In this article, multiple doses of compound 1a  were used for the treatment of mice infected with influenza A/FM/1/47-MA (H1N1), and surprisingly we found that compound 1a significantly increased the number of survivors and prolonged the mean survival time. The preliminary studies on the mechanism of antiviral activity showed no interaction between compound 1a and the neuraminidase or the M2 protein. The novel target to overcome drug resistance combined with its good in vivo profile support compound 1a to be a new lead for further development of antiviral agents.A series of substituted aryl glycoside analogues of gastrodin have been identified as potential anti-influenza agents. The most potent inhibitor  Influenza is a respiratory disease caused by the influenza virus. According to the World Health Organization (WHO), as of April 4, 2010, the 2009-H1N1 influenza A virus have spread to over 213 countries and resulted in over 600,000 laboratory-confirmed cases and at least 17,700 deaths . These numbers represent the tip of the iceberg with regard to the potential morbidity and mortality associated with infection resulting from this virus  exhibited significant inhibitory activity against the tested influenza A strains: A/Hanfang/359/95(H3N2) and A/FM/1/47(H1N1), with IC50 values of 44.4 and 34.4 μM, respectively. Mice infected with influenza A/FM/1/47-MA (H1N1) were treated with multiple doses of compound 1a on a dose regimen of 80 mg/kg/day , which led to a significant increase in the number of survivors and a prolonged mean survival time. Herein, we reported the synthesis and biological evaluation of a series of novel gastrodin analogues as new antiviral agents for the first time, along with a discussion of the preliminary structure-activity relationships (SAR) study and the in vivo activity.In the current work, a series of substituted aryl glycosides were synthesized as analogues of gastrodin and their anti-influenza activity was evaluated. The compounds proved to be a novel class of potential anti-influenza agents. The most potent compound +; HRMS-ESI (m/z): calculated for C22H26FO12 [M+H]+: 501.14083; measured: 501.14078.Methyl 2,6-dimethoxy-4--3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yloxy)benzoate (2a). Compound 2a was synthesized using a method similar to that described above for 1a. Yield: 35%, white solid; m.p.: 121–123 °C; 1H-NMR  δ (ppm): 6.50 , 5.28 , 5.15 , 4.22 , 4.09 , 3.89 , 3.85 , 3.69 , 2.04 , 2.01 ; ESI-MS (m/z): 565 [M+Na]+; HRMS-ESI (m/z): calculated for C24H31O14 [M+H]+: 543.17138; measured: 543.17148. Ethyl 3,5-dichloro-4--3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yloxy)benzoate (3a). Compound 3a was synthesized using a method similar to that described above for 1a. Yield: 32%, white solid; m.p.: 112–114 °C; 1H-NMR  δ (ppm): 7.93 , 6.39 , 5.30 , 4.33 , 4.11 , 2.06 , 2.03 , 1.33 ; ESI-MS (m/z): 587 [M+Na]+; HRMS-ESI (m/z): calculated for C22H25Cl2O12 [M+H]+: 551.07231; measured: 551.07226. Methyl 4-amino-3--3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yloxy)benzoate (4a). Compound 4a was synthesized using a method similar to that described above for 1a. Yield: 29%; brown solid; m.p.: 126–128 °C; 1H-NMR  δ (ppm): 7.84 , 7.77 , 7.10 , 5.54 , 5.44 , 5.14 , 5.02 , 4.21 , 4.05 , 3.90 , 3.74 , 2.19 , 2.10 , 2.07 , 2.00 ; ESI-MS (m/z): 520 [M+Na]+; HRMS-ESI (m/z): calculated for C22H28NO12 [M+H]+: 498.16115; measured: 498.16102. Methyl 4--3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yloxy)benzoate (5a). Compound 5a was synthesized using a method similar to that described above for 1a. Yield: 30%; white solid; m.p.: 147–149 °C; 1H-NMR  δ (ppm): 8.00 , 7.00 , 5.53 , 5.27 , 5.14 , 5.02 , 4.25 , 2.08 , 2.05 , 2.04 , 1.38 ; ESI-MS (m/z): 519 [M+Na]+; HRMS-ESI (m/z): calculated for C23H29O12 [M+H]+: 497.16590; measured: 497.16594. Methyl 3-fluoro-4--3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yloxy)benzoate (7a). Compound 7a was synthesized using a method similar to that described above for 1a. Yield: 31%; white solid; m.p.: 121–123 °C; 1H-NMR  δ (ppm): 7.83 , 6.90 , 6.83 , 5.57 , 5.21 , 4.37 , 3.90 , 2.10 , 2.06 ; ESI-MS (m/z): 523 [M+Na]+; HRMS-ESI (m/z): calculated for C22H26FO12 [M+H]+: 501.14083; measured: 501.14091. Ethyl 4-fluoro-2--3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yloxy)-benzoate (8a). Compound 8a was synthesized using a method similar to that described above for 1a. Yield: 30%; white solid; m.p.: 136–138 °C; 1H-NMR  δ (ppm): 7.80 , 6.95 , 6.84 , 5.92 , 5.48 , 5.08 , 4.17 , 3.85 , 2.16 , 2.09 , 2.05 , 2.01 ; ESI-MS (m/z): 523 [M+Na]+; HRMS-ESI (m/z): calculated for C22H26FO12 [M+H]+: 501.14083; measured: 501.14082.Methyl 2-fluoro-6--3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yloxy)benzoate (9a). Compound 9a was synthesized using a method similar to that described above for 1a. Yield: 35%; white solid; m.p.: 130–132 °C; 1H-NMR  δ (ppm): 7.31 , 6.90 , 6.86 , 5.27 , 5.18 , 5.05 , 4.25 , 3.89 , 2.11 , 2.08 , 2.05 , 2.02 ; ESI-MS (m/z): 523 [M+Na]+; HRMS-ESI (m/z): calculated for C22H26FO12 [M+H]+: 501.14083; measured: 501.14077.-2-(Acetoxymethyl)-6-(3-fluoro-2-methylphenoxy)-tetrahydro-2H-pyran-3,4,5-triyl triacetate (10a). Compound 10a was synthesized using a method similar to that described above for 1a. Yield: 33%; white solid; m.p.: 127–129 °C; 1H-NMR  δ (ppm): 7.62 , 7.21 , 6.90 , 6.33 , 5.72 , 5.50 , 4.57 , 4.22 , 2.40 , 2.07 , 2.02 ; ESI-MS (m/z): 479 [M+Na]+; HRMS-ESI (m/z): calculated for C21H26FO10 [M+H]+: 457.15100; measured: 457.15095. -2-(Acetoxymethyl)-6-(pyridin-4-yloxy)-tetrahydro-2H-pyran-3,4,5-triyl triacetate (11a). Compound 11a was synthesized using a method similar to that described above for 1a. Yield: 27%; colorless solid; m.p.: 132–134 °C; 1H-NMR  δ (ppm): 8.30 , 6.93 , 5.50 , 5.18 , 5.05 , 4.15 , 2.08 , 2.04 , 2.02 ; ESI-MS (m/z): 448 [M+Na]+; HRMS-ESI (m/z): calculated for C19H24NO10 [M+H]+: 426.14002; measured: 426.14006. -2-(Acetoxymethyl)-6-(3-fluoropyridin-2-yloxy)-tetrahydro-2H-pyran-3,4,5-triyl triacetate(12a). Compound 12a was synthesized using a method similar to that described above for 1a. Yield: 31%; pale yellow solid; m.p.: 140–142 °C; 1H-NMR  δ (ppm): 7.92 , 7.39 , 6.99 , 6.18 , 5.35 , 5.24 , 4.30 , 4.14 , 3.94 , 2.10 , 2.07 , 2.04 , 2.02 ; ESI-MS (m/z): 466 [M+Na]+; HRMS-ESI (m/z): calculated for C19H23FNO10 [M+H]+: 444.13060; measured: 444.13051. -2-(Acetoxymethyl)-6-(5-(acetoxymethyl)-2-fluorophenoxy)-tetrahydro-2H-pyran-3,4,5-triyl triacetate (13a). Compound 1a  was added to THF (25 mL), and solid LiAlH4 was added in a portion-wise manner to the resulting solution. Upon completion of the addition, the mixture was stirred at 66 °C for 3 h before being cooled to ambient temperature and quenched by the dropwise addition of CH3OH (10 mL). The reaction mixture was then stirred at room temperature for 10 min and concentrated to dryness under vacuum. The resulting residue was then dissolved in a mixture of acetic anhydride (20 mL) and pyridine (1 mL). The reaction mixture was stirred at 80 °C for 4 h and then concentrated to dryness in vacuo. The residue was added to ice water and stirred until precipitation occurred. The mixture was then filtered, and the filter-cake was collected and purified by flash chromatography over silica gel, affording the title compound . White solid; m.p.: 125–127 °C; 1H-NMR  δ (ppm): 7.80 , 7.74 , 7.10 , 5.89 , 5.30 , 5.07 , 5.19 , 4.36 , 4.14 , 2.11 , 2.08 , 2.06 , 2.04 ; ESI-MS (m/z): 537 [M+Na]+; HRMS-ESI (m/z): calculated for C23H28FO12 [M+H]+: 515.15648; measured: 515.15652. -2-(2-Fluoro-5-(hydroxymethyl)phenoxy)-6-(hydroxymethyl)-tetrahydro-2H-pyran-3,4,5-triol (14a). To a solution of 13a  in CH3OH (10 mL) was added a 0.2 N solution of CH3ONa in CH3OH, and the resulting mixture was stirred at room temperature for 1 h and then allowed to stand at ambient temperature overnight. The reaction mixture was filtered, and the filter cake was collected and recrystallized from anhydrous ethanol to afford the title compound . White solid; m.p.: 134–136 °C; 1H-NMR  δ (ppm): 7.15 , 6.95 , 6.91 , 5.60 , 4.66 , 4.20 , 3.47 , 2.76 , 2.16 , 1.78 , 0.99 ; ESI-MS (m/z): 327 [M+Na]+; HRMS-ESI (m/z): calculated for C13H18FO7 [M+H]+: 305.10366; measured: 305.10362. Methyl 4-fluoro-3--3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yloxy)benzoate (1b). Compound 1b was synthesized using a method similar to that described above for 1a. Yield: 38%; white solid; m.p.: 143–145 °C; 1H-NMR  δ (ppm): 7.88 , 7.78 , 7.14 , 5.53 , 5.46 , 5.11 , 5.01 , 4.20 , 4.07 , 3.90 , 2.19 , 2.10 , 2.07 , 2.00 ; ESI-MS (m/z): 523 [M+Na]+; HRMS-ESI (m/z): calculated for C22H26FO12 [M+H]+: 501.14083; measured: 501.14086.Methyl 3--3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yloxy)benzoate (6b). Compound 6b was synthesized using a method similar to that described above for 1a. Yield: 36%; white solid; m.p.: 154–156 °C; 1H-NMR  δ (ppm): 7.75 , 7.67 , 7.37 , 7.20 , 5.50 , 5.12 , 4.16 , 3.91 , 2.19 , 2.07 , 2.00 ; ESI-MS (m/z): 505 [M+Na]+; HRMS-ESI (m/z): calculated for C22H27O12 [M+H]+: 483.15025; measured: 483.15030. Methyl 3-fluoro-4--3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yloxy)benzoate (7b). Compound 7b was synthesized using a method similar to that described above for 1a. Yield: 32%; white solid; m.p.: 121–123 °C; 1H-NMR  δ (ppm): 7.77 , 7.21 , 5.52 , 5.45 , 5.11 , 5.01 , 4.20 , 4.05 , 3.90 , 2.15 , 2.08 , 2.03 , 1.99 ; ESI-MS (m/z): 523 [M+Na]+; HRMS-ESI (m/z): calculated for C22H26FO12 [M+H]+: 501.14083; measured: 501.14076. Methyl 4-fluoro-2--3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yloxy)benzoate (8b). Compound 8b was synthesized using a method similar to that described above for 1a. Yield: 30%; white solid; m.p.: 136–138 °C; 1H-NMR  δ (ppm): 7.83 , 6.92 , 6.83 , 5.60 , 5.47 , 5.11 , 5.04 , 4.17 , 3.85 , 2.18 , 2.08 , 2.06 , 2.02 ; ESI-MS (m/z): 523 [M+Na]+; HRMS-ESI (m/z): calculated for C22H26FO12 [M+H]+: 501.14083; measured: 501.14074. Methyl 2-fluoro-6--3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yloxy)benzoate (9b). Compound 9b was synthesized using a method similar to that described above for 1a. Yield: 35%; white solid; m.p.: 130–132 °C; 1H-NMR  δ (ppm): 7.31 , 6.92 , 6.84 , 5.47 , 5.07 , 5.00 , 4.16 , 3.90 , 2.21 , 2.12 , 2.06 , 2.03 ; ESI-MS (m/z): 523 [M+Na]+; HRMS-ESI (m/z): calculated for C22H26FO12 [M+H]+: 501.14083; measured: 501.14077. -2-(Acetoxymethyl)-6-(3-fluoro-2-methylphenoxy)-tetrahydro-2H-pyran-3,4,5-triyl triacetate (10b). Compound 10b was synthesized using a method similar to that described above for 1a. Yield: 36%; white solid; m.p.: 127–129 °C; 1H-NMR  δ (ppm): 7.60 , 7.20 , 6.93 , 6.32 , 5.74 , 5.50 , 4.54 , 4.24 , 2.40 , 2.09 , 2.07 , 2.04 , 2.02 ; ESI-MS (m/z): 479 [M+Na]+; HRMS-ESI (m/z): calculated for C21H26FO10 [M+H]+: 457.15100; measured: 457.15098.MDCK cells, influenza A , influenza A  and influenza B (B/Jifang/13/97) were obtained from the Institute of Virology of the Chinese Academy of Preventive Medicine .4/well) were plated into a 96-well plate. After 24 h, the monolayer cells were incubated in the presence of a variety of different concentrations of the test compounds. After 48 h of culture at 37 °C and under a 5% CO2 atmosphere in a carbon dioxide incubator, the cells were monitored by CPE. The median toxic concentrations (TC50) were determined using a Reed and Muench calculation.The cytotoxicities of the compounds in the presence of MDCK cells were monitored by CPE. MDCK cells (2.5 × 1050) of the test strains. Following 2 h of adsorption at 37 °C, the monolayers were washed with phosphate-buffered saline (PBS) and incubated at 37 °C in the maintenance medium either in the presence or in the absence of different concentrations of the test compounds. The viral CPE was observed when the viral control group reached 4, and the antiviral activities (IC50) of the bisaryl amide derivatives were determined using a Reed and Muench calculation. The SI value was calculated from TC50/IC50.Confluent MDCK cells grown in 96-well microplates were infected with 100 median tissue culture infective dose units (100TCID1a on influenza A/FM/1/47-MA (H1N1) virus-infected mice was evaluated during the course of the current study, with ribavirin being used as a reference standard. Mice  were purchased from the National Institute for the Control of Pharmaceutical and Biological Products .The effect of compound 50 dose of mouse-adapted