Cimicifuga foetida was conducted. 113 g (0.17%) actein was purified by recrystallization while eight cycloartane-type triterpenes (1–8) were isolated from the mother liquid. The chemical structures of new compounds (1–4) were elucidated by 1D and 2D NMR and HRESIMS spectroscopic analyses. Moreover, new compounds showed moderate and broad-spectrum cytotoxicity against 5 human cancer cell lines with IC50 values ranging from 4.02 to 15.80 μM.To enrich the bioactive cycloartane triterpenoid glycoside named actein and find out more cytotoxic cycloartane triterpenes, a phytochemical study of The online version of this article (10.1007/s13659-019-0214-1) contains supplementary material, which is available to authorized users. Cimicifuga foetida, commonly known as “shengma”, is a famous traditional chinese medicine native to china. The roots of C. foetida have been widely used to relieve fever and inflammation for thousand years and also have been officially listed in the Chinese Pharmacopoeia -β-d-xylopyranoside (1), 25-O-acetylcimigenol-3-O-[2′-O-3-methoxy-3-oxo-propionyl]-β-d-xylopyranoside (2), 25-O-acetylcimigenol-3-O-[3′-O-3-methoxy-3-oxo-propionyl]-β-d-xylopyranoside (3), 25-O-methoxycimigenol-3-O-[4′-O-acetyl]-β-d-xylopyranoside (4), and four known ones 25-O-acetylcimigenol-3-O-[2′-O-(E)-butenoyl]- α-l-arabinopyranoside (5), Soulieoside A (6), 3′-O-acetylactein (7), 25-O-acetycimigenol-3-O-β-d-xylopyranoside (8) were isolated. New compounds (1–4) were evaluated for their cytotoxic activities against human HepG2, SMMC-7721, A549, MCF-7, and SW-480 cancer cell lines by using the MTT assay. Herein, the recrystallization process of actein, the isolation, structure’s elucidation, and biological activities of new isolates are described.Massive experiments found that triterpenoid saponins which have the similar polar with actein show similar cytotoxic effects –15. Mean1–8) were isolated from the mother liquid. The structure elucidation of new compounds (1–4) are as followed.Actein obtained by simply repeated recrystallization. And eight triterpenoid saponins . IR absorption bands at 3436 and 1734 cm−1 indicated the presence of hydroxy and carbonyl groups. The 1H NMR spectrum of 1 showed the resonances of nonequivalent protons of a cyclopropyl methylene signal [δH 0.18 and 0.51 ], a secondary [δH 0.94 ], and five tertiary methyl groups , an acetyl group [δH 2.13 ], two olefinic protons [δH 6.10  and 7.15 ], an anomeric proton [δH 4.88 ]. The above evidence, together with the characteristic signals for a ketal carbon [δC 105.7 (C-23)] in the 13C NMR spectrum, confirmed that 1 is a 9,19-cimigenol-type monoglycoside with one acetoxy group.Compound δH 5.72 ] to two quaternary carbon resonances [δC 105.7 (C-23) and 65.4 (C-25)], and [δH 1.77 ] to a quaternary carbon resonance at [δC 65.4 (C-25)] and two methine carbon resonances at [δC 98.3 (C-26) and 63.3 (C-24)] indicated that 1 was acteol-type triterpenoid. In the 13C NMR (DEPT) spectra, the signals ascribable to an α, β-unsaturated ketone moiety at δC 165.9 (s), 122.1 (d), 149.7 (d), 31.6 (q) were observed. A comparison of the spectroscopic data of 1 with actein and the olefinic proton [δH 7.15 ], which indicated the tetra-carbon unit to be a 2-butenoyl group. In addition, the coupling constant (J = 15.6 Hz) of the two olefinic protons [δH 6.10 (H-2′′) and 7.15 (H-3′′)] confirmed the E-geometry of a double bond in the 2-butenoyl moiety. Moreover, the observed HMBC correlation of [δH 5.65 ] with the carbonyl of 2-butenoyl suggested that 2-butenoyl was connected to [δC 75.3 (C-2′)]. Meanwhile, [δH 5.07 ] shows HMBC correlation with an ester carbonyl carbon at [δC 170.5 (-OAc)], therefore, a acetyl group is located at C-12. Finally, in the HMBC spectrum ] with the methine carbon [δC 88.2 (C-3)] illustrated that the sugar moiety was located at C-3. After acid hydrolysis, the sugar was identified as D-xylose by comparing its TLC and specific rotation with an authentic sample  in the 1H NMR spectrum. Additionally, when combined with the key correlations observed in the ROESY spectrum of H-1′ , H-2′ , H-3′ , and H-4′ , it indicated that C-2′, C-3′, and C-4′ are α-, β-, and α-configurations, respectively. These findings confirmed that the monoglycoside unit of 1 is a xylopyranoside moiety. Moreover, correlations between H-3/H-5, H-12/H-28, H-16/H-28 suggested α-orientation of H-3, H-12 and H-16.The correlations between H-24/H-27 and H-26/H-27 in ROESY spectrum suggested β-orientation of H-24, Me-27 and H-26 and indicated that compound 1 is stable of α-OH (C-26). The absolute configuration of C-23 was established by comparing to the 13C NMR spectroscopic data of 23-epi-26-deoxyActein (23S) and actein (23R). When the chemical shifts of C-16 and C-20 were δC 73.0 and 26.0, the absolute configuration of C-23 was assigned as R, similar to actein, while signals at δC 74.5 and δC 23.3 were corresponding to S-configuration, similar to 23-epi-26-deoxyActein -β-d-xylopyranoside.In the ROESY spectrum Fig. , the d-xxyActein . Thus, c2 was obtained as a white, amorphous powder, showing an [M−H]− ion at m/z 761.4119 in the HR-ESIMS consistent with the empirical molecular formula C41H62O13 , requiring 11 sites of unsaturation. The IR spectrum showed absorptions of hydroxy and carbonyl groups at 3436 and 1739 cm−1, respectively. In the 13C NMR (DEPT) spectrum, 41 carbon signals could be resolved as nine methyls, ten methylenes, twelve methines, and ten quaternary carbons. In the 1H NMR spectrum of 2, the characteristic cyclopropane methylene resonances at δH 0.23 and 0.46 , an anomeric proton at δH 4.81 , an acetyl methyl at δH 1.66 , a secondary methyl resonances at δH 0.83 , and seven tertiary methyl groups  and a ketal carbon [δC 112.3 (C-16)] in the 13C NMR spectrum, confirmed that 2 is a 9,19-cimigenol-type monoglycoside with one acetoxy group. The 13C NMR spectrum also revealed carbons assignable to a 3-methoxy-3-oxo-propionyl moiety  and a glycosidic moiety .Compound δH 4.81  with the methine carbon at δC 88.5  indicated that the sugar moiety was located at C-3. Aforementioned data indicated that the structure of 2 was similar to 25-O-acetylcimigenol-3-O-β-d-xylopyranoside (8)  instead of 24R-compound [δC 84.1 and δH 3.77 ] -β-d-xylopyranoside.In the ROESY spectra Fig. , the d-x4.4 Hz)] . The con3 gave the same molecular formula as compound 2 by HR-ESIMS. The IR and 1D NMR spectra of compound 3 showed close resemblances to those of 2 except for slight differences resonances of the sugar moiety. In 2, the H-3′ resonance was observed at δH 4.12 m, whereas in 3 it shifted downfield to δH 5.73 . In addition, the C-2′ resonances at δC 75.9 and C-4′ at δC 71.1 in 2 shifted upfield to δC 72.8 and 68.9, respectively, in 3, which may be explained by the presence of the C-3′ 3-methoxy-3-oxo-propionyl moiety of the xylose unit. Which was further proved by the HMBC correlation. And used the same method as compound 2, the configuration at C-23, C-24 were assigned as R and S, respectively. Thus, compound 3 was determined as 25-O-acetylcimigenol-3-O-[3′-O-3-methoxy-3-oxo-propionyl]-β-d-xylopyranoside.Compound 4 gave a molecular formula of C38H60O10 by HR-ESIMS, and the 1H NMR spectra displayed characteristic cycloartane proton signals, including a cyclopropane methylene δH 0.27 and 0.51 , and six methyl proton signals at δH 1.02, 1.12, 1.17, 1.24, 1.26, 1.30 , secondary methyl resonances at δH 0.83  and methoxy group at δH 3.18 . An β-d-xylopyranoside unit was observed by comparison of the 1D NMR data with those of compounds 2 − 3 (Tables 4 were similar to 4′-O-acetylcimigenol-3-O-β-d-xylopyranoside -β-d-xylopyranoside.The ROESY correlations Fig.  of H-3 w6) [O-acetylcimigenol-3-O-[2′-O-butenoyl]-α-l-arabinopyranoside (5) -β-d-xylopyranoside (1): white, amorphous powder; [α]D22 = − 86.9 ; UV (MeOH) λmax (log ε) 197.4 (4.05) nm; IR (KBr) νmax; 3436, 3038, 2728, 1734, 1652, 1457, 1381, 1043, 985 cm−1; 1H and 13C NMR data see Tables m/z 743.4011 [M−H]− .Acteol-3-ranoside : white, O-acetylcimigenol-3-O-[2′-O-3-methoxy-3-oxo-propionyl]-β-d-xylopyranoside (2): white, amorphous powder; [α]D22 = − 22.6 ; UV(MeOH) λmax(log ε) 253.4 (3.05), 196.4 (3.60) nm; IR (KBr) νmax; 3436, 3034, 2727, 1739, 1638, 1457, 1368,1308, 1070, 980 cm−1;1H and 13C NMR data see Tables m/z 761.4119 [M−H]− .25-2.6 c 0.2, MeOH; UO-acetylcimigenol-3-O-[3′-O-3-methoxy-3-oxo-propionyl]-β-d-xylopyranoside (3): white, amorphous powder; [α]D22 = − 31.0 ; UV(MeOH) λmax(log ε) 195.8 (3.62) nm; IR (KBr) νmax; 3467, 3034, 2870, 1737, 1631, 1547, 1475, 1368, 1338, 1037, 992, 865 cm−1;1H and 13C NMR data see Tables m/z 761.4115 [M−H]− .25-O-methoxycimigenol-3-O-[4′-O- acetyl]-β-d-xylopyranoside (4): white, amorphous powder; [α]D22 = − 52.7 ; UV(MeOH) λmax(log ε) 195.6 (3.61) nm; IR (KBr) νmax; 3452, 3035, 2869, 1741, 1631, 1458, 1367, 1308, 1051, 993, 865 cm−1; 1H and 13C NMR data see Tables m/z 721.4165 [M+HCOO]− .25-− 52.7 c .54, MeOH2. Growth inhibition by compounds 1–4 on HL-60, SMMC-7721, A-549, MCF-7 and SW-480 cells was tested by 3--2, 5diphenyltetrazolium bromide (MTT) assay 25 D +21.22 } comparable to those of d-xylose (Sigma-Aldrich).The new compounds Supplementary material 1 (DOCX 11586 kb)Below is the link to the electronic supplementary material.


