English

• Diterpenoids originated from geranylgeranyl pyrophosphate (GGPP) are a large group of highly diversified terpenoids with typical skeletons including acyclic, mono-, bi-, tri-, and tetracyclic systems [1]. A small subset of diterpenoids comprises dimeric diterpenoids featured with at least 40 carbons in their structures, in which the two monomeric blocks are linked together directly or through an oxygen atom [2]. Diterpenoid dimers possess increased complex ring systems and more chiral centers compared to their monomeric analogues, which poses a great challenge for their structural determination.

  The genus Euphorbia (Euphorbiaceae) has been regarded as a valuable resource of bioactive diterpenoids with unique structural diversity [3]. For example, euphopias A−C isolated from E. helioscopia are rearranged jatrophane diterpenoids with anti-inflammatory potentials [4]; pedrolide is a polycyclic diterpene obtained from E. pedroi showed the inhibition effects on P-glycoprotein [5]. Some plants of Euphorbia genus, including E. ebracteolate and E. fischeriana, have been used as traditional Chinese medicines for thousands of years. With the aim of discovering more intriguing diterpenoids from medicinal plants of the genus Euphorbia [6, 7], biseupyiheoid A (1, Fig. 1), an unprecedented dimeric ent–abietane containing spirocyclic 6/6/6/5/6/6/6/6 ring systems, and a decacyclic 6/6/6/3/5/6/5/6/6/6 skeletal ent–abietane dimer, bisfischoid C (2), were identified from the roots of E. fischeriana. The biological assay revealed that 1 showed the significant cytotoxic activity by apoptosis-induced effect in human colon carcinoma LoVo cells.

  Figure 1

  

  Figure 1.  Structures of dimeric diterpenoids 1 and 2.

  DownLoad: Full-Size Img PowerPoint

  Compound 1, obtained as a yellow oil, has a molecular formula of C₄₁H₅₈O₆ with 13° of unsaturation, as deduced from its HRESIMS (m/z 669.4129 [M+Na]⁺, calcd. 669.4126). The ¹H NMR data (Table S1 in Supporting information) exhibited eight tertiary methyls at δ_H 0.56 (s, 3H), 0.80 × 3 (s, 9H), 0.85 (s, 3H), 0.93 (s, 3H), 1.33 (s, 3H), and 1.36 (s, 3H), and a methoxy group at δ_H 3.26, three olefinic signals at δ_H 5.43 (br s), 5.87 (dd, J = 10.2, 2.9 Hz) and 5.99 (dd, J = 10.2, 1.7 Hz). In addition, a proton associated with the oxygenated carbon at δ_H 5.41 (s) and a number of protons at δ_H 1.0−3.0 were also observed, suggesting the existence of a terpenoid backbone. The ¹³C NMR and DEPT spectra (Table S1 in Supporting information) displayed 41 carbon resonances, including nine methyls, ten methylenes, nine methines (three olefinic and one oxygenated), and thirteen nonprotonated carbons (two oxygenated, three olefinic, and two carbonyls). Three double bonds and two carbonyl moieties accounted for five degrees of unsaturation, suggesting the existence of eight rings in the structure of 1.

  The planar structure of 1 was determined by its 2D NMR spectra (Fig. S1 in Supporting information). The ¹H–¹H COSY spectrum shows H₂–1/H₂–2/H₂–3 and H-5/H₂–6/H₂–7 correlations, together with long-range correlations from H₃–20 to C-1, C-5, C-9, and C-10, from H-9 to C-8, C-10, C-11, C-12, and C-14 in the HMBC spectrum, allowing in three fused six-membered rings as shown in Fig. 1. In addition, HMBC correlations of H-14/C-12 and C-13, H₂–17/C-12, C-15, and C-16 were observed, indicating that a block of abietane (part A) was present in 1. The cross-peaks of H₂–1′/H₂–2′/H₂–3′, H-5′/H-6′/H-7′, and H-9′/H₂–10′/H-11′ in the ¹H–¹H COSY spectrum, as well as cross-peaks from H₃–20′ to C-1′, C-5′, C-9′, and C-10′, from H-9′ to C-8′ and C-14′, from H₃–16′ to C-13′, from OCH₃–15′ to C-15′ in the HMBC spectrum, confirmed the presence of another abietane part with a methoxy at C-15′ (part B) in the structure of 1. The ¹H–¹H COSY correlation of H-12′/H₂–17 and HMBC correlations of H-14′/C-15 and H-9′/C-15 suggested that part A was linked with the part B via two single bonds of C-17 and C-15 with C-12′ and C-8′. Taking together, compound 1 was confirmed to be an abietane dimer with a bicyclo[2.2.2]octane moiety.

