Uncaria genus, the phytochemistry and the in-vitro α-glucosidase inhibitory activities of Malaysian Uncaria cordata var. ferruginea were investigated. The phytochemical study of this plant, which employed various chromatographic techniques including recycling preparative HPLC, led to the isolation of ten compounds with diverse structures comprising three phenolic acids, two coumarins, three flavonoids, a terpene and an iridoid glycoside. These constituents were identified as 2-hydroxybenzoic acid or salicylic acid (1), 2,4-dihydroxybenzoic acid (2), 3,4-dihydroxybenzoic acid (3), scopoletin or 7-hydroxy-6-methoxy-coumarin (4), 3,4-dihydroxy-7-methoxycoumarin (5), quercetin (6), kaempferol (7), taxifolin (8), loganin (9) and β-sitosterol (10). Structure elucidation of the compounds was accomplished with the aid of 1D and 2D Nuclear Magnetic Resonance (NMR) spectral data and Ultraviolet-Visible (UV-Vis), Fourier Transform Infrared (FTIR) spectroscopy and mass spectrometry (MS). In the α-glucosidase inhibitory assay, the crude methanolic extract of the stems of the plant and its acetone fraction exhibited strong α-glucosidase inhibition activity of 87.7% and 89.2%, respectively, while its DCM fraction exhibited only moderate inhibition (75.3%) at a concentration of 1 mg/mL. The IC50 values of both fractions were found to be significantly lower than the standard acarbose suggesting the presence of potential α-glucosidase inhibitors. Selected compounds isolated from the active fractions were then subjected to α-glucosidase assay in which 2,4-dihydroxybenzoic acid and quercetin showed strong inhibitory effects against the enzyme with IC50 values of 549 and 556 μg/mL compared to acarbose (IC50 580 μg/mL) while loganin and scopoletin only showed weak α-glucosidase inhibition of 44.9% and 34.5%, respectively. This is the first report of the isolation of 2-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid and loganin from the genus and the first report of the α-glucosidase inhibitory potential of 2,4-dihydroxybenzoic acid.Continuing our interest in the  Uncaria (Rubiaceae) is amongst the genera known for its alkaloid content. The majority of alkaloids founds in Uncaria are of the indole and oxindole type -2”-pyranone, named uncariechin, along with (−)-epiafzelechin and (−)-epicatechin, from the methanol extract of the leaves of Uncaria longiflora var. pteropoda + molecular ion peak at m/z 139, which corresponds to the molecular formula C7H6O3. The UV spectrum of the compound displayed an absorption peak at 308 nm indicative of an aromatic-type compound due to π → π* transition of benzene chromophore. Its IR spectrum exhibited absorption bands at 3237 cm−1 due to a hydroxyl group, 1675 cm−1 for a conjugated carbonyl group, 1613 cm−1 for aromatic C=C and 759.7 cm−1 (C=C-H). The 1H-NMR spectrum of this compound showed that all proton signals appeared in the aromatic region confirming the presence of a benzene ring. Two doublet of doublet signals were observed at δ 6.78  and δ 6.80 . A triplet was observed at δ 7.28  while a doublet of doublets was observed at δ 7.84 . The COSY spectrum of compound 1 showed H-H correlations between H-4, H-5 and H-6. However, no hydroxyl peak was observed in the 1H-NMR spectrum of compound 1. This may be due to deuterium exchange of the MeOD with the acidic hydrogen present in the molecule + peak at m/z 408 which corresponded to the molecular formula C17H26O10. Its UV spectrum displayed absorption bands at 295 and 330 nm, while the IR spectrum exhibited typical absorption frequencies at 3368, 1655, 1420, 1449 and 669 cm−1 for an iridoid-type compound +, C7H6O3; UV (MeOH) λmax nm: 308; IR (KBr) υmax cm−1: 3237 (OH), 1675 (C=O), 1613 (C=C), 759 (C=C-H); 1H-NMR  δ ppm: 7.84 , 7.28 , 6.78 , 6.80 ; 13C-NMR  δ ppm: 174.8 (C-7), 161.2 (C-2), 132.3 (C-4), 130.2 (C-6), 118.9 (C-1) 117.5 (C-3), 115.7 (C-5) +, C7H6O4; UV (MeOH) λmax nm: 293.3; IR υmax cm−1: 3280 (OH), 1655 (C=O); 1H-NMR  δ ppm: 7.45 , 7.43 , 6.80 ; 13C-NMR  δ ppm: 169.7 (C-7), 149.7 (C-3), 144.5 (C-4), 123.0 (C-1), 122.4 (C-6), 116.4 (C-2), 114.3 (C-5) +, C10H8O4; UV (MeOH) λmax nm: 228, 295, 345; IR (KBr) υmax cm−1: 3471 (free OH), 2964 (C-H aromatic);1H-NMR  δ ppm: 7.63 , 6.95 , 6.87 , 6.30 , 6.19 , 3.98 ; 13C-NMR  δ ppm: 161.5 (C=O), 150.3 (C-6), 149.7 (C-9), 144.0 (C-7), 143.3 (C-4), 113.4 (C-3), 111.5 (C-10), 107.5 (C-5), 103.2 (C-8), 56.4 (OCH3) +, C15H10O7; UV (MeOH) λmax nm: 295, 338, 385; IR (KBr) υmax cm−1: 3411 (OH), 1665 (C=O), 1615 (C=C); 1H-NMR  δ ppm : 12.19 , 7.85 , 7.72 , 7.01  6.54 , 6.28 ; 13C-NMR  δ ppm: 175.7 (C=O), 164.2 (C-7), 161.5 (C-5), 156.9 (C-9), 147.5 (C-4’), 146.1 (C-2), 145.0 (C-3’), 135.9 (C-3), 122.9 (C-1’), 120.6 (C-6’), 115.3 (C-5’), 114.9 (C-2’), 103.2 (C-10), 98.3 (C-6), 93.6 (C-8) −, C15H10O6; UV (MeOH) λmax nm: 310, 380; IR (KBr) υmax cm−1: 3324 (OH), 1660 (C=O), 1614 (C=C); 1H-NMR  δ ppm : 8.09 , 6.90 , 6.39 , 6.18 ; 13C-NMR  δ ppm: 175.9 (C=O), 164.2 (C-7), 161.1 (C-5), 159.1 (C-4’), 156.8 (C-9), 146.6 (C-2), 135.7 (C-3), 129.3 , 122.3 (C-1’), 114.9 , 103.1 (C-10), 97.9 (C-6), 93.1 (C-8) +, C17H26O10; UV (MeOH) λmax nm: 295, 330; IR υmax cm−1: 3368, 1655, 1420, 1449, 669; 1H-NMR  δ ppm: 7.41 , 5.29 , 4.67 , 4.06 , 3.92 , 3.70 , 3.69 , 3.39 , 3.32 , 3.31 , 3.22 , 3.13 , 2.25 , 2.04 , 1.89 , 1.64 , 1.11 ; 13C-NMR  δ ppm: 168.2 (C=O), 150.7 (C-3), 112.6 (C-4), 98.7 (C-1’), 96.3 (C-1), 70.2 (C-4’), 76.6 (C-3’), 73.7 (C-7), 73.3 (C-2’), 77.0 (C-5’), 61.4 (C-6’), 50.3 (C-13OCH3), 45.1 (C-9), 41.3 (C-6), 40.8 (C-8), 30.7 (C-5), 12.0 (C-10) [β-sitosterol (10). Transparent needles, wt: 4.5 mg (chloroform), mp 135 °C (mp lit. 136–138 °C). C29H50O; IR (KBr) υmax cm−1: 3426 (OH), 2950 (C-H), 1635 (C=C); 1H-NMR  δ ppm : 5.36 , 3.53 , 2.24 , 2.00 , 1.83 , 1.68 , 1.50 , 1.42 , 1.33 , 1.23 , 1.02 , 0.93 , 0.87 , 0.81 , 0.69 ; 13C-NMR  δ ppm: 140.8 (C-5), 121.7 (C-6), 71.8 (C-3), 56.8 (C-14), 56.0 (C-17), 50.1 (C-9), 45.8 (C-24), 42.3 (C-13), 42.3 (C-4), 39.8 (C-12), 37.2 (C-1), 36.5 (C-10), 36.1 (C-20), 33.9 (C-22), 31.9 (C-7), 31.9 (C-8), 31.6 (C-2), 29.1 (C-25), 28.2 (C-16), 26.1 (C-23), 24.3 (C-15), 23.1 (C-28), 21.1 (C-11), 19.8 (C-26), 19.4 (C-19), 19.0 (C-27), 18.8 (C-21), 12.0 (C-29), 11.9 (C-18) [R CDCl3, 0 MHz δ pet al. [p-nitrophenyl-α-D-glucopyranoside solution was added into the mixture and absorbance at time 0 min was measured by the spectrophotometer (λ = 405 nm). Then, the reaction mixture was incubated at 37 °C for 30 min and the absorbance was measured. For negative control, the sample was replaced with 5% DMSO and acarbose was used as positive control. Experiments were performed in triplicates with three independent experiments. The percentage inhibitory activity was calculated using the following equation:The method of α-glucosidase inhibitory assay was adopted from Ahmad et al.  with minUncaria cordata var. ferruginea has led to the isolation of ten non-alkaloid constituents from four different classes of compounds. In the evaluation of the in vitro α-glucosidase inhibitory activities of this species, the methanolic stem extract and the acetone fraction both exhibited high percentages of α-glucosidase inhibition of 87.7% and 89.2%, at 1 mg/mL, respectively, while the DCM fraction demonstrated moderate inhibition (75.3%) with IC50 values of 102, 360 and 200 μg/mL, respectively, compared to acarbose. 2,4-DHBA and quercetin isolated from the active acetone fraction showed strong in vitro α-glucosidase inhibition with IC50 values of 549 μg/mL (or 3.56 mM) and 556 μg/mL (or 1.84 mM) respectively, against acarbose (IC50 580 μg/mL or 0.89 mM). Based on the literature, the untested compounds isolated from this fraction may contribute significantly to the strong activity observed for the acetone fraction. However, loganin and scopoletin (isolated from the DCM fraction) showed only weak α-glucosidase inhibitory activities of 44.9% and 34.5%, respectively, which is supported by the weak activities of iridoid glycosides and coumarins reported in the literature. This is the first report of the isolation of 2-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid and loganin from the genus and the first report of the α-glucosidase inhibitory potential of 2,4-dihydroxybenzoic acid.A phytochemical study of the methanolic stem extract of Malaysian


New compounds (7–17), with fused aromatic rings at position 5, were designed and synthesized. Crystallographic X-ray analysis proved that the final compounds (7–17) were substituted with tertiary amine-propyl moiety at position 3 and primary amine group at 2 due to intramolecular Dimroth rearrangement. New compounds were evaluated on their antibiotic adjuvant properties in either Gram-positive or Gram-negative bacteria. Efflux pump inhibitor (EPI) properties towards the AcrAB-TolC pump in Enterobacter aerogenes (EA289) were investigated in the real-time efflux (RTE) assay. Docking and molecular dynamics were applied to estimate an interaction of compounds 6–17 with penicillin binding protein (PBP2a). In vitro ADME-Tox properties were evaluated for compound 9. Most of the tested compounds reduced significantly (4-32-fold) oxacillin MIC in highly resistant MRSA HEMSA 5 strain. The anthracene-morpholine derivative (16) was the most potent (32-fold reduction). The tested compounds displayed significant EPI properties during RTE assay (37–97%). The naphthyl-methylpiperazine derivative 9 showed the most potent “dual action” of both oxacillin adjuvant (MRSA) and EPI (E. aerogenes). Molecular modeling results suggested the allosteric mechanism of action of the imidazolones, which improved binding of oxacillin in the PBP2a active site in MRSA.Searching for new chemosensitizers of bacterial multidrug resistance (MDR), chemical modifications of (Z)-5-(4-chlorobenzylidene)-2-(4-methylpiperazin-1-yl)-3H-imidazol-4(5H)-one ( Staphylococcus aureus  to to6–17 w7 with atom numbering is presented in 7 the Z isomer is observed. This isomer also occurred in other crystal structures, determined earlier, containing 5-(p-chlorobenzylidene)-imidazolone moiety  direction . Further7 allowed us to identify the Dimroth rearrangement and real structure of the series of compounds (7–17). It was difficult