A synthetic method for the preparation of 6 H-carbazoles are usually unselective, taking place indifferently at positions 3, 6, or 9 [H-carbazol-3-yl)boronic acid, easily prepared starting from the known 6-bromo-1,4-dimethyl-9H-carbazole, to the corresponding 6-aryl-1,4-dimethyl-9H-carbazoles by a Suzuki-Miyaura cross-coupling reaction.Several alkaloids based on a carbazole structure are known to possess interesting biological activities. Antitumor, antibacterial, anti-inflammatory, psychotropic and anti-histamine properties have been attributed to many of such compounds . More re 6, or 9 , while m1 (2 [3) [Many examples of Suzuki-Miyaura cross-coupling reactions between heterocyclic halides and phenyl boronic acids have been so far described ,6, while1 , which w1  ,11,12.H-carbazole-3-yl)boronic acid with (hetero)aryl halides to give 3-(hetero)arylcarbazole derivatives. Some typically used reaction conditions proved to be ineffective when applied to 6-bromo-1,4-dimethyl-9H-carbazole and (hetero)arylboronic acids. Herein, we describe an efficient method involving a cross-coupling reaction, which would allow the preparation of a library of 6-aryl substituted carbazoles, as potential antitumoral agents. The key step is the reaction of  was prepared by a known procedure [2CO3/Pd(PPh3)4/1,4-dioxane/water/80°C) [I and II proved to be ineffective for the production of the desired 6-aryl-1,4-dimethyl-9H-carbazoles 5 , giving aryl-1,4-dimethyl-3-nitro-9H-carbazoles 7a and 7b, in reasonable yields . Faced with these difficulties and also because the nitro group was considered undesirable, we have studied the reverse strategy, consisting in the preparation of the boronic acid c acid 1  startingA) and p-iodoanisole (B) remained very modest (<10%), the exception being a heteroaryl halide bearing an electron withdrawing group such as 5-bromo-2-thiophenecarboxaldehyde (C), which gave the product 5c in 28% yield aryl halides such as 3-bromopyridine (8% yield .H-carbazole-3-boronic acid (1). Thus, we prepared N-Boc-5,8-dimethyl-9H-carbazol-3-boronic acid (8), which was obtained after N-Boc-protection and successive lithiation-boronation of compound 4. We previously described the use of this reaction sequence for the preparation of 5,8-dimethyl-9H-carbazole-3-ol [These failures prompted us to protect the nitrogen atom of 5,8-dimethyl-98 was then subjected to the Suzuki cross-coupling reaction with a variety of (hetero)aryl halides under standard conditions [2CO3 and Pd(PPh3)4 was slowly added to a mixture of compound 8 and the appropriate (hetero)aryl halide in 1,4-dioxane at room temperature. The resulting mixture was then heated to reflux for the given time. After the usual work-up, compounds 9a-f were obtained in good yields. The N-deprotection of 9a-f was carried out with gaseous HCl in 1,4-dioxane, giving compounds 5a, 5b, 5c, 5d and 5f in quantitative yields  was recorded on a JEOL Lambda 400 spectrometer; 13C-NMR were not recorded due to the low solubility of the compounds. Chemical shifts are expressed in parts per million downfield from tetramethylsilane as an internal standard. Thin layer chromatography (TLC) was performed on silica gel 60F-264 (Merck). Commercial reagents were purchased from Aldrich, Acros Organics or Alfa Aesar and used without additional purification. Melting points were determined on a Kofler melting point apparatus. IR spectra were taken with a Perkin Elmer BX FT-IR. Mass spectra were taken on a JEOL JMS GCMate spectrometer at ionizing potential of 70 eV (EI) or were performed using a LC-MS Waters Alliance 2695 spectrometer (ESI+). H-carbazole (7a) is reported as a representative example: to a solution of 6-bromo-1,4-dimethyl-3-nitro-9H-carbazole  in 1,4-dioxane (70 mL), kept under an argon atmosphere, phenylboronic acid , Na2CO3  in H2O (3.50 mL) and Pd(PPh3)4  were added sequentially. The reaction mixture was heated to reflux for 48h, and then volatiles were evaporated under reduced pressure. The solid formed was recrystallized from acetonitrile to give 1,4-dimethyl-3-nitro-6-phenyl-9H-carbazole (7a). Yellow solid (67 % yield), mp = 226 °C; IR (KBr): 3451, 1584, 1521, 1302, 1301, 817, 758 cm-1; 1H-NMR (DMSO-d6) δ 11.94 ; 8.35 ; 7.86 ; 7.83-7.83 ; 2.96 ; 2.55 ; MS (EI) m/z (%): 316 , 271 (48) (M+ •-NO2). The preparation of 1,4-dimethyl-3-nitro-6-phenyl-9II) in place of I. Yellow solid (45 % yield), mp = 240 °C; IR (KBr): 3309, 1578, 1299, 1205, 1034, 810, 756 cm-1; 1H-NMR (DMSO-d6) δ 12.06 ; 8.36 ; 7.87 ; 7.71-7.65 ; 7.28-7.23 ; 7.17-7.14 ; 6.95-6.93 ; 3.80 ; 2.99 ; 2.58 ; MS (ESI+): 364 (M++1).Prepared following the general procedure but employing 2-fluoro-5-methoxyphenylboronic acid (tert-butoxycarbonyl-9H-carbazole (9a) is reported as a representative example: to a solution of 3-bromopyridine  in 1,4-dioxane (50 mL), kept under an argon atmosphere, 9-tert-butoxycarbonyl-5,8-dimethyl-9H-carbazole-3-boronic acid , Na2CO3  in H2O (2 mL) and Pd(PPh3)4  were added sequentially. The reaction mixture was heated to reflux for 48 h, and then concentrated under reduced pressure. The solid residue was recrystallized from acetonitrile. Compound 9a was obtained as a light brown solid (50 % yield), mp = 167 °C; IR (KBr): 3435, 2972, 1731, 1458, 1288, 1246, 1145, 1084, 809, 721, 619 cm-1; 1H-NMR (DMSO-d6) δ 9.01 ; 8.60-8.56 ; 8.35 ; 8.20 ; 8.11 ; 7.87 ; 7.53-7.50 ; 7.24 ; 7.15 ; 2.84 ; 2.40 ; 1.67 ; MS (EI) m/z (%): 372 , 316 (100) (M+ •- tBu), 272 (100) (M+ •- CO2tBu). MS (ESI+): 373 (M++1).The preparation of 1,4-dimethyl-6-pyridin-3-yl-9-9a, but using 4-iodoanisole , 9b was obtained as a white solid (70 % yield), mp = 122 °C; IR (KBr): 2977, 1728, 1451, 1285, 1246, 1150, 1081, 804, 757, 697 cm-1; 1H-NMR (DMSO-d6) δ 8.36 ; 8.15 ; 7.89-7.83 ; 7.58-7.55 ; 7.46-7.44 ; 7.29 ; 7.20 ; 3.47 ; 2.89 ; 2.45 ; 1.71 ; MS (EI) m/z (%): 401 , 271 (100) (M+ •- OCH3 - CO2tBu).Following a procedure identical to that described for 9a, but using 5-bromo-2-thiophene-carboxaldehyde  and a reflux time of 24 h, compound 9c was obtained as a yellow solid (90 % yield), mp = 168 °C; IR (KBr): 3428, 2971, 1739, 1657, 1428, 1225, 1149, 1061, 797, 665 cm-1; 1H-NMR (DMSO-d6) δ 9.90 ; 8.38  8.07-8.05 ; 7.94 ; 7.82 ; 7.24 ; 7.15 ; 2.81 ; 2.40 ; 1.66 ; MS (ESI+): 405 (M++1).Following a procedure identical to that described for 9a, but using 2-bromopyridine , 9d was obtained as a light brown solid (45 % yield), mp = 212 °C; IR (KBr): 3370, 2977, 1701, 1457, 1290, 1252, 1149, 1088, 803, 780, 670 cm-1; 1H-NMR (DMSO-d6) δ 8.66 ; 8.08 ; 8.07-7.95 ; 7.75 ; 7.71-7.68 ; 7.20-715 ; 7.10 ; 7.02 ; 2.72 ; 2.41 ; 1.62 ; MS (ESI+): 373 (M++1).Following a procedure identical to that described for 9a, but using 5-bromo-2-furaldehyde  and a reflux time of 24 h, compound 9e was obtained as a yellow solid (60 % yield), mp = 110 °C; IR (KBr): 3430, 2975, 1744, 1672, 1450, 1285, 1149, 1084, 795 cm-1; 1H-NMR (DMSO-d6) δ 9.62 ; 8.48  8.10 ; 8.03 ; 7.67 ; 7.34 ; 7.25 ; 7.16 ; 2.82 ; 2.42 ; 1.67 ; MS (ESI+): 390 (M++1).Following a procedure identical to that described for 9f, but using 6-bromo-1,4-dimethyl-9-tert-butoxycarbonyl-9H-carbazole , 9f was obtained as a white solid (40 % yield), mp = 214 °C; IR (KBr): 3439, 2979, 1726, 1453, 1295, 1248, 1154, 1083, 804, 541 cm-1; 1H-NMR (DMSO-6d) δ 8.40 ; 8.20-8.10 ; 8.00-7.90 ; 7.10-7.30 ; 2.85 ; 2.42 ; 1.68 ; MS (EI) m/z (%): 588 , 388 (100) (M+ • - 2 CO2tBu).Following the identical procedure to that described for H-carbazole (5a) is reported as a representative example: to a mixture of 1,4-dioxane (70 mL) and gaseous HCl (saturated solution) cooled to 0 °C compound 9a (2.36 mmol) was added. The reaction mixture was heated to reflux for 48 h, then concentrated under reduced pressure. The oil formed residue was crystallized from acetonitrile. Compound 5a was obtained as a yellow solid (95 % yield), mp > 260 °C; IR (KBr): 3426, 2518, 1470, 1258, 790, 671 cm-1; 1H-NMR (DMSO-6d) δ 11.51 ; 9.30 ; 8.84 ; 8.77 ; 8.49 ; 8.03 ; 7.85 ; 7.68 ; 7.13 ; 6.91 ; 3.42 ; 2.85 ; 2.52 ; MS (ESI+): 273 (M++1).The preparation of 1,4-dimethyl-6-pyridin-3-yl-9The following compounds were similarly obtained:6-(4’-Methoxyphenyl)-1,4-dimethyl-9H-carbazole (5b). White solid (95 % yield), mp = 200 °C; IR (KBr): 3020, 1454, 1320, 1250, 1170, 1076, 799, 770, 676 cm-1; 1H-NMR (DMSO-d6) δ 10.00 ; 7.85 ; 7.58-7.56 ; 7.40 ; 7.30 ; 7.05-7.00 ; 6.98 ; 6.89 ; 3.85 ; 2.84 ; 2.54 ; MS (EI) m/z (%): 301 .5--thiophene-2-carbaldehyde (5c). Yellow solid (90 % yield), mp = 262 °C; IR (KBr): 3435, 3267, 1634, 1430, 1241, 1058, 800, 669 cm-1; 1H-NMR (DMSO-d6) δ 11.58 ; 9.94 ; 8,50 ; 8.11-8.10 ; 7.89 ; 7.83 ; 7.67 ; 7.19 ; 6.97 ; 2.89 ; 2.58 ; MS (ESI+): 306 (M++1).1,4-Dimethyl-6-pyridin-2yl-9H-carbazole (5d). White solid (92 % yield), mp > 260 °C; IR (KBr): 3424, 2516, 1468, 1270, 800, 673 cm-1; 1H-NMR (DMSO-d6) δ 11.48 ; 8.64 ; 8.10 ; 8.09-7.92 ; 7.79 ; 7.70-7.69 ; 7.22-718 ; 7.13 ; 7.07 ; 3.43 ; 2.73 ; 2.45 ; MS (ESI+): 273 (M++1).1,1’,4,4’-Tetramethyl-9H-dicarbazole (5f). White solid (94 % yield), mp = 254 °C; IR (KBr): 3454, 2987, 1457, 1300, 1254, 1187, 1067, 812, 570 cm-1; 1H-NMR (DMSO-d6) δ 10.20 ; 7.99-7.94 ; 7.59-7.38 ; 7.15-6.98 ; 2.85 ; 2.55 ; MS (EI) m/z: 388 .


