1. Introduction

Gymnotheca chinensis Decne, as one of the endemic genera of seed plant in China, is a perennial herb of the family Saururaceae. The whole plants of G. chinensis have long been used as a traditional herbal medicine to treat contusions and strains ^[1]. In a previous investigation on G. chinensis we had isolated a great amount of new lignans belonging to the dibenzocyclooctene, eupomatilone, eupodienone and norlignan series ^[2-7]. In our continuing endeavor to discover unique and new structures from this Chinese endemic plant, two novel 2, 5-diphenyl oxazole derivatives, 2-(3'- hydroxy-2'-methoxybenzene-1'-yl)-5-phenyl oxazole (1) and 2- (3'-hydroxy-2'-methoxybenzene-1'-yl)-5-(2"-methoxybenzene- 1"-yl) oxazole (2) (Fig. 1), named gymnotheoxazoles A and B, were isolated from the whole plants of G. chinensis. This kind of alkaloid was found for the first time from this endemic genus. 2, 5-Diphenyl oxazole (DPO) was a class of substances exhibiting radioprotective and radiosensitizing effects ^{[8, 9]}. This type of compound is very rare in the natural world. Owing to their attractive structures, there have been total syntheses of some of the members of DPO ^[10-12]. Herein, we describe the isolation and structure elucidation of gymnotheoxazoles A (1) and B (2), and their cytotoxicity against A549 cell.

2. Experimental 2.1. 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-ESI-MS spectra were measured on a MicrO TOF-Q Ⅱ mass spectrometer (Bruker, Karlsruhe, Germany). Column chromatography (CC) was performed using 3"-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 - 250 mm, Kromasil) (Akzo Nobel, Amsterdam, Netherlands). 2.2. Plant material

The plant G. chinensis was collected from Jinfoshan in Chongqing city, China, in October 2012, and identified as G. chinensis by Prof. Si-Rong Yi (Chongqing Institute of Pharmaceutical Plant). A voucher specimen (No. 20130125) was deposited at the herbarium of the Chengdu Institute of Biology, Chinese Academy of Sciences.

2.3. Extraction and isolation

Dried and powdered whole plant of G. chinensis (1.45 kg) were 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 - 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 - 460 mm), petroleum ether/acetone 100:1→1:1) to give twelve fractions. Fraction 8 was subjected to fractionation by CC (ODS (40-63 µm, 36×310 mm), MeOH/H₂O 60:40→100:0) to furnish four main fractions, 8A-8D. Compound 1 (3 mg, t_R = 15.1 min) was obtained from fraction ℃ by semi-preparative HPLC (MeCN/H₂O 55:45, flow rate 4.5 mL/min). Compound 2 (4 mg, t_R = 16.4 min) was isolated from fraction 8D by semipreparative HPLC (MeCN/H₂O 56:44, flow rate 4.5 mL/min).

Gymnotheoxazole A (1): White crystal (MeOH); UV (MeOH) λ_max (log ε) nm 236 (2.36), 303 (5.21); IR (KBr, cm^-1): v_max 3675, 1539, 1433, 1282, 1230, 885; ¹H NMR and ¹³C NMR data, see Table 1; HRESIMS m/z 268.0967 [M+H]⁺ (calcd. for C₁₆H₁₄NO₃⁺, 268.0968), 290.0779 [M+Na]⁺ (calcd. for C₁₆H₁₃NO₃Na⁺, 290.0788).

Gymnotheoxazole B (2): White powder (MeOH); UV (MeOH) lmax (log e) nm 235 (2.58), 316 (5.20); IR (KBr, cm^-1): λ_max 3400, 1571, 1493, 1462, 1250, 1023, 749; ¹H NMR and ¹³C NMR data, see Table 1; HRESIMS m/z 298.1071 [M+H]⁺ (calcd. for C₁₇H₁₆NO₄⁺, 298.1074), 320.0898 [M+Na]⁺ (calcd. for C₁₇H₁₅NO₄Na⁺, 320.0893).

Crystal data for 1: C₁₆H₁₃NO₃, M = 267.27, monoclinic, a = 9.717 (4) Å , b = 13.558 (6) Å , c = 9.822 (4) Å , α = 90.008, β = 91.040 (9) 8, γ = 90.008, V = 1293.8 (10) ų, T = 153 (2) K, space group P21, Z = 4, μ(MoKa) = 0.71073 mm^-1, 3930 reflections measured, 2229 independent reflections (R_int = 0.0516). The final R₁ values were 0.0454 (I > 2σ(I)). The final wR(F²) values were 0.0993 (I > 2σ(I)). The final R₁ values were 0.0734 (all data). The final wR(F²) values were 0.1120 (all data). The goodness of fit on F² was 0.999.