influenza A/FM/1/47-MA (H1N1) virus. Compound 1a, prepared as a suspension in 5% tween-80, or 5% tween-80 alone (vehicle group), was administered intragastrically twice daily for seven days, with the dose regimen being initiated one day prior to viral infection. The 10 mice involved in each group were observed twice daily for 14 days during the experiment for the occurrence of death. The mean day-to-death and survival times were then calculated , and their lungs were removed aseptically. Visual evidence of lung consolidation was scored on a blind basis. Lung scores were based on the percentage of the lung displaying signs of consolidation, with 0 being normal and 4 being indicative of 100% consolidation. Lung consolidation scores were evaluated using ranked sum analysis .Hemagglutination inhibition (HI) assay was employed to evaluate the effect of a compound on virus adsorption to target cells according to the reference with slight modification. Briefly, compound in PBS was mixed with an equal volume of influenza virus that was serially diluted by two-fold in PBS. After 30 min incubation at 4 °C, 100 µL of compound-virus preparation was mixed with an equal volume of 1% chicken erythrocyte suspension in PBS in a U bottom 96-well plate. The mixture was incubated for 1 h at room temperature before observing erythrocyte aggregation on the plate.in vitro inhibitory actions using the method according to the reference with slight modification. This spectrofluorometric assay (excitation wavelength: 355 nm and emission wavelength: 460 nm) uses 2′-(4-methylumbelliferyl)-α-d- acetylneuraminic acid (MUNANA) as the enzyme substrate, which is converted to a fluorescent product upon cleavage by NA. The intensity of fluorescence can reflect the activity of NA sensitively.Neuraminidase inhibition assay were evaluated for m MUNAN, 33 mm MES buffer (pH 3.5), 4 mm CaCl2, DDW). The terminal volume was 100 μL. Two-fold serially diluted compound samples and 30 μL enzyme incubated 1 hour at room temperature, and 60 μL substrate buffer mix was added. After 15 min at 37 °C, 100 μL of 14 mm NaOH in 83% ethanol was added to the reaction mixture to terminate the reaction. The intensity of the fluorescence was quantitated in fluorescence read on Enspire  , and substrate blanks were subtracted from the sample readings. The IC50 was calculated by plotting percent inhibition versus the inhibitor concentration, and determination of each point was performed in duplicate.The enzyme reaction system included 10 μL sample, 30 μL enzyme and 60 μL substrate buffer mix  with protease inhibitor cocktail . The lysates were then performed to immunoblotting with M2 andβ-actin antibodies  and β-Actin Mouse mAb from Cell Signaling Technology Inc. . Protein was detected using Immobilon Western Chemiluminescent HRP Substrate  with ChemiDoc XRS and Image Acquisition . Most studies of the activity and drug sensitivity of A/M2 channels have employed electrophysiology techniques, but since establishment of this technology takes time, we just detected M2 protein expression levels.The effect of compounds on M2 protein expression level were detected by immunoboltting assay. The immunoboltting experiment was employed according to the reference with slight modification.MDCK cells were infected with influenza virus A/Wuhan/359/95 at an 300TCIDGastrodin is a traditional Chinese herbal medicine that has been used clinically in China for thousands of years, mainly for the treatment of neurasthenia. To the best of our knowledge, there have been no reports documenting the use of gastrodin or any of its derivatives as antiviral agents. In our study, a novel series of gastrodin analogues was found to exhibit potent anti-influenza activity. A series of substituted aryl glycoside compounds were synthesized and evaluated for their anti-influenza virus activities, and preliminary structure-activity relationships (SAR) were analyzed.1a exhibited moderate inhibitory activity against the virus. Herein, we also report the first total synthesis of 1a and its associated in vivo anti-influenza data. In this experiment, a dose regimen of 80 mg/kg/day of compound 1a  yielded a significant increase in the survival numbers and prolonged the mean survival time of the animals tested.A total of 15 novel glycoside compounds were synthesized and screened for their antiviral activity against influenza A virus, among which the most potent inhibitor 50 and SI values of the most effective compound, 12a, against the influenza A (A/FM/1/47(H1N1)) virus were 25.24 μM and 13.9, respectively. Unfortunately, the SI values for all the compounds tested were much lower than those of the reference compounds. Remarkably, compounds 1a, 2a and 10b also displayed antiviral activity against the oseltamivir-null strain of influenza B. In addition, there was an increasing need for the development of antiviral along with novel antiviral mechanism. Compound 1a had a different mechanism of action from oseltamivir and ribavirin against influenza virus, so future research may be more meaningful with this result.Furthermore, the data were analyzed to produce a preliminary understanding of the structure-activity relationships. Seven compounds exhibited clear antiviral activity against the tested strain of the influenza A virus. The IC1a has been demonstrated that it has no interaction with NA or M2, it probably has novel targets which might be an important breakthrough in viral study.Undoubtedly, further exploration will be needed to produce more potent compounds with higher SI values and to deduce the detailed mechanism of action of this novel class of glycosides compounds against the influenza virus. Besides, since compound


Chloramphenicol and Trimethoprim/sulphamethoxazole. The antimicrobial results of the investigated compounds revealed promising results and some derivatives have activities similar to the references used.In this study, new derivatives of pyrazole, isoxazole, pyrazolylthiazole, and azolopyrimidine having a thiophene ring were synthesized under microwave irradiation. Their pharmacological activity toward bacteria and fungi inhibition was screened and compared to the references  Five-membered heterocyclic ring systems are very significant class of compounds, not only due to their abundance in nature, but also for their chemical and biological value. Thiophene derivatives have been fully-known for their therapeutic applications. They possess antihypertensive , antimic1 was cyclized with different types of nitrogen nucleophiles, namely, thiosemicarbazide, hydrazine derivatives 3a–c, and hydroxylamine hydrochloride which afforded pyrazole derivatives 2, 4a–c and isoxazole derivative 5, respectively prop-2-en-1-one ectively . The pre2 with hydrazonoyl chlorides. Thus, conventional heating or microwave irradiation of mixture of carbothioic acid amide derivative 2 and 2-oxo-N-arylpropanehydrazonoyl chloride 6a–e in dioxane in the existence of a base catalyst yielded in each case only one isolated product . The structure of products 8 was further supported by an alternative synthesis. Thus, reaction of compound 1 with 2-hydrazinyl-4-methyl-5-(phenyldiazenyl)thiazole 9 under reflux in ethanol led to the formation of product 8a , 6.20 , 8.45 , 8.73 .Azolopyrimidines mparison . Similar2, 4a–c, 5, 8a–e, 11a,b, 13,and 15 prepared in the study was carried out using cultures of two fungal strains Aspergillus niger (ATCC) (ASP) and Candida albicans (ATCC10231) (CA), as well as three bacteria species, namely, Gram positive bacteria, Staphylococcus aureus (ATCC 29213) (SA), and Bacillus subtilus (ATCC 6051) (BS) and the Gram negative bacteria is Escherichia coli (ATCC 25922) (EC). Chloramphenicol and Trimethoprim/sulphamethoxazole antibacterial agents were used as references to evaluate the potency of the examined compounds under the same conditions. The activity was investigated by measuring the diameter of inhibition zone (IZD) in mm ± standard deviation beyond well diameter (6 mm) generated on a range of environmental and clinically pathogenic microorganisms (gram-positive and gram-negative bacteria and fungi) utilizing (0.1 g/mL) concentration of tested samples and the outcomes are portrayed in Aspergillus niger (ATCC) (ASP) while, compounds 4c, 8c, and 11b have excellent activity against Candida albicans (ATCC 10231) (CA) with inhibition zones 23, 24, and 25 respectively. For the antibacterial activity: it was found that Gram positive bacteria are more sensitive to the tested compounds especially SA rather than BS as five compounds 2, 4c, 8b, 8d, and 15 have potent activity against SA while for BS only compounds 4a and 4c showed good activity. In the case of Gram negative activity with EC, two derivatives 2 and 8c revealed higher activity. The used solvent DMSO concentration did not exhibit any influence on bacteria or fungi.In vitro antimicrobial screening of compounds 1H-NMR) and run in deuterated dimethylsulfoxide (DMSO-d6). Chemical shifts were related to that of the solvent. Mass spectra were recorded on a Shimadzu GCeMS-QP1000 EX mass spectrometer  at 70 eV. Microwave reactions were performed with a Millstone Organic Synthesis Unit with a touch control terminal  and a continuous focused microwave power delivery system in a pressure glass vessel (10 mL) sealed with a septum under magnetic stirring. The temperature of the reaction mixture was monitored using a calibrated infrared temperature control under the reaction vessel, and control of the pressure was performed with a pressure sensor connected to the septum of the vessel. Elemental analyses were carried out at the Microanalytical Centre of Cairo University, Giza, Egypt. Compounds 10a,b, 12, and 14 were purchased from Sigma-Aldrich and utilized as it is without previous treatments. Compounds 1, 2, 6a–e, and 9 were prepared as previously reported in the respective literature pyrimidin-4(3H)-one (4c). Brown solid, m.p. 188–190 °C; IR: 3435, 3158 (2NH), 3048, 2966 (C-H), 1596 (C=N) cm−1; 1H-NMR : δ 3.07 , 4.15 , 6.16 , 6.92–7.75 , 7.98 , 8.63 ; MS, m/z (%) 543 , 426 (50), 330 (49), 153 (83), 64 (100), 43 (68). Anal. Calcd. For C26H17N5OS4 (543.03): C, 57.44; H, 3.15; N, 12.88; found: C, 57.58; H, 3.10; N, 12.63.1 , hydroxylamine. HCl , and anhydrous sodium acetate (0.3 g) in acetic acid (20 mL) was stirred at room temperature for 6 h. The formed solid was filtered, washed with water, and crystallized from dioxane to give isoxazoline derivative 5.Method A: A mixture of chalcone 1 and hydroxylamine with the same quantity in method A were heated under microwave irradiation at 500 W and 150 °C for 10 min. The reaction mixture was treated similarly to method A to obtain compounds 5 as yellow solid; m.p. 212–214°C; IR: 3091, 2922 (C-H), 1593 (C=N) cm−1; 1H-NMR : δ3.09 , 4.13 , 6.08 , 7.00–8.23 ; MS, m/z (%) 335 , 152 (65), 83 (100), 70 (21). Anal. Calcd. for C11H9NOS2 (235.01): C, 56.14; H, 3.85; N, 5.95; found: C, 56.03; H, 3.72; N, 5.74.Method B: The above reaction of chalcone H-pyrazole-1-carbothioamide 2  and the appropriate hydrazonoyl halides 6a–e (1 mmol) in dioxane (20 mL) containing TEA (0.5 mL) was refluxed for 6–10 h (monitored by TLC), allowed to cool and the solid formed was filtered off, washed with ethanol, dried, and recrystallized from dimethylformamide to give 8a–e.Method A: A mixture of 3,5-di(thiophen-2-yl)-4,5-dihydro-1Method B: Repetition of the same reactions of method A with heating in microwave oven at 500 W and 150 °C for a period of time gave products identical in all respects with those separated from method A.2--4,5-dihydro-1H-pyrazol-1-yl)-4-methyl-5-(phenyldiazenyl)-thiazole (8a). Red solid, m.p. 164–166 °C; IR:2919 (C-H), 1603 (C=N) cm−1; 1H-NMR : δ 2.58 , 3.07 , 4.17 , 6.21 , 7.00–7.84 ; MS, m/z (%) 435 , 339 (14), 205 (50), 75 (42), 50 (100). Anal. Calcd. for C21H17N5S3 (435.06): C, 57.90; H, 3.93; N, 16.08; found: C, 57.74; H, 3.77; N, 15.82.2--4,5-dihydro-1H-pyrazol-1-yl)-4-methyl-5-(o-tolyldiazenyl)-thiazole (8b). Red solid, m.p. 122–124 °C; IR: 2921 (C-H), 1600 (C=N) cm−1; 1H-NMR : δ 2.04 , 2.60 , 3.09 , 4.19 , 6.22 , 6.93–7.79 ; MS, m/z (%) 449 , 218 (12), 110 (48), 91 (100), 65 (52). Anal. Calcd. for C22H19N5S3 (449.08): C, 58.77; H, 4.26; N, 15.58; found: C, 58.52; H, 4.08; N, 15.46.2--4,5-dihydro-1H-pyrazol-1-yl)-5-((4-methoxyphenyl)diazenyl)-4-methylthiazole (8c). Red solid, m.p. 143–145 °C; IR: 2923 (C-H), 1602 (C=N) cm−1; 1H-NMR : δ 2.56 , 