Lysimachia heterogenea Klatt, together with the eight known compounds: palmitic acid, β-stigmasterol, kaempferol, quercetin, hyperin, isorhamnetin, isorhamnetin-3-O-galactopyranoside and anagallisin C. Heterogenoside F possesses acetoxyl groups at the unusual C-21 and C-22 positions of its oleanane skeleton. The cytotoxic activities of anagallisin C, heterogenoside E and F were weak.Two new 13,28-epoxy oleanane-type triterpenoids, namely heterogenoside E and F, were isolated from  Lysimachia heterogenea Klatt . The cytotoxic activities of compounds 8, 9 and 10 were also evaluated.In a previous cytotoxicity-guided phytochemical screening, we found four 12-oleanene derivatives in the cytotoxic fractions of Heterogenoside E (8), obtained as white powder (MeOH), had the molecular formula of C46H74O17 according to its HRESIMS data. The degrees of molecular unsaturation were calculated to be 10, among which three sugar rings, deduced from the reaction products of the chemical analysis and three pairs of anomeric protons and carbons in 1H- and 13C-NMR spectra , 5.01 (J = 7.5 Hz) and 4.89 (J = 6.5 Hz) determined the glycosyl linkage as 3-O-{β-D-xylopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl}. Therefore, it was concluded that 8 was anagalligenone 3-O-{β-D-xylopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl} , white powder (MeOH), had the formula C56H90O25 according to its HRESIMS data, and the DEPT and HMBC spectra revealed two AcO groups (1H- and 13C-NMR spectra (δ 4.78 (J = 6.0 Hz), 5.50 (J = 7.5 Hz), 5.02 (J = 7.5 Hz) and 4.93 (J = 6.0 Hz), and the HMBC correlations from H''-1 to C'-2, H'''-1 to C'-4, H''''-1 to C'''-2 supported the existence of a β-D-xylopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranosyl moiety. The remaining eight degrees of unsaturation were assigned to the AcO moieties and a six-ring triterpenoid aglycon. HMBC correlations from H-21 to carbon at δ 171.1 and C-22; H-23 to C-3, C-4, C-5, C-24; H-24 to C-3, C-4, C-5 and C-23; H-25 to C-1, C-5, C-9 and C-10; H-26 to C-7, C-8, C-9 and C-14; H-27 to C-8, C-13, C-14 and C-15; H-29 to C-19, C-20, C-21 and C-30; H-30 to C-20, C-21 and C-29; led to the construction of 13,28-epoxy-3,16,21,22-tetrol oleanane. Extensive analysis of the correlation of H-29 and H-21 in the NOESY spectrum, the coupling constant of H-21 (J= 10.0 Hz) and the referenced structures in the literature -α-L-arabinopyranosyl } , β-stigmasterol (2), isorhamnetin (3), kaempferol (4), quercetin (5), isorhamnetin-3-O-galactopyranoside (6), hyperin (7) and anagallisin C (10) were determined by comparison to the 1H and 13C-NMR spectral data in the literature − .HeterogenosideF (9): mp 216–217 °C; −24.9 ; IR (KBr); v 3396, 1077 and 1043 (OH), 2922 , and 1721 (ester) cm−1; 1H- and 13C-NMR spectral data were listed in m/z: 1161.5701 [M-H]− .2, and human epithelial carcinoma cell line Hela cells (5 × 103 cells/well) were cultured in 96-well plates for 24 h, respectively, then a buffer solution (100 μL) containing the test compounds at various concentrations  was added to each well. The cells, treated with those compounds and DMSO (as the control), were incubated for 72 h at 37 °C in a humidified chamber. The culture was terminated by adding MTT solution  to each well, and a further incubation for 4 h was performed. After the suspension was removed from each well, DMSO (100 μL) was added and mixed thoroughly. The absorbance was measured by a microplate reader at 492 nm with 620 nm as reference, and mean IC50 values were calculated.The human epidermoid carcinoma cell line KB-3-1, human hepatocellular liver carcinoma cell line HepGβ-stigmasterol, kaempferol, quercetin, hyperin, isorhamnetin, isorhamnetin-3-O-galactopyranoside and anagallisin C), as well as two new triterpenoids, named heterogenoside E and F, were isolated from Lysimachia heterogenea Klatt. Heterogenoside F is a rare 21,22-diacetoxyl triterpenoid. The cytotoxic activities of anagallisin C, heterogenoside E and F were weak.Eight known compounds (palmitic acid,


NMR data from NOEdiff, 2D-NOESY and 1H-NMR spectra at different temperatures, and also the synthesis of isopropylidene hydrazone revealed the nature of duplicated signals of a 4-methyl-2-phenylpyrimidine-N-acylhydrazone derivative as a mixture of two conformers in solution. Further we investigated the stereoelectronic influence of substituents at the ortho position on the pyrimidine ring with respect to the carbonyl group, as well as the electronic effects of pyrimidine by changing it to phenyl. The conformer equilibrium was attributed to the decoplanarization of the aromatic ring and carbonyl group  and/or the electron withdrawing character of the pyrimidine ring. Both effects increased the rotational barrier of the C-N amide bond, as verified by the ΔG≠ values calculated from dynamic NMR. As far as we know, it is the first description of aryl-NAH compounds presenting two CO-NH bond- related conformations.Herein we describe NMR experiments and structural modifications of 4-methyl-2-phenylpyrimidine-N-acylhydrazone compounds (aryl-NAH) in order to discover if duplication of some signals in their  N-acylhydrazone (NAH) moiety has been identified in a great number of lead compounds that act on different types of molecular targets [E/Z)   and as s-NH bond  5]..N-acylhyN-acylhydrazone compounds with antinociceptive and anti-inflammatory activities led to the discovery of LASSBio-1083 -ethyl 2-((dimethylamino)methylene)-3-oxobutanoate (5) and (Z/E)-methyl 2-((dimethylamino)methylene)-3-oxopentanoate (6), respectively, in ethanol (for 4a) and methanol (for 4b) at room temperature  was obtained in 50% yield by condensation between benzamidine and diethyl 2-(ethoxymethylene)malonate (7) in ethanol at room temperature  waH in THF  13,14].,14.8) wa4d was achieved by Suzuki cross-coupling between methyl ester 10 and phenylboronic acid in the presence of potassium carbonate and PdCl2(PPh3)2 at 80 °C in 91% yield, while the ester 4e was obtained commercially , these signals appear to be duplicated  is presented in E/Z) of the imine double bond, we decided to proceed with differential Nuclear Overhauser Effect  experiments to assess the spatial proximity of 1H-1H. The hydrogen atom selected for irradiation was the NH of the amide. Due to the presence of two singlets related to the NH hydrogen, we chose the one that shifted less and that was more prevalent at 11.67 ppm  and Z (C) stereoisomers, obtained through a process of geometric optimization by molecular mechanics followed by conformational analysis using the semi-empirical method AM1 in the PC Spartan Pro 1.0.5 software [B conformer was not as stable as A, the choice was based on observations from other NAH compounds in X-ray diffraction studies  conformers and be more pronounced at N=CH in the B conformer  and a positive one for both signals of N=CH and H3′′/H5′′, presenting a positive NOE of 9% and 8%, respectively . This effect was dependent on the experimental mixing time , with longer times resulting in more intense cross-correlations.1H has a distinct chemical shift in each conformer. The time required for one conformer to become another explains the increase in intensity with longer mixing times.Normally this type of cross-correlation can be attributed to an artifact because of a chemical exchange with solvent. However, this is not the case, as we observed a cross-correlation for all protons including those that were not exchangeable. The cross-correlation is most plausibly due to an exchange among multiple conformers in which a Z stereoisomer because all NOE results are in agreement with the more stable E stereoisomer [H could be synperiplanar (sp) or antiperiplanar (ap) in relation to the carbonyl oxygen atom of the E isomer 2 and CH3 signals, while partial coalescence was observed for N=CH and H2′′/H6′′. The reversibility of these changes was verified when the experimental temperature was returned to 20 °C. This result corroborated the hypothesis of the conformers: the energy required to faster overcome the rotational barrier is reached upon an increase in temperature, leading to a rapid conversion between the conformers. Traditionally, the amide group is represented by the canonical forms 1 and 3.Based on the work of Quattropani and colleagues , who desobserved . A compl2 atom and the carbonyl π bond contributes to the double bond character of the amides in the planar form. In this context, the resonance structure 3 is considered more significant for the double bond character and rotational barrier [The resonance effect between the lone pair on the nitrogen sp barrier . 2 hybridization to a pyramidal arrangement. In the pyramidal geometry, the lone pair of nitrogen is placed in an orbital with high s character for stabilization. The disruption of favorable interactions between N and C raises the rotational barrier. In the case of formamide, the barrier is 18 kcal mol−1 [Converting a conformer into another requires rotation around the C-N bond, which consequently changes the nitrogen spal mol−1 ,23,24,25et al. conducted theoretical studies concerning the amide bond rotation. Their calculations showed that the C-N bond lengthens by 0.08 Ǻ upon rotation from the planar form to the transition state, whereas the C-O bond shortens slightly (by 0.01 Ǻ). Moreover, an insignificant difference in charge density at the oxygen was observed in the planar form and the transition state. Hence, the oxygen is assumed to simply polarize the C-O bond, resulting in a large δ+ on the carbon [Wiberg e carbon .2/3 and 5/6 [As the earlier model with two canonical structures did not explain these results, one that incorporated a carbonyl dipolar canonical form was suggested (2). Therefore, the rotational barrier is attributed mainly to the resonance structures  and 5/6 ,23.N-Methylation of NAH 1, which results in N-methyl-NAH 3a, causes conformational restriction of the amide bond, thus preventing its rotation, as previously described by our group [E/Z stereoisomers. The compound (isopropylidene)-2-phenyl-4-methyl-pyrimidine-5-hydrazide (12) (11a) with acetone. Its 1H-NMR spectrum presented two singlets for each NH, H6 and CH3a and another three for CH3b and CH3c. Regarding CH3a, the signal present to a lesser extent showed the same chemical shift of the solvent DMSO-d6 (2.48 ppm). Likewise, the signal for the smaller proportion of CH3c and that for the greater proportion of CH3b also coincided (both were at 1.9 ppm). This result confirmed the presence of conformers.ur group ,26. In fur group  and Pallur group , to elimide (12)  was syntH matches the conformer ap, based on the work by Palla in which the more deshielded signal was attributed to imine hydrogen of conformer ap in DMSO-d6 and in CDCl3 [ap conformation, the two dipolar moments (Cδ+=Oδ− and Nδ−-Hδ+) are aligned, which does not occur in the sp conformer [Considering the proportion of the conformers, we believe that the more deshielded signal from N=Cin CDCl3 . Furtheronformer . N-acylhydrazones. However, NAH compound 1 does not structurally match the compounds described in previous reports. Perhaps an ortho steric effect, exerted by the methyl group on the carbonyl, and/or an electronic effect induced by the pyrimidine ring could be related to the observed phenomenon [According to literature reports, the presence of conformers for the NAH compounds is typically observed in those with a spacer between the carbonyl group and the aromatic ring or in alkyl-enomenon . Thus, wortho steric effect, the methyl group was replaced by an H atom (2a) and an ethyl group (2b). The first modification represents the