CDKN1A promoter, the recruitment of p53 as well as RNA Polymerase II and levels of acetylation on histone H3 were diminished by NiCur. Specifically, NiCur reduces the levels of acetylation at lysine 27 on histone H3, which concomitantly increases the levels of trimethylation at lysine 27. Finally, NiCur attenuates p53-directed apoptosis by inhibiting the Caspase 3 activity and cleavage of Poly (ADP-ribose) polymerase (PARP) in normal gastrointestinal epithelial cells. Collectively, NiCur demonstrates the potential to reprogram the chromatin landscape and modulate biological outcomes of CBP-mediated acetylation under normal and disease conditions.Tumor suppressor p53-directed apoptosis triggers loss of normal cells, which contributes to the side-effects from anticancer therapies. Thus, small molecules with potential to downregulate the activation of p53 could minimize pathology emerging from anticancer therapies. Acetylation of p53 by the histone acetyltransferase (HAT) domain is the hallmark of coactivator CREB-binding protein (CBP) epigenetic function. During genotoxic stress, CBP HAT-mediated acetylation is essential for the activation of p53 to transcriptionally govern target genes, which control cellular responses. Here, we present a small molecule, NiCur, which blocks CBP HAT activity and downregulates p53 activation upon genotoxic stress. Computational modeling reveals that NiCur docks into the active site of CBP HAT. On  Targeting tumor cells by anticancer therapies, which are effective in controlling the growth of tumors, also generate severe adverse effects . MassiveCyclin Dependent Kinase Inhibitor 1A or p21 (CDKN1A/p21), which induces growth arrest in DNA damaged cells -4-piperidinone (EF24) and a nitrile-curcuminoid were previously demonstrated to exhibit anti-inflammatory +; [M + Na]+; [2M + Na]+, Found 464.94, 462.94, 466.94; 486.92; 950.93.none (1) : Yellow 3,3′--bis(methanylylidene))dibenzonitrile (2): nitrile-curcuminoid/NiCur) +; [M + Na]+; [2M + Na]+, Found 325.16, 326.14; 347.14; 671.25.d/NiCur) : Yellow -2,6-bis(3-Bromo-4-methoxybenzylidene)cyclohexanone (3): Yellow sheets  from dichloromethane/hexanes. Solvent system A, elution at 75% dichloromethane. 1H-NMR (DMSO-d6) δ 1.73 , 2.86–2.88 , 3.90 , 7.20 , 7.54 , 7.57 , 7.78 . LC t = 7.0 min. HRMS m/z Calcd. for C22H21Br2O3 490.9857 [M + H]+, Found 490.9855.-2,6-bis(2-Methylbenzylidene)cyclohexanone (4) +; [M + Na]+; [2M + Na]+, Found 303.21, 304.21; 325.19; 627.32.none (4) : Yellow -2,6-biscyclohexanone (5): Yellow needles  from EtOH. Solvent system A, elution at 50% dichloromethane. 1H-NMR (CDCl3) δ 1.77–1.78 , 2.35 , 2.91–2.93 , 6.98 , 7.08 , 7.74 . LC t = 7.3 min. HRMS m/z Calcd. for C24H27O 331.2062 [M + H]+, Found 331.2054.-2,6-biscyclohexanone (6) +; [M + Na]+; [2M + Na]+, Found 363.21, 364.21; 385.18; 747.38.none (6) : Yellow -2,6-bis(4-Hydroxybenzylidene)cyclohexanone (7) +; [M + Na]+; [2M + Na]+, Found 307.17, 308.17; 329.14; 635.24.none (7) : Green m-2,6-bis(2-Fluorobenzylidene)cyclohexanone (8) +; [M + Na]+; [2M + Na]+, Found 311.16, 312.16; 333.14; 643.23.none (8) : Yellow -2,6-bis(2-Bromobenzylidene)cyclohexanone (9) +; [M + Na]+; [2M + Na]+, Found 432.96, 430.96, 434.96; 454.94; 886.96.none (9) : Yellow -2,6-bis(3-Bromobenzylidene)cyclohexanone (10) +; [M + Na]+; [2M + Na]+, Found 432.95, 430.95, 434.94; 454.93; 886.93.one (10)  Yellow m-2,6-bis(4-Bromobenzylidene)cyclohexanone (11) +; [M + Na]+; [2M + Na]+, Found 432.96, 430.96, 434.96; 454.94; 886.96.one (11) : Yellow -2,6-bis(Pyridin-3-ylmethylene)cyclohexanone (12): Yellow microneedles  from dichloromethane/hexanes. Solvent system B, elution at 5% MeOH. 1H-NMR (CDCl3) δ 1.82–1.87 , 2.95 , 7.34–7.37 , 7.76–7.78 , 8.58 , 8.73 . LCMS t = 5.0 min, m/z Calcd. for C18H17N2O; C18H16N2NaO; C36H32N4NaO2 277.13, 278.14; 299.11; 575.24 [M + H]+; [M + Na]+; [2M + Na]+, Found 277.18, 278.18; 299.15; 575.22.-2,5-bis(3-Bromo-4-hydroxybenzylidene)cyclopentanone (13): Yellow microcrystals  from hexanes/acetone. 1H-NMR (MeOD) δ 3.10 , 6.97 , 7.39 , 7.48–7.50 , 7.79 . LC t = 6.2 min. HRMS m/z Calcd. for C19H15O3Br2 448.9388 [M + H]+, Found 448.9380.-2,6-bis(3-bromo-4-hydroxybenzylidene)-4-methylcyclohexanone (14): Yellow microneedles  from dichloromethane/hexane. Solvent system B, elution at 100% dichloromethane. 1H-NMR (MeOD) δ 1.08–1.11 , 1.84–1.87 , 2.53 , 2.99–3.01 , 6.94 , 7.34 , 7.59 , 7.63 . LC t = 6.2 min. HRMS m/z Calcd. for C21H19Br2O3 476.9701 [M + H]+, Found 476.9701.Ethyl -3,5-bis(3-bromo-4-hydroxybenzylidene)-4-oxocyclohexanecarboxylate (15): Grey-green microneedles  from dichloromethane/hexane. Solvent system B, elution at 5% MeOH. 1H-NMR (MeOD) δ 1.14 , 2.83–2.86 , 3.08–3.31 , 4.08 , 6.95 , 7.34 , 7.62 , 7.64 . LC t = 6.2 min. HRMS m/z Calcd. for C23H21Br2O5 534.9756 [M + H]+, Found 534.9731.-2,6-bis(3-Bromo-4-hydroxybenzylidene)-4-hydroxycyclohexanone (17): Orange microneedles  from dichloromethane/methanol. Solvent system B, elution at 4% MeOH. 1H-NMR (DMSO-d6) δ 2.84–2.88 , 3.00–3.03 , 3.99–4.00 , 7.03 , 7.41 , 7.53 , 7.69 , 10.79 . LC t = 5.6 min. HRMS m/z Calcd. for C20H17Br2O4 478.9494 [M + H]+, Found 478.9511.-3,5-bis(3-Bromo-4-hydroxybenzylidene)dihydro-2H-pyran-4(3H)-one (18) +; [M + Na]+; [2M + Na]2+, Found 466.93, 464.93, 468.92; 488.91; 954.91.one (18) : Yellow -3,5-bis(3-Bromo-4-hydroxybenzylidene)-1-methylpiperidin-4-one (20) +; [M + Na]+; [2M + Na]+, Found 479.96, 477.95, 481.93; 501.92; 980.95.one (20) : Orange Method B:2O (50 mL), EtOH (50 mL), diethyl ether (50 mL), and dried under vacuum +, Found 463.9516.Method C:3E,5E-bis(3-Bromo-4-hydroxybenzylidene)-4-oxocyclohexanecarboxylate  was hydrolyzed in a solution of THF (50 mL), H2O (25 mL), and NaOH (~5 eq), for 48 h. Aqueous HCl was added until pH was at congo red. THF was added until one layer was formed, and the precipitate product was collected by filtration.Ethyl -3,5-bis(3-Bromo-4-hydroxybenzylidene)-4-oxocyclohexanecarboxylic acid (16): Yellow microcrystals  from THF/H2O. 1H-NMR (MeOD) δ 2.78–2.81 , 3.05–3.10 , 3.20–3.31 , 6.96 , 7.38 , 7.64 , 7.67 . LC t = 5.8 min. HRMS m/z Calcd. for C21H17Br2O5 506.9443, [M + H]+, Found 506.9465.Human osteosarcoma U2OS and normal rat intestinal epithelial cells were maintained in Eagle’s minimal essential medium (DMEM)  and RPMI media, which were supplemented with 10% fetal bovine serum  and antibiotics. For p53 activation, Doxorubicin  was used. The compounds were dissolved in DMSO (Sigma). The antibodies used for immunoprecipitation and immunoblotting were p53 (sc-6243), p21 (sc-397), and CBP (sc-7300) from Santa Cruz Biotechnology Inc. Dallas, TX, USA; p53Ser15p (9282), p53K382ac (2525), H2AX S139p (9718), H3S28p (9713), PARP (5625) and all Cyclins (4656 (A2), 12231 (B1), 4132(E2) antibodies from Cell Signaling Technology Inc., Danvers, MA, USA; H3 (ab1791), H3K27me3 (1791), H3K9ac (ab4441) and EZH2 (ab3748) antibodies from Abcam, Cambridge, MA, USA; and Actin (A4700) from Sigma.C5-Curcumionoid analogues by using Dox-induced p53 response element system as well as to determine the respective IC50 values of the potential CBP HAT inhibitor. U2OS cells were co-transfected with p53 reporter element in tandem with luciferase gene (1 μg) and Renilla luciferase (100 ng) vectors in 24-well plate format using Fugene 6 . Briefly, total of 1.1 μg of vector was incubated with 3 mL of Fugene 6 reagent for 30 min. In these experiments, DMSO, transfected cells with empty vector and cells without Dox treatment were used as controls. DMSO concentration was maintained at 0.01%. Transfected cells with Dox treatment were used as positive control. The luciferase activity was estimated by following the manufacturer’s instruction  in a luminometer. Both active and passive lysis of cells yielded consistent results. The inhibitory activity (IC50) of a small molecule on luciferase activity was obtained from the average of three biological replicates using the PRISM software .A luciferase-based reporter assay in U2OS cells was developed to screen and to quantitate the inhibitory effects of newly synthesized Following manufacturer’s instructions , fluorescence-based HAT assay was performed using recombinant CBP and PCAF HAT enzymes ,63. BrieDocking calculations used Autodock-4  that seaU2OS and normal rat intestinal epithelial cells were harvested cells and lysed in lysis buffer (20 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EGTA, 1% Triton X-100, and 50 mM NaF) containing protease inhibitor cocktail (Sigma). The cells were sonicated and spun down at 14,000 rpm for 30 min at 4 °C. After protein estimation, 30–50 μg of lysates were subjected to SDS-PAGE, transferred onto nitrocellulose membranes, blocked with 5% milk/PBS and blotted with a primary antibody. Horseradish peroxidase (HRP)-labeled secondary antibodies (goat anti-mouse or anti-rabbit) were added for 60 min at room temperature, and the blots were washed with TBS  and subjected to autoradiography after development of reaction by ECL  ,17,18.CDKN1A promoter primers . The qPCR data was normalized using H3 antibody as a control with regard to the input.For ChIP-qPCR, EZ-Magna ChIP G  was used and manufacturer’s instruction was followed. For ChIP analyses, U2OS cells were treated with Dox in presence or absence of NiCur. IP was carried out with antibodies to the following proteins: RNA polymerase II (2 μg), p53 (2 μg), CBP (2.5 μg), EZH2 (2.5 μg), H3 (6 μg), H3Kac (6 μg), H4Kac (6 μg), H3Kme3 and H3K27ac (2.5 μg). An aliquot was amplified and analyzed by qPCR using Human To test the effects of NiCur on proliferation of U2OS and normal rat gastrointestinal epithelial cells, BrdU incorporation assay was performed in 96-well plates using calorimetry-based kit from Calbiochem Burlington Massachusetts, USA (Cat # QiA58). One hundred microliters of 5000–10,000 U2OS and rat gastrointestinal epithelial cells were seeded with DMEM and RPMI media  with 10% fetal bovine serum , respectively. After 16 h, cells were treated with 1.5 μM of NiCur and BrdU in presence or absence of 300 ng of Dox. To rule out the possibilities of any background effects, controls included in the assay were media alone, or together with DMSO and with BrdU and the untreated cells. Subsequently, after 24 h, cells were fixed, and then treated with anti-BrdU antibody. After washing, the wells were further incubated with secondary antibody labeled with HRP. After washings, the reaction in each well was developed using TMB as a substrate, and then finally, reaction in each well was stopped by adding stop solution and optical density was measured at 450 nm.To test the effects of NiCur on apoptosis, intracellular levels of cleaved Caspase 3 was determined by using a kit from Thermo Fisher Scientific, Waltham, MA, USA (Cat# 62218) following manufacturer’s instructions with minor modifications. Approximately, 10,000 U2OS and normal rat gastrointestinal epithelial cells were seeded in 96 well plates. After overnight, cells were treated with 300 ng of Dox in presence and absence of 1.5 μM of NiCur. The controls included were media alone and untreated cells with and without NiCur. After 24 h of incubation, cells were fixed with 4.0% formaldehyde. After washing, cells were permeabilized and then each well was incubated in blocking solution to prevent non-specificity. After washing, the each well was incubated with anti-Caspase 3 antibody , which can show reactivity to Caspase 3 proteins from multiple species, including humans and rats. Subsequently, each well was incubated with anti-rabbit HRP-labeled secondary antibodies. Finally, the reaction in each well was developed using TMB and the plate was read at 450 nm.