3.04 , 3.83 , 4.12 , 6.13 , 6.92–7.82 ; MS, m/z (%) 465 , 368 (9), 218 (13), 111 (100), 43 (72). Anal. Calcd. for C22H19N5OS3 (465.08): C, 56.75; H, 4.11; N, 15.04; found: C, 56.53; H, 4.04; N, 14.86.5-((4-Chlorophenyl)diazenyl)-2--4,5-dihydro-1H-pyrazol-1-yl)-4-methylthiazole(8d). Orange solid, m.p. 153–155 °C; IR: 3063, 2922 (C-H), 1605 (C=N) cm−1; 1H-NMR : δ 2.56 , 3.08 , 4.08 , 6.16 , 6.98–7.85 ; MS, m/z (%) 471 , 469 , 368 (6), 264 (15), 111 (59), 77 (57), 43 (100). Anal. Calcd. for C21H16ClN5S3 (469.03): C, 53.66; H, 3.43; N, 14.90; found: C, 53.49; H, 3.40; N, 14.73.2--4,5-dihydro-1H-pyrazol-1-yl)-4-methyl-5-((4-nitrophenyl)-diazenyl)thiazole (8e). Brown solid, m.p. 162–164 °C; IR: 3096, 2920 (C-H), 1590 (C=N) cm−1; 1H-NMR : δ 2.61 , 3.09 , 4.11 , 6.21 , 6.58–7.95 ; MS, m/z (%) 480 , 427 (16), 232 (31), 111 (100), 77 (59), 43 (86). Anal. Calcd. for C21H16N6O2S3 (480.58): C, 52.48; H, 3.36; N, 17.49; found: C, 52.28; H, 3.19; N, 17.33.1  and 2-hydrazinyl-4-methyl-5-(phenyldiazenyl)thiazole (9)  in 2-propanol (10 mL), was refluxed for 2 h then cooled to room temperature. The solid precipitated was filtered off, washed with water, dried, and recrystallized from dimethylformamide to give the corresponding product, 8a which were identical in all aspects  with those obtained from reaction of 2 with 6a but in 70% yield.Equimolar amounts of chalcone 1  and the appropriate heterocyclic amine  (1 mmol) in ethanol (20 mL) in the presence of catalytic drops of acetic acid was refluxed for 10–15 h (monitored through TLC). The reaction mixture was poured into water and the solid product was collected by filtration followed by washing with ethanol. The crude product was then recrystallized from EtOH or DMF to give pure products 11a,b, 13, and 15, respectively.Method A: A mixture of chalcone 11a,b, 13, and 15 with their physical constants and spectral data are depicted as shown below:Method B: Repetition of the same reactions of method A with heating in microwave oven at 500 W and 150 °C for a period of time gave products identical in all respects with those separated from method A. Compounds 5,7-Di(thiophen-2-yl)-4,7-dihydro-triazolopyrimidine (11a). Yellow solid m.p. 241–243 °C (DMF); IR: 3425 (NH), 3091, 2920 (C-H), 1599 (C=N), cm−1; 1H-NMR: δ 5.14 , 6.20 , 6.85–8.04 , 8.45 , 8.73 ; MS m/z (%): 286 , 284 (100), 111 (52), 69 (44). Anal. Calcd. for C13H10N4S2 (286.03): C, 54.52; H, 3.52; N, 19.56; found: C, 54.40; H, 3.64; N, 19.51.5,7-Di(thiophen-2-yl)-4,7-dihydrotetrazolopyrimidine (11b). Yellow solid, m.p. 266–268 °C (DMF); IR: 3402 (NH), 3087, 2924 (C-H), 1636 (C=N), cm−1; 1H-NMR: δ 5.41 , 6.08 , 7.16–8.24 , 8.29 ; MS m/z (%): 287 , 259 (73), 220 (99), 111 (100), 65 (48). Anal. Calcd. for C12H9N5S2 (287.03): C, 50.16; H, 3.16; N, 24.37; found: C, 50.29; H, 3.07; N, 24.39.2-Phenyl-5,7-di(thiophen-2-yl)-4,7-dihydropyrazolopyrimidine (13). Yellow solid m.p. 218–220 °C (DMF); IR: 3429 (NH), 3095, 3071, 2923 (C-H), 1596 (C=N) cm−1; 1H-NMR: δ 4.86 , 6.14 , 6.59 , 6.80–8.25 , 8.72 ; MS m/z (%): 361 , 359 (100), 228 (16), 111 (49), 77 (63). Anal. Calcd. for C20H15N3S2 (361.07): C, 66.45; H, 4.18; N, 11.62; found: C, 66.61; H, 4.09; N, 11.60.2,4-Di(thiophen-2-yl)-1,4-dihydrobenzoimidazopyrimidine (15). Yellow solid, m.p. 230–232 °C (EtOH); IR: 3412 (NH), 3077, 2920 (C-H), 1599 (C=N) cm−1; 1H-NMR: δ 4.79 , 6.12 , 6.69–8.27 , 8.49 ; MS m/z (%): 335 , 333 (100), 224 (23), 111 (50), 64 (53). Anal. Calcd. for C18H13N3S2 (335.06): C, 64.45; H, 3.91; N, 12.53; found: C, 64.68; H, 3.87; N, 12.49.Antimicrobial activity was determined using the agar disc diffusion assay method as described previously by Hossain et al. . The tesSabouraud dextrose agar: The medium used for isolation of pathogenic yeasts has the following composition (g/L): glucose, 20; peptone, 10; agar, 25 and distilled water, 1 L, pH was adjusted at 5.4. The medium was autoclaved at 121 °C for 15 min.Trypticase soya agar (TSA): The medium was used to cultivate tested bacteria. It contains (g/L) Tryptone (Pancreatic Digest of Casein) 15.0 g, Soytone  5.0 g, Sodium Chloride 5.0 g, Agar 15.0 g, and distilled water 1 L. The medium was autoclaved at 121 °C for 15 min.At the end, we have succeeded in the synthesis of new derivatives of pyrazole, isoxazole, pyrazolylthiazole, and azolopyrimidine incorporated with a thiophene ring under microwave irradiation. Different spectroscopic methods and elemental analyses were used to confirm the structures of the newly synthesized compounds. The antimicrobial results of the examined compounds revealed promising results and some derivatives have activities similar to the references used.


The reactions afforded unusual 2-methylene-2,3-dihydrofurochromen-2-ones in good yields.A catalytic system consisting of the ruthenium(II) complex [Ru( An-2-ones  from reaone-pot manner, involves the initial trifluoroacetic acid-promoted propargylation of the 1,3-dicarbonyl compound, and subsequent cycloisomerization of the resulting γ-ketoalkyne A (5-exo cyclization + aromatization) catalyzed by the 16-electron allyl-ruthenium(II) complex [Ru(η3-2-C3H4Me)(CO)(dppf)][SbF6] (1).In the course of our studies focused on the application of ruthenium catalysts for the construction of furan- and pyrrole-ring frameworks ,23,24,25c]chromen-2-ones are also accessible by this route using 4-hydroxychromen-2-one as substrate, representing an appealing one-pot method of synthesis for this type of heterocycles chromen-2-one derivatives 4 HR result in the selective formation of the 2-methylene-2,3-dihydrofurochromen-2-one derivatives starting from 4-hydroxychromen-2-one chromen-2-one  takes place after only 10 min. As shown in 4b–d could also be synthesized in good yields (77%–92%) by reacting 3 with the secondary propargylic alcohols 1-(2-methoxyphenyl)-2-propyn-1-ol (2b), 1-(1-naphthyl)-2-propyn-1-ol (2c) and 1-(2-thienyl)-2-propyn-1-ol (2d) under the same MW conditions.The use of microwave (MW) irradiation represents a convenient alternative to the conventional thermal heating in organic synthesis since a more effective energy transfer to the system takes place, thus shortening considerably the reaction times and improving in many cases the product yields ,28,29. A2 signal at ca. 92 ppm, typical of a terminal olefinic =CH2 unit, in their 13C{1H}-NMR spectra (DEPT experiments). Characteristic 1H-NMR peaks for these units were also observed at 4.5–5.5 ppm .The presence of a 2-methylene-2,3-dihydrofuran moiety in the structure of these compounds was readily identified by the appearance of a high-field CH carbon resonance at 43–49 ppm (CHR unit) and a CHc]chromen-2-ones has been scarcely documented in the literature [c]chromen-2-ones. In this sense, the reluctance shown by compounds 4a–d to aromatize under the acidic conditions employed merits to be highlighted. In fact, only in the case of 4b such aromatization process could be observed after prolonged MW irradiation (3 h) of the reaction mixture at 100 °C. Under this conditions, the novel 8-chloro-substituted furochromen-2-one 5b could be synthesized with an acceptable 63% yield and fully characterized (CO)(dppf)][SbF6] (1) [2a–d [1H) or 75.4 MHz (13C). The chemical shift values (δ) are given in parts per million and are referred to the residual peak of the deuterated solvent used (CDCl3). High-resolution mass spectra (HRMS) were provided by the Mass Spectrometry Service of the Instituto de Investigaciones Químicas . CCDC 831021 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.Solvents were dried by standard methods and distilled under nitrogen before use. The complex [Ru(ηbF6] (1)  and prop1) [SbF6] (1) , CF3CO2H  and a magnetic stirring bar. The vial was then placed inside the cavity of a CEM Discover® S-Class microwave synthesizer and power was held at 300 W until the desired temperature was reached (75 °C). Microwave power was automatically regulated for the remainder of the experiment (10 min) to maintain the temperature (monitored by a built-in infrared sensor). Then, the vial was cooled to room temperature, the volatiles removed under vacuum, and the residue purified by flash chromatography (silica gel) using a mixture EtOAc/hexanes (1:20) as eluent. Characterization data for the novel 8-chloro-2-methylene-2,3-dihydrofurochromen-2-ones 4a–d are as follows:Under a nitrogen atmosphere, a pressure-resistant septum-sealed glass vial was charged with the corresponding propargylic alcohol 8-Chloro-3-(4-methoxyphenyl)-2-methylene-2,3-dihydrofurochromen-2-one (4a). Yellow solid ; m.p. 125–127 °C; IR (Nujol) ν = 1721 (C=O) cm−1; 1H-NMR (CDCl3) δ = 3.78 , 4.52 , 5.11 , 6.87 , 7.23 , 7.32 , 7.53 , 7.75  ppm; 13C-NMR (CDCl3) δ = 47.7, 55.2, 91.7, 107.4, 112.5, 114.2, 118.5, 122.2, 128.8, 129.6, 131.2, 132.7, 153.5, 159.1, 157.8, 162.9, 164.8 ppm; HRMS (EI) m/z = 340.0501, C19H13O4Cl requires 340.0502.8-Chloro-3-(2-methoxyphenyl)-2-methylene-2,3-dihydrofurochromen-2-one (4b). Yellow solid ; m.p. 122–124 °C; IR (Nujol) ν = 1748 (C=O) cm−1; 1H-NMR (CDCl3) δ = 3.79 , 4.55 , 5.12 , 6.82–6.91 , 7.25 , 7.34 , 7.55 , 7.76  ppm; 13C-NMR (CDCl3) δ = 48.5, 55.3, 92.1, 107.2, 112.6, 113.0, 113.8, 118.6, 120.1, 122.3, 129.8, 130.0, 132.9, 140.7, 153.6, 157.9, 160.0, 163.4, 164.3 ppm; HRMS (EI) m/z = 340.0513, C19H13O4Cl requires 340.0502.8-Chloro-3-(1-naphthyl)-2-methylene-2,3-dihydrofurochromen-2-one (4c). Yellow solid ; m.p. 140–142 °C; IR (Nujol) ν = 1719 (C=O) cm−1; 1H-NMR (CDCl3) δ = 4.55 , 5.15 , 5.33 , 7.33–7.49 , 7.81–7.85  ppm; 13C-NMR (CDCl3) δ = 48.8, 92.3, 107.3, 112.6, 118.7, 122.4, 125.3, 126.2, 126.4, 127.0, 127.7, 127.9, 129.0, 129.8, 132.9, 133.0, 133.4, 136.5, 153.6, 157.9, 163.4, 164.5 ppm; HRMS (EI) m/z = 360.0557, C22H13O3Cl requires 360.0553.8-Chloro-3-(2-thienyl)-2-methylene-2,3-dihydrofurochromen-2-one (4d). Orange solid ; m.p. 131–133 °C; IR (Nujol) ν = 1733 (C=O) cm−1; 1H-NMR (CDCl3) δ = 4.72 , 5.18 , 5.48 , 6.98 , 7.12 , 7.25 , 7.35 , 7.56 , 7.75  ppm; 13C-NMR (CDCl3) δ = 43.0, 92.3, 106.3, 112.1, 118.2, 122.0, 124.9, 125.6, 126.7, 129.4, 132.7, 141.2, 153.1, 157.3, 162.9, 163.0 ppm; HRMS (EI) m/z = 315.9971, C16H9O3ClS requires 315.9961.2b, 0.162 g, 1 mmol), 6-chloro-4-hydroxychromen-2-one , THF (0.5 mL), [Ru(η3-2-C3H4Me)(CO)(dppf)][SbF6] , CF3CO2H  and a magnetic stirring bar. The vial was then placed inside the cavity of a CEM Discover® S-Class microwave synthesizer and power was held at 300 W until the desired temperature was reached (100 °C). Microwave power was automatically regulated for the remainder of the experiment (3 h) to maintain the temperature (monitored by a built-in infrared sensor). Then, the vial was cooled to room temperature, the volatiles removed under vacuum, and the residue purified by flash chromatography (silica gel) using a mixture EtOAc/hexanes (1:20) as eluent to give 5b. Yellow solid ; m.p. 120–122 °C; IR (Nujol) ν = 1749 (C=O) cm−1; 1H-NMR (CDCl3) δ = 2.42 , 3.82 , 7.01–7.08 , 7.26–7.43 , 7.85  ppm; 13C-NMR (CDCl3) δ = 12.1, 55.1, 110.6, 113.7, 116.2, 118.0, 118.3, 119.6, 120.0, 129.2, 129.4, 131.0, 132.9, 136.5, 150.1, 152.5, 156.9, 162.5, 163.0 ppm; HRMS (EI) m/z = 340.0496, C19H13O4Cl requires 340.0502.Under nitrogen atmosphere, a pressure-resistant septum-sealed glass vial was charged with 1-(2-methoxyphenyl)-2-propyn-1-ol /Σw(Fo2)]1/2 where w = 1/[σ2(Fo2) + (aP)2 + bP]  with σ2(Fo2) from counting statistics and P = (Max (Fo2 + 2Fc2)/3. Atomic scattering factors were taken from the International Tables for X-ray Crystallography [The most relevant crystal and refinement data are collected in  Pro CCD . Data re Pro CCD . An empi Pro CCD . The sof Pro CCD . The str Pro CCD . Isotroperformed . During lography . The crylography .c]chromen-2-one derivatives, by coupling of secondary propargylic alcohols with commercially available 6-chloro-4-hydroxychromen-2-one, has been developed with the aid of the catalytic system [Ru(η3-2-C3H4Me)(CO)(dppf)][SbF6]/CF3CO2H. Apparently, the presence of the electron-withdrawing Cl substituent on the 4-hydroxychromen-2-one skeleton exerts a marked influence on the behavior of these species since, as previously described by us [c]chromen-2-ones by aromatization of the five-membered ring. Overall, the results reported herein represent a new example of the utility of the allyl-ruthenium(II) complex 1 in synthetic organic chemistry [In summary, an efficient synthesis of unusual and remarkably stable 2-methylene-2,3-dihydrofuro[3,2-ed by us , the samhemistry .


