The genus Euphorbia is the largest in the spurge family, comprising of more than 2000 species,many of which have been used in folk as traditional Chinese medicine for the treatment of skin diseases,edemas,etc. ^[1]. Previous investigations on this genus revealed that the major bioactive constituents are diterpenoids ^{[2, 3, 4, 5]}. Euphorbia dracunculoides Lam.,as an annual or shortlived perennial herb,is distributed in riverbanks,valleys and roadsides of sandy areas in North Africa,South Europe,and Southwest Asia ^[6],and has been used as a folk medicine in India for its purgative and diuretic effects ^[7]. The earlier phytochemical investigations on E. dracunculoides are limited to the presence of flavonoids ^{[7, 8, 9]},triterpenoids ^[9],and coumarins ^[10]. To the best of our knowledge,there are no reports about diterpenoids from E. dracunculoides over the last two decades. In our efforts to search for structurally interesting and potential bioactive diterpenoids from genus Euphorbia,two new myrinsol diterpenoids euphordracunculins A (1) and B (2),together with three known analogues, euphorprolitherin B (3) ^[11],proliferins A (4) and B (5) ^[12],were isolated from the aerial parts of E. dracunculoides. In this paper,the isolation and structural elucidation of two new compounds are presented (Fig. 1).

Fig. 1

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Fig. 1. Structures of compounds 1-5.

The aerial parts of E. dracunculoides Lam. were collected in September 2012 from Xishuang Banna prefecture,Yunnan Province,People’s Republic of China,and identified by Prof. Yao- Wen Yang,Yunnan University of Traditional Chinese Medicine. A voucher specimen (YTCM 20120915) was deposited at Yunnan University of Traditional Chinese Medicine. 2.2. Extraction and isolation

The air-dried and powdered aerial parts of E. dracunculoides Lam. (4.0 kg) were extracted with 70% aqueous acetone (8 L × 2 d × 3) at room temperature. The extracts were concentrated by a rotary evaporator under reduced pressure to remove organic solvent. The aqueous residue was then partitioned with petroleum ether (4 × 1 L),EtOAc (4 × 1 L),and n-BuOH (4 × 1 L) sequentially. The petroleum ether extract (48.0 g) was subjected to column chromatography (CC) on silica gel (200-300 mesh) using a gradient system of increasing polarity with petroleum ether- EtOAc (50:1,20:1,10:1,5:1,2:1,1:1,0:1) to afford six fractions (A- F) based on TLC analysis.

Fraction D (7.2 g) was decolorized on a MCI gel (CHP 20P) CC eluted by MeOH,and then divided into three subfractions (Fr. D-1- Fr. D-3) by silica gel (200-300 mesh) CC eluting with petroleum ether/Me₂CO (15:1,6:1,1:1,respectively). Fr. D-1 was separated by Sephadex LH-20 column (MeOH/CHCl₃,1:1),followed by semipreparative HPLC (HITACHI HPLC system; YMC-Triart C18 column,250 mm × 10 mm; DAD detector,MeOH/H₂O 75:25, 232 nm,3.5 mL/min) to give compounds 3 (18.6 mg,t_R = 25.8 min), 4 (9.0 mg,t_R = 37.1 min),and 5 (3.2 mg,t_R = 40.1 min). Fr. D-2 was chromatographed on Sephadex LH-20 column (MeOH),followed by preparative HPLC (CXTH LC-3000 HPLC system,Kromasil C18 column,250 mm × 20 mm; UV detector,MeOH-H₂O 76:24, 232 nm,12.0 mL/min) to yield compound 2 (14.5 mg, t_R = 13.8 min). Fr. D-3 was subjected to Sephadex LH-20 CC eluted by MeOH,followed by semipreparative HPLC (HITACHI HPLC system; YMC-Triart C18 column,250 mm × 10 mm; DAD detector, MeOH-H₂O 77:23,232 nm,3.5 mL/min) to afford compound 1 (8.6 mg,t_R = 15.9 min).

