Wissadula periplocifolia (L.) C. Presl  is commonly used in Brazil to treat bee stings and as an antiseptic. The antioxidant properties of its extracts have been previously demonstrated, thus justifying a phytochemical investigation for its bioactive phenolic constituents. This has yielded five new sulphated flavonoids: 8-O-sulphate isoscutellarein (yannin) (1a); 4′-O-methyl-7-O-sulphate isoscutellarein (beltraonin) (1b); 7-O-sulphate acacetin (wissadulin) (2a); 4′-O-methyl-8-O-sulphate isoscutellarein (caicoine) (2b) and 3′-O-methyl-8-O-sulphate hypolaetin (pedroin) (3b) along with the known flavonoids 7,4′-di-O-methyl-8-O-sulphate isoscutellarein (4), acacetin, apigenin, isoscutellarein, 4′-O-methyl isoscutellarein, 7,4′-di-O-methylisoscutellarein, astragalin and tiliroside. The compounds were isolated by column chromatography and identified by NMR  and LC-HRMS. A cell based assay was carried out to evaluate the preliminary cytotoxic properties of the flavonoids against UVW glioma and PC-3M prostate cancer cells as well as non-tumour cell lines. The obtained results showed that acacetin, tiliroside, a mixture of acacetin + apigenin and the sulphated flavonoids 2a + 2b exhibited inhibitory activity against at least one of the cell lines tested. Among the tested flavonoids acacetin and tiliroside showed lower IC50 values, presenting promising antitumor effects. Malva sylvestris L. and Sidastrum micranthum (A. St.-Hil.) Fryxell − ion, found at retention time (RT) of 5.34 min, was 365.0049 (C15H9O9S) and for compound 1b also as an [M − H]− ion (RT: 6.05 min) was 379.0206 (C16H11O9S). These results confirm the presence of O-sulphate groups in both molecules. Thus, compound 1a was identified as 8-O-sulphate isoscutellarein (yannin) and compound 1b as 4′-O-methyl-7-O-sulphate isoscutellarein (beltraonin)   and are 1H-NMR of 2 showed a similar pattern to 1. There were also two downfield singlets at δH 12.82 and δH 12.70 integrating for 1.0 and 0.51, respectively. The set of aromatic protons also indicated that 2 was a mixture of two flavonoids. Additionally, a signal corresponding to methoxyl protons was integrated for 4.61, suggesting the presence of one methoxyl for each flavonoid in the mixture. The 13C-NMR spectra also showed 31 signals including a duplicated methoxyl carbon at δc 56.03. By analyzing the HMBC and HMQC spectra of this mixture, it was possible to determine the major constituent (compound 2a) to possess an acacetin-like moiety and the minor one (compound 2b) showed a 4′-O-methyl isoscutellarein skeleton. However, comparison of 2a with acacetin NMR data indicated that C-7 in compound 2a is shielded by 4 ppm and C-6, C-8 and C-10 are deshielded by 3 ppm − ion were obtained as 395.0136 and C16H11O10S (RT 5.41 min), respectively, thus the structure of compound 3b was determined as 3′-O-methyl-8-O-sulphate hypoaletin (pedroin) (Similar to the mixtures of skeleton . When copedroin)  and it i4 showed a skeleton similar to 7,4′-di-O-methylisoscutellarein + ion (RT 6.15 min) of compound 4 was 395.0405 and the molecular formula C17H15O9S allowed its identity to be confirmed as 7,4′-di-O-methyl-8-O-sulphate isoscutellarein  [Compound ellarein . As obseellarein , previouW. periplocifolia were identified by comparisons of their NMR data with literature . They are being reported for the first time from Wissadula genera.The non-sulphated flavonoids isolated from W. periplocifolia against tumour  and non-tumour  cell lines was accomplished by the alamarBlue assay. Among all tested fractions, only acacetin, tiliroside, a mixture of acacetin + apigenin and the sulphated flavonoids 2a + 2b exhibited inhibitory activity against at least one of the cell lines tested. 50) of the tested samples on each cell line. Comparison between IC50 values shows that the treatment with these flavonoids (0.4–100 µg/mL) for 24 h exerted higher anti-proliferative activity against tumour cells (mainly PC-3M prostate carcinoma cells), when compared to both non-cancer cell lines.The cytotoxic evaluation of the flavonoids from W. periplocifolia tested, 7,4′-di-O-methylisoscutellarein and the sulphated flavonoids 2a + 2b presented anti-proliferative effects against UVW and PC-3M cells, respectively, but with higher IC50 values. Acacetin exhibited the best cytotoxicity against UVW and PC-3M cells, with the lowest IC50 values. Most importantly, the anti-proliferative effects of acacetin were higher in tumour cells when compared to non-cancer cell lines. It has been shown that acacetin exhibits anti-proliferative activities against many tumour cell lines [Among the flavonoids from ll lines ,26,27, ill lines . In prosll lines , inhibitll lines , or is all lines .Apigenin is another flavone that decreases viability of many cancer cells through mechanisms that had not been completely explored so far ,35,36,37W. periplocifolia exerted selective anti-proliferative activity against PC-3M prostate carcinoma cells, when compared to glioma and both non-cancer cell lines. Tiliroside is a naturally occurring flavonoid that has toxic effects on some tumour cell lines such as lung cancer A549 [in vitro. Therefore, tiliroside presents promising antitumor effects without a significant toxicity [Tiliroside isolated from cer A549  and no scer A549 ,40. In hcer A549 . Howevercer A549 . Tilirostoxicity .W. periplocifolia on glioma and prostate cancer cell lines. Further analyses are necessary to investigate the mechanisms of action of these compounds.In this study, we showed preliminary cytotoxic characterizations of flavonoids isolated from 1H 400 MHz, 13C 100 MHz), acquired on a Bruker-Avance III spectrometer  using deuterated DMSO.Column chromatography separations (CC) were performed on glass columns packed with silica gel  and gel filtration chromatography (GFC) were carried out using Sephadex LH-20 . Thin layer chromatography (TLC) were performed on silica gel PF 254 plates  and spots were visualized under UV light (254 and 366 nm) and by spraying with vanillin–sulphuric acid reagent. Isolated compounds were identified by 1D and 2D NMR analysis , in August 2005 (SISBIO Authorization Number 46923-2). A voucher specimen (JPB 6498) was authenticated by Prof. Dr. Maria de Fátima Agra and deposited at Prof. Lauro Pires Xavier Herbarium (JPB/UFPB).The aerial parts of 2Cl2), ethyl acetate (EtOAc) and n-butanol to yield 48 g of hexane (HF), 33 g of CH2Cl2 (DF), 28 g of EtOAc (EAF) and 9 g of n-butanol (BF) fractions. The various fractions were analysed by 1H-NMR to detect the presence of phenolic constituents and DP, EAP and BF were chosen for column chromatography (CC).The plant material was dried in an oven at 40 °C for 72 h. After milling, 8.9 kg of powder was macerated with absolute ethanol for 72 h. The obtained ethanol extract was concentrated with a rotatory evaporator yielding 705 g of crude extract (CEE). 200 g of CEE was solvent extracted using hexane, dichloromethane (CHO-methylisoscutellarein (24 mg), identified by NMR and LC-MS techniques and by comparisons with literature data. CC fraction 79–92 on GFC yielded the compounds apigenin, isoscutellarein, and 4′-O-methyl isoscutellarein. Their NMR data match with literature [The fraction DF (15 g) was subjected to silica column chromatography (CC) eluted with hexane, EtOAc and methanol in a gradient manner. The fractions were analysed by TLC and interesting fractions were further subjected to GFC, eluted isocratic wise with methanol to isolate the compounds. CC fraction 58–69 chromatographic separation on GFC led to isolation of acacetin (35 mg) and 7,4′-di-terature ,23.1 and 2 respectively (3 (The CC fraction 206–218 (246 mg) was subjected to GFC yielding 34 fractions. These fractions were analyzed by TLC and combined. Fractions 16–17 (12 mg) and 20–21 (9 mg) showed only one spot at TLC, named as compounds ectively . The CC ively  (12 mg) and kaempferol-3-O-b-d-(6′′-E-p-coumaroyl) glucopyranoside (tiliroside) (95 mg). The purified fractions were analyzed by 1D and 2D NMR techniques and by LC-MS and the data match with literature [EAF (2 g) was subjected to GFC eluted with methanol. Fractions were analyzed by TLC and selected to purification also through GFC. From this process two glucosyl flavonoids were purified: kaempferol 3-terature .4 (32 mg) (BF (2 g) was subjected to consecutives GFC eluted with methanol. From this process it was purified compound  (32 mg)  and the 1H 400 MHz, 13C 100 MHz) using deuterated DMSO, and their mass spectra were obtained by LC-HRMS. The NMR data of compounds 1 to 4 are showed at 1 to 4 can be found at The isolated compounds were identified by 1D and 2D NMR analysis (Acacetin. 1H-NMR (DMSO) δ (ppm): 12.93 , 6.88 , 6.20 , 6.51 , 8.12 , 7.11 , 3.86 . 13C-NMR (DMSO) δ (ppm): 163.8 (C-2), 104.1 (C-3), 182.3 (C-4), 162.0 (C-5), 99.4 (C-6), 164.7 (C-7), 94.60 (C-8), 157.9 (C-9), 104.3 (C-10), 123.3 (C-1′), 128.8 , 115.1 , 162.8 (C-4′), 56.1 (-OCH3-4′). The 1H- and 13C-NMR spectral data are consistent with published data [Apigenin. 1H-NMR (DMSO) δ (ppm): 12.96 , 6.78 , 6.19 , 6.48 , 7.92 , 6.93 . 13C-NMR (DMSO) δ (ppm): 164.3 (C-2), 103.4 (C-3), 182.3 (C-4), 162.0 (C-5), 99.4 (C-6), 164.7 (C-7), 94.5 (C-8), 157.9 (C-9), 104.2 (C-10), 121.7 (C-1′), 129.0 , 116.5 , 161.7 (C-4′). The 1H- and 13C-NMR spectral data are consistent with published data [Isoscutellarein. 1H-NMR (DMSO) δ (ppm): 12.39 , 6.74 , 6.27 , 8.01 , 6.93 . 13C-NMR (DMSO) δ (ppm): 164.1 (C-2), 102.9 (C-3), 182.7 (C-4), 153.6 (C-5), 99.2 (C-6), 153.9 (C-7), 125.6 (C-8), 146.0 (C-9), 103.8 (C-10), 121.9 (C-1′), 129.2 , 116.4 , 161.7 (C-4′). The 1H- and 13C-NMR spectral data are consistent with published data [4′-O-Methyl isoscutellarein. 1H-NMR (DMSO) δ (ppm): 12.40 , 6.83 , 6.28 , 8.12 , 7.12 , 3.86 . 13C-NMR (DMSO) δ (ppm): 163.7 (C-2), 103.6 (C-3), 182.7 (C-4), 153.6 (C-5), 99.2 (C-6), 153.6 (C-7), 125.6 (C-8), 146.0 (C-9), 103.9 (C-10), 123.6 (C-1′), 129.0 , 115.1 , 162.8 (C-4′), 56.1(-OCH3-4′). The 1H- and 13C-NMR spectral data are consistent with published data [7,4′-di-O-Methyl isoscutellarein. 1H-NMR (DMSO) and 13C-NMR (DMSO) δ (ppm). Previously reported [reported .Tiliroside. 1H-NMR (DMSO) δ (ppm): 12.55 , 6.13 , 6.38 , 7.97 , 6.85 , 5.43 , 3.18–3.40 , 4.02–4.27 , 7.34 , 6.78 , 7.31 , 6.08 . 13C-NMR (DMSO) δ (ppm): 157.1 (C-2), 133.5 (C-3), 177.9 (C-4), 161.7 (C-5), 99.3 (C-6), 164.7 (C-7), 94.2 (C-8), 156.9 (C-9), 102.5 (C-10), 121.3 (C-1′), 130.7 (C-2′/6′), 116.3 (C-3′′′/5′′′), 160.5 (C-4′), 101.4 (C-1′′), 74.8 (C-2′′), 76.7 (C-3′′), 70.5 (C-4′′), 74.6 (C- 5′′), 63.4 (C-6′′), 125.4 (C-1′′′), 131.3 (2′′′ and 6′′′), 115.6 (C-3′′′ and 5′′′), 160.3 (C-4′′′), 145.2 (C-7′′′), 114.1 (C-8′′′), 166.7 (C-9′′′). The 1H- and 13C-NMR spectral data are consistent with published data [Astragalin. 