The series of 6-, 7- and 6,7-subtituted isoquinolinequinones were evaluated in vitro for their cytotoxic activity using the MTT colorimetric method. All the newly synthesized compounds showed moderate to high potency against MRC-5 healthy lung fibroblasts and four human tumor cell lines: AGS gastric adenocarcinoma, SK-MES-1 lung, J82 bladder carcinoma, and HL-60 leukemia cells. Among the series, compounds 4b, 12 and 13 exhibited interesting antitumor activity against human gastric adenocarcinoma, human lung and human bladder carcinoma cancer cells. 7-Amino-6-bromoisoquinoline-5,8-quinone (13) was found to be the most promising active compound against the tested cancer cell lines, with IC50 values in the 0.21−0.49 μM range, lower than the anti-cancer agent etoposide used as reference. The synthesis of 4-methoxycarbonyl-3-methylisoquinolinequinone ( WT cell line [50 values ranging from 0.22 to 0.34 μg/mL, whereas caulibugulone D showed less cytotoxic activity (IC50 = 1.67 μg/mL). According to these IC50 values against the tested cell line it can be deduced that insertion of bromine or chlorine atoms at the 6-position in caulibugulone A (0.34 μg/mL), as in caulibugulone B (0.22 μg/mL) and C (0.28 μg/mL), is not an important determinant of cytotoxicity. Furthermore, the replacement of the methylamino group in caulibugulone A by the 2-hydroxyethylamino group, as in caulibugulone D, produces a decrease of the cytotoxic activity. Evidences reported by Wipf and Lazo [in vitro inhibitors of the Cdc25 family of cell cycle-controlling protein phosphatases. The search for biologically active natural products from marine sources continues to be an important scientific field that offers promising opportunities for the development of new compounds endowed with pharmacological properties ,5,6,7,8.ell line  exhibit and Lazo ,12 indic3·7H2O has been simultaneously reported by Wipf [N-chlorosuccinimide and N-bromosuccinimide provides access to caulibugulones B and C, respectively.The access to caulibugulones A–D via regioselective nucleophilic amination substitution at the 7-position in the reaction of isoquinoline-5,8-quinone with methylamine or 2-aminoethanol in ethanol as solvent in the presence of CeCl by Wipf  and Tama by Wipf . These a4) proved to be the most active compound, with an overall potency of 0.089 μM. It was followed by mansouramycin B (mean IC50 = 2.7 μM) and then mansouramycin A, with a mean IC50 value of 13.44 μM. These data indicate that insertion of a methoxycarbonyl group at C-1 or a chlorine atom at C-6 into the 7-methylaminoisoquinolinequinone pharmacophore induces an increase of antitumor activity. Regarding mansouramycins A–C, screening on a panel of up to 36 human tumor cell lines displayed significant cytotoxicity, with pronounced selectivity for non-small cell lung cancer, breast cancer, melanoma, and prostate cancer cells . Mansourin vitro antitumor evaluation of a variety of amino-, alkylamino and alkylamino-haloisoquinolinequinone derivatives, structurally related with the above mentioned marine isoquinolinequinones, as well the SAR of this series.As part of our research program aimed at the synthesis and structure-activity relationship (SAR) of novel aminoquinones as potential antitumor agents ,16,17,181, unsubstituted at the 1-position and containing a methoxycarbonyl group on the heterocyclic ring, as in mansouramycin C, was selected as a suitable precursor of the designed aminoisoquinolinequinones. The synthesis of the target compound 1 was accomplished in 86% yield from commercially available 2,5-dihydroxybenzaldehyde and methyl aminocrotonate, through a one-pot procedure previously developed in our laboratory [Isoquinolinequinone boratory   in 74% yield. Further separation by column chromatography on silica gel afforded pure compound 2a in 52% yield  and extensive decomposition of quinone 1 was detected. According to precedents on the use of a Lewis acid to facilitate the reaction of quinones with amines [1 was allowed to react with 2-aminoethanol in ethanol in the presence of 5 mmol% of CeCl3·7H2O. Under these conditions, the reaction went to completion in 3 h to give an 80:20 mixture of regioisomers 3a and 3b  in 65% yield. Further separation by column chromatography on silica gel afforded the major regioisomer 3a pure in 47% yield  and high resolution mass spectra (HRMS). The position of the nitrogen substituent in these aminoquinones was determined by means of HMBC experiments. For example, the location of the nitrogen group at C-7 in compound 4a was deduced by the 3JC,H couplings between the carbon at C-8 (δ 180.83) with the protons at C-1 (δ 9.17), at C-6 (δ 5.77) and that of the NH group (δ 5.96). In the case of aminoquinone 4b, the location of the nitrogen substituent at C-6 was established by 3JC,H coupling between the carbon at C-5 (δ 181.71) with the proton at C-7 (δ 5.79) and the proton of the NH group (δ 5.75). Also, the 3JC,H coupling between the carbon at C-8 (δ 181.49) with the proton at C-1 (δ 9.29) can be observed  and four human cancer cell lines: AGS (gastric), SK-MES-1 (lung), J82 (bladder), and HL-60 (leukemia), using the conventional microculture tetrazolium reduction assay [50 values collected in 1, as in 2a,3a,4a,4b,5a and 5b, increases the cytotoxic activity in all the evaluated cell lines, compared to those of precursor 1. The initial structure-activity relationship (SAR) was focused on the nature and location of alkylamino at the quinone nucleus of the isoquinolinequinone pharmacophore. The data of 2a, 3a, 4a and 5a reveal that the shape and polarity of the alkylamino group at the 7-position in the isoquinolinequinone moiety does not significantly influence the biological activity. Concerning the effect of the position of the alkylamino groups, the IC50 values for the regioisomers 4a/4b and 5a/5b indicate that substitution of the nitrogen group at the 6-position of the quinone pharmacophore induces a greater effect on the antitumor activity than at the 7-position. It can be seen that the effect of such substitution on the antitumor potency on gastric adenocarcinoma and lung cancer cells reaches values nearly seven times higher compared to regioisomer 4a.Isoquinolinequinones on assay ,26,27. T2a, 3a, 4a and 5a. Comparison of the IC50 values of halo derivatives 6–11 with those of their corresponding precursors indicates that the substitution of halogen atom at the 6-position enhances the antitumor potency in all the evaluated cell lines, except for compound 11 on lung fibroblasts and gastric cancer cells. Next, the SAR analysis was focused on the effects of insertion of chlorine and bromine atoms at the 6-position of the 7-substituted alkylaminoisoquinolinequinones 12 showed more cytotoxic activity (0.93–2.16 µM) on the cancer cell lines than the N-alkyl 7-substituted analogs 2a, 3a, 4a and 5a. It is noteworthy that compound 12 exhibits higher antitumor potency on lung and bladder cell lines than the anticancer drug etoposide.According to the data of 12 (0.93−2.16 µM) with that of its bromoderivative 13 (IC50: 0.21−0.49 µM) reveals that insertion of the bromine atom at the 6-position of 12 induces a high increase of the cytotoxic activity in all the evaluated cell lines. It is noteworthy that compounds 12 and 13 exhibit higher antitumor potency and selectivity index  in almost all cell lines than the anticancer drug etoposide (SI: 0.1–0.6). Among the tested analogs of the series, compounds 4b, 12 and 13 are the most significant antitumor members. Compound 13 was selected as a promising lead compound due to its high potency on the tested tumor cell lines and selectivity index.Comparison of the cytotoxic potency of compound 1H-NMR spectra were recorded on Bruker AM-200 and Avance-400 instruments in deuterochloroform (CDCl3). 13C-NMR spectra were obtained in CDCl3 at 50 and 100 MHz. Bidimensional NMR techniques and DEPT were used for signal assignment. Chemical shifts are expressed in ppm downfield relative to tetramethylsilane and the coupling constants (J) are reported in Hertz. The HRMS spectra were obtained on a Thermo Finnigan spectrometer, model MAT 95XP. Silica gel Merck 60 (70–230 mesh) was used for preparative column chromatography and TLC aluminum foil 60F254 for analytical TLC.All reagents were commercially available reagent grade and were used without further purification. Melting points were determined on a Stuart Scientific SMP3 apparatus and are uncorrected. 4-Methoxycarbonyl-3-methylisoquinoline-5,8-quinone (1). A suspension of 2,5-dihydroxybenzaldehyde (1 mmol), methyl 3-aminocrotonate (1 mmol), Ag2O (2 mmol) and MgSO4 (0.5 g) in CH2Cl2 (25 mL) was stirred at rt for 3 h. The mixture was filtered, the solids were washed with CH2Cl2 and the solvent removed under reduced pressure. The residue was column cromatographed over silica gel (90:10 CH2Cl2/AcOEt) to yield pure quinone 1 (86%) as yellow solid, mp 109–111.5 °C; IR νmax 1731 (C=O ester), 1668 (C=O quinone); 1H-NMR : δ 2.65 , 4.03 , 7.04 , 9.22 ; 13C-NMR (100 MHz): δ 22.69, 53.11, 122.18, 125.15, 133.39, 138.38, 138.58, 148.57, 161.83, 167.68, 183.38, 183.60; HRMS (M+): m/z calcd for C12H9NO4: 231.05315; found: 231.05229.7-Methylamino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (2a). A suspension of quinone 1 (1 mmol), methylamine hydrochloride (2 mmol) and AcONa (2 mmol) in ethanol (20 mL) was stirred at rt for 1.5 h. The solvent was removed under reduced pressure to give an orange solid containing regioisomers 2a and 2b (74%) in 80:20 proportion. The major regioisomer 2a was isolated by column chromatography over silica gel (90:10 CH2Cl2/AcOEt): red solid (52 %), mp 209.5–211 °C; IR νmax 3279 (N-H), 1740 (C=O ester), 1681 (C=O quinone); 1H-NMR: : δ 2.66 , 2.95 , 4.04 , 5.75 , 6.09 , 9.18 ; 13C-NMR (100 MHz) δ: 23.02, 29.36, 53.21, 101.16, 122.08, 126.38, 136.38, 148.10, 148.82, 163.02, 168.94, 180.24, 180.55; HRMS (M+): m/z calcd for C13H12N2O4: 260.0797; found: 260.0731.7-(2-Hydroxyethylamino)-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (3a). A suspension of quinone 1 (1 mmol), 2-aminoethanol (2 mmol), CeCl3.7H2O (0.05 mmol) and ethanol (25 mL) was left with stirring at rt for 3 h. The solvent was removed under reduced pressure to give an orange solid containing regioisomers 3a and 3b (65%) in 80:20 proportion. The major regioisomer 3a was isolated by column chromatography over silica gel (90:10 CH2Cl2/AcOEt); red solid (47%), mp 195.5–197 °C; IR νmax 3399 (O-H), 3186(N-H), 1700 (C=O ester), 1600 (C=O quinone); 1H-NMR : δ 2.52 , 3.27 , 3.59 , 3.88 , 4.89 , 5.79 , 7.75 , 9.06 ; 13C-NMR (100 MHz): δ 22.35, 44.77, 52.63, 58.47, 99.87, 122.43, 125.18, 135.92, 147.36, 149.04, 161.13, 168.25, 178.91, 180.24; HRMS (M+): m/z calcd for C14H14N2O5: 290.0903; found: 290.0891.6- and 7-(Cyclohexylamino)-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone . A suspension of quinone 1 (1 mmol), cyclohexylamine (2 mmol), CeCl3·7H2O (0.05 mmol) and ethanol (25 mL) was left with stirring at rt for 2 h. The solvent was removed under reduced pressure to give an orange solid containing regioisomers 4a and 4b in 86:14 ratio. These compounds were isolated by column chromatography over silica gel (90:10 CH2Cl2/AcOEt).4a : orange solid, mp 161.5–163.5 °C; IR νmax 3,246 (N-H), 2,927 and 2,856 (CH), 1741 (C=O ester), 1685 (C=O quinone); 1H-NMR : δ 1.35 , 1.68 , 1.81 , 2.03 , 2.65 , 3.30 , 4.03 , 5.77 , 5.96 , 9.17 ; 13C-NMR (100 MHz): δ 22.99, 24.54 (2C), 25.42, 31.83 (2C), 51.44, 53.16, 101.25, 122.11, 126.23, 136.34, 146.52, 148.03, 162.89, 168.93, 180.13, 180.83; HRMS (M+): m/z calcd for C18H20N2O4: 328.14227; found: 328.14083.Compound 4b (9.5%): red solid, mp 158.5–160 °C; IR νmax 3246 (N-H); 2927 and 2856 (CH); 1741 (C=O ester); 1685 (C=O quinone); 1H-NMR : δ 1.35 , 1.68 , 1.81 , 2.03 , 2.65 , 3.30 , 4.05 , 5.75 , 5.79 , 9.28 , 13C-NMR (100 MHz): δ 22.64, 24.54 (2C), 25.47, 31.86 (2C), 51.39, 53.26, 101.23, 123.05, 124.92, 132.44, 146.31, 148.73, 159.91, 168.51, 181.49, 181.71;HRMS (M+): m/z calcd for C18H20N2O4: 328.14227; found: 328.14070.Compound 6- and 7-Morpholino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone . A suspension of quinone 1 (1 mmol), morpholine (2 mmol), CeCl3·7H2O (0.05 mmol) and ethanol (25 mL) was left with stirring at rt for 2 h. The solvent was removed under reduced pressure to give an orange solid containing regioisomers 5a and 5b in 87:13 proportion. These compounds were isolated by column chromatography over silica gel (90:10 CH2Cl2/AcOEt).5a : dark orange solid, mp 169.5–171.5 °C; IR νmax 1722 (C=O ester), 1,696 and 