Astrocaryum sciophilum was extracted and screened for antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Four out of five antibacterial ethyl acetate extracts were also cytotoxic for the MRC-5 cells line. Liquid chromatography coupled to tandem mass spectrometry (UPHLC-HRMS/MS) analyses combined with molecular networking data processing were carried out to allow the identification of depsipeptides and cyclopeptides responsible for the cytotoxicity in the dataset. Specific ion clusters from the active Luteibacter sp. extract were also highlighted using an MRSA activity filter. A chemical study of Luteibacter sp. was conducted leading to the structural characterization of eight fatty acid exhibiting antimicrobial activity against MRSA in the tens of µg/mL range.A library of 197 endophytic fungi and bacteria isolated from the Amazonian palm tree  Endophytes are microorganisms that live inside the tissues of a host plant without causing apparent symptoms either for all or a part of their life cycle . This coPlants are widely explored for new chemical entities with therapeutic purposes. Plant endophytes are increasingly regarded as an important and a relatively underrated source of natural products for drug discovery. Recent surveys suggested that tropical forests harbour several million hyperdiverse endophyte species ,5 that aAstrocaryum sciophilum endophytes. A. sciophilum grows as an understorey palm throughout the North-East Amazon  + adducts. Careful analysis of the MS2 data allowed the determination of the double bonds’ position (Z)-hexadecenoic acid (7) and 15-Methyl-(9Z)-hexadecenoic acid (8).Compounds (7) and (8) presented an unsaturated chain. Based on the coupling constant of the multiplet  is a C17 fatty acid with a Z double bond at C-9. Fatty acids with hydroxyl at C-3 (2 – 5) or gem-dimethyl at the final position (8) appear to have lower antibacterial activities .Astrocaryum sciophilum’s associated microorganisms, demonstrated that isolated endophytic strains could produce antibacterial compounds. Indeed, six out of 197 endophytic extracts displayed significant antimicrobial activity.Our study, related to the secondary metabolites of the plant host model Fusarium strains and one Akanthomyces strain) also exhibited cytotoxic activity against MRC-5 cells. Thus, there is a high probability that the antibacterial activity of these extracts depends on the presence of cytotoxic secondary metabolites. Using a molecular networking-based approach, several structurally related analogues of depsipeptide and cyclic peptide - molecules known to be cytotoxic - were highlighted from four active fungal extracts. Depsipeptides have been frequently isolated from fungi belonging to the Fusarium genus + ion (m/z 391.28429) was used as the internal lock mass. The optimized HESI-II parameters were as follows: source voltage, 3.5 kV (pos); sheath gas flow rate (N2), 55 units; auxiliary gas flow rate, 15 units; spare gas flow rate, 3.0; capillary temperature, 275.00 °C (pos), S-Lens RF Level, 45. The mass analyser was calibrated using a mixture of caffeine, methionine−arginine− phenylalanine−alanine−acetate (MRFA), sodium dodecyl sulphate, sodium taurocholate, and Ultramark 1621 in an acetonitrile/ methanol/water solution containing 1% formic acid by direct injection. The data-dependent MS/MS events were performed on the four most intense ions detected in full scan MS (Top3 experiment). The MS/MS isolation window width was 1 Da, and the normalized collision energy (NCE) was set to 35 units. In the data-dependent MS/MS experiments, full scans were acquired at a resolution of 35,000 FWHM (at m/z 200) and MS/MS scans were acquired at 17,500 FWHM both with a maximum injection time of 50 ms. After being acquired in a MS/MS scan, the parent ions were placed on the dynamic exclusion list for 2.0 s.Chromatographic separation was performed using an Acquity UHPLC system interfaced to a Q-Exactive Plus mass spectrometer , using a heated electrospray ionization (HESI-II) source. Thermo Scientific Xcalibur 2.1 software was used for instrument control and data analysis. The LC conditions were as follows: column, Waters BEH (Ethylene Bridget Hybrid) C18 50 × 2.1 mm, 1.7 μm; mobile phase, (A) water with 0.1% formic acid; (B) acetonitrile with 0.1% formic acid; flow rate, 600 μL/min; injection volume, 1 μL; gradient, linear gradient of 5−100% B over 7 min and isocratic at 100% B for 1 min. An Acquity UPLC photodiode array detector was used to acquire the PDA spectra which were collected in the 200–500 nm range. In the positive ion mode, the di-isooctyl phthalate Cm/z tolerance of 0.002 or 5 ppm. The wavelets (ADAP) algorithm was used for the chromatogram deconvolution with the following settings: S/N threshold of 10, an intensity window SN, a minimum feature height of 1000, a coefficient area threshold of 100, a peak duration range between 0.01 and 0.5 and an RT wavelet range between 0.001 and 0.05. The m/z and RT range for MS2 scan pairing were set to 0.001 Da and 0.3 min, respectively. Chromatograms were deisotoped using the isotopic peaks grouper algorithm with an m/z tolerance of 0.003 (5 ppm), RT tolerance of 0.1 (absolute), maximum charge of 2 and the representative isotope used was the most intense. Peak alignment was performed using the join aligner method: m/z tolerance of 0.001 or 5.0 ppm, weight for m/z of 0.001, RT tolerance of 0.3 min, weight for RT of 0.1. Adduct search  was conducted on the peak list with an RT tolerance set to 1.0 min and the maximum relative peak height at 50%. The found adducts were then removed from the peak list. The peak list was gap-filled with the peak finder module: intensity tolerance of 90%, m/z tolerance of 0.001 or 5.0 ppm and RT tolerance of 0.1 min. For further analysis, the peak list was reduced to the ions with the m/z values between 200 and 900, in order to decrease the number of data.Raw files were converted into MzXML (mass spectrometry data format) files using the MSConvert software. Then, MzXML files were processed using Mzmine 2.37 . Mass dem/z values of 200–900 and/or selfloop nodes can be removed. Networks were generated using the following parameters: m/z tolerance set to 0.02, Minimum Matched Peaks set to 6, topK set to 10, Minimal Cosine score Value of 0.7 and Max. Connected Component Size of 100. Then, the associated CSV file was loaded. For the mapping process, the relative quantification of each ion was represented by pie chart-diagrams for which the proportions were based on the respective areas of the corresponding extracted ion chromatograph areas (XIC). Then, the analogues of the spectra in the network were searched in the available spectral libraries. The library spectra were filtered in the same manner as the input data. All of the matches between network spectra and library spectra were required to have a score above 0.7 and at least 6 matched peaks. The m/z tolerance for the analogues’ search was set to 100. For a more advanced retreatment of the network, the MN was also exported to the Cytoscape 3.7.0 software (https://cytoscape.org/).After preprocessing the UHPLC-HRMS/MS data with MZmine 2.33, the output mgf file was processed with the MetGem software  to give Strains were cultivated 15 days at 28 °C in 14 cm Petri dishes of PDA (potato dextrose agar) media. Culture media was extracted three times consecutively with ethyl acetate (EtOAc) at room temperature (the organic phase after 24 h and replacing the remaining agar in EtOAc). The combined organic solution was washed as described above.Luteibacter sp. was conducted on 210 14 cm Petri dishes to yield 1.96 g of a brown-yellow crude extract. This crude extract (1.8 g) was fractionated by reverse flash chromatography on a C18 column with a 5-min-step gradient of water mixed with an increasing proportion of acetonitrile . Six fractions were generated based on the UV and ELSD detection: F1 , F2 , F3 , F4 , F5  and F6 . A step gradient of acetonitrile—methylene chloride  was conducted to generate 2 additional fractions: F7 , F8 .Large-scale cultivation of 18, mobile phase H2O + 0.1% FA/CH3CN + 0.1% FA, isocratic elution 60:40 during 6 min then linear gradient from 60:40 to 0:100 over 13 min, flow rate 21 mL/min) to obtain (R)-3-hydroxy-13-methyltetradecanoic acid (5) . F5 (41.4 mg) was purified by preparative HPLC  to obtain (R)-2-hydroxy-13-methyltetradecanoic acid (1) , (R)-3-hydroxy-14-methylpentadecanoic acid (2) , (S)-β-hydroxypalmitic acid (3)  and 9Z-hexadecenoic acid (7) . F6 (258.0 mg) was purified by preparative HPLC  to obtain (R)-3-hydroxy-15-methylhexadecanoic acid (4) , 9Z-hexadecenoic acid (7) , 13-methyltetradecanoic acid (6)  and 15-methyl-9Z-hexadecenoic acid (8) .F4 (35.0 mg) was purified by preparative HPLC (Luna CR)-2-hydroxy-13-methyltetradecanoic acid (1). 1H-NMR : 4.07 , 1.75 , 1.63 , 1.52 , 1.44 , 1.30 , 1.17 , 0.88 . 13C-NMR : 178.6 (C-1), 71.9 (C-2), 40.4 (C-12), 35.7 (C-3), 31.2–30.8 , 30.7 (C-5), 29.3 (C-13), 28.7 (C-11), 26.3 (C-4), 23.2 . HR-ESI-MS: 257.2109 , 515.4294 .R)-3-hydroxy-14-methylpentadecanoic acid (2). 1H-NMR : 3.96 , 2.42 , 2.34 , 1.52 , 1.47 , 1.30 , 1.18 , 0.88 . 13C-NMR : 176.7 (C-1), 69.6 (C-3), 43.7 (C-2), 40.4 (C-13), 38.2 (C-4), 31.2-30.9 , 29.3 (C-14), 28.7 (C-12), 26.8 (C-5), 23.2 . HR-ESI-MS: 271.2274 , 543.4611 .S)-β: White powder. [α]D20 = +18 . 1H-NMR : 3.97 , 2.43 , 2.36 , 1.47 , 1.30 , 0.90 . 13C-NMR : 176.1 (C-1), 69.5 (C-3), 43.5 (C-2), 38.3 (C-4), 33.2 (C-14), 30.9-30.6 , 26.8 (C-5), 23.9 (C-15), 14.6 (C-16). HR-ESI-MS: 271.2274 , 543.4611 .R)-3-hydroxy-15-methylhexadecanoic acid (4). 1H-NMR : 3.96 , 2.43 , 2.35 , 1.53 , 1.47 , 1.30 , 1.18 , 0.88 . 13C-NMR : 176.5 (C-1), 69.7 (C-3), 43.7 (C-2), 40.4 (C-14), 38.3 (C-4), 31.2-30.9 , 29.3 (C-15), 28.7 (C-12), 26.8 (C-5), 23.2 . HR-ESI-MS: 285.2431 , 571.4926 .R)-3-hydroxy-15-methylhexadecanoic acid (4). 1H-NMR : 3.96 , 2.43 , 2.35 , 1.53 , 1.47 , 1.30 , 1.18 , 0.88 . 13C-NMR : 176.5 (C-1), 69.7 (C-3), 43.7 (C-2), 40.4 (C-14), 38.3 (C-4), 31.2-30.9 , 29.3 (C-15), 28.7 (C-12), 26.8 (C-5), 23.2 . HR-ESI-MS: 285.2431 , 571.4926 .R)-3-hydroxy-13-methyltetradecanoic acid (5). 1H-NMR : 3.89 , 2.33 , 2.24 , 1.52 , 1.45 , 1.30 , 1.18 , 0.88 . 13C-NMR : 176.5 (C-1), 70.4 (C-3), 45.3 (C-2), 40.4 (C-12), 38.2 (C-4), 31.2-30.9 , 29.3 (C-13), 28.7 (C-11), 26.8 (C-5), 23.1 . HR-ESI-MS: 257.2101 , 515.4285 .13-methyltetradecanoic acid (6): White powder. 1H-NMR : 2.27 , 1.60 , 1.52 , 1.30 , 1.18 , 0.88 . 13C-NMR : 177.9 (C-1), 40.4 (C-11), 35.2 (C-2), 31.2-30.4 and 28.7 , 29.3 (C-13), 26.3 (C-3), 23.2 . HR-ESI-MS: 241.2168 , 483.4432 .Z-hexadecenoic acid (7)9: yellow oily liquid. 1H-NMR : 5.35 , 2.27 , 2.03 , 1.60 , 1.33 , 0.90 . 13C-NMR : 178.0 (C-1), 131.1 (C-10), 130.9 (C-11), 35.3 (C-2), 33.1 (C-14), 31.0 , 31.0-30.2 , 28.3 , 26.3 (C-3), 23.9 (C-15), 14.6 (C-16). HR-ESI-MS: 255.2326 , 253.2164 , 507.4426 .Z-hexadecenoic acid (8)15-methyl-9: yellow oily liquid. 1H-NMR : 5.35 , 2.27 , 2.04 , 1.60 , 1.53 , 1.33 , 1.19 , 0.88 . 13C-NMR : 177.9 (C-1), 131.0 , 40.3 (C-14), 35.2 (C-2), 31.3-30.3 , 29.3 (C-15), 28.3 , 28.3 (C-13), 26.3 (C-3), 23.2 . HR-ESI-MS: 269.2484 , 267.2334 , 535.4756 .w/v) HCl was obtained by diluting conc. HCl  in methanol (40.9 mL). Each fatty acid was dissolved in toluene to reach a concentration of 0.005 g/mL. Then, methanol (7.5-fold) and 8.0% HCl solution (1.5-fold) were added sequentially to this solution. The solution was stirred at 45°C overnight. After cooling at room temperature, hexane (5-fold) and water (5-fold) were added for the extraction of FAMEs. HCl  was purchased from Carlo Erba©.Preparation of FAMEs was carried out based on methanolysis/methylation using conc. Hydrochloric acid (HCl) as described by Ichihara and Fukubayashi . A solut2Cl2 to a concentration of 2 mg/mL and then were diluted in CH3CN to the concentration 200 µg/mL. These solutions were analysed by MS/MS on a Q-ToF 6540 mass spectrometer  by direct introduction at a flow rate of 10 µL/min and using an APCI  ion source in the positive mode. The corona current was set to 2 µA, the nebulizer pressure was 60 psig and 8 L/min nitrogen flow heated at 300 °C was used for desolvation. Capillary, fragmentor and skimmer voltages were set to 3000 V, 100 V, and 45 V, respectively. CH2Cl2 and CH3CN were purchased from J.T. Baker©. The MS/MS collision energy was 15 (arbitrary units).FAMEs were then diluted in CHS. aureus ATCC33591. Extracts, fractions and pure compounds were tested according to the reference protocol of the European Committee on Antimicrobial Susceptibility Testing [The ATCC strains were purchased from the Pasteur Institute. The strain used in this study was methicillin-resistant  Testing . The sta Testing . Crude eThe crude extract for each strain was tested to determine its cytotoxicity using the MRC-5 (ATCC CCL-171) normal human lung fibroblast cells . MRC-5 cells were seeded into 96-well microplates at 2000 cells per well. The assay was conducted according to the procedure described by Tempête et al. . After 2