1H-NMR (DMSO) δ (ppm): 12.549 , 6.20 , 6.00 , 8.01 , 6.85 , 5.37 , 3.15–3.55 . 13C-NMR (DMSO) δ (ppm): 155.6 (C-2), 133.5 (C-3), 177.1 (C-4), 160.5 (C-5), 101.9 (C-6), 163.1 (C-7), 94.9 (C-8), 155.7 (C-9), 103.5 (C-10), 122.7 (C-1′), 131.2 (C-2′/6′), 115.5 (C-3′′′/5′′′), 160.5 (C-4′), 101.9 (C-1′′), 74.7 (C-2′′), 77.9 (C-3′′), 70.3 (C-4′′), 76.9 (C- 5′′), 61.3 (C-6′′). The 1H- and 13C-NMR spectral data are consistent with published data [hed data .In order to confirm the compounds structures, the high-resolution mass spectra was obtained by LC-MS analysis performed on an Accela 600 HPLC system combined with an Exactive (Orbitrap) mass spectrometer from Thermo Fisher Scientific  in negative or positive mode using method developed to separate phenolic compounds .2O (solvent A) and acetonitrile (solvent B) was used in a gradient mode as summarized in the Each sample was dissolved in methanol (HPLC grade) to obtain a final concentration of 1 mg/mL. The injection volume was 20 μL and an ACE C-18 column  from HiChrom  was used. A flow rate of 300 μL/min and a mobile phase composed of 0.1% formic acid in Hv/v fetal bovine serum, penicillin (100 units/mL), streptomycin (100 μg/mL), Fungizone (2.5 μg/mL of amphotericin B), and L-glutamine (2 mM). The PC-3M cells were cultured in the same MEM medium as described for UVW cells with the addition of 1% v/v MEM Non-essential Amino Acids Solution, 1% v/v MEM Vitamin Solution, and 1mM sodium pyruvate. The cells were cultured at 37 °C in a humidified atmosphere of 5% CO2.All media and supplements were obtained from Invitrogen . The following human cell lines were used in the current study: the human glioma cancer cell line (UVW) previously described , the pro3cells/well). After 24 h incubation, the medium was replaced by fresh media containing the flavonoid fractions at 0.4, 1.2, 3.7, 11.1, 33.3, and 100 μg/mL or DMSO at 0.5% v/v, and then cultured for 24 h at 37 °C in a humidified atmosphere of 5% CO2. Untreated cells were used as negative controls. After treatment, the culture medium was removed and replaced by fresh media containing alamarBlue (10% v/v), and the dye was incubated in the plates with the cells for 4 h. Fluorescence was measured on a micro plate fluorescence reader  using an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Cell viability was calculated and plotted as the percentage of metabolically active cells relative to the control. The 50% inhibitory concentrations (IC50) were calculated graphically from the individual concentration–response curves by non-linear curve fitting. Two-way analysis of variance (two-way ANOVA) with Bonferroni post-hoc test was used to compare the 50% inhibitory concentration values. The level of significance was set at p < 0.05 and statistical analysis was performed using GraphPad Prism version 5.0 for Windows .Each sample tested was dissolved in DMSO at 50 mg/mL and kept at −20 °C until extract preparation was proceeded. For the biological assays, the samples were diluted in culture medium to get the selected concentrations. The toxicity of the flavonoids on UVW, PC-3M, PNT2A, and Hs27 cells was assessed by the fluorometric measurement of metabolic activity using the AlamarBlue assay (Invitrogen) . BrieflyWissadula periplocifolia: 8-O-sulphate isoscutellarein (1a); 4′′-O-methyl-7-O-sulphate isoscutellarein (1b); 7-O-sulphate acacetin (2a); 4′-O-methyl-8-O-sulphate isoscutellarein (2b) and 3′-O-methyl-8-O-sulphate hypolaetin (3b) along with the known flavonoids 7,4′-di-O-methyl-8-O-sulphate isoscutellarein (4), acacetin, apigenin, isoscutellarein, 4′-O-methyl isoscutellarein, 7,4′-di-O-methyl isoscutellarein, astragalin and tiliroside. Besides, the cytotoxic properties of the isolated compounds were evaluated demonstrating the potential cytotoxicity of acacetin, 7,4′-di-O-methyl isoscutellarein, apigenin and the new compounds 7-O-sulphate acacetin (2a) and 4′-O-methyl-8-O-sulphate isoscutellarein (2b).The present study has led to the identification of five new sulphated flavonoids from


O-methylscutellarein (3), the major metabolite of the natural medicine scutellarin, is reported. Two hydroxyl groups at C-4′ and C-7 in 2 were selectively protected by chloromethyl methyl ether after the reaction conditions were optimized, then 6-O-methyl-scutellarein (3) was produced in high yield after methylation of the hydroxyl group at C-6 and subsequent deprotection of the two methyl ether groups.In this paper, a new and efficient synthesis of 6- To a stirring mixture of 2  and K2CO3  in dry acetone (100 mL) at room temperature was added chloromethyl ether , the reaction mixture was refluxed gently for 8 h. After cooling to room temperature, the reaction mixture was filtered. Removal of the solvent in vacuo followed by purification of the residue by column chromatography on silica gel with 20% ethyl acetate in petroleum ether as eluent afforded 7 , 8  and 9  as yellow solids, respectively.5,6-Dihydroxy-2-(4-hydroxyphenyl)-7-(methoxymethoxy)-4H-chromen-4-one (7). 1H-NMR (DMSO-d6) δ 3.50 , 5.35 , 6.60 , 6.66 , 7.04 , 8.02 , 10.33 , 12.40 ; ESI-MS: m/z 329 [M − H]−.5,6-Dihydroxy-7-(methoxymethoxy)-2-(4-(methoxymethoxy)phenyl)-4H-chromen-4-one (8). 1H-NMR (DMSO-d6) δ 3.52 , 3.61 , 5.22 , 5.35 , 6.61 , 6.69 , 7.05 , 8.01 , 12.76 ; ESI-MS: m/z 373 [M − H]−.5-Hydroxy-6,7-bis(methoxymethoxy)-2-(4-(methoxymethoxy)phenyl)-4H-chromen-4-one (9). 1H-NMR (DMSO-d6) δ 3.66 , 3.76 , 3.87 , 5.35 , 5.48 , 5.60 , 6.95 , 7.09 , 7.15 , 8.06 , 12.93 ; ESI-MS: m/z 419 [M + H]+.5-Hydroxy-6-methoxy-7-(methoxymethoxy)-2-(4-(methoxymethoxy)phenyl)-4H-chromen-4-one (10). To a stirring solution of 8  in dry DMF (20 mL) was added K2CO3  and iodomethane  at room temperature. After 12 h, the reaction mixture was then partitioned between ethyl acetate (100 mL) and water (100 mL). The ethyl acetate layer was then washed with brine (100 mL), dried over Na2SO4, filtered and concentrated. The crude material was purified by column chromatography (25% ethyl acetate in petroleum ether) to yield 10  as a yellow solid. 1H-NMR (DMSO-d6) δ 1H-NMR  δ 3.46 , 3.51 , 3.96 , 5.31 , 5.34 , 6.48 , 6.62 , 7.13 , 8.02 , 12.71 ; ESI-MS: m/z 389 [M + H]+.5,7-Dihydroxy-2-(4-hydroxyphenyl)-6-methoxy-4H-chromen-4-one (3). Hydrochloric acid (1 mL) was added to a stirred solution 10  in CH2Cl2 (5 mL) and ether (5 mL) at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for a further 1 h. The reaction mixture was diluted with a large amount of ethyl acetate and washed with water and brine. The ethyl acetate layer was dried over MgSO4, filtered, then concentrated and the crude material purified by column chromatography (50% ethyl acetate in petroleum ether) to yield 3  as a yellow solid. 1H-NMR (DMSO-d6) δ 3.72 , 6.58 , 6.89 , 7.11 , 7.87 , 10.19 , 10.72 , 12.76 ; ESI-MS: m/z 299 [M − H]−.


All the compounds were characterized by 1H-NMR, mass spectroscopy and elemental analysis. Preliminary bioassays indicated that all the compounds acted against the tobacco mosaic virus (TMV) with different in vivo and in vitro modes at 500 μg/mL and were found to possess promising activity. Especially, compound 3p showed the most potent biological activity against tobacco mosaic virus (TMV) compared to ningnanmycin, and a molecular docking study was performed and the binding model revealed that the pyrazole amide moiety was tightly embedded in the binding sites of TMV PC (PDB code: 2OM3).A series of novel pyrazole amide derivatives  Tobacco mosaic virus (TMV) is one of the most extensively studied among a number of plant viruses. Its infections are very widely distributed ,2,3 and Recently, different targets essential to plant viruses that could be new weapons to design and discover new antiviral drugs have been studied and validated. Most antiviral drugs exhibit their activities through interaction with viral proteins ,6. Coat Pyrazole derivatives have great importance in the medicinal and pesticide fields ,14,15, det al. [Modification of the structural profile by the change of substituents at the 1-, 3-, or 4-positions in the pyrazole ring can bring about significant changes in bioactivity , and linet al. , the reset al. ,32,33, aet al. . We describe herein the synthesis and antiviral activity of a series of new pyrazole amide derivatives . Molecul1a–1d were prepared in accordance with the literature procedure [3. Finally, the products were oxidized with KMnO4, to give the 5-chloro-1-aryl-3-methyl-1H-pyrazole-4-carboxylic acids 1 (The synthetic route is shown in rocedure  from the acids 1 .2a–2d were obtained from substituted phenyl hydrazine hydrochlorides and 2-(ethoxymethylene) malononitrile in ethanol medium, which was refluxed for 3 h to produce the 5-amino-1-aryl-1H-pyrazole-4-carbonitriles 2a–2d [Compounds es 2a–2d  (Scheme 1 and 2 were reacted using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and N-hydroxybenzotriazole (HOBt) in DMF medium in the presence of triethylamine as catalyst at room temperature to produce the 5-chloro-N-(4-cyano-1-aryl-1H-pyrazol-5-yl)-1-aryl-3-methyl-1H-pyrazole-4-carboxamides 3a–3p  spectra of the compounds, the N-H proton was hard to distinguish in the spectra. To prove its existence we used DMSO as solvent to dissolve compounds 3e, 3f, 3n and then recorded the NMR spectra, whereupon the signal of compounds 3e, 3n appears at 12.88 ppm and the signal of compounds 3f appears at 12.89 ppm. The NH group of compound 3n appears at 115.11 ppm in the corresponding 13C-NMR spectrum taken in DMSO. The IR spectrum of compound 3b has been investigated in detail to exactly indicate the presence of CN groups and the main observed absorption bands were those located at: 3180.66 cm−1 (N-H), 2230.44 cm−1 (C≡N), 1636.96 cm−1 (C=O), 1560.03 cm−1 (C=O), 1404.77 cm−1 (C-N), 821.27 cm−1 (C-Cl arom), and 587.59 cm−1 (C-Cl pyrazole), which all support the proposed structure. The structures of the newly synthesized compounds were confirmed by 3a–3p, the commercially available plant virucide ningnanmycin was used as the control. The antiviral bioassay against TMV was assayed by the method reported by Thorson et al. [To judgme the antiviral potency of the synthesized compounds n et al. . in vivo and in vitro bioassay results of compounds 3a–3p against TMV are given in 3p showed the most potent biological activity against TMV and it exhibited slightly higher activities compared to the commercial agent ningnanmycin.The 4p into the three dimensional X-ray structure of the TMV PC protein-ligand