1,638 (C=O quinone); 1H-NMR : δ 2.65 , 3.59 , 3.87 , 4.03 , 6.01 , 9.14 ; 13C-NMR (100 MHz): δ 22.90, 49.21 (2C), 53.28, 66.49 (2C), 110.92, 123.98, 125.36, 134.86, 148.72, 152.78, 161.98, 168.72, 181.16, 181.75; HRMS (M+): m/z calcd for C16H16N2O5: 316.10593; found: 316.10521. Compound 5b (17%): dark red solid, mp 159.5–162 °C; IR νmax 1724 (C=O ester), 1,678 and 1,634 (C=O quinone); 1H-NMR : δ 2.66 , 3.47 , 3.85 , 4.03 , 6.01 , 9.23 ; 13C-NMR (100 MHz): δ 22.89, 49.10 (2C), 53.25, 66.44 (2C), 111.30, 122.43, 125.41, 135.43, 148.35, 153.21, 160.76, 168.54, 182.10, 182.30; HRMS (M+): m/z calcd for C16H16N2O5: 316.10593; found: 316.10445. Compound N-bromosuccinimide (NBS) or N-chlorosuccinimide (NCS) (1 mmol) and methanol (15 mL) was left with stirring at rt after completion of the reaction as indicated by TLC. The solvent was removed under reduced pressure and the residue was column cromatographed over silica gel (90:10 CH2Cl2/AcOEt) to yield the corresponding 7-amino-6-haloisoquinolinequinone derivative.A solution of the 7-aminoisoquinolinequinone derivative (1 mmol), the corresponding 6-Bromo-4-methoxycarbonyl-7-methylamino-3-methylisoquinoline-5,8-quinone (6). Prepared from 2a and NBS : dark red solid, mp 170–172 °C; IR νmax 3331 (N-H); 1730 (C=O ester), 1687 (C=O quinone); 1H-NMR : δ 2.65 , 3.48 , 4.05 , 6.39 , 9.16 ; 13C-NMR (100 MHz): δ 23.09, 33.34, 53.39, 121.29, 126.56, 134.72, 146.86, 148.62, 163.23, 168.42, 174.40, 178.84 ; HRMS (M+): m/z calcd for C13H11N2O4Br: 337.99023; found: 337.98951.6-Chloro-4-methoxycarbonyl-7-methylamino-3-methylisoquinoline-5,8-quinone (7). Prepared from 2a and NCS : dark red solid, mp 158–160 °C; IR νmax 3331 (N-H); 1773 (C=O ester), 1693 (C=O quinone); 1H-NMR : δ 2.65 , 3.48 , 4.05 , 6.30 , 9.15 ; 13C-NMR (100 MHz): δ 23.09, 32.82, 53.37, 121.25, 126.52, 135.11, 144.83, 148.50, 163.41, 168.36, 174.83, 179.27 ; HRMS (M+): m/z calcd for C13H11N2O4Cl: 294.0407; found: 294.040026-Bromo-2-(hydroxyethylamino)-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (8). Prepared from 3a and NBS : dark red solid, mp 134–136 °C; IR νmax 3463 (O-H), 3320 (N-H), 1720 (C=O ester), 1685 (C=O quinone); 1H-NMR : δ 2.63 , 3.92 , 4.06 , 4.11 , 6.65 , 9.13 ; 13C-NMR (100 MHz): δ 22.98, 47.25, 53.40, 61.36, 121.49, 126.41, 134.60, 146.84, 148.61, 163.02, 168.52, 174.19, 178.66, ; HRMS (M+): m/z calcd for C14H13N2O5Br: 368.00079; found: 368.00005.6-Chloro-2-(hydroxyethylamino)-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (9). Prepared from 3a and NCS : dark red solid, mp 123–125 °C; IR νmax 3461 (O-H), 3325 (N-H), 1716 (C=O ester), 1687 (C=O quinone); 1H-NMR : 1H-NMR : δ 2.65 , 3.92 , 4.05 , 4.07 , 6.58 , 9.16 ; 13C-NMR (100 MHz): δ 23.09, 4.82, 53.42, 61.71, 121.38, 126.48, 134.99, 144.53, 148.59, 163.35, 168.41, 174.67, 179.22, ; HRMS (M+): m/z calcd for C14H13N2O5Cl: 324.0513; found: 324.05073.6-Bromo-7-(cyclohexylamino)-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (10). Prepared from 4a and NBS : dark red solid, mp 132.5–134.5 °C; IR νmax 3312 (N-H), 3008 and 2858 (C-H), 1735 (C=O ester), 1687 (C=O quinone); 1H-NMR : δ 1.34 , 1.68 , 1.81 , 2.03 , 2.64 , 4.05 , 4.58 , 6.24 , 9.15 ; 13C-NMR (100 MHz): δ 23.05, 24.52 (2C), 25.29, 34.50 (2C), 53.32, 61.58, 121.36, 126.59, 134.80, 148.65, 149.99, 163.16, 168.38, 174.32, 178.90, ; HRMS (M+): m/z calcd for C18H19N2O4Br: 406.05280; found: 406.05196.6-Bromo-7-morpholino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (11). Prepared from 5a and NBS : violet solid, mp 127.5–129 °C; IR νmax 2955 and 2856 (C-H), 1722 (C=O ester), 1696 and 1638 (C=O quinone); 1H-NMR : δ 2.66 , 3.70 , 3.90 , 4.05 , 9.19 ; 13C-NMR (100 MHz): δ 22.87, 52.59 (2C), 53.42, 67.51 (2C), 115.51, 122.43, 126.05, 133.51, 149.00, 152.08, 162.25, 168.21, 176.25, 180.24; HRMS (M+): m/z calcd for C16H15N2O5Br: 394.01643; found: 394.01457.7-Amino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (12). A solution of sodium azide (1.2 mmol) in water (5 mL) was added, with stirring, to a previously heated solution (~40 °C) of quinone 1 (1 mmol) in glacial acetic acid (10 mL). The mixture was stirred at rt for 3.5 h, diluted with water and then extracted with AcOEt (3 × 15 mL). The organic phase was washed with saturated aqueous solution of NaHCO3, dried (MgSO4) and the solvent was removed under reduced pressure. The residue was column cromatographed over silica gel (80:20 CH2Cl2/AcOEt) to yield aminoquinone 12 as an orange solid (51%), mp 189–191 °C; IR νmax 3376 and 3140 (N-H), 1732 (C=O ester), 1691 (C=O quinone); 1H-NMR : δ 2.66 , 4.05 , 5.38 , 6.03 , 9.20 ; 13C-NMR (100 MHz): δ 23.37, 52.68, 102.44, 122.47, 125.17, 135.92, 147.19, 150.88, 161.00, 168.34, 179.30, 180.65; HRMS (M+): m/z calcd for C12H10N2O4: 246.06406; found: 246.06207.7-Amino-6-bromo-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (13). Prepared from 12 and NBS : mp 146–148 °C; IR νmax 3423 and 3326 (N-H), 1726 (C=O ester), 1683 (C=O quinone); 1H-NMR : δ 2.67 , 4.06 , 5.45 , 6.26 , 9.22 ; 13C-NMR (100 MHz): δ 23.18, 53.48, 105.07, 121.08, 126.91, 134.81, 147.20, 148.50, 163.58, 168.26, 174.53, 177.24; HRMS (M+): m/z calcd for C12H9N2O4Br: 323.97455; found: 323.97419.2 in air at 37 °C. For the experiments, cells were plated at a density of 50,000 cells/mL in 96-well plates. One day after seeding, the cells were treated with the medium containing the compounds at concentrations ranging from 0 up to 100 μM during 3 days and finally the 3--2,5-diphenyltetrazolium bromide (MTT) reduction assay was carried out. The final concentration of MTT was 1 mg/mL. The compounds were dissolved in DMSO  and complete medium. Untreated cells (medium containing 1% DMSO) were used as controls. Each experiment was carried out in sextuplicate.The cell lines used in this work were obtained from the American Type Culture Collection . They included MRC-5 normal human lung fibroblasts (CCL-171), AGS human gastric adenocarcinoma cells (CRL-1739), SK-MES-1 human lung cancer cells (HTB-58) and J82 human bladder carcinoma cells (HTB-1). After the arrival of the cells, they were proliferated in the corresponding culture medium as suggested by the ATCC. The cells were stored in medium containing 10% glycerol in liquid nitrogen. The viability of the cells after thawing was higher than 90%, as assessed by trypan blue exclusion test. Cells were sub-cultured once a week and the medium was changed every two days. Cells were grown in the following media: MRC-5, SKMES-1, and J82 in Eagle's minimal essential medium (EMEM) and AGS cells in Ham F-12. The EMEM medium contained 2 mM L-glutamine, 1 mM sodium pyruvate and 1.5 g/L sodium hydrogen carbonate. Ham F-12 was supplemented with 2 mM L-glutamine and 1.5 g/L sodium hydrogen carbonate. All media were supplemented with 10% heat-inactivated FBS, 100 IU/mL penicillin and 100 μg/mL streptomycin in a humidified incubator with 5% CO1) from 2,5-dihydroxybenzaldehyde, methyl aminocrotonate and silver (I) oxide by employing a highly efficient one-pot procedure. A variety of new isoquinolinequinones substituted with amino, alkylamino and halogen groups were prepared from quinone 1. The members of this series expressed moderate to high in vitro cytotoxic activity against MRC-5  and four human cancer cell lines: AGS (gastric), SK-MES-1 (lung), J82 (bladder), and HL-60 (leukemia) cell lines. From the current investigation, structure activity relationships of the aminoisoquinolinequinone series demonstrate that nitrogen and halogen substituents at the quinone nucleus of pharmacophore show increased antitumor activity compared with precursor 1. The effect of such substitutions is more significant in enhancing the antitumor activity for those members containing the cyclohexylamino group at C-6 (compound 4b), the amino group at C-7 (compound 12) and the amino and bromine substituents at the 6- and 7-positions (compound 13). Due to the high incidence of undesirable side-effects induced by the majority of current anticancer drugs and by considering the selectivity index values of aminoquinones 4b (SI: 0.7–3.7), 12 and 13 (We have carried out the synthesis of 4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (2 and 13 , they ap


Correction to: Oncogenesis10.1038/oncsis.2017.76 Published online 11 September 2017The FAB classification of OCI-M2, GF-D8, CMK-11-5, and THP-1 cell lines in Table


Perilla frutescens (L.) Britton (Lamiaceae) and its hexane fraction on Aβ aggregation. Here, the hexane fraction of P. frutescens was subjected to diverse column chromatography based on activity-guided isolation methodology. This approach identified five asarone derivatives including 2,3-dimethoxy-5-(1E)-1-propen-1-yl-phenol (1), β-asarone (2), 3--(2E)-2-propen-1-ol (3), asaronealdehyde (4), and α-asarone (5). All five asarone derivatives efficiently reduced the aggregation of Aβ and disaggregated preformed Aβ aggregates in a dose-dependent manner as determined by a Thioflavin T (ThT) fluorescence assay. Furthermore, asarone derivatives protected PC12 cells from Aβ aggregate-induced toxicity by reducing the aggregation of Aβ, and significantly reduced NO production from LPS-stimulated BV2 microglial cells. Taken together, these results suggest that asarone derivatives derived from P. frutescens are neuroprotective and have the prophylactic and therapeutic potential in AD.Alzheimer’s disease (AD) is a progressive, neurodegenerative brain disorder associated with loss of memory and cognitive function. Beta-amyloid (Aβ) aggregates, in particular, are known to be highly neurotoxic and lead to neurodegeneration. Therefore, blockade or reduction of Aβ aggregation is a promising therapeutic approach in AD. We have previously reported an inhibitory effect of the methanol extract of  Alzheimer’s disease (AD) is a chronic, progressive neurodegenerative brain disorder associated with declining memory and cognitive function, occurring in middle or late life . The etiAβ has been shown to be a major component of the senile plaques of AD patients. The amyloid precursor protein (APP) found on chromosome 21 is cleaved by proteases, β-secretase (BACE1) and γ-secretase to form the Aβ peptide. Aβ is very prone to aggregation, and this process is key to subsequent AD-related pathologies including tau phosphorylation, neurotoxicity, and clinical dementia ,4. AdditPerilla frutescens (L.) Britton var. acuta Kudo is a perennial member of the Lamiaceae or mint family. It is primarily cultivated in China, Japan, India, Korea, and other Asian countries. It has been used as a traditional medicine to treat inflammatory diseases, depression, and anxiety-related disorders -phenol (1) C11H14O3 MS m/z 195 [M + H]+. 1H NMR (CD3OD) δ: 6.50 , 6.50 , 6.27 , 6.14 , 1.85 , 3.78 , 3.83 . 13C-NMR (CD3OD) δ: 150.1 (C-1), 135.5 (C-2), 153.1 (C-3), 101.3 (C-4), 134.1 (C-5), 106.3 (C-6), 130.8 (C-7), 124.1 (C-8), 17.1 (C-9), 54.9 (C-10), 59.6 (C-11). logP (o/w): 2.370 (est).β-asarone (2) C12H16O3 MS m/z 209 [M + H]+. 1H NMR (CD3OD) δ: 6.68 , 6.88 , 6.44 , 5.71 , 1.83 , 3.88 , 3.81 , 3.80 . 13C-NMR (CD3OD) δ: 118.2 (C-1), 152.0 (C-2), 97.9 (C-3), 149.0 (C-4), 142.3 (C-5), 114.9 (C-6), 124.7 (C-7), 124.6 (C-8), 13.5 (C-9), 56.2 (C-10), 55.4 (C-11), 55.3 (C-12).logP (o/w): 3.406 (est).3--(2E)-2-Propen-1-ol (3) C12H16O3 MS m/z 225 [M + H]+. 1H NMR (CD3OD) δ: 6.66 , 7.08 , 6.85 , 6.25 , 4.22 , 3.88 , 3.84 , 3.82 . 13C-NMR (CD3OD) δ: 117.7 (C-1), 153.1 (C-2), 97.4 (C-3), 150.3 (C-4), 144.1 (C-5), 110.7 (C-6), 125.8 (C-7), 128.6 (C-8), 62.9 (C-9), 56.0 (C-10), 55.6 (C-11), 55.2 (C-12). logP (o/w): 1.290 (est).asaronealdehyde (4) C10H12O4 MS m/z 197 [M + H]+. 1H NMR (CD3OD) δ: 6.73 , 7.28 , 10.2 , 3.97 , 3.96 , 3.82 . 13C-NMR (CD3OD) δ: 116.7 (C-1), 159.4 (C-2), 96.3 (C-3), 156.8 (C-4), 143.6 (C-5), 109.0 (C-6), 188.1 (C-7), 55.5 (C-8), 55.4 (C-10), 55.4 (C-11). logP (o/w): 1.634 (est).α-asarone (5) C12H16O3 MS m/z 209 [M + H]+. 1H NMR (CD3OD) δ: 6.62 , 7.01 , 6.11 , 4.89 , 1.86 , 3.85 , 3.81 , 3.80 . 