N-protection. 3-Vinylindazoles could be interesting synthetic intermediates allowing access to biologically active molecules.Herein we report an expeditive C-3 vinylation of unprotected 3-iodoindazoles under microwave irradiation. Ten C-5 substituted 3-vinylindazole derivatives, nine of them novel, were synthesized through this method, which proceeds in moderate to excellent yields starting from C-5 substituted 3-iodoindazole derivatives. In all cases, the C-3 vinylated derivative was the only isolated product. This methodology allows access to 3-vinylated indazoles selectively and directly without the need of  Yield: 20%.Method i: Prepared from 3-iodoindazole , 2 equivalents of vinyl boronic acid pinacol ester , tetrakistriphenylphosphine palladium , an aqueous solution of sodium carbonate 2N (2 mL) and dioxane (7 mL) using microwave method described above, but at a reaction temperature of 180 °C, to obtain 80 mg of a crystalline plates. Yield: 68%.Method j: Prepared from 3-iodoindazole , 2 equivalents of vinyl boronic acid pinacol ester , tetrakistriphenylphosphine palladium , an aqueous solution of sodium carbonate 2N (2 mL) and dioxane (7 mL) using microwave method described above, but with a reaction time of 60 min, to obtain 40 mg of a crystalline plates. Yield: 34%.Method k: Prepared from 3-iodoindazole , 2 eq. of vinyl boronic acid pinacol ester , tetrakistriphenylphosphine palladium , an aqueous solution of sodium carbonate 2N (1 mL) and dioxane (3.5 mL) using method a described above, but employing conventional heating, to obtain 26 mg of a crystalline plates: Yield: 44%.−1): 3047 (NH); 1620 (C=C). 1H-NMR (CDCl3) δ (ppm): 11.32 ; 7.95 ; 7.48 ; 7.40 ; 7.21 ; 7.13 ; 6.16 ; 5.58 . 13C-NMR (CDCl3) δ (ppm): 144.18; 141.59; 128.90; 127.03; 121.48; 121.14; 121.00; 117.18; 110.30; HRMS calculated for C9H8N2: 144.0687, Found: 144.0681.m.p.: 104–105 °C (lit.:  115.5–115-Nitro-3-vinyl-1H-indazole (3b). Method a: Prepared from 3-iodo-5-nitro-1H-indazole , 2 equivalents of vinyl boronic acid pinacol ester , tetrakis- triphenylphospine palladium , aqueous solution of sodium carbonate 2N (2 mL) and dioxane (7 mL), to give 0.113 g of a pale brown solid. Yield: 87%.Method b: Prepared from tert-butyl 3-iodo-5-nitro-1H-indazole-1-carboxylate , 2 eq. of vinyl boronic acid pinacol ester , tetrakistriphenylphosphine palladium , an aqueous solution of sodium carbonate 2N (2 mL) and dioxane (7 mL) using microwave method described above to obtain 13 mg of a pale brown solid: Yield: 13%.Method c: Prepared from 3-iodo-5-nitro-1H-indazole , 2 equivalents of vinyl boronic acid pinacol ester , tetrakistriphenylphospine palladium , aqueous solution of sodium carbonate 2N (2 mL) and dioxane (7 mL) using method a described above, but employing conventional heating, to give 24 mg of pale brown solid. Yield: 21%.−1): 3179 (NH); 1620 (C=C); 1528 ; 1335 . 1H-NMR δ (ppm): 13.75 ; 8.93 ; 8.20 ; 7.71 ; 7.14 ; 6.19 ; 5.58 .13C-NMR δ (ppm): 145.12; 142.97; 141.89; 127.65; 121.14; 119.61; 118.21; 117.95; 111.31; HRMS calculated for C9H7N3O2: 189.0538, Found: 189.0535.m.p.: 166–168 °C; IR (KBr) ν (cm5-Bromo-3-vinyl-1H-indazole (3c). Method a: Prepared from 5-bromo-3-iodo-1H-indazole , 2 equivalent of vinyl boronic acid pinacol ester , tetrakis- triphenylphospine palladium , aqueous solution of sodium carbonate 2N (2 mL) and dioxane (7 mL), to give 82 mg of a pale yellow solid. Yield: 60%.Method b: Prepared from tert-butyl 5-bromo-3-iodo-1H-indazole-1-carboxylate , 2 eq. of vinyl boronic acid pinacol ester , tetrakistriphenylphosphine palladium , an aqueous solution of sodium carbonate 2N (2 mL) and dioxane (7 mL) using microwave method described above to obtain 45 mg of a pale yellow solid: Yield: 43%.−1): 3112 (NH); 1689 (C=C); 424 (C-Br). 1H-NMR (CDCl3) δ (ppm): 11.68 ; 8.03 ; 7.44 ; 7.32 ; 7.01 ; 6.07 ; 5.57 . 13C-NMR (CDCl3) δ (ppm): 143.45;140.15; 130.21; 128.12; 123.48; 122.54; 117.83; 114.66; 111.77; HRMS calculated for C9H7BrN2: 221.9793, Found: 221.9797.m.p.: 250–251 °C; IR (KBr) ν (cm5-Methoxy-3-vinyl-1H-indazole (3d). Method a: Prepared from 3-iodo-5-methoxy-1H-indazole , 2 equivalents of vinyl boronic acid pinacol ester , tetrakis- triphenylphospine palladium , aqueous solution of sodium carbonate 2N (2 mL) and dioxane (7 mL), to give 74 mg of a pale yellow solid. Yield: 58%.Method b: Prepared from tert-butyl 3-iodo-5-methoxy-1H-indazole-1-carboxylate , 2 eq. of vinyl boronic acid pinacol ester , tetrakistriphenylphosphine palladium , an aqueous solution of sodium carbonate 2N (2 mL) and dioxane (7 mL) using microwave method described above to obtain 29 mg of a pale yellow solid. Yield: 46%.Method c: Prepared from 3-iodo-5-methoxy-1H-indazole , 2 equivalents of vinyl boronic acid pinacol ester , tetrakistriphenylphosphine palladium , an aqueous solution of sodium carbonate 2N (2 mL) and dioxane (7.0 mL) using method described above, but employing conventional heating, to obtain 96 mg of a pale yellow solid. Yield: 75%.−1): 3155 (NH); 1635 (C=C); 1219 (C-O). 1H-NMR δ (ppm): 12.95 ; 7.44 ; 7.33 ; 7.06–6.96 ; 6.02 ; 5.41 ; 3.82 . 13C-NMR δ (ppm): 154.50; 141.71; 137.02; 129.38; 120.70; 118.05; 114.90; 111.47; 100.02; 55.50; HRMS calculated for C10H10N2O: 174.0793, Found: 174.0788.m.p.: 88–89 °C; IR (KBr) ν (cm5-Fluoro-3-vinyl-1H-indazole (3e). Method a: Prepared from 5-fluoro-3-iodo-1H-indazole , 2 equivalents of vinyl boronic acid pinacol ester , tetrakis- triphenylphospine palladium , aqueous solution of sodium carbonate 2N (2 mL) and dioxane (7 mL), to give 58 mg of a pale yellow solid. Yield: 47%.Method b: Prepared from 5-fluoro-3-iodo-1H-indazole , 2 eq. of vinyl boronic acid pinacol ester , tetrakistriphenylphosphine palladium , an aqueous solution of sodium carbonate 2N (1 mL) and dioxane (3.5 mL) using method a described above, but employing conventional heating, to obtain 50 mg of the precursor 5-fluoro-3-iodo-1H-indazole: Yield of recovery: 63%.−1): 3163 (NH); 1636 (C=C); 1118 (C-F). 1H-NMR (CDCl3) δ (ppm): 11.73 ; 7.52 ; 7.41 ; 7.17 ; 7.05 ; 6.05 ; 5.56 . 13C-NMR (CDCl3) δ (ppm): 158.39 , 144.04 , 138.52, 128.38, 121.01 , 117.25, 111.49 , 105.21 ; HRMS calculated for C9H7FN2: 162.0593, Found: 162.0590.m.p.: 104–105 °C; IR (KBr) ν (cm5-Chloro-3-vinyl-1H-indazole (3f). Prepared from 5-chloro-3-iodo-1H-indazole , 2 equivalents of vinyl boronic acid pinacol ester , tetrakistriphenylphospine palladium , aqueous solution of sodium carbonate 2N (2 mL) and dioxane (7 mL), to give 77 mg of a yellow solid. Yield: 60%; m.p.: 131–132 °C; IR (KBr) ν (cm−1): 3181 (NH); 1628 (C=C); 649 (C-Cl). 1H-NMR δ (ppm): 13.28 ; 8.05 ; 7.56 ; 7.37 ; 7.01 ; 6.08 ; 5.46 . 13C-NMR δ (ppm): 142.00; 139.72; 128.57; 126.56; 125.45, 121.23, 119.60, 116.29, 112.17; HRMS calculated for C9H7ClN2: 178.0298, Found: 178.0296.5-Methyl-3-vinyl-1H-indazole (3g). Method a: Prepared from 3-iodo-5-methyl-1H-indazole , 2 equivalent of vinyl boronic acid pinacol ester , tetrakis- triphenylphospine palladium , aqueous solution of sodium carbonate 2N (2 mL) and dioxane (7 mL), to give 60 mg of a pale brown solid. Yield: 49%.Method b: Prepared from 3-iodo-5-methyl-1H-indazole , 2 equivalents of vinyl boronic acid pinacol ester , tetrakistriphenylphosphine palladium , an aqueous solution of sodium carbonate 2N (1 mL) and dioxane (3.5 mL) using method a described above, but employing conventional heating, to obtain 10 mg of a pale brown solid. Yield: 17%.−1): 3179 (NH); 2916 (C-H); 1628 (C=C). 1H-NMR (CDCl3) δ (ppm): 11.63 ; 7.72 ; 7.37 ; 7.24–7.11 ; 6.16 ; 5.57 ; 2.50 . 13C-NMR (CDCl3) δ (ppm):143.28; 140.28; 130.84; 129.02; 128.96; 121.38; 119.96; 116.69; 110.12; 21.58; HRMS calculated for C10H10N2: 158.0844, Found: 158.0840.m.p.: 88–89 °C; IR (KBr) ν (cm5-Cyano-3-vinyl-1H-indazole (3h). Prepared from 5-cyano-3-iodo-1H-indazole , 2 equivalents of vinyl boronic acid pinacol ester , tetrakistriphenylphospine palladium , aqueous solution of sodium carbonate 2N (2 mL) and dioxane (7 mL), to give 77 mg of a white solid. Yield: 61%; m.p.: 156–157 °C; IR (KBr) ν (cm−1): 3209 (NH); 2222 (CN); 1612 (C=C). 1H-NMR δ (ppm): 13.59 ; 8.63 , 7.68 ; 7.04 , 6.21 , 5.53 . 13C-NMR δ (ppm): 143.56; 142.01; 128.38; 128.01; 127.43; 119.99; 119.82; 117.82; 111.98; 103.29; HRMS calculated for C10H7N3: 169.0640, Found: 169.0637.tert-Butyl (3-vinyl-1H-indazol-5-yl)carbamate (3i). Prepared from tert-butyl (3-iodo-1-H-indazol-5-yl) carbamate , 2 equivalents of vinyl boronic acid pinacol ester , tetrakistriphenylphospine palladium , aqueous solution of sodium carbonate 2N (2 mL) and dioxane (7 mL), to give 80 mg of a White solid. Yield: 55%; m.p.: 189–190 °C; IR (KBr) ν (cm−1): 3356 (NH); 1700 (C=O); 1636 (C=C); 1165 (C-O). 1H-NMR δ (ppm): 12.96 ; 9.30 ; 8.13 ; 7.45–7.37 ; 6.98 ; 5.92 ; 5.44 ; 1.49 . 13C-NMR δ (ppm): 153.18; 141.83; 137.80; 133.36; 129.70; 120.33; 119.92; 114.88; 110.54; 107.98; 78.91; 28.22; HRMS calculated for C14H17N3O2: 259.1321, Found: 259.1330.5-Amino-3-vinyl-1H-indazole (3j). Method a: Prepared from 5-amino-3-iodo-1H-indazole , 2 equivalents of vinyl boronic acid pinacol ester , tetrakis- triphenylphospine palladium , aqueous solution of sodium carbonate 2N (2 mL) and dioxane (7 mL), to give 44 mg of a brown solid. Yield: 36%.Method b: A solution of tert-butyl (3-vinyl-1H-indazol-5-yl) carbamate  and trifluoroacetic acid  in CH2Cl2 (2 mL) was stirred vigorously for 2 h at room temperature. After stirring, the reaction was neutralized to pH = 7 using NaOH 1M and the organic layer was extracted with ethyl acetate (3 × 20 mL) The combined organic layers were dried with anhydrous sodium sulfate and removal of the solvent under vacuum afforded a 80 mg of pure product as a brown solid. Yield: 100%; m.p.: 157–158 °C; IR (KBr) ν (cm−1): 3461 (NH); 1682 (C=O). 1H-NMR δ (ppm):12.64 ; 7.24 ; 7.00 ; 6.91 ; 6.80 ; 5.84 ; 5.34 ; 4.87 . 13C-NMR δ (ppm): 143.20; 140.39; 135.83; 130.33; 121.46; 118.02; 113.61; 110.81; 100.76; HRMS calculated for C9H9N3: 159.0796, Found: 159.0792.N-protection herein presented shows tolerance concerning: (i) the electronic properties of the C-5 substituent on the indazole nucleus, (ii) the use of conventional heating, and (iii) the use of diverse catalytic systems. Despite such tolerance, the best conditions for this reaction were identified as: microwave irradiation, Pd(PPh3)4 as a catalyst, 120 °C, and 40 min of reaction time. In all reported cases the main isolated product was the C-3 vinyl derivative without detection of the any other competing product. With this methodology in hand, vinylated indazoles may be directly and selectively obtained without the need of N-protection.We have prepared ten, nine of them novel, C-5 substituted 3-vinylindazole derivatives through an easy, simple, fast and reproducible Suzuki cross-coupling reaction between unprotected 3-iodoindazoles and pinacol vinyl boronate with yields ranging from moderate to excellent. The vinylation reaction without