complex crystal structure (PDB code: 2OM3) was carried out using the Discovery Studio (DS)  software [http://www.rcsb.org/pdb/home/home.do) and prepared by Discovery Studio 3.1 with all bound waters and ligands eliminated from the protein and the polar hydrogen added to the protein. The molecular docking procedure was performed by using CDOCKER protocol for receptor-ligand interactions section of DS 3.1.Molecular docking of compound software  as implein vitro antiviral activity data were used to build the 3D-QSAR models, and the 3D-QSAR study was performed by means of the DS 3.5 software. The correlation coefficient r2 between observed and predicted activity of training set was found to be 0.899, which meant the dependability of linear fit. The molecules aligned with the iso-surfaces of the 3D-QSAR model coefficients on electrostatic potential grids  have similar inhibition as 3p. The 3D QSAR models fitted the inhibitory activity well and thus provide us the directions for further modification.As shown in 1H-NMR spectra were recorded on a Bruker PX400 spectrometer  at 25 °C with TMS and solvent signals assigned as internal references, 13C-NMR spectra and 1H-NMR spectra of compound 3e, 3n were recorded on an Agilent DD2 PX600 spectrometer . Chemical shifts were reported in ppm (δ). Elemental analyses were performed on a CHN-O-Rapid instrument  and were within 0.4% of the theoretical values.Melting points (uncorrected) were determined on an XT4 MP apparatus . ESI mass spectra were obtained on a Mariner System 5304 mass spectrometer . H-pyrazole-4-carboxylic acids 1a–1d were synthesized as the following: para-substituted phenylhydrazines (0.025 mol) were reacted with ethyl acetoacetate (0.025 mol) in anhydrous ethanol to form a solid, which was then dissolved in a cold mixed solution of DMF (20 mL) and POCl3 (16 mL), and stirred at 50–60 °C. The resulting mixture was poured into ice-cold water, a saturated solution of sodium hydroxide was added to neutralize the mixture, and the solid precipitate was filtered, and washed with water. Then above product was oxidized by KMnO4 solution, stirred at 70–80 °C. After cooling to room temperature the pH of the reaction mixture was adjusted to pH 7–8 by the dropwise addition of KOH solution, and the solution was filtered, HCl solution was added to the solution and solid 1a–1d eventually separated out. The crude product obtained was recrystallized from DMF to afford the pure product. All the reactions were monitored by TLC.The intermediate 5-chloro-1-aryl-3-methyl-1para-substituted phenylhydrazine hydrochloride (0.025 mol) was dissolved in H2O (30 mL), then the pH of the mixture was adjusted to pH 7–8 by the dropwise addition of 10% NaOH solution to form the free para-substituted phenyl hydrazines, which were then refluxed for 3 h with ethoxymethylene malononitrile in an ethanol medium. After completion of the reaction, the reaction mixture was allowed to cool at room temperature, and the solid 2a–2d were filtered under vacuum. The crude products obtained were recrystallized from DMF to afford the pure products. A stirred mixture of 1a–1d (1 mmol) and triethylamine (2 mmol) in DMF (12 mL) medium, a mixture of EDCI (1 mmol) and HOBt (1mmol) was added and the reaction mixture was stirred at room temperature for 30 min, then a mixture of compounds 2a–2d (1 mmol) and DMF (5 mL) was added, the reaction was stirred at room temperature. And the reaction progress was monitored by TLC. After completion of the reaction, the product was added into chloroform, then extracted from chloroform with water, and washed successively with 0.2 mol/L hydrochloric acid, water, 2 mol/L sodium hydroxide, water, saturated sodium chloride, then dried, concentrated and purified by preparative thin layer chromatography (PE:EA = 8:1) followed by recrystallization from ethanol.To a stirred solution of the intermediate compounds 5-Chloro-N-(4-cyano-1-phenyl-1H-pyrazol-5-yl)-3-methyl-1-phenyl-1H-pyrazole-4-carboxamide (3a). White crystals, yield 68%, mp: 133–135 °C. 1H-NMR  δ: 8.12 , 7.61–7.43 , 2.66 . MS (ESI): 403.1 . Anal. Calcd for C21H15ClN6O: C, 62.61; H, 3.75; N, 20.86; Found: C, 62.82; H, 3.84; N, 21.04.5-Chloro-1-(4-chlorophenyl)-N-(4-cyano-1-phenyl-1H-pyrazol-5-yl)-3-methyl-1H-pyrazole-4-carbox-amide (3b). White crystals, yield 68%. mp: 171–172 °C, IR (cm−1): 3180.66, 2230.44, 1636.96, 1560.03, 1404.77, 821.27, 587.59. 1H-NMR  δ: 8.12 , 7.55–7.43 , 2.65 . MS (ESI): 437.1 . Anal. Calcd for C21H14Cl2N6O: C, 57.68; H, 3.23; N, 19.22; Found: C, 57.72; H, 3.31; N, 19.11.5-Chloro-N-(4-cyano-1-(p-tolyl)-1H-pyrazol-5-yl)-3-methyl-1-phenyl-1H-pyrazole-4-carboxamide (3c). White crystals, yield 62%, mp: 123–125 °C; 1H-NMR  δ: 8.12 , 7.63–7.41 , 2.66 , 2.42 . MS (ESI): 417.1 . Anal. Calcd for C22H17ClN6O: C, 63.39; H, 4.11; N, 20.16; Found: C, 63.71; H, 4.42; N, 20.22.5-Chloro-N-(4-cyano-1-(4-fluorophenyl)-1H-pyrazol-5-yl)-3-methyl-1-phenyl-1H-pyrazole-4-carbox-amide (3d). White crystal, yield 71%, mp: 181–183 °C. 1H-NMR  δ: 8.12 , 7.51–7.21 , 2.66 . MS (ESI):421.0 . Anal. Calcd for C21H14ClFN6O: C, 59.54; H, 3.35; N, 19.97; Found: C, 59.72; H, 3.41; N, 20.06.5-Chloro-1-(4-chlorophenyl)-N-(4-cyano-1-phenyl-1H-pyrazol-5-yl)-3-methyl-1H-pyrazole-4-carbox-amide (3e). White crystals, yield 66%, mp: 172–174 °C. 1H-NMR  δ: 8.12 , 7.61–7.43 , 2.65 . 1H-NMR  δ: 12.88 , 7.97 , 7.62–7.41 , 2.48 . MS (ESI): 437.1 . Anal. Calcd for C21H14Cl2N6O: C, 57.68; H, 3.23; N, 19.22; Found: C, 57.74; H, 3.41; N, 19.42.5-Chloro-1-(4-chlorophenyl)-N-(1-(4-chlorophenyl)-4-cyano-1H-pyrazol-5-yl)-3-methyl-1H-pyrazole-4-carboxamide (3f). White crystals, yield 79%, mp: 178–180 °C. 1H-NMR  δ: 12.89 , 7.98 , 7.63–7.42 , 2.65 . MS (ESI): 471.0 . Anal. Calcd for C21H13Cl3N6O: C, 53.47; H, 2.78; N, 17.82; Found: C, 53.56; H, 2.81; N, 17.91.5-Chloro-1-(4-chlorophenyl)-N-(4-cyano-1-(p-tolyl)-1H-pyrazol-5-yl)-3-methyl-1H-pyrazole-4-carboxamide (3g). White crystals, yield 69%, mp: 130–132 °C. 1H-NMR  δ: 8.12 , 7.60–7.43 , 2.65 , 2.42 . MS (ESI): 451.1 . Anal. Calcd for C22H16Cl2N6O: C, 58.55; H, 3.57; N, 18.62; Found: C, 58.58; H, 3.71; N, 18.74.5-Chloro-1-(4-chlorophenyl)-N-(4-cyano-1-(4-fluorophenyl)-1H-pyrazol-5-yl)-3-methyl-1H-pyrazole-4-carboxamide (3h). White crystals, yield 69%, mp: 177–178 °C. 1H-NMR  δ: 8.12 , 7.53–7.20 , 2.66 . MS (ESI): 455.1 . Anal. Calcd for C21H13Cl2FN6O: C, 55.40; H, 2.88; N, 18.46; Found: C, 55.51; H, 2.93; N, 18.48.5-Chloro-N-(4-cyano-1-phenyl-1H-pyrazol-5-yl)-3-methyl-1-(p-tolyl)-1H-pyrazole-4-carboxamide (3i). White crystals, yield 64%, mp: 124–126 °C. 1H-NMR  δ: 8.12 , 7.62–7.41 , 2.65 , 2.42 . MS (ESI): 417.1 . Anal. Calcd for C22H17ClN6O: C, 63.39; H, 4.11; N, 20.16; Found: C, 63.44; H, 4.14; N, 20.18.5-Chloro-N-(1-(4-chlorophenyl)-4-cyano-1H-pyrazol-5-yl)-3-methyl-1-(p-tolyl)-1H-pyrazole-4-carboxamide (3j). White crystals, yield 77%, mp: 155–157 °C. 1H-NMR  δ: 8.11 , 7.61–7.41 , 2.65 , 2.46 . MS (ESI): 451.1 . Anal. Calcd for C22H16Cl2N6O: C, 58.55; H, 3.57; N, 18.62; Found: C, 58.58; H, 3.61; N, 18.66.5-Chloro-N-(4-cyano-1-(p-tolyl)-1H-pyrazol-5-yl)-3-methyl-1-(p-tolyl)-1H-pyrazole-4-carboxamide (3k). White crystals, yield 63%, mp: 133–135 °C. 1H-NMR  δ: 8.12 , 7.61–7.43 , 2.65 , 2.42 . MS (ESI): 431.1 . Anal. Calcd for C23H19ClN6O: C, 64.11; H, 4.44; N, 19.50; Found: C, 64.17; H, 4.47; N, 19.56.5-Chloro-N-(4-cyano-1-(4-fluorophenyl)-1H-pyrazol-5-yl)-3-methyl-1-(p-tolyl)-1H-pyrazole-4-carbox-amide (3l). White crystals, yield 62%, mp: 156–158 °C. 1H-NMR  δ: 8.12 , 7.61–7.22 , 2.65 , 2.42 . MS (ESI): 435.1 . Anal. Calcd for C22H16ClFN6O: C, 60.76; H, 3.71; N, 19.33; Found: C, 60.77; H, 3.74; N, 19.46.5-Chloro-N-(4-cyano-1-phenyl-1H-pyrazol-5-yl)-1-(4-fluorophenyl)-3-methyl-1H-pyrazole-4-carbox-amide (3m). White crystals, yield 69%, mp: 118–120 °C. 1H-NMR  δ: 8.12 , 7.61–7.23 , 2.65 . MS (ESI): 421.1 . Anal. Calcd for C22H16ClN6O: C, 59.94; H, 3.35; N, 19.97; Found: C, 60.07; H, 3.43; N, 20.04.5-Chloro-N-(1-(4-chlorophenyl)-4-cyano-1H-pyrazol-5-yl)-1-(4-fluorophenyl)-3-methyl-1H-pyrazole-4-carboxamide (3n). White crystals, yield 81%, mp: 149–151 °C. 1H-NMR  δ: 8.12 , 7.62–7.22 , 2.65 . 1H-NMR  δ: 12.88 , 7.97 , 7.62–7.21 , 2.48 . 13C-NMR  δ 163.47, 163.14, 151.88, 151.73, 142.43, 136.76, 133.97, 132.68, 131.25, 128.64, 128.58, 116.71, 116.56, 115.11, 110.46, 73.96, 14.86. MS (ESI): 455.1 . Anal. Calcd for C21H13Cl2FN6O: C, 55.40; H, 2.88; N, 18.46; Found: C, 55.53; H, 2.91; N, 18.49.5-Chloro-N-(4-cyano-1-(p-tolyl)-1H-pyrazol-5-yl)-1-(4-fluorophenyl)-3-methyl-1H-pyrazole-4-carbox-amide (3o). White crystals, yield 65%, mp: 127–129 °C. 1H-NMR  δ: 8.12 , 7.61–7.22 , 2.65 , 2.42 . MS (ESI): 435.1 . Anal. Calcd for C22H16ClFN6O: C, 60.76; H, 3.71; N, 19.33; Found: C, 60.80; H, 3.77; N, 19.49.5-Chloro-N-(4-cyano-1-(4-fluorophenyl)-1H-pyrazol-5-yl)-1-(4-fluorophenyl)-3-methyl-1H-pyrazole-4-carboxamide (3p). White crystals, yield 62%, mp: 182–183 °C. 1H-NMR  δ: 8.12 , 7.61–7.21 , 2.65 . MS (ESI): 439.1 . Anal. Calcd for C21H13ClF2N6O: C, 57.48; H, 2.99; N, 19.15; Found: C, 57.53; H, 3.03; N, 19.17.et al. [in vitro and to test the protective effect, the inactivation effect and the curative effect in vivo against TMV.The anti-TMV activity of the synthesized pure compounds were tested by using the method reported by Thorson et al. . This meNicotianatabacum var. XanthiNC) were selected for the test. The tobacco was inoculated by the juice-leaf rubbing method, and the concentration of TMV was 5.88 × 10−2 μg/mL. The leaves was cut into halves along the main vein, and a solution of the compounds with a concentration of 500 μg/mL was smeared on the halves, and then cultured at 25 °C for 72 h. Each compound was tested three times.Fresh 5–6 growth stage of tobacco leaves (Nicotianatabacum var. XanthiNC). After 12 h, TMV at a concentration of 6.0 μg/mL was inoculated on the leaves which were previously scattered with silicon carbide through the above juice-leaf rubbing method. Then the leaves were again immersed into water and rubbed softly along the nervation once or twice. The local lesion numbers showing 3–4 days after inoculation were counted. Each compound was tested in three replicates.The test compound solution was smeared on the left side, and the solvent served as control on the right side of growing tobacco (Nicotianatabacum var. XanthiNC), and a mixture of solvent and the virus was inoculated on the right side to serve as control. The local lesion numbers showing 3–4 days after inoculation were counted. Each compound was tested in three replicates.The virus was inhibited by mixing with the target compound solution at the same volume for 30 min. Then the