13C-NMR (CD3OD) δ: 119.0 (C-1), 151.1 (C-2), 98.1 (C-3), 149.0 (C-4), 143.2 (C-5), 110.5 (C-6), 125.0 (C-7), 123.1 (C-8), 17.5 (C-9), 56.0 (C-10), 55.6 (C-11), 55.2 (C-12). logP (o/w): 2.794 (est).1–42 was dissolved in DMSO at 1 mg/mL concentration and five asarone derivatives were diluted in DMSO. To monitor the effects of asarone derivatives on the aggregate of Aβ, 20 µM of Aβ1–42 was incubated together with various concentrations  of asarone derivatives at 37 °C for 24 h. Then, 3 µM of ThT was added (protected from light) and fluorescence was measured after 30 min using an Emax precision microplate reader (black plate)  with excitation at 442 nm and emission at 485 nm. The Aβ treated with DMSO was used as a control and each assay was performed in triplicate.To evaluate the aggregate formation of Aβ, a ThT assay was performed . The Aβ11–42 was incubated at 37 °C for 24 h. Various concentrations  of asarone derivatives were then added for an additional incubation at 37 °C for additional 24 h (protected from light). Then, 3 µM of ThT was added and fluorescence was measured after 30 min using an Emax precision microplate reader (black plate) with excitation at 442 nm and emission at 485 nm. Aβ only-treated group was used as a control and each assay was performed in triplicate.To monitor the disaggregation effects of asarone derivatives on pre-aggregated Aβ, a ThT assay was performed. Briefly, 20 µM of Aβ2 atmosphere at 37 °C. The cells growing exponentially were harvested from flasks and plated in 96-well plates with approximately 6 × 104cells per 100 µL of medium per well. Plates were incubated at 37 °C for 3 h to allow the cells to attach to the plates.PC12 cells (rat pheochromocytoma cells) were obtained from Korea Cell Line Bank  and grown in RPMI 1640 medium supplemented with 5% heat-inactivated FBS and 15% heat-inactivated horse serum. The cells were incubated in a humidified 5% COIn order to determine the possible cytotoxicity of asarone derivatives themselves, compounds were added to individual wells . The plates were then incubated for 24 h at 37 °C. Cell viability was determined using a MTT toxicity assay by adding 10 µL of 5 mg/mL MTT to each well. After 3 h of incubation at 37 °C, 100 µL of DMSO was added to each well. Plates were incubated at room temperature for 30 min to dissolve the MTT formazan crystals and the absorbance at 570 nm was measured using a Emax precision microplate reader. Averages from three replicate wells were used for each sample and each assay was performed in triplicate.25–35, 10 µM). The plates were then incubated for an additional 24 h at 37 °C. Cell viability was determined using a MTT toxicity assay. Averages from three replicate wells were used for each sample and each assay was performed in triplicate.In order to determine the protective effect of asarone derivatives against Aβ, the cells in 96-well plates were pre-treated with asarone derivatives  for 1 h and then followed by the treatment with Aβ (Aβ1–42 (10 µM) were mixed together in 96-well plates and incubated at 37 °C for 24 h to inhibit the formation of Aβ aggregates. Then, the mixture was added to the cells in 96-well plates and incubated at 37 °C for an additional 24 h. Cell viability was determined using a MTT toxicity assay. Averages from three replicate wells were used for each sample and each assay was performed in triplicate.To investigate whether the inhibition of Aβ aggregation by asarone derivatives affects the viability of PC12 cells against Aβ aggregate-induced toxicity, compounds  and Aβ2 atmosphere at 37 °C. BV2 cells growing exponentially were harvested from flasks and plated in 96-well plates with approximately 6 × 104cells per 100 µL of medium per well. Plates were incubated at 37 °C for 3 h to allow the cells to attach to the plates. Compounds  were added to individual wells. After 1 h, LPS was added in each culture well with 1 µg/mL of concentration. The plates were then incubated for an additional 24 h at 37 °C. The NO production was determined using Griess reagents [BV2 cells  were grown in a DMEM medium supplemented with 5% heat-inactivated FBS. The cells were incubated in a humidified 5% COreagents . The supp < 0.05.All data in the text and figures are expressed as means ± SD. Two or more group comparisons were evaluated by one-way analysis of variance followed by Tukey post hoc text . Differences were considered statistically significant at


Tagetes minuta L. (Asteraceae) leaves resulted in the isolation and identification of two new compounds: 5-methyl-2,2',5',2'',5'',2''',5''',2''''-quinquethiophene (1) and quercetagetin-6-O-(6-O-caffeoyl-β-d-glucopyranoside) (9), in addition to seven known compounds: quercetin-3,6-dimethyl ether (2), quercetin-3-methyl ether (3), quercetin (4), axillarin-7-O-β-d-glucopyranoside (5), quercetagetin-3,7-dimethoxy-6-O-β-d-glucopyranoside (6), quercetagetin-7-methoxy-6-O-β-d-glucopyranoside (7), and quercetagetin-6-O-β-d-glucopyranoside (8). The compounds were identified by UV, IR, 1D, 2D NMR, and HRESIMS spectral data. They showed significant antioxidant activity, comparable with that of propyl gallate. Compounds 8 and 3 showed weak to moderate antileishmanial and antimalarial activities, with IC50 values of 31.0 μg/mL and 4.37 μg/mL, respectively.Phytochemical investigation of the methanolic extract of  Tagetes (Asteraceae) is mainly native to the central and southern part of America. It consists of approximately 30 species + and m/z 318.0299 [M+H-(caffeoyl+hexose)]+. The 1H-NMR spectrum revealed the presence of six singlets signals at δH 6.59 (H-8), 8.50 (3-OH), 9.35 (3'-OH), 9.35 (4'-OH), 10.89 (7-OH), and 12.24 (5-OH). Also, it showed three coupled protons at δH 6.88 , 7.57 , and 7.74  for a tri-substituted B-ring  indicated β-configuration of the glycosidic linkage D −176 . UV (MeOH): λmax 275, 365 nm; +NaOMe: 282, 405 nm; +AlCl3: 295, 410 nm; +AlCl3/HCl: 293 388 nm; +NaOAc: 296, 385 nm; +NaOAc/H3BO3: 280, 385 nm. IR (KBr): maxν 3460, 2976, 1668, 1608, 1565, 1058 cm−1. NMR data: see m/z 643.1223 .9 (3 mg) was refluxed in 10 mL of 1 N HCl for 4 h. The aglycone was extracted with CHCl3. The sugar in the aqueous layer was identified by co-paper chromatography (PC) with authentic materials using solvent system (S5) and aniline phthalate spray as detection reagent [Compound  reagent .2–9 were tested for antimicrobial activity against Candida albicans ATCC 90028, Candida glabrata ATCC90030, Candida krusei ATCC 6258, Asperigillus fumigates ATCC 90906, methicillin-resistant Staphylococcus aureus ATCC 33591, Cryptococcus neoformans ATCC 90113, Staphylococcus aureus ATCC 2921, Escherichia coli ATCC 35218, Pseudomonus aeruginosa ATCC 27853, and Mycobacterium intracellulare ATCC 23068 as described previously [All the isolated compounds eviously ,23,24. CPlasmodium falciparum using previously reported method [The isolated compounds were tested on chloroquine sensitive  and resistant  strains of d method ,25. Artein vitro against a culture of L. donovani promastigotes as previously outlined [The anti-leishmanial activity of the isolated metabolites was tested outlined . Pentami2–9 (20 μM) in DPPH solution (4 mg was dissolved in HPLC MeOH 50 mL to obtain a concentration 80 μg/mL) was determined as previously outlined [The antioxidant activity of the isolated compounds outlined ,28,29.T. minuta L. two of them (compounds 1 and 9) are new. The antioxidant, antimicrobial, antimalarial, and antileishmanial activities of the isolated compounds were evaluated. They showed antioxidant activity ranging from 91.6% to 68.3%. Compound 8 showed weak antileishmanial activity with an IC50 31.0 μg/mL, while compound 3 showed moderate antimalarial activity against chloroquine sensitive (D6) clones of P. falciparum with an IC50 4.37 μg/mL.In conclusion, in this study nine compounds were isolated and elucidated from


Ganoderma resinaceum is usually used as ethnomedicine for immune-regulation, hyperglycemia, and liver disease. To date, only a few chemical constituents have been reported from G. resinaceum. In this study, fifteen nortriterpenoids including six new nortriterpenoids (1–6) and nine known analogs (7–15), were separated and purified from the fruiting bodies of G. resinaceum. New compounds were identified as lucidone I (1), lucidone J (2), lucidone K (3), lucidone I (4), ganosineniol B (5), and ganosineniol C (6), based on analysis of extensive spectroscopic data (high resolution mass spectrometry (HRMS), nuclear magnetic resonance (NMR), infrared (IR), and ultraviolet (UV)). The known compounds were assigned as lucidone A (7), lucidone B (8), lucidone H (9), lucidone E (10), lucidone F (11), lucidone D (12), lucidone C (13), ganoderense F (14), and ganosineniol A (15), by comparing their spectroscopic data with those reported in the literature. Compounds 3, 4, and 7–13 were examined for α-glucosidase inhibitory activity and display no significant activity, but the finding may support that the side chain of ganoderma triterpenoids played an important role in α-glucosidase inhibitory activity. Ganoderma − . Its IR spectrum showed the presence of hydroxy group (3429 cm−1) and carbonyl group (1717 cm−1). 1H-NMR spectrum showed the presences of six singlet methyl signals . Heteronuclear single quantum coherence (HSQC) and 13C-NMR spectra displayed 24 carbon resonances, including six methyls, five methylenes, five methines including three oxygenated carbon resonances at δC 79.5, 66.9 and 65.3, and eight quaternary carbons including a tetrasubstituted olefinic carbon δC 136.8 and 146.2, and two ketone carbons δC 220.2 and 209.3. The above spectroscopic data suggested 1 to be nortriterpenoid similar to lucidone A (7). However, a comparison of their 13C-NMR data showed that 1 possesses an additional oxygenated methine at δC 65.3 but absent of one ketone group signal at δC 196.7 in 7. Moreover, the chemical sifts of tetrasubstituted olefinic carbons of 1 in 13C-NMR spectrum were significantly different from those of 7 due to the disappearance of α,β-unsaturated ketone conjugated system at C-8/C-9/C-11. Accordingly, it was assumed that 1 was the 11-OH derivative of 7. The 11-OH was further confirmed on the basis of the heteronuclear multiple bond correlation (HMBC) correlations from H-11 (δH 4.48) to C-8 (δH 136.8), C-9 (δC 146.2) and C-13 (δC 43.8), and H-12β (δH 2.00) to C-11 (δH 65.3), and the hydrogen-hydrogen correlation spectroscopy (1H–1H COSY) correlation of H-11 (δH 4.48) with H-12α (δH 2.51). Finally, the planar structure of 1 was established by 1D and 2D NMR spectra.Compound 1 was established by analyses of rotating-frame nuclear overhauser effect correlation spectroscopy (ROESY) experiment and coupling constant. The ROESY correlations (H 1.08) with H-12α (δH 2.51) and H-7 (δH 4.54), H-12α with H-11 (δH 4.48), and H-3 (δH 3.08) with H-28 (δH 0.90) and H-5 (δH 0.93) suggested 3-, 7- and 11-OH to be β-orientation, which were also supported by the larger coupling constant . Detail analyses of 13C-NMR  to C-20 (δC 69.9) and C-17 (δC 48.9), as well as the 1H–1H COSY correlations of H-20 (δH 3.79) with H-21 (δH 1.20) and H-17 (δH 2.28). The 3- and 7-hydroxy groups were assigned as β- and β-orientation, respectively, on the basis of ROESY correlations of H-3 with H-28 and H-5, H-7 with H-5 and H-30. The ROESY correlations of H-17 with H-30, and H-20 with H-18 . The IR and UV spectra revealed the presence of hydroxy group (3484 cm−1), carbonyl group (1722 cm−1) and α,β-unsaturated carbonyl group (1689 cm−1 and 259 nm). Comparison of spectroscopic data of 3 with that of 2 displayed their structural similarities except for the presence of ketone group to replace oxygenated methine. The ketone group at C-7 was confirmed by the key HMBC correlations from H-5 (δH 1.50) and H-6 (δH 2.60 and 2.40) to C-7 (δC 202.3). The 20α-OH was confirmed on the basis of ROESY correlations of H-17 (δH 2.28) with H-30 (δH 1.52), and H-18 (δH 0.80) with H-20 (δH 3.65)  . Accordi4 was obtained as pale yellow powder. Its molecular formula was assigned as C24H34O4 by HRESIMS at m/z 445.2583 [M + CH3COOH − H]− . The hydroxy group (3441 cm−1), carbonyl group (1743 cm−1) and α,β-unsaturated carbonyl group (1680 cm−1) were observed in IR spectrum. The UV absorption at λmax 260 nm showed the presence of α,β-unsaturated carbonyl. A comparison of NMR data of 4 with those of 3 revealed their structural similarities. The major differences were the existence of triplet methyl signal but absent of an oxygenated methine at δC 69.9, suggesting the disappearance of 20-OH in 4. The HMBC correlations from H-21 (δH 0.99) to C-20 (δC 32.9) and C-17 (δC 46.2), combined with the 1H–1H COSY correlations of H-20 (δH 2.13) with H-17 (δH 2.34) and H-21 (δH 0.99), confirmed that the ethyl group was attached to C-17. The 3-OH was assigned as β-orientation by the ROESY correlations of H-3 with H-28 and H-5. Therefore, compound 4 was determined as 3β-hydroxy-4,4,14α-trimethyl-7,11,15-trioxo-5α-pregn-8-ene, named lucidone L.Compound 5 was isolated as white powder. Its molecular formula was established as C25H36O5 based on HRESIMS at m/z 415.2488 [M − H]− . The IR spectrum showed the presence of hydroxy group (3436 