  The relative configuration of 1 was partially fixed by the NOESY spectrum (Fig. S1). The correlation of H₃–20/H-14 indicated their same orientation. The relative configuration of the rigid bicyclo[2.2.2]octane was established by correlations of H-9′/H-17 and H₃–20′/H-14′. As for the C-8 and C-15, their configuration was a challenge, due to the lack of corresponding signals. Fortunately, a signal crystal of 1 was successfully obtained from a mixed solvent system (MeOH/CH₂Cl₂, 5:1), and X-ray diffraction was carried out with Cu Kα radiation (Fig. 2), which not only confirmed the proposed structure but also determined the absolute configuration of 1 as 5R, 8S, 9S, 10R, 14R, 15S, 5′R, 8′R, 9′S, 10′S, 12′S. Finally, 1 was conducted as the first example of a novel class of diterpenoid dimer based on ent–abietane, and it was named biseupyiheoid A.

  Figure 2

  

  Figure 2.  X-ray crystallographic structure of 1 (CCDC number: 2121291).

  DownLoad: Full-Size Img PowerPoint

  The molecular formula of compound 2 was deduced as C₄₀H₄₈O₇ by analysis of the HRESIMS (m/z 641.3477 [M + H]⁺, calcd. 641.3473), ¹³C NMR, and DEPT spectra (Table S1). Compound 2 was also a dimeric diterpenoid that shares the same skeleton with bisfischoids A and B [8], as evidenced by its detailed 2D NMR analysis (Fig. S2 in Supporting information). The relative configuration of C-12 in 2 was determined by the cross-peak of H-12/H₃–20 in the NOESY spectrum (Fig. S2). Although the correlations of H₂–17′/H-14 were observed, the relative configuration of C-15′ and C-8 (C-14) could not be clearly assigned. Therefore, four possible isomers of 2 that differed in the configurations of C-8 (C-14) and C-15′ (Fig. S3 in Supporting information) have resorted to quantum chemical calculations of NMR and DP4+ analysis [9-11]. The results show that isomer 8S*, 14S*, 15′R*−2 with DP4+ possibility of 100% agrees well with the experimental data. Finally, the structure of 2 was elucidated as shown in Fig. 1 by ECD calculations (Fig. 3).

  Figure 3

  

  Figure 3.  Calculated and experimental ECD spectra of 2 at the CAM-B3LYP/def2-tzvp level.

  DownLoad: Full-Size Img PowerPoint

  Although 1 and 2 have different backbones, it should be noted that the linkage connecting the two blocks of dimers 1 and 2 are both cyclohexene rings. It is reasonable to speculate that the dimerization reaction in the formation of 1 and 2 was the Diels-Alder-like [4 + 2] cycloaddition [12]. A biosynthetic pathway starting from GGPP is proposed in Scheme 1. The first key intermediate produced from GGPP was i with a triene moiety in the ent–abietane framework. Enzyme-dependent oxidation and esterification reactions in plants could yield caudicifolin, a major diterpenoid monomer exited in E. fischeriana [6], from intermediate ⅰ. The oxidation at C-12 and the dehydration at C-17 would be able to give ⅱ with a diene fragment. The highly reactive epoxy moiety could be cleaved and oxidized to form intermediate ⅲ. Further dehydration reactions can give ⅳ with an aromatic ring. The [4 + 2] cycloaddition between ⅲ and ⅳ affords ⅴ that readily to form 2 [13, 14]. The key intermediate ⅳ may undergo hydrolysis and oxidation to give ⅵ. The oxidation of OH-12 and the subsequent keto-enol tautomerism and esterification reactions formed ⅷ. Another [4 + 2] reaction between ⅰ and ⅷ produced ⅸ, which produced 1 eventually.

  Scheme 1

  

  Scheme 1.  Proposed biogenetic pathway for 1 and 2.

  DownLoad: Full-Size Img PowerPoint

  ent–Abietanes in the genus Euphorbia have long been known for their promising cytotoxic activities [15, 16]. The cytotoxic activities of 1 and 2 against four human cancer cells derived from diverse organs were evaluated. The results (Table 1) revealed that dimer 1 (IC₅₀ = 6.7 µmol/L) possesses an anti-proliferative effect against colon carcinoma LoVo cells, while dimer 2 displayed no cytotoxicity. Subsequently, the apoptosis-inducing activity for compound 1 was evaluated, and the result revealed that 1 could induce the apoptotic cell death of LoVo cells in a dose-dependent manner (Fig. 4A). Dimer 1 was further investigated for the expression levels of apoptosis-related markers in LoVo cells. As shown in Fig. 4B, compound 1 up-regulated levels of Bax, cleaved caspase 3, and cleaved caspase 9, and down-regulated the level of Bcl-2 dose-dependently.

  Table 1

  

  Table 1.  Cytotoxic activities of 1 and 2.

  DownLoad: CSV

  Figure 4

  

  Figure 4.  (A) Cell apoptosis induction effects of dimer 1 analyzed by annexin V-FITC/PI flow cytometry. (B) Compound 1 regulated the expression of apoptosis-related proteins in LoVo cells.

