A new thiophene and two new monoterpenoids from Xanthium sibiricum

Three new compounds (1–3), together with six known compounds (4–9), were isolated from the fruits of Xanthium sibiricum. The structures and the absolute configurations of sibiricumthionol (1), (+)-(5Z)-6-methyl-2-ethenyl-5-hepten-1,2,7-triol [(+)-2], ( − )-(5Z)-6-methyl-2-ethenyl-5-hepten-1,2,7-triol [( − )-2], (2E,4E,1′S, 2′R, 4′S, 6′R)-dihydrophaseic acid (3), (+)-xanthienopyran [(+)-4] and ( − )-xanthienopyran [( − )-4] were established by extensive spectroscopic analyses, X-ray crystallographic analysis, ECCD analysis and ECD calculations. Caffeic acid (7) and caffeic acid ethyl ester (8) weekly inhibited α-glucosidase enzymatic activity by 44.5% and 40.2%, respectively, at 40 μM. Protocatechuic acid (9) selectively exhibited cytotoxicity against HepG2 cell lines, with an IC50 value of 2.92 μM.


Introduction
The fruits of Xanthium sibiricum L. have been used as traditional Chinese medicine for hundreds of years to treat leucoderma, fever, scrofula, sinusitis, headache, herpes, and cancer [1 -4]. In a previous study [5], we have reported the isolation and structural elucidation of a pair of rare racemic spirodienone sesquineolignans from the extract of this medicinal plant. As part of a program to systematically study the chemical diversity of traditional Chinese medicines and their biological effects, the remaining fractions of the EtOAc extract were investigated, leading to the isolation of a new thiophene, two new monoterpenoids, together with six known compounds. The isolation and structural elucidation of three new compounds 1, (þ )-2, and (2 )-2, the absolute configurations of compounds 3, (þ )-4, and (2 )-4, and the bioactivities of the isolated compounds are reported in this paper.

Results and discussion
Compound 1 was isolated as a yellow amorphous powder and was assigned a molecular formula of C 9 Figure 2) from H-3 to C-2/C-4/C-5 indicated a fragment of C(2) -C(3)H -C (4) -C(5). HMBC correlations from H-3 to C-11 and from Me-11 to C-2/C-3 indicated that C-11 and C-2 were directly connected. The HMBC correlation from Me-11 to C-5 and the chemical shifts of C-2 (d C 152.8) and C-5 (d C 133.5) together with the molecular formula of C 9 H 11 NO 2 S showed that C-2 was connected with C-5 through a sulfur atom to form ring A. The signal intensity of HMBC correlation from Me-11 to C-5 is weaker than HMBC correlations from Me-11 to C-2 and from Me-11 to C-3, which indicated the fourbond correlation from Me-11 to C-5 is reasonable. The HMBC correlation from H-8 to C-6 and the chemical shifts of H-8 (d H 4.41) and C-8 (d C 51.9) showed that C-8 was connected with C-6 through a nitrogen atom. Analyses of the degrees of unsaturation of compound 1 and the HMBC correlations from H-8 to C-4/C-5 indicated that another five-membered ring (ring B) was fused to ring A at C-4 and C-5. The fragment of C(9)H 2 -C(10)H 2 confirmed by 1 H-1 H COSY correlation ( Figure 2) was connected with N-7 by HMBC correlations from H 2 -9 to C-6/C-8. In addition, one hydroxyl group, which was determined by IR data (3370 cm 21 ) was located at C-10 by the chemical shifts of C-10 (d C 61.7) and H 2 -10 (d H 3.70). Thus, the structure of compound 1 was characterized as shown in Figure 1 and named sibiricumthionol.
Compound 3 was obtained as yellow crystals (in CH 3    , which indicated that the hydroxy methylene group at C-3 in 6-hydroxydihydrophaseic acid was replaced by a methyl group in compound 3. This was further confirmed by HMBC correlations from Me-6 to C-2/C-3/C-4 ( Figure 2). The relative configuration of compound 3 was deduced from a NOESY experiment, in which the correlations of Me-6/H-5 and H-2/H-4 verified that the conjugated diene was 2E,4E-configur-ation. The cross-peaks between H-4 0 and H-3 0 a/H-5 0 a indicated that H-4 0 , H-3 0 a, and H-5 0 a were on the same face of the sixmembered ring. Moreover, cross-peaks were observed for H-5/Me-7 0 and H-3 0 b, H-5/Me-9 0 , and H-5 0 b indicated that bond C1 0 -C5, Me-7 0 , Me-9 0 , H-3 0 b and H-5 0 b were on the opposite face of the sixmembered ring as shown in Figure 3. According to the established relative configuration of compound 3, the possible absolute configurations of compound 3 were proposed to be (1 0 S, 2 0 R, 4 0 S, 6 0 R) (3a) or (1 0 R, 2 0 S, 4 0 R, 6 0 S) (3b). The calculated ECD spectrum of 3a displayed a CD curve similar to the experimental spectrum of compound 3 ( Figure 4). Meanwhile, the absolute configuration of compound 3 was also supported by the single crystal that was obtained for X-ray diffraction. The crystal structure ( Figure 5) obtained by anomalous scattering of CuKa radiation allowed the unambiguous assignment of the absolute configuration of compound 3 (1 0 S, 2 0 R, 4 0 S, 6 0 R). Thus, the structure of compound 3 was characterized as shown in Figure 1 and named (2E,4E,1 0 S, 2 0 R, 4 0 S, 6 0 R)-dihydrophaseic acid. Compound 3 has been detected and the structure was assigned on the basis of LC-MS n [7]. However, no spectral NMR data were reported earlier. In this paper, the NMR spectral data of compound 3 were presented in Table 3, and the relative and absolute configurations were determined for the first time. Compound 4 possessed identical NMR data with (þ )-xanthienopyran [8] and (2 )-xanthienopyran [9]. Compound 4 was optically inactive, ½a 25 D < 0 (c 0.3, CH 3 OH), indicating that it was obtained as a racemate. Subsequent HPLC separation of compound 4 on a chiral column yielded two compounds whose NMR data were identical to those of compound 4 prior to HPLC separation. However, the isolated compounds showed opposite optical rotation, and their CD spectra displayed mirror curves. This confirmed the successful separation of enantiomers (þ )-4 and (2 )-4. Each enantiomer was obtained with enantiomeric excess (ee) $ 99% (Supporting Information, Figure S35). The absolute configuration of the stereogenic center at C-8 position in (þ )-4 was assigned on the basis of circular dichroism (CD) spectroscopic evidence. In excitoncoupled circular dichroism (ECCD) [10,11], the interaction between two strong electronic transition dipoles leads to "split" Cotton effects (CE), the signs of which are directly related to the chiral twist between the corresponding chromophores. The positive helicity observed for (þ )-4 is in agreement with observed positive exciton-coupled CD experimental data showing a positive Cotton effect at 255 nm (D1 þ 4.92) and negative effect at 225 nm (D1 2 4.50). For (2 )-4, the CD curve showed opposite signs at 255 nm (D1 2 6.84) and 225 nm (D1 þ 6.44) (Supporting Information, Figures S38 and  S39). Thus, the absolute configurations of (þ )-4 [(þ )-xanthienopyran] and (2 )-4 [(2 )-xanthienopyran] were assigned to be 8R and 8S, respectively. The known compounds were identified as caffeic acid (7) [12], caffeic acid ethyl ester (8) [13] and protocatechuic acid (9) [14], by NMR analysis and comparison with literature data.
Compounds 7 and 8 weekly inhibited a-glucosidase enzymatic activity by 44.5% and 40.2% at concentration of 40 mM, respectively (acarbose was used as a positive control, inhibition rate of 99.6%).
The cytotoxic activities of the isolated compounds were tested in vitro against five human cell lines (HCT-116, HepG2, BGC-823, NCI-H1650, and A2780) using the MTT method. However, only compound 9 selectively exhibited cytotoxicity against HepG2 cell lines, with an IC 50 value of 2.92 mM. The other compounds were inactive (IC 50 . 10 mM) against the cell lines tested (taxol was used as a positive control, IC 50 ¼ 3.0 £ 10 22 mM).

Plant material
The fruits from Xanthium sibiricum

Extraction and isolation
The extraction and isolation procedures were reported previously [3]. Fraction ED (20.8 g) was chromatographed over RP-C 18 silica gel (3.5

In vitro cytotoxicity assays
The isolates were tested for their cytotoxic activity against five human cell lines (HCT-116, HepG2, BGC-823, NCI-H1650, and A2780) by established colorimetric MTT assay protocols [15]. Taxol was used as the positive control.

a-Glucosidase inhibitory activity assays
Rat small intestinal brush border membrane vesicles were prepared and its suspension in 0.1 M phosphate buffer (pH 6.0) was used as the small intestinal aglucosidase of maltase, sucrase, isomaltase, and trehalase. The enzyme suspension was diluted to hydrolyse sucrose to produce D-glucose in the following reaction. Reaction was performed in a 96-well plate. The substrate (sucrose: 100 mg/dl), test compound and the enzyme in 0.1 M phosphate buffer (pH 6.0, 0.2 ml) were incubated together at 378C. After 30 min of incubation, the plate was immediately heated to 80 -858C for 3 min to stop the reaction, and then cooled. Glucose concentration was determined by the glucose-oxidase method. The assay was performed in triplicate with five different concentrations around the IC 50 values. The IC 50 values were calculated from the dose -response curves thus obtained in the experiments [16]. Acarbose was used as the positive control.

Disclosure statement
No potential conflict of interest was reported by the authors. Y.-S. Shi et al. 8 .