to recognize this structure only on the basis of the results of both, NMR and LC-MS analyses, since the molecular mass and number of protons is the same in the expected compound 7′ and the real Dimroth product (7). Before the crystallography results, we had identified the bright singlet (7.80 ppm) occurring in 1H-NMR spectrum of basic form of 7  singlet at 9–10 ppm , which indicated a probable transformation of NH2 (at C2) into the tautomeric form of N1-H and C2 = NH . Thus, the crystallography studies allowed us to right assign 1H-NMR peaks to the suitable protons of the Dimroth rearrangement product. Similar trends that occurred in the 1H-NMR spectra of the rest of the series confirm the Dimroth rearrangement structure for the rest of the compounds (8–17), although the hydrochloric form and more extended aromatic areas disturb the detection of such clear and regular peaks as those for the phenyl symmetric-substituted 7 in its basic form.Results of the crystallographic studies for compound of 7 see  as that = NH see  in the civatives , whereasStaphylococcus aureus strains. Selected compounds  were also tested in Gram-negative E. aerogenes strains, by employing both the microdilution and the real-time efflux (RTE) assays. In the first step of the study, direct antibacterial activity of compounds against aforementioned bacteria was evaluated. Next, the influence of compounds (at the concentrations corresponding to 25% of their intrinsic MICs) on MICs of antibiotics was investigated. Finally, real-time efflux (RTE) assays were performed in order to determine efflux pump inhibitory properties of compounds towards AcrAB-TolC in E. aerogenes.All compounds were investigated on their adjuvant action in the reference and MDR Gram-positive S. aureus: the reference strain S. aureus ATCC 25923 and extremely-drug resistant (XDR) MRSA HEMSA 5 clinical isolate. This step of the study was necessary for: (i) elucidation whether molecules tested are devoid of antistaphylococcal activity and thus cannot become antimicrobial agents by themselves; and (ii) determination of the concentrations of compounds suitable for the further assay on their antibiotic adjuvant potency. In addition, the antistaphylococcal efficacy of oxacillin, which was paired with compounds tested, in the following assays was also assessed . The MICs of remaining compounds were in the range of 0.25 mM to more than 2 mM.The results obtained for arylideneimidazolone derivatives E. aerogenes. Three following strains of this bacterial species were employed in the studies: (i) the clinical isolate Ea-289 overexpressing the AcrAB-TolC efflux pump and exhibiting a porin-deficient phenotype; (ii) the CM-64 strain which also overexpresses the AcrAB-TolC efflux pump but has no changes in porin content; (iii) the Ea-294 and Ea-298 that are Ea-289 strain derivatives which are devoid of AcrAB and TolC, respectively . For these molecules, the MICs could not be determined precisely.Then, MIC values of the compounds were evaluated with Gram-negative ectively . For mos10, 12 and 13 did not show any significant differences when assayed in efflux pump overexpressing strains (CM-64 and Ea-289) and efflux pump deficient strains (Ea-294 and Ea-308), these compounds were not considered as substrates for the AcrAB-TolC transporter. Furthermore, the absence of porins in Ea-289, Ea-298 and Ea-294 has no significant effect on the activity. Compounds 11 and 17 influenced the strains growth in corresponding manner to that of known EPI, PAβN, increasing MICs at least in 4-fold for Ea-289 and CM-64 in comparison to Ea-298 and Ea-294. Similar trends can be expected for 9 and 16, but it was impossible to determine it exactly due lack of growth inhibition for Ea-289 and CM-64 caused by both compounds at their highest tested concentration.As summarized in 6–16) were able to significantly improve the susceptibility of MRSA to oxacillin. At the same time, compounds did not exert any activity against the reference S. aureus strain (A < 4). The strongest chemosensitizing effect was demonstrated by compounds 16 and 9, which, at a concentration of 0.125 mM, reduced the MIC of oxacillin in 16- to 32-fold against MRSA HEMSA 5. Slightly lower adjuvant-like activity was determined for the imidazolone derivatives 7 and 10 (A = 8–16). The efficacy of the remaining compounds was slightly less marked, however, also significant. Although compounds tested improved noticeably the antibacterial activity of oxacillin against MRSA clinical isolate, none of them restored the activity of the antibiotic (MIC < 2 µg/mL) against this highly resistant strain.The lack of a direct antibacterial activity of arylideneimidazolone derivatives tested allowed analyzing their anti-resistance potency in combination with the β-lactam antibiotic, oxacillin. Compounds studied were examined at the concentrations corresponding to 25% of their respective MIC values or at the highest concentrations, at which they did not precipitate. The potential of compounds to become antibiotic adjuvants was evaluated by comparing the drug effectiveness in the presence and absence of compounds tested, thereby, by determining activity gain parameter A . Most ofE. aerogenes strains, their ability to enhance the activity of several antibiotics was assessed. The following four antibiotics were selected for the assays: chloramphenicol, erythromycin, doxycycline, and norfloxacin. The compounds were tested at the concentration corresponding to ¼ of their MIC. For compounds 9, 11, 16 and 17, for which determination of the exact intrinsic MIC value was not possible  to previously found potent efflux pump inhibitors for AcrAB-TolC , the representative structures  were also evaluated for their ability to inhibit the activity of AcrAB-TolC pump in E. aerogenes by measuring the efflux of a dye-substrate in the real-time efflux assay (RTE). The RTE assay is a dynamic tool by which the ability of a potential EPI to inhibit substrate transport in a drug efflux pump can be measured as a function of time and energy. Moreover, the test cannot discriminate for a possible outer membrane-destabilizing action of compounds. The dye, 1,2′-dinaphthylamine , a substrate of the AcrAB-TolC efflux pump, is a sensitive to highly lipophilic environment where it is fluorescent, but it is almost non-fluorescent in an aqueous solution  305.04. 1H-NMR δ [ppm]: 8.94–8.63 , 8.30 , 8.03–7.87 , 7.80–7.61 , 7.12 , 6.76 .(Z)-5-(4-phenoxybenzylidene)-2-thioxoimidazolidin-4-one (23) 4-Phenoxybenzaldehyde  and thiohydantoin  were used. Yellow solid. Yield 81%; mp 233–235 °C. C16H12N2O2S MW 296.34. LC/MS ±: purity 99.11% tR = 6.52, (ESI) m/z [M+H]+ 297.07. 1H-NMR δ [ppm]: 12.00 , 7.80–7.77 , 7.41–7.39 , 7.20–6.97 , 6.42 , 3.52 .(Z)-5-((9H-fluoren-2-yl)methylene)-2-thioxoimidazolidin-4-one (24) 9H-Fluorene-2-carbaldehyde  and thiohydantoin  were used. Yellow solid. Yield 86%; mp 276–278 °C. C17H12N2OS MW 292.35. LC/MS ±: purity 94.88% tR = 6.45, (ESI) m/z [M+H]+ 293.01. 1H-NMR δ [ppm]: 12.38 , 12.38 , 12.22 , 8.04 , 7.96–7.92 , 7.71 , 7.60 , 7.43–7.32 , 6.57 , 3.96 .Sodium  was put into ethanol (21.00–37.77 mL). Sodium ethoxide mixed with appropriate 5–arylidenethiohydantion (21.00–37.77 mmol) in flat–bottom flask for 3 min. Then, iodomethane  was added and whole were mixed for 5–24 h. Reaction was controlled by TLC-chloroform/ethyl acetate: 1/1. Purification was performed using crystallization from acetone wherever necessary.(Z)-2-(methylthio)-4-(phenanthren-9-ylmethylene)-1H-imidazol-5(4H)-one (31) (Z)-5-(Phenanthren-9-ylmethylene)-2-thioxoimidazolidin-4-one (21)  with iodomethane  was used. Yellow solid. Yield 99%; mp 271–274 °C. C19H14N2OS MW 318.39. LC/MS±: purity 97.70% tR = 6.77, (ESI) m/z [M+H]+ 319.06. 1H-NMR δ [ppm]: 9.24–9.10 , 8.90–8.81 , 8.36–8.29 , 8.02–7.99 , 7.67–7.40 , 6.87 , 2.71 .(Z)-4-(4-phenoxybenzylidene)-2-(methylthio)-1H-imidazol-5(4H)-one (33) (Z)-5-(4-Phenoxybenzylidene)-2-thioxoimidazolidin-4-one (23)  with iodomethane  was used. Yellow solid. Yield 92%; mp 177–179 °C. C17H14N2O2S MW 310.37. LC/MS±: purity 96.15% tR = 7.39, (ESI) m/z [M+H]+ 311.09. 1H-NMR δ [ppm]: 11.80 , 8.29–8.18 , 7.44–7.38 , 7.21–7.13 , 7.09–6.99 , 6.71 , 2.62 .(Z)-5-((9H-fluoren-2-yl)methylene)-2-(methylthio)-3H-imidazol-4(5H)-one (34) (Z)-5-((9H-Fluoren-2-yl)methylene)-2-thioxoimidazolidin-4-one (24)  with iodomethane  was used. Yellow solid. Yield 90%; mp 240–242 °C. C18H14N2OS MW 306.38. LC/MS±: purity 91.26% tR = 7.58, (ESI) m/z [M+H]+ 307.10. 1H-NMR δ [ppm]: 8.39 , 8.27–8.18 , 7.99–7.87 , 7.64–7.54 , 7.45–7.20 , 6.80 , 3.96 , 2.70 .(Z)-5-(3-phenoxybenzylidene)-2-(methylthio)-3H-imidazol-4(5H)-one (37) (Z)-5-(3-Phenoxybenzylidene)-2-thioxoimidazolidin-4-one (27)  with iodomethane  was used. Yellow solid. Yield 95%; mp 214–216 °C. C17H14N2O2S MW 310.37. LC/MS±: purity 95.24% tR = 7.32, (ESI) m/z [M+H]+ 311.02. 1H-NMR δ [ppm]: 11.80 , 8.06 , 7.64–7.61 , 7.43–7.38 , 7.19–7.05 , 6.67 , 2.35 .H-imidazol-4(5H)-one (2–10 mmol) with appropriate amine (3–15 mmol) derivative in 50 mL flat-bottom flask were heated in oil bath with controlled temperature (120–130 °C) for 15 min. Then, ethanol (15-30 ml) was added and mixture was heated for 5–7 h and mixed for the next 20 h. Then, compounds were converted into hydrochloride forms by gaseous hydrochloride acid obtained from reaction between sodium chloride and sulphuric acid. Purification was performed using crystallization from ethanol wherever necessary.(Z)-2-(methylthio)-5-arylidene-3(Z)-5-(4-chlorobenzylidene)-2-amino-3-(3-(4-methylpiperazin-1-yl)propyl)-3H-imidazol-4(5H)-one hydrochloride (7); (Z)-5-(4-Chlorobenzylidene)-2-(methylthio)-3H-imidazol-4(5H)-one (28)  and 3-(4-methylpiperazin-1-yl)propan-1-amine  were used. White solid. Yield 7.5%; mp 294 °C. C18H24ClN5Ox3HClxH2O MW 486.25. LC/MS ±: purity 100.00% tR = 3.34, (ESI) m/z [M+H]+ 362.26. 1H-NMR δ [ppm]: 11.82 , 9.22 , 7.84 , 7.49 , 6.77 , 3.78 , 3.48 , 3.19 , 2.80 , 2.02 .(Z)-5-(4-Methoxybenzylidene)-2-amino-3-(3-(4-methylpiperazin-1-yl)propyl)-3H-imidazol-4(5H)-one hydrochloride (8); (Z)-5-(4-Methoxybenzylidene)-2-(methylthio)-3H-imidazol-4(5H)-one (29)  and 3-(4-methylpiperazin-1-yl)propan-1-amine  were used. Yellow solid. Yield 15%; mp 262 °C. C19H27N5O2x3HCl·0.5H2O MW 475.93. LC/MS±: purity 100.00% tR = 2.58, (ESI) m/z [M+H]+ 358.34. 