The structures of these new compounds were characterized by NMR, HRESIMS, quantum chemical computation, and X-ray diffraction analysis. Compound 1 features a 7-oxabicyclo[2.2.1]heptane-2-ene core, representing the first example of germacrane-type sesquiterpene dimer fused via a spiro ring system. Compound 2 is a novel sesquiterpene with a completely new carbon skeleton, which is characteristic of an additional carbon attaching to the cadinane backbone via a carbon–carbon bond. Additionally, compounds 2 and 4 exert acceptable cytotoxicity toward normal cells and high selectivity in cancer cells, especially in HepG2 cells.A novel sesquiterpene dimer, spirocommiphorfuran A ( Commiphora genus in the family Burseraceae comprises over 150 species, spreading over the tropical and subtropical regions, especially occurring in Eastern Africa, Arabia, and India heptane-2-ene core and a spiro ring system  allows one to clarify the absolute configurations at each chiral center in the architecture (Δ2′(3′) double bond could be concluded from the coupling constant of H-2′ or H-3′. However, this is impossible in the present structure due to the overlapped signals for these protons. The X-ray data also indicate an E configuration for the Δ2′(3′) double bond. Collectively, the structure of 1 was deduced as 2R,3R,6R,10S,12S,1′S,4′S,10′R.The ROESY correlations  of H-2/Hitecture . In gene2), obtained as yellow gums, was found to have the molecular formula C18H20O4 by analysis of its HRESIMS, 13C-NMR, and DEPT spectra, indicating nine degrees of unsaturation. The 1H-NMR spectrum of 2 exhibits four methyl groups (δH 2.71 (s), 2.47 (s), 2.10 (s), and 1.18 ), and one olefinic/aromatic proton. The 13C-NMR and DEPT spectra , H-4/C-2, C-6 and H-5/C-1, and C-6 reveals the presence of a six-membered ring (C-1−C-2−C-3−C-4−C-5−C-6) with a ketone-carbonyl group at C-2 and a methyl group at C-4, as shown (C 126.8), C-6 (δC 140.9) and C-7 (δC 124.7), H-9/C-1, C-7, C-8 (δC 156.2), C-10 (δC 139.5) and C-15 (δC 24.3), and H3-15/C-1, C-9 (δC 113.1) and C-10 reveal a pentasubstituted benzene ring (C-6−C-7−C-8−C-9−C-10−C-1) with a methyl group at C-10. The associations of H3-13/C-7, C-11 (δC 116.7), C-12 (δC 147.7) and H2-16/C-11, and C-12 indicate the presence of a chain consisting of C-13−C-11−C-12−C-16 connected with the pentasubstituted benzene ring via the carbon bond C-7−C-11. Furthermore, the presence of an acetoxyl group positioned at C-16 is supported by the chemical shift of C-16 (δC 56.3), as well as the HMBC correlations of H2-16/C-2′ and H3-1′/C-2′. In consideration of the chemical shifts of C-8 (δC 156.2) and C-12 (δC 147.7), the requirement of the molecular formula, and the remaining one degree of unsaturation, the presence of C-8−O−C-12 is secured. Taken together, the planar structure of 2 was identified.Commiphorene A  calculation using the TDDFT method. The experimental ECD spectrum of 2 is similar to the calculated one of (4R)-2 -2 . Thus, t3) was obtained as white gums. Its molecular formula was deduced as C15H16O3 (8 degrees of unsaturation) by its HREIMS , 13C-NMR, and DEPT spectra. The 1H-NMR spectrum of 3 (H 2.25 (s) and 1.79 (s)), one olefinic methylene ), and one olefinic methine. The 13C-NMR and DEPT spectra of 3  double bond is present in 3, instead of a methyl group at C-4; the drastic downfield shifts of C-4 (δC 143.7) and C-14 (δC 109.0) and the HMBC correlations  calculation. The Merck Molecular Force Field (MMFF) conformational search of (11-R)-3 and (11S)-3 resulted in 2 and 2 conformers in a 17 kJ/mol energy window, respectively. These conformers were further optimized by TDDFT at the B3LYP/6-31G(d) level, as implemented in the Gaussian 09 program package + . 