cm−1) and α,β-unsaturated carbonyl group (1664 cm−1). The UV absorption brand λmax 244 nm also supported the presence of α,β-unsaturated carbonyl group. Detail comparison of 1 D NMR spectroscopic data with those of ganoderense F (14) suggested their structural similarities except that the hydroxy group at C-3 replaced the ketone group in 5. The 3-OH was confirmed by the HMBC correlations from H-28 (δH 1.07) and H-29 (δH 0.88) to C-3 (δC 78.2), and H-3 (δH 3.26) to C-28 (δC 28.2) and C-29 (δC 15.6), as well as the 1H–1H COSY correlation of H-3 (δH 3.26) with H-2 (δH 1.70). The hydroxy groups at C-3 and C-7 were all assigned as β-orientation on the basis of the ROESY correlations (H 3.26) with H-28 (δH 1.07) and H-5 (δH 0.98), and H-7 (δH 4.84) with H-5 (δH 0.98) and H-30 (δH 1.58), Thus, compound 5 was elucidated as 20-carbinol-3β,7β-dihydroxy-4,4,14α-trimethyl-11,15-trioxo-5α-pregn-8,16-dien, named ganosineniol B.Compound elations  of H-3 . The presence of hydroxy group (3432 cm−1), carbonyl group (1722 cm−1) and α,β-unsaturation group (1655 cm−1 and 254 nm) were confirmed by IR and UV spectra. Detail analyses of 1H, 13C-NMR and HSQC spectra revealed that the structure of 6 was highly similar to that of ganosineniol A (15). The difference between those two constituents was that one oxymethine was converted into ketone in 6. The 13C-NMR spectrum of 6 did not show any signal at around δC 72.6, but revealed an additional downfield shift at δC 218.9. Hence, it was inferred that the ketone was located at C-15. The hypothesis was proved by the key HMBC correlations from H-30 (δH 1.30) and H-16 (δH 2.68 and 2.10) to C-15 (δC 218.9). The ROESY correlations of H-3 (δH 3.08) with H-28 (δH 0.95) and H-5 (δH 0.87), and H-7 (δH 4.78) with H-5 (δH 0.87) and H-30 (δH 1.30) assigned 3-OH and 7-OH as β-orientation. Accordingly, compound 6 was determined as 20-carbinol-3β,7β-dihydroxy-4,4,14α-trimethyl-11,15-dioxo-5α-pregn-8-dien, named ganosineniol C.Compound Ganoderma. The above-mentioned nortriterpenoids are considered to originate from triterpenoid due to the degradation of its side chain. A possible biogenetic pathway for C24 nortriterpenoids is proposed as shown in 1, and 7–13). Compounds 7 and 12 were further transformed into 2 and 3, respectively, through an addition reaction. Finally, compound 3 (intermediate B) generated 4 (D) via elimination reaction and addition reaction.Triterpenoids, biogenetically derived via mevalonic acid pathway, are the main secondary metabolites of G. resinaceum has been used as ethnomedicine for lowering blood sugar in Nigeria − .White powder; c 0.38, MeOH); UV (MeOH) λmax (log ε) 202 (3.45), 253 (3.76) nm; IR (KBr) vmax 3432, 2964, 2925, 2866, 1731, 1654, 1457, 1383, 1306, 1172, 1058, 1035 cm−1; 1H- and 13C-NMR spectroscopic data see m/z 403.2481 [M − H]− .White powder; c 0.17, MeOH); UV (MeOH) λmax (log ε) 201 (3.53), 259 (3.68) nm; IR (KBr) vmax 3484, 2982, 2932, 2862, 1742, 1689, 1457, 1426, 1379, 1338, 1300, 1232, 1200, 1163, 1082, 1034, 976 cm−1; 1H- and 13C-NMR spectroscopic data see m/z 401.2352 [M − H]− . Pale yellow powder; c 0.74, MeOH); UV (MeOH) λmax (log ε) 202 (3.49), 260 (3.58) nm; IR (KBr) vmax 3441, 2974, 2935, 2875, 1743, 1680, 1461, 1383, 1199, 1088, 1035, 928 cm−1; 1H- and 13C-NMR spectroscopic data see m/z 445.2583 [M + CH3COOH − H]− .Pale yellow powder; c 0.26, MeOH); UV (MeOH) λmax (log ε) 201 (3.62), 244 (3.83), 341(2.05) nm; IR (KBr) vmax 3436, 2967, 2928, 2872, 1664, 1599, 1457, 1379, 1247, 1171, 1102, 1037 cm−1; 1H- and 13C-NMR spectroscopic data see m/z 415.2488 [M − H]− .White powder; c 1.44, MeOH); UV (MeOH) λmax (log ε) 202 (3.44), 254 (3.66), 341(2.04) nm; IR (KBr) vmax 3432, 2968, 2928, 2866, 1722, 1655, 1459, 1380, 1270, 1176, 1034 cm−1; 1H and 13C-NMR spectroscopic data see m/z 417.2629 [M − H]− .White powder; α-Glucosidase inhibitory activity was examined by the method described by Dengqiang Li et al. . Acarbos1–6), together with nine known analogs (7–15), were isolated and identified from the fruiting bodies of Ganoderma resinaceum. The possible biogenetic pathway for C24 nortriterpenoids was proposed Compounds 3, 4, and 7–15 displayed no significant α-glucosidase inhibitory activity, however, this finding may support that the side chain of ganoderma triterpenoids is critical for α-glucosidase inhibitory activity, especially the presence of carboxyl acid group.In summary, six new nortriterpenoids (


Sangre de drago), a viscous red sap derived from Croton lechleri Muell-Arg (Euphorbiaceae), is extensively used by indigenous cultures of the Amazonian basin for its wound healing properties. The aim of this study was to identify the minor secondary metabolites and test the antioxidant activity of this sustance. A bio-guided fractionation of the n-hexane, chloroform, n-butanol, and aqueous extracts led to the isolation of 15 compounds: three megastigmanes, four flavan-3-ols, three phenylpropanoids, three lignans, a clerodane, and the alkaloid taspine. In addition to these known molecules, six compounds were isolated and identified for the first time in the latex: blumenol B, blumenol C, 4,5-dihydroblumenol A, erythro-guaiacyl-glyceryl-β-O-4’-dihydroconiferyl ether, 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-propane-1,3-diol and floribundic acid glucoside. Combinations of spectroscopic methods , ESI-MS, and literature comparisons were used for compound identification. In vitro antioxidant activities were assessed by DPPH, total antioxidant capacity and lipid peroxidation assays. Flavan-3-ols derivatives (as major phenolic compounds in the latex) exhibited the highest antioxidant activity. Dragon’s blood ( Croton lechleri (Muell-Arg) (Euphorbiaceae), is a tree which grows in the low mountainous areas of the Peruvian Andean regions, as well as in Colombia, Ecuador and Bolivia and it is known for its therapeutic properties. The bark, when slashed, releases a reddish or yellowish latex called “sangre de drago” or “sangre de grado” or “dragon’s blood”. The blood-red latex or sap is a common household remedy in Peru and in other Latin American countries, where indigenous tribes use it internally and externally to stop bleeding, help heal wounds, and treat intestinal ailments -propane-1,3-diol (5) and 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-propane-1,3-diol. (6). Lignans were reported in the latex of various oton sp. . Along w5, also termed erythro-guaiacyl-glycerol-β-O-4’-dihydroconiferyl ether, was identified on the basis of its spectroscopic data. The EI-MS spectrum showed a molecular ion peak at m/z 378 [M]+ which is consistent with a molecular formula of C20H26O7.Compound 1H-NMR , a doublet at δ 6.77 (J= 8.1 Hz) and at δ 6.87 . The second aromatic system was 1’,3’,4’-trisubstituted and characterized by signals at δ 6.98 , δ 6.73  and δ 6.87 . The COSY spectrum also indicated the presence of a glycerol-type moiety from C-7 to C-9 . In accordance with the erythro relative configuration, a coupling constant value of 4.7 Hz between H-7 and H-8, was observed -propane-1,3-diol, also known with the trivial name erythro-guaiacyl-glycerol-β-O-4’-dihydroconiferyl ether. In the literature, this compound has only been previously reported following isolation from Pinus silvestris + peak at m/z 256. The 1H-NMR (1H-1H COSY spectra showed two one-proton doublets at δΗ 6.99 (J=8.1Hz) and δΗ 6.86 (J=1.5 Hz) and a one-proton double doublet at δΗ 6.74 , suggesting a typical 1,2,4-trisubstituted aromatic ring. These spectra also included a singlet at δ 3.84, ascribable to a methoxyl function, a sequence of three methylene groups , indicating a propanol side chain and a quintuplet at δ 4.15 (H-2) coupled with four protons resonating at δ 3.75 (H2-1 and H2-3). The structure of 6 and the complete assignment of its 1H- and 13C-NMR data was accomplished by HSQC and HMBC experiments. The key correlations in the HMBC allowed the arrangement of the three substituent groups on the aromatic ring. The correlation between H-2 (δ 4.15) and C-1’ (146.5 ppm) revealed that the glycerol moiety was linked to C-1’; the correlation of H-3’ (δ 6.86) and C-2’ (151.6 ppm) showed a 2-OMe position; the correlation of H-3’ (δ 6.86) with C-7’ (32.4 ppm) established the location of propanol side chain in C-4’. Most relevant ROESY connectivity were observed between –OCH3 (δ 3.84) and H-3’ and H2-1 (δ 3.75) with H-6’ (δ 6.99). Thus the structure of 6 was defined as 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-propane-1,3-diol. This compound was previously identified in wine from Vitis vinifera and has been described as the corresponding peracetylated derivative  and purified by HPLC with MeOH-H2O (15:85) as the mobile phase. Fractions 2, 3, and 4 contained mainly 7 (5.2 mg), 10  and 9 (3.0 mg), respectively. The CHCl3 extract was chromatographed by DCCC using CHCl3-MeOH-H2O (7:13:8) in ascending mode (the lower phase was the stationary phase). Six fractions (A-F) were obtained and purified by HPLC as follows. Fraction A was purified with MeOH-H2O (1:9) and contained 7 (2.9 mg) and 8 (4 mg); fraction B, eluted with MeOH-H2O (15:85), contained 6 (1.6 mg); fraction C contained mainly 14 (1.2 mg) and was eluted with MeOH-H2O (4:6); fraction D, eluted with MeOH-H2O (2:8), contained 3 (9.5 mg), 2 (2.2 mg), 12 (0.8 mg), and 5 (1.3 mg); Compounds 4 (3.9 mg), 11 (2.8 mg) and 13 (1.7 mg) were obtained from fraction E, eluted with MeOH-H2O (25:75); fraction F, eluted with MeOH-H2O (3:7), contained mainly 1 (8.6 mg).A small portion (150 mL) of sap was dissolved in MeOH-Hg method . The watC. lechleri latex (50 mL) was lyophilized to yield 7.6 g of reddish-powdered latex. A portion (3.0 g) of the powdered latex was combined with distilled H2O (150 mL) and acidified with concentrated HCl. The aqueous layer was continuously extracted with CHCl3 for 12 h and its pH was then adjusted to 8 with concentrated NH4OH. This fraction was then continuously extracted with CHCl3 over 12 h to yield the first basic fraction. Following adjustment of pH to 10 with NH4OH, the aqueous layer was extracted for an additional 12 h with CHCl3 to yield the second basic fraction. TLC examination of the two basic fractions with Dragendorff’s reagent showed reagent-positive component in the second basic fraction. This fraction was dissolved in MeOH with warming, and 15 (11.3 mg) was obtained as a white precipitate upon refrigeration.EI-MS data: Blumenol B (1): m/z 224 [M]+ (C13H20O3); Blumenol C (2): m/z 208 [M]+ (C13H20O2); 4,5-Dihydroblumenol A (3): m/z 226 [M]+ (C13H22O3); 3’,4-O-Dimethylcedrusin (4): m/z 374 [M]+ (C21H26O6); (–)-Epicatechin (7): m/z 290 [M]+ (C15H14O6); (+)-Catechin (8): m/z 290 [M]+ (C15H14O6); (–)-Epigallocatechin (9): m/z 306 [M]+ (C15H14O7); (+)-Gallocatechin (10): m/z 306 [M]+ (C15H14O7); Compound11: m/z 224 [M]+ (C12H16O4); Compound12: m/z 196 [M]+ (C11H16O3); Compound13: m/z 226 [M]+ (C12H18O4); Floribundic acidglucoside (14): m/z 529 [M+Na]+ (C26H34O10); Taspine (15): m/z 369 [M]+ (C20H19NO6).(±) erythro-Guaiacyl-glycerol-β-O-4’-dihydroconiferyl ether (5):  = 0° ; EI-MS m/z 378 [M]+ (C20H26O7); 1H- and 13C-NMR see 2-[4-(3-Hydroxypropyl)-2-methoxyphenoxy]-propane-1,3-diol (6): EI-MS m/z 256 [M]+ (C13H20O5); 1H- and 13C-NMR see Total phenols of the extracts and fractions were quantified by the Folin-Ciocalteau spectrophotometric assay, using gallic acid as the reference standard . Therefo2+ to Cu+).  [The total antioxidant capacity of the samples at concentrations of 10 and 1 μM was determined by a validated method based upon the copper reduction  . The ref50 was calculated by employing Prism® 4 (GraphPad Software Inc.). Suitable aliquots of a methanolic solution containing each sample, at increasing concentrations from 1 μM to 100 μM, were added to 15 μM ethanol solution of 2,2-diphenyl-2-picrylhydrazyl radical (DPPH). Absorbance was read at 517 nm after 15 min of incubation in the dark . The IC5