mixture was inoculated on the left side of the host tobacco leaves (Nicotianatabacum var. XanthiNC) of the same age growing at the six-leaf stage were selected for the test. TMV at a concentration of 6.0 μg/mL was inoculated on the whole leaves. The leaves were washed with water, and dried in a greenhouse. The target compound solution was smeared on the left side, and the solvent served as control on the right side. The local lesion numbers showing 3–4 days after inoculation were counted. Each compound was tested in three replicates. The inhibition rates of the compound in vitro and in vivo were calculated according to the following formula (controls were not treated with compound):Host plant tobacco leaves  in the A-ring showed more potent activities than those with electron-donating substituents . In comparison the activity of compound with para substituents on the phenyl ring showed that an electron-withdrawing group gave slightly improved antiviral activity and the potency order is F > Cl. Among all the compounds, 3p with a para-F group showed more activity, while 3k showed the lowest activity.Structure-activity relationships in these pyrazole amide derivatives were evaluated using the DS approach to proceed with structure optimization, The data demonstrated that compounds with 3p, an H-bond (2.49 Å and 109.02°) was established between its fluorine atom and Gin47, and it also formed a π-cation interaction (4.65 Å). The result of molecular docking suggests that introduction of a fluorine atom facilitates the adoption of a complementary shape of the designed ligands with TMV PC, and this possibly improves the anti-TMV inhibitory potency.For the binding model of compound


Alstonia yunnanensis afforded five new monoterpenoid indole alkaloids: 11-hydroxy-6,7-epoxy-8-oxo-vincadifformine (1), 14-chloro-15-hydroxy-vincadifformine (2), perakine N4-oxide (3), raucaffrinoline N4-oxide (4), and vinorine N1,N4-dioxide (5), together with three known compounds: 11-methoxy-6,7-epoxy-8-oxo-vincadifformine (6), vinorine N4-oxide (7) and vinorine (8). The structures of the isolated compounds were established based on 1D and 2D  NMR spectroscopy, in addition to high resolution mass spectrometry. The isolated compounds were tested in vitro for cytotoxic potential against seven tumor cell lines and anti-inflammatory activities. Compounds 3, 4 and 7 exhibited weak cytotoxicity against the tested cell lines and selective inhibition of Cox-2 (>85%).The 80% ethanol extract of  Alstonia (Apocynaceae), which are usually shrubs or trees, are widely distributed throughout the tropical areas of the World, including Central America, Africa, Indo-Malaya, Australia and Asia  , three methylene carbons [two oxygenated (δC 51.2 and 57.1)], four quarternary carbons , and a NH signal at δH 8.96. The 14,15-epoxy group was established by the HMBC correlations of δH 3.48 (H-15) with δC 165.0 (C-3), 26.2 (C-19) and 63.4 (C-21) and of δH 3.62 (H-14) with δC 40.7 (C-20) , the α-orientation of the 14,15-epoxy group was determined by the correlation between H-15 and H-17β, which was possible only if the orientation of H-15 was β  . Besides7 (C-20) . These dl with 6 . The dis15 was β ,33. Thus2 gave two isotopic peaks at m/z 388.1634 [M−1+H]+ and 390.1634 [M+1+H]+ in its HR-ESI-MS, accounting for a molecular formula of C21H25ClN2O3, suggesting 10 degrees of unsaturation. The existence of the chlorine atom was identified by the appropriate 13C-NMR chemical shift at δC 59.6  and EIMS analysis. The EI mass spectrometry showed one molecular and one quasi-molecular-ion peaks at m/z 387  and 389  with ratio of relative intensity approximating 3:1. The 1H-NMR spectrum exhibited four A2B2-system protons at δH 7.09 , 6.80 , 7.06 , and 7.61 , indicating the OH group at C-11 in 1 was absent in 2. The NMR data were similar to those of compound 1, except for a hydroxy group at C-15 (δC 76.0) and a chlorine atom at C-14 (δC 59.6) in 2 taking place the 14,15-epoxy group in 1 and 6, as evidenced by the HMBC correlations and the 1H-1H-COSY correlations of proton signals at δH 4.20 (H-14) with δH 3.29 (H-3) and δH 3.92 (H-15). The α-orientation of H-14 and the β-orientation of H-15 were deduced from the NOESY correlations of H-14/H-21 and H-15/H-17β, respectively. Accordingly, compound 2 was identified as 14-chloro-15-hydroxyvincadifformine.Compound 3, a colorless oil, exhibited a molecular formula of C21H22N2O4, based on the HRESIMS spectrum which showed a pseudomolecular ion at m/z 389.1475 [M+Na]+ . The UV absorptions at 220 and 264 nm showed the presence of an indolenine chromophore, which was confirmed by the characteristic chemical shift of C-2 at δC 179.7 in the 13C-NMR spectrum. The IR spectrum indicated the presence of aldehyde group (1725 cm−1), and benzene ring (1650 cm−1). The NMR data of 3 was almost identical with those of alstoyunine C . The stereochemistry of 4 was expected to be the same as 3 on the basis of the NOESY data. Thus, compound 4 was elucidated as raucaffrinoline N4-oxide.Compound 5 was assigned as C21H22N2O4 on the basis of the quasimolecular ion peak [M+H]+ at m/z 367.1653 in the HR-ESI-MS. The 1D and 2D-NMR data were closely related to those of 7. The only difference was compound 5 contains one more oxygen atom compared to 7. The upfield chemical shift of C-2 from 178.1 in 7 to δC 147.4 in 5, combined with the HMBC correlations of δH 5.15 , 2.10 , and 2.43  with δC 147.4 (C-2), indicated that 5 was an N1-oxide derivative of 7. The E configuration of the double bond between C-19 and C-20 was determined by the NOESY correlations of H-18 at δH 1.70  with H-15 at δH 3.37  and of H-19 at δH 5.45  with H-21 at δH 4.03 . The NOESY analysis showed that the relative configurations of the C-3, C-5, C-15, C-16, and C-17 were in good agreement with those of compounds 3 and 4. Therefore, compound 5 was identified as vinorine N1,N4-dioxide.The molecular formula of the compound in vitro evaluated for their cytotoxic potential using BEN-MEN-1 (meningioma), CCF-STTG1 (astrocytoma), CHG-5 (glioma), SHG-44 (glioma), U251 (glioma), SK-MEL-2 (human skin cancer), and MCF-7 cells (human breast cancer). Alkaloids 3, 4 and 7 exhibited cytotoxicity against all the tested tumor cell lines except BEN-MEN-1, while other compounds were noncytotoxic (IC50 > 50 μM). Furthermore, The IC50 value  with lower IC50 value than against human skin cancer (SK-MEL-2) and human breast cancer (MCF-7 cells).All these compounds were 50 value  indicate1–8 were tested in vitro for their anti-inflammatory activities. The results of the anti-inflammatory assay were summarized in 3, 4 and 7 displayed selective inhibition of Cox-2 (>85%).The compounds 5 and 8 had no cytotoxic activities or selective inhibition of Cox-2 comparable to those of 3, 4 and 7 although they possess the same monoterpene indole skeleton. The observations indicated that a N4-oxide functionality was essential for cytotoxic and anti-inflammatory properties, while a N1-oxide maybe weaken the cytotoxic and anti-inflammatory activities for this type of alkaloids.Alkaloids Optical rotations were measured on a Perkin-Elmer 341 polarimeter . UV spectra were obtained on a Shimadzu UV-2550 spectrophotometer, whereas IR spectra were recorded on a Perkin-Elmer 577 spectrometer with KBr disks. NMR spectra were measured on a Bruker AM-600 spectrometer. EIMS and HREIMS (70 eV) were carried out on a Finnigan MAT 95 mass spectrometer. All solvents used were of analytical grade . Silica gel (200–300 mesh), silica gel H , C18 reversed-phase silica gel , and MCI gel  were used for column chromatography. HPLC separation was performed on an instrument consisting of a Waters 600 controller, a Waters 600 pump, and a Waters 2487 dual λ absorbance detector, with a Prevail (250 × 10 mm i.d.) preparative column packed with C18 (5 μm). Precoated thin-layer chromatography (TLC) plates with silica gel GF254  were used for TLC.A. yunnanensis were collected in Chuxiong, Yunnan Province, China, in September 2010. The sample was identified by one of the authors (G. B. Shi). A specimen (JGCS201009001) was deposited in the Herbarium of Shengyang Medicine College, Shengyang, China.The whole plants of A. yunnanensis (15 kg) were extracted with 80% ethanol (40 L) three times under reflux for 15 h and then concentrated under reduced pressure to give a crude extract. The crude extract dissolved in 7% HCl (2 L) and filtered. The filtrate was made alkaline using 10% ammonia-water to pH 9–10. The basic solution was partitioned with EtOAc to give a total alkaloidal extract (42 g). The total alkaloidal extract was further fractionated on a silica gel column, eluted with CHCl3-Me2CO (from 1:0 to 0:1), to obtain eight fractions (1–10). Fraction 3  was applied to an ODS MPLC column and eluted with MeOH-H2O  to yield four subfractions (Fr. 3-1 and 3-4). Fr. 3-2  was purified by preparative RP-HPLC  using MeOH/H2O (25:75) as mobile phase to obtain 6 (67 mg). Fr. 3-3  was chromatographed on a Sephadex LH-20 column eluted with MeOH/H2O (50:50), and purifed by preparative RP-HPLC  using MeOH/H2O (34:66) as mobile phase to yield 1 (68 mg) and 2 (57 mg). Separation of fraction 5 (4.2 g) by silica gel column chromatography, eluted with petroleum ether-Me2CO (from 8:1 to 1:1), afforded six subfractions (Fr. 5-1 and Fr. 5-6). Fr. 5-2 (278 mg) was subjected to RP-18  and Sephadex LH-20 (MeOH) column chromatography to yield 7 (43 mg). Fr. 5-3  was repeatedly chromatographed on silica gel  and then purifed on a Sephadex LH-20 column eluted with MeOH/H2O (50:50) to afford 3 (78 mg). Fraction 8 (2.7 g) was subjected to Sephadex LH-20  column chromatography to afford four subfractions (Fr. 8-1 and 8-4). Fr. 8-3 (120 mg) was purified by preparative RP-HPLC  using MeOH/H2O  to afford 4 (44 mg) and 5 (52 mg), respectively.The dried whole plants of 11-Hydroxy-6,7-epoxy-8-oxo-vincadifformine (1). colorless oil. = −129.6 . UV (CDCl3) λmax(log ε) 325 (3.81), 245 (3.85), 228 (3.83), 197 (3.79) nm. IR (KBr) νmax 3375, 1715, 1660, 1620, 1437, 1105, 755 cm−1. 1H-NMR  and 13C-NMR  data see m/z: 382 ([M]+). HR-ESI-MS (pos.) m/z: 405.1423 .14-Chloro-15-hydroxy-vincadifformine (2). colorless oil. = −341.9 . UV (CDCl3) λmax(logε) 326 (3.85), 244 (3.76), 228 (3.50), 223 (3.82), 198 (3.44) nm. IR (KBr) νmax 3425, 1675, 1618, 1265, 1115, 760 cm−1. 1H-NMR  and 13C-NMR  data see m/z: 387 ([M−1]+), 389 ([M+1]+). HR-ESI-MS (pos.) m/z: calc. 388.1634 [M−1+H]+, 390.1634 [M+1+H]+, C21H26ClN2O3. calc. 388.1632, 390.1634).4-oxidePerakine N (3). colorless oil. = −99.8 . UV (CDCl3) λmax(logε) 264 (3.80), 220 (3.83) nm. IR (KBr) νmax 3435, 2950, 1725, 1650, 1235, 1040, 765 cm−1. 