Hypericum sampsonii Hance (Clusiaceae) is a folk medicine used in Taiwan to treat blood stasis, relieve swelling, and as an anti-hepatitis drug. Two new polyprenylated phloroglucinol derivatives, hypersampsone R (1) and hypersampsone S (2), and a known prenylated benzophenone, hyperibone K (3) were isolated from the aerial parts of H. sampsonii. Their structures were determined by extensive 1D and 2D NMR, and MS spectral analyses. Hypericum sampsonii Hance (Guttiferae) is a folk herbal medicine used in Taiwan for treating blood stasis, to relieve swelling, and as an anti-hepatitic drug , eight methyls , a benzoyl [δH 7.36  and 7.39 ], and a conjugated hydroxyl group [δH 16.25 (s)]. The 13C-NMR, DEPT and HMQC spectra indicated the presence of 32 carbons, including two carbonyls (δC 195.2 and 209.4), eight quaternary carbons with one conjugated oxygenated quaternary carbon (δC 185.5), five fully substituted aromatic and olefinic quarternary carbons  and two quaternary carbons (δC 42.4 and 65.4), eight double-bond methine carbons , three methine carbons , three methylene carbons , and eight methyl carbons . The 1H-1H COSY indicated the correlations of H-12 (δH 4.00) and H-13 (δH 4.63) and H-8 (δH 1.50); H-6 (δH 2.50) and H-7  and H-24 (δH 2.32). Comparison of the 1H- and 13C-NMR data (1 with those of hyperibone K (3) , seven tertiary carbons [including three olefinic  and four other tertiary carbons ], and six methylene carbons , and nine methyl carbons . Comparison of the 1H- and 13C-NMR data  and C-13 (δC 58.1); between H-20 (δH 4.99) and C-6 (δC 69.2); and between H-30 (δH 2.45) and C-2 (δC 88.8). The NOESY cross-peaks , H-32/Me(11), H-7/Me(10), H-7/H-14, H-7/H-19, suggested that Hα-4, Hα-13, Me(11), and the 2-isobutyryl groups are α-oriented, and 6-geranyl, Me(10), and 13-isoprenyl groups are β-oriented. On the basis of the evidence above, the structure of hypersamsone S was elucidated as 2, which was further substantiated through 2D-experiments, including HMQC, 1H-1H COSY, HMBC (Hypersamsone S (2) was obtained as a colorless amorphous powder. The molecular formula was determined to be CNMR data  of 2 witdata 2a,  18]. Th. Th32H46data 2a,  between ss-peaks  between SY, HMBC , and NOESY, HMBC  spectra.3) was readily identified by a comparison of physical and spectroscopic data  with the literature values + (8), 446 (15), 128 (23), 105 (100). HR-EI-MS: m/z = 474.3128 [M]+ .HypersamsoneS (2). Colorless amorphous powder. c 0.3, CHCl3). IR (KBr): υmax 2969, 2927, 1718, 1703, 1441, 1136, 1067, and 826 cm−1. 1H- and 13C-NMR spectroscopic data, see m/z (%) = 494 [M]+ (10), 466 (16), 397 (25), 355 (35), 189 (38), 135 (45), 109 (52), 91 (55), 71 (100). HR-EI-MS: m/z = 494.3396 [M]+ .1 and 2, were isolated from the aerial parts of H. sampsonii. The structures of these compounds were established on the basis of spectroscopic data. The discovery of new compounds from the genus Hypericum may not only provide more spectroscopic data about these isolates, but may also contribute to enhancing our understanding of the taxonomy and evolution of the genus Hypericum.Three compounds, including two new compounds


H-imidazol-2-yl group at R2 position as an important feature for producing potent AR agonists. Moreover, the nature of the R1 substituent highly affects not only affinity/activity at the hA1 and hA2B ARs but also selectivity versus the other subtypes. Potent hA1 and hA2B AR ligands were developed, and among them, the 2-amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-[4-(prop-2-en-1-yloxy)phenyl]pyridine-3,5-dicarbonitrile (3) is active in the low nanomolar range at these subtypes and shows a good trend of selectivity versus both the hA2A and hA3 ARs. This combined hA1/hA2B partial agonist activity leads to a synergistic effect on glucose homeostasis and could potentially be beneficial in treating diabetes and related complications.The amino-3,5-dicyanopyridine derivatives belong to an intriguing series of adenosine receptor (AR) ligands that has been developed by both academic researchers and industry. Indeed, the studies carried out to date underline the versatility of the dicyanopyridine scaffold to obtain AR ligands with not only a wide range of affinities but also with diverse degrees of efficacies at the different ARs. These observations prompted us to investigate on the structure–activity relationships (SARs) of this series leading to important previously reported results. The present SAR study has helped to confirm the 1 The role of the A clearer ,8. In fandidates ,6. Sinceications . For thihologies . While t to date . However to date ; most ofle P453,  turns ouH-imidazol-2-yl group in the R2 pendant as an important feature for producing potent AR agonists. Certain 2-amino-4-aryl-6-(1H-imidazol-2-yl-methylsulfanyl)-pyridine-3,5-dicarbonitrile compounds, belonging to the LUF series methyl)thio)-6-amino-4-(4-ethoxyphenyl)pyridine-3,5-dicarbonitrile 5, previously reported in reference -derivative thesized  startingized  cells, and were also studied as hA2B agonists by evaluating their stimulatory effect on cAMP production in CHO cells, stably expressing the hA2B AR. Compounds 3, 8, and 11, the most interesting in terms of combined affinity/activity at hA1 and hA2B ARs, were evaluated for their A1 pharmacological profile in the cAMP assay, where each compound was tested to assess its capability to modulate Forskolin-stimulated cAMP levels in the absence or presence of the A1AR agonist 2-chloro-N6-cyclopentyladenosine (CCPA) (1 nM). All pharmacological data are reported in The amino-3,5-dicyanopyridines 1 and hA2B ARs, molecular docking studies were performed on the cryo-EM structure of the agonist-bound hA1AR  [To simulate the binding mode of the herein reported dicyanopyridines at hAsolution ) and on solution ). The ob19.0101)  by inspe19.0101) . For eac1 and hA2A ARs of selected dicyanopyridine compounds are reported in Docking conformations at hA1–20 are reported in H-imidazol-2-ylmethyl)sulfanyl]-4-phenylpyridine-3,5-dicarbonitrile) ethyl]purine-2-carboxamide) [N-ethyl-β-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid) pyridine-3,5-dicarbonitrile (8) emerging as the most active compound with a partial agonist profile. The compounds showing a combined hA1/hA2B AR partial agonist activity, as derivatives 3, 8, and 11, deserve the greatest attention. In particular, the 2-amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-[4-(prop-2-en-1-yloxy)phenyl]pyridine-3,5-dicarbonitrile (3) is active in the low nanomolar range at both these subtypes and shows a good trend of selectivity versus both the hA2A and hA3 ARs. This combined hA1/hA2B partial agonist activity, leading to a synergistic effect on glucose homeostasis, could be of interest in the development of potentially useful agents for treating diabetes and its complications. The in silico calculated ADMET profiles indicated that some bioavailability and toxicity parameters fall in the safety range, while others suggest some points of concerns for compound 3. Thus, further studies will be devoted in the future to thoroughly investigate on the toxicity of compound 3. We are however confident in considering 3 as a promising lead for the development of new antidiabetics with improved pharmacokinetics and safer therapeutic potential. This study, which has led to some amino-3,5-dicyanopyridines differently substituted at R-1. NMR spectra were recorded on a Bruker Avance 400 spectrometer  (400 MHz for 1H NMR and 100 MHz for 13C NMR). The chemical shifts are reported in δ (ppm) and are relative to the central peak of the residual non-deuterated solvent, which was DMSO-d6. The following abbreviations are used: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad, Ar = aromatic protons. Compounds 28–32 were synthesized as reported in reference -6-[(1H-imidazol-2-ylmethyl)sulfanyl]pyridine-3,5-dicarbonitrile (1H NMR (DMSO-d6) 11.87 ; 8.10 ; 7.46 ; 7.09 ; 7.08 ; 6.96 ; 4.49 ; 4.03 ; 2.70-2.60 ; 2.08-2.07 ; 1.91-1.88 . IR 3385; 3320; 3217; 2210. Anal. Calc. for C22H20N6OSThe crude product was purified by preparative TLC using EtOAc/cyclohexane/MeOH 6:4:1 as eluting system. Yield 20%; mp 214-216 °C. 2)2-Amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-[4-(2-methylpropoxy)phenyl]pyridine-3,5-dicarbonitrile (1H NMR (DMSO-d6) 11.84 ; 8.14 , 7.47 ; 7.10 ; 7.08 ; 6.85 ; 4.50 ; 3.84 ; 2.09-2.02 ; 1.00 ; IR 3387; 3315; 3215; 2208. Anal. Calc. for C21H20N6OSThe crude product was purified by preparative TLC using EtOAc/cyclohexane /MeOH 5:5:1 as eluting system. Yield 31%; mp 230-232 °C. 3)2-Amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-[4-(prop-2-en-1-yloxy)phenyl]pyridine-3,5-dicarbonitrile (1H NMR (DMSO-d6) 11.86 ; 8.09 ; 7.48 ; 7.12 ; 7.07 ; 6.85 ; 6.11-6.04 ; 5.40 ; 5.30 ; 4.62 ; 4.48 . Anal. Calc. for C20H16N6OSThe crude product was purified by preparative TLC using DCM/MeOH 9:1 as eluting system. Yield 21%; mp 216-218 °C. 4)2-Amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-{4-[(2-methylprop-2-en-1-yl)oxy]phenyl} pyridine-3,5-dicarbonitrile (1H NMR (DMSO-d6) 11.85 ; 8.08 ; 7.48 ; 7.12 ; 7.07 ; 6.85 ; 5.09 ; 4.99 ; 4.56 ; 4.49 ; 1.79 ; 13C NMR (DMSO-d6) 167.19; 161.03; 160.95; 159.11; 143.71; 141.67; 131.75; 131.28; 126.93; 116.58; 115.93; 115.80; 113.79; 94.22; 87.00; 72.14; 27.75; 20.32; IR 3404; 3377; 3336; 2210. Anal. Calc. for C21H18N6OSThe crude product was purified by column chromatography using EtOAc/cyclohexane /MeOH 6:3:1 as eluting system. Yield 36%; mp 224-226 °C. 6)N-sulfanyl]pyridin-4-yl}phenyl) acetamide (1H NMR (DMSO-d6) 11.84 ; 10.22 ; 8.11 ; 7.73 ; 7.46 ; 7.05 ; 6.86 ; 4.49 ; 2.09 ; IR 3387; 3211; 2214; 1683. Anal. Calc. for C19H15N7OSThe crude product was purified by column chromatography using EtOAc/cyclohexane /MeOH 8.2:1.5:0.9 as eluting system. Yield 61%; mp 275-278 °C. 7)2-Amino-4-(4-chlorophenyl)-6-[(1H-imidazol-2-ylmethyl)sulfanyl]pyridine-3,5-dicarbonitrile (1H NMR (DMSO-d6) 11.87 ; 8.2 ; 7.65 ; 7.59 ; 7.08 ; 6.85 ; 4.50 ; IR 3336; 3244; 2220; 1683. Anal. Calc. for C17H11ClN6SYield 42%; mp 229-231 °C dec (EtOAc). 8)2-Amino-4-(4-fluorophenyl)-6-[(1H-imidazol-2-ylmethyl)sulfanyl]pyridine-3,5-dicarbonitrile (1H NMR (DMSO-d6) 11.9 ; 8.2 ; 7.60 ; 7.45 ; 7.0 ; 4.50 ; IR 3314; 3145; 2229. Anal. Calc. for C17H11FN6SYield 80%; mp 223-225 °C dec (EtOAc). 9)2-Amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-[4-(trifluoromethyl)phenyl]pyridine-3,5-dicarbonitrile (1H NMR (DMSO-d6) 11.85 ; 8.22 ; 7.97 ; 7.79 ; 7.08 ; 6.86 ;4.55 ; IR 3527; 3185; 2437. Anal. Calc. for C18H11F3N6SYield 80%; mp 233-235 °C dec (EtOH). 10)2-[(1H-Imidazol-2-il)metiltio]-6-ammino-4(4-metilfenil)piridina-3,5-dicarbonitrile (1H NMR (DMSO-d6) 11.85 ; 8.14 ; 7.40 ; 7.38 ; 7.07 ; 4.50 ; 2.39 . IR 3518; 3129; 2213. Anal. Calc. for C18H14N6SYield 67%; mp 259-261 °C dec (EtOH). 11)2-Amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-[4-(methylsulfanyl)phenyl]pyridine-3,5-dicarbonitrile (1H NMR (DMSO-d6) 11.87 ; 8.12 ; 7.47 ; 7.42 ; 7.08 ; 6.85 ; 4.50 ; 2.51 . 13C NMR (DMSO-d6) 166.65; 160.26; 158.33; 143.00; 142.31; 130.19; 129.43; 125.69; 115.81; 93.54; 86.35; 27.23; 14.47. IR 3420; 3390; 2223. Anal. Calc. for C18H14N6S2Yield 95%; mp 257-259 °C dec (MeOH). 12)2-Amino-4-benzyl-6-[(1H-imidazol-2-ylmethyl)sulfanyl]pyridine-3,5-dicarbonitrile (1H NMR (DMSO-d6) 11.82 ; 8.14 ; 7.34 ; 7.24 ; 6.95 ; 4.47 ; 4.08 . 13C NMR (DMSO-d6) 166.73; 160.33; 159.10; 142.89; 136.61; 129.38; 128.78; 127.64; 115.61; 93.99; 86.95; 38.84; 27.15; 14.47. IR 3431; 3357; 2377. Anal. Calc. for C18H14N6SYield 70%; mp 246-248 °C dec (EtOH). 13)2-Amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-methylpyridine-3,5-dicarbonitrile (1H NMR (DMSO-d6) 11.87 ; 8.12 ; 7.02 ; 4.51 , 2.43 . IR 3455; 3363; 2473. Anal. Calc. for C12H10N6SYield 66%; mp 267-269 °C dec (EtOH). 20)Methyl{sulfanyl}acetate (1H NMR (DMSO-d6) 7.96 ; 7.51 ; 7.12 ); 4.20 , 3.84 , 3.70 . IR 3426; 3397; 3325; 3224; 2209. Anal. Calc. for C17H14N4O3SYield 80%; mp 218-220 °C (EtOH). 14; ethanolamine, for 15; 2-(4-methoxyphenyl)ethanamine, for 16; or imidazole (for compound 17 (1.18 mmol), and triethylamine (1.18 mmol) were sequentially added to a solution of the 2-chloro-derivative 42 -4-phenylpyridine-3,5-dicarbonitrile (1H NMR (DMSO-d6) 11.64 ; 8.39 ; 7.88 ; 7.61-7.45 ; 7.04 ; 6.83 ;4.56 . IR 3360; 3322; 3186; 2207. Anal. Calc. for C17H13N7Yield 57%; mp 208-210 °C (EtOH). 15) 2-Amino-6-[(2-hydroxyethyl)amino]-4-phenylpyridine-3,5-dicarbonitrile (1H NMR (DMSO-d6) 7.55-7.53 ; 7.47-7.45 ; 7.37 ; 7.20 ; 4.74 ; 3.54 ; 3.47 . IR 3472; 3345; 3223; 2204. Anal. Calc. for C15H13N5OYield 64%; mp 230-231 °C (EtOH). 