Leonurus japonicus Houtt., named “Yimucao” and “Chongweizi”, respectively, in Chinese, have been widely used in China as gynecological medicines. The components of the essential oils obtained by hydrodistillation were investigated by GC-MS. The antibacterial activity of the essential oils was determined by micro-dilution assay. The results showed large variations in the chemical composition and antibacterial activity of the oils. The oil of “Yimucao” showed antibacterial activity against various Gram-positive bacteria and consisted mainly of sesquiterpenes and diterpenes, with phytone, phytol, caryophyllene oxide and β-caryophyllene being the most significant constituents, whereas the oil of “Chongweizi”, mainly made up of bornyl acetate and aliphatic hydrocarbons, was inactive in the antibacterial assay. Further study of the main compounds in “Yimucao oil” showed that β-caryophyllene had wide-spectrum activity against Gram-positive bacteria.The herb and fruits of  Leonurus japonicus Houtt. is an annual herbaceous plant of the Lamiaceae family, widely distributed in East Asia. The herb harvested in summer before blossoming and the ripe fruits picked in autumn, commonly referred to as “Yimucao” and “Chongweizi”, respectively, in Traditional Chinese Medicine, are both used for regulating menstrual disturbances and invigorating blood circulation +, 243.2 [M+Na]+; HRESI-MS: m/z 243.1729 [M+Na]+ ; 1H-NMR  δ: 4.96 , 4.85 , 2.86 , 2.61 , 1.19 , 0.99 , 0.97 ; 13C-NMR  δ: 51.0 (C-1), 27.3 (C-2), 39.4 (C-3), 59.7 (C-4), 63.8 (C-5), 30.0 (C-6), 29.7 (C-7), 151.8 (C-8), 48.8 (C-9), 39.9 (C-10), 34.1 (C-11), 17.0 (C-12), 112.8 (C-13), 29.7 (C-14), 21.7 (C-15).Phytol (38): Colorless oil; ESI-MS m/z 319.3 [M+Na]+; HRESI-MS m/z 319.2978 [M+Na]+ ; 1H-NMR  δ: 5.41 , 4.15 , 1.99 , 1.67 , 0.87, 0.86 , 0.85, 0.84 ; 13C-NMR  δ: 59.4 (C-1), 123.1 (C-2), 140.3 (C-3), 39.9 (C-4), 25.1 (C-5), 36.7 (C-6), 32.8 (C-7), 37.4 (C-8), 24.5 (C-9), 37.4 (C-10), 32.7 (C-11), 37.3 (C-12), 24.8 (C-13), 39.4 (C-14), 28.0 (C-15), 22.7 , 19.7 , 16.2 (C-20).β-caryophyllene is in fact the main antibacterial component in “Yimucao oil”. However, it is very difficult to attribute the biological activities of a total essential oil to one or a few active principles, because the minor compounds also may be effective. In addition, it is known that the synergistic or antagonistic effect of a compound present in minor percentage in a mixture has to be considered as well [It can be concluded that  as well . Thus, f


H-pyridines were generated in a Michael addition–cyclocondensation with ethyl cyanoacetate. The peculiar product formation depends on the reaction conditions as well as on the electronic substitution pattern of the alkynone. While electron-donating groups furnish α-pyrones as main products, electron-withdrawing groups predominantly give the corresponding 1H-pyridines. Both heterocycle classes fluoresce in solution and in the solid state. In particular, dimethylamino-substituted α-pyrones, as donor–acceptor systems, display remarkable photophysical properties, such as strongly red-shifted absorption and emission maxima with daylight fluorescence and fluorescence quantum yields up to 99% in solution and around 11% in the solid state, as well as pronounced emission solvatochromism. Also a donor-substituted α-pyrone shows pronounced aggregation-induced emission enhancement.Starting from substituted alkynones, α-pyrones and/or 1 A high sensitivity and precise tuneability of fluorescence colors are prerequisites for the application of fluorescent substances in chemistry, medicine and materials science . With thIn recent years, we have coined diversity-oriented syntheses of functional chromophores by multicomponent strategies –18, openH-pyridines starting from diversely substituted alkynones and cyanoethylacetate. This bifurcating domino process furnishes small chromophore libraries which were characterized by photophysical studies (absorption and emission spectroscopy) and the studies on the electronic structure were accompanied by TD-DFT calculations for assigning the dominant longest-wavelength absorption bands.While most α-pyrones neither fluoresce in solution nor in the solid state specific substitution patterns have been identified for fluorophore design for this heterocyclic family. Tominaga and co-workers synthesized a series of α-pyrone derivatives with emission maxima between 400 and 675 nm in the solid state and between 486 and 542 nm in chloroform ,24–26, iRecently, we reported a straightforward access to α-pyrones through a consecutive alkynylation–Michael addition–cyclocondensation (AMAC) multicomponent synthesis . The rea1a), phenylacetylene (2a), and ethyl cyanoacetate (4) within the AMAC sequence (H-pyridine derivative 5a (2% yield) and on the other hand an aniline derivative with two ester groups (4% yield). Both compounds indicate that two molecules of ethyl cyanoacetate (4) were incorporated in the final structure.With this sequence in hand, we envisioned the variation of CH-acidic esters to generate differently 3-substituted α-pyrones. For introducing a cyano substituent we employed benzoyl chloride , while 1H-pyridine 5a can also be isolated in around 15% yield  the yields drops  and reaction temperature exert a significant impact on which heterocyclic compound is formed, we also tried to change the electronic nature of the starting material. Therefore, an electron-donating substituent was introduced in the alkynone yrone 6b .H-pyridine 5b was the only product  a reaction between α-pyrone 6a and ethyl cyanoacetate (4) under the same reaction conditions as for the 1H-pyridine from alkynone 3a was conducted, but only starting material could be isolated. Another option for the formation of the 1H-pyridine 5a was envisioned by an in situ generation of a dimer of ethyl cyanoacetate (4). The dimer 7 can be synthesized by iridium catalysis : 3092 (w), 2992 (w), 2963 (w), 2943 (w), 2876 (w), 2806 (w), 2193 (m), 1625 (m), 1620 (m), 1597 (m), 1577 (m), 1506 (w), 1466 (w), 1413 (w), 1396 (w), 1369 (w), 1308 (m), 1300 (m), 1283 (s), 1258 (m), 1206 (w), 1165 (m), 1115 (s), 1092 (m), 1082 (m), 1071 (m), 1045 (s), 1030 (m), 1015 (m), 980 (m), 976 (m), 968 (w), 920 (w), 885 (w), 874 (w), 837 (s), 829 (m), 764 (s), 745 (w), 727 (w), 685 (m), 662 (w), 655 (w), 650 (w); UV–vis (CH2Cl2) λmax [nm] (ε [L·mol−1·cm−1]): 274 (32000), 321 (18100), 428 (10000); emission (CH2Cl2) λmax [nm] (Stokes shift [cm−1]): 565 (5700); quantum yield (CH2Cl2) Φf: 0.01; Anal. calcd for C23H17F3N2O2 (410.1): C, 67.31; H, 4.18; N, 6.83; found: C, 67.50; H, 4.32; N, 6.70.1-Phenyl-3-[4-(trifluoromethyl)phenyl]prop-2-yn-1-one  was placed in a dry Schlenk tube and ethanol (2 mL) was added. Sodium carbonate , sodium acetate , water (1 mL), and ethyl cyanoacetate  were added and the mixture was stirred at 75 °C for 16 h. After the addition of CH2Cl2 (5.00 mL) and NaOH/FeSO4 solution (5.00 mL), the solution was extracted with CH2Cl2 (3 × 50.0 mL). The combined organic layers were dried (anhydrous MgSO4) and the solvent was removed in vacuo. The residue was purified by flash chromatography on silica gel (n-hexane/EtOAc 5:1 to 1:1 to 0:1) and washed with hot ethanol (2.00 mL) and compound 6c  was obtained as deep purple solid. Mp 224–253 °C; 1H NMR  δ 3.10 , 6.69 , 6.69–6.75 , 7.51–7.58 , 7.67–7.73 , 7.78–7.85 ; 13C NMR  δ 40.2 (CH3), 91.1 (Cquat), 100.1 (CH), 111.8 (CH), 115.7 (Cquat), 116.5 (Cquat), 128.0 (CH), 128.8 (CH), 129.3 (CH), 131.6 (CH), 135.3 (Cquat), 153.4 (Cquat), 160.3 (Cquat), 164.1 (Cquat), 164.9 (Cquat). EIMS ): 317 (15), 316 , 293 (11), 289 , 288 , 287 (19), 167 , 150 (11), 149 , 148 (20), 144 (13), 127 , 85 (13), 71 (22), 57 (18), 43 ; IR (ATR) ν̃ [cm−1]: 3092 (w), 3048 (w), 2901 (w), 2864 (w), 2812 (w), 2739 (w), 2212 (w), 1708 (m), 1706 (m), 1609 (m), 1589 (m), 1570 (m), 1530 (m), 1497 (m), 1491 (m), 1482 (m), 1478 (m), 1473 (m), 1467 (m), 1458 (m), 1433 (m), 1375 (m), 1360 (m), 1333 (m), 1252 (m), 1209 (m), 1171 (m), 1159 (m), 1125 (m), 1111 (m), 1082 (m), 1059 (m), 1020 (m), 995 (m), 953 (m), 945 (m), 924 (w), 853 (m), 818 (s), 795 (m), 750 (m), 748 (m), 692 (s), 669 (m), 640 (m); UV–vis (CH2Cl2) λmax [nm] (ε [L·mol−1·cm−1]): 294 (18800), 482 (47300); emission (CH2Cl2) λmax [nm] (Stokes-shift [cm−1]): 567 (3100); quantum yield (CH2Cl2) Φf: 0.99; emission (solid) λmax [nm]: 694; quantum yield (solid) Φf: 0.11; Anal. calcd for C20H16N2O2 (316.1): C, 75.93; H, 5.10; N, 8.86; found: C, 75.74; H, 5.19; N, 8.56.1-[4-(Dimethylamino)phenyl]-3-phenylprop-2-yn-1-one  was placed in a dry Schlenk tube and ethanol (1.00 mL) was added. Sodium carbonate , sodium acetate , water (50.0 µL) and diethyl (Z)-3-amino-2-cyanopent-2-endioate  were added and the mixture was stirred at 75 °C for 16 h. After the addition of CH2Cl2 (5.00 mL) and NaOH/FeSO4 solution (5.00 mL), the solution was extracted with CH2Cl2 (3 × 50.0 mL). The combined organic layers were dried (anhydrous MgSO4) and the solvent was removed in vacuo. The residue was purified by flash chromatography on silica gel (n-hexane/EtOAc 5:1 to 1:1 to 0:1) and washed with hot ethanol (5.00 mL) to furnish compound 8a  as yellow solid. Mp 140–146 °C; 1H NMR  δ 1.08 , 1.37 , 4.22 , 4.31 , 6.94 , 7.40–7.48 , 7.55–7.62 , 7.75–7.84 , 15.60 ; 13C NMR  δ 13.7 (CH3), 14.7 (CH3), 60.9 (CH2), 62.2 (CH2), 62.7 (Cquat), 112.2 (CH), 118.2 (Cquat), 122.8 (Cquat), 126.3 (CH), 127.9 (CH), 128.8 (CH), 129.6 (CH), 130.1 (CH), 131.77 (CH), 131.84 (Cquat), 137.4 (Cquat), 146.1 (Cquat), 151.9 (Cquat), 153.0 (Cquat), 165.1 (Cquat), 172.1 (Cquat); EIMS ): 415 , 414 , 369 , 342 (28), 341 , 340 , 314 (20), 313 , 298 (24), 297 , 296 , 287 (10), 286 (25), 271 (24), 270 , 269 (23), 268 , 266 (12), 258 , 245 (18), 241 (13), 240 (26), 231 (15), 230 , 203 (19), 202 (31), 164 (13); IR (ATR) ν̃ [cm−1]: 2978 (w), 2895 (w), 2197 (m), 1722 (m), 1636 (m), 1593 (s), 1578 (m), 1501 (m), 1489 (w), 1462 (w), 1441 (w), 1420 (w), 1364 (w), 1308 (m), 1288 (m), 1248 (s), 1188 (w), 1169 (m), 1134 (m), 1113 (s), 1092 (m), 1067 (m), 1047 (m), 1028 (w), 1001 (w), 885 (m), 854 (m), 847 (w), 775 (w), 758 (s), 746 (m), 694 (m), 658 (w); UV–vis (CH2Cl2) λmax [nm] (ε [L·mol−1·cm−1]): 274 (20300), 324 (20100), 417 (7700); emission (CH2Cl2) λmax [nm] (Stokes shift [cm−1]): 557 (6000); quantum yield (CH2Cl2) Φf = < 0.01; Anal. calcd for C25H22N2O4 (414.2): C, 72.45; H, 5.35; N, 6.76; found: C, 71.97; H, 5.45; N, 6.52.1,3-Diphenylprop-2-yn-1-one (3, 5, 6, and 8, 1H and 13C NMR, and absorption and emission spectra of compounds 5, 6, and 8, solid state emission spectra of compounds 5 and 6, X-ray structural data of compound 5a, and DFT/TDDFT calculations of compounds 5 and 6, see below.For experimental details of the synthesis and analytical data of compounds File 1Additional experimental and calculated data.


Kalanchoe daigremontiana turned out to be a rich source of bufadienolides. The existing literature data relate mainly to the aerial parts of Kalanchoe but there is no information about the metabolic profile of the roots, which are also used in traditional medicine. Our investigation concerning the roots of K. daigremontiana led to the isolation and characterization of eight new bufadienolides, namely 1β,3β,5β,14β,19-pentahydroxybufa-20,22-dienolide (1), 19-(acetyloxy)-1β,3β,5β,14β-tetrahydroxybufa-20,22-dienolide (2), 3β-O-α-l-rhamno-pyranosyl-5β,11α,14β,19-tetrahydroxybufa-20,22-dienolide (3), 19-(acetyloxy)-3β,5β,11α,14β-tetrahydroxybufa-20,22-dienolide (4), 3β,5β,11α,14β,19-pentahydroxy-12-oxo-bufa-20,22-dienolide (5), 19-(acetyloxy)-3β,5β,11α,14β-tetrahydroxy-12-oxo-bufa-20,22-dienolide (6), 19-(acetyloxy)-1β,3β,5β,11α,14β-pentahydroxy-12-oxo-bufa-20,22-dienolide (7) and 1β-(acetyloxy)-3β,5β,11α,14β,19-pentahydroxy-12-oxo-bufa-20,22-dienolide (8), together with seven known compounds: 11α,19-dihydroxytelocinobufagin (9), bersaldegenin-1-acetate (10), daigredorigenin-3-acetate (11), bersaldegenin-1,3,5-orthoacetate (12), bryotoxin B (13), bryophyllin B (14) and bersaldegenin (15). The structures were established applying extensive 1D- and 2D-NMR and MS spectroscopic analyses.An aqueous extract from the roots of  Kalanchoe daigremontiana (Bryophyllum daigremontianum) Raym.-Hamet & H. Perrier (common name: Mother of Thousands), from the Crassulaceae family, is a succulent herb native to the dry zones of Madagascar − in its HR-ESI-TOF-MS suggested that the molecular formula of 1 was C24H34O7 and indicated eight degrees of unsaturation. Its IR spectrum indicated the presence of hydroxyl (3423 cm−1), carbonyl (1714 cm−1), and olefin (1631 cm−1) functionalities. The UV spectrum implied the presence of a 2H-pyran-2-one moiety (λmax 299 nm). In the 1H-NMR spectrum of 1, a signal for one angular methyl [δH 0.74 ], one hydroxymethylene [δH 4.06  and δH 4.40 ] and a 2H-pyran-2-one unit  were observed. Analysis of 13C-NMR and DEPT spectra revealed that 1 possessed 24 carbon atoms, composed of six quaternary carbons, eight methines, nine methylenes and one methyl. The carbon signals at δC 115.5 (C-23), 125.0 (C-20), 149.3 (C-22), 150.5 (C-21) and 164.8 (C-24) were typical for a 2H-pyran-2-one moiety. This evidence indicated that 1 possessed a bufadienolide skeleton. All the 13C- and 1H-NMR signals  and C-5 (δC 76.5) suggested the existence of 5-OH, and correlation between Me-18 (δH 0.74) and C-14 (δC 85.9) suggested the existence of 14-OH. Furthermore, the correlation between H2-19 and C-1 (δC 71.7) indicated that the hydroxyl group was also located at C-1 . Small coupling constants measured between H-1 and both H-2α  and H-2β  suggested that the hydroxyl group at C-1 should be β-oriented. This was further supported by the NOE effect visible in the ROESY spectra between H-1 and H-11α . Additionally, the correlation visible in the HMBC spectra between H-1 and C-3 (δC 68.1), suggested that 1 was a 1,3,5,14,19-pentahydroxybufadienolide. The NOE correlations observed in the ROESY spectrum between H-8 (δH 1.80), Me-18 and H-19 suggested their 1,3-diaxial configuration and β-orientation. The cross-peaks in ROESY spectra between H-4α (δH 2.32)/H-2α (δH 1.98) and H-9 (δH 1.46) indicated that these protons were α-oriented. Additionally, the NOE correlations between Me-18 (δH 0.74) and H-21 (δH 7.42)/H-22 (δH 7.98) confirmed the β-orientation of the 2H-pyran-2-one unit  and 14-OH  suggested that the relative configuration of 14-OH was β, indicating C/D cis ring juncture, which was in line with the biogenesis pathway. However, to be more precise, Electronic Circular Dichroism (ECD) spectra were acquired . The UV spectrum indicated the presence of a 2H-pyran-2-one moiety. Comparison of the 1H- and 13C-NMR data of 2 with those of 1 revealed their structural similarity, except for the presence of the signals for a methyl  and carbonyl (δC 173.0) group in 2, characteristic of an acetoxy group. It was supported by a long-range correlation visible in the HMBC spectrum between downfield shifted H2-19 (δH 4.62 and 4.81) and the carbonyl group. Additionally, γ-effect was observed for C-5 (δC 75.3), which was shifted upfield by Δδ 1.2 ppm comparing to 1. In order to confirm relative stereochemistry of 2 ROESY spectra were acquired and presented similar to compound 1 NOE enhancements. Based on these results, the structure of 2 was deduced to be 19-(acetyloxy)-1β,3β,5β,14β-tetrahydroxybufa-20,22-dienolide.Compound 3 was obtained as a white powder. The molecular formula of 3 was determined to be C30H44O11 by its HR-ESI-TOF-MS data . The UV spectrum indicated the presence of a 2H-pyran-2-one moiety. The 1H- and 13C-NMR spectra of the genin part of 3 was very similar to that of 11α,19-dihydroxytelocinobufagin , which was correlated in HSQC spectra with the methyl carbon signal at δ 18.0 (C-6′) and five other oxymethine signals in TOCSY spectra at δH 3.40 , 3.63 , 3.65 , 3.78  and δC 70.6 (C-5′), 72.5 (C-3′), 72.6 (C-2′), 73.8 (C-4′). The α-orientation of an anomeric proton was confirmed by the 1JCH coupling constant (168 Hz). An HMBC correlation between H-1′ (δH 4.85) and C-3 (δC 76.0) and a NOE correlation visible in the ROESY spectra between H-1′ and H-3 (δH 4.15) established that the glycosidic moiety was linked to C-3 of the aglycone. The structure of 3 was thus established as 3β-(O-α-l-rhamnopyranosyl)-5β,11α,14β,19-tetrahydroxybufa-20,22-dienolide.Compound 4 was obtained as a white powder. The molecular formula of 4 was determined to be C26H36O8 by its HR-ESI-TOF-MS data . The UV spectrum indicated the presence of a 2H-pyran-2-one moiety. Comparison of the 1H- and 13C-NMR data of 4 with those of 9 revealed their structural similarity, except for the presence of the signals for a methyl  and carbonyl (δC 173.0) group in 4, characteristic for an acetoxy group. It was supported by a long-range correlation visible in the HMBC spectrum between downfield shifted H2-19 (δH 4.45 and 4.42) and the carbonyl group. Additionally, γ-effect was observed for C-5 (δC 75.9), which was shifted upfield by Δδ 2.8 ppm comparing to 9. Based on these results, the structure of 4 was deduced to be 19-(acetyloxy)-3β,5β,11α,14β-tetrahydroxybufa-20,22-dienolide.Compound 5 was obtained as a white powder. The molecular formula of 5 was determined to be C24H32O8 by its HR-ESI-TOF-MS data . The UV spectrum indicated the presence of a 2H-pyran-2-one moiety. Comparison of the 1H- and 13C-NMR spectra of 5 with those of a known group of compounds—the lucibufagins  instead of a methyl group. Thus, 5 was proposed to be a lucibufagin derivative. In the HMBC spectrum of 5, the correlation between H2-19 (δH 4.01 and 4.19) and C-1 (δC 21.9)/C-9 (δC 43.9)/C-10 (δC 45.4)/C-5 (δC 78.1) suggested the methylene group was located at C-19. Furthermore, the correlations between Me-18 (δH 0.92)/H-9 (δH 1.80) and C-12 (δC 214.5) indicated that oxo- group was located at C-12. It was supported by the downfield shift of H-11/C-11 at δH/δC 4.71/75.1, downfield shift of C-13 at δC 63.6, downfield shift of H-17 at δH 4.11, and γ-effect observed for C-17 (δC 42.1), which was shifted upfield by Δδ 10 ppm comparing to 1–4 . The UV spectrum indicated the presence of a 2H-pyran-2-one moiety. Comparison of the 1H- and 13C-NMR data of 6 with those of 5 revealed their structural similarity, except for the presence of the signals for a methyl  and carbonyl (δC 172.8) group in 6, characteristic for an acetoxy group. It was supported by a long-range correlation visible in the HMBC spectrum between downfield shifted H2-19 (δH 4.50 and 4.60) and the carbonyl group. Additionally, γ-effect was observed for C-5 (δC 75.9), which was shifted upfield by Δδ 2.2 ppm comparing to 5. Based on these results, the structure of 6 was deduced to be 19-(acetyloxy)-3β,5β,11α,14β-tetrahydroxy-12-oxo-bufa-20,22-dienolide.Compound 7 was obtained as a white powder. The molecular formula of 7 was determined to be C26H34O10 by its HR-ESI-TOF-MS data . The UV spectrum indicated the presence of a 2H-pyran-2-one moiety. The NMR spectroscopic features of 7 were similar to the compound 6. A detailed examination of 1D and 2D NMR data of 7 revealed that a methylene group located at C-1 in 6 was replaced by an oxygenated methine . In the 1H-1H COSY spectrum of 7, the methine proton  was correlated with H-2α (δH 2.14) and H-2β (δH 2.04), indicating that a hydroxyl group was attached to C-1. It was further supported by a correlation visible in the HMBC spectra between H2-19 (δH 4.85 and 5.00) and C-1 (δC 71.0). Appearance of H-1 as a broad singlet was observed, suggesting that the hydroxyl group at C-1 was β-oriented. Thus the structure of 7 was established as 19-(acetyloxy)-1β,3β,5β,11α,14β-pentahydroxy-12-oxo-bufa-20,22-dienolide.Compound 8 was obtained as a white powder. The molecular formula of 8 was determined to be C26H34O10 by its HR-ESI-TOF-MS data , which was the same as 7. The UV spectrum indicated the presence of a 2H-pyran-2-one moiety. Comparison of the 1H- and 13C-NMR spectra of 8 and 7 implied that these two compounds were almost the same, and differed only in the position of an acetoxy group. According to the HMBC spectral analysis, correlation between downfield shifted H-1 (δH 6.52) and a carbonyl group at δC 172.6, indicated the location of the acetoxy group at C-1 (δC 74.1). It was further supported by the correlation visible between shifted upfield H2-19 (δH 4.29 and 4.37) and C-9 (δC 46.5)/C-10 (δC 49.3)/C-1 (δC 74.1). The relative stereochemistry of 8 was determined to be the same as 7 based on the ROESY spectral analysis. The NOE correlations between H-8 (δH 2.49), Me-18 (δH 0.95) and H2-19, suggested their 1,3-diaxial configuration and β-orientation, as in 7. Thus, the structure of compound 8 was elucidated as 1β-(acetyloxy)-3β,5β,11α,14β,19-pentahydroxy-12-oxo-bufa-20,22-dienolide.Compound Kalanchoe daigremontiana. Wagner isolated bersaldegenin-1,3,5-orthoacetate, bersaldegenin-1-acetate, daigredorigenin-3-acetate and daigremontianin − ; ESI-MS/MS (TOF), m/z: 479 [M − H]−, 433 [M − H − 46]−; 13C- and 1H-NMR data are shown in 19-(Acetyloxy)-1β,3β,5β,14β-tetrahydroxybufa-20,22-dienolide (2): White powder (11.2 mg); c 0.39, MeOH); ECD  λmax (Δε) 220 (+1.90), 247 (-0.83), 300 (+0.46); IR υmax: 3430 (OH), 2945 and 2878 (CH), 1715 (C=O), 1632 (C=C), 1539, 1247, 1124, 1083, 1038 cm−1; UV (UPLC-PDA) λmax (nm): 299; HR-ESI-TOF-MS, m/z: 475.2338 [M − H]− ; ESI-MS/MS (TOF), m/z: 521 [M − H]−, 475 [M − H − 46]−, 433 [M − H − 46 − 42]−; 13C- and 1H-NMR data are shown in l-Rhamnopyranosyl)-5β,11α,14β,19-tetrahydroxybufa-20,22-dienolide3β-(O-α- (3): White powder (11 mg); c 0.27, MeOH); ECD  λmax (Δε) 220 (+2.39), 249 (−0.75), 300 (+0.22); IR υmax: 3413 (OH), 2934 (CH), 1705 (C=O), 1631 (C=C), 1539, 1126, 1037 cm−1; UV (UPLC-PDA) λmax (nm): 297; HR-ESI-TOF-MS, m/z: 579.2808 [M − H]− ; ESI-MS/MS (TOF), m/z: 625 [M − H]−, 579 [M − H − 46]− 433 [M − H − 46 − 146]−; 13C- and 1H-NMR data are shown in 19-(Acetyloxy)-3β,5β,11α,14β-tetrahydroxybufa-20,22-dienolide (4): White powder (6.1 mg); c 0.30, MeOH); ECD  λmax (Δε) 225 (+1.25), 251 (−0.25), 279 (+0.41), 300 (+0.25); IR υmax: 3412 (OH), 2932 and 2876 (CH), 1712 (C=O), 1630 (C=C), 1251, 1125, 1037 cm−1; UV (UPLC-PDA) λmax (nm): 299; HR-ESI-TOF-MS, m/z: 475.2339 [M − H]− ; ESI-MS/MS (TOF), m/z: 521 [M − H]−, 475 [M − H − 46]−, 433 [M − H − 46 − 42]−, 415 [M − H − 46 − 60]−; 13C- and 1H-NMR data are shown in 3β,5β,11α,14β,19-Pentahydroxy-12-oxo-bufa-20,22-dienolide (5): White powder (15.4 mg); c 0.54, MeOH); ECD  λmax (Δε) 217 (+1.09), 245 (−0.05), 284 (+0.85); IR υmax: 3413 (OH), 2929 and 2879 (CH), 1709 (C=O), 1632 (C=C), 1539, 1250, 1131, 1019 cm−1; UV (UPLC-PDA) λmax (nm): 299; HR-ESI-TOF-MS, m/z: 447.2021 [M − H]− ; ESI-MS/MS (TOF), m/z: 493 [M − H]−, 447 [M − H − 46]−; 13C- and 1H-NMR data are shown in 19-(Acetyloxy)-3β,5β,11α,14β-tetrahydroxyl12-oxo-bufa-20,22-dienolide (6): White powder (14.7 mg); c 0.43, MeOH); ECD  λmax (Δε) 218 (+1.01), 245 (−0.21), 279 (+0.98); IR υmax: 3423 (OH), 2941 and 2879 (CH), 1711 (C=O), 1634 (C=C), 1540, 1247, 1133, 1029 cm−1; UV (UPLC-PDA) λmax (nm): 301; HR-ESI-TOF-MS, m/z: 489.2125 [M − H]− ; ESI-MS/MS (TOF), m/z: 535 [M − H]−, 489 [M − H − 46]−; 447 [M − H – 46 − 42], 429 [M − H – 46 − 60]−; 13C- and 1H-NMR data are shown in 