Compound 1,[α]_D^26:5 -134.0 (c 0.21,MeOH),UV(MeOH) λ_max (log e): 271 (2.98),230 (4.08) and 201 (4.07) nm,obtained as colorless needles from MeOH,mp 197-200 ℃. Its molecular formula was determined to be C₃₉H₄₈O₁₅ based on the HR-ESI-MS data (m/z 779.2887 [M+Na]⁺,calcd. 779.2891),corresponding to 16 degrees of unsaturation. Its IR spectrumshowed absorption bands for carbonyl groups at 1742 cm^-1. The ¹H NMR spectrum (Table 1) showed 10 3H-singlets at δ_H 2.15,2.10,2.06,2.00,1.98,1.71,1.64,1.55,1.33 and 1.22,of which six may be assigned for acetate groups and four be assigned for tertiary methyl groups. A mono-substituted benzene ring [δ_H 8.08 (d,2H,J = 7.8 Hz),7.58 (t,1H,J = 7.8 Hz),7.45 (t,2H, J = 7.8 Hz)] was also evident in the ¹H NMR spectrum. Additionally, the signals of two vicinal olefinic protons [δ_H 6.18 (ddd,1H,J = 9.9, 6.6,1.5 Hz),5.91 (dd,1H,J = 9.9,5.6 Hz)] and an oxygenated methylene group [δ_H 4.16 (d,1H,J = 8.7 Hz),3.53 (dd,1H,J = 8.7, 1.6 Hz)] were also observed. Seven carbonyl signals at δ_C 170.9, 170.7,170.6,169.6,169.5,168.7 and 165.9,were obvious in the ¹³C NMR spectrum of 1. Accordingly,1 was presumably a highly oxidized tetracyclic diterpenoid substituted by six acetoxy and one benzoyloxy groups. Four oxymethine protons geminal to ester functions [δ_H 5.95 (dd,1H,J = 11.1,1.5 Hz),5.81 (1H,s),5.36 (br d, 1H,J = 4.1 Hz),4.86 (d,1H,J = 6.6 Hz)] suggested that the other three ester groups were located at quaternary carbons. Comparison the above NMR data with those of euphorprolitherin B (3),a myrsinol diterpene ^[11] which was also isolated in our present study, indicated that they are quite structurally similar. The possible differencewas that a propionyloxy groupatC-3 in euphorprolitherin B is replaced by an acetoxy group in 1,which was supported by the disappearance of theNMR signals of propionyloxyl and presence of a typical acetoxyl signals (δ_C 170.7,s,21.4,q; δ_H 2.06,s) in 1. The hypothesis was further verified by the HMBC correlations (Fig. 2) from a methyl signal at δ_H 2.06 (s,3H) and an oxymethine proton signal at δ_H 5.36 (br d,1H,J = 4.1 Hz,H-3) to an ester carbonyl signal at δ_C 170.7,respectively. The accurate assignments of all protons and carbons were preformed through the correlations in 2D-NMR spectra (¹H-¹H COSY,HSQC anδ_HMBC) of 1 (Fig. 2),fromwhich the positions of the ester groups were also clarified. The correlations of the protons at δ_H 5.95 (H-5),4.86 (H-7),and 5.81 (H-14) with the carbonyl carbons at δ_C169.6,170.7,and165.9 in theHMBCspectrum demonstrated the presences of two acetoxy and one benzoyloxy groups at C-5,C-7,and C-14,respectively. In addition,three slightly weak correlations from methyl signals in acetoxy groups at δ_H 1.71 (s,3H,2-OAc),2.10 (s,3H,10-OAc),2.15 (s,3H,15-OAc) to three quaternary carbons at δ_C 87.1 (s,C-2),86.0 (s,C-10),90.2 (s,C-15) (Fig. S5 in Supporting information),respectively,indicated that the three acetoxy groups were located at C-2,C-10 and C-15, respectively.

Table 1

Table 1 NMR data for compounds 1 and 2 (TMS as the internal standard,δ in ppm,J in Hz).a,b

Table 1 NMR data for compounds 1 and 2 (TMS as the internal standard,δ in ppm,J in Hz).a,b

Fig. 2

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Fig. 2. Key COSY,HMBC,and ROESY correlations of 1.

The relative configurations of 1 were decided by the ROESY experiment (Fig. 2) as well as biosynthetic consideration. For the reported natural myrsinol diterpenes,the three rings (5/7/6) forming the myrsinol diterpenoids’ skeleton are trans-fused,H-4 and H2-17 are a-oriented,and Me-16,H-12,the side chain at C-11, and the C-15 acyloxy group are β-oriented ^[13]. In addition,the ROESY correlations: H-4α with H-3 and H-7 with H-17a,b supported the a-orientations for H-3 and H-7,respectively,and the ROESY correlations between H-12β with H-5,H-14 and Me-20 were in agreement with the β-orientations of H-5,H-14 and Me-20 (Fig. S8 in Supporting information). Consequently,compound 1 was elucidated as 14-desoxo-2α,3β,5α,7β,10,15β-O-hexacetyl- 14α-O-benzoyl-10,18-dihydromyrsinol and given the name euphordracunculin A.

Compound 2,colorless crystals from MeOH,mp 199-203 ℃, [α]_D^26:5 -119.0 (c 0.18,MeOH),UV (MeOH) λ_max (log e): 272 (3.06), 231 (4.12) and 201 (4.10) nm,was found to possess the molecular formula C₃₈H₄₈O₁₄ from the HR-ESI-MS data (m/z 751.2946 [M+Na]⁺,calcd. 751.2942),indicating 15 degrees of unsaturation. The 1Hand ¹³CNMRspectra of 2 were similar to those of the known compound 3,except for the absence of an acetoxyl group at C-2. A hydroxyl group at the C-2 position was evident for 2 on the basis of the observation of an upfield shifted quaternary carbon signal (δ_C 78.4) in the ¹³C NMR spectrum and HMBC correlations of H-1 (δ_H 2.45),H-3 (δ_H 5.12),and Me-16 (δ_H 1.10) with C-2 (δ_C 78.4) (Fig. S15 in Supporting information). Further 2D NMR experiments allowed a determination of 2 as 14-desoxo-5α,7b,10,15β-Otetraacetyl- 14α-O-benzoyl-2a-hydroxy-3β-O-propionyl-10,18- dihydromyrsinol and given the name euphordracunculin B. Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (No. 21162044) and the Mid-aged and Young Academic and Technical Leader Raising Foundation of Yunnan Province (No. 2010CI040).

Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2014.08.005.