16)2-Amino-6-{[2-(4-methoxyphenyl)ethyl]amino}-4-phenylpyridine-3,5-dicarbonitrile (1H NMR (DMSO-d6) 7.54-7.53 ; 7.51-7.45 ; 7.39 ; 7.20 ; 6.87 ; 3.73 ; 3.57-3.51 ; 2.80 . IR 3451; 3342; 3244; 2201. Anal. Calc. for C22H19N5OYield 80%; mp ˃ 300 °C (EtOH/DMF). 17)2-Amino-6-(1H-imidazol-1-yl)-4-phenylpyridine-3,5-dicarbonitrile (1H NMR (DMSO-d6) 8.40 , 8.36 , 7.81 , 7.63 , 7.18 . IR 3399; 2220; 1681;1466. Anal. Calc. for C16H10N6Yield 77%; mp 253-255 °C (EtOH). 42 pyridine-3,5-dicarbonitrile (23) 1HNMR (DMSO-d6) 7.98 ; 7.92 ; 7.82 ; 7.63-7.61 ; 7.52-7.51 . IR 3320; 3211; 2243. Anal. Calc. for C20H11F3N4SYield 64%; mp 199-201 °C (EtOH). 24) Yield 74%; mp 220-221 °C (lit mp 215-216 °C).25) [2-Amino-4-[4-(methylsulfanyl)phenyl]-6-(phenylsulfanyl)pyridine-3,5-dicarbonitrile (25) Yield 75%; mp 244-246 °C (lit mp 254-256 °C).26)2-Amino-4-benzyl-6-(phenylsulfanyl)pyridine-3,5-dicarbonitrile (1HNMR (DMSO-d6) 7.81 ; 7.60-7.57 ; 7.47-7.50 ; 7.39-7.35 ; 7.29-7.25 ; 4.12 . IR 3329; 3225; 2198. Anal. Calc. for C20H14N4SYield 70%; mp 247-249 °C (MeOH). 27) pyridine-3,5-dicarbonitrile (35)  1HNMR (DMSO-d6) 13.15 ; 8.07 ; 7.8 . IR 3310; 3193; 2225.Yield 90%; mp 212-214 °C (MeOH). 36) -6-sulfanylpyridine-3,5-dicarbonitrile (1HNMR (DMSO-d6) 13.1 ; 8.03 , 7.46 ; 7.41 ; 2.51 . IR 3311; 3195; 2222. Anal. Calc. for C14H10N4S2Yield 82%; mp 188-190 °C (EtOH). 38)2-Amino-4-benzyl-6-sulfanylpyridine-3,5-dicarbonitrile (1HNMR (DMSO-d6) 12.96 ; 7.82 ; 7.37-7.35 ; 7.30-7.28 ; 4.01 . IR 3319; 3280; 2229. Anal. Calc. for C14H10N4SYield 80%; mp 231-233 °C (EtOH). 40) -8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX) with membrane suspension (50 μg of protein/100 μL) and different concentrations of the examined compounds at 25°C for 90 min in 50 mM Tris HCl, pH 7.4. Non-specific binding was defined as binding in the presence of 1 μM DPCPX and was always < 10% of the total binding [2A ARs were performed incubating the radioligand [3H]-ZM241385 (4-(2-triazolotriazin-5-ylamino]ethyl)phenol) (1 nM) with the membrane suspension (50 μg of protein/100 μL) and different concentrations of the examined compounds for 60 min at 4°C in 50 mM Tris HCl (pH 7.4), 10 mM MgCl2. Non-specific binding was determined in the presence of ZM241385 (1 μM) and was about 20% of the total binding [3 ARs were carried out incubating the membrane suspension (50 μg of protein/100 μL) with 0.5 nM [125I]-N6-(4-aminobenzyl)-N-methylcarboxamidoadenosine ([125I]-ABMECA) in the presence of different concentration of the examined compounds for an incubation time of 120 min at 4°C in 50 mM Tris HCl (pH 7.4), 10 mM MgCl2, and 1 mM EDTA. Non-specific binding was defined as binding in the presence of 1μM ABMECA and was always < 10% of the total binding /KD*), where [C*] is the concentration of the radioligand and KD* its dissociation constant [Ki and IC50 values were calculated by non-linear regression analysis using the equation for a sigmoid concentration-response curve .The protein concentration was determined according to a Bio-Rad method with bovine albumin as a standard reference. Inhibitory binding constant  gradient of the potential energy was less than 0.05 kJ mol−1 Å−1. Reliability and quality of the model were checked using the Protein Geometry Monitor application within MOE, which provides a variety of stereochemical measurements for inspection of the structural quality in a given protein, like backbone bond lengths, angles and dihedrals, Ramachandran φ-ψ dihedral plots, and quality of side chain rotamer and non-bonded contact. The cryo-EM structure of the agonist-bound hA1 AR  suite . A homolsolution ). A multsolution ) was add2B and hA1 ARs structures using as docking tools the Induced Fit docking protocol of MOE and the genetic algorithm docking tool of CCDC Gold [Conformational Analysis of ligands. The algorithm generated conformations from a single 3D conformation by conducting a systematic search. In this way, all combinations of angles were created for each ligand. Placement. A collection of poses was generated from the pool of ligand conformations using Alpha Triangle placement method. Poses were generated by superposition of ligand atom triplets and triplet points in the receptor binding site. The receptor site points are alpha sphere centres which represent locations of tight packing. At each iteration, a random conformation was selected, a random triplet of ligand atoms and a random triplet of alpha sphere centres were used to determine the pose. Scoring. Poses generated by the placement methodology were scored using the Alpha HB scoring function, which combines a term measuring the geometric fit of the ligand to the binding site and a term measuring hydrogen bonding effects. Induced Fit. The generated docking conformations were subjected to energy minimization within the binding site and the protein sidechains are included in the refinement stage. In detail, the protein backbone is set as rigid while the side chains are not set to “free to move” but are set to “tethered”, where an atom tether is a distance restraint that restrains the distance not between two atoms but between an atom and a fixed point in space.Rescoring. Complexes generated by the Induced Fit methodology stage were scored using the Alpha HB scoring function. Gold tool was used with default efficiency settings through MOE interface, by selecting GoldScore as scoring function [function .All compound structures were docked into the binding site of the hACDC Gold . The Ind


The structure of the new compound was elucidated by extensive NMR  and high resolution mass spectrometry analysis. The three lignans 1, 3, and 4 did not exhibit significant cytotoxicity against human gastric cancer Ags cells or HepG2 liver cancer cells. This is the first report of the isolation of a lignan skeleton from the genus Viola L.A new lignan glycoside, tianshanoside A ( Viola tianshanica Maxim (Violaceae) is a perennial herb widely distributed in Central Asia, especially in the Xinjiang Uygur Autonomous Region (XUAR) of China + in combination with its 1H-NMR, 13C-NMR and DEPT spectra. Maximum absorption bands at 230 and 280 nm in the UV spectrum suggested the existence of conjugated groups in 1. The IR spectrum exhibited absorption bands for free hydroxyl groups (3387 cm−1) and benzene rings (1637 and 1513 cm−1). Detailed analysis of 1H-NMR , 6.68 , and 6.82 ; and 6.69 , 6.62 , and 6.73 .Repeated column chromatography of the ethyl acetate fraction of the ethanolic extract of f 1H-NMR  and HSQC1H-NMR chemical shifts observed for these two benzene systems together with the presence of two oxygenated methyl proton signals at δH 3.72  suggested the presence of two guaiacyl (3-methoxy-4-hydroxyphenyl) groups in this compound , three methylene , three methine , twelve signals of two glucose moieties, and twelve aromatic carbons, of which six were quaternary , and the remaining six were unsubstituted aromatic carbons  (1 as a tetrahydrofuran lignan diglucoside  of 1 was in good agreement with that of -5,5′-dimethoxylariciresinol 9′-O-β-d-glucopyranoside [Δε: +11.1 (211 nm)] [1 was similar with that of (+)-lariciresinol 4′-O-β-d-glucopyranoside [Δε: −0.47 (280 nm)]   and the 280 nm)]  and (+)-280 nm)] . In summdigotica . This is2–4 were identified as syringin (2) +; HRESIMS m/z 707.2531 [M + Na]+ .Syringin (2). Amorphous powder. 1H-NMR (CD3OD): δH 6.77 , 6.57 , 6.35 , 4.24 , 3.88 . 13C-NMR (CD3OD): δC 154.3 , 135.2 (C-1), 131.2 (C-7), 130.0 (C-8), 105.4 , 105.3 (C-1′), 78.3 (C-5′), 77.8 (C-3′), 75.7 (C-2′), 71.3 (C-4′), 63.5 (C-9), 62.5(C-6′). ESI-MS: m/z 395 [M + Na]+, 411 [M + K]+.Picraquassioside C (3). Amorphous powder. 1H-NMR (CD3OD): δH 6.81 , 6.73 , 6.55 , 6.35 , 5.03 , 4.80 , 4.25 , 3.86 , 3.85 , 3.2–3.6 . 13C-NMR (CD3OD): δC 154.2 , 153.7 , 139.2 (C-1′), 136.8 (C-1′′), 135.4 (C-4′), 134.7 (C-4′′), 131.2 (C-2′′′), 129.9 (C-1′′′), 105.7 , 105.6 (C-1-Glc), 104.7 , 87.9 (C-2), 78.3 (C-5-Glc), 78.1 (C-3-Glc), 75.7 (C-2-Glc), 74.0 (C-1), 71.3 (C-4-Glc), 63.5 (C-3′′′), 62.5 (C-6-Glc), 61.9 (C-3), 56.9 , 56.6 . ESI-MS: m/z 621 [M + Na]+, 637 [M + K]+.Aketrilignoside B (4). Colorless gum. 1H-NMR (CD3OD): δH 7.36 , 6.78 , 4.84 , 4.38 , 4.30 , 4.26 , 4.21 , 4.09 , 3.94 , 3.88 , 3.62 , 3.20-3.85 , 2.75 . 13C-NMR (CD3OD): δC 200.5 (C-7′), 149.3 , 149.2 , 142.9 (C-4′), 136.3 (C-4), 132.6 (C-1), 128.7 (C-1′), 107.8 , 105.3 , 105.1 (C-1-Glc), 85.3 (C-7), 78.2 (C-3-Glc), 78.1 (C-5-Glc), 75.2 (C-2-Glc), 71.7 (C-9′), 71.6 (C-4-Glc), 69.1 (C-9), 62.8 (C-6-Glc), 57.1 , 56.8 , 53.3 (C-8), 50.1 (C-8′). ESI-MS: m/z 597 [M + H]+, 619 [M + Na]+.2 atmosphere. The cytotoxicity of the compounds was determined in vitro using the Cell Counting Kit-8 (CCK-8) assay kit , according to the manufacturer′s instructions. The sample compounds were dissolved in DMSO, and then further diluted. Cells were plated at a density of 1.0 × 105 per well in a 96-well microplate and incubated at 37 °C for 24 h. The cells were treated with various concentrations of test compounds  in quadruplicate for 72 h. Then, 20 μL of the solution of CCK-8 was added into each well, and the plates were further incubated for an additional 1 h. The absorbance was measured at 450 nm with a Varioskan Flash microplate reader .Two human cancer cell lines, the gastric cancer Ags cells and HepG2 liver cancer cells were obtained from the Cell Bank of the Chinese Academy of Sciences . The Ags cell line was cultured in a Ham’s/F12 medium , supplemented with 10% fetal bovine serum (Gibco). The HepG2 cell line was cultured in DMEM medium (Hyclone), supplemented with 10% fetal bovine serum (Gibco). Cells were maintained at 37 °C in a 95% air-5% COV. tianshanica. This is the first report of lignans from Viola species. Three lignan glycosides exhibited no significant inhibitory activities against two human tumor cell lines, gastric cancer Ags or liver cancer HepG2.Three lignan glycosides including a new one, tianshanoside A, and a phenylpropanoid glycoside were isolated for the first time from the dried whole plant of


It is noteworthy that the sulfide function reacts under mild conditions without over-oxidation to sulfone. The role of NaNO2 as an efficient NO equivalent for the activation of molecular oxygen was identified. Under the optimal conditions, a broad range of sulfide substrates were converted into their corresponding sulfoxides in high yields by molecular oxygen. The present catalytic system utilizes cheap and readily available agents as the catalysts, exhibits high selectivity for sulfoxide products and releases only innocuous water as the by-products.A highly efficient transition-metal-free catalytic system Br The oxidation of sulfides into the corresponding sulfoxides is one of the most important functional group transformations in organic synthesis . Sulfoxiet al. +.ulfoxide , entry 14-Methoxyphenyl methyl sulfoxide (1H-NMR (DMSO-d6): δ 2.71, 3.83 , 7.13 , 7.63 ; IR : ν 1027, 831; MS (EI), m/z: 170 [M]+.ulfoxide , entry 22-Methoxyphenyl methyl sulfoxide (1H-NMR (DMSO-d6): δ 2.70 , 3.86 , 7.14 , 7.21 , 7.52 , 7.63; IR : ν 1038, 757; MS (EI), m/z: 170 [M]+.ulfoxide , entry 34-Methylphenyl methyl sulfoxide (1H-NMR (DMSO-d6): δ 2.35 , 2.69 , 7.4 , 7.56 ; IR : ν 1046, 810; MS (EI), m/z: 154 [M]+.ulfoxide , entry 44-Fluorophenyl methyl sulfoxide (1H-NMR (DMSO-d6): δ 2.73 , 7.40 , 7.74 ; IR : ν 1048 , 834; MS (EI), m/z: 158 [M]+.ulfoxide , entry 52-Chlorophenyl methyl sulfoxide ( 1H-NMR (DMSO-d6): δ 2.81 , 7.56-7.59 , 7.62~7.68 , 7.81-7.86 ; IR : ν 1067, 759; MS (EI), m/z: 174 [M]+.ulfoxide , entry 61-(4-Methansulfinylphenyl)-ethanone (1H-NMR (DMSO-d6): δ 2.63 , 2.80 , 7.83 , 8.13 ; IR : ν 1674, 1047, 830; MS (EI), m/z: 182 [M]+.ethanone , entry 74-Chlorophenyl methyl sulfoxide (1H-NMR (DMSO-d6): δ 2.78 , 7.67 , 7.73 ; IR : ν 1051, 822; MS (EI), m/z: 174 [M]+.ulfoxide , entry 84-Methanesulfinylbenzoic acid (1H-NMR (DMSO-d6): δ 2.79 , 7.81 , 8.11 , 13.30 ; IR : ν 2773, 1690, 1083, 856; MS (EI), m/z: 184 [M]+.oic acid , entry 9Dibutyl sulfoxide (1H-NMR (DMSO-d6): δ 0.91 , 1.41 , 1.608 , 2.657 ; IR : ν 737, 923, 1021, 1085; MS (EI), m z: 162 [M]+.2 as co-catalysts. The results showed that reaction with a low concentration of Br2 and NaNO2 in a small amount of water is highly selective to the desired products without over-oxidation. This reliable non-metal catalytic system for aerobic sulfide oxidation is applicable to a wide range of sulfides, including methyl aryl sulfides and their analogues. In conclusion, we have successfully developed a highly efficient, transition-metal-free catalytic process for the aerobic oxidation of sulfides with molecular oxygen. It should be noted that the quantitative oxidation of methyl phenyl sulfide to the corresponding sulfoxides with excellent yields can be achieved without acid under mild conditions. A catalyst system is based on molecular bromine as an active catalyst in the presence of very inexpensive NaNO