Platycodon grandiflorum. On the basis of acid hydrolysis, comprehensive spectroscopic data analyses and comparison with the spectral data of the known compounds, its structure was elucidated as 3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-2β,3β,16α,23-tetrahydroxyolean-12-en-28-oic acid 28-O-β-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside. The six known compounds were platycodin D (2), deapioplatycodin D (3), platycodin D3 (4), deapio- platycodin D3 (5), platycoside E (6) and deapioplatycoside E (7).A new oleanane-type triterpenoid saponin, named platycoside N (1), together with six known saponins, was isolated from the roots of  Platycodon grandiflorum A. DC (Campanulaceae), Platycodi Radix, are traditionally used as food and a herbal medicine in the treatment of a wide range of diseases, including bronchial asthma, hepatic fibrosis, bone disorders -, HR-ESI-MS m/z 1105.5408 [M-H]- . 1H-NMR  δ: 0.89 × 2, 0.98, 1.17, 1.58 × 2 , 1.59 , 4.36 , 3.82, 4.60 , 4.72, 5.10 × 2 , 5.68 , 6.27 , 5.46  5.03 . 13C-NMR  data: see Dry and powdered roots of 1 (2.0 mg) was refluxed with 4.0 M HCl (5.0 mL) for 1 h at 95 °C, and the reaction mixture was extracted with ethyl acetate. The aqueous layer was then adjusted to pH 7.0 with NaHCO3. After evaporating to dryness, the sugar mixture was dissolved in pyridine and developed on silica gel TLC [CHCl3-MeOH-H2O , n-BuOH-AcOH-H2O . Three spots were seen on the TLC after spraying with 4% α-naphthol-EtOH-5% H2SO4. Through comparison with authentic sugar standards (purchased from Sigma), it was found that compound 1 possessed D-glucose, L-rhamnose and L-arabinose units.Compound 1), together with six known saponins from the roots of Platycodon grandiflorum. In summary, we have isolated a new oleanane-type triterpenoid saponin, named platycoside N (


Four new compounds, phaindole (1), -phaithrene (2), methyl 3-hydroxy-4,5-dimethoxypropiophenone (3), and methyl hematinate (4), as well as 44 known compounds were isolated from the MeOH extract of Phaius mishmensis. The structures of the compounds were determined using spectroscopic methods.The partitioned  Phaius mishmensis (Orchidaceae), a native orchid of Taiwan D −18.5°) was obtained as a colorless powder and determined to have the molecular formula C47H67O7, owing to the pseudo-molecular ion peak at m/z 743.4883 in HR-FABMS. The IR spectrum showed a broad absorption at 3399 cm−1 corresponding to a hydroxyl group and a strong absorption at 1728 cm−1 corresponding to a carbonyl group. The 1H-NMR (300 MHz) and 13C-NMR (75 MHz) spectral data of compound 2 were shown in 1H-NMR and COSY spectra, three mutually coupled proton signals at δ 6.69 ppm , 6.71 ppm , and 8.09 ppm  corresponding to a trisubstituted benzene ring and singlet proton signal at δ 6.53 ppm  for a pentasubstituted benzene ring were observed. In the aliphatic region of the 1H-NMR spectrum, an ethylene proton signal at δ 2.66 ppm  was observed. Using HMQC, HMBC, and NOESY, key long-range 1H-13C correlations of H-1 with C-10 (δ 29.7 ppm); H-4 with C-4b (δ 116.8 ppm); H-9 with C-4b , C-8 (δ 113.9 ppm), C-10a (δ 139.3 ppm); H-10 with C-1 (δ 114.1 ppm) and C-8a (δ 136.8 ppm), as well as a key NOE correlation between H-1 and H-10 revealed a 2,5,7,8-tetrasubstituted 9,10-dihydrophenanthrene nucleus. Three very down-field–shifted 13C- signals at C-2 (δ 153.4 ppm), C-5 (δ 158.5 ppm), and C-7 (δ 159.0 ppm) together with the NOE correlations between a hydroxyl group (δ 4.76 ppm) and H-1, -3, a methoxyl group (δ 3.87 ppm) and H-4, -6, revealed a hydroxyl (2-OH), a methoxyl (5-OCH3), and oxygenated substituents (7-OR). The remaining three unresolved aromatic proton signals at δ 6.88 ppm , a hydroxyl signal at δ 5.63 ppm (4′-OH), a methoxyl signal at δ 3.88 ppm (3′-OCH3), -CHCH- proton signals at δ 4.21 ppm  and 5.93 ppm , together with a carbonyl signal at δ 172.4 ppm (C-9'), revealed a 7′-oxygenated-8′-alkyl dihydroferulate moiety. The HMBC correlations of 4′-OH with C-3′ (δ 146.7 ppm) and C-5′ (δ 114.5 ppm); 3′-OCH3 with C-3′ (δ 146.7 ppm); H-7′ with C-2′ (δ 108.0 ppm), C-6′ (δ 118.6 ppm), and C-9′; H-8′ with C-1′ (δ 132.8 ppm) and C-9 confirmed the presence of a ferulate moiety. A 22-long chain alcohol at δ 0.88 ppm , 1.25 ppm , 1.62 ppm , and 4.15 ppm  formed docosyl ferulate, as noted by the HMBC correlation of H-1′′ with C-9′. Finally, the HMBC correlation of H-7′ with C-7, -8; H-8′ with C-7; and the NOE correlation between H-8′ and H-9 revealed that the ferulate was fused to the phenanthrene ring to form a furanophenanthrene. Based on a report by Juhasz , 3,3 + .-Phaithrene (2): colorless amorphous powders; [α]D − 18.5° ; UV (CHCl3) λmax (log ε) 240 (3.94), 282 (4.01) nm; IR (KBr) νmax 3399, 1728, 1609 cm−1; FABMS m/z (rel. int.) 743 , 389 (100); HR-FABMS m/z 743.4883 [M + H]+ .3-Hydroxy-4,5-dimethoxypropiophenone (3): colorless needles, UV (CHCl3) λmax (log ε) 238 (3.47), 274 (3.82) nm; IR (KBr) νmax 3390, 1680 cm−1; EIMS m/z (rel. int.) 210 , 181 (100); HR-EIMS m/z 210.0894 [M]+ .Methyl hematinate (4): white powder; UV (MeOH) λmax (log ε) 230 (3.80), 270 (3.06) nm; IR (film) νmax 3295, 2955, 1776, 1731, 1714 cm−1; FABMS m/z (rel. int.) 198 , 149 (100); HR-FABMS m/z 198.0767 [M + H]+ .3 cells per well in 96-well plates overnight and then treated with different concentrations of the isolated compounds. After 48 h, MTS cell proliferation assay kit  was added to each well; then, the experiment was performed as the manufacturer recommended (Promega). The absorbance was measured at 490 nm on a MQX200R microplate reader .The cytotoxicity assay was carried out according to a procedure described previously . Carcino1), -phaithrene (2), methyl 3-hydroxy-4,5-dimethoxypropiophenone (3), and methyl hematinate (4), and 44 known compounds were isolated from P. mishmensis. Most of the isolated compounds, except tryptanthrin (13) and phaitanthrin-A (14), did not exhibit any significant cytotoxicity against the MCF-7, NCI-H460, and SF-268 cell lines. Phaitanthrin-A (14), an aldol adduct of tryptanthrin with acetone, exhibited better solubility than compound 13 in commonly used solvents . Derivatives using tryptanthrin as a template are being produced in our laboratories to obtain tryptanthrin derivatives with better solubilities and stronger tumor-selective toxicities.Four new compounds, phaindole (


Synthesis, biological evaluation, and structural studies of a kinase targeted 3-aminopyridin-2-one-based library identified ligand efficient inhibitors of MPS1 and Aurora kinases. 1H-pyrazol-4-yl)pyridin-2(1H)-one (2) and 3-amino-5-(pyridin-4-yl)pyridin-2(1H)-one (3) as ligand efficient inhibitors of the mitotic kinase Monopolar Spindle 1 (MPS1) and the Aurora kinase family. These kinases are well recognised as attractive targets for therapeutic intervention for treating cancer. Elucidation of the binding mode of these fragments and their analogues has been carried out by X-ray crystallography. Structural studies have identified key interactions with a conserved lysine residue and have highlighted potential regions of MPS1 which could be targeted to improve activity and selectivity.Screening a 3-aminopyridin-2-one based fragment library against a 26-kinase panel representative of the human kinome identified 3-amino-5-(1-methyl- This represents the ratio of compounds in a screening library that inhibit the particular kinase > 50%.Owing to the successful application of structural biology to the development of small-molecule protein kinase inhibitors from fragments, fragment-based drug design (FBDD) has become an area of sustained interest.T-cell kinase (Itk) that are based on the 3-aminopyridin-2-one fragment 1 have been reported.1 yielding potent Itk inhibitors,The identification of novel kinase inhibitor scaffolds is highly desirable in order to develop selective kinase inhibitors. Small-molecule inhibitors of Interleukin-2-inducible 2in situ (15) was synthesised in two steps from the phosphodiesterase inhibitor Milrinone (13). Hydrolysis of the nitrile 13 to the carboxamide intermediate 14 by treatment with concentrated sulfuric acid at 120 °C, was followed by a Hofmann rearrangement to give compound 15  gave high percentage inhibition against a large number of kinases in the panel, resulting in a S(50%) of 0.77 (3), which contains a 4-pyridyl substitution, showed reasonable activity against four members of the panel , resulting in a S(50%) score of 0.08. Introduction of a methyl group at the C6-position of the aminopyridinone 3 gave compound 15 which showed low potency against the 26-kinase panel, only one kinase, Aurora B, was inhibited by>50% (77%)  .Fig. 3Li15 clashes with the protein-binding site, or induces an unfavourable conformation of the biaryl system. Retention of the exocyclic 3-NH2 hydrogen bond donor in 3, accompanying by the removal of the hydrogen bond donor of the pyridone ring by conversion to the 3-amino-2-methoxy-pyridine analogue 5 resulted in a similar inhibitory profile against MPS1 and Aurora B compared to compound 3  maintained activity against some members of the panel  resulting in a selectivity score S(50%) of 0.35 (7 and 8 is possibly due to the aminopyrimidine acting as a hinge binding motif as previously reported.kinase(50%) against this compound library  resulted in an increase in potency compared to that observed for 3  generally demonstrated an increase in activity compared to compound 3 (16 demonstrated >50% inhibition against eight members of the panel including MPS1 and Aurora A (S(50%) = 0.31; 17 also exhibited a selectivity score S(50%) of 0.31  of 0.38  was not well tolerated and equivalent fluorine substitutions (compounds 19 and 20) led to significant loss of activity  and piperidine (compound 23) retained good activity against all three kinases but resulted in a reduction in ligand efficiency due to the increase in the number of heavy atoms  as shown in N-methyl piperazine groups present in compounds 23 and 22, respectively, pointed out towards the solvent exposed surface as hypothesised , Aurora A and B have a leucine residue (L210 in Aurora A) as shown in 32 and compounds 22 and 23 with MPS1 was elucidated using X-ray crystallography providing structural information that could prove valuable in improving potency and obtaining a desirable selectivity profile. This scaffold may prove an attractive starting point for the development of potent inhibitors of MPS1 by providing vectors suitable for probing the P-loop, as in NMS-P715, or positioning of a hydrophobic group in a manner that further orders the activation loop.44In summary, screening a small compound library based on the 3-aminopyridin-2-one motif in a target-blind manner against a 26-kinase panel, representative of the kinome, identified hits that possess good activity and excellent ligand efficiency against MPS1, Aurora A and Aurora B. The binding mode of fragment 4Chemistry: All anhydrous solvents and reagents were obtained from commercial suppliers  and used without further purification. Reactions were performed in oven-dried, round-bottom flasks fitted with rubber septa under an atmosphere of argon unless otherwise stated. Analytical thin layer chromatography (TLC) was performed on pre-coated aluminium sheets of silica  and visualised by short-wave UV light. Reactions using microwave irradiation were performed in a Biotage® Initiator Sixty. Flash column chromatography was performed on a FlashMaster personal unit using isolute Flash silica columns or a Biotage SP1 purification system using Biotage Flash silica cartridges. Ion exchange chromatography was performed using acidic Isolute Flash SCX-II cartridges. Silicon carbonate columns used were Isolute Si-Carbonate from Biotage.Semi-Preparative HPLC Method A: 1000 μL standard injections (with needle rinse) of the sample, at 20 mg/mL concentration in DMSO, were made onto a Phenomenex Gemini column . Chromatographic separation at room temperature was carried out using Gilson GX-281 liquid handler system combined with a Gilson 322 HPLC pump  over a 15 min gradient elution from 10:90 to 100:0 methanol:water (both modified with 0.1% formic acid) at a flow rate of 20 mL/min. UV-Vis spectra were acquired at 254 nm on a Gilson 156 UV-Vis detector . Collection was triggered by UV signal, and collected using a Gilson GX-281 liquid handler system . Raw data was processed using Gilson Trilution Software.1H NMR spectra were recorded at 500 MHz on a Bruker Avance-500 using an internal deuterium lock. Chemical shifts were measured in parts per million (ppm) relative to tetramethylsilane (δ = 0) using the following internal references for residual protons in the solvent: CDCl3 (δ 7.26), CD3OD (δ 3.32) and (CD3)2SO (δ 2.50). Data is presented as follows: chemical shift, integration, multiplicity, coupling constant (J) in Hz. 13C NMR spectra were recorded at 126 MHz on a Bruker Avance-500 using an internal deuterium lock. Chemical shifts were measured in parts per million (ppm) relative to tetramethylsilane (δ = 0) using the following internal references: CDCl3 (δ 77.0), CD3OD (δ 49.0) and (CD3)2SO (δ 39.5). Data is presented as follows: chemical shift, integration, multiplicity, coupling constant (J) in Hz.LC-MS analysis was performed on a Waters LCT with a Waters Alliance 2795 separations module and Waters 2487 dual wavelength absorbance detector coupled to a Waters/Micromass LCT time of flight mass spectrometer with ESI source. Analytical separation was carried out at 30 °C either on a Merck Chromolith SpeedROD column  using a flow rate of 2 mL/min in a 3.5 min gradient elution with detection at 254 nm or on a Merck Purospher STAR column  using a flow rate of 1.5 mL/min in a 3.5 min gradient elution with detection at 254 nm. The mobile phase was a mixture of methanol (solvent A) and water (solvent B) both containing formic acid at 0.1%. Gradient elution was as follows: 1:9 (A/B) to 9:1 (A/B) over 2.25 min, 9:1 (A/B) for 0.75 min, and then reversion back to 1:9 (A/B) over 0.3 min, finally 1:9 (A/B) for 0.2 min.