecanoate in NIST MS library ( Figure S12 in Supplemental Material), implying that the structure of the aliphatic chain was n-pentadecanoate.
The relative configuration of 1 was assigned to be the same as that of 4 by a NOESY experiment and comparison of their 1 D NMR data. In particular, NOE correlations of H-3/H-5, H-13/H-14 and Me-16, and H-13/Me-18 implied that H-3, H-5, H-13, H-14, and Me-18 were assigned as a-orientations. Thus, the cross-peaks of H-8/Me-17 and H-11/Me-17 indicated that H-8 and H-11 were b-oriented ( Figure S2 in Supplemental Material). Thus, compound 1 was determined as depicted, and was named ingenol-20-pentadecanoate.
Compound 2 exhibited the molecular formula C 37 H 42 O 9 as determined by pseudomolecular ion in HRESIMS at m/z 653.2726 for [M þ Na þ ] (calcd 653.2721). The 1 D NMR data of 2 bore a resemblance to those of coisolated known compound 8-Omethyl-ingol-3,12-diacetate-7-benzoate (7), except for the replacement of an acetyl group in 7 by a benzoyl group in 2. The location of the additional benzoyl group was assigned at OH-3 by the HMBC correlation from H-3 (d H 5.38) to the benzoyl carbonyl (d C 166.1) ( Figure S1 in Supplemental Material).
All the isolates were screened for their cytotoxicities against A549 cancer cells by MTT method (Alley et al. 1988), and doxorubicin was used as a positive control (IC 50 ¼ 0.53 ± 0.07 mM). As a result, compounds 12 and 13 showed moderate cytotoxicities with IC 50 values of 14.84 ± 0.56 and 27.11 ± 1.65 mM, respectively, while other compounds were inactive (IC 50 s > 100 mM).

Plant material
The plants of E. royleana were collected from Yunnan Province, P. R. China in March 2019 and authenticated by Prof. You-Kai Xu (Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences). A voucher specimen (accession number: LD1903) was deposited at the School of Pharmaceutical Sciences, Sun Yat-Sen University.

Alkaline hydrolysis of 1 and GC-MS analysis
To a solution of 1 (1 mg, 0.002 mmol) in methanol was added K 2 CO 3 . After the mixture was stirred at rt for 2 h, the corresponding concentrated residue was redissolved in EtOAc and analysed by GC-MS using an DB-5MS capillary column (30 m Â 250 mm i.d. Â 0.25 mm) under the following conditions [injector temperature, 250 C; initial temperature, 100 C, increased at 10 C/min to 250 C, held for 10 min; Helium was used as the carrier gas with flow rate of 1 mL/min, operated in a split mode with split flow of 10 mL/min; injection size, 1 mL; MS conditions: EI voltage, 70 eV; scanned-mass range, m/z 50-650]. Identification of pentadecanoic acid, methyl ester was carried out for 1, giving a peak at 10.96 min and m/z 256.

Alkaline hydrolysis of 2 and 7
To a solution of 2 (3 mg, 0.005 mmol) in methanol was added K 2 CO 3 . After the mixture was stirred at 45 C for 8 h, the corresponding concentrated residue was purified with semi-preparative HPLC (MeCN/H 2 O ¼ 60:40) to afford 1 mg of 2a (45%, t R 7 min). To a solution of 7 (3 mg, 0.005 mmol) in methanol was added K 2 CO 3 . After the mixture was stirred at rt for 3 h, the corresponding concentrated residue was purified via the same procedure to obtain 1.6 mg of 7a (80%, t R 7 min). On the basis of their 1 H NMR data, 2a and 7a were identical to 5 ( Figure S23 in Supplemental Material).

Cytotoxicity assay
In vitro cytotoxic activity against the A549 cancer cells were tested by using the MTT method (Alley et al. 1988), and with doxorubicin as a positive control.

Conclusion
In the present study, two new diterpenoids and 11 known compounds were isolated from the EtOAc fraction of the whole plants of E. royleana. Among them, compounds 1 and 2 were new ingenane and ingol diterpenoids, respectively. Their structures were elucidated by a combination of extensive spectroscopic analysis, ECD analysis, and chemical correlations. Compounds 1-13 were investigated for the cytotoxicities against human cancer cell line A549, and 12 and 13 showed moderate cytotoxicities. The current research expanded the structural diversity of the diterpenoids family.

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