absence of any steric effect while the second one increases the steric effect. Both compounds showed duplicated signals in the 1H-NMR spectra, as shown in 2c and 2d. Thus, we could study the influence of the heterocyclic ring both in the presence (2c) and absence (2d) of an ortho steric effect. Only 2d did not show duplicated signals. Therefore, we found that the steric effect and/or the pyrimidine ring induced the formation of conformers in this class of NAH.To evaluate the ortho-alkyl group) as well as the electron withdrawing effect exerted by the pyrimidine ring on the carbonyl group increase polarization. Both effects are disadvantageous in regard to the transition state between one conformer and the other. The transition state between amide rotamers is when the C-N bond is turned 90° and the nitrogen is not conjugated with the carbonyl . As stated by Olsen and colleagues for nicotinamide and picolinamide, in the transition state, the π electrons from the aromatic ring can stabilize the resonance forms B, lowering the transition state energy and facilitating amide bond rotation , leading to increases in the transition state energy and rotational barrier  are present in the stable conformers. The HMBC correlations show that for compounds 1 and 2a–c, the signals of the amide N-1H of each conformer are coupled differently to the aromatic 13C (13C-CO-N-1H). Only the minor conformer, which has a trans correlation between these atoms, shows a 3JCH coupling . Because of this small barrier, 2d does not possess the fixed trans conformation necessary for 3JCH coupling in this series. In addition, 3JCH coupling of the amide N-1H and imine 13C=N was present in all compounds, indicating the similar chemical environments with no great differences in conformation for this moiety of NAH compounds.The higher spcoupling . These rd to cis ,30. On tG≠) related to the transition state among one conformer and another can be calculated using Equation 1. Through a dynamic NMR process, we could estimate the coalescence temperature (Tc), which provides, in association with the maximum peak separation (Δv in Hz) at low-temperature , the energy of the rotational barrier (∆G≠) [The Gibbs free energy of activation (∆er (∆G≠) :∆G≠ = 4G≠ values showed that the steric effect exerted by the methyl group on the ring decoplanarization and the electron withdrawing effect of pyrimidine have similar effects on the rotational barrier because compounds 2a and 2c have almost the same ∆G≠  are given in ppm and coupling constants (J) in Hertz. Melting points were determined with a Quimis Q340.23 apparatus and are uncorrected. Elemental analyses were carried out on a Thermo Scientific Flash EA 1112 Series CHN-Analyzer. Thin layer chromatography was performed on Merck Kieselgel 60 HF 254 plates and detection took place using UV (254 and 365 nm). All reagents and solvents were purchased from commercial suppliers and used without further purification.NMR spectra were recorded on a 200/50 MHz Bruker DPX-200, 250/62.5 MHz Bruker DPX-250, 400/100 MHz Bruker DPX-400, 500/125 MHz Bruker Avance-500, 400/100 MHz Varian 400-Mr, 300/75 MHz Varian Unity-300 spectrometer. The spectra were performed in DMSO-E/Z)-ethyl 2-((dimethylamino)methylene)-3-oxobutanoate . The reaction mixture was stirred at room temperature for 2 h. The solvent was evaporated under vacuum and the resulting residue was filtered and washed with cold water. The title compound was obtained as a white crystalline solid in 80% yield. 1H-NMR  δ 9.17 ; 8.46–8.44 ; 7.58–7.55 ; 4.36 ; 2.80 ; 1.35 .  13C-NMR  δ 168.13 (C=O); 164.50 (C2het); 164.31 (C4het); 158.90 (C6het); 136.11 (C1′Ph); 131.76 (C4′Ph); 128.84 ; 128.37 ; 121.30 (C5het); 61.30 (OCH2); 24.28 (CH3het); 14.01 (CH3Et).In a round bottomed flask under argon atmosphere were successively added anhydrous ethanol (20 mL), sodium metal , benzamidine hydrochloride  and (E/Z)-methyl 2-((dimethylamino)methylene)-3-oxopentanoate . The reaction mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure. The precipitate was filtered and washed with cold water, affording the title compound as a white amorphous solid in 73% yield. 1H-NMR  δ 9.12 ; 9.48–9.46 ; 7.46–7.41 ; 3.89 ; 3.18 ; 1.32 . 13C-NMR  δ 172.98 (C=O); 165.42 (C4het); 159.19 (C6het); 136.88 (C1′Ph); 132.50 (C4′Ph); 128.88 ; 128.50 ; 120.16 (C5het); 52.14 (OCH3); 29.84 (CH2); 12.36 (CH3).To a solution of benzamidine hydrochloride  and sodium hydroxide  in methanol (2 mL), was added (3 (1:1:1) (45 mL), giving the ethyl 4-hydroxy-2-phenylpyrimidine-5-carboxylate (8) as a white crystalline solid in 50% yield. The derivative 8  and POCl3  were refluxed at 100oC for 1 h. The excess of POCl3 was removed under vacuum, ice was added on the resulting solid into the reaction flask, followed by filtration and washing with cold water. The chloride compound 9, obtained as a white amorphous solid in 98% yield, was successively dehalogenated with zinc powder  in anhydrous THF (2 mL). The reaction mixture was stirred at 60 °C for 1 h, and then 5 drops of acetic acid were added to the reaction vessel. After stirring at 60 °C for 23 h, the reaction mixture was cooled to room temperature, followed by addition of CH2Cl2 (3 mL), filtration and evaporation of solvent. Purification by silica gel column chromatography (n-hexane-EtOAc 0.6%) afforded the title compound as a white crystalline solid in 50% yield. Compound 8: 1H-NMR  δ 12.22 ; 8.64 ; 8.16 ; 7.68–7.51 ; 4.25 ; 1.28 . 13C-NMR  δ 163.9; 161.3; 160.1; 159.0; 133.0; 131.9; 129.1; 128.8; 115.0; 60.8; 14.5. Compound 9: 1H-NMR  δ 9.19 ; 8.50 ; 7.61–7.46 ; 4.46 ; 1.45 . 13C-NMR  δ 166.57; 162.94; 160.61; 160.52; 135.32; 132.48; 129.29; 128.85; 121.61; 62.28; 14.23. Compound 4c: 1H-NMR  δ 9.27 ; 8.45–8.41 ; 7.59–7.52 ; 4.36 ; 1.33 . 13C-NMR  δ 166.30 (C=O); 163.84 (C2het); 158.67 ; 136.49 (C1′Ph); 132.45 (C4′Ph); 129.37 ; 128.90 ; 122.28 (C5het); 61.87 (OCH2); 14.47 (CH3).In a round bottomed flask under argon atmosphere were successively added anhydrous ethanol (5 mL), sodium metal , benzamidine hydrochloride  and diethyl 2-(ethoxymethylene)malonate . The reaction mixture was stirred at room temperature for 9 h, and then poured into ice. The precipitate was filtered, washed with coldwater and recrystallized in EtOH-MeOH-CHCl2Cl2. The organic layer was washed with 10% sodium carbonate solution, dried over anhydrous Na2SO4, filtered and evaporated, giving the methyl 4-bromo-2-methylbenzoate 10 as a colorless oil in 86%, which was directly used in cross coupling reaction. The compound 10  was transferred to a round bottomed flask with a mixture of toluene-methanol , followed by addition of phenylboronic acid , PdCl2(PPh3)2  and potassium carbonate . The reaction mixture was stirred at 60 °C for 3 h, and after cooling to room temperature, was filtered over Celite. The resulting oil was purified by silica gel column chromatography (n-hexane-EtOAc 0%–1%), affording the title compound as a white crystalline solid in 91% yield, after evaporation under vacuum. Compound 10: 1H-NMR  δ 7.73 ; 7.58 ; 7.51 ; 3.82 ; 2.49 . 13C-NMR  δ 167.18; 142.31; 134.65; 132.51; 129.54; 129.16; 126.33; 52.58; 21.18. Compound 4d: 1H-NMR  δ 7.92 ; 7.55–7.51 ; 7.40–7.25 ; 3.83 ; 2.60 . 13C-NMR  δ 167.97 (C=O); 144.75 (C1Ph); 140.91 (C1′Ph); 140.14 (C3Ph); 131.36 (C5Ph); 130.49 (C2Ph); 128.95 ; 128.30 (C4Ph); 128.09 C4′Ph); 127.33 ; 124.47 (C6Ph); 51.89 (OCH3); 22.08 (CH3).In a round bottomed flask into ice-bath, were added methanol (15 mL), acetyl chloride  and 4-bromo-2-methylbenzoic acid . The reaction mixture was stirred at room temperature for 24 h, and then concentrated under reduced pressure. The resulting residue was extracted with CH4a, 0.710 g, 2.92 mmol) in ethanol (5 mL) was added hydrazine monohydrate 100% . The reaction mixture was stirred at 55 °C for 4 h. After cooling the reaction to room temperature, the round bottomed flask was placed in an ice-bath and the solid, filtered and washed with cold water. The title compound was obtained as a white crystalline solid in 83% yield. 1H-NMR  δ 9.80 ; 8.76 ; 8.44–8.39 ; 7.56–7.52 ; 4.62 ; 2.63 . 13C-NMR  δ 165.43 (C=O); 164.80 (C2het); 163.02 (C4het); 155.56 (C6het); 136.54 (C1′Ph); 131.16 (C4′Ph); 128.72 ; 127.92 ; 126.32 (C5het); 22.63 (CH3).To a solution of ethyl 4-methyl-2-phenylpyrimidine-5-carboxylate  in ethanol (3 mL) was added hydrazine monohydrate 100% . The reaction mixture was stirred at 50 °C for 5 h, cooled to room temperature and poured into ice. The solid was filtered and washed with cold water, giving the title compound as a white amorphous solid in 79% yield. 1H-NMR  δ 9.77 ; 8.72 ; 8.42–8.40 ; 7.54–7.51 ; 4.59 ; 2.90 ; 1.27 . 13C-NMR  δ 170.12 (C=O); 165.40 (C4het); 163.66 (C2het); 156.09 (C6het); 137.23 (C1′Ph); 131.71 (C4′Ph); 129.31 ; 128.44 ; 126.68 (C5het); 28.62 (CH2); 13.14 (CH3). To a solution of 1H-NMR  δ 10.12 ; 9.20 ; 8.43–8.39 ; 7.55–7.52 ; 4.64 . 13C-NMR  δ 165.04 (C=O); 162.88 (C2het); 156.71 ; 136.78 (C1′Ph); 131.99 (C4′Ph); 129.31 ; 128.56 ; 125.03 (C5het). To a solution of ethyl 2-phenylpyrimidine-5-carboxylate (4c)  in ethanol (2 mL) was added hydrazine monohydrate 100% . The reaction mixture was stirred at room temperature for 2 h. Ice was added to the reaction flask and the solid, filtered and washed with cold water. The title compound was obtained as a white amorphous solid in 85% yield. 1H-NMR  δ 9.46 ; 7.69–7.66 ; 7.54–7.37 ; 4.49 ; 2.42 . 13C-NMR  δ 168.85 (C=O); 141.68 (C1Ph); 140.04 (C1′Ph); 136.98 (C3Ph); 135.06 (C4Ph); 129.50 ; 129.28 (C5Ph); 128.54 (C2Ph); 128.28 (C4′Ph); 127.26 ; 124.25 (C6Ph); 20.08 (CH3).To a solution of methyl ester (4d)  in ethanol (5 mL) was added hydrazine monohydrate 100% . The reaction mixture was stirred at 50 °C for 15 h, and then concentrated under reduced pressure. Ice was added to the reaction flask and the solid, filtered and washed with cold water. The title compound was obtained as a white amorphous solid in 95% yield. 4e, 0.50 g, 2.36 mmol) in ethanol (5 mL) was added hydrazine monohydrate 100% . The reaction mixture was stirred at 80 °C for 8 h, and then concentrated under reduced pressure. Ice was added to the reaction flask and the solid, filtered and washed with cold water. The title compound was obtained as a pale amorphous solid in 85% yield. 1H-NMR  δ 9.85 ; 7.93 ; 7.77–7.69 ; 7.52–7.36 ; 4.55 . 13C-NMR  δ 166.13 (C=O); 143.17 (C1Ph); 139.74 (C1′Ph); 132.66 (C4Ph); 129.57 ; 128.55 (C4′Ph); 128.17 ; 127.37 ; 127.08 .To a solution of methyl biphenyl-4-carboxylate  in ethanol (5 mL) were added HCl catalytic (2 drops) and the appropriate aromatic aldehyde. The reaction mixture was stirring at room temperature for N′-(4-(Dimethylamino)benzylidene)-4-methyl-2-phenylpyrimidine-5-carbohydrazide (1): The condensation of 4-methyl-2-phenylpyrimidine-5-carbohydrazide (11a) and 4-dimethylamino- benzaldehyde, afforded the title compound as a yellow solid (mp. 206 °C) in 99% yield. 1H-NMR  δ 11.79, 11.67 ; 8.91, 8.81 ; 8.45–8.41 ; 8.17, 7.98 ; 7.56–7.54 ; 7.24 ; 6.76, 6.64 ; 2.97, 2.88 2); 2.66, 2.53 . 13C-NMR  δ 167.42, 165.96 (C=O); 164.37, 163.48 (C2het); 162.95, 161.59 (C4het); 156.37, 156.10 (C6het); 151.97, 151.73 (C4′′); 149.60, 145.96 (N=CH); 136.97, 136.84 (C1′Ph); 131.47, 131.34 (C4′Ph); 128.99 ; 128.26 ; 127.64, 126.72 (C5het); 121.09, 121.03 (C1′′); 111.73 ; 39.87 (N(CH3)2); 23.02, 22.89 (CH3). Anal. Calcd for C21H21N5O: C: 70.18; H: 5.89; N: 19.48. Found: C: 70.17; H: 5.89; N: 19.62.N′-(4-(Dimethylamino)benzylidene)-2-phenylpyrimidine-5-carbohydrazide (2a): The condensation of 2-phenylpyrimidine-5-carbohydrazide (11c) and 4-dimethylaminobenzaldehyde, afforded the title compound as a yellow amorphous solid (mp. 269 °C) in 99% yield. 1H-NMR  δ 11.79 ; 9.28 ; 8.47–8.44 ; 8.30, 8.01 ; 7.57–7.55 ; 7.39 ; 6.77–6.69 ; 2.97, 2.92 2). 13C-NMR  δ 165.47, 165.33 (C=O); 164.66, 160.02 (C2het); 158.90, 157.43 ; 152.45, 152.22 (C4′′); 150.42, 146.48 (N=CH); 137.11 (C1′Ph); 132.26 (C4′Ph); 129.54 ; 129.35 ; 128.90 ; 126.70, 125.96 (C5het); 121.88 (C1′′); 112.50 ; 40.56 (N(CH3)2). Anal. Calcd for C20H19N5O: C: 69.55; H: 5.54; N: 20.28. Found: C: 69.20; H: 5.54; N: 20.02.N′-(4-(Dimethylamino)benzylidene)-4-ethyl-2-phenylpyrimidine-5-carbohydrazide (2b): The condensation of 4-ethyl-2-phenylpyrimidine-5-carbohydrazide (11b) and 4-dimethylamino- benzaldehyde, afforded the title compound as a yellow solid (mp. 240 °C) in 85% yield. 1H-NMR  δ 11.86, 11.74 ; 8.90, 8.79 ; 8.47–8.43 ; 8.15, 7.96 ; 7.56–7.53 ; 7.21 ; 6.75 ; 6.62 ; 2.99–2.94 2); 2.87 2); 2.81 ; 1.32–1.25 .  13C-NMR  δ 169.75, 167.93 (C=O); 167.22, 163.24 (C4het); 162.65, 161.25 (C2het); 155.93, 155.80 (C6het); 151.61, 151.36 (C4′′); 149.18, 145.50 (N=CH); 136.81, 136.67 (C1′Ph); 131.15, 131.00 (C4′Ph); 128.70, 128.56 ; 127.92, 127.08 ; 126.19 ; 121.03 (C1′′), 120.95 (C5het); 111.69 ; 39.64, 39.46 (N(CH3)2); 28.30, 28.08 (CH2); 12.49, 12.03 (CH3). Anal. Calcd for C22H23N5O: C: 70.76; H: 6.21; N: 18.75. Found: C: 70.62; H: 6.19; N: 18.80. (E)-N′-(4-(Dimethylamino)benzylidene)-3-methylbiphenyl-4-carbohydrazide (2c): The condensation of 3-methyl-biphenyl-4-carbohydrazide (11d) and 4-dimethylaminobenzaldehyde, afforded the title compound as a yellow amorphous solid (mp. 238 °C) in 94% yield. 1H-NMR  δ 11.41 ; 8.17, 7.94 ; 7.72–7.67 ; 7.58–7.35 ; 7.22 ; 6.74, 6.62 ; 2.96, 2.87 2); 2.45, 2.33 . 13C-NMR  δ 164.83 (C=O); 151.74, 151.48 (C4′′); 148.44, 144.70 (N=CH); 141.66, 140.62 (C1Ph); 139.69 (C1′); 136.84, 135.66 (C3Ph); 134.82 (C4Ph); 129.17, 129.06 ; 128.63, 128.32 ; 127.85 (C4′Ph); 126.97 ; 124.03, 123.46 (C6Ph); 121.84 (C1′′); 112.03, 111.29 ; 39.95 (N(CH3)2); 19.69 (CH3). Anal. Calcd for C23H23N3O: C: 77.28; H: 6.49; N: 11.76. Found: C: 76.92; H: 6.47; N: 11.59.(E)-N′-(4-(Dimethylamino)benzylidene)-biphenyl-4-carbohydrazide (2d): The condensation of biphenyl-4-carbohydrazide (11e) and 4-dimethylaminobenzaldehyde, following the general procedure for the synthesis of N-acylhydrazones, afforded the title compound as a yellow amorphous solid (mp. 289 °C) in 99% yield. 1H-NMR  δ 11.58 ; 8.32 ; 7.98 ; 7.8 ; 7.74–7.72 ; 7.55–7.47 ; 7.42–7.38 ; 6.75 ; 2.96 2). 13C-NMR  δ 162.27 (C=O); 151.52 (C4′′); 148.64 (N=CH); 142.97 (C1Ph); 139.12 (C1′Ph); 132.53 (C4Ph); 129.02 ; 128.42 ; 128.14 ; 126.85 ; 126.58 ; 121.62 (C1′′); 111.81 . Anal. Calcd for C22H21N3O: C: 76.94; H: 6.16; N: 12.24. Found: C: 76.65; H: 6.15; N: 11.96. N-acylhydrazone 1 (0.14 mmol) and potassium carbonate (0.42 mmol) in acetone (5 mL) in an ice-bath was added methyl iodide (0.42 mmol). After, the reaction mixture was stirred at 43 °C for 24 h. The addition of 0.42 mmol of methyl iodide was needed, and then the reaction mixture was stirred at the same temperature for more 20 h. The reaction mixture was concentrated under vacuum, ethanol (1 mL) was added and the resulting mixture poured into ice. After filtering, the solid was heated into petroleum ether, and then refiltered to give the title compound as a yellow amorphous solid (mp. 188–189 °C) in 77% yield. 1H-NMR  δ 8.73 ; 8.46–8.42 ; 7.94 ; 7.54–7.52 ; 7.22 ; 6.61 ; 3.50 ; 2.85 2); 2.43 . 13C-NMR  δ 167.81 (C=O); 164.18 (C2het); 163.12 (C4het); 156.09 (C6het); 152.13 (C4′′); 143.46 (N=CH); 137.53 (C1′Ph); 131.65 (C4′Ph); 129.57 ; 129.36 ; 128.76 ; 128.62 (C5het); 122.34 (C1′′); 112.50 ; 40.32 (N(CH3)2; 28.92 (N-CH3); 23.29 (CH3). Anal. Calcd for C22H23N5O: C: 70.76; H: 6.21; N: 18.75. Found: C: 70.41; H: 6.05; N: 18.43. To a solution of 1H-NMR  δ 10.9, 10.6 ; 8.83, 8.70 ; 8.42–8.40 ; 7.54–7.53 ; 2.60, 2.48 ; 1.99, 1.91, 1.77 2). 13C-NMR  δ 168.57, 165.73 (C=O); 164.32, 163.47 (N=C); 162.97, 162.39 (C2het); 159.30, 153.61 (C4het); 156.47, 156.30 (C6het); 137.15, 137.12 (C1′Ph); 131.72, 131.60 (C4′Ph); 129.31, 129.25 ; 128.43, 128.38 ; 127.43 (C5het); 25.62, 25.56 (N=C-CH3); 23.14, 23.09 (CH3het); 18.55, 17.98 (N=C-CH3). Anal. Calcd for C15H16N4O: C: 67.15; H: 6.01; N: 20.88. Found: C: 66.93; H: 6.05; N: 20.52.A mixture of 4-methyl-2-phenylpyrimidine-5-carbohydrazide (11a) 0.07 g, 0.31 mmol) in acetone (7 mL) was stirred for 15 h at 45 °C. After, the solvent was evaporated to give a white solid in 85% yield (mp. 186–188 °C). The sketching, geometry optimization and conformational search of compounds were performed in PC Spartan Pro 1.0.5 software. They were subjected to structural minimization by the use of molecular mechanics, using the base MMFF. Subsequently, the conformational analysis was performed, using the semi-empirical AM1.E/Z), and additionally the election of a second conformer (B) for the E isomer which ΔHf = 114.68 kcal/mol. Conformers that have the carbonyl group synperiplanar related to NH hydrogen were obtained by a dihedral angle search in 12 steps of 30° on the amide bond CO-NH, leaving the other angles free. Then we selected the lowest energy conformer, generated by the conformational analysis with the semi-empirical AM1 method, which presented the carbonyl synperiplanar related to the NH. The NOE calculations were carried out in the MSpin 1.03 software from the selected structures from the conformational analysis.The NOE prediction study was performed by selecting the minimum conformation of each stereoisomer  among them, which results from both the decoplanarization of the aromatic ring and carbonyl group  and the electron withdrawing nature of the pyrimidine ring.In this work, we revealed that duplicated signals observed in NMR spectra of 4-methyl-2-phenylpyrimidine-N-acylhydrazone correspond to the presence of two CO-NH bond- related


Their structures were elucidated on the basis of extensive spectroscopic data . The cytotoxic activities of compounds 1–18, 20, and 22 were evaluated by MTT assay. Compound 6 showed the most potent cytotoxicity in MCF-7, and HepG2 cell lines, with IC50 values of 6.25 and 3.83 μM, respectively.Sixteen new prenylated flavonoids, sinoflavonoids P–Z ( Sinopodophyllum hexandrum, belonging to the family of Berberidaceae, are widely distributed in the Southwest of China , and one olefinic carbon δ 93.3 at upper field instead of C-8 (δ 97.7) in 1. The above data suggested that compound 2 was 6,2′-diprenyl-3-methoxyquercetin, which was also supported by HR-ESI-MS and the HMBC spectrum. The HR-ESI-MS gave an [M + H]+ ion peak at m/z 453.1910 , being 14 mass units more than that of 1. Two 3-methyl-2-butenyls were linked to C-6 and C-2′, respectively, due to the long range correlations of methylene group δ 3.21  with C-5 (δ 158.5), C-6 (δ 111.1), and C-7 (δ 162.3), and of δ 3.24  with C-1′ (δ 123.2), C-2′ (δ 128.2), and C-3′ (δ 143.7) in the HMBC spectrum -5,7,3′,4′-tetrahydroxy-3-methoxyflavone, and named sinoflavonoid Q.Compound e 1H-NMR  and 13C-NMR data  of compospectrum . The met3 and 4 were obtained as yellow, amorphous powders. Their HR-ESI-MS showed the same molecular formula of C26H28O7, according to an [M + H]+ quasi-molecular ion peak  in 3; m/z 453.1912  in 4). Their 1H , 1.74 , δ 16.7, 30.8 in 3; δ 2.62 , 1.81 , δ 15.7, 30.9 in 4), two tertiary-methyl groups , δ 26.4 (×2) in 3; δ 1.30 , δ 26.3 (×2) in 4), and one oxygen-bearing aliphatic quaternary carbon . The methylene group  in 3; δ 2.62  in 4) showed the long range correlations , C-6 , and C-7 , indicating that the 2,2-dimethyldihydropyrano group was attached to C-6 and C-7, or C-5 and C-6. The presence of the phenolic hydroxyl group  in 3; δ 13.00  in 4) supported the linking position of the 2,2-dimethyldihydropyrano group . Thus, the structures of compounds 3 and 4 were deduced respectively as 5,6--2′-(3-methyl-2-butenyl)-7,3′,4′-trihydroxy-3-methoxyflavone (3) and 6,7--2′-(3-methyl-2-butenyl)-5,3′,4′-trihydroxy-3-methoxyflavone (4), and named sinoflavonoids R and S.Compounds Their 1H  and 13C- instead of the 3-methyl-2-butenyl in 3, suggesting the absence of 3-methyl-2-butenyl at C-2′ in 5. This was further supported by the HR-ESI-MS of 5 which gave an [M + H]+ ion peak at m/z 385.1262 , being 68 mass units less than that of 3. Thus, compound 5 was deduced as 5,6--7,3′,4′-trihydroxy-3-methoxyflavone, and named sinoflavonoid T.Compound . Its 1H  and 13C- spectra  was quit6 was obtained as a yellow, amorphous powder. The molecular formula was found to be C21H20O8, as deduced by analysis of the [M + Na]+ molecular ion peak at m/z 423.1056  in the HR-ESI-MS. Its 1H dihydrofurano group was observed instead of one 2,2-dimethyldihydropyrano group in 5. 2-(1-Hydroxy-1-methylethyl)dihydrofurano group was deduced by the HMBC correlations  to C-1″ (δ 25.7), C-3″ (δ 70.0), C-4″ (δ 24.8), and C-5″ (δ 25.9), from methylene group δ 3.06  to C-2″ (δ 91.5). 2-(1-Hydroxy-1-methylethyl)dihydrofurano group was located at C-6 and C-7 from the HMBC correlations of oxymethine group δ 4.75  with C-7 (δ 166.2), of methylene group δ 3.06  with C-5 (δ 155.3), C-6 (δ 108.8), C-7 (δ 166.2), and in combination with the chelated phenolic hydroxyl group δ 12.92 . Thus, compound 6 was deduced as 6,7-[2-(1-hydroxy-1-methylethyl)dihydrofurano]-5,3′,4′-trihydroxy-3-methoxyflavone, and named sinoflavonoid U.Compound . Its 1H  and 13C- spectra  were simelations  from two7 was obtained as a yellow, amorphous powder and possessed a molecular forma of C23H20O7, derived from its HR-ESI-MS analysis . The 1H  and 8.09  instead of one 3-methyl-2-butenyl in 1. This was also further supported by HMBC spectrum  and 8.09  showed the HMBC correlations with C-1′ (δ 113.4), C-2′ (δ 128.1), C-3′ (δ 143.1), and C-2′ (δ 128.1), C-3′ (δ 143.1), respectively, indicating the disubstituted furan ring was attached to C-2′ and C-3′. The methoxy group was located at C-3 by the HMBC correlation of the methoxy group δ 3.65  with C-3 (δ 137.7). Thus, compound 7 was deduced as 2′,3′-furano-5,3′,4′-trihydroxy-3-methoxyflavone, and named as sinoflavonoid V.Compound . The 1H  and 13C- spectra  of compospectrum . The ole8 was obtained as a yellow, amorphous powder. Its 1H , 2.95 , δ 38.2, one dioxymethine group δ 6.03 , δ 101.0. Those were further supported by its HR-ESI-MS, which gave an [M + H]+ quasi-molecular ion peak m/z 427.1390 , being 18 mass units more than that of 7. Thus, compound 8 was identified as 8-(3-methyl-2-butenyl)-2′,3′-(2-hydroxydihydrofurano)-5,7,4′-trihydroxy-3-methoxyflavone, and named as sinoflavonoid W.Compound . Its 1H  and 13C- spectra  were qui9 and 10 were obtained as yellow, amorphous powders. Their molecular formulae were assigned as C26H28O8 by HR-ESI-MS  in 9; m/z 491.1669 [M + Na]+  in 10). Their 1H , 1.20 , δ 25.1, 20.8 in 9; δ 1.32 , 1.17 , δ 25.3, 19.8 in 10), one methylene group , 2.47 , δ 24.9 in 9; δ 2.75 , 2.55 , δ 29.5 in 10), and one oxymethine group , δ 66.8 in 9; δ 3.60 , δ 67.8 in 10) were observed, implying the presence of one 2,2-dimethyl-3-hydroxydihydropyrano group in 9 and 10 instead of 2-hydroxydihydrofurano ring in 8, respectively. The 2,2-dimethyl-3-hydroxydihydropyrano group and 3-methyl-2-butenyl in 9 were attached to C-7 and C-8, and C-2′ and C-3′, respectively, by the HMBC correlations  with C-7 (δ 161.5), C-8 (δ 105.9) and C-9 (δ 154.1), and of H-1‴  with C-1′ (δ 120.5), C-2′ (δ 120.6) and C-3′ (δ 141.0). Thus, compounds 9 and 10 were elucidated respectively as 7,8--2′-(3-methyl-2-butenyl)-5,3′,4′-trihydroxy-3-methoxyflavone (9), 8-(3-methyl-2-butenyl)-2′,3′--5,7,4′-trihydroxy-3-methoxyflavone (10), and named sinoflavonoids X and Y.Compounds Their 1H  and 13C- spectra  were simelations  from H-111 was obtained as a yellow, amorphous power and possessed a molecular forma of C26H26O8, derived from its HR-ESI-MS analysis . Its 1H-NMR  and 3-methyl-2-butenyl in 7, respectively. The isopropenyl was deduced by two olefinic protons δ 5.77 , 5.28 , one tertiary-methyl group δ 2.10 , δ 18.9, two olefinic carbons δ 114.1, 132.5. The furan ring was inferred from one olefinic proton δ 7.01 , and two characteristic olefinic carbons δ 104.2, 156.5. In the HMBC spectrum (11 was elucidated as 8-(3-hydroxy-3-methylbutyl)-2′,3′-(2-isopropenylfurano)-5,7,4′-trihydroxy-3-methoxyflavone, and named sinoflavonoid Z.Compound s 1H-NMR  and 13C NMR data  were clospectrum , the lon12 was obtained as a yellow, amorphous powder and possessed a molecular forma of C21H20O6, derived from its HR-ESI-MS analysis . Its 1H and 13C-NMR spectra were similar to kaempferol + in 9). The 2,2-dimethyldihydropyrano group was attached to C-2′ and C-3′, by the HMBC correlations  with C-1′ (δ 120.3), C-2′ (δ 121.3), and C-3′ (δ 142.0). Thus, compound 14 was elucidated as 7,8-bis-2′,3′--5,4′,2″-trihydroxy-3-methoxyflavone, and named sinoflavonoid NC.Compound . Its 1H  and 13C- spectra  were quielations  of the m15 was obtained as a yellow, amorphous powder. Its 1H , δ 56.9 in 15, suggesting 15 to be a further methyl ether derivative of 14. This was further confirmed by the HR-ESI-MS, which gave the molecular formula C27H30O9 by the quasi-molecularion peak m/z 521.1792 [M + Na]+ , being 14 mass units more than that of 14. In the HMBC spectrum -5,4′,2″-trihydroxy-3,1″-dimethoxyflavone, and named sinoflavonoid ND.Compound . Its 1H  and 13C- spectra  were quispectrum , the add16 was obtained as a yellow, amorphous powder. Its 1H dihydrofurano group in 16 instead of 3-hydroxy-2,2-dimethyldihydropyrano group in 14. 2-(1-Hydroxy-1-methylethyl)dihydrofurano group was deduced by the HMBC correlations  to C-1″ (δ 25.2), C-3″ (δ 69.9), C-4″ (δ 24.8), and C-5″ (δ 25.7), from methylene group δ 3.13  to C-2″ (δ 91.5). 2-(1-Hydroxy-1-methylethyl)dihydrofurano group was located at C-7 and C-8, based on the HMBC correlation of methylene group δ 3.13  with C-7 (δ 166.2), C-8 (δ 104.4), C-9 (δ 151.3). Thus, compound 16 was deduced as 7,8-[2-(1-hydroxy-1-methylethyl)dihydrofurano]-2′,3′--5,4′-dihydroxy-3-methoxyflavone, and named sinoflavonoid NE.Compound . Its 1H  and 13C- spectra  were quielations  from two1–18, 20, 22 were tested for their in vitro cytotoxic activities against MCF-7 and HepG2 cell lines using the MTT assay + , m/z 461.1576 [M + Na]+ ; NMR data (DMSO-d6), see Sinoflavonoid P (2): yellow, amorphous powder; UV (MeOH) λmax (log ε) 262 (1.24), 321 (0.73) nm; IRνmax 3354, 2961, 2926, 2855, 1659, 1611, 1573, 1468, 1358, 1293, 1234, 1155, 1092 cm−1; HR-ESI-MS (positive): m/z 453.1910 [M + H]+ ; NMR data (DMSO-d6), see Sinoflavonoid Q (3): yellow, amorphous powder; UV (MeOH) λmax (log ε) 262 (1.24), 321 (0.73) nm; IRνmax 3374, 2959, 2926, 2854, 1650, 1597, 1451, 1359, 1293, 1228, 1162, 1092 cm−1; HR-ESI-MS (positive): m/z 453.1910 [M + H]+ ; NMR data (DMSO-d6), see Sinoflavonoid R (4): yellow, amorphous powder; UV (MeOH) λmax (log ε) 261 (1.24), 325 (0.60) nm; IRνmax 3524, 2975, 2930, 2856, 1651, 1604, 1462, 1373, 1301, 1258, 1161, 1091 cm−1; HR-ESI-MS (positive): m/z 453.1912 [M + H]+ ; NMR data (DMSO-d6), see Sinoflavonoid S (5): yellow, amorphous powder; UV (MeOH) λmax (log ε) 256 (0.66), 345 (0.75) nm; IRνmax 3421, 2974, 2941, 1655, 1617, 1571, 1454, 1287, 1241, 1159, 1088 cm−1; HR-ESI-MS (positive): m/z 385.1262 [M + H]+ , (negative): m/z 383.1116 [M − H]− ; NMR data (DMSO-d6), see Sinoflavonoid T (6): yellow, amorphous powder; + , (negative): m/z 399.1084 [M − H]− ; NMR data (DMSO-d6), see Sinoflavonoid U (7): yellow, amorphous powder; UV (MeOH) λmax (log ε) 269 (0.11), 359 (0.07) nm; IRνmax 3330, 2957, 2925, 2870, 2855, 1650, 1610, 1562, 1511, 1496, 1453, 1424, 1364, 1334, 1307, 1230, 1154, 1098 cm−1; HR-ESI-MS (positive): m/z 409.1282 [M + H]+ , (negative): m/z 407.1111 [M − H]− ; NMR data (DMSO-d6), see Sinoflavonoid V (8): yellow, amorphous powder; + , m/z 449.1210 [M + Na]+ ; NMR data (DMSO-d6), see Sinoflavonoid W (9): yellow, amorphous powder; + ; NMR data (DMSO-d6), see Sinoflavonoid X (10): yellow, amorphous powder; + ; NMR data (DMSO-d6), see Sinoflavonoid Y (11): yellow, amorphous powder; UV (MeOH) λmax (log ε) 263 (0.14), 332 (0.09) nm; IRνmax 3403, 2957, 2922, 2851, 1654, 1578, 1488, 1457, 1375, 1361, 1302, 1229, 1161, 1076 cm−1; HR-ESI-MS (positive): m/z 465.1544 [M − H]− ; NMR data (DMSO-d6), see Sinoflavonoid Z (12): yellow, amorphous powder; UV (MeOH) λmax (log ε) 268 (0.53), 355 (0.60) nm; IRνmax 3421, 2974, 2929, 2853, 1647, 1603, 1507, 1489, 1448, 1358, 1306, 1160, 1089 cm−1; HR-ESI-MS (positive): m/z 369.1336 [M + H]+ , m/z 391.1144 [M + Na]+ ; NMR data (DMSO-d6), see Sinoflavonoid NA (13): yellow, amorphous powder; UV (MeOH) λmax (log ε) 260 (0.23), 376 (0.18) nm; IRνmax 3419, 2955, 2924, 2852, 1651, 1596, 1556, 1456, 1363, 1323, 1159, 1084 cm−1; HR-ESI-MS (positive): m/z 371.0989 [M + H]+ , (negative): m/z 369.0972 [M − H]− ; NMR data (DMSO-d6), see Sinoflavonoid NB (14): yellow, amorphous powder; + ; NMR data (DMSO-d6), see Sinoflavonoid NC (15): yellow, amorphous powder; + , m/z 521.1792 [M + Na]+ ; NMR data (DMSO-d6), see Sinoflavonoid ND (16): yellow, amorphous powder; + ; NMR data (DMSO-d6), see Sinoflavonoid NE , streptomycin (100 ug/mL) under humidified air with 5% COprotocol .S. hexandrum [S. hexandrum resulted in the isolation of 16 new prenylated flavonoids and eight known analogues. Their cytotoxic activity was evaluated against MCF-7 and HepG2 cell lines. Compound 6 was the most valuable of all tested compounds. Further research is necessary to elucidate the antitumor mechanism. This study also enriches the chemical and pharmacological diversity of prenylated flavonoids.Prenylated flavonoids are characterized by the presence of lipophilic prenylated group on the parent skeleton. Their structure diversity is most attributed to the different position of prenylation, and various length, further cyclization and hydroxylation of prenyl chain. With diverse chemical structure, prenylated flavonoids exhibit extensive pharmacological actions, including antioxidant, anti-inflammatory, anticoagulant, antiviral, antimicrobial anticancer , antigenexandrum ,9,10,11.exandrum ,9,11. Fu


Zanthoxylum rhetsa is an aromatic tree, known vernacularly as “Indian Prickly Ash”. It has been predominantly used by Indian tribes for the treatment of many infirmities like diabetes, inflammation, rheumatism, toothache and diarrhea. In this study, we identified major volatile constituents present in different solvent fractions of Z. rhetsa bark using GC-MS analysis and isolated two tetrahydrofuran lignans (yangambin and kobusin), a berberine alkaloid (columbamine) and a triterpenoid (lupeol) from the bioactive chloroform fraction. The solvent fractions and purified compounds were tested for their cytotoxic potential against human dermal fibroblasts (HDF) and mouse melanoma (B16-F10) cells, using the MTT assay. All the solvent fractions and purified compounds were found to be non-cytotoxic to HDF cells. However, the chloroform fraction and kobusin exhibited cytotoxic effect against B16-F10 melanoma cells. The presence of bioactive lignans and alkaloids were suggested to be responsible for the cytotoxic property of Z. rhetsa bark against B16-F10 cells. The squamous and basal skin cancers, which are non-melanoma cancers, are common and not life threatening. These types of non-melanoma skin cancers can be cured if treated at an early stage. Clinical data show that skin cancer occurs primarily based on geographical areas. Basal cell carcinoma (BCC) is usually reported among Hispanics, Caucasians, Japanese and Chinese Asian, whereas squamous cell carcinoma (SCC) is more common among African, Americans and Asian Indians +, 177 [(CH3O)2-C6H3-CH-CH=CH2]+, 165 [(CH3O)2-C6H3-CO]+ + ,27. Prev6H3-CO]+ ,13. To tMTT assay was performed to evaluate the toxic effects of the crude methanolic extract, solvent fractions and the isolated compounds, against HDF and B16-F10 melanoma cells. The extract and solvent fractions showed minimal effect on HDF cell and exhibited no toxicity against normal skin cells .Zanthoxylum species have also been reported to be non-toxic to normal cell lines + . 1H-NMR  δ: 0.76, 0.79, 0.83, 0.95, 0.97, 1.03, 1.68 , 3.18 , 4.57 , 4.69 . 13C-NMR  δ: 151.00 (C-20), 109.3 (C-29), 79.0 (C-3), 55.3 (C-5), 50.4 (C-9), 48.3 (C-18), 48.0 (C-19), 43.0 (C-17), 42.8 (C-14), 40.8 (C-8), 40.0 (C-22), 38.9 (C-13), 38.7 (C-4), 38.0 (C-1), 37.2 (C-10), 35.6 (C-16), 34.3 (C-7), 29.8 (C-21), 28.0 (C-23), 27.4 (C-12), 25.1 (C-2), 20.9 (C-11), 19.3 (C-30), 18.3 (C-6), 18.0 (C-28), 16.1 (C-25), 16.0 (C-24), 15.4 (C-27), 14.5 (C-26). Compound A was identified as lupeol [Compound s lupeol .B (Colourless gum): EI-MS: m/z 370 [M]+ . 1H-NMR  δ: 6.91–6.77 , 5.95 , 4.73 , 4.26 , 3.80 , 3.87 , 3.08  13C-NMR  δ: 149.2 (C-3′), 148.6 (C-4′), 147.8 (C-3′′), 147.1 (C-4′′), 135 (C-1′′), 133.2 (C-1′), 119.3 (C-6′′), 118.2 (C-6′), 111.1 (C-5′), 109.2 (C-2′), 108 (C-5′′), 106.4 (C-2′′), 100.9 (-OCH2O-), 85.8 (C-2), 85.7 (C-6), 71.7 (C-4), 71.6 (C-8), 56 (-OCH3), 55.9 (-OCH3), 54.3 (C-5), 54.2 (C-1). Compound B was identified as kobusin + . 1H-NMR  δ: 6.57 , 4.75 , 4.31 , 3.93 , 3.88 , 3.84 , 3.11  13C-NMR  δ: 153.4 , 137.5 (C-4/4′), 136.7 (C-1/1′), 102.9 , 85.9 (C-7/7′), 72.1 (C-9/9′), 60.8 , 56.2 , 54.4 (C-8/8′). Compound C was identified as yangambin + . 1H-NMR  δ: 9.72 , 8.75 , 8.09 , 7.98 , 7.65 , 6.86 , 4.91, 4.20 , 4.10 , 4.02 , 3.20  13C-NMR  δ: 150.2 (C-3), 148.3 (C-2), 144.6 (C-8), 144.3 (C-10), 138.9 (C-14), 134 (C-12a), 128.9 (C-4a), 126.7 (C-11), 122.9 (C-12), 121.7 (C-8a), 119.4 (C-13), 117.8 (C-14a), 114.5 (C-1), 108.6 (C-4), 61.1 (9-OCH3), 56.3 (10-CH3), 56.0 (C-6), 55.5 (3-OCH3), 26.3 (C-5). Compound D was identified as columbamine [Compound Zanthoxylum species, especially the bark material, have been previously reported to be rich in tetrahydrofuran lignans. These included six lignans from Z. nitidium bark [Z. schinifolium stem [Z. armatum bark [Z. budrunga bark [Z. planispinum [Zanthoxylum species. For example, the alkaloids N-methyl corydine, magnoflorine and berberine have been reported to be major constituents in the stem and branches of Z. punctatum and Z. monophyllum [Z. rhetsa have also reported the presence of different types of alkaloids. This included 6-acetonyldihydrochelerythrin and arnottianamide in the conical prickles, and the quinolone-terpene alkaloids chelerybulgarine, 2,11-didemethoxyvepridimerine B, rhetsidimerine, simulanoquinoline and 2′-episimulanoquinoline in the root bark [Z. rhetsa.ium bark , twelve ium stem , seven ltum bark , three lnga bark , and eignispinum . Apart fophyllum . Previouoot bark ,37. In a50 values of 112.2, 195.6, 377.8 and 442.4 µg/mL, respectively. Quercetin, the positive control, gave an IC50 value of 22.72 µg/mL. The results of the cytotoxicity test on the isolated compounds are shown in Z. alatum bark, showed strong cytotoxic effect, with IC50 values of 34.71 µg/mL and 32.86 µg/mL, against A549 (lung) and MIA-PaCa (pancreatic) cell lines, respectively [Rhizoma coptidis, has been found to be moderately cytotoxic with an IC50 value of 226.54 µg/mL against HepG2 cells [Grewia tiliaefolia, was reported to be weakly cytotoxic with an IC50 value of 330 µg/mL against B16-F10 cells [The isolated compounds were evaluated for cytotoxicity against HDF and B16-F10 melanoma cells. The isolated compounds were found to be non-toxic to HDF cells . Howeverectively . On the G2 cells . Meanwhi10 cells . It was 10 cells . In the 10 cells .d (CDCl3) and methanol-d4 (CD3OD) were used as NMR solvents, and TMS was utilized for internal referencing. Solvents used for extraction and isolation were of analytical grade and obtained from R & M chemicals . Kieselgel 60 (0.040–0.063 mm) and Lichroprep RP-18 (40–63 µm) were purchased from Merck  while Sephadex LH-20 was purchased from Sigma . Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin–EDTA, penicillin/streptomycin were purchased from GIBCO . Trypan blue and MTT -2,5-biphenyl tetrazolium bromide) were purchased from Sigma. A SpectraMax Plus  microplate reader was used in the bioassays.Melting points were recorded on a Koffler hot-stage apparatus  and were uncorrected. GC-MS analysis were carried out on a QP-2010 Ultra GCMS spectrometer  equipped with a flame ionization detector (FID). 1D and 2D NMR spectra were recorded on a Unity INOVA 500 MHz spectrometer  using standard pulse programs. Chloroform-Z. rhetsa were collected from Pangkor Island, Malaysia. A voucher specimen (SK2226/13) was deposited at the Herbarium of the Institute of Bioscience, Universiti Putra Malaysia.The trunk barks of Z. rhetsa was subjected to ultrasound-assisted extraction with 100% methanol and dried under vacuum at 40 °C to yield the crude methanolic extract (65 g). The extract was then subjected to solvent-solvent extraction using organic solvents of increasing polarities. The resulting solvent fractions were dried under vacuum, lyophilized to yield 14 g hexane, 17.4 g chloroform, 2 g ethyl acetate and 5.3 g butanol fractions. All the samples were stored at −20 °C prior to further analysis [The air dried, powdered bark material (910 g) of 2 incubator at 37 °C. Only the cells that attained more than 80% confluence (growth phase) were used for cell seeding [Both HDF and B16-F10 cells were provided by the Laboratory of Vaccines and Immunotherapeutics (LIVES), Institute of Bioscience, UPM. Human dermal fibroblasts (HDF) cells (passage 11–15) were maintained in DMEM medium containing 5% fetal bovine serum, 1% penicillin (100 U/mL) and streptomycin (100 μg/mL). Mouse melanoma (B16-F10B) cells (passage 11–15) were also maintained in DMEM medium with 10% fetal bovine serum, 1% penicillin (100 U/mL) and streptomycin (100 μg/mL). Cells were sub-cultured, incubated and maintained in a humidified 5% CO seeding .4 cells/well) and incubated in 5% CO2 incubator at 37 °C for 24 h. The cells were treated with serial dilutions of Z. rhetsa extract and positive control ranging from 500, 250, 125, 62.5, 31.25, 15.625, 7.81 µg/mL. The cells treated with medium without the sample served as negative control. Quercetin which is known to inhibit cell growth, induce apoptosis and is cytotoxic against B-16 melanoma cells, served as positive control [Cells were seeded in 96 well plates . The oven temperature program was set to 50 °C for 0 min, heated to 330 °C at the rate of 3 °C/min and held constant at 330 °C for a further 5 min. Helium was used as carrier gas with the following conditions: total flow—11.8 mL/min, column flow—0.8 mL/min, linear velocity—32.4 cm/s, purge flow—3.0 mL/min, split ratio—10. Mass spectra were recorded with ion source temperature of 200 °C and interface temperature of 250 °C. The mass scan parameters included a start time of 2.5 min and end time of 95.0 min. The acquisition (ACQ) parameters were as follows: Scan event time—0.10 s, scan speed—10000, mass range—40 The scheme for the isolation of chemical constituents from the chloroform fraction is illustrated in A (lupeol) was isolated as white powder (45 mg) from sub-fraction C1 using normal phase CC, where hexane and ethyl acetate were used as eluent at the ratio of 3:7. Compound B  was isolated from sub-fraction C5 by first passing it through a Sephadex LH-20 column, eluted with methanol. Sub-faction C6 was similarly pretreated with Sephadex LH-20 to give thirteen sub-fractions. White needle like crystals were filtered from sub-fractions 5–8, which yielded 70 mg of Compound C (yangambin) after further purification by recrystallization in methanol. The remaining sub-fraction C6 was subjected to CC over reverse phase silica (40–63 µm), eluted with solvent system water:methanol at varying ratios , to give 18 sub-fractions. Sub-fractions 13–15 were pooled and further purified via repeated CC and recrystallization steps to yield 28 mg of Compound D (columbamine).The dried chloroform extract (14 g) was subjected to column chromatography (CC) over silica gel (0.040–0.063 mm) with solvent systems hexane:ethyl acetate  followed by ethyl acetate:methanol (9:1) and 100% methanol, to give 44 sub-fractions (~50 mL each). Sub-fractions with similar TLC profiles were pooled together and relabeled as C1, C2, C3, C4, C5, C6. Compound p values < 0.05.Data were expressed as mean ± SD of three independent experiments. Statistical analysis were performed through GraphPad Prism software Version 5 using one way ANOVA followed by Turkey’s t- test. Differences were considered to be significant when rhetsa bark extract towards the melanoma B16-F10 cell line. In contrast, all the solvent fractions of the bark extract and the isolated compounds were found to be relatively non-toxic to HDF cells. The selective cytotoxicity of the extract and isolated compounds may be due to the metabolic defects in the cancerous cells or the peculiar reaction of the compounds against the melanoma cells [Z. rhetsa bark extract may have potential use as a dermo protective ingredient in skincare or cosmeceutical products.Our findings suggest that the presence of compounds such as lignans and alkaloids plays an important role in the overall cytotoxic effects of Z. ma cells ,45,46,47


H-chromen-2-one (4), 5-[(2-oxo-2H-chromen-7-yloxy)methyl]-1,3,4-thiadiazol-2(3H)-one (5), 2-[2-(2-oxo-2H-chromen-7-yloxy)acetyl]-N-phenylhydrazinecarbothioamide (7), 7--2H-chromen-2-one (8) and 7-[methoxy]-2H-chromen-2-one (9) were prepared starting from the natural compound umbelliferone (1). The newly synthesized compounds were characterized by elemental analysis and spectral studies .New coumarin derivatives, namely 7-[methoxy]-2 For the synthesis of new umbelliferone derivatives, the reaction sequences outlined in ensation . Synthes2 yield compound 6 that was converted to compound 7 by addition of phenyl isothiocyanate. The cyclization of compound 7 by sulphuric acid yielded compound 8, while reaction with sodium hydroxide produced compound 9. The addition of hydrazine to compound 2) the lactone carbonyl stretching frequency was observed at 1759 cm−1, whereas the ester carbonyl stretching appeared at 1717 cm−1, and a C-H aliphatic stretching frequency appeared at 2987 cm−1that was not seen in the IR spectrum of the starting material umbelliferone, in addition of the disappearance of the hydroxyl group. In the 1H-NMR spectrum of compound 2, a 3H triplet was observed at 3.126 ppm due to the ester CH3 protons and a quartet at 3.81 ppm due to the ester CH2 protons. The isolated CH2 protons were observed downfield as a singlet (2H) at 4.770 ppm and triplet (2H) at 5.260, 5.239 and 5.27. The coumarin alkene C-H appeared at 5.416 ppm. The protons of the aromatic ring were observed as quartet , quintet  and doublet, 7.895 ppm, 7.871 ppm for C8-H. The 13C-NMR spectrum analysis for compound 2, combined with the information from 1H-NMR experiments, can be considered enough to guide future synthetic work. In the IR spectrum of ethyl 2-(2-oxo-2H-chromen-7-yloxy)acetate (3) the lactone carbonyl stretching frequency was observed at 1755 cm−1, whereas the carboxylic acid carbonyl stretching appeared at 1724 cm−1, and the hydroxyl group appeared as a broad band at 2975–3170 cm−1. In the 1H-NMR spectrum, the CH2 protons were observed downfield as a singlet (2H) at 4.83 ppm, and triplet (2H) at 5.311, 5.3 and 5.28. The C-H  of coumarin appeared at 5.418 ppm. The protons of the aromatic ring were observed as quartet , triplet  and doublet  for C8-H. The 13C-NMR spectrum analysis for compound 3, combined with the information from 1H-NMR experiments, can be considered enough to prove the structure of compound 3. In the IR spectrum of 2-(2-oxo-2H-chromen-7-yloxy)acetic acid  at 4.94 ppm and the C-H  of the coumarin appeared at 5.76 ppm and 5.891. The protons of the aromatic ring were observed as multiplet (7.51–7.28). In the 13C-NMR, 163.5 and 176.4 were new and due to the heterocyclic ring carbons. The IR spectrum is good evidence for formation of compound 5 showed a carbonyl group at 1713 cm−1, in addition to an amino group at 3297 cm−1. In the 1H-NMR; the coumarin C-H  protons appeared at 5.81 ppm (singlet) and 6.43 ppm (singlet) and the singlet (NH) at 5.31 ppm. In the 13C-NMR the thiadiazole ring carbonyl carbon atom appears at very low field (171.2). The IR spectrum of compound 2 with hydrazine hydrate afforded 2-(2-oxo-2H-chromen-7-yloxy)acetohydrazide (6) in good yield. The IR spectra of compound 6 showed absorption bands in the 3351.3, 3287.1 cm−1 region (hydrazide NH-NH2), 1689.2 cm−1 (amide-C=O carbonyl stretching), and 1761.5 cm−1 (lactone-C=O carbonyl stretching). The 1H-NMR spectrum exhibited a singlet due to the –CO-NH-NH2 proton at δ 7.93 ppm. For compound 7, the IR spectrum has the following characteristic absorption bands: νN-H ; νC=O , νC=S (1258.5 cm−1). In the IR spectrum of compound 8, no absorption band at 1692.7 cm−1 was detected, indicating the absence of the amide carbonyl group, which is evidence for the conversion of compound 7 to compound 8. Also, in the IR spectrum of the new heterocyclic compound 8 a stretching band characteristic of the C=N group from the thiadiazole nucleus appeared at 1620.6 cm−1. Although two types of tautomers, thione or thiole, could be expected from the cyclization of compound 7, under basic conditions, only the thione type compound 9 was observed. The existence of the thione form predominantly in the solid state is demonstrated by the presence of two absorption bands at 1257 cm−1 and 3389.3 cm−1 belonging to the νC=S and νNH groups, respectively, and by absence of νSH. In the 13C-NMR spectra of new heterocyclic compounds 8 and 9 the absence of the signals for the ester carbonyl and the absence of a thiocarbonyl carbon in compound 8, confirmed that cyclization of compound 7 took place. In compound 9 the signal at 168.4 ppm indicated that in solution this compound exists predominantly in the thione tautomeric form .The minimized geometry for the compound 5 and 8. Optimized molecular structures of the most stable forms are shown in 5 and 8 are shown in 5 and 8 are listed in 5 and 8, respectively. The lower value in the HOMO and LUMO energy gap explain the eventual charge transfer interaction taking place within the molecules. The dipole moments of compounds 5 and 8, were also calculated and listed in 5 is oriented inwards, while the dipole moment for compounds 8, is oriented outwards.DFT calculations were performed for compounds 1H-NMR spectra were obtained on a Jeol jnm-ECP400 FT-NMR system. Elemental microanalysis was carried out using a model 5500-Carlo Erba C.H.N elemental analyzer instrument. A Gallenkamp M.F.B.600.010 F melting point apparatus was used to measure the melting points of all the prepared compounds.The chemicals used for the synthesis were supplied by Sigma-Aldrich. Purity of the compounds was checked on thin layer chromatography (TLC) plates (Silicagel G) using the solvent systems benzene-ethyl acetate-methanol  and toluene-acetone . The spots were located under UV light (254 and 365 nm). The IR spectra were obtained on a Thermo Scientific, Nicolet 6700 FT-IR spectrometer (without KBr or CsI pellets). The Ethyl 2-(2-oxo-2H-chromen-7-yloxy)acetate (2). A suspension of 7- hydroxycoumarin (6.17 mmol) in acetone (30 mL) was refluxed with ethyl bromoacetate (9.15 mmol) and K2CO3  for 12 h. After cooling, the mixture was evaporated to dryness and the residue was partitioned between CHCl3 (50 mL) and water (50 mL). The organic phase was dried (Na2SO4), filtered and evaporated to dryness. The residue was recrystallized from acetone, yield 82%, m.p. 112.5 °C; 1H-NMR (CDCl3): δ 3.126 , δ 3.81 , δ 4.77  and δ 5.260, δ 5.239, δ 5.270 , δ 5.416 , δ 7.472, δ 7.457, δ 7.413, δ 7.216 ; 13C-NMR (DMSO-d6): 22.3, 58.9, 65.1, 103.8, 108, 108.9, 110.1, 123.7, 154.6, 157.1, 160.8, 161.2, 166.5; IR: 2987 cm−1 , 1759 cm−1 , 1717 cm−1 ; Anal. Calcd. for C13H12O5: C 62.90%, H 4.87%. Found: C 61.81% H 4.01%.2-(2-Oxo-2H-chromen-7-yloxy)acetic acid3. A solution of the compound 2 (2.7 mmol) and 5% sodium hydroxide (2.16 mL) in ethanol (15 mL) was stirred under reflux for 2 h. After removal of the solvent, the residue was dissolved in water and acidified with HCl 6 M. The white solid collected by filtration was washed with cool water, dried and recrystallized from ethanol, yield 92%, m.p. 210.5 °C; 1H-NMR (CDCl3): δ 4.83  and δ 5.311, δ 5.300, δ 5.280 , δ 5.418 , δ 7.470, δ 7.455, δ 7.411, δ 7.207 , δ 7.555, δ 7.531, δ 7.527  for C6-H aromatic ring, δ 7.90, δ 7.876 ; 13C-NMR (DMSO-d6): 105.2, 110.3, 112.9, 113.9, 124.1, 153.9, 156.3, 161.2, 161.9, 167.1; IR: 3170-2975 cm−1 (hydroxyl), 1755 cm−1 , 1724 cm−1 ; Anal. Calcd. for C11H8O5: C 60.00%, H 3.66%. Found: C 58.40% H 2.94%.7-[methoxy]-2H-chromen-2-one (4). Phosphorus oxychloride (20 mL) was added to compound 3 (0.05 mol) and the mixture was stirred for 1 h. at room temperature. Thiosemicarbazide  was added and the mixture was heated under reflux for 5 h. On cooling, the mixture was poured on to ice. After 4 h. the mixture was stirred for 15 min. to decompose the excess phosphorus oxychloride, then heated under reflux for 30 min, cooling, the mixture was neutralized by 5% potassium hydroxide, the precipitate was filtered, washed with water, dried and recrystallized from ethanol, yield 51%, m.p. 105 °C; 1H-NMR (CDCl3): δ 4.94 , δ 5.760, 5.891 , δ 7.510–7.283 ; 13C-NMR (DMSO-d6): 101.9, 111.0, 113.1, 114.1, 128.1, 149.5, 156.3, 160.8, 161.2, 163.5, 168.3, 176.4; IR: 3302 and 3343 cm−1 , 1747 cm−1 ; Anal. Calcd. for C12H9N3O3S: C 52.36%, H 3.30%, N 15.26%. Found: C 50.49% H 2.76%, N 16.20%.5-[(2-Oxo-2H-chromen-7-yloxy)methyl]-1,3,4-thiadiazol-2(3H)-one (5). 10% Aqueous sodium nitrite solution (10 mL) was added dropwise with continuous stirring over a period of 20 min to an cooled (ice-bath) suspension of compound 4 (0.01 mol) and hydrochloric acid (5 mL) in cold water (20 mL). The temperature was then allowed to rise to room temperature and the mixture was heated to boiling for 10 min, cooled and allowed to stand overnight. The separated crude product was filtered, washed with water, dried and recrystallized from ethanol, yield 47%, m.p. 133 °C; 1H-NMR (CDCl3): δ 5.31 , δ 5.810, 6.430 , δ 7.811–7.299 ; 13C-NMR (DMSO-d6): 103.2, 115.1, 117.5, 117.9, 123.2, 155.3, 155.9, 156.4, 160.8, 161.1, 166.2, 171.2; IR: 3397 cm−1 , 1713 cm−1 ; Anal. Calcd. for C12H8N2O4S: C 52.17%, H 2.29%, N 10.14%. Found: C 50.34% H 3.01%, N 9.51%.2-(2-Oxo-2H-chromen-7-yloxy)acetohydrazide (6). A solution of compound 2 (10 mmol) in ethanol (25 mL) was refluxed with hydrazine hydrate (25 mmol) for 4 h. After concentrating the reaction mixture a solid mass separated out and was recrystallized from ethanol, yield 75%, m.p. 234.5 °C; 1H-NMR (CDCl3): δ 7.93 (-CO-NHNH2), δ 4.61  and δ 5.251, δ 5.222, δ 5.214 , δ 5.716 ,  δ 7.61–7.110 ; IR: 3351.3 and 3287.1 cm−1 , 1761.5 cm−1 , 1689.2 cm−1 ; Anal. Calcd. for C11H10N2O4: C 56.40%, H 4.38%, N 11.96%. Found: C 54.44%, H 4.04%, N 10.79%.2-[2-(2-Oxo-2H-chromen-7-yloxy)acetyl]-N-phenylhydrazinecarbothioamide (7). A mixture of hydrazide 6 (2 mmol) and phenyl isothiocyanate (2 mmol) in ethanol (15 mL) was refluxed for 12 h. The reaction mixture was cooled and the separated product was filtered off, dried and recrystallized from ethanol, yield 61%, m.p. 199 °C; IR: 3367.6, 3301.2 and 3278.9 cm−1 , 1763 cm−1 , 1692.7 cm−1 , 1258.5 cm−1 (C=S); Anal. Calcd. for C18H15N3O4S: C 58.53%, H 4.09%, N 11.38%. Found: C 56.49%, H 3.78%, N 10.22%. 7--2H-chromen-2-one (8). A mixture of compound 7 (0.15 mmol) and concentrated H2SO4 (5 mL) was stirred in ice path for 5 h and then, at room temperature, for another 5 h. The reaction mixture was neutralized with a diluted solution of ammonium hydroxide, in ice bath. The precipitated was filtered off, washed with water, dried and recrystallized from ethanol, yield 57%, m.p. 244 °C; IR: 3291 cm−1 (N-H), 1762.8 cm−1 , 1620.6 cm−1 (C=N); 13C-NMR (DMSO-d6): 100.3, 107.2, 111.9, 114.2, 122.0, 125.4, 126.2, 126.8, 129.2, 129.7, 141.6, 156.2, 156.9, 157.3, 158.1, 160.5, 169.3; Anal. Calcd. for C18H13N3O3S: C 61.53%, H 3.73%, N 11.96%. Found: C 61.01%, H 3.11%, N 11.32%.7-[methoxy]-2H-chromen-2-one (9). Compound 7 (0.5 mmol) was added to 8% NaOH solution (4 mL) and the reaction mixture was heated under reflux for 4 hours. After cooling, the solution was acidified with a diluted solution of HCl. The crude product was precipitated, filtered off and washed with water. The solid thus separated was dried and recrystallized from chloroform, yield 44%, m.p. 251 °C; IR: 3389.3 cm−1 (N-H), 1767.7 cm−1 , 1618.1 cm−1 (C=N), 1257 cm−1 (C=S); 13C-NMR (DMSO-d6): 99.6, 109.8, 112.3, 114.1, 122.3, 127.1, 129.3, 129.8, 130.1, 130.9, 134.4, 154.0, 155.8, 156.5, 157.2, 163.6, 168.4; Anal. Calcd. for C18H13N3O3S: C 61.53%, H 3.73%, N 11.96%. Found: C 61.01%, H 3.11%, N 11.32%.3 model was employed to obtain quantum chemical parameters and to optimize the molecules’ geometry. These calculations employed an ab initio, generalized gradient approximation (GGA) with the Lee-Yang-Parr correlation functional (BLYP) functional and Double Numerical d-functions (DND) basis set. This approach is shown to yield favorable geometries for a wide variety of systems. The following quantum chemical indices were calculated: the energy of the highest occupied molecular orbital (HOMO), the energy of the lowest unoccupied molecular orbital (LUMO) and dipole moment. The molecular drawings of the nine thio compounds were plotted using Visualization Materials Studio 5.5. All quantum chemical calculations were performed using Density Functional Theory (DFT) as implemented in the Materials Studio 5.5 software. DMol2–9 were synthesized, and characterized using various spectroscopic methods and elemental analysis. The synthesized compounds were studied theoretically and the atomic charges, heat of formation and stereochemistry were estimated, and it was found that compounds 5 and 8 are not planar.In this study, the compounds


Indane dimer 5 was obtained for the first time by direct transformation from isorhapontigenin. The structures and relative configurations of the dimers were elucidated using spectral analysis, and their possible formation mechanisms were discussed. The results indicate that this reaction could be used as a convenient method for the semi-synthesis of indane dimers because of the mild conditions and simple reaction products.Using potassium hexacyanoferrate (III)–sodium acetate as oxidant, the oxidative coupling reaction of isorhapontigenin and resveratrol in aqueous acetone resulted in the isolation of three new indane dimers  Owing t7 could be determined by analyzing the NOESY spectrum b and upfield shift of H-14a in comparison to those of 6, the interaction between H-14a and H-2(6)a suggests that ring A1 must be located near ring A2, whereas the interaction between H-7a and H-8b indicates that H-7a and H-8b are situated in a cis-orientation. Therefore, the structure of 7 is characterized as shown in The relative stereochemistry of spectrum , in whic3, 5, 8, 9, 10, and 11 were identified as shegansu B  yielded 10. Evidently, all reactions mentioned above should be carried out concertedly. In general, free radical reaction is not stereoselective. In the course of nucleophilic attack reaction, the chances of attack to Re or Si face of intermediates are equal. Therefore, all products are found to be enantiomer pairs.The coupling of radicals Mectively . Meanwhic attack . In the 6 and 7 results from the high nucleophilicity of the cyano anion, and the low yield of 10 results from the low nucleophilicity of the phenyl group. The dimeric structures indicate that, during the long reaction time, the oxidation reactions of hydroxystilbene through K3Fe(CN)6/NaOAc in aqueous acetone, mainly generate M5 and M8. Product coupling involving M4 and M10 was not detected in the course of the reaction. The sterically less hindered radical M5 would be easily involved in the reaction, possibly accounting for the higher yield of benzofuran products 3 and 11. However, an appropriate account for the distinction of reactivity cannot be proposed based only on this evidence.Moreover, the comparatively high yield of 1H and 125 or 150 MHz for 13C, respectively, on INOVA 500 MHz , or Bruker AVANCE III HD 600 MHz spectrometers , in acetone-d6 or methanol-d4, with the solvent peaks as references. ESI-MS and HR-ESI-MS data were measured using an AccuToFCS JMST100CS spectrometer . Column chromatography (CC) was performed with silica gel . HPLC separation was performed on an instrument consisting of a Waters 515 pump and a Waters 2487 dual λ absorbance detector  with a YMC semi-preparative column (250 mm × 10 mm ID) packed with C18 (5 μM). TLC was carried out with glass precoated silica gel GF254 plates . Spots were visualized under UV light or by spraying with 7% H2SO4 in 95% EtOH followed by heating.Optical rotations were measured on a P2000 polarimeter . UV spectra were obtained on a JASCO P650 spectrometer. IR spectra were recorded on a Nicolet 5700 FT-IR microscope instrument . 1D and 2D NMR spectra were acquired at 500 or 600 MHz for E)-isorhapontigenin  in acetone cooled to 0 °C in an ice bath, a mixed solution of K3Fe(CN)6  and NaOAc  in 25 mL of water was added under stirring. The reactant was stirred at 0 °C for 1 h under a N2 atmosphere, and subsequently stirred for another 15 days at room temperature. The reaction mixture was extracted with ethyl acetate and water, the organic layer was washed with brine, water and dried over anhydrous Na2SO4 for 24 h. Then it was concentrated in vacuo to yield a residue that was subjected to silica gel column chromatography eluting with CHCl3–MeOH  to give 3  and fraction Fr-1 (14.0 mg). Fr-1 was subsequently subjected to semi-preparative Rp-18 HPLC  eluting with acetonitrile in water  to yield compounds 4  and 5 , respectively.To a solution of ; UV (MeOH) λmax (log ε): 229 , 281 (4.08) nm; IR (film) νmax: 3360, 2973, 1605, 1516, 1464, 1344, 1276, 1151, 1127, 1032, 1004, 841, 822, 699 cm−1; 1H-NMR  and 13C-NMR , see m/z: 555.3 [M + Na]+, 571.2 [M + K]+, 531.2 [M − H]−, 567.2 [M + Cl]−; HR-ESI-MS m/z: 531.1662 [M − H]− .Gnetuhainin I (5): brown amorphous powder; c = 0.70, MeOH); 1H-NMR  and 13C-NMR , see m/z: 555.2 [M + Na]+, 571.2 [M + K]+, 531.2 [M − H]−, 567.2 [M + Cl]−; HR-ESI-MS m/z: 531.1661 [M − H]− .1, 1000 mg) in acetone (100 ml), a mixed solution of K3Fe(CN)6 (2885.0 mg) and NaOAc (2660.0 mg) in water (125 mL) was added under stirring. The reactant was refluxed for 35 h, after removal of the acetone under reduced pressure, the solution was extracted with ethyl acetate (100 mL × 3), the organic layer was washed with brine, water and dried over anhydrous Na2SO4 for 24 h. The solution was concentrated in vacuo to yield a reaction mixture that was subjected to a silica gel  column and eluted with a gradient of increasing MeOH in CHCl3  to provide seven fractions, namely, FNR-A~FNR-G. FNR-B (102.1 mg) was unchanged resveratrol, and FNR-C was the main product 11 . 