Gymnotheca chinensis Decne,as one of the endemic genera of seed plants in China,is a perennial herb of family Saururaceae. The whole plants of G. chinensis have long been used as traditional herbal medicine to treat contusions and strains ^[1]. Our previous investigations on the constituents of G. chinensis had led to the isolation of 15 new lignans belonged to three kinds of lignan skeletons,namely dibenzocyclooctene,eupomatilone and eupodienone ^[2]. The latter two rare types were only previously isolated from the Australian shrub Eupomatia bennettii and Eupomatia laurina ^{[3, 4, 5, 6, 7]},respectively. Further investigation on the chemical constituents of G. chinensis resulted in the isolation of two new eupodienone lignans (Fig. 1). Compound 1 was tested for cytotoxic activity in HCT15,HCT116,A549,MCF-7 and HepG2 cells.

Fig. 1

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Fig. 1.The structures of compounds 1 and 2.

Dried and powdered whole plant of G. chinensis (1.45 kg) was extracted with MeOH at r.t. to give an extract (152 g),which was suspended in H₂O (1 L) and extracted with petroleum ether and AcOEt (3 × 1 L,3 h each) successively. The petroleum ether extract (8 g) and AcOEt extract (19 g) were combined and then subjected to CC over MCI gel (85 mm × 100 mm,MeOH/H₂O 90:10). In total, two fractions (A and B) were obtained based on TLC analysis. Fraction A (15 g) was further separated by medium pressure CC (sio₂ (49 mm × 460 mm),petroleum ether/acetone 100:1→1:1) to give twelve fractions. Fraction 11 was subjected to reversedphase CC with MeOH-H₂O (70→100%) as the solvent system to yield four subfractions (11A–D). Compound 1 (25mg,tR = 19.0 min) was obtained from fraction 11A by semi-preparative HPLC (MeOH/H₂O 60:40,flow rate 3.2mL/min). Fraction 9 was subjected to reversed-phase CC with MeOH-H₂O (70→100%) as the solvent system to yield four subfractions (9A–D). Compound 2 (4 mg, tR = 21.7min) was obtained from fraction 9A by semi-preparative HPLC (CH₃CN/H₂O 50:50,flow rate 4.8mL/min). 3. Results and discussion

Compound 1 was obtained as a yellow gum,[α]_D²⁰ D t 20 (c 0.35, MeOH). Its molecular formula of C₂₃H₂₈O₇,which indicated 10 degrees of unsaturation,was established from the quasi-molecular ion peak at m/z 439.1730 [M + Na]⁺ (calcd. for C₂₃H₂₈O₇Na⁺: 439.1727) in the HRESIMS. Its IR spectrum exhibited strong absorption bands at 3436 and 1670 cm^-1 due to hydroxyl and carbonyl functional groups. The ¹H NMR and ¹³C NMR spectra (Table 1) showed that it probably belonged to the eupodienone family ^{[2, 5]}. The ¹H NMR,¹³C NMR and HSQC spectra of 1 showed 23 carbon resonances due to two methyls [δ_H 1.03 (d,3H, J = 7.4 Hz),1.07 (d,3H,J = 7.2 Hz); δ_C 15.5,15.1],two oxygenated methines [δ_H 4.74 (s,1H),3.43 (d,1H,J = 2.7 Hz); δ_C 92.3,85.7] and other two methines [δ_H 2.33 (m,1H),2.77 (m,1H); δ_C 45.7,36.7], five methoxy groups [δ_H 3.57,3.71,3.84,3.56,3.69 (s,each 3H); δ_C 60.9,60.5,56.3,55.1,55.4],a quaternary carbon (δ_C 46.4),an aromatic ring moiety [δ_H 6.70 (s,1H); δ_C 154.1,153.8,142.4,135.7, 120.1,105.4],a diketene moiety [δ_H 6.20 (d,1H,J = 2.3 Hz),6.10 (d, 1H,J = 2.3 Hz); δ_C 118.8,150.3,175.6,153.9,121.9]. The HMBC (Fig. 2) correlations ofH-6/C-9 anδ_H-9/C-6 confirmed an epoxide at C-6 and C-9; key HMBC and HSQC correlations revealed that five methoxy groups were attached to the C-2,C-3,C-4,C-13 and C-15, respectively. The NOESY correlations of H-6/H3-17 and H-9/H3-18 supported the hypothesis that H-6,H-9,H3-17 and H3-18 existed in the same face of the molecule ^[2]. Detailed analysis of ¹H NMR,¹³CNMR and HMBC spectra suggested that the structure of 1 was very similar to those of gymnothelignan O ^[2] except for the replacement of the methylenedioxy by two methoxy groups at C-2 and C-3. In summary,the structure of 1 was determined to be 6,9-epoxy- 7,8-dimethyl-2,3,4,13,15- pentamethoxy-10,11-benzospiro ^[5.6] dodec-13,15-dien-14-one and named as gymnothelignan T.

Fig. 2

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Fig. 2.Key HMBC correlations of compound 1 and key NOESY correlations of compound 2.

Compound 2 was obtained as a white powder,[α]_D²⁰ D +17 (c 0.30, MeOH). The molecular formula was determined to be C₂₁H₂₂O₆ by analysis of the HRESIMS ion peak atm/z 393.1300 [M + Na]⁺ (calcd. for C₂₁H₂₂O₆Na⁺: 393.1309),which indicated 11 degrees of unsaturation. The ¹H NMR and ¹³C NMR spectra (Table 1) were similar to those of 1. The HMBC correlations showed the presence of a methylenedioxy group at C-2 and C-3. HMBC correlations of H- 6/C-9 and H-9/C-6 displayed the connection of C-6 to C-9 via an ether bond. The vicinal coupling constants (H-9/H-8) value was 4.9 Hz for 2,whereas that of 1 was 0 Hz,indicating an epimerization at C-8. In a NOESY experiment (Fig. 2),the crosspeaks of H-7/H-16 indicated that H-7 and H-16 were cis to each other. Detailed HMBC analysis suggested that the structure of 2 was very similar to those of gymnothelignan N ^[2] except for the absence of the OCH₃ at C-15,which was revealed by the ¹H NMR ABX spin system (δ_H 7.08 (dd,1H,J = 10.1,2.4 Hz,H-16),6.39 (d, 1H,J = 10.1 Hz,H-15),6.13 (d,1H,J = 2.4 Hz,H-12). Compound 2 was then assigned as 6,9-epoxy-7,8-dimethyl-2,3-methylenedioxy- 4,13- dimethoxy-10,11-benzospiro ^[5.6] dodec-13,15- dien-14-one and named as gymnothelignan U.

Table 1

Table 1 1H NMR (400MHz) and 13C NMR (100 MHz) data of compounds 1 and 2 (acetoned6).

Table 1 1H NMR (400MHz) and13C NMR (100 MHz) data of compounds 1 and 2 (acetoned6).

Two new eupodienone lignans,named gymnothelignan T (1) and gymnothelignan U (2) were isolated from the whole plant of G. chinensis. Compound 1 was tested for cytotoxic activity in HCT15, HCT116,A549,MCF-7 and HepG2 cells and exhibited no appreciable activity against these tested cell lines with IC₅₀ values above 50 mmol/L. Acknowledgment

This work was financially supported by the grant from the National Natural Sciences Foundation of China (No. 30973634).