1H-NMR  and 13C-NMR  data see m/z: 366 ([M]+). HR-ESI-MS (pos.) m/z: 389.1475 .4-oxideRaucaffrinoline N (4). colorless oil. = −30.2 . UV (CDCl3) λmax(logε) 258 (3.85), 220 (3.82) nm. IR (KBr) νmax 3425, 2935, 1740, 1590, 1233, 1033, 750 cm−1. 1H-NMR  and 13C-NMR  data see M]+). HR-ESI-MS (pos.) m/z: 391.1635 .1,N4-dioxideVinorine N (5). colorless oil. = −62.5 . UV (CDCl3) λmax(logε) 265 (3.85), 220 (3.83), 195 (3.83) nm. IR (KBr) νmax 3423, 2964, 1745, 1650, 1230, 1035, 753 cm−1. 1H-NMR  and 13C-NMR  data see m/z: 366 ([M]+). HR-ESI-MS (pos.) m/z: 367.1653 .1–8 were subjected to cytotoxic evaluation against BEN-MEN-1 (meningioma), CCF-STTG1 (astrocytoma), CHG-5 (glioma), SHG-44 (glioma), U251 (glioma), SK-MEL-2 (human skin cancer), and MCF-7 cells (human breast cancer) employing the revised MTT method [−1 penicillin and 100 μg/mL streptomycin in 25-cm2 culture flasks at 37 °C in humidified atmosphere with 5% CO2. For the cytotoxicity tests, cells in exponential growth stage were harvested from culture by trypsin digestion and centrifuging at 180 × g for 3 min, then resuspended in fresh medium at a cell density of 5 × 104 cells per ml. The cell suspension was dispensed into a 96-well microplate at 100 μL per well, and incubated in humidified atmosphere with 5% CO2 at 37 °C for 24 h, and then treated with the compounds at various concentrations . After 48 h of treatment, 50 μL of 1 mg/mL MTT solution was added to each well, and further incubated for 4 h. The cells in each well were then solubilized with DMSO (100 μL for each well) and the optical density (OD) was recorded at 570 nm. All drug doses were tested in triplicate and the IC50 values were derived from the mean OD values of the triplicate tests versus drug concentration curves. All cell lines were purchased from the Cell Bank of the Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences .The isolated compounds T method . AdriamyThe anti-inflammatory activities were determined according to a literature method with minor modifications . The reaA. yunnanensis led to the isolation of five new monoterpenoid indole alkaloids: 11-hydroxy-6,7-epoxy-8-oxovincadifformine (1), 14-chloro-15-hydroxyvincadifformine (2), perakine N4-oxide (3), raucaffrinoline N4-oxide (4), and vinorine N1,N4-dioxide (5), together with three known compounds: 11-methoxy-6,7-epoxy-8-oxo-vincadifformine (6), vinorine N4-oxide (7) and vinorine (8). All the alkaloids were evaluated in vitro for cytotoxic activities against seven tumor cell lines and anti-inflammatory properties. Alkaloids 3, 4 and 7 showed particular cytotoxic activities against astrocytoma and glioma cell lines  with low IC50 value (<20 μM) and selective inhibition of Cox-2 (>85%).In summary, a chemical investigation of the 80% EtOH extract of the dried whole plant of


The data displayed that all the tested compounds (at 20 μM) significantly decreased the intracellular accumulation of a P-gp fluorescent substrate (rhodamine 123) when incubated simultaneously for 60 min, demonstrating an increased activity of the efflux, when compared to control cells. Additionally, all of them except ATx 3 (+), caused similar results when the accumulation of the P-gp fluorescent substrate was evaluated after pre-incubating cells with the test compounds for 24 h, significantly reducing the rhodamine 123 intracellular accumulation as a result of a significant increase in P-gp activity. However, ATx 2 (−) was the only derivative that, after 24 h of incubation, significantly increased P-gp expression. These results demonstrated a significantly increased P-gp activity, even without an increase in P-gp expression. Therefore, ATxs 1–8 were shown to behave as P-gp activators. Furthermore, no significant differences were detected in the activity of the protein when comparing the enantiomeric pairs. Nevertheless, ATx 2 (−) modulates P-gp expression differently from its enantiomer, ATx 1 (+). These results disclosed new activators and inducers of P-gp and highlight the existence of enantioselectivity in the induction mechanism.Recently, thioxanthone derivatives were found to protect cells against toxic P-glycoprotein (P-gp) substrates, acting as potent inducers/activators of this efflux pump. The study of new P-gp chiral modulators produced from thioxanthone derivatives could clarify the enantioselectivity of this ABC transporter towards this new class of modulators. The aim of this study was to evaluate the P-gp modulatory ability of four enantiomeric pairs of new synthesized chiral aminated thioxanthones (ATxs)  MDR1 (ABCB1) and MDR3 (ABCB4), being the multidrug resistance (MDR) phenotype associated with the ABCB1 isoform. Also, the term P-gp refers, usually, to the ABCB1 isoform  − [S]/([R] + [S]) or 100 × ([S] − [R]/([S] + [R]), where [S] and [R] are the peak areas of each enantiomer.HPLC analysis were performed on a Finnigan Surveyor  equipped with an autosampler (AutoSampler Plus) and a TSP UV8000LP diode array detector (Thermo Electron Corporation). The treatment of the chromatographic data was performed using the XcaliburH-thioxanthen-9-one  and a suitable chiral amino alcohol  were dissolved in methanol (30 mL) and CuI (0.15 mmol) and K2CO3 (1.92 mmol) were added. The reaction mixture was heated at 100 °C in a muffle furnace for 48 h. After the completion of the reaction, the crude material was filtrated, washed with dichloromethane, and the organic solvents were evaporated under reduced pressure. Then, the obtained solid was dissolved in 50 mL of dichloromethane and extracted with HCl 1 M (3 × 50 mL). The aqueous layer was basified with NaOH 20% and extracted with dichloromethane (3 × 100 mL). The organic layers were gathered, washed with water (3 × 50 mL), dried over anhydrous sodium sulphate and the solvent was evaporated under reduced pressure. Then, a solid phase extraction using a cation exchange cartridge Discovery® DSC-SCX was applied to further purify the extracted material. First, an activation of the cartridge with dichloromethane (50 mL) was performed followed by loading the cartridge with the sample (previously incorporated in silica). Then, elution was carried out with the following solvents: dichloromethane, a mixture of dichloromethane/methanol 5:5 (v/v), methanol 100% and NH3 2% in methanol. The fractions containing the chiral ATxs were gathered and the solvent was evaporated under reduced pressure. A flash column chromatography with n-hexane/ethyl acetate in gradient and crystallization from chloroform and petroleum ether (4:1) were also performed to obtain pure compounds.1-Chloro-4-propoxy-9(S)-1-((1-Hydroxypropan-2-yl)amino)-4-propoxy-9H-thioxanthen-9-one (ATx 1 (+)). Yield: 1.97%. m.p.: 116–118 °C (dec.); −3 g/mL in dichloromethane). IR (KBr) vmax: 3419, 3270, 2962, 2925, 2873, 2359, 2341, 1618, 1568, 1507, 1435, 1293, 1269, 1252, 1225, 746 cm−1. 1H-NMR : δ: 8.50 , δ: 7.56 , δ: 7.43 , δ: 7.13 , δ: 6.85 , δ: 4.03 , δ: 3.81 , δ: 1.90 , δ: 1.32 , δ: 1.12 . 13C-NMR : δ: 183.55 (C-9), 146.50 (C-4), 144.02 (C-1), 136.85 (C-6), 131.92 (C-8a), 129.85 (C-4a), 129.24 (C-9a), 126.08 (C-8), 125.98 (C-7), 125.20 (C-5), 119.12 (C-3), 114.10 (C-10a), 109.80 (C-2), 72.48 (C-a), 65.96 (C-CH2OH), 51.91 (C-1′), 22.66 (C-b), 16.86 (C-2′), 10.71 (C-c). e.e. > 99% , Mobile phase: n-hexane:ethanol (70:30 v/v), 0.5 mL/min, λmax 254 nm).(R)-1-((1-Hydroxypropan-2-yl)amino)-4-propoxy-9H-thioxanthen-9-one (ATx 2 (−)). Yield: 5.91%. m.p.: 118–120 °C (dec.); −3 g/mL in dichloromethane). IR (KBr) vmax: 3447, 3282, 2963, 2927, 2874, 1611, 1595, 1569, 1506, 1436, 1290, 1270, 1252, 1226, 1072, 795, 746 cm−1. 1H-NMR : δ: 8.50 , δ: 7.56 , δ: 7.42 , δ: 7.13 , δ: 6.82 , δ: 4.04 , δ: 3.78 , δ: 1.90 , δ: 1.33 , δ: 1.11 . 13C-NMR: δ: 183.56 (C-9), 146.80 (C-4), 144.00 (C-1), 136.82 (C-6), 131.87 (C-8a), 129.91 (C-4a), 129.22 (C-9a), 126.05 (C-8), 125.97 (C-7), 125.20 (C-5), 119.35 (C-3), 114.00 (C-10a), 109.02 (C-2), 72.52 (C-a), 66.04 (C-CH2OH), 51.55 (C-1′), 22.70 (C-b), 17.00 (C-2′), 10.71 (C-c). e.e. > 99% , Mobile phase: n-hexane:ethanol (70:30 v/v), 0.5 mL/min, λmax 254 nm).(S)-1-((2-Hydroxypropyl)amino)-4-propoxy-9H-thioxanthen-9-one (ATx 3 (+)). Yield: 7.86%. m.p.: 149–152 °C (dec.); −3 g/mL in dichloromethane). IR (KBr) vmax: 3460, 3320, 2965, 2933, 2875, 1607, 1566, 1556, 1502, 1450, 1434, 1286, 1269, 1249, 1227, 1158, 1133, 1060, 744 cm−1. 1H-NMR: δ: 8.52 , δ: 7.56 , δ: 7.42 , δ: 7.13 , δ: 6.78 , δ: 4.26 , δ: 4.03 , δ: 3.30 , δ: 1.90 , TM: 1.35 , δ: 1.12 . 13C-NMR : δ: 183.46 (C-9), 146.18 (C-4), 144.01 (C-1), 136.98 (C-6), 133.27 (C-8a), 129.54 (C-4a), 129.43 (C-9a), 127.19 (C-8), 126.32 (C-7), 126.10 (C-5), 117.94 (C-3), 115.36 (C-10a), 111.83 (C-2), 72.31 (C-a), 65.58 (C-2′), 54.12 (C-1′), 22.74 (C-b), 20.97 (C-CH3), 14.15 (C-c). e.e. > 99% , Mobile phase: methanol, 0.5 mL/min, λmax 254 nm).(R)-1-((2-Hydroxypropyl)amino)-4-propoxy-9H-thioxanthen-9-one (ATx 4 (−)). Yield: 23.62%. m.p.: 145–150 °C (dec.); −3 g/mL in dichloromethane). IR (KBr) vmax: 3460, 3323, 2966, 2932, 2875, 1606, 1566, 1556, 1501, 1451, 1434, 1328, 1286, 1270, 1249, 1227, 1158, 1133, 1102, 1060, 972, 744 cm−1. 1H-NMR : δ: 8.51 , δ: 7.50 , δ: 7.41 , δ: 7.12 , δ: 6.73 , δ: 4.22 , δ: 4.03 , δ: 3.29 , δ: 1.90 , δ: 1.35 , δ: 1.12 . 13C-NMR : δ: 183.46 (C-9), 147.48 (C-4), 143.91 (C-1), 136.81 (C-6), 131.83 (C-8a), 129.92 (C-4a), 129.85 (C-9a), 129.26 (C-8), 126.02 (C-7), 125.95 (C-5), 119.25 (C-3), 113.95 (C-10a), 108.42 (C-2), 72.47 (C-a), 66.11 (C-2′), 52.07 (C-1′), 22.85 (C-b), 21.02 (C-CH3), 10.71 (C-c). e.e. > 99% , Mobile phase: methanol, 0.5 mL/min, λmax 254 nm).(S)-1-((1-Hydroxy-4-methylpentan-2-yl)amino)-4-propoxy-9H-thioxanthen-9-one (ATx 5 (+)). Yield: 19.59%. m.p.: 82–85 °C (dec.); −3 g/mL in dichloromethane). IR (KBr) vmax: 3457, 3290, 2955, 2923, 2870, 1613, 1595, 1566, 1505, 1464, 1435, 1386, 1265, 1224, 1107, 1067, 1018, 745 cm−1. 1H-NMR : δ: 8.51 , δ: 7.57 , δ: 7.42 , δ: 7.13 , δ: 6.93 , δ: 4.04 , δ: 3.88 , δ: 3.71 , δ: 1.85 , δ:1.78 , δ: 1.57 , δ: 1.12 , δ: 0.94 , δ: 0.89 . 13C-NMR : δ: 183.61 (C-9), 145.00 (C-4), 142.80 (C-1), 136.7 (C-6), 131.98 (C-8a), 129.97 (C-4a), 129.83 (C-9a), 129.28 (C-8), 126.11 (C-7), 126.00 (C-5), 119.05 (C-3), 114.43 (C-10a), 111.02 (C-2), 72.45 (C-a), 64.62 (C-1′), 55.01 (C-CH2OH), 40.44 (C-2′), 24.90 (C-3′), 22.86 (C-4′a), 22.83 (C-4′b), 22.61 (C-b), 10.71 (C-c). e.e. > 99% , Mobile phase: acetonitrile, 0.5 mL/min, λmax 254 nm).(R)-1-((1-Hydroxy-4-methylpentan-2-yl)amino)-4-propoxy-9H-thioxanthen-9-one (ATx 6 (−)). Yield: 10.40%. m.p.: 80–82 °C (dec.); −3 g/mL in dichloromethane). IR (KBr) vmax: 3459, 3291, 2954, 2925, 2871, 1613, 1595, 1566, 1505, 1480, 1435, 1386, 1266, 1224, 1108, 1072, 1018, 746 cm−1. 