3. Results and discussion

Compound 1 was obtained as a white crystal. Its molecular formula, C16H13NO3 which indicated 11 degrees of unsaturation, was established from the quasi-molecular ion peak at m/z 290.0779 [M+Na]⁺ (calcd. for C₁₆H₁₃NO₃Na⁺: 290.0788) in the HRESIMS. Its IR spectrum exhibited strong absorption bands at 3324 cm^-1 due to hydroxyl functional group. The ¹H NMR, ¹³C NMR andHSQCspectra of 1 showed 16 carbon resonances due to a monosubstituted benzene ring [δ_H 7.85 (d, 2H, J = 7.6 Hz), 7.51 (t, 2H, J = 7.6 Hz), 7.39 (t, 1H, J = 7.6 Hz); δ_C 129.9, 129.9, 124.9, 124.9, 129.3, 129.1], a 1, 2, 3-trisubstituted phenyl group [δ_H 7.55 (dd, 1H, J = 7.8, 1.7 Hz), 7.12 (t, 1H, J = 7.8 Hz), 7.07 (dd, 1H, J = 7.8, 1.7 Hz); δ_C 152.2, 147.0, 125.4, 122.6, 121.2, 119.3], a oxazole moiety [δ_H 7.71 (1H, s); δ_C 160.1, 152.0, 124.5], and a methoxyl group [δ_H 3.97 (3H, s); δ_C 61.6]. The HMBC correlations (Fig. 2) of OCH3 (δ_H 3.97) to C-2' (δ_C 147.0), H-6' to C-2' and H-4' to C-2', indicated that the methoxyl located at C-2'. The HMBC correlations of H-6' to C-1', C-2' and C-2 confirmedthat the 1, 2, 3-trisubstituted phenyl group located at C-2; the HMBC correlations of H-2" to C-1" and C-5 revealed that the mono-substituted benzene ring was attached to C-5. A single crystal of 1 was obtained from MeOH, and X-ray crystallographic analysis with molybdenum radiation was successfully performed (CCDC 996979), which unambiguously determined the complete structure of 1 as deduced (Fig. 3). In summary, the structure of compound 1 was determined to be 2-(3'-hydroxy-2'-methoxybenzene- 1'-yl)-5-phenyl oxazole, named gymnotheoxazole A.

Compound 2 was obtained as white powder. The molecular formula was determined to be C₁₇H₁₅NO₄ by analysis of the HRESIMS ion peak at m/z 320.0898 [M+Na]⁺ (calcd. for C₁₇H₁₅NO₄Na⁺, 320.0893), indicating 11 degrees of unsaturation. The ¹H, ¹³C NMR and HSQC spectra of 2 showed the presence of a 1, 2-disubstituted benzene ring [δ_H 7.92 (d, 1H, J = 7.6 Hz), 7.51 (t, 1H, J = 7.6 Hz), 7.37 (t, 1H, J = 7.6 Hz), 7.18 (d, 1H, J = 7.6 Hz); δ_C 156.8, 130.1, 126.4, 121.7, 118.0, 112.2], a 1, 2, 3-trisubstituted phenyl group [δ_H 7.56 (dd, 1H, J = 7.8, 1.7 Hz), 7.11 (t, 1H, J = 7.8 Hz), 7.07 (dd, 1H, J = 7.8, 1.7 Hz); δ_C 152.2, 147.0, 125.4, 122.6, 121.2, 119.1], a oxazole moiety [δ_H 7.69 (s, 1H); δ_C 159.1, 148.7, 128.2], two methoxyl groups [δ_H 4.05, 3.96 (each 3H, s); δ_C 61.6, 55.9]. Detailed HMBC analysis suggested that the structure of 2 was very similar to those of 1 except for the replacement of the mono-substituted benzene ring by a 1, 2-disubstituted benzene ring at C-5. The HMBC correlations of OCH₃-2' to C-2', H-3' to C-2' and C-1', H-60 to C-2' and C-1', confirmed that the methoxyl group located at C-2'. Compound 2 was then assigned as 2-(3'-hydroxy- 2'-methoxybenzene-1'-yl)-5-(2"-methoxybenzene-1"-yl) oxazole, named gymnotheoxazole B.

In the MTT assay, compounds 1 and 2 were evaluated in vitro for the cytotoxic activity against A549 cell line, with the IC₅₀ values above 50 μmol/L.

4. Conclusion

Two novel 2, 5-diphenyl oxazole derivatives were isolated from the endemic medicinal plant of G. chinensis. The results of the cell viability assay indicated that these novel compounds showed no detectable cytotoxicity against the A549 cell.

Acknowledgment

This work was financially supported by grants from the National Natural Sciences Foundation (Nos. 21572219 and 21302180) of China. We are grateful to Prof. Sirong Yi for the collection and identification of the plant. We also appreciate Prof. Kaibai Yu for the sing-crystal X-ray experiments.