1H-NMR δ [ppm]: 11.8 , 9.5 , 7.76 , 7.01 , 6.84 , 3.81 , 3.70–3.20 , 2.79 , 2.00 .(Z)-2-Amino-3-(3-(4-methylpiperazin-1-yl)propyl)-5--3H-imidazol-4(5H)-one hydrochloride (9) (Z)-2-(methylthio)-4--1H-imidazol-5(4H)-one (30)  and 3-(4-methylpiperazin-1-yl)propan-1-amine  were used. Yellow solid. Yield 74%; mp 215-217 °C. C22H28ClN5O MW 413.94. LC/MS±: purity 100.00% tR = 3.34, (ESI) m/z [M+H]+ 378.28. 1H-NMR δ [ppm]: 8.39–8.34 , 7.85–7.78 , 7.47–7.45 , 6.50 , 3.54 , 2.45–2.20 , 2.09 , 1.68 .(Z)-2-Amino-3-(3-(4-methylpiperazin-1-yl)propyl)-5-(phenanthren-9-ylmethylene)-3H-imidazol-4(5H)-one hydrochloride (10) (Z)-2-(Methylthio)-5-(phenanthren-9-ylmethylene)-3H-imidazol-4(5H)-one (31)  and 3-(4-methylpiperazin-1-yl)propan-1-amine  were used. Yellow solid. Yield 17%; mp 266–268 °C. C26H30ClN5O MW 464.00. LC/MS±: purity 99.00% tR = 4.03, (ESI) m/z [M+H]+ 428.19. 1H-NMR δ [ppm]: 8.89–8.86 , 8.80–8.78 , 8.24–8.21 , 7.89 , 7.73–7.69 , 7.68–7.64 , 6.93 , 3.60–3.20 , 2.45–2.15 , 2.09 , 1.75 .(Z)-2-Amino-5-(anthracen-10-ylmethylene)-3-(3-(4-methylpiperazin-1-yl)propyl)-3H-imidazol-4(5H)-one hydrochloride (11) (Z)-5-(Anthracen-10-ylmethylene)-2-(methylthio)-3H-imidazol-4(5H)-one (32)  and 3-(4-methylpiperazin-1-yl)propan-1-amine  were used. Yellow solid. Yield 61%; mp 246-248 °C. C26H30ClN5O MW 464.00. LC/MS±: purity 100.00% tR = 3.89, (ESI) m/z [M+H]+ 428.26. 1H-NMR δ [ppm]: 8.61 , 8.11-8.01 , 7.53–7.52 , 7.00 , 3.40–3.15 , 2.56–2.06 , 1.58–1.52 .(Z)-5-(4-Phenoxybenzylidene)-2-amino-3-(3-(4-methylpiperazin-1-yl)propyl)-3H-imidazol-4(5H)-one (12) (Z)-5-(4-Phenoxybenzylidene)-2-(methylthio)-1H-imidazol-5(4H)-one (33)  and 3-(4-methylpiperazin-1-yl)propan-1-amine  were used. Yellow solid. Yield 68%; mp 220–230 °C. C24H30ClN5O2 MW 455.98. LC/MS±: purity 100.00% tR = 4.1, (ESI) m/z [M+H]+ 420.28. 1H-NMR δ [ppm]: 7.62–7.57 , 7.46–7.36 , 7.15–6.95 , 6.72 , 3.77 , 3.70–3.14 , 2.79 , 1.99 . 13C-NMR : δ 157.23, 156.98, 130.56, 124.01, 118.93, 40.77, 40.49, 40.20, 39.92, 39.64, 39.36, 39.08.(Z)-5-((9H-Fluoren-2-yl)methylene)-2-amino-3-(2-(4-methylpiperazin-1-yl)ethyl)-3H-imidazol-4(5H)-one hydrochloride (13) (Z)-5-((9H-Fluoren-2-yl)methylene)-2-(methylthio)-3H-imidazol-4(5H)-one (34)  and 3-(4-methylpiperazin-1-yl)propan-1-amine  were used. Orange solid. Yield 69.27%; mp 265–267 °C. C25H30ClN5O MW 451.99. LC/MS±: purity 99% tR = 3.92, (ESI) m/z [M+H]+. 1H-NMR δ [ppm]: 12.00 , 9.35 , 8.32–7.70 , 7.67–7.55 , 7.49–7.24 , 6.82 , 4.08 , 3.90–3.01 , 2.90 , 2.06 . 13C-NMR : δ 144.20, 143.96, 127.42, 126.88, 125.74, 125.51, 121.18, 121.00, 40.94, 40.89, 40.52, 40.24, 39.68, 39.39, 39.24, 38.95.(Z)-2-Amino-5-(biphen-4-ylmethylene)-3-(2-(4-methylpiperazin-1-yl)ethyl)-3H-imidazol-4(5H)-one hydrochloride (14) (Z)-5-(Biphen-4-ylmethylene)-2-(methylthio)-3H-imidazol-4(5H)-one (35)  and 3-(4-methylpiperazin-1-yl)propan-1-amine  were used. Yellow solid. Yield 49%; mp 258–260 °C. C24H30ClN5O MW 439.98. LC/MS±: purity 96.39% tR = 3.78, (ESI) m/z [M+H]+. 1H-NMR δ [ppm]: 12.00 , 8.24–8.05 , 7.80 , 7.79–7.61 , 7.52–7.30 , 6.83 , 4.26–2.97 , 2.80 , 2.04 . 13C-NMR : δ 129.51, 127.11, 40.77, 40.49, 40.21, 39.92, 39.64, 39.36, 39.08.(Z)-2-Amino-3-(3-(4-methylpiperazin-1-yl)propyl)-5--3H-imidazol-4(5H)-one hydrochloride (15) (Z)-2-(Methylthio)-5--3H-imidazol-4(5H)-one (36)  and 3-(4-methylpiperazin-1-yl)propan-1-amine  were used. Yellow solid. Yield 24%; mp 187–191 °C. C22H28ClN5O MW 413.94. LC/MS±: purity 99.07% tR = 3.21, (ESI) m/z [M+H]+ 378.21. 1H-NMR δ [ppm]: 8.83 , 8.19 , 7.96–7.88 , 7.85–7.75 , 7.61–7.44 , 6.98 , 3.32 , 2.47–2.17 , 2.12 , 1.70 . 13C-NMR : δ 133.79, 131.93, 131.62, 129.16, 128.53, 127.90, 126.89, 126.19, 126.05, 123.27, 55.09, 53.02, 46.12, 40.81, 40.52, 40.24, 39.96, 39.68, 39.20, 39.11.(Z)-2-Amino-5-(anthracen-10-ylmethylene)-3-(3-morpholinopropyl)-3H-imidazol-4(5H)-one hydrochloride (16) (Z)-5-(Anthracen-10-ylmethylene)-2-(methylthio)-3H-imidazol-4(5H)-one (33)  and 3-morpholinopropan-1-amine  were used. Orange solid. Yield 70%; mp 244–246 °C. C25H27ClN4O2 MW 450.96. LC/MS±: purity 98.71% tR = 4.19, (ESI) m/z [M+H]+ 415.17. 1H-NMR δ [ppm]: 11.25 , 10.21 , 9.39 , 8.72 , 8.25–7.95 , 7.68–7.43 , 4.16–2.69 , 2.12 , 1.80 . 13C-NMR : δ 131.49, 129.42, 129.28, 126.77, 125.96, 66.60, 55.99, 53.71, 40.79, 40.50, 40.22, 39.94, 39.66, 39.37, 39.09.(Z)-5-(3-phenoxybenzylidene)-2-amino-3-(3-morpholinopropyl)-3H-imidazol-4(5H)-one hydrochloride (17) (Z)-5-(3-Phenoxybenzylidene)-2-(methylthio)-3H-imidazol-4(5H)-one (37)  and 3-morpholinopropan-1-amine  were used. Yellow solid. Yield 41%; mp 227–230 °C. C23H27ClN4O3 MW 442.94. LC/MS±: purity 96.49% tR = 4.37, (ESI) m/z [M+H]+ 407.19. 1H-NMR δ [ppm]: 11.35 , 9.45 , 7.82-7.27 , 7.26–6.85 , 6.74 , 4.03 , 3.66–2.65 , 2.16–1.88 . 13C-NMR : δ 157.67, 157.41, 130.55, 124.13, 118.97, 63.54, 51.44, 40.81, 40.53, 40.25, 39.97, 39.68, 39.40, 39.12.2. The hydrogen atoms bonded to carbons were included in the structure at idealized positions and were refined using a riding model with Uiso(H) fixed at 1.2 Ueq of C and 1.5 Ueq for methyl groups. Hydrogen atoms attached to nitrogen atom were found from the difference Fourier map and refined without any restraints. Refinement and further calculations were carried out using SHELXL , R1 = 0.0499, wR2 = 0.1197 .Crystallographic data: CThe supplementary for these crystallographic data can be obtained free of charge from the Cambridge Crystallographic Data Centre, deposited as CCDC 1,886,697.S. aureus and E. aerogenes bacteria were maintained and grown on Columbia agar  or Trypticase Soy Agar II  supplemented with 5% sheep blood, respectively. Cation-adjusted Mueller-Hinton (MH II) broth used in the microbiological assays was obtained from bioMérieux. Oxacillin, norfloxacin, chloramphenicol, doxycycline, PAβN, and INT were purchased from Sigma Aldrich , whereas erythromycin lactobionate was purchased from Amdipharm (United Kingdom). The compounds tested were dissolved in DMSO  and the solutions obtained were stored at −20 °C until used. Furthermore, the following chemical compounds were used to perform RTE assay: K2HPO4 (Sigma Aldrich) and MgCl2 (Sigma Aldrich) to prepare potassium phosphate buffer (PPB), CCCP (Sigma Aldrich), 1,2′-dNA , glucose (Sigma Aldrich).® France, SA-Lyon, France) after overnight incubation at 37 °C. Experiments were carried out in triplicate and the resulting medians were presented.Susceptibility testing was performed by the 2-fold standard microdilution method in MH II broth following the Clinical and Laboratory Standards Institute (CLSI) and the Comité de l’Antibiogramme de la Société Française de Microbiologie (CA-SFM) recommendations ,48. MIC S. aureus and E. aerogenes strains. Then, MICs of selected antibiotics were determined in the absence and in the presence of compounds in order to measure the adjuvant-like effect of these structures. In the latter case, compounds tested were evaluated for the ability to enhance antibacterial activity of β-lactam antibiotic oxacillin in methicillin-resistant and methicillin-susceptible S. aureus (MRSA and MSSA) strains as well as chloramphenicol, erythromycin, doxycycline, and norfloxacin in AcrAB-TolC-overexpressing and AcrAB-TolC-deficient mutants of E. aerogenes. The concentrations of imidazolidine derivatives used in the MIC reduction assay were not greater than 25% of their respective MICs, therefore, cell viability was not affected by the direct antibacterial activity of compounds. For compounds for which precipitation was observed after the addition of bacterial suspension in MH II broth, the highest concentration at which they did not precipitate was chosen. The final concentration of DMSO in the assays never exceed 2.5% v/v and had no influence on bacteria. In tests with E. aerogenes, a well-known efflux pump inhibitor, PAβN, was used as reference compound. The MICs of imidazolidine derivatives and antibiotics selected for experiments were recorded as the lowest concentrations of compounds inhibiting the growth of bacteria after 18-h incubation at 37 °C. Due to the heterogeneity of MRSA strains, the incubation of compounds with oxacillin was extended to 24 h. The growth of bacterial cells was visualized by iodonitrotetrazolium chloride (INT). The results of the MIC reduction assay were presented by employing activity gain parameter (A) calculated as the ratio of the MIC value of an antibiotic alone to its MIC in the presence of the compound analyzed (Equation (1)). All MIC determinations were repeated in at least two independent experimentsFirst, minimum inhibitory concentration (MIC) values of a series of imidazolidine derivatives were examined against +-consuming). After the addition of compounds, at the final concentration of 100 µM, an efflux was initiated by automated injection of glucose that provides pump energy (H+-donating compound) to the final concentration of 50 μM. The fluorescence intensity was measured using a microplate reader (Tecan) with an excitation wavelength of λex = 370 nm and an emission wavelength of λem = 420 nm. In order to analyze and quantitatively compare the EPI activity of compounds tested, the pre-energization fluorescence intensity was adjusted to 100 relative fluorescence units and the inhibition efficiency (IE) of each compound was calculated according to Equation (2):1 corresponds to the difference between the fluorescence of the dye in the presence and absence of compound tested after the addition of a source of energy for the pump (glucose) and Δi2 is the difference between the fluorescence of the dye in the presence and absence of compound tested before the addition of a source of energy for the pump.In the first step, bacteria were loaded with 1,2′-dNA, a fluorescent membrane probe which is a substrate of the AcrAB-TolC efflux pump, in the presence of a well-known EPI carbonyl cyanide m-chlorophenylhydrazone (CCCP) that inactivates the pump in the pump energy-collapsing mode  were generated with the use of LigPrep . At firsThe poses with the best Glide docking score were use as starting points for molecular dynamic (MD) simulations. MD simulations