  DownLoad: Full-Size Img PowerPoint

  In conclusion, biseupyiheoid A (1) and bisfischoid C (2), two spiro ent–abietane dimers with 6/6/6/5/6/6/6/6 and 6/6/6/3/5/6/5/6/6/6 fused ring systems, respectively, were obtained from E. fischeriana. Dimeric diterpenoids 1 and 2 possessed the skeletons derived from Diels-Alder cycloaddition in different patterns, which further expanded the structural diversity of diterpenoids from the genus Euphorbia. Dimer 1 displayed prominent cytotoxicity and showed significant apoptosis-inducing activities in LoVo cells mediated by regulating Bax, caspase 3, caspase 9, and Bcl-2.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  Acknowledgments

  The authors thank the National Natural Science Foundation of China (No. 81930112), Distinguished Professor Program of Liaoning Province (No. XLYC2002008), Natural Science Foundation of Liaoning Province (Nos. 2020-BS-203 and 2020-MS-256), and Natural Science Foundation of Fujian Province (No. 2021J01509).

  
  
• 1. [1]

     J.R. Hanson, T. Nichols, Y. Mukhrish, M.C. Bagley, Nat. Prod. Rep. 36 (2019) 1499–1512. doi: 10.1039/c8np00079d

  2. [2]

     L.G. Lin, C.O.L. Ung, Z.L. Feng, et al., Planta Med. 82 (2016) 1309–1328. doi: 10.1055/s-0042-114573

  3. [3]

     A. Vasas, J. Hohmann, Chem. Rev. 114 (2014) 8579–8612. doi: 10.1021/cr400541j

  4. [4]

     Q.Q. Shi, X.J. Zhang, T.T. Wang, et al., Org. Lett. 22 (2020) 7820–7824. doi: 10.1021/acs.orglett.0c02676

  5. [5]

     R.J. Ferreira, G. Spengler, A. Orthaber, et al., Org. Lett. 23 (2021) 274–278. doi: 10.1021/acs.orglett.0c03647

  6. [6]

     C.J. Wang, Q.L. Yan, Y.F. Ma, et al., J. Nat. Prod. 80 (2017) 1248–1254. doi: 10.1021/acs.jnatprod.6b00786

  7. [7]

     Y.L. Wei, C. Wang, Z.B. Cheng, et al., J. Nat. Prod. 80 (2017) 3218–3223. doi: 10.1021/acs.jnatprod.7b00595

  8. [8]

     C.P. Sun, Y.B. Chang, C. Wang, et al., Bioorg. Chem. 116 (2021) 105356. doi: 10.1016/j.bioorg.2021.105356

  9. [9]

     N. Grimblat, M.M. Zanardi, A.M. Sarotti, J. Org. Chem. 80 (2015) 12526–12534. doi: 10.1021/acs.joc.5b02396

  10. [10]

      W.Y. Wang, J. Yang, Y.Y. Liao, et al., J. Nat. Prod. 83 (2020) 1998–2003. doi: 10.1021/acs.jnatprod.0c00189

  11. [11]

      W.Y. Wang, J. Yang, Y.Y. Liao, et al., J. Nat. Prod. 83 (2020) 1157–1166. doi: 10.1021/acs.jnatprod.9b01165

  12. [12]

      B. Liu, S.M. Fu, C.Y. Zhou, Nat. Prod. Rep. 37 (2020) 1627–1660. doi: 10.1039/c9np00037b

  13. [13]

      L. Gao, C. Su, X.X. Du, et al., Nat. Chem. 12 (2020) 620–628. doi: 10.1038/s41557-020-0467-7

  14. [14]

      Q.B. Chen, J. Gao, C. Jamieson, et al., J. Am. Chem. Soc. 141 (2019) 14052–14056. doi: 10.1021/jacs.9b06592

  15. [15]

      X.L. Yan, J.S. Zhang, J.L. Huang, et al., Phytochemistry 166 (2019) 112064. doi: 10.1016/j.phytochem.2019.112064

  16. [16]

      Y. Wang, X.Q. Ma, S.S. Yan, et al., Cancer Res. 69 (2009) 7302. doi: 10.1158/0008-5472.CAN-09-0462

• 

  Figure 1  Structures of dimeric diterpenoids 1 and 2.

  下载: 全尺寸图片 幻灯片
  

  Figure 2  X-ray crystallographic structure of 1 (CCDC number: 2121291).

  下载: 全尺寸图片 幻灯片
  

  Figure 3  Calculated and experimental ECD spectra of 2 at the CAM-B3LYP/def2-tzvp level.

  下载: 全尺寸图片 幻灯片
  

  Scheme 1  Proposed biogenetic pathway for 1 and 2.

  下载: 全尺寸图片 幻灯片
  

  Figure 4  (A) Cell apoptosis induction effects of dimer 1 analyzed by annexin V-FITC/PI flow cytometry. (B) Compound 1 regulated the expression of apoptosis-related proteins in LoVo cells.

  下载: 全尺寸图片 幻灯片

  Table 1.  Cytotoxic activities of 1 and 2.

  下载: 导出CSV
•