Ailanthus altissima (Mill.) Swingle (Simaroubaceae), together with one known phenylpropionamide, 13 known phenols, and 10 flavonoids. The structures of the new compounds were elucidated as 2-hydroxy-N-[(2-O-β-d-glucopyranosyl)phenyl]propionamide (1), 2-hydroxy-N-[(2-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl)phenyl]propionamide (2), 2β-carboxyl-piperidine-4β-acetic acid methyl ester (4), and 4-hydroxyphenyl-1-O-[6-(hydrogen-3-hydroxy-3-methylpentanedioate)]-β-d-glucopyranoside (5) based on spectroscopic analysis. All the isolated compounds were evaluated for their inhibitory activity against Tobacco mosaic virus (TMV) using the leaf-disc method. Among the compounds isolated, arbutin (6), β-d-glucopyranosyl-(1→6)-arbutin (7), 4-methoxyphenylacetic acid (10), and corilagin (18) showed moderate inhibition against TMV with IC50 values of 0.49, 0.51, 0.27, and 0.45 mM, respectively.Four novel compounds—two phenylpropionamides, one piperidine, and one phenolic derivatives—were isolated and identified from the fruit of a medicinal plant,  Ailanthus altissima (Mill.) Swingle (Simaroubaceae), a deciduous tree (6–20 m in height), is native to Mainland China and now naturalized in many temperate regions of the world + . The IR spectrum (−1). The 1H-NMR , 7.20 , 7.05 , and 7.02 ], an oxygenated methine [4.15 ], a methyl [δH 1.33 ], an amide proton [δH 9.42 ], and a glucopyranosyl moiety . Its 13C-NMR (δC 173.1), C-1′ (δC 128.6), C-2′ (δC 146.5), and C-6′ (δC 119.6) and from H-2 to C-1, C-2 (δC 67.8), and C-3 (δC 20.8) indicated the presence of a 2-hydroxypropionamide moiety, which was attached to C-1′ of the benzene ring via an NH linkage. The anomeric proton appearing as a doublet at δH 4.85 with a diaxial coupling constant of 7.5 Hz suggested that the glucopyranosyl moiety must be a β-anomer. Additionally, it was attached to C-2′ of the benzene ring through an oxygen, as indicated from the HMBC correlations from the anomeric proton to C-2′, which was further confirmed by the NOESY (nuclear Overhauser effect spectroscopy) correlation between H-3′/H-1‴. The acid hydrolysis of 1 afforded d-glucose, which was identified using TLC by comparison with standard sugars. Therefore, the structure of Compound 1 was established as 2-hydroxy-N-[(2-O-β-d-glucopyranosyl)phenyl]propionamide.Compound spectrum  exhibitee 1H-NMR  and HSQC 13C-NMR  and DEPT 13C-NMR  observed2 was obtained as a white amorphous powder. Its molecular formula was deduced to be C21H31NO13 by an HR-ESI-MS ion peak at m/z 528.1726 [M + Na]+ . The IR spectrum (−1). Analysis of the 1H- and 13C-NMR data  and 4.22 , which correlated to the corresponding anomeric carbon signals at δC 101.5 (C-1″) and 103.2 (C-1‴) in the 13C-NMR spectra, respectively, indicating the presence of two glucopyranosyl units with β-form. The HMBC correlations from an anomeric proton at δH 4.22 (H-1‴) to δC 68.3 (C-6″) and from another anomeric proton at δH 4.86 (H-1″) to δC 146.4 (C-2′) indicated that two sugars were connected through 1→6 linkage, and the sugar chain was attached at C-2′ of the aglycone through an oxygen. Therefore, Compound 2 was determined as 2-hydroxy-N-[(2-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl)phenyl]propionamide.Compound spectrum  exhibiteNMR data  suggeste3 was obtained as a white amorphous powder, with a molecular formula of C9H12NO3 as indicated by an ion peak at m/z 182.0840 [M + Na]+  in its HR-ESI-MS. Comparison of the 1H- and 13C-NMR data with that of Compounds 1 and 2, as well as with those reported in the literature + . Its IR spectrum (−1), a carbonyl (1729 cm−1), and a strong peak for a hydroxyl group (3428 cm−1). The 1H-NMR , two methines at δH 3.47  and 2.11 , as well as four methylene protons. The 13C-NMR spectrum and DEPT showed the presence of two methine carbons at δC 60.6 and 32.9, one methoxyl carbon at δC 52.1, two carbonyl carbons at δC 173.7 and 173.8, as well as four methylene carbons. The key HMBC correlations (1H-1H COSY (correlated spectroscopy) correlations between H-2/H-3, H-3/H-4, H-4/H-5, H-5/H-6, and H-4/H-8. An acetic acid methyl ester group was established by the HMBC correlations from the methoxyl protons to C-8 and C-9, and it was attached to C-4 of the piperidine ring as indicated by the HMBC correlations from H-4 to C-8 and C-9. The HMBC correlation from H-2 to C-7 proved that a carbonyl group was located at C-2. The NOESY correlations . The IR spectrum  and 6.66 ], a glucopyranosyl moiety , one methyl [1.22 ], and two methylene groups . The 13C-NMR and DEPT of 5 displayed resonances of two carbonyls [δC 174.4 (C-5″) and 170.5 (C-1″)], one oxygenated quaternary carbon [δC 68.9 (C-3″)], two methylenes [δC 46.1 (C-4″) and 45.9 (C-2″)], and one methyl [δC 27.6 (C-6″)] carbons, besides signals for a typical glucopyranosyl moiety and a p-hydroxyphenyl group. The HMBC correlations  group. The HMBC correlations from the anomeric proton H-1′ to C-1 (δC 150.1) and from H2-6′ to C-1″ indicated that the p-hydroxyphenyl and HMG group were connected with C-1′ and C-6′ of the glucopyranosyl moiety, respectively. The anomeric proton appearing as a doublet at δH 4.65 with a diaxial coupling constant of 7.7 Hz suggested that the glucopyranosyl moiety must be a β-anomer. Furthermore, the NOESY correlations (5 was identified as 4-hydroxyphenyl-1-O-[6-(hydrogen 3-hydroxy-3-methylpentanedioate)]-β-d-glucopyranoside.Compound spectrum  of 5 shospectrum  showed selations  observedelations  observed6–28 were identified by a comparison of their spectroscopic data with those reported in the literatures as arbutin (6) pyran-6-one (17) propionamide (1). White amorphous powder. IR (KBr) υmax: 3478, 3388, 2907, 1674, 1603, 1534, 1456, 1256, 1118, 1074, 1023 cm−1; HR-ESI-MS m/z: 366.1185 ; 1H-NMR  and 13C-NMR  data (see data see .2-Hydroxy-N-[(2-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl) phenyl]propionamide (2). White amorphous powder. IR (KBr) υmax: 3423, 3346, 2928, 2891, 1662, 1602, 1532, 1454, 1260, 1070 cm−1; HR-ESI-MS m/z: 528.1726 ; 1H-NMR  and 13C-NMR  data (see data see .2-Hydroxy-N-(2-hydroxyphenyl)propionamide (3). White amorphous powder. HR-ESI-MS m/z: 182.0840 ; 1H-NMR  δ 10.06 , 9.25 , 8.17 , 6.89 , 6.77 , 6.11 , 4.14 , 1.32 . 13C-NMR  δ 172.7 (C-1), 146.1 (C-2′), 126.2 (C-1′), 123.6 (C-4′), 119.1 (C-5′), 118.9 (C-6′), 114.7 (C-3′), 67.7 (C-2), 20.8 (C-3).2β-Carboxyl-piperidine-4β-acetic acid methyl ester (4). White amorphous powder. IR (KBr) υmax: 3428, 2958, 1729, 1624, 1439, 1402, 1299, 1216, 1180, 1137, 1010 cm−1; HR-ESI-MS m/z: 224.0907 ; 1H-NMR  δ 3.66 , 3.47 , 3.34 , 2.97 , 2.34 , 2.34 , 2.11 , 1.90 , 1.41 , 1.32 . 13C-NMR  δ 173.8 (C-9), 173.7 (C-7), 60.6 (C-2), 52.1 (C-OCH3), 44.3 (C-6), 40.9 (C-8), 34.0 (C-3), 32.9 (C-4), 29.1 (C-5).d-glucopyranoside4-Hydroxyphenyl-1-O-[6-(hydrogen 3-hydroxy-3-methylpentanedioate)]-β- (5). Pale-yellow solid. IR (KBr) υmax: 3420, 1718, 1510, 1399, 1216, 1073, 1042 cm−1; HR-ESI-MS m/z: 439.1234 ; 1H-NMR  δ 6.84 , 6.66 , 4.65 , 4.32 , 4.00 , 3.50 , 3.26 , 3.19 , 3.14 , 2.57 , 2.49 , 2.40 , 2.33 , 1.22 . 13C-NMR  δ 174.4 (C-5″), 170.5 (C-1″), 152.3 (C-4), 150.1 (C-1), 117.7 (C-2 and C-6), 115.5 (C-3 and C-5), 101.6 (C-1′), 76.3 (C-3′), 73.7 (C-5′), 73.2 (C-2′), 70.0 (C-4′), 68.9 (C-3″), 63.4 (C-6′), 46.1 (C-4″), 45.9 (C-2″), 27.6 (C-6″). 1, 2, and 5 (2 mg each) were hydrolyzed in 1 M HCl  at 95 °C for 2 h, respectively. After evaporated to dryness, the reaction mixtures were diluted in H2O and extracted with Et2O (3 × 2 mL). The aqueous layer was neutralized with NaHCO3 and evaporated under vacuum to furnish a neutral residue, from which d-glucose was identified by TLC comparison with standard sugars.Compounds 260 × dilution ration)/Nicotiana tabacum cv. K326, which were cultivated and grown to a 5–6-leaf stage in an insect-free greenhouse, were used for anti-TMV assay. Purified TMV (strain U1) was obtained from Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, China, and concentration was determined as 15 mg/mL using an ultraviolet spectrophotometer method  × 100%.Pure compounds were dissolved in DMSO and diluted with 0.01 M PBS to a certain concentration for test. The final concentration of DMSO in the test solution (≤2%) showed no adverse effect on the plants. Anti-TMV assay was carried out using the leaf-disc method as described previously in our paper . Growingterature . Virus cN-[(2-O-β-d-glucopyranosyl)phenyl]propionamide (1), and 2-hydroxy-N-[(2-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl)phenyl]propionamide (2), one piperidine, 2β-carboxyl-piperidine-4β-acetic acid methyl ester (4), and one phenol derivative, 4-hydroxyphenyl-1-O-[6-(hydrogen 3-hydroxy-3-methylpentanedioate)]-β-d-glucopyranoside (5), as well as one known phenylpropionamide (3), 13 phenols (6–18), and 10 flavonoids (19–28), were identified from the n-BuOH-soluble fraction from MeOH extract of Ailanthus altissima fruit. All the compounds obtained were evaluated for their antiviral activity against TMV; however, only weak to moderate activity was observed. These results provide us with general knowledge of these diverse phenolic constituents and suggest the distribution of novel nitrogenous analogues in the metabolites of this Simaroubaceae plant.Two phenylpropionamides, 2-hydroxy-


Five sulfur-containing flavor compounds were synthesized for the first time by the reaction of 4-ethyloctanoyl chloride with sulfur-containing alcohols or mercaptans. The synthesized compounds are 3-(methylthio)propyl 4-ethyloctanoate, 2-methyl-3-tetrahydro-furanthiol 4-ethyloctanoate, 4-methyl-5-thiazoleethanol 4-ethyloctanoate, 2-furan-methanethiol 4-ethyloctanoate and 2-methyl-3-furanthiol 4-ethyloctanoate. These five synthetic sulfur-containing ester flavor compounds all have meaty odor and might be used in foods if approved for this purpose in the future.  Esters are important flavor compounds because of the large number of accessible ester compounds, their occurrence in a wide range of natural sources, various odor characteristics and their wide range of uses in flavourings. The odor of esters is related to the organic acid and alcohol from which they derived. Moreover, the odor intensity of esters decreases with the increase of molecular weight; however, the flavor industry needs esters with strong odors and high molecular weight to prolong the duration of the odor of flavorings. Esters containing sulfur can be synthesized to yield flavor substances with strong odor and high molecular weight, as flavor compounds containing sulfur exhibit very low odor threshold levels and very good odor characteristics.3SCH2CH2COOCH3, and the other in which the sulfur atom is from an alcohol or mercaptan (AOM), such as CH3COOCH2CH2SCH3 or CH3COSCH2CH2CH3. The first sulfur-containing ester class can be synthesized by the thia-Michael addition reaction, which is facile and efficient [p-toluenesulfonic acid. A second method is treatment of AOM with an anhydride yielding the corresponding ester [The sulfur-containing esters can be divided into two classes; one in which the sulfur atom belongs to the organic acid, such as CHfficient ,2,3. Thefficient  or acidifficient ,6 like sng ester ,8,9; merng ester ,13,14,15ng ester ; these s2) was synthesized by the reaction of thionyl chloride with 4-ethyloctanoic acid (1), and then 2 reacted with either 3-(methylthio)propanol, 2-methyl-3-tetrahydrofuranthiol, 4-methyl-5-thiazoleethanol, 2-furanmethane-thiol and 2-methyl-3-furanthiol, respectively  to 4-ethyloctanoyl chloride increased, while the yield of 3c decreased as the ratio of alcohol to 4-ethyloctanoyl chloride increased. This phenomenon was related to the characteristics of the starting materials involved in each reaction. In the reaction producing 3c, the 4-methyl-5-thiazoleethanol used as a starting material has multiple nucleophilic centers , which may react with 4-ethyloctanoic chloride to give the product or byproducts. Although the reaction temperature and steric-hindrance effect were not appropriate to form byproducts, the yield increased as the ratio of 4-methyl-5-thiazoleethanol to 4-ethyloctanoyl chloride increased. This may account for the low yield of 3c. The yields of 3d and 3e induces the breakage of furan ring structure found in the starting material and the formation of resin-like byproducts. Pyridine was used in these reactions to trap the hydrogen chloride [The hydrogen chloride generated in the reactions to produce chloride . Pyridinchloride .3e is lower than that of 3d.2-Methyl-3-furanthiol and 2-furanmethanethiol are isomers, where the latter one is more reactive than the former. The mercapto group of 2-methyl-3-furanthiol is adjacent to the conjugated system, which weakens its nucleophilicity. Consequently, the yield of 3b, 3d and 3e. Amongst those five compounds, the three derived from mercaptan and 4-ethyloctanyl chloride  have stronger odors than the two derived from a sulfur-containing alcohol and 4-ethyloctanyl chloride (3a and 3c). All five flavor compounds have relatively high molecular weights (> 260) and are therefore less volatile, which can prolong the flavoring lifetime to improve the flavoring quality. The odor evaluation results are shown in 1H-NMR and 13C-NMR spectra were obtained on a Bruker DRX-300 nuclear magnetic resonance spectrometer. IR spectra were acquired on a Nicolet Avater 370 Fourier transform infrared spectrometer. Odor evaluation was carried out by gas chromatography-olfactometry.The samples were analyzed by Varian CP3800 gas chromatography. Mass spectra were obtained using a Waters Micromass Q-TOF mass spectrometer. in vacuo to collect the distillate at 74 ºC/0.4 kPa and give the 4-ethyloctanoyl chloride, yield 97%. 