were performed in Desmond ,55, usinThe interactions between ligands and PBP2a protein were analyzed manually and with the use of the Simulation Interaction Diagram from the Schrodinger Suite.N-oxide (NQNO) and were provided by Sigma-Aldrich . HepG2 (ATCC® HB-8065™) hepatoma cell line was kindly donated by the Department of Pharmacological Screening, Jagiellonian University Medical College. Salmonella typhimurium TA100 strain with base pair substitution  was purchased from Xenometrix, Allschwil, Switzerland.The references used in ADMET studies included caffeine (CFN), norfloxacin (NFX), doxorubicin (DX), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), nonyl-4-hydroxyquinoline-9 and the references (200 µM) were prepared first in PBS buffer (pH = 7.4) and added to the donor wells of PAMPA Plate System. PBS was added to the acceptor wells. The plate was incubated for 5 h at room temperature. The compounds’ as well as references’ concentrations in acceptor and donor wells were estimated by the UPLC-MS analyses with use of internal standard. The permeability coefficients  were calculated according to formulas provided by the manufacturer.Pre-coated PAMPA Plate System Gentest™ was purchased from Corning . The tested compound S. typhimurium TA100 in the indicator medium without histidine after 72 h incubation at 37 °C temperature. NQNO (0.5 µM) was used as a positive control. Compound 9 was tested in triplicate at the final concentrations 1 and 10 µM. The revertants’ growth induced the colour change of medium which was analysed next colourimetrically with a microplate reader  at 420 nm.Ames microplate fluctuation protocol (MPF) assay was obtained from Xenometrix AG . The occurrence of mutagen-induced or spontaneous reversion events to histidine prototrophy was determined as a growth of 4 cells/well and incubated for 24 h at 37 °C in 5% CO2 atmosphere to reach 50% of confluence. Compound 9 was diluted into fresh growth medium and added to the cells at the final concentrations 0.1 μM–100 μM. The positive controls DX and CCCP were added at 1 μM and 10 μM, respectively, and the cells were incubated for 72 h. The MTS reagent  was added next to the each well and incubated for 4 h. The absorbance was measured next using a microplate reader  at 490 nm to determine cells’ viability.The HepG2 cells were seeded in 96-well plates at a concentration of 1 × 10Z)-5-(4-chlorobenzylidene)-2-(4-methylpiperazin-1-yl)-3H-imidazol-4(5H)-one (6) have provided a series of new, and even unexpected, chemical structures due to Dimroth rearrangements within the imidazolone ring. Most of those original compounds significantly improved oxacillin activity in MRSA, while they did not influence the activity of oxacillin in the reference S. aureus strain, suggesting an impact on MDR mechanisms associated with PBP2a. Docking and molecular dynamic simulations have confirmed this hypothesis, indicating the ability of the most active compounds to interact with the allosteric site of PBP2a, and to enhance the binding of oxacillin to the active site. On the other hand, results of the RTE assay for the new 5-arylideneimidazolones towards AcrAB-TolC in E. aerogenes (EA289) have confirmed their potent capacity to inhibit this important MDR efflux system. The performed SAR analysis indicated the anthracene-morpholine derivative (16) as the most active oxacillin adjuvant in the MRSA, and the naphthalene-methylpiperazine imidazolone (9) as the most potent “dual-action” compound displaying both oxacillin potentiating action in MRSA in the range of compound 16 and efflux pump inhibitory properties in E. aerogenes. The primary ADMET screening in vitro for 9 showed a rather moderate “drugability” profile for this active compound. In the context of the obtained results, further comprehensive studies for this interesting chemical family are needed. Overall, compound 9, and then 16, seem to be new lead structures for further modifications in order to discover therapeutically useful adjuvants, able to restore the effectiveness of common antibiotics against MDR pathogens.In order to search for successful antibiotic adjuvants, useful against both Gram-positive and Gram-negative MDR pathogens, the performed chemical modifications of the lead structure (


We obtained both cross coupling and homocoupling products of pyridine and some side products such as dimethylaminopyridine and unsubstituted pyridine.A number of novel benzimidazole derivatives  Some of them also need very reactive phenyl lithium or phenyl magnesium halide as a homocoupling partner , NCHN in benzimidazolium salts 1–4 were observed at δ = 143.5, 143.2, 142.9 and 142.7 ppm, respectively. These values are in good agreement with other recently reported results [(C=N), for benzimidazolium salts 1–4 were observed at 1,559, 1,559, 1,567 and 1,558 cm−1, respectively.In this work the synthesis of the benzimidazole salts  from 1-4, and I  is shown results ,35,36,372O (1:1) mixture was chosen as a best solvent for the both the Suzuki-Miyaura and the Ullmann type coupling reactions.In the present work 2-bromopyridine was chosen as a model coupling partner for both the Suzuki-Miyaura type C-C cross-coupling and the Ullmann type homocoupling reaction to determine the best solvent for these reactions. The values of other parameters such as base, temperature and catalyst loading true catalyst amount were based on our recently reported optimal parameters . DMF/H2OThe Suzuki-Miyaura reaction is one of the most versatile and utilized reactions for the selective construction of carbon-carbon bonds, in particular for the formation of biaryl and heterobiaryl derivatives ,55.I and 1–4 in the coupling reactions between 2- or 3-halopyridines and phenylboronic acid under optimized conditions. The solvent effects were investigated using 2-bromopyridine and phenylboronic acid in terms of the main product yield (Suzuki-Miyaura product) in EtOH/H2O (1:1) 57.6% (2O (1:1) 63.4% (2O (1:1) with a 70.0% (2O (1:1) mixture was chosen as a best solvent system for the coupling reaction. The catalytic reactivity was also investigated in pure water as a green solvent, but the coupling product yield decreased drastically to 2%  at 120 °C/300 W microwave heating given rise to the best conversation within 10 min. Apart from the expected Suzuki-Miyaura cross coupling products, some side products such as biphenyl, pyridine, 2,2'-bipyridine or 3,3'-bipyridine, and 2-dimethylaminopyridine were also obtained varying amounts from the reaction mixtures. All of the results for the Suzuki-Miyaura type coupling reactions with side products were given in tBu and a long reaction time, except without phenylboronic acid, was reported in the literature recently [In this work, we aimed to determine efficiencies of benzimidazolium salts 1) 57.6% , entry 31) 63.4% , entry 4 a 70.0% , entry 5ly to 2% , entry 2ly to 2% , entry 1recently ,57.4 having only an ethyl group was found to be the less reactive for the Suzuki-Miyaura type reactions  (2O (1:1) (2O (1:1) (2O (1:1) was found the good solvent systems but the homocoupling yield was still low. Hence, the reaction time was increased to 30 min. Running the reaction for 30 min at DMF/H2O (1:1) mixture as a solvent we obtained reasonable homocoupling yield  at 120 °C/300 W microwave heating were chosen as an optimum reaction conditions. All of the results obtained using the optimum parameters for the Ullmann type coupling reactions, along with some side products, were given in Efficiencies of benzimidazolium salts in homocoupling reactions were examined using 2- or 3-halopyridines. Similar to the Suzuki-Miyaura cross-coupling reaction, we first tried different solvent systems such as pure water , entry 12O (1:1) , entry 22O (1:1) , entry 32O (1:1) , entry 4ng yield , entry 5It is noteworthy that 2- or 3-chloropyridines -Im complexes has been reported [The coupling products of halopyridines are given in the literature as a Suzuki-Miyaura type or the Ullmann type coupling product only. In our work, we also determined some side products such as dimethylaminopyridine, and pyridine in addition of the main coupling products. To the best of knowledge, there is limited information about these types of products in the literature. Catalytic dimethylamino group transfer from some amides to aryl halides using Ni(phen)Clreported ,57.1–4. The use of a palladium catalyst system including benzimidazolium salts/base and solvent in the Suzuki-Miyaura and the Ullmann type coupling reactions gives low to moderate yields under microwave-assisted conditions and relatively short reaction times compared with those reported in the literature [In conclusion, we prepared four new NHC precursor benzimidazolium salts containing substituted benzyl and phenethyl or ethyl groups terature ,49,50,511H-NMR (300.13 MHz) and 13C-NMR (75.47 MHz) spectra were determined using a Bruker DPX-300 high-performance digital FT-NMR spectrometer. FT-IR spectra were recorded on a Perkin-Elmer spectrophotometer in the range 4,000–400 cm−1. Elemental analyses were performed by LECO CHNS-932 elemental analyzer. Melting points were recorded using an Electrothermal 9200 melting point apparatus, and are uncorrected.All of using starting chemicals were supplied commercially by Merck, Fluka or Aldrich Chemical Co. All catalytic activity experiments were carried out in a microwave oven system manufactured by Milestone (Milestone Start S Microwave Labstation for Synthesis) under aerobic conditions. Both m/z 50–550.GC-MS analysis were performed on an Agilient 6890 N GC and 5973 Mass Selective Detector system using by an HP-Innowax column of 60 m length, 0.25 mm diameter and 0.25 μm film thickness. GC-MS parameters for the S-M and homocoupling reactions were as follows: initial temperature 60 °C; initial time, 15 min; temperature ramp 1.30 °C/min; final temperature, 200 °C; ramp 2, 20 °C/min; final temperature 230 °C; total run time 72 min; injector port temperature 250 °C; detector temperature 250 °C; injection volume, 1.0 μL; carrier gas, helium; mass range between I [1–4 are given below.All coupling product yields are based on aryl halides and determined as follows: the percent of the aryl halides were determined from the aryl halide chromatograms using the normalized peak areas method taken at the optimized chromatographic conditions. After completion of the reaction, the % coupling products were determined based on aryl halides. 