19-(Acetyloxy)-1β,3β,5β,11α,14β-pentahydroxy-12-oxo-bufa-20,22-dienolide (7): White powder (4.8 mg); c 0.27, MeOH); ECD  λmax (Δε) 213 (+1.18), 246 (−0.12), 285 (+0.75); IR υmax: 3429 (OH), 2944 and 2880 (CH), 1712 (C=O), 1633 (C=C), 1539, 1247, 1124, 1036 cm−1; UV (UPLC-PDA) λmax (nm): 298; HR-ESI-TOF-MS, m/z: 505.2090 [M − H]− ; ESI-MS/MS (TOF), m/z: 551 [M − H]−, 505 [M − H − 46]−, 463 [M − H − 46 − 42], 445 [M − H – 46 − 60]−; 13C- and 1H-NMR data are shown in 1β-(Acetyloxy)-3β,5β,11α,14β,19-pentahydroxy-12-oxo-bufa-20,22-dienolide (8): White powder (4.6 mg); c 0.13, MeOH); ECD  λmax (Δε) 222 (+1.33), 284 (+0.85); IR υmax: 3441 (OH), 2947 and 2880 (CH), 1712 (C=O), 1632 (C=C), 1539, 1247, 1126, 1033 cm−1; UV (UPLC-PDA) λmax (nm): 298; HR-ESI-TOF-MS, m/z: 505.2088 [M − H]− ; ESI-MS/MS (TOF), m/z: 551 [M − H]−, 505 [M − H − 46]−, 463 [M − H – 46 − 42], 445 [M − H – 46 − 60]−; 13C- and 1H-NMR data are shown in 3 (0.50 mg) was hydrolysed in 1 mL solution of 3 M HCl  at 90 °C for 2 h. After dioxane was removed from the hydrolysis reaction mixture under a stream of nitrogen, aglycones were extracted with EtOAc (3 × 1 mL) and dried. The aqueous layer containing sugar was neutralized using an Amberlite IRA-400 ion exchange resin in its hydroxide form, concentrated under a stream of nitrogen at 60 °C and afterwards used for the sugar determination. To determine the absolute configuration of monosaccharide of the isolated compound, a modified method of Tanaka et al. was used [l-cysteine methyl ester hydrochloride was added. The mixture was kept at 60 °C for 1 h. 0.2 µL of o-tolyl isothiocyanate was added to the mixture, which was heated at 60 °C for 1 h. After cooling, the reaction mixture was directly analysed by UPLC-ESI-MS/MS. Chromatographic separation was carried out on an Acquity BEH C18 column . Mass spectrometry analyses were performed in a positive ionization mode, using the SRM method. Details of the analysis can be found in the work of Pérez, et al. [l-Rhamnose (Rha) was identified on the basis of the retention time (Rt) and m/z value of an authentic standard, derivatized in the same way as the sample .Compound was used . Sugars , et al. . l-RhamnKalanchoe deigremontiana are a rich source of various bufadienolides. In the present study, eight bufadienolides which have not been reported previously in the scientific literature, were isolated from the aqueous extract and named: 1β,3β,5β,14β,19-pentahydroxybufa-20,22-dienolide (1), 19-(acetyloxy)-1β,3β,5β,14β-tetrahydroxybufa-20,22-dienolide (2), 3β-(O-α-l-rhamnopyranosyl)-5β,11α,14β,19-tetrahydroxybufa-20,22-dienolide (3), 19-(acetyloxy)-3β,5β,11α, 14β-tetrahydroxy-bufa-20,22-dienolide (4), 3β,5β,11α,19-pentahydroxy-12-oxo-bufa-20,22-dienolide (5), 19-(acetyloxy)-3β,5β,11α,14β-tetrahydroxy-12-oxo-bufa-20,22-dienolide (6), 19-(acetyloxy)-1β,3β,5β,11α,14β-pentahydroxy-12-oxo-bufa-20,22-dienolide (7), and 1β-(acetyloxy)-3β,5β,11α,14β, 19-pentahydroxy-12-oxo-bufa-20,22-dienolide (8), together with seven known compounds: 11α,19-dihydroxytelocinobufagin (9), bersaldegenin-1-acetate (10), daigredorigenin-3-acetate (11), bersaldegenin-1,3,5-orthoacetate (12), bryotoxin B (13), bryophyllin B (14), and bersaldegenin (15). Most of these compounds are acetylated in position 19 or 1, and only one contains a sugar moiety—the bufadienolide 2. The discovery of these new compounds from K. daigremontiana provides more insight into the bufadienolides of the Kalanchoe genus.In conclusion, roots of


The scope and limitations of the protocol were investigated, showing good tolerance of acyl, cyano, and ester functional groups in the amide counterpart while halo group competed via the classical Suzuki coupling. The trialkylboranes generated in situ by hydroboration of olefins with BH3 or 9-BBN performed similarly to those separately prepared, making this protocol more practical.A highly efficient acylative cross-coupling of trialkylboranes with activated amides has been effected at room temperature to give the corresponding alkyl ketones in good to excellent yields by using 1,3-bisphenylimidazolylidene and 3-chloropyridine co-supported palladium chloride, the PEPPSI catalyst, in the presence of K N-acyl imides + 253.1568, found 253.1569.3-Phenyl-1-(thiophen-2-yl)butanone (3m) [1H-NMR  δ(ppm): 7.70–7.69 , 7.64–7.63 , 7.36–7.28 , 7.25–7.23 , 7.14–7.12 , 3.56–3.51 , 3.24 , 3.14 , 1.38 . 13C-NMR  δ(ppm): 191.94 (C=O), 146.18 (C1Ph), 144.60 (C2 thiophenyl), 133.56 (thiophenyl), 131.81 (thiophenyl), 128.49(CPh), 128.01(thiophenyl), 126.78 (CPh), 126.30 (C4Ph), 47.78 (COCH2), 35.93 (CH), 21.64 (CH3).one (3m) : Colorle1-Phenylnonanone (3n) [1H-NMR  δ(ppm): 7.97–7.95 , 7.57–7.53 , 7.47–7.44 , 2.96 , 1.77–1.69 , 1.39–1.25 5), 0.88 . 13C-NMR  δ(ppm): 200.62 (C=O), 137.05 C1Ph), 132.83 (C4Ph), 128.51 (CPh), 128.02 (CPh), 38.61 (COCH2), 31.81 (CH2), 29.42 (CH2), 29.36 (CH2), 29.15 (CH2), 24.36 (CH2), 22.63 (CH2), 14.08 (CH3).one (3n) : Colorle2CO3 performed best in MTBE at room temperature. The reaction proceeded to give alkyl ketones in good to excellent yields, tolerating a variety of functional groups in the amide counterpart. Unlike the high-order arylboron compounds, in which all the aryl groups react effectively, only one of the three primary alkyl groups in trialkylboranes could be used as alkyl source for the acyl alkylation. The trialkylboranes generated in situ by hydroboration of olefins with BH3 or 9-BBN performed comparably to those separately prepared. This protocol complements ketone synthesis via palladium-catalyzed acylative cross-coupling of amides, providing a feasible access to both monoalkyl and dialkyl ketones.In summary, we have developed a highly efficient acylative cross-coupling of trialkylboranes with activated amides by using 1,3-bisphenylimidazolylidene and 3-chloropyridine co-supported palladium chloride, the PEPPSI catalyst, under mild conditions. Bases appeared to play a key role in the reaction, among which K


Their structures were determined on the basis of extensive spectroscopic analyses and comparison with literature data. Compound 1 was evaluated for in vitro cytotoxicity activity against HL-60, SMMC-7721, A549, MCF-7, SW480, and BEAS-2B cell lines.A new sesquilignan glucoside, urariasinoside A ( Uraria (family Leguminosae), consists of 35 species, distributed throughout the tropical regions of Africa, Asia, and Australia + at m/z 743.2863 in the HRESIMS spectrum. The UV spectrum suggested the existence of conjugated groups on the basis of maximum absorption bands at 228 nm and 280 nm. The IR spectrum exhibited the presence of hydroxyl groups (3364 cm−1) and benzene rings (1603 cm−1 and 1513 cm−1). Its 1H-NMR spectrum , 6.63 , and 6.50 ; 6.61 , 6.70 , and 6.53 ; 7.12 , 6.74 , and 6.86 . Its 13C-NMR and DEPT spectral data  and three methoxy groups . Subsequently, 27 carbons were assigned to a sesquilignan moiety, including three groups of aromatic carbons: nine quaternary carbons , and nine unsubstituted aromatic carbons . The remaining 9 carbons were ascribed to five methylene carbons , and four methine carbons . These before mentioned 1H- and 13C-NMR data implied that compound 1 should be a sesquilignan glucoside.Compound spectrum  showed tral data  revealed1H-1H COSY spectrum  and 2.62  to methine proton at δH 1.87 , and from H-8 to the methylene protons at δH 3.56  and 3.82  suggested the linkage of the C3 fragment of 7CH2-8CH-9CH2OH. The 1H–1H COSY correlation of CH2-7' with CH-8' and the correlation of CH-8' with CH2-9' revealed the connectivity of the C3 unit 7'CH2-8'CH-9'CH2OH. Additionally, the 1H-1H COSY correlation of the oxygenated methine proton at δH 5.17  with the oxygenated methine proton at δH 4.30 , and the correlation of H-8'' with the oxygenated methylene protons at δH 3.51  and 3.84  disclosed the fragment of the third the C3 unit 7''CH(O)-8''CH(O)-9''CH2OH. These three C3 units were connected to the above three sets of 1,3,4-trisubstituted phenyl groups respectively, according to the HMBC correlations , corresponding to six carbon signals at δc 101.1, 75.3, 77.8, 71.9, 77.8, 62.8 in 13C-NMR spectrum. The β-glucose was located at C-7'' as elucidated by the HMBC correlation between Glc-H-1''' and the oxygenated methine carbon C-7'', which was further confirmed by the NOESY correlation between H-1''' and H-7''. Herein, 1 was identified as a secoisolariciresinol-sesquilignan glucoside. Furthermore, the presence of β-D-glucose was determined by GC analysis of its trimethylsilyl thiazolidine derivative after acid hydrolysis  (1). Pale yellow oil. [α]20D: –29 ; UV (MeOH) λmax (logε) nm: 280 (3.32), 228 (3.68), 207 (4.03); CD  λmax (Δε) 211 (+ 11.48), 229 (+ 1.30), 239 (− 3.74), 281 (+ 2.71), 297 (+ 1.37), 322 (+ 1.83); IR (KBr) νmax 3364, 2935, 1603, 1513, 1453, 1423, 1369, 1268, 1224, 1155, 1127, 1075, 1029 cm−1; 1H- and 13C-NMR: see m/z: 743.2863 .1 (1.0 mg), in 2 M aqueous CF3COOH (2.0 mL) was heated at 100 °C for 3 h in a water bath. The reaction mixture was diluted in H2O (4.0 mL) and extracted with EtOAc (4.0 mL × 3), then the aqueous layer was concentrated to remove CF3COOH. The residue was dissolved in pyridine (1.0 mL), to which L-cysteine methyl ester hydrochloride (2.0 mg) in pyridine (1.0 mL) was added. Then, the mixture was kept at 60 °C for 2 h. After the reaction mixture was dried in vacuo, the residue was trimethylsilylated with 1-trimethylsilylimidazole (0.2 mL) at 60 °C for 2 h in a water bath. Finally, the mixture was partitioned between hexane and H2O (0.3 mL each) and the hexane extract was analyzed by gas chromatography (GC) under the following conditions: column temperature, 250 °C; injection temperature, 250 °C; carrier N2 gas; flow rate 1.0 mL/min. In the acid hydrolysate of 1, D-Glucose was confirmed by comparison of the retention times of their derivatives with those of D-glucose and L-glucose derivatives prepared in a similar way, which showed retention times of 13.66 and 14.34 min, respectively.A solution of 1 (2.67 mg) in 0.1 M acetate buffer  was treated with cellulase (3.0 mg) and then the reaction mixture was stirred at 40 °C for 12 h. The reaction mixture was diluted in H2O (4.0 mL) and extracted with EtOAc (4.0 mL × 3). After that, the EtOAc extract was further purified by semi-preparative HPLC  to obtain compounds 1a .A solution of Erythro-(–)-secoisolariciresinol-sesquilignan (1a). Pale yellow oil. [α]20D: –7.1 ; 1H-NMR  δH: 7.03 , 6.93 , 6.86 , 6.76 , 6.68 , 6.66 , 6.64 , 6.63 , 6.54 , 4.88 , 4.20 , 3.86 , 3.82 , 3.77 , 3.75 , 3.72 , 3.69 , 3.58 , 3.46 , 2.67 , 2.60 , 2.06 ; HRESIMS m/z: 581.2344 .1 was tested against five human cancer cell lines  and a normal cell line (BEAS-2B). The antiproliferative assay was performed by the MTT colorimetric method as described previously [5 cells/mL. After 12 h, cells were treated with the medium containing different concentrations of test compounds for 48 h. Then, attached cells were incubated with MTT  and subsequently solubilized in DMSO. The optical density of absorbency at 595 nm was then measured using a microplate reader. Experiments were performed in triplicate, and the values are the averages of three (n = 3) independent experiments. DDP  and taxol were used as the positive control.Compound eviously . BrieflyU. sinensis, revealed the presence of a new sesquilignan glucoside, urariasinoside A (1), was isolated from the aerial parts of U. sinensis together with eight known compounds, including two lignans (–)-syringaresinol (2), (–)-medioresinol (3); a sesquilignan (–)--4',4''-dihydroxy-3,3',3'',5,5'-pentamethoxy-7,9':7',9-diepoxy-4,8''-oxy-8,8'-sesquineolignan-7'',9''-diol (4); a dilignan (+)--4'',4'''-dihydroxy-3,3',3'',3''',5,5'-hexamethoxy-7,9':7',9-diepoxy-4,8'':4',8'''-bisoxy-8,8'-dineolignan-7'',7''',9'',9'''-tetraol (5), and four flavonoids derivatives 5,4'-dihydroxy-3'-methoxyflavanone-7-(6''-O-β-l-rhamnopyranosyl)-β-d-glucopyranoside (6), naringenin (7), quercetin (8), and kaempferol (9). Compounds 2–9 were isolated from U. sinensis for the first time. Cytotoxicity assays revealed that compound 1 was inactive (IC50 > 40 μM) against the HL-60, SMMC-7721, A549, MCF-7, and SW480 cell lines.Studies carried out on the EtOH extracts of