45 mg FNR-D (132.2 mg) was then separated by semi-preparative Rp-HPLC  eluted using methanol/water  to afford 6  and 7 . FNR-F (110.7 mg) was dealt with in the same manner to give 8 , 9  and 10 , respectively.To a solution of resveratrol ; UV (MeOH) λmax (log ε): 206 (4.48), 228 , 282 (3.59) nm; IR (film) νmax: 3393.9, 2920.3, 2849.7, 2248.6, 1610.6, 1513.2, 1468.4, 1344.1, 1247.6, 1157.3, 1003.3, 835.7 cm−1; 1H-NMR (500 MHz in acetone-d6) and 13C-NMR (125 MHz in acetone-d6), see m/z: 482 [M + H]+, 504 [M + Na]+, 520 [M + K]+; HR-ESI-MS m/z: 504.1409 [M + Na]+ .Compound7: a light brown amorphous powder. c = 0.66, MeOH); UV (MeOH) λmax (log ε): 204 (4.55), 230 , 283 (3.70), nm; IR (film) νmax: 3337.0, 2916.6, 2850.1, 1610.9, 1513.5, 1468.2, 1342.0, 1247.8, 1155.6, 1006.9, 834.0 cm−1; 1H-NMR (500 MHz in acetone-d6) and 13C-NMR (125 MHz in acetone-d6), see m/z: 482 [M + H]+, 504 [M + Na]+, 520 [M + K]+; HR-ESI-MS m/z: 504.1431 [M + Na]+ .Leachianol G (8): a brown amorphous powder. c = 1.05, MeOH); 1H-NMR (500 MHz in acetone-d6) data: 7.07 a), 6.73 a), 4.45 , 3.38 , 6.22 , 5.71 , 6.87 b), 6.71 b), 4.27 , 3.50 , 6.14 b), 6.16 . 13C-NMR (125 MHz in acetone-d6): 135.81s (C-1a), 129.48d (C-2(6)a), 115.18d (C-3(5)a), 157.23s (C-4a), 77.25d (C-7a), 62.69d (C-8a), 147.38s (C-9a), 123.04s (C-10a), 154.84s (C-11a), 102.17d (C-12a), 158.49s (C-13a), 105.55d (C-14a), 138.09s (C-1b), 129.31d (C-2(6)b), 115.55d (C-3(5)b), 156.18s (C-4b), 55.93d (C-7b), 59.04d (C-8b), 151.63s (C-9b), 106.07d (C-10(14)b), 159.21s (C-11(13)b), 100.87d (C-12b). (+)-ESI-MS m/z: 473 [M + H]+, 495 [M + Na]+, 511 [M + K]+; HR-ESI-MS m/z: 495.1419 [M + Na]+ .Leachianol F (9): a brown amorphous powder. c = 1.05, MeOH); 1H-NMR (500 MHz in acetone-d6) data: 6. 86 a), 6.68 a), 4.46 , 3.37 , 6.32 , 6.59 , 6.85 b), 6.74 b), 4.24 , 2.95 , 5.93 , 6.13 , 4.01 . 13C-NMR (125 MHz in acetone-d6): 136.28s (C-1a), 128.87d (C-2(6)a), 115.34d (C-3(5)a), 156.26s (C-4a), 76.58d (C-7a), 61.91d (C-8a), 148.84s (C-9a), 122.46s (C-10a), 154.96s (C-11a), 102.19d (C-12a), 158.75s (C-13a), 106.12d (C-14a), 137.46s (C-1b), 129.35d (C-2(6)b), 115.51d (C-3(5)b), 157.06s (C-4b), 55.61d (C-7b), 59.47d (C-8b), 150.75s (C-9b), 105.90d (C-10(14)b), 159.14s (C-11(13)b), 100.99d (C-12b). (+)-ESI-MS m/z: 472 [M]+, 495 [M + Na]+, 511 [M + K]+; HR-ESI-MS m/z: 495.1425 [M + Na]+ .Pallidol (10): a brown amorphous powder. c = 1.05, MeOH); 1H-NMR (500 MHz in acetone-d6) δ: 6. 99 a), 6.71 a), 4.58 , 3.82 , 6.20 , 6.63  , 3.32 ; 13C-NMR (125 MHz in acetone-d6) δ: 137.74s (C-1a), 129.05d (C-2(6)a), 115.74d (C-3(5)a), 159.27s (C-4a), 60.52d (C-7a), 53.97d (C-8a), 123.19 (C-9a), 102.35s (C-10a), 155.24s (C-11a), 103.28d (C-12a), 156.27s (C-13a), 150.53d (C-14a). (+)-ESI-MS m/z: 455 [M + H]+, 477 [M + Na]+, 493 [M + K]+. Resveratrol trans-dehydrodimer  (11): a brown amorphous powder. 1H-NMR (500 MHz in acetone-d6), δ: 7.24 , 6.83 , 5.44 , 4.45 , 6.18 , 6.27 , 7.25 , 6.87 , 7.42 , 7.04 , 6.89 , 6.53 , 6.27 ; 13C-NMR (125 MHz in acetone-d6) δ: 131.71 (C-1a), 128.57 , 116.08 , 158.33 (C-4a), 94.03 (C-7a), 57.80 (C-8a), 145.21 (C-9a), 107.33 (C-10a), 159.66 (C-11a), 102.24 (C-12a), 159.66 (C-13a), 107.33 (C-14a), 132.10 (C-1b), 123.90 (C-2b), 132.52 (C-3b), 160.60 (C-4b), 110.12 (C-5b), 128.61 (C-6b), 129.09 (C-7b), 127.19 (C-8b), 140.76 (C-9b), 105.63 , 159.45 , 102.57 (C-12b); (+) ESI m/z: 455 [M + H] +, 477 [M + Na]+, 493 [M + K]+; (−) ESI m/z: 453 [M − H]−, 489 [M + Cl]−.3Fe(CN)6/NaOAc in aqueous acetone as oxidant, in combination with silica gel column chromatography and preparative Rp-HPLC resulted in the isolation of nine stilbene dimers. The structures of the nine dimers were determined on the basis of spectral analysis and chemical properties. Products 4, 6, and 7 are new dimers with indane skeletons, product 5 with an indane skeleton was obtained for the first time by direct transformation from isorhapontigenin, and all products would be rather difficult to obtain by common organic reactions.The oxidative coupling reaction of isorhapontigenin and resveratrol with K3 and 11 with the highest yields, and the indane dimers 4, 5, and 6–10 in comparatively low yields. Compared with other non-enzymatic oxidants , this reaction seems to generate only two types of radical intermediates, namely, M5 and M8, and only two coupling mechanisms occur to form benzofuran and indane stilbene dimers. Thus, the reaction could be used as a convenient method of synthesizing indane stilbene dimers because of its mild conditions, long reaction time and simple products. To the best of our knowledge, up to now, this is the most detailed report on the potassium hexacyanoferrate (III)-sodium acetate catalyzed biomimetic synthesis of stilbene dimers.Results indicated that, under the reaction conditions, the oxidative coupling reaction of two stilbenes yields only two types of stilbene dimers, namely, the benzofuran dimers


Plantago asiatica L.), which showed significant antioxidative activity. The new compound was characterized as 2-ethyl 3-O-β-D-allopyranosyl-6-O-caffeoyl-β-D-glucopyranoside on the basis of spectral and chemical evidence, and its antioxidant activity was comparable to that of tea catechins.Seven phenolic compounds, including a new phenylethanoid glycoside, were isolated from the ethyl acetate fraction of an aqueous ethanol extract of Plantago Herb (whole part of  Plantago consists of more than 200 species, most of which are small plants with elliptic leaves and small spikes of very small flowers. Plantago asiatica L. (Plantaginaceae) is a weed widely distributed in eastern Asia. The aerial parts of P. asiatica, ‘Plantago Herba’, are used as a crude drug in China, Korea, and Japan for diuretic, antitussive, expectorant, and antiphlogistic purposes  on a tetramethylsilane scale. The standard pulse sequences programmed for the instrument (AVANCE 500) were used for each 2D measurement . JCH was set at 8 or 10 Hz in HMBC. Column chromatography was carried out with Diaion HP-20, MCI-gel CHP-20P , YMC gel ODS , and Sephadex LH-20 , respectively. Normal-phase (NP) HPLC conducted on a YMC-Pack SIL A-003  column (4.6 i.d. × 150 mm) developed with n-hexane-MeOH-tetrahydrofuran-formic acid (55:33:11:1) containing oxalic acid (450 mg/L) . Reversed-phase (RP) HPLC conditions were as follows: (Condition 1) column, L-column ODS  ; mobile phase, solvent A was 5% acetic acid and solvent B was acetonitrile ; injection volume, 2 μL; column temperature, 40 °C; flow-rate, 0.3 mL/min; detection, 200–400 nm. (Condition 2) column, YMC-Pack ODS AQ  (YMC Co. Ltd.); mobile phase, 10 mM H3PO4-10 mM KH2PO4-acetonitrile (41:41:18); column temperature, 40 °C; flow-rate, 0.2 mL/min; detection, 280 nm. (Condition 3) column, COSMOSIL Cholester Waters  ; mobile phase, 10 mM H3PO4-10 mM KH2PO4-MeOH (37.5:37.5:25); column temperature, 40 °C; flow-rate, 0.2 mL/min; detection, 280 nm. Microplate reader was used an Infinite F200 microplate reader .Optical rotations were measured with a JASCO P-1020 digital polarimeter. UV spectra were recorded on a Shimadzu UVmini-1240 . Electrospray ionization (ESI)-MS, and high-resolution (HR) ESI-MS spectra were obtained using a micrOTOF-Q  mass spectrometer using acetonitrile as the solvent. Plantago asiatica L) was obtained from Uchida Wakanyaku Ltd. . N-Methylmorpholine-N-oxide, 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH), L-cysteine methyl ester hydrochloride and o-tolyl isothiocyanate were purchased from Wako Pure Chemical Industries . Fluorescein sodium salt and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were obtained from Sigma-Aldrich . All other reagents were of analytical grade. Plantago Herb (dried whole plant of 2O (8:2)] (5 L) and the homogenate was filtered. The filtrate was concentrated and extracted with n-hexane (0.6 L) and ethyl acetate (1.2 L), to give the respective n-hexane (338.8 mg), ethyl acetate (8.6 g), and water (60.7 g) extracts. The ethyl acetate extract (4 g) was chromatographed over MCI-GEL CHP-20P with MeOH-H2O (0:100→10:90→20:80→30:70→40:60→100:0) in stepwise gradient mode. The fractions showing similar HPLC patterns were combined and further purified by column chromatography over Sephadex LH-20 with EtOH and/or YMC GEL ODS-AQ with aqueous MeOH to afford vanillic acid (1) (26.1 mg), p-hydroxybenzoic acid (2) (2.1 mg), -dehydrodiconiferyl alcohol 9'-β-D-glucopyranoside (3) (7.9 mg), plantamajoside (4) (1.16 g), desrhamnosyl acteoside (5) (29.0 mg), calceorioside B (6) (109.3 mg), 2-ethyl 3-O-β-D-allopyranosyl-6-O-caffeoyl-β-D-glucopyranoside (7) (29.4 mg). On the other hand, the water extract (60 g) was separated by column chromatography over Diaion HP-20 with aqueous MeOH to give 4 (6.6 g). These compounds were identified by direct comparison with authentic specimens or by comparison of their spectral data with those reported in the literature. The physical data of new compound 7 are as follows.The Plantago Herb (500 g) was homogenized in 80% EtOH 23D −22.1° . 1H-NMR  δH: 7.55 , 7.02 , 6.88 , 6.76 , 6.66 , 6.62 , 6.53 , 6.25 , 4.87 , 4.51 , 4.38 , 4.33 , 4.06 , 3.95 , 3.85 , 3.76 , 3.62 , 3.20–3.60 , 3.41–3.49 , 2.78 . 13C-NMR δC: 36.7 (C-7), 63.0 , 64.6 (glc C-6), 68.9 , 70.4 (glc C-4), 72.4 (C-8), 72.8 , 72.9 , 74.3 (glc C-2), 75.1 (glc C-5), 75.9 , 88.1 (glc C-3), 103.1 , 104.1 (glc C-1), 114.8 (C-8'), 115.7 (C-2'), 116.5 , 117.1 (C-2), 121.3 (C-6), 123.2 (C-6'), 127.7 (C-1'), 131.4 (C-1), 144.7 (C-3), 146.1 , 147.2 (C-7'), 149.1 (C-4'), 169.1 (C-9'). HR-ESI-MS m/z: 639.1939 .7 (1.0 mg) were hydrolyzed by heated in 0.5 M HCl (0.2 mL) and neutralized with Amberlite IRA400. After drying, the residue was dissolved in pyridine (0.2 mL) containing L-cysteine methyl ester hydrochloride (1.0 mg) and heated at 60 °C for 1 h. After heating, o-tolyl isothyocyanate (1.0 mg) in pyridine (0.2 mL) was added to the mixture and heated at 60 °C for 1h. The reaction mixture was directly analyzed by RP-HPLC. The peaks were coincided with derivatives of D-allose and D-glucose.Sugar configuration was determined using previous described methods. Compound Antioxidant activity was estimated by the ORAC method. Measurement of the ORAC values was performed using previous described methods. Briefly, the ORAC assay was performed in 75 mM phosphate buffer (pH 7.4) with a final reaction volume of 200 μL. Trolox (20 μL) and fluorescein  solutions were pipetted into each well of a 96-well microplate. The mixture was pre-incubated in a microplate reader for 15 min at 37 °C. A solution of AAPH  was added rapidly to the microplate, and after shaking for 15 s, the fluorescence was recorded every minute for 90 min at excitation and emission wavelengths of 485 and 528 nm, respectively. The area under the curve (AUC) was calculated, and the net AUC was calculated by subtracting the AUC of the blank (phosphate buffer only) from that of each sample. ORAC values were expressed as trolox equivalents (μmol TE/g) using the calibration curve generated in each assay.O-β-D-allopyranosyl-6-O-caffeoyl-β-D-glucopyranoside (7), was isolated from Plantago Herb (whole part of Plantago asiatica L.), together with six known phenolic compounds. Among them, vanillic acid (1), plantamajoside (4), desrhamnosyl acteoside (5), and calceorioside B (6) showed potent antioxidant activity. The potency of 7 was comparable to that of EGCG. The new phenylethanoid glycoside, 2-ethyl-3-