1-(4-Bromobenzyl)benzimidazole  and its I  were preSynthesis of 1-(4-bromobenzyl)-3-(4-chlorobenzyl)benzimidazolium chloride (1). A mixture of 1-(4-bromobenzyl)benzimidazole  and 4-chlorobenzyl chloride  in DMF (5 mL) was refluxed for 4 h. After mixture was cooled solvent was removed under reduced pressure. The precipitate was crystallized from EtOH/Et2O (1:1) (30 mL). The product was obtained as white colored crystals. Yield: 2.28 g, 73%; mp 249–250 °C, IR, υ(C=N) = 1559 cm−1. Anal. found: C, 55.87; H, 3.48; N, 5.91%. Calcd for C21H17N2BrCl2 (448.18): C, 56.28; H, 3.82; N, 6.25%. 1H-NMR (DMSO-d6) δ 5.83 , 5.84 , 7.49-8.00 , 10.23 . 13C-NMR (DMSO-d6) δ 49.8 (CH2C6H4Br and CH2C6H4Cl), 114.5, 122.6, 127.3, 129.5, 130.9, 131.2, 131.5, 132.4, 133.3, 133.8, 134.0 (Ar-C), 143.5 (NCHN). The compounds 2, 3, and 4 were similarly prepared from 1-(4-bromobenzyl)benzimidazole and the appropriate alkyl halides.1-(4-Bromobenzyl)-3-(4-methylbenzyl)benzimidazolium bromide (2). Yield: 2.57 g, 78%; mp 255–256 °C, IR, υ(C=N) = 1559 cm−1. Anal. found: C, 56.19; H, 4.34; N, 5.80%. Calcd for C22H20N2Br2 (472.21): C, 55.96; H, 4.27; N, 5.93%. 1H-NMR (DMSO-d6) δ 2.30 ; 5.77 , 5.82 , 7.23–8.01 , 10.16 . 13C-NMR (DMSO-d6) δ 21.2 (CH2C6H4CH3), 49.8 (CH2C6H4CH3), 50.4 (CH2C6H4Br), 114.5, 114.6, 122.6, 127.3, 128.8, 128.9, 130.0, 131.2, 131.3, 131.4, 131.5, 132.4, 133.8, 138.7 (Ar-C), 143.2 (NCHN). 1-(4-Bromobenzyl)-3-(2-phenylethyl)benzimidazolium bromide (3). Yield: 2.83 g, 86%; mp 144–145 °C, IR, υ(C=N) = 1567 cm−1. Anal. found: C, 55.39; H, 4.44; N, 5.74%. Calcd for C22H20N2Br2 (472.21): C, 55.96; H, 4.27; N, 5.93%. 1H-NMR (DMSO-d6) δ 3.27 , 4.82 , 5.72 , 7.17–8.13 , 9.77 . 13C-NMR (DMSO-d6) δ 34.8 (CH2CH2C6H5), 48.4 (CH2CH2C6H5), 49.5 (CH2C6H4Br), 114.3, 114.5, 122.5, 127.2, 127.3, 127.4, 129.1, 129.3, 130.9, 131.1, 131.6, 132.3, 133.8, 137.3 (Ar-C), 142.9 (NCHN).1-(4-Bromobenzyl)-3-ethylbenzimidazolium iodide (4). Yield: 2.07 g, 67%; mp 148–150 °C, IR, υ(C=N) = 1558 cm−1. Anal. found: C, 44.03; H, 3.88; N, 6.04%. Calcd for C16H16N2BrI (443.12): C, 43.37; H, 3.64%; N, 6.32. 1H-NMR (DMSO-d6) δ 1.57 , 4.54 , 5.76 , 7.48-8.13 , 9.88 . 13C-NMR (DMSO-d6) δ 14.5 (CH2CH3), 42.8 (CH2CH3), 49.7 (CH2C6H4Br), 114.3, 114.4, 122.5, 127.1, 127.2, 131.1, 131.3, 131.7, 132.3, 133.8 (Ar-C), 142.7 (NCHN).2 (1 mmol %), benzimidazole salt  (2 mmol %), halopyridine (1 mmol), phenylboronic acid (1.2 mmol), K2CO3 (2 mmol) and mixture of solvent, water (3 mL)-DMF (3mL) were added in apparatus of microwave equipment in aerobic conditions. The mixture was heated at 120 °C, by microwave irradiation (300 Watt) for 10 min. At the end of reaction, the mixture extracted by ethyl acetate/n-hexane (1:5), filtered through 3 cm length column of silica gel. The solution was given to GC-MS equipment. The yields were determined by GC-MS based on halopyridine using the normalized peak areas method.Pd(OAc)2 (1 mmol %), benzimidazole salt I, 1–4 (2 mmol %), halopyridine (2 mmol), K2CO3 (2 mmol) and mixture of solvent, water (3 mL)-DMF (3 mL) were added in apparatus of microwave equipment in aerobic conditions. The mixture was heated at 120 °C, by microwave irradiation (300 Watt) for 30 min. After competition of the reaction, work-ups similar to those of the Suzuki-Miyaura reactions were followed to obtain the appropriate products.Pd(OAc)2/benzimidazolium salt and K2CO3 for the Suzuki-Miyaura cross coupling and the Ullmann type homocoupling reactions of 2- or 3-halopyridines. We also obtained some side products such as dimethylaminopyridine dervivatives and pyridine with low yield in both the coupling reactions under microwave irradiation.In summary, we have synthesized several novel benzimidazolium salts through nucleophlic substitution reactions. Catalytic studies were done using catalytic systems consisting of Pd(OAc)


Four novel bisquaternary aldoxime cholinesterase reactivators differing in their chemical structure were prepared. Afterwards, their biological activity was evaluated for their ability to reactivate acetylcholinesterase  and butyryl-cholinesterase  inhibited by paraoxon. Their reactivation activity was compared with standard reactivators—pralidoxime, obidoxime and HI-6—which are clinically used at present. As it resulted, none of the prepared compounds surpassed obidoxime, which is considered to be the most potent compound if used for reactivation of AChE inhibited by paraoxon. In case of BuChE reactivation, two compounds (K053 and K068) achieved similar results as obidoxime. Acetylcholinesterase AChE; EC 3.1.1.7) reactivators are a group of drugs originally developed as antidotes for the treatment of nerve agent poisonings . With th.1.1.7 reStandard therapy of such intoxications consists of administration of anticholinergic drugs (mostly atropine), AChE reactivators  could neutralize nerve agents in the blood stream before they reach their physiological targets. The most investigated, human serum BuChE (EC 3.1.1.8), can be used successfully with relatively high protective potency ,7.In this study, we have prepared four novel bisquaternary aldoxime cholinesterase reactivators  with the aim of obtaining new more promising oxime candidates which could in future replace the clinically used reactivators . ParaoxoAll obtained results are summarized in If the obtained results are compared, the new AChE reactivators are not better than the clinically used ones, so that their further investigation cannot be recommended. Due to this, novel structurally different oximes derived from clinically used ones  should be designed and tested for their reactivation potency against POX. On the contrary, if BuChE reactivation is considered, only two oximes (K053 and K068) achieved 10% reactivation. Due to this, if novel BuChE reactivators are to be designed in the future, the results of this study could be used as first approximation to the desired structure with higher BuChE reactivation potency.O,O-diethyl-O-4-nitrophenylphosphate, 95% purity) was purchased from Dr. Ehrenstorfer . 1H- and 13C-NMR spectra were recorded at 300 and 75 MHz, respectively, on a Varian Mercury 300 spectrometer, using D2O as solvent. All experiments were carried out in compliance with the current law of Czech Republic.All chemicals used in this study were of reagent grade. They were obtained from commercial sources . Paraoxon methyl]-1,1′-bispyridinium dibromide (K053): yield 68%; m.p. 194-196 °C; 1H-NMR δ: 5.52 , 5.89 , 6.03 , 6.34 , 8.07 , 8.21 , 8.24 , 8.38 , 8.61 , 8.63 , 8.85 , 8.87 . The signals of =NOH disappeared in deuterated solvent; 13C-NMR δ: 48.37 (CH2), 62.23 (CH2), 126.94 (CH=), 126.96 (CH=), 127.02 , 129.90 (CH-3), 132.85 (CH-5), 146.95 , 146.99 (CH-6), 148.18 (C-4′), 148.21 (CH-4), 148.74 (CH=N), 151.41 (CH=N), 157.37 (C-2).trans-3,4-Dicarbamoyl-2′-(hydroxyimino)methyl-1,1′-bispyridinium dibromide (K054): yield 55%; m.p. 209-211 °C; 1H-NMR δ: 5.40 , 5.54 , 6.04 , 6.42 , 8.09 , 8.34 , 8.43 , 8.59 , 8.64 , 8.86 , 9.11 , 9.25 . The signals of CONH2 and =NOH disappeared in deuterated solvent; 13C-NMR δ: 48.48 (CH2), 61.25 (CH2), 127.51 (CH=), 129.08 (CH=), 129.30 (CH-5), 129.91 (CH-3′), 130.27 (CH-5′), 135.86 (C-3), 144.12 (CH-6), 146.76 (CH-6′), 148.37 (CH-4′), 148.76 (CH-2), 149.88 (CH=N), 152.04 (C-4), 157.32 (C-2′), 167.90 (CONH2), 169.49 (CONH2).trans-2,2′-Bis[(hydroxyimino)methyl]-1,1′-bispyridinium dibromide (K068): yield 60%; m.p. 196-199 °C; 1H-NMR δ: 5.28 , 6.06 , 8.08 , 8.38 , 8.60 , 8.78 , 8.87 . The signal of =NOH disappeared in deuterated solvent; 13C-NMR δ: 48.53 (CH2), 127.45 (CH=), 129.80 (CH-3), 130.53 (CH-5), 145.01 (CH-6), 148.57 (CH-4), 148.74 (CH=N), 157.31 (C-2).trans-4′-tert-Butyl-2-(hydroxyimino)methyl-1,1′-bispyridinium dibromide (K071): yield 47%; m.p. > 300 °C; 1H-NMR δ: 1.25 , 5.22 , 5.84 , 6.04 , 6.32 , 8.02-9.25 , 8.64 . The signal of =NOH disappeared in deuterated solvent; 13C-NMR δ: 31.55 (CH3), 38.45 (C), 48.38 (CH2), 64.37 (CH2), 126.61 , 127.47 (CH=), 127.54 (CH=), 128.30 (CH-3), 129.84 (CH-5), 142.96 , 146.05 (CH-6), 146.69 (CH-4), 148.70 (CH=N), 157.94 (C-2), 166.88 (C-4′).in vitro reactivation test [1/2) was calculated from experimentally determined half life (T1/2) of reaction between enzyme and paraoxon. Then, the inhibited enzyme was incubated for 10 min with a solution of reactivator at concentration 10-4 M and 10-5 M. Activity of AChE (BuChE) was measured spectrophotometrically by modified method according to Ellman with acetylthiocholine (butyrylthiocholine) as substrate [0 is activity of intact enzyme, ai is activity of inhibited enzyme and ar is activity of reactivated enzyme minus oximolysis and spontaneous hydrolysis. Each measurement was repeated three times and was conducted under standard laboratory temperature (25 °C). Calculations were performed using software GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego California USA (www.graphpad.com). Purity of novel compounds was checked once again using TLC technique and HPLC technique immediately prior the experiment ,16. Reacion test . A shortubstrate . The rea


