Euphorbia prolifera Buch-Ham, belonging to the family Euphorbiaceae, is a perennial herbaceous plant distributed in southwest China [1]. Its roots have been used as a folk medicine to treat inflammations and tumors [2]. Previous phytochemical investigations on E. prolifera revealed that the main and characteristic constituents are myrsinol-type diterpenes [3,4,5,6,7,8,9], which are rare in natural products and whose studies of biological activities are limited [6]. In the course of our survey on new and pharmacologically active substances in medicinal plants [10,11], considerable attention has been given to the occurrence of compounds with neuroprotective effects, since these substances are expected to be useful for the treatment of nervous system diseases, such as Parkinson’s disease [12]. As a continuation of the search for bioactive natural products, our further chemical investigation on the roots of E. prolifera led to the isolation of three new myrsinol diterpenes 1-3 (Figure 1). Their structures were elucidated as 2α-O-isobutyryl-3β,5α,7β,10,15β-penta-O-acetyl-14α-O-benzoyl-10,18-dihydromyrsinol (1), 2α-O-isobutyryl-3β-O-propionyl-5α,7β,10,15β-tetra-O-acetyl-10,18-dihydro-myrsinol (2), and 2α,14α-di-O-benzoyl-3β,5α,7β,10,15β-penta-O-acetyl-10,18-dihydromyrsinol (3) on the basis of spectroscopic data analyses (IR, ESI-MS, HR-ESI-MS, and 1D and 2D NMR). The neuroprotective activities against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPP⁺)-induced neuronal cell death in SH-SY5Y cells were also evaluated. Herein, we describe the isolation and structural elucidation of the three new natural myrsinol diterpenes and their neuroprotective activities.

Compound 1 was isolated as colorless needles. Its molecular formula was determined as C₄₁H₅₂O₁₅ on the basis of HR-ESI-MS (m/z 807.3195 [M + Na]⁺, calcd. for C₄₁H₅₂O₁₅Na 807.3204). The ¹H-NMR spectrum of 1 exhibited two olefinic protons [δ_H 6.17 (1H, dd, J = 10.2, 6.5 Hz, H-8), and 5.90 (1H, dd, J = 10.2, 5.6 Hz, H-9)], four oxygenated methines [δ_H 5.48 (1H, d, J = 3.9 Hz, H-3), 5.92 (1H, d, J = 11.0 Hz, H-5), 4.84 (1H, d, J = 6.5 Hz, H-7), and 5.78 (1H, s, H-14)], and one oxygenated methylene [δ_H 4.09 and 3.49 (each 1H, d, J = 8.8 Hz, H₂-17)]. In addition, 11 methyls and one monosubstituted benzene ring were also revealed in the ¹H-NMR spectrum. The ¹³C-NMR spectrum of 1 exihibited 41 carbon resonances. From the ¹H- and ¹³C-NMR spectra of 1, five acetyl groups [δ_H 2.13 s, 2.09 s, 2.05 s, 1.98 s, and 1.96 s; δ_C 170.6, 170.3, 170.2, 169.2, and 168.4 (CO), and 22.4, 22.2, 21.0, 20.9, and 20.8 (CH₃)], and one benzoyl group (δ_H 8.09 × 2 d, 7.42 × 2 t, and 7.56 t; δ_C 129.6, 129.9 × 2, 128.3 × 2, 133.2, and 165.6) (Table 1 and Table 2) were confirmative, and one isobutyryl group (δ_H 2.24 q, 0.83 d, and 1.08 d; δ_C 18.2, 18.8, 34.3, and 175.3) (Table 1 and Table 2) was also deduced based on those reported diterpenes with isobutyryl groups from the genus Euphorbia [4,5,7]. Apart from the above 21 signals for the substituents (five acetoxy groups, one isobutyryloxy group, and one benzoyloxy group), there are additional 20 resonances exhibited for the parent skeleton in the ¹³C-NMR spectrum, which comprised four methyls (C-16, C-18, C-19, and C-20), two methylenes (C-1 and C-17), nine methines (C-3, C-4, C-5, C-7, C-8, C-9, C-11, C-12, and C-14), and five quaternary carbons (C-2, C-6, C-10, C-13, and C-15). By comparing the chemical shifts of C-1-C-20 of compound 1 with those of myrsinol diterpenes reported in the literature [4,5,7], the presence of the 10,18-dihydromyrsinol skeleton was obvious. In order to confirm the 10,18-dihydromyrsinol skeleton and determine the positions of the acyloxy groups, the following HMQC and HMBC experiments were performed. By the interpretation of 1D and 2D NMR spectra, the 10,18-dihydromyrsinol skeleton was defined (Figure 1). The HMBC correlations of the carbonyl signal at δ_C 170.3 with the proton signal at δ_H 5.48 (H-3) indicated the presence of the one acetoxy group at C-3. Similarly, the long-range couplings of the carbonyl carbon signals at δ_C 169.2, 170.2, and 165.6 with the proton signals at δ_H 5.92 (H-5), 4.84 (H-7), and 5.78 (H-14) demonstrated the presence of the two acetoxy groups and one benzoyloxy group at C-5, C-7, and C-14, respectively. The positions of these remaining acyloxy groups were further elucidated by a NOESY experiment. The NOESY correlations of H-14 (δ_H 5.78) and H-1 β (δ_H 2.44) to the methyl protons (δ_H 2.09) of the C-15 acetoxy group, and H₃-19 (δ_H 1.53) to the methyl protons (δ_H 2.13) of the C-10 acetoxy group, implied that the two acetoxy groups were located at C-15 and C-10, respectively. Consequently, the isobutyryloxy group could be assigned only to C-2 based on the chemical shift for C-2. By further analyzing the HMQC, HMBC, and ¹H-¹H COSY spectra (Figure 2), all the proton and carbon signals were assigned unambiguously. Thus, the planar structure of 1 was established.

The relative configuration of 1 was elucidated based on the NOESY spectrum and the comparison of NMR data with those of reported myrsinol diterpenes in the literature [4,5,7,13,14]. For all of the reported natural myrsinol diterpenes, the three rings (5/7/6) comprising the myrsinol-skeleton are trans-fused with each other, H₃-16, H-12, the side chain at C-11, and the acyloxy group at C-15 are biogenetically β-oriented, and H-4 and H₂-17 are biogenetically α-oriented [7,13]. NOESY correlations observed for H-3/H-4, H-5/H-12, H-12/H-14, H-7/H₂-17, and H-12/H₃-20, suggested that H-3, H-4, and H-7 were in α-positions, and H-5, H-12, H-14, and H₃-20 were in β-positions. These assignments were consistent with the configuration of reported 10,18-dihydromyrsinol diterpenes [7,13]. Therefore, the structure of compound 1 was characterized as 2α-O-isobutyryl-3β,5α,7β,10,15β-penta-O-acetyl-14α-O-benzoyl-10,18-dihydromyrsinol.

Compound 2 possessed a molecular formula of C₃₅H₅₀O₁₄ based on the HR-ESI-MS (m/z 717.3101 [M + Na]⁺, calcd. for C₃₅H₅₀O₁₄ Na 717.3098). The ¹H- and ¹³C-NMR spectra of 2 were very similar to those of compound 1, which implied that compound 2 should also be a 10,18-dihydromyrsinol diterpene. Close similarities of the chemical shifts from C-1 to C-20 in 2 with those in 1 (Table 2) suggested that compounds 2 and 1 had the same parent skeleton [4,5,7]. In addition to the 20 skeletal carbons, the ¹³C-NMR spectrum revealed additional 15 carbons for the substituent groups, which were deduced and defined as four acetoxy groups, one isobutyryloxy group, and one propinoyloxy group based on the analyses of HMQC and HMBC spectroscopic data of 2 [4,5,7]. The locations of the six acyloxy groups were elucidated by the interpretation of the HMBC spectrum as in the case of 1. By the HMBC correlations of the protons H-3 (δ_H 5.22), H-5 (δ_H 5.83), and H-7 (δ_H 4.79), with the corresponding carbonyl signals at δ_C 173.4, 169.1, and 170.2, the two acetoxy groups and the propinoyloxy group were attributed to C-5, C-7, and C-3, respectively. There were no long-range correlations observed for H-14 (δ_H 4.05) to the carbonyl carbons of the acyloxy groups, which indicated that the substituent at C-14 was a hydroxy group based on the chemical shift for C-14. The remaining two acetoxy groups and one isobutyryl group were assigned to C-10, C-15, and C-2, respectively, which was supported by the NOESY correlation of the H-14 (δ_H 4.05) and H-1β (δ_H 2.13) to the methyl protons (δ_H 1.91) of the C-15 acetoxy group and the H₃-19 (δ_H 1.48) to the methyl protons (δ_H 2.04) of the C-10 acetoxy group. The similar myrsinol-type diterpene skeleton of 2 compared to those of compound 1 implied the same trans-fusion of the three rings (5/7/6) [7,13]. The NOESY correlations of H-3/H-4, H-5/H-12, H-14/H-12, H-7/H₂-17, and H-12/H₃-20 suggested that the C-3 propinoyloxy group and the C-7 acetoxy group were β-oriented, while the C-5 acetoxy group and C-14 hydroxy group were α-oriented. Thus, compound 2 was elucidated as 2α-O-isobutyryl-3β-O-propionyl-5α,7β,10,15β-tetra-O-acetyl-10,18-dihydromyrsinol.

Compound 3 was obtained as a white powder. The molecular formula of 3 was determined to be C₄₄H₅₀O₁₅ by HR-ESI-MS (m/z 841.3040 [M + Na]⁺, calcd. for C₄₄H₅₀O₁₅Na 841.3047), which was compatible with the NMR data. On comparing the chemical shifts for the skeletal carbons in 3 with those for C-1-C-20 in compound 1, the almost coincidence implied that compounds 3 and 1 had the same characteristic 10,18-dihydromyrsinol skeleton [4,5,7]. The only difference between 3 and 1 was that the one isobutyryloxy group in 1 was replaced by a benzoyloxy group in 3, which was deduced based on the ¹³C-NMR spectrum and the chemical shifts for the benzoyloxy groups in the reported myrsinol diterpenes [7,13]. Following the same NMR procedures used for 1, the locations of the acyloxy groups in 3 were determined by the analysis of the HMBC spectrum, which revealed that the five acetoxy groups were attached at C-3, C-5, C-7, C-10, and C-15, and the two benzoyloxy groups were at C-2 and C-14, respectively. The same relative configuration to be inferred for compound 3 and compound 1 was revealed by the careful comparison of the NOESY spectra of 3 and 1. The structure of compound 3 was therefore characterized as 2α,14α-di-O-benzoyl-3β,5α,7β,10,15β-penta-O-acetyl-10,18-dihydromyrsinol.

In order to explore the potential biological activities of these new diterpenes isolated from the roots of E. prolifera, the neuroprotective effects against MPP⁺-induced neuronal cell death in human dopaminergic neuroblastoma SH-SY5Y cells were evaluated as described previously [11,15]. Bakkenolide H was used as a positive control [16]. Compounds 1 and 2 exhibited neuroprotective activities against MPP⁺-induced neuronal cell death in SH-SY5Y cells. The neuroprotective effects of the two compounds were shown in Figure 3. MTT assay indicated that compounds 1 and 2 (3–30 μM) neither affected the cell viability nor showed significant cytotoxicity with the absence of MPP⁺ (data not shown). Compound 3 was not investigated for the neuroprotective effects because of inadequate amount.

In summary, three new myrsinol diterpenes were successfully isolated from the roots of E. prolifera. Their structures were elucidated on the basis of extensive spectroscopic data analyses. Biological evaluation disclosed that two of them exhibited strong neuroprotective effects against MPP⁺-induced neuronal cell death in human dopaminergic neuroblastoma SH-SY5Y cells. The results of our chemical investigation further revealed the chemical constituents of E. prolifera. While, the current biological data suggest that myrsinol diterpenes with strong neuroprotective activities may probably be useful for the treatment of nervous system diseases, such as Parkinson's disease [12]. Further biological studies on these compounds are still underway by our group.

Supplementary data (1D and 2D NMR spectra and HR-ESIMS spectra) associated with this article can be accessed at: http://www.mdpi.com/1420-3049/17/8/9520/s1.