+ 195.087652; hexakisphosphazene [M + H]+ 922.009798, and hexakis phosphazene [M + H]+ 622.02896 or reserpine [M + H]+ 609.280657.HRMS analysis was performed on an Agilent 1200 series HPLC and diode array detector coupled to a 6520 Quadrupole-Time of flight mass spectrometer with dual multimode APCI/ESI source. Analytical separation (Method A) was carried out at 30 °C on a Merck Purospher STAR column  using a flow rate of 1.5 mL/min in a 4 min gradient elution with detection at 254 nm. The mobile phase was a mixture of methanol (solvent A) and water (solvent B) both containing formic acid at 0.1%. Gradient elution was as follows: 1:9 (A/B) to 9:1 (A/B) over 2.5 min, 9:1 (A/B) for 1 min, and then reversion back to 1:9 (A/B) over 0.3 min, finally 1:9 (A/B) for 0.2 min. The following references masses were used for HRMS analysis: caffeine [M + H]4.1H-pyrazol-4-ylboronic acid , potassium phosphate , 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl  and tris (dibenzylideneacetone)dipalladium(0)  were dissolved in n-butanol (4 mL) and stirred at 110 °C for 3 h. The solution was then cooled and filtered through a pad of Celite, which was then washed with methanol. The solution was concentrated and purified by SCX column (eluting at room temperature with 2 M ammonia in methanol) and concentrated. The residue was then dissolved in acetonitrile and sodium iodide  was added, followed by dropwise addition of trimethylsilyl chloride  and the reaction mixture stirred for 16 h. The solution was concentrated, taken up in methanol and purified by SCX column (eluting at room temperature with 2 M ammonia in methanol) to give a grey solid . Rf = 0.36 (5% MeOH in EtOAc). mp: 264 °C. 1H NMR  3.81 , 5.09 , 6.63 , 6.83 , 7.59 , 7.83 , 11.38 . 13C NMR  157.3 (C), 139.2 (C), 135.6 (CH), 127.0 (CH), 119.7 (C), 115.8 (CH), 112.3 (C), 110.1 (CH), 39.0 (CH3). HRMS: Found 191.0932, calculated for C9H11N4O (M + H)+: 191.0927.5-Bromo-2-methoxypyridin-3-amine , 1-methyl-14.24.2.1n-butanol (10 mL) and heated to reflux for 3 h. The reaction mixture was then cooled, filtered through a pad of Celite, which was subsequently washed with methanol and concentrated. The residue was then purified using a SCX column, eluting at room temperature with 2 M ammonia in methanol and concentrated to give a brown oil that crystallized upon standing to give a red solid . Rf = 0.45 (10% MeOH in CHCl3). mp: 204 °C. 1H NMR  3.92 , 5.17 , 7.24 , 7.58 , 7.82 , 8.58 .13C NMR  153.1 (C), 150.6 (CH), 145.6 (C), 133.3 (C), 131.2 (CH), 127.5 (C), 121.1 (CH), 116.6 (CH), 53.6 (CH3). HRMS: Found 202.0986, calculated for C11H12N3O (M + H)+: 202.0975.2-Dicyclohexylphosphino-2,4,6-triisopropylbiphenyl , tris(dibenzylideneacetone)dipalladium(0) , 4-pyridinylboronic acid  and potassium phosphate , were added to 5-bromo-2-methoxypyridin-3-amine  in 4.2.2n-butanol (7 mL). Rf = 0.35 (DCM:EtOAc 1:1). mp: 218 °C. 1H NMR  3.87 , 4.05 , 7.13 , 7.32–7.37 , 7.42–7.46 , 7.51–7.54 , 7.82 .13C NMR  152.6 (C), 138.5 (C), 133.3 (CH), 131.0 (C), 130.7 (C), 128.8 (CH), 127.1 (CH), 126.8 (CH), 119.2 (CH), 53.5 (CH3). HRMS: Found 201.1030, calculated for C12H13N2O (M + H)+: 201.1022.Prepared using general procedure A to give a brown solid : 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl , tris(dibenzylideneacetone)dipalladium(0) , 4-phenylboronic acid , potassium phosphate , 5-bromo-2-methoxypyridin-3-amine  and 4.34.3.1f = 0.51 (5% MeOH in CHCl3). mp: 291 °C. 1H NMR  5.16 , 6.81 , 6.93 , 7.25 , 7.36–7.40 , 7.44–7.48 , 11.53 .13C NMR  157.5 (C), 139.2 (C), 138.0 (C), 129.3 (CH), 126.9 (CH), 125.7 (CH), 119.6 (C), 117.8 (CH), 110.2 (CH). HRMS: Found 187.0877, calculated for C11H11N2O (M + H)+: 187.0866.Trimethylsilyl chloride  was added dropwise to a solution of 2-methoxy-5-phenylpyridin-3-amine  and sodium iodide  in acetonitrile (6 mL) and stirred for 2 h at room temperature. The solvent was then removed under reduced pressure and the residue purified using a SCX column to give a grey solid . R4.3.2n-butanol (7 mL). Rf = 0.3 (DCM: EtOAc 1:1). mp: 233 °C. 1H NMR  3.92 , 5.19 , 7.21 , 7.78 , 9.02 , 9.16 .13C NMR  157.4 (CH), 154.6 (CH), 152.9 (C), 133.4 (C), 132.0 (C), 131.0 (CH), 124.2 (C), 116.7 (CH), 53.6 (CH3). HRMS: Found 203.0929, calculated for C10H11N4O (M + H)+: 203.0927.Prepared using general procedure A to give a brown solid : 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl , tris(dibenzylideneacetone)dipalladium(0) , pyrimidinyl-5-boronic acid , potassium phosphate , 5-bromo-2-methoxypyridin-3-amine  and 4.3.3f = 0.57 (DCM:EtOAc 1:1). mp: 240 °C. 1H NMR  5.26 , 6.82 , 7.20 , 8.94 , 9.06 .13C NMR  157.9 (C), 156.7 (CH), 153.7 (CH), 139.6 (C), 131.6 (C), 119.5 (CH), 113.2 (C), 108.6 (CH). HRMS: Found 189.0711, calculated for C9H9N4O (M + H)+: 189.0771.Prepared using general procedure B to give a brown solid : 2-methoxy-5-(pyrimidin-5-yl)pyridin-3-amine , trimethylsilyl chloride  and sodium iodide  in acetonitrile (5 mL). R4.44.4.1f = 0.58 (1:1 DCM:EtOAc). mp: 291 °C. 1H NMR  7.54–7.67 , 7.82 , 7.90–8.00 , 8.52–8.65 , 8.77 , 9.39 , 12.60 .13C  165.6 (C), 157.8 (C), 150.7 (CH), 144.0 (C), 134.2 (C), 132.7 (CH), 129.5 (C), 129.3 (CH), 128.0 (CH), 127.7 (CH), 122.5 (CH), 120.2 (CH), 115.7 (C). HRMS: Found: 292.1075, calculated for C17H14N3O2 (M + H)+: 292.1086.Benzoyl chloride  was added dropwise to 3-amino-5-(pyridin-4-yl)pyridin-2-one  in pyridine (3 mL) and the mixture was stirred for 16 h at room temperature. The solvent was removed under reduced pressure and the residue taken up in methanol and filtered to give the desired product as a light-yellow solid . R4.4.2f = 0.69 (DCM:EtOAc 1:1). mp: 287 °C. 1H NMR  3.85 , 7.21 , 7.43–7.60 , 8.17–8.31 , 8.79–8.88 , 9.46 , 13.01 .13C NMR  165.6 (C), 159.9 (C), 158.1 (C), 152.3 (C), 142.8 (CH), 135.6 (C), 132.3 (CH), 130.5 (CH), 129.6 (C), 122.6 (CH), 122.0 (CH), 119.8 (CH), 118.5 (CH), 113.1 (CH), 55.9 (CH3). HRMS: Found 322.1182, calculated for C18H16N3O3 (M + H)+: 322.1186.Prepared using general procedure C to give a yellow solid : 3-methoxybenzoylchloride , 3-amino-5-(pyridin-4-yl)pyridin-2-one . R4.4.3f = 0.67 (DCM:EtOAc 1:1). mp: 280 °C. 1H NMR  3.83 , 6.75 , 7.06 , 7.61 , 7.83 , 8.59 , 8.70 , 9.36 , 12.58 .13C NMR  165.4 (C), 161.1 (C), 157.9 (C), 150.7 (CH), 144.0 (C), 136.5 (C), 129.6 (C), 128.4 (CH), 123.3 (CH), 120.1 (CH), 115.5 (C), 105.6 (CH), 104.3 (CH), 56.0 (CH3). HRMS: Found 352.1291, calculated for C19H18N3O4 (M + H)+: 352.1292.Prepared using general procedure C to give a grey solid : 3,5-dimethoxybenzoylchloride , 3-amino-5-(pyridin-4-yl)pyridin-2-one . R4.4.4f = 0.51 (DCM:EtOAc 1:1). mp: 292 °C. 1H NMR  7.50 , 7.62 , 7.77 , 7.82 , 8.23 , 8.26 , 8.82 , 8.83 , 9.62 , 13.01 . 13C  164.8 (C), 162.5 , 158.1 (C), 152.2 (CH), 142.8 (C), 136.5 , 132.7 (CH), 131.5 , 129.5 (C), 124.0 , 123.5 (CH), 121.9 (CH), 119.6 , 114.9 , 113.0 (C). HRMS: Found 310.0984, calculated for C17H13N3O2F (M + H)+: 310.0986.Prepared using general procedure C to give a yellow solid : 3-fluorobenzoylchloride , 3-amino-5-(pyridin-4-yl)pyridin-2-one . R4.4.550 values for MPS1, Aurora A and Aurora B inhibition were determined as previously described,i values were estimated from the mean IC50 values using the Cheng-Prusoff equation.IC4.4.622 and 23 with MPS1 were produced by vapour diffusion using the hanging-drop method. The well buffer contained 0.2 M NaCO2H, 0.1 M BTP (pH 7.5) and 15% (w/v) PEG 3350. The protein solution contained 11.59 mg/mL MPS1, 50 mM HEPES (pH 7.5), 150 mM NaCl and 5 mM DTT, supplemented with 1 mM compound (1% DMSO). Drops contained a 1:1 mixture of well buffer and protein solution. Crystals grew over 3 days at 18 °C. Harvested crystals were briefly transferred to cryoprotectant  PEG 3350 and 0.08 M Bis-Tris propane pH 7.5) and flash cooled in liquid nitrogen. Crystals of MPS1 in complex with 2 were obtained by back-soaking a crystal of MPS1 with 23 in a new solution of 0.19 M NaCO2H, 0.095 M BTP (pH 7.5), 14.25% PEG 3350 supplemented with 2 mM 2 for 48 h prior to harvesting. Structure solution and refinement data are presented in supplementary material.Co-crystals of


The antibacterial activities of the synthesized compounds were screened against standard strains of six Gram positive and four Gram negative bacteria using the microbroth dilution assay. Most of the compounds studied showed promising activities against both types of bacteria.Several new substituted sulfonamide compounds were synthesized and their structures were confirmed by  Heterocycles containing sulfonamido moieties have attracted obvious attention due to their significant biological properties and their role as pharmacophores ,3,4,5,6.In our study, new promising bioactive compounds based on the sulfonamide moiety were designed and synthesized by a simple and efficient method, followed by the evaluation of their biological activities. The synthesis emphasizes a strategy that combines two or more pharmacologically compatible moieties in one molecule by attaching a sulfonamide moiety to an imidazole, benzimidazole or another sulfonamide moiety. We believe this route has a wide range of applications and we have high expectations for the future development of new compounds.1 as shown in Bis-benzimidazole and bis-imidazole sulfonamides were synthesized from the diol Three bis-benzimidazole sulfonamides and three bis-imidazole sulfonamides compounds were obtained by treating imidazole (or benzimidazole) with tris-(4-substituted benzensulfonate)- diethanolamine under basic conditions to form the corresponding bis-imidazole (or bis-benzimidazole) sulfonamides. The reaction of tris-(4-substituted benzensulfonate) with either imidazole or benzimidazole has produced symmetrical products and it is in agreement with literature  to synth3a–c and 4a–c showed absorption bands at 1351–1375 cm−1 and 1150–1185 cm−1 which were assigned to the O=S=O group. The same compounds showed stretching absorption bands at 3100–3047 cm−1, 2975–2855 cm−1, 1590–1457 cm−1, and 1666–1584 cm−1 attributed to (C-H)Aromatic, (C-H)Aliphatic, (C=C)Aromatic, and (C=N), respectively. The target compounds 3b and 4b showed characteristic stretching absorption bands at 1,220 cm−1 and 1,238 cm−1 which were assigned to C-O-C, while the bands at 1529–1520 cm−1 and 1355–1340 cm−1 for compounds 3c and 4c were assigned to Ar-NO2.The FTIR spectra for compounds 1H-NMR spectra of compounds 3a, 4a, 3b, and 4b showed singlets at δ 2.33 ppm and δ 2.80 ppm which were assigned to the 4-methyl and 4-methoxy protons of the arylsulfonyl groups, respectively. A triplet recorded at δ 3.31–3.54 ppm and δ 3.95–4.27 ppm was assigned to the two methylene groups protons of compounds 3a–c and 4a–c. The aromatic protons of compounds 3a–c and 4a–c were recorded as multiplets in the δ 6.89–7.83 ppm range. A singlet which was observed at δ 7.42–8.16 ppm corresponds to the isolated C-H of the imidazole and benzimidazole rings. The 13C-NMR spectra of compounds 3a–c and 4a–c showed characteristic peaks in the δ 163.23–163.34 ppm, δ 149.03–153.12 ppm and δ 129.78–144.28 ppm ranges which were assigned to CAr-O, CAr-NO2 and CAr-S, respectively. The peaks recorded at δ 43.09–45.77 ppm, δ 48.20–50.37 ppm and δ 56.18–56.24 ppm were attributed to the methylene and methoxy carbon atoms, correspondingly.The 3a and 4a showed various characteristic peaks. Those at m/z 459.2 and 359.1 were assigned to the molecular ions of 3a and 4a, respectively. The base peak of 3a at m/z 328.1 was assigned to the N-(benzimidazol-1-yl)ethyl-N-4-dimethylbenzenesulfonamido radical, while the base peak of 4a at m/z 278.1 was assigned to the N-(imidazole-1-yl)ethyl-(4-methylbenzene) sulfonamide-methyliumyl ion. The characteristic peaks at m/z 155.0 and 91.0 for both 3a and 4a were due to (4-methylphenyl)dioxosulfanium and 4-methylbenzene-1-ylium ions, respectively 3a and 4a, respectively.The mass spectra of compounds 9 from 2-((benzimidazol-2-yl)methylthio)-benzimidazole. A simple method was adopted to synthesize a pure heterocyclic product in good yield. 2-Mercaptobenzimidazole and sodium methoxide were stirred with 2-chloromethylbenzimidazole. A pale-yellow solid precipitated instantly due to the reactivity of -SH group then it was treated with tosyl chloride in pyridine.8 indicated the absence of a free -SH absorption band and the appearance of S-C stretching at 748 cm−1, while the absorption bands at 1,365 and 1,170 cm−1 were assigned to the O=S=O group. The 1H-NMR spectrum of compound 9 showed two singlet peaks at δ 2.31, and 2.37 ppm integrating for six protons for the two methyl protons of the sulfonamido moieties. The protons of the methylene group appeared as a singlet at δ 5.18 ppm, whereas the aromatic protons appeared as multiplets and doublet peaks in the δ 7.19–7.98 ppm range. The 13C-NMR of compound 9 showed peaks at δ 21.77 and 21.80 ppm which was assigned to two non-corresponding methyls for two tosyl groups. The CH2-S carbon atom was observed at δ 31.20 ppm.The infrared spectrum of compound 11 from 2,2-(ethylenedioxy)bis(ethylamine) by treating with tosyl chloride and triethylamine in dry dichloromethane that produced a significant yield of pure bis-sulfonamide compound 11.11 showed absorption bands at 3276 cm−1 indicating the presence of a N-H group, 1088 cm−1 for a C-O-C group, 1124 cm−1 for C-C-O vibrations, and the peaks at 1317, 1152 cm-1 were assigned to the O=S=O group. The 1H-NMR spectrum of compound 11 showed characteristic doublet peaks at δ 7.27 ppm and 7.73 ppm which were assigned to the aromatic protons. The methylene groups were recorded as a quartet at δ 3.09 ppm and a triplet at δ 3.50 ppm integrating for eight and four protons, respectively. The amino protons of compound 11 appeared as a triplet at δ 5.50 ppm. The corresponding methyls for two tosyl groups were observed as a single peak at δ 2.39 ppm integrating for six protons. The 13C-NMR of compound 11 showed aromatic carbon peaks at δ 143.45, 137.07, 129.78 and 127.18 ppm. The peak at δ 21.59 ppm was assigned to the two corresponding methyl groups while, peaks of 4 × CH2-O were observed at δ 69.78 and 70.43 ppm. The structures of compounds 3a, 4a, 9 and 11 were further characterized by single crystal X-ray diffraction which indicated tetragonal and triclinic crystal systems for 3a and 4a, respectively, while the crystal structures of 9 and 11 have been reported .The FTIR spectrum for compound reported ,31. Crysin vitro antibacterial bioassay, the eight compounds 3a–c, 4a–c, 9 and 11 were evaluated by microbroth dilution assays using representative standard strains of Gram-positive and Gram-negative bacteria, and the results are listed in 9 and 11, followed by 3c and 4c then 3b and 4b. Compounds 3a and 4a showed the least antibacterial activity for the selected concentration range, as shown in 3a–c and 4a–c. Studies have also shown that the presence of a sulfur atom as a sulfide in drugs provides a greater stability to the three dimensional structure of the molecule -4-methylbenzenesulfonamide (3a). White solid; Yield: 94%; a single crystal was obtained properly for X-ray structural determination by using DMF; m.p.192–194 °C; FTIR (cm−1): 3098, 3047 (C-H)Ar, 2927 (C-H)Aliph, 1666, 1615, 1598 (C=N)Ar, 1498, 1460 (C=C)Ar, 1362, 1150 (O=S=O); 1H-NMR (DMSO-6d) δ ppm: 8.09 , 7.64–7.60 , 7.44 , 7.30–7.19 , 4.27 , 3.49 , 2.33 ; 13C-NMR (DMSO-d6) δ ppm: 144.57 (2 × CHBImidazole), 144.20 (CAr-S), 143.84 (2 × CBImidazole), 135.36 (CAr-CH3), 134.04 (2 × CBImidazole), 130.43 (2 × CHAr), 127.49 (2 × CHBImidazole), 123.07 (2 × CHBImidazole), 122.15 (2 × CHAr), 120.06 (2 × CHBImidazole), 110.59 (2 × CHBImidazole), 48.71 (2 × CH2-N), 43.77 (2 × CH2-NAr), 21.51 (CH3); EIMS (m/z): 459 , 278 (100%), 304 (10%), 278 (13%), 172 (36%), 155 (24%), 131 (87%), 91 (75%).N,N-bis[2-(Benzimidazol-1-yl)ethyl]-4-methoxybenzenesulfonamide (3b). White solid; Yield: 84%; m.p. 138–140 °C; FTIR (cm−1): 3096, 3055 (C-H)Ar, 2910 (C-H)Aliph, 1666, 1615, 1597 (C=N)Ar, 1494, 1460 (C=C)Ar, 1375, 1164 (O=S=O), 1220 (C-O-C); 1H-NMR (DMSO-d6) δ ppm: 8.09 , 7.68–7.62 , 7.45 , 7.28–7.15 , 7.00–6.96 , 4.27 , 3.80 , 3.48 ; 13C-NMR (DMSO-d6) δppm 163.23 (CAr-O), 144.51 (2 × CHBImidazole), 143.83 (2 × CBImidazole), 134.07 (2 × CBImidazole), 129.78 (CAr-S), 129.70 (2 × CHAr), 123.05 (2 × CHBImidazole), 122.19 (2 × CHBImidazole), 120.08 (2 × CHBImidazole), 115.10 (2 × CHAr), 110.60 (2 × CHBImidazole), 56.18 (O-CH3), 48.66 (2 × CH2-N), 43.75 (2 × CH2-N); EIMS (m/z): 475 , 344 (100%), 304 (10%), 278 (13%), 172 (44%), 107 (35%), 131(75%), 91(80%).N,N-bis[(Benzimidazol-1-yl)ethyl]-4-nitrobenzenesulfonamide (3c). Pale green solid; Yield: 62%; m.p. 80–82 °C; FTIR (cm−1): 3102, 3055 (C-H)Ar, 2950 (C-H)Aliph, 1660, 1615, 1598 (C=N)Ar, 1510, 1485 (C=C)Ar, 1360, 1178 (O=S=O), 1529, 1340 (Ar-NO2); 1H-NMR (DMSO-d6) δ ppm: 8.16 , 7.78–7.73 , 7.52 , 7.31–7.22 , 6.99–6.94 , 4.23 , 3.54 ; 13C-NMR (DMSO-d6) δ ppm: 153.12 (CAr-NO2), 145.57 (2 × CHBImidazole), 143.65 (2 × CBImidazole), 134.12 (2 × CBImidazole), 130.04 (CAr-S), 128.44 (2 × CHAr), 124.95 (2 × CHBImidazole), 120.34 (2 × CHBImidazole), 118.88 (2 × CHBImidazole), 117.10 (2 × CH), 111.07 (2 × CHBImidazole), 50.37 (2 × CH2-N), 44.75 (2 × CH2-N); EIMS (m/z): 490 , 359 (100%), 304 (20%), 278 (30%), 186 (45%), 22 (75%).N,N-bis[(Imidazol-1-yl)ethyl]-4-methylbenzenesulfonamide (4a). White solid; Yield: 82%; a single crystal was obtained for X-ray analysis by using acetonitrile; m.p. 92–94 °C; FTIR (cm−1) 3093 (C-H)Ar, 2975 (C-H)Aliph, 1597 (C=N)Ar, 1510, 1457 (C=C)Ar, 1351, 1159 (O=S=O); 1H-NMR (DMSO-6d) δ ppm: 7.72 , 7.54 , 7.41, 7.12 , 6.89 , 3.96 , 3.32 , 2.39 ; 13C-NMR (DMSO-d6) δ ppm 144.28 (CAr-S), 137.87 (2 × CHImidazole), 135.53 (CAr-CH3), 130.49 (2 × CHAr), 128.99 (2 × CHImidazole), 127.56 (2 × CHAr), 119.95 (2 × CHImidazole), 50.30 (2 × CH2-N), 45.75 (2 × CH2-N), 21.49 (-CH3); EIMS (m/z): 359 , 278 (100%), 204 (20%), 155 (22%), 122 (39%), 91 (97%).N,N-bis[2-(Imidazol-1-yl)ethyl]-4-methoxybenzenesulfonamide (4b). Off-white solid; Yield: 90%; m.p. 66–68 °C; FTIR (cm−1): 3100 (C-H)Ar, 2855 (C-H)Aliph, 1584 (C=N)Ar, 1530, 1460 (C=C)Ar, 1360, 1170 (O=S=O), 1238 (C-O-C); 1H-NMR (DMSO-6d) δ ppm: 7.76 , 7.55 , 7.14–7.10 , 6.89 , 3.97 , 3.82 , 3.31 ; 13C-NMR (DMSO-d6) δ ppm: 163.34 (CAr-O), 137.92 (2 × CHImidazole), 129.95 (2 × CHImidazole), 129.86 (CAr-S), 129.01 (2 × CHAr), 120.06 (2 × CHImidazole), 115.23 (2 × CHAr), 56.24 (O-CH3), 50.24 (2 × CH2-N), 45.67 (2 × CH2-N); EIMS (m/z): 375 , 294 (100% ), 204 (20%), 171 (36%), 122 (35%), 107 (35%).N,N-bis[(Imidazol-1-yl)ethyl]-4-nitrobenzenesulfonamide (4c). Pale green solid; Yield: 54%; m.p. 46–48 °C; FTIR (cm−1): 3080 (C-H)Ar, 2890 (C-H)Aliph, 1620 (C=N)Ar, 1574, 1460 (C=C)Ar, 1375, 1185 (O=S=O), 1520, 1355 (Ar-NO2); 1H-NMR (DMSO-6d) δ ppm 7.96 , 7.78 , 7.36–7.32 , 6.95 , 3.95 , 3.55. 13C-NMR (DMSO-d6) δ ppm 149.03 (CAr-NO2), 138.23 (2 × CHImidazole), 130.11 (2 × CHImidazole), 129.78 (CAr-S), 128.12 (2 × CHAr), 122.06 (2 × CHImidazole), 112.14 (2 × CHAr), 48.20 (2 × CH2-N), 43.09 (2 × CH2-N); EIMS (m/z): 390 , 309 (60%), 204 (20%), 186 (30%), 122 (100%), 81 (75%).o-Phenylenediamine  was dissolved in absolute ethanol (40 mL) in a 250 mL flask. Carbon disulfide (10 mL) was then added to the solution followed by the addition of a solution of potassium hydroxide  in water (25 mL). The reaction mixture was thoroughly stirred and refluxed for 5 h. It was initially yellow, then turned to brown as the reaction progressed. Evolution of hydrogen sulfide gas was observed. After completion of the reaction, the mixture was poured into a beaker with ice-water and acidified with 4N hydrochloric acid to pH 4–5 to obtain a white precipitate. The precipitate was then filtered and recrystallized from ethanol. White solid; Yield 84%; m.p. 303–305 °C; FTIR (cm−1): 3,250 (N-H), 1,557 (C=C)Ar, 1,633 (C=O), 1,517 (C=N).Prepared according to the modified procedure in . o-Pheny−1): 3372 (N-H), 3090 (C-H)Ar, 2975 (C-H)Aliph, 1621 (C=N)Ar, 1590 (C=C)Ar, 748 (C-S); 1H-NMR (DMSO-6d) δ ppm: 12.52 , 12.23 , 7.41–7.32 , 6.97–6.90 , 4.93 . 13C-NMR (DMSO-d6) δ ppm: 153.11 (CAr-S), 149.73 (CAr-S-CH2), 140.32, 139.66 (4 × CAr), 125.34, 123.92 (4 × CHAr), 114.53, 116.03 (4 × CHAr) 33.12 (CH2).Prepared according to modified procedure in . Sodium p-toluenesulfonyl chloride  in pyridine (25 mL) was added dropwise to a solution of 2-((benzimidazol-2-yl)methylthio)-benzimidazole (8)  in pyridine (25 mL) at 0 °C, within 3 h. The mixture was stirred at room temperature and left overnight. It was then quenched with ice-water, stirred for another 20 minutes, extracted with dichloromethane (5 × 30 mL) and washed with distilled water. The organic layer was dried over anhydrous magnesium sulfate and solvent evaporated off. The crude product was purified by using flash chromatography with hexane-ethyl acetate  as eluent. The obtained solid was recrystallized from acetonitrile to give colorless crystals of compound (9). Colorless crystals; Yield 52%; m.p. 204–206 °C; FTIR (cm−1) 3087 (C-H)Ar, 2990 (C-H)Aliph, 1660, 1,616 (C=N)Ar, 1593, 1462 (C=C)Ar, 1365, 1170 (O=S=O), 752 (C-S); 1H-NMR (CDCl3) δ ppm 7.98–7.89 , 7.64–7.49 , 7.38–7.24 , 7.19 , 5.18 , 2.31, 2.37 ; 13C-NMR (CDCl3) δ ppm 151.81 (CBImidazole-S), 149.43 (CBImidazole-CH2-S), 146.42, 146.18 (2 × CAr-S), 143.22, 141.80 (2 × CBImidazole-N), 135.04, 134.57 (2 × CAr-CH3), 133.98, 133.16 (2 × CBImidazole-N), 130.38, 130.15 (4 × CHAr), 127.54, 127.45 (4 × CHAr), 125.52, 124.91, 124.69, 124.14, 120.56, 118.95, 113.56, 112.95 (8×CHBImidazole), 31.20 (CH2-S), 21.80, 21.77 (2 × CH3); EIMS (m/z): 588 , 433 (80%), 278 (35%), 155 (85%), 148 (12%), 91 (100%).A solution of p-Toluenesulfonyl chloride  was dissolved in dry dichloromethane (25 mL) and added dropwise to a stirring solution of 1,8-diamino-3,6-dioxaoctanebis-(ethyl amine)  and triethylamine  in dichloromethane (25 mL) at 0 °C. The mixture was stirred further at room temperature for overnight and extracted with water and saturated solution of NaHCO3 (3 × 15 mL). The organic layer was dried over anhydrous magnesium sulfate and the solvent was evaporated off. Colorless crystals were obtained through slow evaporation of methanolic solution at room temperature. Yield: 92%; m.p. 88–90 °C; FTIR (cm−1) 3276 (NH), 3090 (C-H)Ar, 2921, 2898, 2872 (C-H)Aliph, 1596 (C=C)Ar, 1317, 1152 (O=S=O), 1088 (C-O-C), 1124 (C-C-O); 1H-NMR (CDCl3) δ ppm: 7.73 , 7.27 , 5.50 , 3.50 , 3.09 , 2.39 ; 13C-NMR (CDCl3) δ ppm: 143.45 (2 × CAr-S), 137.07 (2 × CAr-CH3), 129.78 (4 × CHAr), 127.18 (4 × CHAr), 70.43 (2 × CH2-O), 69.78 (2 × CH2-O), 42.99 (2 × CH2-N), 21.59 (2 × CH3); EIMS (m/z): 456 , 301 (100%), 155 (70%), 146 (35%), 91 (90%).3a–c, 4a–c, 9 and 11 was tested against standard strains of ten bacteria. They were obtained from the collection of the School of Biosciences and Biotechnology, Faculty of Science and Technology, University Kebangsaan, Malaysia. These strains included Gram positive bacteria: Streptococcus pyogenes ATCC19615, Staphylococcus aureus ATCC 29213, Bacillus subtilis ATCC6051, Rodococcus Ruber ATCC27863, Enterococcus faecalis ATCC 29212, Staphylococcus epidermidis ATCC12228 and Gram negative bacteria: Escherichia coli ATCC10538, Salmonella typhimurium ATCC14028, Pseudomonas aeruginosa ATCC15442, Acinetobacter calcoaceticus ATCC 23055.The antibacterial activity of synthesized compounds The antibacterial activity was assessed in terms of minimum inhibitory concentrations (MICs) by using microbroth dilution assays according to the CLSI guidelines . All thei.e., 3a–c, 4a–c, and 9 in addition to novel bis-sulfonamide compound i.e., 11 were successfully synthesized through simple methods. The structures for the synthesized compounds were confirmed by FTIR, NMR, and HRMS studies. These compounds were evaluated for in vitro antibacterial activities against ten strains of bacteria. Compounds 3c, 9 and 11 demonstrated the highest bioactivities among the compounds, however, most of them showed significant activities for both Gram-positive and Gram-negative bacteria. Such results are encouraging for synthesis of promising new complexes from these compounds with several metals to evaluate their biological activity in the near future. Further studies of these new synthesized sulfonamide derivatives by the same authors are in progress related to their use as ligands and their complexes.Novel imidazole and benzimidazole compounds with a sulfonamido moiety as substituent,