1H-NMR  2.75-2.80 , 1.58-1.60 , 1.16-1.22 , 0.77-0.83 ; 13C-NMR  174.10 (C=O), 44.81 (CH2), 37.89 (CH), 32.31 (CH2), 28.67 (CH2), 28.25 (CH2), 25.41 (CH2), 22.98 (CH2), 14.06 (CH3), 10.60 (CH3); IR  2960, 2929, 2860, 1801, 1458, 1404, 1380, 956, 709, 686, 596. To 4-ethyloctanoic acid (0.2 mol) was added thionyl chloride (0.28 mol) at 30 ºC. The resulting solution was refluxed for 6 hours at 80 ºC, and the excess thionyl chloride was evaporated 2CO3 aqueous solution and saturated NaCl aqueous solution to pH 6-7, then the sample was dried with anhydrous NaSO4 and filtered. The ethyl ether was evaporated in vacuo and the mixture was weighed. The composition of the product mixture was determined by gas-chromatography, and the yield was calculated. The product was further purified using distillation under the reduced pressure. To alcohol (or mercaptan) (0.1 mol) was added 4-ethyloctanoyl chloride (0.05 mol) at 30 ºC, and the resulting solution was refluxed for 4 hours at 70 ºC. After cooling, ethyl ether (30 mL) was added. The mixture was washed with 10% Na3-(Methylthio)propyl 4-ethyloctanoate (3a). Yield 94%; b.p. 142 ºC/0.4 kPa; refractive index (nD20) 1.4654; MS (EI), m/z, 260.1812 (M+), formula C14H28O2S, . 1H-NMR  4.14-4.18 , 2.53-2.58 , 2.25-2.30 , 2.10 , 1.87-1.96 , 1.55-1.60 , 1.23-1.30 , 0.84-0.90 ; 13C-NMR  173.82 (C=O), 62.65 (OCH2), 38.25 (CH), 32.33 (CH2), 31.60 (CH2), 30.50 (CH2), 28.66 (CH2), 28.15 (CH2), 28.12 (CH2), 25.42 (CH2), 22.91 (CH2), 15.30 (SCH3), 13.94 (CH3), 10.57 (CH3); IR  2958, 2925, 2864, 1737, 1460, 1383, 1349,1241, 1168, 1108, 1024, 960. 2-Methyl-3-tetrahydrofuranthiol 4-ethyloctanoate (3b). Yield 94%; b.p. 140 ºC/0.2 kPa; refractive index (nD20) 1.4830; MS (EI), m/z, (M+) 272.1813, formula C15H28O2S, . 1H- NMR  4.09-4.13 , 3.95-4.03 , 3.89-3.93 , 3.70-3.82 , 3.48-3.53 , 2.41-2.53 , 1.82-1.86 , 1.59-1.61, 1.16-1.26 , 0.80-0.88 ; 13C-NMR  199.48 (C=O), 80.19 (CH-O), 66.81 (CH2-O), 46.47 (CH-S), 41.68 (CH2), 38.33 (CH), 33.39 (CH2), 32.51 (CH2), 29.05 (CH2), 28.83 (CH2), 25.60 (CH2), 23.11(CH2), 17.00 (CH3), 14.19 (CH3),.10.77 (CH3); IR  2959, 2928, 2872, 1692, 1456, 1380, 1352, 1315, 1188, 1111, 1063, 1022, 990, 858. 4-Methyl-5-thiazoleethanol 4-ethyloctanoate (3c). Yield 93%; b.p. 175 ºC/0.4 kPa; refractive index (nD20) 1.4920; MS (EI), m/z, 297.1766 (M+), formula C16H27O2SN, ; 1H-NMR  8.55 , 4.17-4.21 , 3.03-3.08 , 2.38 , 2.22-2.27 , 1.53-1.55 , 1.19-1.24 , 0.78-0.87 ; 13C-NMR  173.90 (C=O), 149.86 (S-CH=N), 149.77 (N-C), 126.75 (S-C), 63.81 (O-CH2), 38.28 (CH), 32.36 (CH2), 31.65 (CH2), 28.72 (CH2), 28.07 (CH2), 25.77 (CH2), 25.45 (CH2), 23.03 (CH2), 14.90 (CH3), 14.10 (CH3), 10.68 (CH3); IR  2958, 2926, 2860, 1738, 1543, 1458, 1416, 1378, 1238, 1167, 1106, 1035, 915, 847, 786. 4 and filtered. Dichloromethane and pyridine were evaporated in vacuo and the mixture weighed. The composition of the product was determined by gas-chromatography and the yield calculated. The product was further purified via distillation under reduced pressure. A mixture of 2-furanmethanethiol(or 2-methyl-3-furanthiol) (0.1 mol), pyridine (0.1 mol) and dichloromethane (40 mL) was prepared and to this was added 4-ethyloctanoyl chloride (0.05 mol) at 30 ºC. The resulting solution was refluxed for 6 hours at 45 ºC. At the end of this period, the mixture was filtered to remove solids, and the filtrate was washed with saturated NaCl aqueous solution. The organic phase was dried with anhydrous NaSO2-Furanmethanethiol 4-ethyloctanoate (3d). Yield 95%; b.p. 142 ºC/0.3 kPa; refractive index (nD20) 1.5005; MS (EI), m/z, 268.1500 (M+), formula C15H24O2S, ; 1H-NMR  7.31 , 6.27 , 6.19-6.20 , 3.03-3.08 , 4.13 , 2.51-2.56 , 1.63 , 1.23-1.28 , 0.81-0.90 ; 13C-NMR  198.54 (C=O), 150.61 (=C-O), 142.13 (=CH-O), 110.53 (CH=), 107.82 (CH=), 41.41 (CH2), 38.24 (CH), 32.42 (CH2), 28.73 (CH2), 25.63 (CH2), 25.55 (CH2), 25.51 (CH2), 23.03 (CH2), 14.10 (CH3), 10.68 (CH3); IR  2958, 2927, 2859, 1694, 1596, 1502, 1460, 1380, 1246, 1151, 1071, 1010, 935, 885, 807, 735, 597. 2-Methyl-3-furanthiol 4-ethyloctanoate (3e). Yield 86%; b.p. 134 ºC/0.2 kPa; Refractive index (nD20) 1.4960; MS (EI), m/z, 268.1499 (M+), formula C15H24O2S, ; 1H-NMR  7.35-7.36 , 6.31-6.32 , 2.59-2.62 , 2.26 , 1.63-1.70 , 1.26-1.32 , 0.84-0.92 ; 13C-NMR  197.49 (C=O), 155.92 (=C-O), 140.88 (=CH‑O), 114.78 (CH=), 104.30 (S-C), 40.70 (CH2), 38.25 (CH), 32.41 (CH2), 28.71 (CH2), 28.68 (CH2), 25.50 (CH2), 22.95 (CH2), 13.99 (CH3), 11.78 (CH3), 10.62 (CH3); IR  2959, 2926, 2859, 1715, 1588, 1514, 1387, 1228, 1129, 1090, 1020, 940, 889, 728, 648, 603, 525. These compounds were purified by column chromatography. Odor assessments were carried out by using a Agilent 6890 GC coupled with a Sniffer 9000 system. Separations were achieved with a HP-5 capillary column, 30 m × 0.250 mm; with a programmed temperature ramp,  detector (FID) temperature at 300 ºC. By one ‘Y’ shape glass splitter, the column effluent was divided (1:1) between the flame ionization detector (FID) and the olfactometer (Sniffer 9000). The effluent to the odor port was enclosed with a stream of humidified air of 6 mL/min and transferred to the glass detection cone by one long capillary at the temperature of 280 ºC. Five trained assessors were selected for smelling and recording the odor characteristics .1H-NMR, 13C-NMR and IR, and odor evaluation indicated they all have meaty odors. This research will be helpful to develop new flavor compounds based on organic acid and sulfur-containing alcohols (or mercaptans). We report the synthesis and odor evaluation of sulfur-containing esters derived from 4-ethyl-octanoic acid. The structures of five new flavor compounds were characterized by MS,


For most of the investigated compounds, the inhibition of Aβ40 aggregation (expressed as pIC50) was found to be correlated with lipophilicity, as assessed by a reversed-phase HPLC method, through a bilinear relationship. The N1-cyclopropyl derivative 28 was tested in cell-based assays of Aβ42 oligomer toxicity and oxidative stress induced by hydrogen peroxide, showing significant cytoprotective effects. This study confirmed the versatility of isatin in preparing multitarget small molecules affecting different biochemical pathways involved in AD.Despite the controversial outcomes of clinical trials executed so far, the prevention of β-amyloid (Aβ) deposition and neurotoxicity by small molecule inhibitors of Aβ aggregation remains a target intensively pursued in the search of effective drugs for treating Alzheimer’s disease (AD) and related neurodegeneration syndromes. As a continuation of previous studies, a series of new 3-(2-arylhydrazono)indolin-2-one derivatives was synthesized and assayed, investigating the effects of substitutions on both the indole core and arylhydrazone moiety. Compared with the reference compound  Alzheimer’s disease (AD) is the most common cause of age-related neurodegenerative pathologies. AD represents a serious challenge for health systems, physicians and caregivers, because of its disabling course and the limited efficacy of pharmacological therapies . The tre40) or 42-mer (Aβ42). Aβ monomers in AD brains aggregate to soluble oligomers, that in turn lead to intermediate protofibrils and finally to the deposition of amyloid plaques    1,  as a hit6.25 µM) .iPr substituent with phenyl groups bearing polar and apolar electron-withdrawing substituents , which resulted however 25-fold less potent than compound 1.An early objective of the present study was to replace the 4-7) resulted in a much lower activity (IC50 90 μM), whereas the 1-naphthyl moiety in compound 8 did recover a fair potency (IC50 28 µM), indicating that additional aromatic interactions may improve the antiaggregating potency. In contrast, quinolyl analogues 9 and 10, (similarly to the pyrid-2-yl congener 7), showed lower potency, and the 7-chloroquinolin-4-yl derivative 11 resulted a very weak inhibitor.The replacement of the hydrazone phenyl ring with a number of other aromatic or heteroaromatic rings was carried out for investigating the effects of bulkiness and/or additional π-π interactions on the anti-aggregating potency. The bioisosteric replacement of the phenyl group with pyrid-2-yl , apparently due to additional hydrophobic/aromatic interactions attained by the phenyl group at the C4 position of the thiazole ring.Further information on the bioisosteric replacement of phenylhydrazone ring was achieved with thiazolylhydrazones 13–18 . An expl16–18, regardless the physicochemical feature of the meta substituent, retained antiaggregating activity in the micromolar range, but resulted 8-to-30-fold less potent than 15, thereby suggesting critical steric requirements for these derivatives. In contrast, smaller alkyl substituents, namely methyl or chloromethyl in compounds 13 and 14, respectively, displayed contrasting effects, with the 4-chloromethyl derivative 13 retaining a fair anti-aggregating potency (IC50 13 µM), and 4,5-dimethyl analogue resulting a very weak inhibitor.The 3′-substituted congeners of 4-phenylthiazol-2-yl derivatives According to our investigation strategy , the fol1 revealed a 100-fold decrease of potency (from 0.43 to 41 µM), when linker was changed in the 1H-indole derivative 19, likely due to the lack of intramolecular hydrogen bond (IMHB) between the hydrazone NH and the carbonyl O at position 2 of isatin, which favors co-planarity between the two aromatic moieties. The inhibitory activity was maintained in the low micromolar range by the aza-derivatives 21 (IC50 6.7 µM), that is a strict analog of 1, and 22 (IC50 11 µM), that is the 3-chloro congener of 21, whereas a sharp drop of activity was observed for the carboxyhydrazide derivative 23. The antiaggregating potency of compounds 21–23 appears to be related to their lipophilicity, as assessed by logP values calculated with the ACDLab software .A comparison of the data in 1 were then investigated , N-cyclopropyl (28) and N-phenylalkyl (32–34) derivatives resulted almost equipotent with the hit compound 1 in the submicromolar range (IC50s ranging from 0.53 to 0.83 µM). A nonlinear relation between the antifibrillogenic potency and lipophilicity of the N-alkyl groups exists for these compounds, the N-benzyl derivative 32 being the most potent one within the subset (IC50 0.53 µM). In contrast, methylation of the hydrazone nitrogen (24) resulted in a more than 10-fold decrease in the inhibition potency compared with the respective non-methylated compound (26), which proves that N-methylation of hydrazone N may prevent IMHB formation with carbonyl O at 2-position 1H-NMR sp 1H-NMR sp 1H-NMR sp−. IR (KBr): 3359, 3218, 2217, 1686, 1606, 1571, 1515, 1488, 1151 cm−1. Mp 246–249 °C from isopropanol.(Z)-5-Methoxy-3-(2-(4-(trifluoromethyl)phenyl)hydrazono)indolin-2-one (5). Yield: 50%. 1H-NMR δ 3.76 , 6.80–6.87 , 7.15 , 7.60 , 7.68 , 10.90 , 12.80 . ESI-MS m/z = 333.8 (100%) [M − H]−. IR (KBr): 3166, 1684, 1563, 1487, 12887, 1161 cm−1. Mp 228–230 °C from methanol.(Z)-3-phenyl)hydrazono)-5-methoxyindolin-2-one (6). Yield: 84%. 1H-NMR δ 3.77 , 6.82 , 6.87 , 7.24 , 7.61 , 8.12–8.14 , 10.89 , 12.86 . ESI-MS m/z = 401.8 (100%) [M − H]−. HRMS (ESI) m/z [M − H]− calcd for C17H10F6N3O2− 402.0683; found, 402.0685. IR (KBr): 3164, 1684, 1564, 1487, 1374, 1279, 1163 cm−1. Mp 240–242 °C (dec.) from methanol.(Z)-5-Methoxy-3-(2-(pyridin-2-yl)hydrazono)indolin-2-one (7). Yield: 80%. 1H-NMR δ 3.78 , 6.76 , 6.87 , 7.02 , 7.43–7.45 , 7.77–7.80 , 8.27 , 10.41 , 11.07 . ESI-MS, m/z = 266.8 (100%) [M − H]−, 291.0 (100%) [M + Na]+. IR (KBr): 1715, 1691, 1570, 1484, 1269, 1197 cm−1. Mp > 250 °C from methanol.(Z)-5-Methoxy-3-(2-(quinolin-2-yl)hydrazono)indolin-2-one (9). Yield: 94%. 