1H-NMR : δ: 8.51 , δ: 7.55 , δ: 7.42 , δ: 7.12 , δ: 6.86 , δ: 4.03 , δ: 3.85 , δ: 3.72 , δ: 1.83 , δ: 1.76 , δ: 1.57 , δ: 1.12 , δ: 0.96 , δ: 0.89 . 13C-NMR : δ: 183.61 (C-9), 145.00 (C-4), 142.80 (C-1), 136.82 (C-6), 131.87 (C-8a), 129.93 (C-4a), 129.24 (C-9a), 128.80 (C-8), 126.03 (C-7), 125.97 (C-5), 119.42 (C-3), 113.94 (C-10a), 108.80 (C-2), 72.49 (C-a), 64.87 (C-1′), 40.71 (C-2′), 24.92 (C-3′), 22.96 (C-4′a), 22.86 (C-4′b), 22.55 (C-b), 10.71 (C-c). e.e. > 99% , Mobile phase: acetonitrile, 0.5 mL/min, λmax 254 nm).(S)-1-((1-Hydroxy-3-methylbutan-2-yl)amino)-4-propoxy-9H-thioxanthen-9-one (ATx 7 (+)). Yield: 10.12%. m.p.: 98–102 °C (dec.); −3 g/mL in dichloromethane). IR (KBr) vmax: 3385, 3256, 2960, 2926, 2873, 2360, 1614, 1569, 1514, 1463, 1434, 1386, 1329, 1267, 1249, 1225, 1165, 1070, 1021, 744 cm−1. 1H-NMR : δ: 8.54 , δ: 7.59 , δ: 7.45 , δ: 7.14 , δ: 7.07 , δ: 4.06 , δ: 3.86 ,δ: 3.53 , δ: 2.08 , δ: 1.91 , δ: 1.05 , δ: 0.87 , δ: 1.12 . 13C-NMR : δ: 183.43 (C-9), 145.0 (C-4), 142.8 (C-1), 136.43 (C-6), 131.66 (C-8a), 129.53 (C-4a), 129.03 (C-9a), 128.84 (C-8), 125.70 (C-7), 125.51 (C-5), 117.82 (C-3), 114.71 (C-10a), 111.6 (C-2), 71.80 (C-a), 63.93 (C-1′), 61.56 (C-CH2OH), 29.15 (C-2′), 22.21 (C-b), 18.53 (C-3′a), 18.41 (C-3′b), 10.14 (C-c). e.e. > 99% , Mobile phase: acetonitrile, 0.5 mL/min, λmax 254 nm).(R)-1-((1-Hydroxy-3-methylbutan-2-yl)amino)-4-propoxy-9H-thioxanthen-9-one (ATx 8 (−)). Yield: 10.24%. m.p.: 98–104 °C (dec.); −3 g/mL in dichloromethane). IR (KBr) vmax: 3423, 3272, 2959, 2925, 2872, 1611, 1570, 1511, 1463, 1435, 1385, 1251, 1226, 1161, 1068, 1022, 747 cm−1.1H-NMR: δ: 8.56 , δ: 7.64 , δ: 7.49 , δ: 7.34 , δ: 7.17 , δ: 4.10 , δ: 3.90 , δ: 3.50 , δ: 2.10 , δ: 1.91 , δ: 1.02 , δ: 0.98 , δ: 1.14 ; 13C-NMR : δ: 183.65 (C-9), 147.74 (C-4), 141.53 (C-1), 137.16 (C-6), 132.60 (C-8a), 130.43 (C-4a), 129.52 (C-9a), 129.21 (C-8), 126.52 (C-7), 126.19 (C-5), 117.12 (C-3), 116.69 (C-10a), 115.38 (C-2), 72.19 (C-a), 67.02 (C-1′), 61.17 (CH2OH), 28.87 (C-2′), 22.65 (C-b), 19.31 (C-3′a), 18.58 (C-3′b), 10.69 (C-c). e.e. > 99% , Mobile phase: acetonitrile, 0.5 mL/min, λmax 254 nm).2 flasks and maintained in a 5% CO2–95% air atmosphere, at 37 °C. Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 μM nonessential amino acids (NEAA), 100 U/mL penicillin, 100 µg/mL streptomycin and 6 μg/mL transferrin was used and changed every 2 to 3 days. Cultures were passaged weekly by trypsinization (0.25% trypsin/1 mM EDTA). For all the experiments, the cells were taken between the 27th and 36th passages. The cells were always seeded at a density of 60,000 cells/cm2 and used 3 days after seeding, when confluence was reached. For the cytotoxicity assays the cells were seeded in 96-well plates, while for the evaluation of P-gp expression and P-gp transport activity, the cells were seeded in 12-well plates.For the in vitro assays, the Caco-2 cell model was used, which derived from human colorectal adenocarcinoma. The cells were routinely cultured in 75 cm1–8 (0–50 μM) was accessed by the NR uptake assay, 24 h after exposure. The NR uptake assay provides a quantitative estimation of the number of viable cells in a culture based on the ability of viable cells to incorporate and bind the supravital dye NR in the lysosomes . In order to detect a possible contribution from cells autofluorescence to the analyzed fluorescence signals, non-labelled cells  were also analyzed in each experiment by a 585 ± 40 nm band-pass filter (FL2). Five independent experiments were performed in duplicate.P-gp expression was evaluated by flow cytometry using a P-gp monoclonal antibody [UIC2] conjugated with phycoerythrin (PE) . For tha1–8 (20 μM) only during the RHO 123 accumulation phase; a RHO 123 accumulation assay, in the presence or absence of elacridar (10 μM), with or without pre-exposure of Caco-2 cells to ATxs 1–8 (20 μM) for 24 h.The effect of the tested compounds on P-gp transport activity was accessed by flow cytometry using RHO 123 (2 µM) as a P-gp fluorescent substrate. Two different approaches were tested: a RHO 123 accumulation assay in the presence (IA) and absence (NA) of elacridar (10 μM), a known P-gp inhibitor, and with or without simultaneous exposure to ATxs 2 flasks and, after reaching confluence, harvested by trypsinization to obtain a cellular suspension, which was then divided into several aliquots of 300,000 cells. After centrifugation (300× g for 10 min), the cells were suspended in HBSS containing 10% heat-inactivated FBS and 2 μM RHO 123, with and without 20 μM ATxs 1–8, and incubated at 37 °C, for 60 min, in the presence and absence of elacridar (10 μM). The cells were then washed twice with ice-cold HBSS with 10% heat-inactivated FBS, centrifuged (300× g for 10 min at 4 °C) and kept on ice until flow cytometry analysis. The fluorescence measurements of isolated cells were performed as described in the section “Evaluation of P-gp expression”, being the RHO 123 fluorescence measured by a 530 ± 15 nm band-pass filter (FL1). The activity of the efflux pump was accessed by the RHO 123 accumulation ratio (Equation (1)) and expressed as percentage of control cells (0 μM ATXs):For this purpose, the cells were seeded onto 75 cmP-gp activity was assessed by ratio between the amount of RHO 123 accumulated under P-gp inhibition (with 10 μM elacridar) and the amount of RHO 123 accumulated in the absence of the P-gp inhibitor.An increase in P-gp activity results in an increased RHO 123 efflux from the cells, which results in a decrease in the intracellular fluorescence intensity (decreased intracellular RHO 123 content). Therefore, a higher RHO 123 accumulation ratio (Equation (1)) is a consequence of a smaller MFI NA, which results from a higher P-gp activity since the dye is being effluxed out of the cells during the accumulation phase. Five independent experiments were performed in triplicate.g for 10 min) and suspended in HBSS buffer (pH 7.4) containing 10% heat-inactivated FBS, 2 µM RHO 123 and the P-gp inhibitor elacridar (10.0 µM), while for the NA accumulation a similar incubation was performed but in the absence of the P-gp inhibitor. In both cases, the cells were incubated with the P-gp substrate at 37 °C for 60 min to allow RHO 123 accumulation. After this accumulation period, the cells were washed twice with ice-cold HBSS buffer containing 10% heat-inactivated FBS and suspended in ice-cold HBSS buffer immediately before analysis. The evaluation of RHO 123 intracellular content was performed as described in the “Flow cytometry analysis of P-gp expression” section, being the RHO 123 fluorescence measured by a 530 ± 15 nm band-pass filter (FL1). The results were calculated according to the ratio defined in Equation (1) and expressed as percentage of control cells. Four independent experiments were performed in duplicate.Caco-2 cells, seeded onto 12-well plates, were exposed to chiral thioxanthones (20 μM), in fresh cell culture medium, for 24 h, prior to the evaluation of P-gp activity. After exposure, the cells were harvested by trypsinization to obtain a cell suspension, being the cells of each well divided into two aliquots. One aliquot was submitted to a RHO 123 accumulation phase under inhibited conditions (RHO 123 accumulation performed in IA conditions) and the second aliquot was submitted to a RHO 123 accumulation phase performed under normal conditions (RHO 123 accumulation performed in NA conditions). For IA accumulation, the cells were centrifuged , according to the manufacturer’s instructions.The effects of the tested ATxs Briefly, recombinant human P-gp-enriched membranes (25 μg/well) were incubated with 20 μM chiral thioxanthones or sodium vanadate at 100 μM (positive control for P-gp ATPase activity inhibition) or verapamil at 200 μM (positive control for P-gp ATPase activity stimulation) in assay buffer, and 5 mM MgATP, for exactly 60 min, at 37 °C. After incubation, the reaction was stopped and the remaining ATP detected in a multi-well plate reader (BioTek Synergy™ HT), after a 20 min signal-developing period at room temperature, as a luciferase-generated luminescent signal.3VO4-treated incubations reflects the basal P-gp ATPase activity. The decreased luminescence of incubations performed with the positive control drug verapamil relative to Na3VO4-treated reactions reflects verapamil-stimulated P-gp ATPase activity. The luminescence of the chiral thioxanthones incubations relative to that of the Na3VO4-treated reactions reflects the effect, if any, of that compounds on P-gp ATPase activity . Three independent experiments were performed in triplicate for this assay and the results expressed as mean ± SEM.The decreased luminescence of the untreated (NT) incubations relative to Nap values lower than 0.05 were considered significant.All statistical calculations were performed with the GraphPad Prism version 6.00 for Windows . Normality of the data distribution was assessed by three different tests . Data obtained from the thioxanthones cytotoxicity assays are expressed as mean ± SEM from at least five independent experiments (performed in triplicate) and the statistical comparisons were estimated using the nonparametric method of Kruskal–Wallis, followed by the Dunn’s post hoc test. Data from the P-gp expression assays are presented as mean ± SEM from five independent experiments (performed in duplicate) and the statistical comparisons were made using One-way ANOVA, followed by the Tukey’s multiple comparisons test. Results from the P-gp transport activity assays performed in the presence of chiral thioxanthones during the RHO 123 accumulation phase are presented as mean ± SEM from five independent experiments (performed in triplicate) and the statistical comparisons were estimated using One-way ANOVA, followed by the Tukey’s multiple comparisons test. Results from the P-gp transport activity assays performed in cells pre-exposed to chiral thioxanthones for 24 h are presented as mean ± SEM from four independent experiments (performed in duplicate) and the statistical comparisons were estimated using One-way ANOVA, followed by the Tukey’s multiple comparisons test. Results from the P-gp ATPase activity is presented as mean ± SEM from three independent experiments (performed in triplicate) and the statistical comparisons were estimated using One-way ANOVA, followed by the Tukey’s multiple comparisons test. In all cases, 1–8 demonstrated that these compounds have the ability to immediately increase P-gp activity without interfering with its levels of expression and, therefore, can