All these compounds were isolated for the first time from this source. Their structures were elucidated on the basis of extensive spectroscopic analysis and chemical methods. In addition, the in vitro cytotoxic activity of these compounds was evaluated. The results showed that none of the compounds had any significant inhibitory activities on the proliferation of HCT-116 and SW-620 cells.  Jasminum giraldii Diels (Oleaceae), is an endemic plant which is found distributed at an altitude of 300–1,500 m, in valleys and shrubbery of the Qinba Mountains in China’s Shaanxi Province 20D: +47.2, indicating that it was D-glucose + , The UV absorption bands of 2 appeared at 278, 216 and 203 nm in CH3OH. The IR spectrum of 2 showed the presence of hydroxyl , conjugated-ester carbonyl (1714 cm−1), α,β-unsaturated olefinic  and aromatic ring  functionalities. The 1H-NMR data of 2 , 6.85 , 6.61  and 6.90 , 6.78 , 6.77 ; and a set of cinnamyl proton signals at δ 7.61 , 7.43 , 7.68 , 6.56  in the aromatic region. In addition, resonances assignable to two aromatic methoxyl proton signals at δ 3.86 , 3.85 , and an anomeric proton signal at δ 4.88  were observed. Furthermore, in the 1H-NMR spectrum, an O-bearing methine proton signals at δ 4.72 , an aliphatic methylene proton signals at δ 2.85 , 2.38 , two oxygenated methylene proton signals at δ 3.88, 3.62 , 3.76 , 3.60 , and two aliphatic methine proton signals at δ 2.59 , 2.31  were indicated. Besides the carbon resonances corresponding to the above units, the 13C-NMR spectrum (δ 168.37 (C-9''); a set of a β-glucose moiety carbon signals at δ 102.87 (C-1'''), 77.95 (C-3'''), 75.61 (C-5'''), 75.03 (C-2'''), 72.13 (C-4'''), 64.96 (C-6'''). The key HMBC correlations  exhibited a HMBC correlation with C-9'', which confirmed that the cinnamyl moiety was linked to C-6'''. In the NOESY spectrum of 2 -lariciresinol 4'-(6''-O-feruloyl-β-D-glucopyranoside) + ; 1H-NMR  and 13C-NMR  data, see (−)-Lariciresinol 4-(6'''-O-cinnamyl-β-D-glucopyranoside) (2): white powder; C35H40O12; max (MeOH) λmax 278, 216 and 203 nm; IR(KBr) νmax 3498, 3458, 3384, 2925, 2854, 1714, 1645, 1635, 1512, 1456, 1269, 1226, 1161, 1124, 1105, 1072, 1035 cm−1; HR-TOF-MS m/z 653.2590 [M+H]+ ; 1H-NMR  and 13C-NMR  data, see (+)-Pinoresinol(3): yellow powder; C20H22O6; max (MeOH) λmax 280, 231 and 207 nm; IR (KBr) νmax 3504, 3438, 3379, 2958, 2939, 2860, 1604, 1514, 1460, 1438, 1271, 1029 cm−1; ESI-MS m/z 357 [M−H]−; 1H-NMR  δ 7.47 , 6.99 , 6.84 , 6.79 ,4.67 , 4.20 , 3.84, 3.81 , 3.08 ; 13C-NMR  δ 148.40 , 146.94 , 134.27 , 119.69 , 115.62 , 110.67 , 86.72 , 72.29 , 56.33 (3-OCH3 and 3'-OCH3), 55.32 .(+)-Cycloolivil (4): yellow powder; C20H24O7; max (MeOH) λmax 283, 230 and 204 nm; IR (KBr) νmax 3517, 3444, 3313, 3201, 2939, 2900, 2835, 1699, 1598, 1517, 1465, 1446, 1433, 1373, 1269, 1236, 1215, 1149, 1124, 1022, 881, 858, 838, 811 cm−1; ESI-MS m/z 375 [M−H]−; 1H-NMR  δ 6.76 , 6.76 , 6.67 , 6.63 , 6.18 , 4.03 , 3.80 , 3.77 , 3.74 , 3.59 , 3.23 , 2.62 , 2.04 ; 13C-NMR  δ 149.27 (C-3), 147.64 (C-3'), 146.25 (C-4), 145.44 (C-4'), 138.62 (C-1'), 133.69 (C-1), 126.57 (C-6'), 123.71 (C-6), 117.48 (C-5), 116.15 (C-5'), 114.06 (C-2), 113.09 (C-2'), 75.10 (C-8'), 69.55 (C-9), 60.98 (C-9'), 56.52 (OCH3), 56.49 (OCH3), 47.69 (C-8), 45.03 (C-7), 40.06 (C-7').Nobiletin (5): white powder; C9H12O2; max (MeOH) λmax 328, 269, 249 and 208 nm. IR (KBr) νmax 2947, 2854, 1629, 1598, 1514, 1461, 1377, 1278, 1149, 1107, 1078, 1012, 973, 838 cm−1; ESI-MS m/z 403 [M+H]+; 1H-NMR  δ 7.66 , 7.59 , 7.15 , 6.62 , 4.07 , 4.04 , 3.96 , 3.92 , 3.89 , 3.85 ; 13C-NMR  δ 176.74 (C-4), 161.67 (C-2), 153.38 (C-4'), 152.4 (C-7), 150.72 (C-3'), 149.22 (C-9), 148.64 (C-8), 145.15 (C-5), 139.24 (C-6), 124.85 (C-1'), 120.39 (C-6'), 115.91 (C-10), 112.69 (C-5'), 110.01 (C-2'), 107.29 (C-3), 62.44, 62.37, 62.04, 61.98, 56.34, 56.31.1 in H2O (3 mL) was hydrolysed with β-glucosidase  at 37 °C for 24 or 36 h. The reaction mixture was extracted with EtOAc for three times (3 × 3 mL) to yield the EtOAc exrract and H2O phase after removing the solvents. The aqueous phase of the hydrolysate of compound 1 was subjected to CC over silica gel eluted with MeCN/H2O (8:1) to yield glucose with positive optical rotations, and the value of 2O). The solvent system MeCN/H2O (6:1) was used for the TLC analysis of glucose and authentic sugar samples.A solution of compound 2 in 0.5 M H2SO4 (2.0 mL) was heated under reflux for 3 h. After cooling, the reaction mixture was diluted with H2O, neutralized with BaCO3, then filtered. The solution was extracted with EtOAc for three times (3 × 3 mL) to obtained the EtOAc extract and H2O layer after removing the solvents. The aqueous layer of the hydrolysate of compound 2 was evaporated and to yield glucose with positive optical rotations, and the value of c 0.11, H2O). The solvent system MeCN/H2O (6:1) was used for the TLC analysis of glucose and authentic sugar samples.A solution of compound 2. Cell proliferation of HCT-116 cells was assessed by conducting colorimetric MTT cell proliferation assay. Briefly, a limited number of growing cells were seeded into 96-well cell culture plate and were maintained for 24 h so that they became attached to the bottom of the well. The medium was aspirated and new medium (200 µL) including three groups of concentrations  of compounds were added to parallel wells. The cells were incubated at 37 °C in a humidified atmosphere for 24 h. Twenty µL of MTT solution was added and the incubation continued for 4 h. The pure formazan product was then solubilized by 150 µL DMSO. The plates were read at 570 nm using a microtiter plate reader.The human colon cell line HCT-116 and SW620 was obtained from the Shanghai Institute of Cell Biology. The cells were maintained in DMEM medium supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 mg/mL) at 37 °C in a humidified atmosphere with 5% CO1, 2 were new glycosides and together with compounds 3–5 were isolated from the Jasminum genus for the first time. Compounds 1–5 were assayed for their cytotoxic activity, and the data proved that none of the compounds had any significant inhibitory activities on proliferation of HCT-116 cells and SW-620 cells at the concentrations of 1–10 μM.Compounds


When L-proline is used as an additional promoter to obtain coumarins from o-hydroxybenzaldehydes, the reaction also proceeds in high yields. Work-up is very simple and the ionic liquid can be reused several times. Some of the coumarins obtained are described for the first time.The ionic liquid 1,3-dimethylimidazolium methyl sulfate, [MMIm][MSO The Knoevenagel condensation  is one o4] containing a small amount of water, without any other promoter, can act efficiently as the reaction medium and catalyst of the Knoevenagel condensation of 4-substituted benzaldehydes. We report here the results of optimizing the amount of water in the medium and of applying this reaction to the synthesis of coumarins.Over the past decade, ionic liquids (ILs) have attracted great interest because of their unusual properties, including negligible vapour pressure, broad liquid temperature range, and high capacity as specific solvents . Their u4] could act as both solvent and catalyst  was obtained as a pure white solid that was identified by comparison of its physical and spectroscopic data with those reported in the literature  absorbs 2.16% of water when exposed to the atmosphere (water content was determined using a Karl Fischer 756 coulometer), we then used the IL without drying and without adding either water or any other promoter. To evaluate the influence of recycling on the efficiency of undried [MMIm][MSO4], the ethyl acetate used to extract the reaction product was evaporated under vacuum and the medium was reused for the same reaction. After a total of six such cycles, yield was still high (96%), though the reaction time had increased to 4 h coumarin  was stirred in a round-bottomed flask until completion of the reaction as indicated by TLC (eluent: 4:1 hexane:AcOEt). The reaction mixture was then extracted with ethyl acetate (3 × 2 mL), and the pooled organic extract was concentrated under vacuum, affording a pure solid.A mixture of benzaldehyde  and malononitrile  in [MMIm][MSO4] (2 mL) was stirred in a round-bottomed flask until completion of the reaction as indicated by TLC (eluent: 8:2 hexane:AcOEt). The reaction mixture was then extracted with ethyl acetate (3 × 2 mL), and the pooled organic extract was concentrated under vacuum, affording a pure solid.A mixture of salicylaldehyde , dimethyl malonate  and Reaction and work-up as above afforded a solid that required no further purification. The reaction medium was recovered by evaporation of ethyl acetate under vacuum, and was reused.3-(Methoxycarbonyl)-6-methyl-2H-chromen-2-one  in undried [MMIm][MSO4] (2 mL) afforded the corresponding product; yield: 0.220 g (87%); white crystals (EtOH); mp 127–128 °C; 1H-NMR  δ 8.53 , 7.47 , 7.40 , 7.28 , 3.97 , 2.44 ; 13C-NMR  δ 163.8, 157.0, 153.4, 149.2, 135.7, 134.8, 129.2, 117.7, 117.6, 116.5, 52.9, 20.7; νmax(NaCl)/cm−1 3034, 2942, 2844, 1755; Electrospray MS (micrOTOF Focus) m/z (%) 241 [M + Na+] (52), 220 [M + H+ + 1] (13), 219.06575 [M + H+] , 203 (30), 189 (18), 171 (17).Entry 5, : The rea3-(Methoxycarbonyl)-8-methyl-2H-chromen-2-one  in undried [MMIm][MSO4] afforded the corresponding product; yield: 0.245 g (94%); yellow needles (EtOH); mp 106–107 °C; 1H-NMR  δ 8.56 , 7.51 , 7.45 , 7.24 , 3.97 , 2.48 ; 13C-NMR : 163.9, 156.9, 153.6, 149.6, 135.7, 127.2, 126.4, 124.4, 117.6, 117.4, 52.9, 15.4; νmax(NaCl)/cm−1 3051, 2953, 2847, 1761; Electrospray MS (micrOTOF Focus) m/z (%) 257 [M + K+] (7), 242 [M + Na+ + 1] (8), 241 [M + Na+] (65), 220 [M + H+ + 1] (17), 219.06524 [M + H+] , 201 (9), 187 (29), 175 (4).Entry 7, : The rea6-Bromo-8-methoxy-3-(methoxycarbonyl)-2H-chromen-2-one  in undried [MMIm][MSO4] (2 mL) afforded the corresponding product; yield: 0.310 g (99%); white powder (EtOH); mp 209–210 °C; 1H-NMR  δ 8.45 , 7.32 , 7.26 , 3.98 , 3.96 ; 13C-NMR  δ 163.4, 155.5, 148.0, 147.7, 144.0, 122.5, 119.3, 119.2, 118.8, 117.1, 56.6, 53.1; νmax(NaCl)/cm−1 3080, 2965, 1761; Electrospray MS (micrOTOF Focus) m/z (%) 337 [M + Na+ + 2] (59), 335 [M + Na+] (60), 315 [M + H+ + 2] (43), 312.97004 [M + H+] , 259 (17), 233 (11), 203 (100), 189 (84), 175 (50), 171 (26), 159 (15), 157 (9).3-(Methylsulfonyl)-2H-chromen-2-one  in undried [MMIm][MSO4] (2 mL) afforded the corresponding product; yield: 0.3214 g (96%); white needles (EtOH); mp 184–185 °C; 1H-NMR  δ 8.67 , 7.76 , 7.45 , 3.36 ; 13C-NMR  δ 156.0, 155.3, 147.6, 135.5, 130.4, 127.6, 125.6, 117.2, 117.1, 41.7; νmax(NaCl)/cm−1 3052, 2933, 1743; Electrospray MS (micrOTOF Focus) m/z (%) 248 [M + Na+ + 1] (14), 247 [M + Na+] (87), 226 [M + H+ + 1] (10), 225.02098 [M + H+] .7-Hydroxy-3-(methylsulfonyl)-2H-chromen-2-one  in undried [MMIm][MSO4] (2 mL) afforded the corresponding product; yield: 0.260 g (99%); yellow needles (EtOH); mp 295–296 °C; 1H-NMR  δ 8.65 , 7.73 , 6.91 , 6.80 , 3.29 ; 13C-NMR  δ 166.8, 159.3, 158.4, 149.4, 133.7, 123.4, 115.9, 111.6, 103.5, 42.0; νmax(NaCl)/cm−1 3297, 3062, 2933, 1712; Electrospray MS (micrOTOF Focus) m/z (%) 263 (M + Na+) (100), 241.01573 (M + H+), , 225 (22), 201 (60).7-Bromo-3-(methylsulfonyl)-2H-chromen-2-one  in undried [MMIm][MSO4] (2 mL) afforded the corresponding product; isolated yield: 0.141 g (93%); yellow powder (EtOH); mp 254–255 °C; 1H-NMR  δ 8.61 , 7.65 , 7.58 , 3.35 ; 13C-NMR  δ 155.3, 155.2, 146.8, 131.0, 130.3, 129.3, 127.7, 120.5, 115.9, 41.7; νmax(NaCl)/cm−1 3066, 2927, 1749; Electrospray MS (micrOTOF Focus) m/z (%) 327 (M + Na+ + 2) (84), 325 (M + Na+) (64), 305 (M + H+ + 2) (88), 302.93216 (C10H8O4SBr requires 302.93212) (100), 201 (56).4], an inexpensive, easily prepared, low-viscosity ionic liquid with low cytotoxicity, can act efficiently as the solvent and catalyst of Knoevenagel condensation reactions between 4-substituted benzaldehydes and active methylene compounds without any promoter other than a small amount of water (≈2%) such as can be introduced simply by exposure to air; these reactions are clean and are completed at room temperature within a few minutes, work-up is simple, and the reaction medium can be reused several times without significant reduction in yield. When used to cyclize o-hydroxybenzaldehydes with active methylene compounds to obtain coumarins it was necessary to use L-proline as promoter: Heating the reagents and promoter at 90 °C in undried [MMIm][MSO4] afforded high yields of the desired coumarins. Some of the coumarins are described for the first time.1,3-Dimethylimidazolium methyl sulfate, [MMIm][MSO