1H-NMR δ 3.76 , 6.73 , 6.80–6.88 , 7.23–7.29 , 7.44–7.76 , 7.80–8.10 , 10.24 , 12.00 . ESI-MS m/z = 316.8 (100%) [M − H]−, 341.0 (100%) [M + Na]+. IR (KBr): 3430, 2919, 1688, 1634, 1476, 1302, 1186 cm−1. Mp 227–234 °C from ethanol.(Z)-3-(2-(7-Chloroquinolin-4-yl)hydrazono)-5-methoxy-indolin-2-one (11). Yield: 82%. 1H-NMR δ 3.79 , 6.60–6.64 , 6.80–6.90 , 7.25–7.26 , 7.68–7.76 , 8.03–8.12 , 8.30 , 9.67 , 11.20 . ESI-MS m/z = 350.8 (100%) [M − H]−, 352.8 (34%) [M − H]− + 2, 353.0 (39%) [M + H]+, 355.0 (14%) [M + H]+ + 2. IR (KBr): 3403, 3166, 1703, 1630, 1381, 1185 cm−1. Mp > 250 °C from ethanol.8 and 10 were prepared from azo coupling of aryldiazonium salts 3a,b with 5-methoxyindolin-2-one 2, previously described −. IR (KBr): 3014, 1672, 1562, 1484, 1194, 1156 cm−1. Mp > 250 °C from hexane/ethyl acetate 70:30 (v/v).(Z)-5-Methoxy-3-(2-(quinolin-8-yl)hydrazono)indolin-2-one (10). Yield: 50%. 1H-NMR δ 3.78 , 6.84–6.85 , 7.21–7.22 , 7.61–7.67 , 7.96 , 8.39 , 8.91–8.93 , 10.88 , 13.92 . ESI-MS m/z = 341.0 (64%) [M + Na]+, 316.8 [M − H]–. IR (KBr): 3435, 1675, 1649, 1522, 1194 cm−1. Mp > 250 °C from dichloromethane/methanol 95:05 (v/v).13–18 were prepared adding to a solution of 12 −, 322.7 (48%) [M − H]− + 2. IR (KBr): 3436, 2946, 1683, 1556, 1476, 1168 cm−1. Mp 153–155 °C (dec.) from chloroform/methanol 96:04 (v/v).(Z)-3-hydrazono)-5-methoxyindolin-2-one (14). Yield: 40%. 1H-NMR δ 2.12 , 2.25 , 3.76 , 6.85 , 6.89 , 7.00 , 10.98 , 13.19 . ESI-MS m/z = 300.8 (100%) [M − H]−, 325.0 (94%) [M + Na]+. IR (KBr): 3446, 1686, 1544, 1486, 1162 cm−1. Mp 160–164 °C from ethanol.(Z)-3-(2-(4-(3-Hydroxyphenyl)thiazol-2-yl)hydrazono)-5-methoxyindolin-2-one (16). Yield: 44%. 1H-NMR δ 3.84 , 6.69–6.72 , 6.86 , 6.91 , 7.06 , 7.19 , 7.29–7.31  7.53 , 9.47 , 11.06 , 13.41 . ESI-MS m/z = 364.8 (100%) [M − H]−. HRMS (ESI) m/z [M + H]+ calcd for C18H15N4O3S+ 367.0859; found, 367.0833. IR (KBr): 3431, 1551, 1523, 1163 cm−1. Mp > 250 °C from ethanol.(Z)-5-Methoxy-3-(2-(4-(3-methoxyphenyl)thiazol-2-yl)hydrazono)indolin-2-one (17). Yield: 91%. 1H-NMR δ 3.78 , 3.80 , 6.87–6.93 , 7.06 , 7.32 , 7.43–7.48 , 7.66 , 11.07 , 13.39 . ESI-MS m/z = 378.8 (100%) [M − H]−. IR (KBr): 3171, 2926, 1679, 1551, 1171 cm−1. Mp > 250 °C from ethanol.(Z)-3-(2-(4-(3-Bromophenyl)thiazol-2-yl)hydrazono)-5-methoxyindolin-2-one (18). Yield: 30%. 1H-NMR δ 3.77 , 6.87 , 6.92 , 7.06 , 7.37 , 7.49–7.53 , 7.78 , 7.89–7.91 , 8.07 , 11.07 , 13.40 . ESI-MS m/z = 426.8 (99%) [M − H]−, 428.7 (100%) [M − H]− + 2. IR (KBr): 1683, 1552, 1483, 1155, 718 cm−1. Mp 240–242 °C (dec.) from dichloromethane/methanol 92:8 (v/v).4-isopropylphenylhydrazine hydrochloride (1.8 mmol) was added to a solution of 5-methoxy-indole-3-carbaldehyde (1 mmol) in methanol (5 mL). After stirring overnight at room temperature, the resulting precipitate was filtered and crystallized.(E)-3-(2(4-Isopropylphenyl)hydrazonomethyl))-5-methoxy-1H-indole (19). Yield: 30%; 1H-NMR δ 1.23 2CH), 2.97 2), 3.82 , 6.80 , 6.94 , 7.07 , 7.28 , 7.54 , 7.78 , 8.05 , 9.68 , 11.14 . ESI-MS m/z = 305.9 (100%) [M − H]−. IR (KBr): 3340, 1485, 1296, 1196 cm−1. Mp 175–177 °C from ethanol.20 −, 344.0 (100%) [M + Na]+. HRMS (ESI) m/z [M + H]+ calcd for C19H20N3O2+ 322.1537; found, 322.1550. IR (KBr): 3390, 2956, 1733, 1485, 1296, 1196 cm−1. Mp 223–225 °C from chloroform/methanol 95:5 (v/v).(Z)-3-((E)-(3-chlorobenzylidene)hydrazono)-5-methoxyindolin-2-one (22). Yield: 30%. 1H-NMR δ 3.78 , 6.86 , 6.95 , 7.15 , 7.64 , 7.70–7.75 , 7.80–7.87 , 7.90–7.93 , 8.70 , 10.20 . ESI-MS m/z = 313.05 (100%) [M − H]−, 315.04 (34%) [M − H]− + 2. IR (KBr): 3465, 1745, 1626, 1487 cm−1. Mp 230–235 °C from ethyl acetate/hexane 60:40 (v/v).5-Methoxyisatin (1 mmol) was dissolved in methanol (5 mL), then 3-chloro-benzohydrazide (2 mmol) was added at room temperature and, after stirring overnight, the reaction mixture was evaporated to dryness and the residue purified by column chromatography.3-Chloro-N’-(5-methoxy-2-oxoindolin-3-ylidene)benzohydrazide (23). Yield: 40%. 1H-NMR δ 3.77 , 6.87 , 6.97 , 7.12 , 7.64 , 7.74–7.77 , 7.80–7.83 , 7.87–7.89 , 11.26 , 13.83 . ESI-MS m/z = 351.9 (45%) [M + Na]+, 353.9 (15%) [M + Na]+ + 2. IR (KBr): 3246, 1681, 1540, 1486, 1252, 1152 cm−1. Mp 236–238 °C from dichloromethane/methanol 96:4 (v/v).1 +. IR (KBr): 2924, 1673, 1500, 1278, 1247, 1097 cm−1. Mp 66–69 °C from ethyl acetate/hexane 20:80 (v/v).5-Methoxy-1-phenetylindolin-2,3-dione (30). Yield: 60%. 1H-NMR δ 2.88 , 3.75 , 3.85 , 7.08–7.27 . ESI-MS m/z = 280.0 (30%) [M − H]−. IR (KBr): 3084, 2964, 1739, 1626, 1491, 1287 cm−1. Mp 122–127 °C.5-Methoxy-1-(3-phenylpropyl)indolin-2,3-dione (31). Yield: 60%. 1H-NMR δ 1.87 , 2.64 , 3.66 , 3.75 , 7.06–7.27 . ESI-MS m/z = 318.0 (100%) [M + Na]+. IR (KBr): 2926, 1722, 1596, 1491 cm−1. Mp 130–137 °C.1-Cyclopropyl-5-methoxyndolin-2,3-dione 27. Yield: 60%. 1H-NMR δ 0.76–0.85 , 0.94–1.04 , 2.66 , 3.75 , 7.09 , 7.14 , 7.26 . ESI-MS m/z = 240.0 (100%) [M + Na]+. IR (KBr): 2962, 2924, 1722, 1490, 1281 cm−1. Mp 121–123 °C.5-Methoxyisatin (2 mmol), cyclopropylboronic acid (4 mmol) and sodium carbonate (4 mmol) were mixed in dichloroethane (3 mL). Then a solution of copper(II) acetate (2 mmol) and bipyridine (2 mmol) in dichloroethane (6.6 mL) at 50 °C was added and the mixture was heated for 4 h. After cooling, a solution of ammonium chloride was added and the resulting aqueous phase was extracted with dichloromethane. 25 −. IR (KBr): 2952, 2872, 1683, 1487, 1246, 1223 cm−1. Mp 101–103 °C from ethanol.(Z)-1-Cyclopropyl-3-(2-(4-isopropylphenyl)hydrazono)-5-methoxyindolin-2-one (28). Yield: 50%. 1H-NMR δ 0.84–0.86 , 1.00–1.02 , 1.18 2CH), 2.77 , 2.85 2), 3.77 , 6.89 , 7.09 , 7.12 , 7.23 , 7.35 , 12.74 . ESI-MS m/z = 347.9 (100%) [M − H]−, 372.1 (82%) [M + Na]+. IR (KBr): 3436, 1668, 1553, 1480, 1248, 1031 cm−1. Mp 106–109 °C from ethanol.(Z)-1-Benzyl-3-(2-(4-isopropylphenyl)hydrazono)-5-methoxyindolin-2-one (32). Yield: 80%. 1H-NMR δ 1.19 2CH), 2.86 2), 3.75 , 4.97 , 6.80 , 6.93 , 7.16 , 7.25 , 7.31–7.33 , 7.40 , 12.74 . ESI-MS m/z = 397.9 (100%) [M − H]−, 422.1 (34%) [M + Na]+. HRMS (ESI) m/z [M + H]+ calcd for C25H26N3O2+ 400.2020; found, 400.2028. IR (KBr): 3436, 2964, 1676, 1487, 1162 cm−1. Mp 105–109 °C from ethanol.(Z)-3-(2-(4-Isopropylphenyl)hydrazono)-5-methoxy-1-phenethylindolin-2-one (33). Yield: 90%. 1H-NMR δ 1.18 2CH), 2.85 2), 2.92 , 3.77 , 3.96 , 6.83 , 7.06 , 7.14 , 7.16–7.28 , 7.36 , 12.68 . ESI-MS m/z = 412.0 (100%) [M − H]−, 436.1 (88%) [M + Na]+. IR (KBr): 3431, 2950, 1674, 1557, 1487, 1249, 1160, 1149 cm−1. Mp 96–98 °C from ethanol.(Z)-3-(2-(4-Isopropylphenyl)hydrazono)-5-methoxy-1-(3-phenylpropyl)indolin-2-one (34). Yield: 70%. 1H-NMR δ 1.18 2CH), 1.92 , 2.62 , 2.85 2), 3.74–3.79 , 3.77 , 6.85 , 7.02 , 7.14–7.28 , 7.36 , 12.73 . ESI-MS m/z = 426.0 (100%) [M − H]−, 450.1 (100%) [M + Na]+. HRMS (ESI) m/z [M + H]+ calcd for C27H30N3O2+ 428.2333; found, 428.2332. IR (KBr): 3435, 2957, 1669, 1559, 1485, 1248, 1158 cm−1. Hygroscopic compound from dichloromethane/hexane 65:35 (v/v).35a–d (1 mmol) (prepared using the Sandmayer method −. IR (KBr): 3298, 2952, 2915, 1683, 1649, 1557, 1513, 1240, 1159 cm−1. Mp 164–166 °C from ethanol.7-Bromo-3-(2-(4-isopropylphenyl)hydrazono)indolin-2-one (37). Yield: 80%. 1H-NMR δ 1.18 2CH), 2.86 2), 6.98 , 7.24 , 7.36–7.41 , 7.52 , 11.26 , 12.78 . ESI-MS m/z = 355.9 (95%) [M − H]−, 357.7 (100%) [M − H]− + 2. HRMS (ESI) m/z [M + Na]+ calcd for C17H16BrN3NaO+ 380.0369; found, 380.0378. IR (KBr): 3154, 2959, 1672, 1551, 1170 cm−1. Mp 240–242 °C from ethanol.Sodium 3-(2-(4-isopropylphenyl)hydrazono)-2-oxoindolin-5-sulfonate (38). Yield: 60%. 1H-NMR δ 1.18 2CH), 2.85 2), 6.83 , 7.22 , 7.35 , 7.48 , 7.73 , 11.04 , 12.69 . ESI-MS m/z = 357.8 (100%) [M − Na]−. IR (KBr): 3201, 2959, 1560, 1189 cm−1. Mp > 250 °C from ethanol.(Z)-7-(2-(4-isopropylphenyl)hydrazono)-5H-dioxoloindol-6(7H)-one (39). Yield: 50%. 1H-NMR δ 1.17 2CH), 2.84 2), 5.98 , 6.55 , 7.07 , 7.20 , 7.29 , 10.79 , 12.59 . ESI-MS m/z = 321.9 (100%) [M − H]−. IR (KBr): 3177, 1675, 1557, 1464, 1156 cm−1. Mp 229–234 °C ethyl acetate/hexane 50:50 (v/v).The lipophilicity parameters were determined by an RP-HPLC technique ,35. Methk′ = log (tR − t0)/t0) of each compound at different mobile phase compositions have been calculated; tR represents the retention time of the solute and t0 is the column dead time, measured as the elution time of a KNO3 solution in MeOH. For each compound, the log k′ values increased linearly with decreasing MeOH volume fraction. Logarithms of capacity factors extrapolated to the 100% aqueous mobile phase (log wk′) were calculated from the linear regressions on at least five data points (r2 > 0.975).The logarithm of capacity factors , and BioLoom software, vers. 1.7 . The SAR equation (1) has been obtained by using the Prism software .The spectrofluorimetric assays, measuring ThT fluorescence in the presence of Aβ, were done as previously described . Briefly42-induced neurotoxicity and from ROS generation was assessed in SH-SY5Y cells as already described [n ≥ 18) were carried out, and the results were averaged. Briefly, for MTT assay, SH-SY5Y cells were cultured in DMEM-Dulbecco’s modified Eagle’s medium (Sigma-Aldrich), grown to 70% confluence and seeded for experiment in 96-well plates at a density of 10,000 cells/well. Cultured cells were added with a DMEM solution of Aβ42, alone or in mixture with 28  and incubated at 37 °C in 5% CO2. At the time points of 24 and 48 h, the culture medium was replaced by DMEM supplemented with a solution of MTT in PBS . After removal of solution and addition of DMSO, the absorbance of product formazan produced from MTT was measured at 570 nm using a multilabel plate counter Victor3 V , with DMSO medium as the blank solution.Cytoprotection from Aβescribed . At leas28 for 1 h. After washing, DCFH-DA  was added directly to each well, and the plate was incubated at 37 °C in 5% CO2 for 0.5 h. After washing, 100 μM H2O2/well in DMEM was added and the cells were incubated for an additional 30 min. The formation of fluorescent dichlorofluorescein (DCF) due to oxidation of DCFH in the presence of ROS was read directly in each well at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a multilabel plate reader Victor3 V (Perkin Elmer), with DMSO medium as the control.Intracellular ROS production was evaluated using an oxidation-sensitive fluorescent probe, 2′,7′-dichlorodihydrofluorescin diacetate . Viable SHSY5Y cells were seeded in a black 96-well cell culture plate for 24 h, then the cells were incubated in DMEM supplemented with different concentration (0 to 20 μM) of 1 [15 and N1-substituted indolin-2-ones 26, 28, 32–34) were found almost equipotent in vitro with the hit compound 1. Among them, compound 28, i.e., the N1-cyclopropyl derivative of 1, emerged from cell-based assays as a multitarget agent with strong cytoprotective effects in both Aβ42 oligomer toxicity and oxidative stress. These results were in full agreement with those already reported for congeners of 28, particularly for cytoprotection from amyloid insult that resulted well comparable with reference compound 1 [50 and log k’w determined by RP-HPLC was derived which hold for those compounds that do not undergo detrimental steric effects in binding Aβ peptides.The SAR studies on the indoline-2-one 3-arylhydrazone derivatives reported herein warranted further gain of knowledge in our ongoing study of inhibition of Aβ aggregation. Rooting on previous results ,20,21 an1  as a hitmpound 1 . Aβ anti28 could deserve for this compound a preliminary pharmacological investigation in animal models of Aβ-dependent neurodegeneration, provided that further efforts in ameliorating aqueous solubility would be beneficial. The potency and versatility of indolin-2-one derivatives may still encounter the interest of research and development of small molecules as pharmacological tools for treating AD and related neurodegenerative syndromes.The present work adds new information to the potential of indolin-2-one 3-arylhydrazones as privileged structures for the inhibition of Aβ aggregation and neurotoxicity, and pleiotropic agents for AD therapy. Despite the claims of disengagement of pharmaceutical industry from AD research , because