be characterized as P-gp activators. Chiral thioxanthones 2 (−) and 7 (+) demonstrated to be the most efficient P-gp activators, among all the thioxanthones tested.Thioxanthone derivatives and, particularly, aminated thioxanthones, have been characterized as P-gp modulators. Since the pharmacokinetics, efficacy and safety of drugs that are P-gp substrates depend on the level of expression and functionality of P-gp, and can be affected by the chirality of compounds, it is important to better understand the effect that this important property may have in P-gp modulation. Therefore, in order to accomplish the main aim of this study, four enantiomeric pairs of thioxanthones were synthesized through a copper catalyzed Ullman asymmetric coupling reaction that provided the desired chiral thioxanthones with an e.e. > 99%. The assays carried out for the evaluation of the biological activity of the tested chiral ATxs MDR1 gene expression involves the activation of the pregnane X receptor (PXR) [Comparing the results obtained with both enantiomers of each enantiomeric pair, no significant enantioselectivity was observed for the direct modulation of P-gp. Nevertheless, the introduction of a chiral center close to the thioxanthonic scaffold favored their activation effect. Differences were noted in what concerns the induction effect, which may be related to their mechanism of induction, which deserves to be further explored. However, it is well known that P-gp expression is regulated by multiple signaling pathways, each involving different molecular targets and transcription factors, making the elucidation of the induction mechanism underlying ATX2-mediated P-gp induction a complex task. One of the possible pathways involved in the transcriptional activation of the or (PXR) ,66, and or (PXR) ,70,71,72


Two BZS compounds showed strong antibacterial activity against methicillin-resistant Staphylococcus aureus and Bacillus subtilis. Quantitative studies of their structure-activity relationship using a simple linear regression analysis were applied to explore the correlation between the biological activity and the charges on acidic hydrogen atoms in the synthesized compounds. A series of benzimidazolylbenzenesulfonamide compounds containing electron-releasing and electron-withdrawing substituents were synthesized and tested for their  Streptococcus pneumoniae continues to increase worldwide, except in cities that apply good antibiotic use policies. An additional problem is the emergence of multi-drug resistant strains in America, Europe and Asia [Enterococcus. These infections have emerged as severe complications, mainly in critically ill patients in tertiary care hospital wards [Staphylococcus aureus is one of the most prominent and widespread human pathogens, causing skin and tissue infections, deep abscess formation, pneumonia, endocarditis, osteomyelitis, toxic shock syndrome and bacteremia. Methicillin-resistant Staphylococcus aureus (MRSA) has been recognized as one of the main pathogenic causes of nosocomial infections throughout the world [Drug resistance in Gram-positive bacteria is a worldwide public health problem with serious morbidity and mortality consequences. For example, the incidence of penicillin-resistant and Asia ,2,3, foral wards ,5. Staphhe world ,7. Infeche world . SolvingN-methyl-D-aspartate antagonists [β-hydroxysteroid dehydrogenate [Some benzimidazoles ,11 and sagonists , inhibitrogenate , and therogenate . Benzimidazolylbenzenesulfonamide (BZS) compounds fulfill the structural requirements for affinity toward metalloproteinases: a ligand group that may be directed toward a metal center, a sulfonamide group that may provide a hydrogen bonding interaction with the amino acids of the enzyme backbone, and an aryl group that may have effective Van der Waals interactions with enzyme subsites . Based oThe present work establishes the regio-chemical, stereo-chemical, and electronic requirements of the benzimidazole ring of BZS for achieving activity toward Gram-positive bacteria, such as MRSA. The electronic requirements are modulated by the presence (or absence) of electron-releasing or electron-withdrawing substituents at positions 4, 5 and 7 of the benzimidazole ring of novel BZS derivatives.. The synthesis of benzimidazol-amines 10-13 proceeded from the corresponding R3-dinitroaniline compounds 1-4. The formation of the imidazole ring was carried out by reductive cyclization with acetic acid and tin (II) chloride dihydrate at reflux temperature, followed by acid hydrolysis in the presence of 10% hydrochloric acid at reflux temperature. The primary amine of benzimidazolamine 14 was obtained by nitration of benzimidazole 8 using a mixture of nitric and sulfuric acids, followed by reduction with tin (II) chloride dihydrate in hydro-chloric acid at reflux temperature. Synthesis of benzimidazolamine 15 proceeded from the starting material nitroaniline 5. The second nitro group necessary for formation of the imidazole ring was obtained by nitration of 5 with fuming nitric acid. Formation of the imidazole ring was done by reductive cyclization, similar to the procedure for formation of the imidazole ring in derivatives 10-13. Finally, the benzimidazolamine 15 was obtained by acid hydrolysis of the amide group  chloride dihydrate in a 1:1 ethyl acetate/ethanol mixture , Staphylococcus aureus (ATCC 25923), methicillin-resistant S. aureus (MRSA) (ATCC 43300) and Enterococcus fecalis (ATCC 29212) were assayed. All microorganisms were grown in brain hear infusion (BHI) broth . Solid media was obtained by adding 1.5% (w/v) agar to the liquid media.The Gram-positive microorganisms 5 colony forming units (CFU mL-1). The MIC was measured after 20 h incubation at 37 °C with each substance tested. Growth inhibition of the bacterial strains was assessed by visual inspection. The antibacterial activities of the synthesized compounds were compared to oxacillin and ciprofloxacin as positive control. All assays were carried out in triplicate.The antimicrobial activity of the synthesized compounds was determined on 96-well microdilution plates according to the protocols  for det16-31 in the gas phase were performed using GAUSSIAN 03 [Quantum chemical calculations of BZS SSIAN 03  in conju1H-NMR, and mass spectra agreed with the proposed structures.All of the BZS compounds described above have not been previously reported. The structures of all synthesized compounds were confirmed by spectral data, and the IR, 16-31 were tested in vitro against four Gram-positive bacteria using a standard microdilution method and the control drugs (oxacillin and ciprofloxacin), as shown in 16-23 inhibited growth of the screened microorganisms, yielding MIC values in the range 125-500 μg/mL. However, the synthesized compounds exhibited low antibacterial potencies. The synthesized compounds 24-31 showed antibacterial activity against S. aureus, MRSA, E. fecalis and B. subtilis with MIC values in the range 2-500 μg/mL.16-31 was determined using the data presented in S. aureus, MRSA and B. subtilis. The dependence of compounds efficacy  on the position of the nitro group did not appear to be important. The transformation of the nitro group in position 7 to an amino group led to loss of antibacterial activity. The substitution and region of a third atom or group in the nitro-BZS was decisive for increasing activity in MRSA and B. subtilis. The highest result antibacterial activity was exhibited by BZS containing a nitro group at position 5 and either chlorine or bromine atoms at position 4 of the benzimidazole ring. The importance of nitro group in enhancing the antimicrobial activity against Gram-positive bacteria is supported by similar results observed by Ören et al. [et al. [E. fecalis.The structure-activity relationship (SAR) of compounds n et al.  and Moro [et al.  ExchangeThe SAR study suggested that the acidity of hydrogens 1 and 1’ in the BZS molecule plays an important role in activity of against Gram-positive bacteria. Romero and Mendez showed that the charge on the hydrogen atom, calculated using DFT, is indicative of the gas phase acidity of substituted phenols, in agreement with the local hard and soft acids-bases principle (HSAB).  Based on this antecedent and with the purpose of quantifying a substituent’s effect on antibacterial activity, a study was carried out to investigate the quantitative structure-activity relationship (QSAR) by linear regression analysis of the –log MIC (μM) as a function of the charge on acidic hydrogen atoms and on BZS hydrogen bond acceptors.B. subtilis. The main differences between the conformations of BZS and with a NO2 group lay in the orientation of the NHSO2 hydrogen atom with respect to the NO2 group. The charge on hydrogen atoms (qH) that were hydrogen-bonded to the nitrogen atoms of BZS, as determined from the energetically favorable conformers of each BZS structure at the B3LYP/6-31+G level, showed a larger charge for compounds containing Cl or Br atoms at position 4 and a nitro group at position 5  of the bB. subtilis and MRSA, with r > 0.6 , showed that the best correlation between charge and antibacterial activity occurred between the qN3 and the –log MIC in  r > 0.6 . B. subtilis, and between qH1, qN3, qO2’, and the –log MIC in MRSA, both for the BZS substituent positions 4 and 5. In general, the parameters showed a high co-linearity with a correlation coefficient of r > 0.8 if the linear regression was performed by splitting the compounds into two groups: the first group comprised BZS with substituents in the 4 and 5 positions, and the second group comprised BZS with substituents in the 5 and 7 positions. This method yielded the best r values between qH1, qH1’, qO2’, and the –log MIC in r, the squared correlation, 2r, the standard error, S, and the Fisher ratio, F.The best correlation models for the BZS derivatives that showed the greatest antibacterial activity are described in Equations 1-8  along wiB. subtilis and MRSA.The results suggested that the higher charges on H1 and H1’ in conjunction the lower charges on N3 and O2 promoted the activity of BZS derivatives against  1H-NMR spectra were recorded on Varian Gemini-200 MHz and Eclipse 300 MHz JEOL spectrometers in deuterated dimethyl sulfoxide (DMSO-d6) solutions using an internal TMS standard (0 ppm). The chemical shifts are reported in parts per million (δ/ppm). The peak patterns are indicated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. The coupling constants (J) are reported in Hertz (Hz). Mass spectra were detected by electronic impact (EI) and recorded using a JEOL JEM-AX505HA spectrometer by electronic impact (EI) of lower resolution at 70 eV. Flash column chromatography was carried out using silica gel 60 (230-400 mesh ASTM) from Merck.Melting points were determined using a Mel-Temp melting point apparatus in open capillary tubes and are uncorrected. IR spectra were collected on a Nexus 470 FT-IR spectrophotometer.3-nitroaniline 1-4 (13.06 mmol) and tin (II) chloride dihydrate (58.77 mmol for every nitro group) in acetic acid (20 mL) was heated under reflux for 4 h. The excess acetic acid was removed under reduced pressure, and the crude product was dissolved in 10% hydrochloric acid. The reaction mixture was heated under reflux for 1 h, was made alkaline by NaOH addition, and was extracted with ethyl acetate. The organic layer was dried over Na2SO4, and the solvent was evaporated under reduced pressure to given a crude product, which was purified by flash column chromatography on silica gel, using ethyl acetate:ethanol (90:10) as the eluent.A mixture of R2-Methyl-1H-benzimidazol-6-amine (10). Yield: 84%; m.p. 86-88 °C. FT-IR : 3399 (N-H), 3299 (N-H), 1639 . 1H-NMR (200 MHz): δ 2.37 , 3.45 , 6.43 , 6.59 , 7.11 . MS, m/z (Rel. Int.): 147 , 119(8), 105 (12), 79 (13), 52 (13).5-Fluoro-2-methyl-1H-benzimidazol-6-amine (11). Yield: 72%; m.p. 157-158 °C. FT-IR : 3417 (N-H), 3371 (N-H), 1645 . 1H-NMR (300 MHz): δ 2.36 , 4.69 , 6.76 , 7.08 . MS, m/z (Rel. Int.): 165 , 137(5), 124(6), 97(9), 83 (7).4-Bromo-2-methyl-1H-benzimidazol-6-amine (12). Yield: 66%; oil. FT-IR : 3379 (N-H), 3338 (N-H), 1633 . 1H-NMR (200 MHz): δ 2.38, 4.99 , 6.54, 6.65, , 11.87 . MS, m/z (Rel. Int.): 225 , 146 (19), 119 (9), 77 (10), 52 (7).4-Chloro-2-methyl-1H-benzimidazol-6-amine (13). Yield: 69%; oil. FT-IR : 3338 (N-H), 3391 (N-H), 1637 . 1H-NMR (200 MHz): δ 2.38 , 5.07 , 6.5 , 11.84 . MS, m/z (Rel. Int.): 181 , 146 (13), 105(5), 78(11), 52 (5).3 followed by extraction with ethyl acetate. The organic layers were dried over Na2SO4 and removed by evaporation under vacuum. The pure benzimidazole 8 was obtained in 74% yield. To a suspension of 4-chloro-2-nitroaniline (4.7 mmol) in acetic acid (14.0 mL), was added tin (II) chloride dihydrate (0.021 mmol). The mixture was heated under reflux for 4 h. To the reaction solution was added NaHCO8 (1.8 mmol) was added a mixture of H2SO4/HNO3 . The reaction was stirred at room temperature for 1 h. To the mixture was added cold water. The resulting solid was filtered, washed with cold water, and dried under vacuum to afford pure benzimidazole 9 in 98% yield.To the benzimidazole 9 (1.8 mmol) in HCl (5 mL) was added tin (II) chloride dihydrate (8.0 mmol). The mixture was heated under reflux for 4 h. The resulting solid was filtered, dissolved in aqueous 10% NaOH and the product extracted with ethyl acetate. The extract was dried and under vacuum to gave the desired benzimidazolamine 14. Yield: 78%; m.p. 108 °C. FT-IR : 3429 (N-H), 3354 (N-H), 1639 . 1H-NMR (200 MHz): δ 2.40 , 4.94 , 6.85 , 7.32 . MS, m/z (Rel. Int.): 181 , 146 (11), 105(4), 78(11), 52 (8).To a suspension of benzimidazole 5 (29.8 mmol) was added acetic anhydride (9.2 mL) and acetic acid (11.0 mL). The reaction was stirred under reflux for 2 h. To the mixture was added cold water, and the resulting solid was filtered, washed with cold water, and dried under vacuum. To this solid was added fuming HNO3 (12.0 mL), the reaction was stirred at 0 to 5 °C for 1 h. To the mixture was added cold water, and the resulting solid was filtered, washed with cold water, and dried under vacuum, to afford the pure acetamide 6 in 77% yield. From the acetamide 6, the benzimidazolamine 15 was obtained following the general procedure for preparing benzimidazolamines 10-13. Yield: 54%; oil. FT-IR : 3417 (N-H), 3342 (N-H), 1637 , 1140 (O-CH3). 1H-NMR (200 MHz): δ 2.36 , 3.76 , 4.4 , 6.68 , 6.86 . MS, m/z (Rel. Int.): 177 , 162 (78), 134 (37), 89 (6), 52 (4).To 2-methoxy-4-nitroaniline 10-15, 6.8 mmol) and pyridine  in anhydrous acetone was added benzenesulfonyl chloride . The reaction mixture was stirred at room temperature under a N2 atmosphere for 24 h. Aqueous NaHCO3 was then added, and the mixture was extracted with ethyl acetate. The organic layer was dried over Na2SO4, filtered, concentrated, and purified by flash column chromatography on silica gel (gradient elution 50:50 hexane/ethyl acetate to 70:30 ethyl acetate/ethanol) to give 16-21 and 22-23.To a solution of benzimidazolamine (N-(2-methyl-1H-benzimidazol-6-yl)benzenesulfonamide (16). Yield: 68%; m.p. 221-222 °C. FT-IR : 3302 (N-H), 1633 , 1340 and 1155 (O2S-NH). 1H-NMR (300 MHz): δ 2.41 , 6.83 , 7.14 , 7.5 , 7.54 , 7.68 . MS, m/z (Rel. Int.): 287 , 146 (100), 119 (7), 105 (4), 77 (5).N-(5-fluoro-2-methyl-1H-benzimidazol-6-yl)benzenesulfonamide (17). Yield: 45%; m.p. 152 °C. FT-IR : 3253 (N-H), 1635 , 1323 and 1167 (O2S-NH). 1H-NMR (200 MHz): δ 2.43 , 7.17 , 7.17 , 7.57 , 9.91 , 12.27 . MS, m/z (Rel. Int.): 305 , 164 (100), 137 (12), 96 (7), 77 (11).N-(4-bromo-2-methyl-1H-benzimidazol-6-yl)benzenesulfonamide (18). Yield: 66%; m.p. 272-273 °C. FT-IR : 3290 (N-H), 1631 , 1317 and 1163 (O2S-NH). 1H-NMR (200 MHz): δ 2.43 , 7.02 , 7.15 , 7.53 , 7.69 , 10.19 , 12.45 . MS, m/z (Rel. Int.): 365 , 224 (100), 145 (14), 104 (5), 77 (17).N-(4-chloro-2-methyl-1H-benzimidazol-6-yl)benzenesulfonamide (19). Yield: 57%; m.p. 358 °C. FT-IR : 3284 (N-H), 1633 , 1319 and 1162 (O2S-NH). 1H-NMR (200 MHz): δ 2.43 , 6.89 , 7.11 , 7.53 , 7.70 , 10.2 , 12.41 . MS, m/z (Rel. Int.): 321  180 (100), 153 (6), 103 (2), 77 (8).N-(5-chloro-2-methyl-1H-benzimidazol-6-yl)benzenesulfonamide (20). Yield: 53%; m.p. 224 °C. FT-IR : 3275 (N-H), 1631, 1292 and 1151 (O2S-NH). 1H-NMR (200 MHz): δ 2.45 , 7.20 , 7.46 , 7.6 , 9.82 , 12.36 . MS, m/z (Rel. Int.): 321 , 180 (100), 153 (7), 103 (2), 77 (7).N-(2-methyl-5-methoxi-1H-benzimidazol-6-yl)benzenesulfonamide (21). Yield: 22%; m.p. 206-207 °C. FT-IR : 3282 (N-H), 1633 , 1325 and 1167 (O2S-NH). 1H-NMR (300 MHz): δ 2.42 , 3.38 , 6.86 , 7.26 , 7.47 , 7.57 , 7.61, 9.30 , 12.06 . MS, m/z (Rel. Int.): 317 (36), 176 (100), 148 (32), 107 (6), 77 (7).N-(2-methyl-1-phenylsulfonyl-1H-benzimidazol-6-yl)benzenesulfonamide (22). Yield: 2%; m.p. 146-148 °C. FT-IR : 3427 (N-H), 1622 , 1163 (O2S-NH). 1H-NMR (300 MHz): δ 2.76 , 7.23 , 7.14 , 7.07 , 10.12 . MS, m/z (Rel. Int.): 427 , 286 (100), 141 (41), 97 (6), 77 (45).N-(2-methyl-5-methoxi-1-phenylsolfonyl-1H-benzimidazol-6-yl)benzenesulfonamide (23). Yield: 15%; m.p. 186-187 °C. FT-IR : 3261 (N-H), 1601 , and 1176 (O2S-NH), 1091 (O-CH3). 1H-NMR (200 MHz): δ 2.73 , 3.48 , 7.09 , 7.49 , 7.63 , 7.78 , 7.81 , 7.91 , 9.73 . MS, m/z (Rel. Int.): 457 , 316 (100), 175 (19), 141 (26), 77 (98).16-21, 3.5 mmol) was added fuming HNO3 (2.0 mL). The reaction mixture was stirred at 0-5 °C for 1 h. Cool water was then added to provide a yellow solid. The solid was filtered, washed twice with water and dried. The mixture of 24 and 26 was purified by flash column chromatography on silica gel, using gradient elution, 75:25 to 55:45 hexane/ethyl acetate.To benzimidazolylbenzenesulfonamide (N-(2-methyl-7-nitro-1H-benzimidazol-6-yl)benzenesulfonamide (24). Yield: 76%; m.p. 182 °C. FT-IR : 3410 (N-H), 3222 (N-H), 1630 , 1493 and 1356 (NO2), 1311 and 1161 (O2S-NH). 1H-NMR (300 MHz): δ 2.71 , 7.19 , 7.54 , 7.67 , 7.93 , 10.47 . MS, m/z (Rel. Int.): 332 , 191 (100), 131 (29), 104 (12), 77 (34).N-(2-methyl-5-nitro-1H-benzimidazol-6-yl)benzenesulfonamide (26). Yield: 22%; m.p. 184 °C. FT-IR : 3261 (N-H), 1643 , 1464 and 1358 (NO2), 1307 and 1174 (O2S-NH). 1H-NMR (200 MHz): δ 2.49 , 7.13 , 7.58 , 8.05 , 10.04 , 12.72 . MS, m/z (Rel. Int.): 332 , 191 (100), 131 (26), 104 (6), 77 (59).N-(5-fluoro-2-methyl-7-nitro-1H-benzimidazol-6-yl)benzenesulfonamide (27). Yield: 75%; m.p. 242 °C. FT-IR : 3348 (N-H), 3265, 1639 , 1522 (NO2), 1325 and 1167 (O2S-NH). 1H-NMR (200 MHz): δ 2.51 , 7.55 , 10.35 , 12.95 . MS, m/z (Rel. Int.): 350 , 209 (100), 192 (51), 122 (8), 77 (12).N-(4-bromo-2-methyl-5-nitro-1H-benzimidazol-6-yl)benzenesulfonamide (28). Yield: 71%; m.p. 251 °C. FT-IR : 3373 (N-H), 3215 (N-H), 1624 , 1463 (NO2), 1309 and 1161 (O2S-NH). 1H-NMR (300 MHz): δ 2.53 , 7.13 , 7.54 , 7.64 , 10.33 , 13.11 . MS, m/z (Rel. Int.): 410 , 269 (82), 239 (21), 132 (14), 77 (34).N-(4-chloro-2-methyl-5-nitro-1H-benzimidazol-6-yl)benzenesulfonamide (29). Yield: 92%; m.p. 140-141 °C. FT-IR : 3394 (N-H), 3255 (N-H), 1624 , 1369 (NO2), 1338 and 1172 (O2S-NH). 1H-NMR (300 MHz): δ 2.55 , 6.99 , 7.54 , 7.65 , 10.39 . MS, m/z (Rel. Int.): 366 , 224 (97), 208 (33), 132 (12), 77 (37).N-(5-chloro-2-methyl-7-nitro-1H-benzimidazol-6-yl)benzenesulfonamide (30). Yield: 67%; m.p. 277-278 °C. FT-IR : 3379 (N-H), 3288 (N-H), 1631 , 1520 (NO2), 1338 and 1163 (O2S-NH). 1H-NMR (300 MHz): δ 2.53 , 7.60 , 7.82 , 10.41 , 13.04 . MS, m/z (Rel. Int.): 366 , 224 (100), 208 (74), 132 (13), 77 (16). N-(5-methoxy-2-methyl-7-nitro-1H-benzimidazol-6-yl)benzenesulfonamide (31). Yield: 94%; m.p. 182 °C. FT-IR : 3290 (N-H), 1622 , 1544 (NO2), 1346 and 1140 (O2S-NH). 1H-NMR (300 MHz): δ 2.65 , 3.26 , 7.35 , 7.53 , 10.16 . MS, m/z (Rel. Int.): 362 , 221 (100), 173 (78), 133 (3), 77 (11).24, 2.9 mmol) and tin (II) chloride II dihydrate (13.0 mmol) in ethyl acetate/ethanol 1:1 (100 mL) was heated under reflux for 4 h. The solvent was removed under reduced pressure and the crude product was added to aqueous NaHCO3.The mixture was subsequently extracted with ethyl acetate. The organic layer was dried over Na2SO4, filtered, and concentrated to give 25. Yield: 65%; m.p. 225 °C. FT-IR : 3471 (N-H), 3319 (N-H), 1628 , 1311 and 1159 (O2S-NH). 1H-NMR (300 MHz): δ 2.39 , 4.89 , 6.31 , 6.43 , 7.49 , 7.59 , 7.64 , 9.14 , 11.91. MS, m/z (Rel. Int.): 302 , 161 (100), 134 (9), 107 (4), 77 (14).A mixture of benzimidazolilbenzenesulfonamide (28 and 29, have shown antibacterial activity against Gram-positive bacteria, which suggests that they may be members of a promising new class of BZS anti-MRSA agents. These compounds present a good starting point for modelling antimicrobial activity. Future work will focus on calculation of the absolute gas phase acidity of the hydrogens H1 and H1’ of BZS and a QSAR between the acidity of these hydrogens and antimicrobial activity.We have synthesized and evaluated the antibacterial activities of BZS analogs containing electron-releasing and electron-withdrawing substituents on the benzimidazole ring. SAR and theoretical studies of this series of compounds have shown that the antibacterial activity of BZS compounds may be related to the charges on the acidic hydrogen atoms in the BZS structure. Two compounds,