This nitration  can be carried out selectively to give mainly β,β-dinitro-compounds in yields of up to 73%. From the above mixtures of five possible regioisomers that can be formed, usually two or three of them were isolated, for which the structures were assigned on the basis of 1H NMR spectra including COSY and NOESY measure-ments. These types of products are attractive starting materials for synthesis of potential anticancer PDT agents with unique structures, being practically unavailable by any other alternative method.The synthesis, chromatographic isolation, and structure elucidation of  Thus, nowadays the selective functionalization of porphyrins is being studied intensively.A number of porphyrin derivatives are of significant importance due to their potential use in many fields of chemistry, medicine, pharmacology, new materials, etc. . The des1a  copper(II) (5) for this experiment and its treatment with the above nitric acid in CHCl3 gave similar results. Interestingly, all the possible dinitro-isomers 6a–10a were successfully isolated . Thus, we solved this problem indirectly. From the mixture of dinitrated porphyrins three of them were isolated as pure individual compounds by preparative TLC. Their demetallation using H2SO4/CF3CO2H gave copper-free isomers, which were examined spectroscopically in details.The correct assignment of the corresponding structures to the obtained products was not trivial. It was mainly accomplished on the basis of 3da  was simple (see above) contrary to the assignment of the structure of the next isomer. In the spectrum of the latter we found a singlet δ = 8.88 ppm originating from two β-protons, and an AB-like system (4H) at 8.63 ppm and 8.66 ppm (with J = 5.2 Hz). Unfortunately, these data are in agreement with the three structures . On the basis of two-dimensional COSY and NOESY measurements structure of 3db was assigned. There is a diagnostic correlation between the signals at 8.06–8.02 ppm (2H) and the triplet at 7.58 ppm   see . These s3dc could also give such a correlation pattern. Thus, the structure 3db was definitively confirmed by the NOESY technique. In the spectrum several correlations between protons due to their spatial proximity  from one side, and the two β-protons neighbouring the NO2 groups , from the other side. Additionally, the β-protons of the remaining part of the AB-like system  correlate with the ortho-protons of the last phenyl ring . Finally, no correlation between the ortho-protons at 8.06–8.02 ppm with any β-protons was observed. This is an additional unambiguous evidence for the structure 3db. Moreover, demetallation of 3db and comparison of the 1H NMR spectrum of the free base porphyrin obtained with the data observed for 4b (in the light of the information given below), allowed us to find the same 3,7-disubstitution pattern (the products were the same). By this way, we proved that the analyzed product, obtained in the nitration of TPP(Ni) (1d), has structure 3db.Theoretically, the 2,8-dinitro-isomer 4b, obtained from 3cb, was performed earlier by Wyrębek . Mass spectra were measured with a GCT Premier FD-TOF instrument  (FD method), a MARINER ESI-TOF spectrometer , and Synapt G2-S HDMS ESI-TOF spectrometer (ESI method) (Waters); m/z intensity values for peaks are given as % of relative intensity. UV-vis spectra were measured with a DU-68 , SP-8001 , UV-3600 , and V-730  spectrophotometers. TLC analysis was performed on aluminium foil plates pre-coated with silica gel . All the products were isolated by column chromatography . Some dinitro-isomers were additionally rechromatographed using preparative TLC plates . Molecular formulas of new compounds were confirmed by HR-MS . Starting porphyrinates were obtained according to known procedures described in the previous literature  (log ε): 559 (3.58), 438 . MS (ESI); m/z (% rel. int.): 769 (14), 768 (42), 767 (78), 766 (81), 765 (100) [isotope M+• and (M + H)+]. HR-MS (ESI): m/z calcd for C44H26N6O4Cu [M+]: 765.1312; found: 765.1327. MS (FD), m/z (% rel. int.): 769 (8), 768 (24), 767 (58), 766 (49), 765 (100) [isotope M+•]. HR-MS (FD): m/z calcd for C44H26N6O4Cu [M+]: 765.1312; found: 765.1344.copper(II) (3cb): m.p. > 300 °C. UV-vis (CHCl3); λmax [nm] (log ε): 607 (4.02), 560.5 (4.19), 437.5 , 388 (4.52), 326 (4.44). MS (FD), m/z (% rel. int.): 769 (7), 768 (24), 767 (58), 766 (49), 765 (100) [isotope M+•]. HR-MS (FD): m/z calcd for C44H26N6O4Cu [M+]: 765.1312; found: 765.1327.copper(II) (3cc): m.p. > 300 °C. UV-vis (CHCl3); λmax [nm] (log ε): 601.5 (4.21), 561 (4.43), 436.5 . MS (ESI); m/z (% rel. int.): 769 (17), 768 (38), 767 (61), 766 (79), 765 (100) [isotope M+• and (M + H)+]. HR-MS (ESI): m/z calcd for C44H26N6O4Cu [M+]: 765.1312; found: 765.1260.4a–c (see below).The above products were also fully characterized as their corresponding decomplexed forms meso-tetra-phenylpor-phyrinato)nickel(II) (1d), was prepared from 0.11 mmol of TPP according to procedure described earlier for a similar compound : 8.89 , 8.81 , 8.69 and 8.58 , 8.64 and 8.56 , 8.02–7.95 , 7.76–7.62 . UV-vis (CHCl3); λmax [nm]: 556.5, 449 (Soret band), 389.5, 329. MS (FD), m/z (% rel. int.): 765 (4), 764 (12), 763 (23), 762 (50), 761 (51), 760 (100) [isotope M+•]. The above data were in agreement with those described earlier : 8.88 , 8.66 and 8.63 , 8.06–7.96 , 7.78–7.68 , 7.58 . UV-vis (CHCl3); λmax [nm]: 596, 553, 446 (Soret band), 382, 319. MS (FD), m/z (% rel. int.): 765 (4), 764 (12), 763 (22), 762 (50), 761 (53), 760 (100) [isotope M+•]. HR-MS (ESI): m/z calcd for C44H26N6O4Ni [M+]: 760.1369; found: 760.1403.3CO2H/H2SO4, 1:1; r.t.; 15–30 min); yields: 4a from 3ca, 83%; 4b from 3cb, 93%; 4c from 3cc, 61%; b) Ni-complex (under conditions as above); yield of 4b from 3db, 80%. The analytically pure products were fully characterized and the spectra confirmed the structures. The compounds obtained from 3cb and from 3db exhibited identical spectral properties as assigned to compound 4b.Demetallation reactions to give free base porphyrins were carried out according to modified known procedures ,13,22: a2,7-Dinitro-5,10,15,20-tetraphenylporphyrin (4a): m.p. > 300 °C. 1H NMR ; δ [ppm]: 9.04 , 9.03 , 8.84 and 8.75 , 8.79 and 8.75 , 8.28–8.15 , 7.84–7.68 , −2.17 . UV-vis (CHCl3); λmax [nm] (log ε): 685 (3.42), 538.5 (3.55), 438 . MS (FD), m/z (% rel. int.): 707 (3), 706 (11), 705 (49), 704 (100) [isotope M+•]. HR-MS (EI): m/z calcd for C44H28N6O4 [M+]: 704.2172; found: 704.2182.3,7-Dinitro-5,10,15,20-tetraphenylporphyrin (4b): m.p. > 300 °C. 1H NMR ; δ [ppm]: 8.97 , 8.79 and 8.76 , 8.29–8.26 , 8.22–8.15 , 7.86–7.75 , 7.67 , −2.25 . UV-vis (CHCl3); λmax [nm] (log ε): 685 (3.76), 584 (3.92), 540 (3.90), 444.5 . MS (FD), m/z (% rel. int.): 707 (4), 706 (14), 705 (52), 704 (100) [isotope M+•]. HR-MS (EI): m/z calcd for C44H28N6O4 [M+]: 704.2172; found: 704.2149.2,8-Dinitro-5,10,15,20-tetraphenylporphyrin (4c): m.p. > 300 °C. 1H NMR ; δ [ppm]: 9.01 , 8.93 , 8.32–8.15 , 7.87–7.69 , −2.49 and −2.58 . UV-vis (CHCl3); λmax [nm]: 682, 537.5, 434 (Soret band). MS (ESI); m/z (% rel. int.): 707 (13), 706 (52), 705 (100), 704 (7) [isotope M+• and (M + H)+]. HR-MS (ESI): m/z calcd for C44H29N6O4 [(M + H)+]: 705.2250; found: 705.2276.4b and 4c, obtained in small amounts as by-products, were inversely assigned.In the previous paper  the stru5  in CHCl3 (130 mL), a solution of 33% aqueous nitric acid  was added dropwise via syringe (using septum inlet) during ca 15 min, under argon, at room temperature. The reaction was vigorously stirred for additional 10 min. Then, the mixture was washed with water (4 × 70 mL), and the combined water layers were extracted with CHCl3 (25 mL). The combined organic layers were dried over anhydrous MgSO4/Na2CO3. After evaporating the solvent, the residue was subjected to column chromatography (CHCl3/n-hexane—1:1) to give: (a) copper(II) ; (b) copper(II) ; (c) copper(II) ; (d) copper(II) ; (e) copper(II) .In a round-bottomed flask, to a stirred solution of complex ca 1%, identified by MS). The structures of the above compounds could not be elucidated due to the paramagnetic copper cation inside the core ring, thus their 1H NMR spectra are not readable. Additionally, the compounds 8a,9a were isolated as an inseparable mixture, so to determine the structures, all the compounds obtained and the mixture 8a/9a were demetallated [6b–10b via column chromatography (eluent: CHCl3/n-hexane). The spectral data of the products 6a–10a are given below. Also the porphyrins 6b–10b were fully characterized. The yield of 8a and 9a was calculated on the basis of the ratio of the separated 8b and 9b.In some experiments traces of trinitro-isomers were detected porphyrinato]copper(II) (6a): m.p. > 300 °C. UV-vis (CHCl3); λmax [nm]: 606, 561.5, 440.5 (Soret band), 313. MS (ESI); m/z (% rel. int.): 847 (3), 846 (7), 845 (8), 844 (14) [isotope (M + Na)+], 825 (8), 824 (26), 823 (55), 822 (58), 821 (100) [isotope M+•]. HR-MS (ESI): m/z calcd for C48H34N6O4Cu [M+]: 821.1938; found: 821.1925.copper(II) (7a): m.p. > 300 °C. UV-vis (CHCl3); λmax [nm] (log ε): 608.5 (3.53), 561.5 (3.67), 441.5 , 271.5 (4.30). MS (ESI); m/z (% rel. int.): 847 (7), 846 (14), 845 (14), 844 (25) [isotope (M + Na)+], 825 (9), 824 (29), 823 (61), 822 (63), 821 (100) [isotope M+•]. HR-MS (ESI): m/z calcd for C48H34N6O4Cu [M+]: 821.1938; found: 821.1924.copper(II) (8a) and copper(II) (9a); inseparable mixture: MS (ESI); m/z (% rel. int.): 847 (6), 846 (13), 845 (12), 844 (23) [isotope (M + Na)+], 825 (8), 824 (29), 823 (58), 822 (59), 821 (100) [isotope M+•].copper(II) (10a): m.p. > 300 °C. UV-vis (CHCl3); λmax [nm]: 606, 562.5, 435.5 (Soret band). MS (ESI); m/z (% rel. int.): 847 (6), 846 (12), 845 (12), 844 (23) [isotope (M + Na)+], 825 (8), 824 (29), 823 (58), 822 (59), 821 (100) [isotope M+•]. HR-MS (ESI): m/z calcd for C48H34N6O4Cu [M+]: 821.1938; found: 821.1925.2,7-Dinitro-5,10,15,20-tetrakis(3-methylphenyl)porphyrin (6b): m.p. > 300 °C. 1H NMR ; δ [ppm]: 9.05 , 9.03 , 8.86 , 8.78 and 8.75 , 8.74 , 8.08–7.95 , 7.69–7.56 , 2.66 , 2.64 and 2.63 , −2.22 . UV-vis (CHCl3); λmax [nm]: 683, 582, 540, 444 (Soret band), 366. MS (ESI); m/z (% rel. int.): 764 (2), 763 (11), 762 (48), 761 (100) [isotope (M + H)+]. HR-MS (ESI): m/z calcd for C48H37N6O4 [(M + H)+]: 761.2876; found: 761.2855.3,7-Dinitro-5,10,15,20-tetrakis(3-methylphenyl)porphyrin (7b): m.p. > 300 °C. 1H NMR ; δ [ppm]: 8.98 , 8.79 and 8.76 , 8.11–7.95 , 7.69–7.52 , 2.67 , 2.65 , 2.61 , −2.28 . UV-vis (CHCl3); λmax [nm] (log ε): 684 (3.86), 587.5 (4.02), 539 (3.97), 446 , 396.5 (4.49). MS (ESI); m/z (% rel. int.): 764 (2), 763 (11), 762 (45), 761 (100) [isotope (M + H)+]. HR-MS (ESI): m/z calcd for C48H37N6O4 [(M + H)+]: 761.2876; found: 761.2855.2,8-Dinitro-5,10,15,20-tetrakis(3-methylphenyl)porphyrin (8b): m.p. > 300 °C. 1H NMR ; δ [ppm]: 9.06 , 8.85 , 8.73 , 8.06–7.95 , 7.68–7.55 , 2.67 , 2.62 , NH—undetected. UV-vis (CHCl3); λmax [nm] (log ε): 679 (3.83), 580.5 (4.00), 538 (4.08), 439.5 , 363.5 (4.44), 328 (4.37). MS (ESI); m/z (% rel. int.): 764 (2), 763 (13), 762 (48), 761 (100) [isotope (M + H)+]. HR-MS (ESI): m/z calcd for C48H37N6O4 [(M + H)+]: 761.2876; found: 761.2860.2,12-Dinitro-5,10,15,20-tetrakis(3-methylphenyl)porphyrin (9b): m.p. > 300 °C. 1H NMR ; δ [ppm]: 9.02 and 8.92 , 9.01 , 8.11–7.98 , 7.70–7.57 , 2.68 , 2.63 , −2.61 . UV-vis (CHCl3); λmax [nm] (log ε): 685 (3.72), 538 (3.73), 436 , 361.5 (4.01). MS (ESI); m/z (% rel. int.): 764 (1), 763 (8), 762 (38), 761 (100), 760 (16) [isotope M+• and (M + H)+]. HR-MS (ESI): m/z calcd for C48H37N6O4 [(M + H)+]: 761.2876; found: 761.2862.2,13-Dinitro-5,10,15,20-tetrakis(3-methylphenyl)porphyrin (10b): m.p. > 300 °C. 1H NMR ; δ [ppm]: 9.02 , 8.97 , 8.92 , 8.12–7.97 , 7.69–7.57 , 2.67 , 2.64 , −2.50 , −2.59 . UV-vis (CHCl3); λmax [nm]: 688.5, 539.5, 441 (Soret band), 370, 276.5. MS (ESI); m/z (% rel. int.): 785 (1), 784 (4), 783 (8) [isotope (M + Na)+], 764 (2), 763 (12), 762 (46), 761 (100), 760 (9) [isotope M+• and (M + H)+]. HR-MS (ESI): m/z calcd for C48H37N6O4 [(M + H)+]: 761.2876; found: 761.2870.1H NMR spectra) copper(II)). In the case of copper complexes, the products were treated with H


Lycoris radiata resulted in the isolation of five new Amaryllidaceae alkaloids: (+)-5,6-dehydrolycorine (1), (+)-3α,6β-diacetyl-bulbispermine (2), (+)-3α-hydroxy-6β-acetyl-bulbispermine (3), (+)-8,9-methylenedioxylhomolycorine-N-oxide (5), and 5,6-dihydro-5-methyl-2-hydroxyphenanthridine (7), together with two known compounds, (+)-3α-methoxy-6β-acetylbulbispermine (4) and (+)-homolycorine- N-oxide (6). Structural elucidation of all the compounds were performed by spectral methods such as 1D and 2D  NMR spectroscopy, in addition to high resolution mass spectrometry. Alkaloid 1 showed potent cytotoxicity against astrocytoma and glioma cell lines , as well as HL-60, SMMC-7721, and W480 cell lines with IC50 values of 9.4–11.6 μM. Additonally, compound 1 exhibited antimalarial activity with IC50 values of 2.3 μM for D-6 strain and 1.9 μM for W-2 strain of Plasmodium falciparum.Phytochemical investigation of the 80% ethanol extract of the bulbs of  Lycoris (Amaryllidaceae) consists of more than 20 species which are mainly distributed in the temperate woodlands of eastern Asia, particularly in China and Japan + peak at m/z 286.1075 , corresponding to the molecular formula C16H16NO4+. Its UV absorption at λmax 374, 309, 253, and 212 nm showed an extended chromophore and a methylenedioxyl substituted benzene ring. The IR absorption bands at 3,410, 3,355, 1,645, 1,605 and 923 cm−1 indicated OH groups and phenyl functions. The 1H-NMR spectrum of 1 exhibited two singlets for two para-located aromatic protons at δH 7.28 (H-7) and 7.18 (H-10), a methylenedioxy signal at δH 6.17 and a downfield singlet corresponding to the proton of an iminium salt (δH 8.84) +  consistent with a molecular formula of C20H21NO7, corresponding to 11 degrees of unsaturation. The IR absorption bands at 3385 and 1713 cm−1 are ascribable to the OH and the ester C=O groups, respectively. The 1H-NMR spectrum exhibited two singlets at δH 6.64 (s) and 6.83 (s) assigned to two para-position aromatic protons, a broad singlet at δH 5.88 ascribed to a methylenedioxy, two olefinic signals at δH 6.36 and 5.85 assigned to the H-1 and H-2, and two singlets at δH 2.11 (s) and 2.07 (s) ascribed to two acetoxyl Me groups. The 13C-NMR spectrum displayed 20 carbon resonances, including a phenyl , two Ac , a methylenedioxy (δC 101.2), three oxygenated methins , two olefinic carbons (δC 127.5 and 129.9), two CH2 (sp3), one CH (sp3) and a quaternary carbon (sp3) (α-methoxy-6β-acetyl- bulbispermine (4) +, consistent with the molecular formula C17H17NO, accounting for 10 degrees of unsaturation. The IR absorption bands at 1705 and 1655 cm−1 indicated the existence of ketones, while the UV absorption bands at 265 and 212 nm suggested a conjugated moiety. The lH-NMR spectrum showed singlet signals for two aryl protons (δH 7.18 and 7.58), one olefinic proton (δH 5.80), one methylenedioxy (δH 6.07), and two vicinal methylenes . The 13C-NMR spectrum displayed one N-CH3, four CH2 and six CH groups (including three sp3 carbons and three sp2 carbons), and six sp2 quaternary carbons. The NMR data and the characteristic downfield signals of the carbon resonances for C-4a (δC 79.1), C-12 (δC 71.0), and N-CH3 (δC 56.2) indicated that 5 was a derivative of homolycorine-α-N-oxide + . Its 13C-NMR spectrum showed 15 carbon signals . These spectral data showed similarities to those of 5,6-dihydro-5-methylphenanthridine D23.3 = +438.1 . UV (CDCl3) λmax(log ε): 374 (4.05), 309 (3.83), 253 (4.22), 212 (4.65) nm. IR (KBr) νmax 3410, 3355, 1645, 1605, 1590, 1502, 1275, 1035, 923 cm−1. 1H-NMR and 13C-NMR data, see m/z: 286 ([M]+). HR-ESI-MS (pos.) m/z: 286.1075 .: Colorless oil. [α]D23.3 = +33.7 . UV (CDCl3) λmax(log ε): 292 (3.73), 240 (3.90) nm. IR (KBr) νmax 3385, 2902, 1713, 1483, 1250, 1060, 933, 870 cm−1. 1H-NMR and 13C-NMR data, see m/z: 387 ([M]+). HR-ESI-MS (pos.) m/z: calc. 410.1212 .(+)-3α-Hydroxy-6β-acetylbulbispermine (3): Colorless oil. [α]D23.3 = +23.6 . UV (CDCl3) λmax(log ε): 291 (3.78), 240 (3.87) nm. IR (KBr) νmax 3386, 2898, 1712, 1485, 1248, 1055, 930 cm−1. 1H-NMR and 13C-NMR data see m/z: 345 ([M]+). HR-ESI-MS (pos.) m/z: calc. 368.1112 .(+)-8,9-Methylenedioxyl-homolycorine-N-oxide (5): Colorless oil. [α]D23.3 = +143.9 . UV (CDCl3) λmax(log ε): 380 (2.72), 308 (3.55), 265 (3.78), 212 (4.56) nm. IR (KBr) νmax2945, 1705, 1655, 1600, 1453, 1311, 1224, 1064, 911 cm−1. 1H-NMR and 13C-NMR data, see m/z: 315 ([M]+). HR-ESI-MS (pos.) m/z: calc. 316.1183 .(+)-5,6-Dihydro-5-methyl-2-hydroxyphenanthridine (7): Colorless needles. [α]D23.3 = +43.1 . UV (CDCl3) λmax(log ε): 380 (2.65), 308 (3.55), 263 (3.70), 210 (4.40) nm. IR (KBr) νmax 3410, 1710, 1640, 1602, 1460, 1255, 1035, 915 cm−1. 1H-NMR δH: 6.48 , 6.32 , 6.63 , 4.21 , 7.02 , 6.69 , 2.75 , 5.99 ; 13C-NMR δC: 113.6 (C-1), 154.1 (C-2), 117.2 (C-3), 113.0 (C-4), 138.9 (C-4a), 63.8 (C-6), 133.7 (C-6a), 109.9 (C-7), 147.7 (C-8), 147.6 (C-9), 107.3 (C-7), 110.4 (C-10), 131.0 (C-10a), 117.5 (C-10b), 31.1 (N-Me). EI-MS m/z: 255 ([M]+). HR-ESI-MS (pos.) m/z: 256.0977 .3 cells) suspended in 100 μL/well of DMEM medium containing 10% fetal calf serum were seeded onto a 96-well culture plate. After 24 h pre-incubation at 37 °C in a humidified atmosphere of 5% CO2/95% air to allow cellular attachment, various concentrations of test solution were added and cells were incubated for 48 h under the above conditions. At the end of the incubation, 10 μL of tetrazolium reagent was added into each well followed by further incubation at 37 °C for 4 h. The supernatant was decanted, and DMSO (100 μL/well) was added to allow formosan solubilization. The concentrations of the assayed compounds were 0.04, 0.2, 1.0, 5, 25, and 125 μM, respectively. The optical density (OD) of each well was detected using a microplate reader at 550 nm and for correction at 595 nm. Each determination represented the average mean of six replicates. The 50% inhibition concentration  was determined by non-linear regression with Graphpad Prism software version 4.0  and was used as criteria to judge the cytotoxicity. All the IC50 results represent an average of a minimum of three experiments and were expressed as means ± standard deviation (SD). All cell lines were purchased from the Cell Bank of the Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences . Other reagents were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd . The cytotoxic activities of the isolated compounds were determined using the revised MTT method ,27 againP. falciparum strains D-6 (drug-resistant) and W-2 (drug-sensitive) were cultured in human erythrocytes in RPMI medium  supplemented with 10% human plasma at 37 °C, under 93% N2, 4% CO2, and 3% O2 [2, 4% CO2, and 3% O2, the microplate was immediately frozen at −20 °C for 18 h. Then, the microplate was thawed at 37 °C and 20 μL of the hemolyzed parasite suspension was transferred to another microplate containing 100 μL of Malstat reagent. The plate was further incubated for 15 min at room temperature, and 20 μL of a 1:1 mixture of nitroblue tetrazolium and phenazine ethosulfate  was added to each well. After incubation for 2 h at room temperature in the dark, the blue formazan product was measured at 655 nm with iEMS microplate reader MF . The 50% inhibitory concentration (IC50) value was determined by non-linear regression with Graphpad Prism software version 4.0 . It was used as criteria to judge the activity . P. falciparum strains were purchased from the Cell Bank of the Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences.1), (+)-3α,6β-diacetylbulbispermine (2), (+)-3α-hydroxy-6β-acetylbulbispermine (3), (+)-8,9-methylenedioxyl-homolycorine-N-oxide (5), and 5,6-dihydro-5-methyl-2-hydroxyphenanthridine (7), and two known compounds, (+)-3α-methoxy-6β-acetylbulbispermine (4) and (+)-homolycorine- N-oxide (6), were isolated from the 80% ethanol extract of the bulbs of Lycoris radiata. All the alkaloids were evaluated in vitro for cytotoxic properties against BEN-MEN-1 (meningioma), CCF-STTG1 (astrocytoma), CHG-5 (glioma), SHG-44 (glioma), U251 (glioma), HL-60 (human myeloid leukemia), SMMC-7721 (hepatocellular carcinoma), and W480 (colon cancer) and antimalarial activity against two strains of P. falciparum (D-6: drug-resistant and W-2: drug-sensitive). Alkaloids 1–4 showed significant cytotoxic activities against HL-60 . Lycorine-type alkaloid 1 exhibited the most potent cytotoxicities and crinine-type alkaloids 2–4 possessed moderate cytotoxicities against astrocytoma and glioma cell lines, CCF-STTG1, CHG-5, SHG-44 and U251 . Homolycorine-type alkaloids 5–6 and 7 had no cytotoxic activities . In addition, lycorine-type alkaloid 1 has antimalarial activities against the two strains of P. falciparum.Five new Amaryllidaceae alkaloids: (+)-5,6-dehydrolycorine (


The chemical structure of the new compound 1 was elucidated by extensive nuclear magnetic resonance (NMR) data , heteronuclear single-quantum correlation (HSQC), heteronuclear multiple bond correlation (HMBC), and nuclear overhauser effect spectroscopy (NOESY)) and HR-FABMS analysis. The anti-inflammatory effects of 1–8 were evaluated in lipopolysaccharide (LPS)-stimulated murine microglia BV-2 cells. Compounds 4 and 7 exhibited moderate inhibitory effects on nitric oxide production in LPS-activated BV-2 cells, with IC50 values of 17.80 and 27.91 µM, respectively.A new sesquilignan glycoside  Capsella bursa-pastoris (L.) Medik (Cruciferae), commonly known as ‘shepherd’s purse’ is an annual plant which is widely distributed throughout the world + at m/z: 723.2626 , 7.15 , 7.05 , 7.01 , 6.95 , and 6.90 ), a 1,3,4,5-tetrasubstitued aromatic ring , 6.78 ), two oxygenated methines  and 4.85 ), two oxygenated methylenes , 3.78 , and 3.88 , 3.68 ), a methylene 2.96 , and 2.57 ), three methines , 2.75 , and 2.38 ), three methoxy groups , 3.87  and 3.84 ), and a glucopyanosyl unit , 3.69 , 3.51 , 3.41  and 3.39 . The 13C-NMR spectrum displayed 36 carbon signals, including 18 aromatic carbons δC 151.1 (C-3″), 147.9 , 147.8 (C-3), 147.5 , 145.5 (C-5′), 139.7 (C-1″), 135.7 (C-1), 134.9 (C-1′), 119.7 (C-6″), 119.5 (C-6), 118.4 (C-6′), 118.2 (C-5″), 118.1 (C-5), 114.7 (C-2′), 111.5 (C-2″) and 111.3 (C-2), three oxygenated carbons δC 88.6 (C-7), 83.9 (C-7″), and 73.5 (C-9′), three methylene carbons δC 65.1 (C-9), 60.5 (C-9″), 33.9 (C-7′) and three methine carbons δC 55.7 (C-8), 54.2 (C-8″), 44.0 (C-8′), and, three methoxy carbons (δC 56.9 and 56.8 (×2)), and glucose carbons δc 103.1 (C-1′′′), 78.5 (C-3′′′), 78.4 (C-5′′′), 75.1 (C-2′′′), 71.6 (C-4′′′), and 62.6 (C-6′′′) (Compound (C-6′′′) . 1H- and 13C-NMR spectra (1. The positions of three methoxy groups were confirmed as 3-OCH3 (δH 3.88)/C-3 (δC 147.8), 5′-OCH3 (δC 3.87)/C-5′ (δC 145.5), and 3″-OCH3 (δH 3.84)/C-3″ (δC 151.1). HMBC correlation (H-1′′′ to C-4″) indicated that a D-glucose moiety was linked to C-4″ (J = 7.3 Hz) . HM. HM1H- aectively  reconfir = 0.21)  and the  MS data . Thus, tNitric oxide (NO) is one of the pro-inflammatory mediators and also a signaling molecule responsible for the induction of inflammation and pain through the pronounced induction of pro-inflammatory cytokines . It is b1–8 were evaluated by measuring the levels of nitric oxide (NO) produced in lipopolysaccharide (LPS)-activated microglia BV-2 cells + .Capselloside (1 (1.0 mg) in H2O (3.0 mL) was hydrolyzed with crude β-glucosidase  at 37 °C for 48 h. The reaction mixture was extracted with CHCl3 (3 × 3 mL). The CHCl3 layer (0.6 mg) was chromatographed over a silica gel Waters Sep-Pak Vac 12 cc eluted with CHCl3–MeOH (30:1) to give the aglycone 1a (0.4 mg), which was identified by 1H‑NMR and MS data. The sugar (d-glucose) from the aqueous phase of the hydrolysate of 1 was identified by silica gel TLC  comparison with a d-glucose standard. The specific rotation of the sugar obtained from 1 was c = 0.03, H2O).Compound 2), a soluble oxidation product of NO, in the culture medium was measured using Griess reagent (0.1% N-1-naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid). Fifty microliters of supernatant was mixed with an equal volume of Griess reagent. Absorbance was measured after 10min using a microplate reader  at a wavelength of 570 nm. L-NMMA, a well-known nitric oxide synthase (NOS) inhibitor, was used as a positive control. Graded sodium nitrite solution was used as a standard to calculate nitrite concentration. Cell viability was evaluated by observing the ability of viable cells to reduce the yellow-colored MTT to purple formazan, using an MTT assay.The inhibitory effect of the test compounds on LPS-stimulated NO production was studied using BV-2 cells. BV-2 cells were seeded on a 96-well plate (4 × 104 cells/well) and treated with or without different concentrations of the compounds. The cells were stimulated with LPS (100 µg/mL) and incubated for 24 h. The concentration of nitrite -Pinoresinol-β-d-glucoside (4) showed the most potent anti-inflammatory effects and β-hydroxypropiovanillone 3-O-β-d-glucopyranoside (7) showed moderate NO production in an LPS-activated microglia BV-2 cell line. Compound 4 showed better potency than that of the commercial NOS inhibitor to inhibit LPS-induced NO production and neuroinflammation. In conclusion, our results collectively support the notion that phenolic glycosides from Capsella bursa-pastoris (L.) Medik may be potentially responsible for the observed in vitro anti-inflammatory activity in a LPS-induced neuroinflammatory model.We have isolated and identified from


The last two compounds were isolated for the first time from Alaska cedar in this research. The absolute stereochemistry at C-2 of nootkatol was shown to have the (S) configuration using the Mosher ester method. Assignment of stereochemistry for valencene-13-ol (3) was established by synthesis from valencene (6). Finally, two related sesquiterpenoids were synthesized from nootkatone and valencene. These sesquiterpenoids were nootkatone-1,10-11,12-diepoxide (5) and valencene-13-aldehyde (4), respectively.The essential oil of Alaska cedar heartwood is known to contain compounds which contribute to the remarkable durability of this species. While previous research has identified several compounds, a complete description of this oil has not been undertaken. In this research a profile of the oil is given in which the major components are identified by GC, isolation and spectroscopic techniques. The major components of the steam distilled essential oil were identified as nootkatin, nootkatone, valencene, nootaktene, carvacrol, methyl carvacrol, nootkatol ( Chamacyparis nootkatensis), also known as yellow cedar or Nootka cypress, is an important timber and ecological species of the costal Pacific Northwest of the United States which is known for its heartwood durability. In chemical investigations of the heartwood, carvacrol and the tropolone nootkatin were isolated and shown to be prominent components that contributed to wood durability. Subsequently the tropolone chanootin, monoterepene acids, and the eremoplilane sesquiterpenes nootkatone, nootkatene and valencene were found in early research + 218.16711 calculated for C15H22O, requires 218.16706. Selected 1H-NMR spectral data  δ 9.45 , 6.20 , 5.85 , 5.25 . 13C-NMR data  δ 120.9 (C-1), 26.3 (C-2), 27.5 (C-3), 41.3 (C-4), 38.4 (C-5), 45.3 (C-6), 31.8 (C-7), 32.9 (C-8), 33.5 (C-9), 142.9 (C-10), 155.6 (C-11), 133.2 (C-12), 195.0 (C-13), 16.0 (C-14), 18.6 (C-15).Data for 2O2  followed by 6.0N KOH (2.5 mL) over a period of 10 min and the resultant mixture was stirred for 3 h. The reaction mixture was then quenched with cold deionized water (30 mL), and methanol was eliminated in vacuum. The product was extracted from the aqueous solution by diethyl ether (3 × 20 mL). The combined ethereal layer was dried (Na2SO4), filtered, and concentrated under vacuum to yield the title compound . NMR spectrum showed a mixture of diastereomers with high purity.To a cold solution of nootkatone-11,12-epoxide  in anhydrous methanol (50 mL) was added H5: Semisolid residue; ; HRCIMS [M]+ 250.15670 calculated for C15H22O3, requires 250.15689. Selected 1H-NMR data  δ 3.08 , 1.28 , 0.93 , 0.78 . 13C-NMR data  δ 63.0 (C-1), 207.3 (C-2), 42.3 (C-3), 39.9 (C-4), 36.8 (C-5), 38.0 (C-6), 32.8 (C-7), 30.1 (C-8), 26.7 (C-9), 69.2 (C-10), 54.3 (C-11), 60.0 (C-12), 18.3 (C-13), 15.3 (C-14), 14.8 (C-15).Data for 2) and valencene-13-ol (3) as major components of this essential oil.An analysis of the steam distilled essential oil from the heartwood of Alaska cedar was undertaken in this paper. The oil was shown to contain, among others, nine major compounds: 4-Terpineol, methyl carvacrol, carvacrol, nootkatin, valencene, nootkatene, nootkatone, nootkatol (S) configuration using the Mosher ester method. Assignment of stereochemistry for valencene-13-ol (3) was established by synthesis from valencene (6). Additionally, two related sesquiterpenoids were synthesized from nootkatone and valencene. These sesquiterpenoids were respectively nootkatone-1,10-11,12-diepoxide (5) and valencene-13-aldehyde (4).The absolute stereochemistry at C-2 of nootkatol was shown to have the (


Datura metel L. collected in the Jiangsu Province of China resulted in the isolation of two new withanolides, baimantuoluoline G (1) and baimantuoluoside H (2). Their structures were elucidated as -1,10-seco-6,12,27-trihydroxy-26-oxo-witha-3,5,24-trienolide-1-oic acid-ε-lactone (1) and -5,6,12,27-tetra-hydroxy-1,26-dioxo-witha-2,24-dienolide-27-O-β-glucopyranoside (2) on the basis of extensive spectroscopic analysis  and chemical studies.Chemical investigation of the 50% ethanol eluate fraction from a macroporous resin of flowers of  Datura metel L. (Solanaceae), which widely distributed in China  were unusually shifted to a low field, suggesting the methylene to be situated between an ester carbonyl group and carbon-carbon double bond. Based on this finding and HMBC correlations, between C-1 (δC 175.5) and H-2α, H-2β, and H-3, between H-4  and C-2, C-5, C-6, and C-10, and between C-5 and Me-19, as shown in β, γ-unsaturated lactonic moiety was determined in ring A. The β-configuration of the lactone bond at C-6 was established by a NOESY experiment (br. s) and H-2α, H-7α and H-7β, indicating that H-6 has the same configuration as H-2α. Since a small coupling between H-2α and H-3 in the 1H NMR spectrum was observed due to an approximate 90°, a β-configuration of the lactone bond at C-6 was inferred, which is in agreement with those of 1,10-seco steroids 25D = +17.0 . IR (KBr): νmax = 3426, 2925, 1708, 1387, 1286, 1135, 1089, 995 cm−1. HRESIMS (positive): m/z = 493.2578  and 509.2327 . 1H- and 13C-NMR: see 25D = +24 . IR (KBr): νmax = 3411, 3326, 2942, 2923, 2865, 2590, 1670, 1070, 1030 cm−1. HRESIMS (positive): m/z = 673.3224  and 689.2948 . 1H- and 13C-NMR: see Baimantuoluoside H (2): White amorphous powder, [α]α,β-unsaturated δ-lactone ring formed in the side chain [D. metel, we have isolated two new withanolide lactones. Their structures were established on the basis of spectroscopic evidence. Compound 1 is a novel withanolide lactone obtained from a D. metel with an unusual seven-membered β, γ-unsaturated lactone of ring A.Withanolide lactones constitute a group of C28 steroidal lactones isolated from several genera of Solananceae . A charade chain , which hde chain . As a pa