The aminodi(hetero)arylamines thus obtained were evaluated for their growth inhibitory effect on four human tumor cell lines MCF-7 (breast adenocarcinoma), A375-C5 (melanoma), NCI-H460  and HepG2 (hepatocellular carcinoma). The toxicity to non-tumor cells was also evaluated using a porcine liver primary cell culture (PLP1), established by us. The aminodi(hetero)arylamine with the NH2 group in the ortho position and an OMe group in the para position to the NH of the di(hetero)arylamine, is the most promising compound giving the lowest GI50 values (1.30–1.63 µM) in all the tested human tumor cell lines, presenting no toxicity to PLP1 at those concentrations. The effect of this compound on the cell cycle and induction of apoptosis was analyzed in the NCI-H460 cell line. It was observed that it altered the cell cycle profile causing a decrease in the percentage of cells in the G0/G1 phase and an increase of the apoptosis levels. Three aminodi(hetero)arylamines were prepared  Despite the great amount of research and rapid development of new therapies seen during the past decade, cancer continues to be a leading cause of death worldwide. It is estimated that by 2020 approximately 15 million new cancer cases will be diagnosed and 12 million patients will die every year [in vitro tumoral cell line assays [b]pyridine-2-carboxylate derivatives functionalized at the pyridine ring by Pd-catalyzed C-N  and C-C (Suzuki and Sonogashira) couplings and some of them presented cell growth inhibitory activity in tumor cell lines [b]pyridine derivatives obtained by C-N coupling the most promising compounds bear a benzothiazole (GI50 3.5–6.4 µM) or an indole (GI50 15.8–18.1 µM) moiety pyridine series functionalized at the thiophene ring were synthesized from the corresponding nitro compounds. The aminodi(hetero)arylamines were evaluated for their in vitro growth inhibitory effect on four human tumor cell lines  and also on the non-tumor primary cells PLP1 (porcine liver primary cell culture) established by us. For the most active and less toxic compound, its effects on cell cycle and induction of apoptosis were evaluated using the NCI-H460 cell line.In the present work, three aminodi(hetero)arylamines in the thienopyridine-2-carboxylate (1) pyridine-2-carboxylate (1), and the mixture was heated under Ar at 120 °C for 2 h. After cooling water (5 mL) and ethyl acetate (5 mL) were added. The phases were separated and the aqueous phase was extracted with more ethyl acetate (2 × 5 mL). The organic phase was dried (MgSO4) and filtered. Removal of the solvent gave a brown solid which was submitted to dry flash using ether and a solid was obtained.A dry Schlenk tube was charged under Ar with dry dioxane (3 mL), the bromonitrobenzene (1.1 equiv.), Pd(OAc)Methyl 3-(4-nitrophenylamino)thienopyridine-2-carboxylate (2a): From thienopyridine 1  and 1-bromo-4-nitrobenzene , compound 2a was isolated as a brownish solid , m.p. 203–205 °C. 1H-NMR : δ = 3.96 , 7.08 , 7.43 , 8.13 , 8.19 , 8.67 , 8.76  ppm. 13C-NMR : δ = 52.5 (OMe), 113.8 (C-2), 118.2 (2′ and 6′-CH), 122.1 (6-CH), 124.9 (3′ and 5′-CH), 131.5 (7-CH), 134.2 (C), 141.7 (C), 141.8 (C), 147.2 (5-CH), 147.6 (C-3), 148.0 (C-1’), 164.5 (C=O) ppm. HRMS (EI) Calcd. for C15H11N3O4S [M+] 329.0470; found 329.0479. Methyl 3-(2-nitrophenylamino)thienopyridine-2-carboxylate (2b): From thienopyridine 1  and 1-bromo-2-nitrobenzene , compound 2b was isolated as a reddish solid , m.p. 202–204 °C. 1H-NMR : δ = 3.97 , 6.93–7.00 , 7.32–7.37 , 7.42 , 8.19-8.22 , 8.67 , 10.59  ppm. 13C-NMR : δ = 52.7 (OMe), 118.6 (C-2), 119.9 (5′-CH), 120.1 (6′-CH), 121.8 (6-CH), 125.9 (3′-CH), 131.4 (7-CH), 133.9 (4′-CH), 134.00 (C-1′), 135.9 (C), 139.2 (C), 139.3 (C), 147.4 (5-CH), 148.3 (C), 163.3 (C=O) ppm. HRMS (EI) Calcd. for C15H11N3O4S [M+] 329.0470; found 329.0478. Methyl 3-(4-methoxy-2-nitrophenylamino)thienopyridine-2-carboxylate (2c): From thieno-pyridine 1  and 4-bromo-3-nitroanisole , compound 2c was isolated as a reddish solid , m.p. 213–214 °C. 1H-NMR : δ = 3.82 , 3.87 , 7.02 , 7.14 , 7.53–7.58 , 8.55 , 8.60 , 9.72  ppm. 13C-NMR : δ = 52.5 (OMe), 55.8 (OMe), 107.2 (3′-CH), 114.3 (C-2′), 122.5 (6-CH), 122.9 (5′-CH), 123.5 (6′-CH), 132.1 (C), 132.4 (7-CH), 133.5 (C), 136.8 (C-1′), 139.9 (C), 147.1 (C), 147.4 (5-CH), 153.2 (C-2), 163.3 (C=O) ppm. HRMS (EI) Calcd. for C16H13N3O5S [M+] 359.0576; found 359.0559. 2O (3:1:0.5), compounds 2a–c, NH4Cl (1 equiv.), Fe (8 equiv.) were heated at 100 °C for 2 h. After cooling the mixture was evaporated to give a dark solid. This was submitted to dry-flash using ether and the product was obtained as a yellow or greenish solid.In a round bottom flask with 4.5 mL EtOH/THF/HMethyl 3-(4-aminophenylamino)thienopyridine-2-carboxylate (3a): From nitrodiarylamine 2a  compound 3a was isolated as a greenish solid , m.p. 200–202 °C. 1H-NMR : δ = 3.77 , 4.84 , 6.45 , 6.77 , 7.46 , 8.39 , 8.51 , 8.60  ppm. 13C-NMR : δ = 51.9 (OMe), 103.6 (C), 113.7 (3′ and 5′-CH), 122.3 (6-CH), 123.0 (2′ and 6′-CH), 130.9 (C), 131.8 (7-CH), 133.7 (C), 144.9 (C-4′), 145.3 (C), 146.4 (5-CH), 147.2 (C), 164.4 (C=O) ppm. HRMS (EI) Calcd. for C15H13N3O2S [M+] 299.0728; found 299.0732.Methyl 3-(2-aminophenylamino)thienopyridine-2-carboxylate (3b): From nitrodiarylamine 2b  compound 3b was isolated as a greenish solid , m.p. 204–206 °C. 1H-NMR : δ = 3.79 , 4.97 , 6.39 , 6.63–6.83 , 7.48 , 8.12 , 8.42–8.52  ppm. 13C-NMR : δ = 52.0 (OMe), 105.9 (C-2), 114.9 (CH), 115.9 (CH), 122.2 (CH), 122.3 (6-CH), 124.1 (CH), 127.9 (C), 131.8 (7-CH), 133.6 (C), 141.2 (C), 145.2 (C), 146.7 (5-CH), 147.2 (C), 164.2 (C=O) ppm. HRMS (EI) Calcd. for C15H13N3O2S [M+] 299.0728; found 299.0726.Methyl 3-(2-amino-4-methoxhyphenylamino)thienopyridine-2-carboxylate (3c): From nitro-diarylamine 2c  compound 3c was isolated as a greenish solid , m.p. 172–174 °C. 1H-NMR : δ = 3.65 , 3.79 , 5.00 , 5.99 , 6.31 , 6.61 , 7.43 , 8.09 , 8.38 , 8.47  ppm. 13C-NMR : δ = 51.9 (OMe), 54.8 (OMe), 100.0 (3′-CH), 101.2 (5′-CH), 103.4 (C), 120.9 (C), 122.2 (6-CH), 124.9 (6′-CH), 131.7 (7-CH), 133.68 (C), 143.7 (C), 146.5 (C), 146.5 (5-CH), 147.0 (C), 157.2 (C-4’), 164.6 (C=O) ppm. HRMS (EI) Calcd. for C16H15N3O3S [M+] 329.0834; found 329.0827.L-glutamine, hank’s balanced salt solution (HBSS), trypsin-EDTA (ethylenediaminetetraacetic acid), penincillin/streptomycin solution  and RNAse A were from Gibco Invitrogen Co. . RPMI-1640 medium was from Cambrex  and DMEM medium was from Hyclone . Acetic acid, ellipticine, doxorubicin, sulforhodamine B (SRB), trypan blue, trichloroacetic acid (TCA), Tris and propidium iodide were from Sigma Chemical Co. . Water was treated in a Mili-Q water purification system .DMSO (dimethyl sulfoxide) was analytical grade from Fisher Scientific . Fetal bovine serum (FBS), Stock solutions of the tested aminodiarylamines were prepared in DMSO and kept at −20 °C. Appropriate dilutions were prepared in the corresponding solvent prior to the assays. The effect of DMSO on the growth of the cell lines was evaluated by treating cells with the maximum concentration of DMSO used in the assays (0.25%). No influence was found (data not shown).2 cells), at 37 °C in a humidified atmosphere containing 5% CO2.Four human tumor cell lines, MCF-7 (breast adenocarcinoma), A375-C5 (melanoma), NCI-H460  and HepG2 (hepatocellular carcinoma) were used. MCF-7, A375-C5 and HepG2 were obtained from the European Collection of Cell Cultures  and NCI-H460 was kindly provided by the National Cancer Institute . Cells were routinely maintained as adherent cell cultures in RPMI-1640 medium supplemented with 5% FBS  or in DMEM supplemented with 10% FBS, 2 mM glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin  was prepared from freshly harvested porcine liver, obtained from a local slaughter house. Briefly, the liver tissue was rinsed in HBSS solution containing 100 U/mL penicillin, 100 µg/mL streptomycin and divided into 1 × 1 mmly by us .in vitro growth of human tumor cell lines and non-tumor porcine liver primary cells were evaluated according to the procedure adopted by the NCI (USA) in their “In vitro Anticancer Drug Discovery Screen”, using the SRB assay to assess cell growth [The effects of the compounds on the l growth ,6,7.5 cells/mL for MCF-7, 0.75 × 105 cells/mL for A375-C5 and NCI-H460, and 5.0 × 105 cells/mL for HepG2, followed by 24 h of incubation. For the primary porcine liver cells, cells were incubated in 96-well plates at a density of 5.0 × 105 cells/mL, and cultivated in DMEM medium with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. For the cell lines, exponentially growing cells were obtained by plating 1.5 × 10in situ, washed and stained with SRB. The bound stain was solubilized and the absorbance was measured at 492 nm in the case of MCF-7, NCI-H460 and A375-C5 cells and at 515 nm in the case od HepG2 and PLP1 primary cells, in a plate reader . Dose response curves were obtained and the concentration of the diarylamine that inhibited 50% of the net cell growth (GI50) was calculated as described elsewhere [Following a 24 h period to allow cells to adhere, cells were treated for 48 h with six serial dilutions of each test compound, starting from a maximum concentration of 125 µM. Following the 48 h treatment period, adherent cells were fixed 5 cells/well and incubated at 37 °C for 24 h. Cells were then treated with compound 3c at its GI50 concentration (1.4 µM). As controls, cells were also incubated with medium only (blank) and with the compound’s vehicle (DMSO). Following 48 h of treatment, cells were fixed overnight in ice-cold 70% ethanol and re-suspended in PBS containing 0.1 mg/mL RNase A and 5 µg/mL propidium iodide. Cellular DNA content was analysed by flow cytometry using an Epics XL-MCL Coulter Flow cytometer  plotting at least 20,000 events per sample, as previously described [NCI-H460 cells were plated in 6-well plates at a final density of 1.5 × 103c, as described above, the levels of apoptosis were analysed by flow cytometry using the Human Annexin V-FITC/PI apoptosis kit , according to the manufacturer’s instructions. Flow cytometry was carried out using an Epics XL-MCL Coulter flow cytometer plotting at least 20,000 events per sample, as previously described [Following 48 h treatment of NCI-H460 cells with compound escribed . The dat3c, with the NH2 group in ortho and an OMe group in para to the NH of the diarylamines 3a–c presented lower toxicity to non-tumor cells than the positive control, ellipticine (GI50 = 4 µM). Moreover, compound 3c caused a change in the cell cycle profile of the NCI-H460 cells, leading to a decrease in the percentage of cells in the G0/G1 phase of the cell cycle and caused an increase in the percentage of NCI-H460 apoptotic cells.From the three aminodi(hetero)arylamines synthesized, compound


H-pyrazol-4-yl)-1-(thiophen-2-yl)prop-2-en-1-ones, the thiophene unit is disordered over two sets of atomic sites and a combination of C—H⋯N and C—H⋯O hydrogen bonds link the molecules into sheets.In each of two isostructural 3-(5-aryloxy-3-methyl-1-phenyl-1 H-pyrazol-4-yl)-1-(thiophen-2-yl)prop-2-en-1-one, C23H18N2O2S (I), and 3-[3-methyl-5-(2-methylphenoxy)-1-phenyl-1H-pyrazol-4-yl]-1-(thiophen-2-yl)prop-2-en-1-one, C24H20N2O2S (II), are isomorphous as well as isostructural, and in each the thiophene substituent is disordered over two sets of atomic sites having occupancies 0.844 (3) and 0.156 (3) in (I), and 0.883 (2) and 0.117 (2) in (II). In each structure, the molecules are linked into sheets by a combination of C—H⋯N and C—H⋯O hydrogen bonds. Comparisons are made with some related compounds.Two new chalcones containing both pyrazole and thiophene substituents have been prepared and structurally characterized. 3-(3-Methyl-5-phenoxy-1-phenyl-1 The chaet al., 2003et al., 2009et al., 2012In view of the similarities in the hydrogen bonds formed by (I)Z′ = 2 in space group P21/m   chains by means of C—H⋯N hydrogen bonds. The molecules of 2--N′-(thiophen-2-ylmethylidene)acetohydrazide (V) -ethyl 2-cyano-2-{2-pyridin-3-yl]hydrazinylidene}acetate (VI) H-pyrazoloquinolin-7(6H)-one ethanol monosolvate (VII)  methanone (IX) -8,9-dihydro-3H-pyrazole (A)  under Vilsmeier–Haack conditions, followed by nucleophilic substitution . Condensation with 2-acetylethiophene then gave the products (I)H-pyrazole 4-carbaldehydes , to give crystals suitable for single-crystal X-ray diffraction. Compound (I)−1) 1667 (C=O), 1591 (C=N). Analysis: found C 71.5, H 4.7, N 7.2%: C23H18N2O2S requires C 71.5, H 4.7, N 7.3%. Compound (II)−1) 1671 (C=O), 1564 (C=N). Analysis: found C 72.0, H 5.1, N 7.1%: C24H20N2O2S requires C 72.0, H 5.0, N 7.0%.Compounds (I)Uiso(H) = kUeq(C), where k = 1.5 for the methyl groups, which were permitted to rotate but not to tilt, and 1.2 for all other H atoms. The H atoms in the minor disorder components were included on the same basis. Subject to these conditions, the occupancies of the disorder components refined to 0.844 (3) and 0.156 (3) in (I)Crystal data, data collection and structure refinement details are summarized In Table 310.1107/S205698901901658X/zl2765sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S205698901901658X/zl2765Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S205698901901658X/zl2765IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S205698901901658X/zl2765Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S205698901901658X/zl2765IIsup5.cmlSupporting information file. DOI: 1970925, 1970924CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: