1. Introduction

The Chinese endemic genus Gymnotheca (Saururaceae) com prises two species, namely Gymnotheca chinensis Decne and Gymnotheca involucrata Pei, which are distributed mainly in the territory of Southwest China. Plants of this genus have long been used as traditional medicinal herbs to treat contusions and strains [1]. Our previous investigation on Gymnotheca had isolated a few of new lignans, including the dibenzocyclooctene, eupomatilone, eupodienone, norlignan, and polycyclic spiro lignan series [2-9]. In our continuing endeavor to discover novel and new structures from this unique Chinese endemic plant, two new biphenyl butyl neolignan derivatives, named gymnothebutyllignans A (1) and B (2) (Fig. 1), were isolated from the whole plants of G. involucrata. This type of compound has not previously been identified from a natural resource, to the best of our knowledge [10, 11]. Herein, we describe the isolation and structure elucidation of gymnothebu tyllignans A (1) and B (2) (Fig. 2).

2. Results and discussion

Compound 1 was obtained as a white crystal. Its molecular formula, C₂₃H₂₈O₈ which indicated 10° of unsaturation, was established from the quasi-molecular ion peak at m/z 433.18570 [M+H]⁺ (calcd. for C₂₃H₂₉O₈⁺: 433.18569) in the HR-ESIMS. Its IR spectrum exhibited strong absorption bands at 3358 and 1675 cm^-1 due to hydroxyl and carbonyl functional groups. The ¹³C NMR spectrum of 1 displayed 20 carbon resonances showed there are symmetry elements in the molecule. The ¹H NMR, ¹³C NMR and HSQC spectra of 1 showed two methyl groups [δ_H 0.59, 0.64 (d, each 3H, J = 7.0 Hz); δ_C 9.6, 11.5], two methine moieties [δ_H 1.75 (m, 1H), 2.55 (qd, 1H, J = 6.8, 3.5 Hz); δ_C 37.6, 46.0], one oxygenated methylene [δ_H 3.12-3.22 (m, 2H); δ_C 65.7], four methoxy groups [δ_H 3.75, 3.79, 3.79, 3.84 (s, each 3H); δ_C 56.4, 56.4, 60.2, 60.6], one methylenedioxy group [δ_H 6.12 (d, 2H, J = 5.5 Hz); δ_C 102.8], one carbonyl (δ_C 209.0), and two aromatic ring moieties [δ_H 6.64 (s, 1H), 6.30-6.65 (br., 2H); δ_C 153.9, 153.9, 149.5, 141.4, 139.6, 138.7, 137.2, 132.2, 127.9, 109.2, 109.2, 103.3]. Its ¹H-¹H COSY revealed the existence of a HOCH₂CH(CH₃)CH(CH₃) fragment. The HSQC data and the δ_C 153.9, 109.2, and 56.4 signals enhancement means the presence of a 3, 4, 5-trimethoxy phenyl moiety [9]. The methylenedioxy group was assigned by the HMBC correlations of OCH₂O to C-4 and C-5, as well as H-6 to C-4 and C-5. Similarly, the methoxy group at C-3 was also assigned. The connection between carbonyl and C-8 was established based on the HMBC correlations of H-8 to C-7, H-9 to C-7 and H-80' to C-7. The connection of C-1-C-7 was achieved by HMBC correlations of H-6 to C-7 and H-8 to C-1. The 3, 4, 5-trimethoxy phenyl attached to C-2 was deduced by the analysis of the HMBC correlations of H-6 to C-2, H-6 to C-7, and H-6 to C-5. Accordingly, the planar structure of compound 1 was established. Fortunately, a single crystal of 1 was obtained from MeOH, and X-ray crystallographic analysis with copper radiation was successfully performed (CCDC 1503704), which unambigu ously determined the absolutely structure (8S, 8'S) of 1 as deduced (Fig. 3). In summary, the structure of compound 1 was established and named gymnothebutyllignan A.

Compound 2 was obtained as white powder. It had the same molecular formula of C₂₃H₂₈O₈ as 1 which was established by HRESIMS. The ¹H, ¹³C NMR, HSQC and HMBC spectra (see Supporting information) indicated that it was a diastereoisomer of 1. By comparing the ¹³C NMR spectrum with those of 1, the chemical shift of C-9 was shifted (Δδ = +3.2), and the chemical shift of C-9' was shifted (Δδ = +4.2). Similarly, the signals of C-8 (δ_C 48.1), C-7' (δ_C 64.6) and C-80 (δ_C 38.8) were shifted (Δδ = +2.1, -1.1 and +1.2, respectively). This indicated that epimerization occurred at C-8 or C-80 in the molecule. In summary, the relative configuration of compound 2 was determined and named gymnothebutyllignan B.

3. Conclusion

In this paper, two new biphenyl butyl neolignan derivatives were isolated from the endemic medicinal plant of G. involucrata. To the best of our knowledge, gymnothebutyllignans A (1) and B (2), are the first examples of neolignans obtained from a natural resource bearing a biphenyl and a butyl moiety. This finding could shed new light on the further understanding of the family of lignan.

4. Experimental 4.1. General experimental procedures

UV spectra were recorded on a Perkin-Elmer Lambda 35 UV-vis spectrophotometer (Perkin-Elmer, Waltham, USA). IR spectra were measured on a Perkin-Elmer one FT-IR spectrometer (KBr) (Perkin-Elmer, Waltham, USA). 1D and 2D-NMR spectra were recorded on a Bruker-AVANCE Ⅲ-400 instrument using TMS as the internal reference (Bruker, Karlsruhe, Germany). HR-ESIMS spectra were measured on a Scientific LTQ Orbitrap XL (Thermo, Massachusetts, USA). Column chromatography (CC) was per formed using 300-400 mesh silica gel (Qingdao Marine Chemical Inc., Qingdao, China), 75-150 mm MCI CHP-20 gel (Mitsubishi, Kyoto, Japan). Semi-preparative HPLC was performed on LC3000 system (Beijing Chuang Xing Tong Heng Science and Technology Co., Ltd., Beijing, China) equipped with ODS column (5 mm, i.d. 10 mm × 250 mm, Kromasil) (Akzo Nobel, Amsterdam, Netherlands).

4.2. Plant material

G. involucrata Pei was collected from Emeishan in Sichuan Province, China, in September 2013. A voucher specimen was deposited at the herbarium of the Chengdu Institute of Biology, Chinese Academy of Sciences, No. 20131026. Prof. Shi-Jie Zhu, Chengdu University of Traditional Chinese Medicine, identified the plant species.

4.3. Extraction and isolation

The air-dried and powdered whole plants of G. involucrata (2.6 kg) were extracted with MeOH at room temperature. After filtration, the MeOH extract was concentrated to give a residue (374 g), which was suspended in H₂O (2 L) and extracted successively with petroleum ether, AcOEt, and n-BuOH. The AcOEt extract (31 g) was subjected to column chromatography (CC) over MCI gel (85 mm × 100 mm), eluting with MeOH-H₂O (90:10), to yield two fractions (A and B) based on TLC analysis results. Fraction A (18 g) was further isolated by medium pressure CC (SiO₂ (49 × 460 mm), petroleum ether-acetone 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10) and to furnish six fractions (Fr.1-Fr.6). Compounds 1 (5 mg, t_R = 16.9 min) and 2 (6 mg, t_R = 15.8 min) were obtained from Fr.4 by semi-preparative HPLC (MeOH/H₂O 75:25, flow rate 3.0 mL/min).

Gymnothebutyllignan A (1): White crystal (MeOH); [α]_D²⁰ = +37 (c = 0.11, MeOH); UV (MeOH) λ_max (log ε) nm 297 (3.78); IR (KBr, cm^-1): ν_max 3358, 2971, 1677, 1629, 1444, 1380, 1332, 1244, 1041, 927; ¹H NMR and ¹³C NMR data, see Table 1; HRESIMS m/z 433.18570 [M+H]⁺ (calcd. for C₂₃H₂₉O₈⁺, 433.18569).

Gymnothebutyllignan B (2): White powder (MeOH); [α]_D²⁰ = -44 (c = 0.32, MeOH); UV (MeOH) λ_max (log ε) nm 297 (3.77); IR (KBr, cm^-1): ν_max 3356, 2970, 1675, 1630, 1434, 1386, 1330, 1248, 1042, 917, 749; ¹H NMR and ¹³C NMR data, see Table 1; HRESIMS m/ z 433.18570 [M+H]⁺ (calcd. for C₂₃H₂₉O₈⁺, 433.18569).

Crystal data for 1: C₂₃H₂₈O₈, M = 432.45, triclinic, a = 10.6222 (6) Å, b = 14.5924 (8) Å, c = 14.8889 (9) Å, α = 95.368 (3)°, β = 99.501 (2)°, γ = 105.234 (2)°, V = 2173.3 (2) ų, T = 100 (2) K, space group P1, Z = 4, μ(CuKα) = 0.832 mm^-1, 30613 reflections measured, 10729 independent reflections (R_int = 0.0494). The final R₁ values were 0.0985 (I > 2σ(I)). The final wR (F²) values were 0.2771 (I > 2σ (I)). The final R₁ values were 0.1166 (all data). The final wR (F²) values were 0.3112 (all data). The goodness of fit on F² was 1.405. Flack parameter = 0.0(2). The Hooft parameter is -0.17(9) for 3832 Bijvoet pairs.

Acknowledgment

This work was financially supported by grants from the National Natural Sciences Foundation of China (No. 31560102).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2016.12.011.