1H- and 13C-NMR data, see Compound 2: Yellow gums, c 0.36, CH3OH); CD (CH3OH), ∆ε203 −2.41, ∆ε222 +0.74, ∆ε240 −0.98, ∆ε259 +1.10, ∆ε276 +1.46, ∆ε333 –2.68, ∆ε381 −0.40; UV (CH3OH) λmax (log ε) 276 (5.86) nm; HRESIMS: m/z 301.1428 [M + H]+ . 1H- and 13C-NMR data, see Compound 3: White gums, c 0.07, CH3OH); UV (CH3OH) λmax (log ε) 290 (3.23), 253 (3.48), 206 (4.18) nm; HREIMS: m/z 244.1103 [M]+ . 1H- and 13C-NMR data, see Compound 1: Data were collected using a Sapphire CCD with a graphite monochromated Cu Kα radiation, λ = 1.54184 Å at 100 K. Crystal data: C31H38O6, M = 506.61, space group P1 21 1; unit cell dimensions were determined to be a = 10.4149(2) Å, b = 9.5899(2) Å, c = 14.2271(2) Å, α = 90.00°, β = 109.362(2)°, γ = 90.00°, V = 1340.61(5) Å3, Z = 2, Dx = 1.255 g/cm3, F (000) = 544.0, and μ (Cu Kα) = 0.692 mm−1, and 8122 reflections were collected until θmax = 73.448°, in which independent unique 4692 reflections were observed (F2 > 4σ (F2)). The structure was solved by direct methods using the SHELXS-97 program and refined by the program SHELXL-97 and full-matrix least squares calculations. In the structure refinements, nonhydrogen atoms were placed on the geometrically ideal positions by the “ride on” method. Hydrogen atoms bonded to oxygen were located by the structure factors with isotropic temperature factors. The final refinement gave R = 0.0402(4692), Rw = 0.1061(4831), S = 1.045, and Flack = 0.00(11). Crystallographic data for structure 1 has been deposited at the Cambridge Crystallographic Data Centre (CCDC 1878959), www.ccdc.cam.ac.uk/data_request/cif.Crystal data for compound 2 and 3 were performed using Gaussian 09 heptane-2-ene core (1); a sesquiterpene with an additional carbon in the backbone (2); and a new cadinane-type sesquiterpene (3), adding the structural diversity of sesquiterpenes in nature. Due to the attractive construction features of the isolated compounds, their cytotoxic activities were tested. As a result, compounds 2 and 4 exert low cytotoxicity against normal cells and high selectivity in cancer cells, especially in HepG2 cells, which will be helpful for gaining deep insight into the biological role of such terpenoids in human diseases.


Burkholderia as an under-tapped resource of bioactive molecules resulted in the isolation of two new antimicrobial 2-alkyl-4-quinolones. (E)-2-(Hept-2-en-1-yl)quinolin-4(1H)-one (1) and (E)-2-(non-2-en-1-yl)quinolin-4(1H)-one (3) were isolated from the culture broth of strain MBAF1239 together with four known alkylquinolones (2 and 4–6), pyrrolnitrin (7), and BN-227 (8). The structures of 1 and 3 were unambiguously characterized using NMR spectroscopy and mass spectrometry. Compounds 1–8 inhibited the growth of the marine bacterium Tenacibaculum maritimum, an etiological agent of skin ulcers in marine fish, offering new opportunities to develop antibacterial drugs for fish farming.Exploration of rhizobacteria of the genus  Burkholderia within the family Burkholderiaceae  to afford two new 2-alkylquinolones 1 (0.5 mg) and 3 (0.7 mg), together with four known 2-alkylquinolones, Pyo Ib or 2-heptylquinolin-4(1H)-one , (E)-2-(hept-2-en-1-yl)-3-methylquinolin-4(1H)-one , PSC-C or 2-heptyl-3-methylquinolin-4(1H)-one , PSC-D or (E)-3-methyl-2-(non-2-en-1-yl)quinolin-4(1H)-one , pyrrolnitrin , and BN-227 .For the extraction of secondary metabolites, 100 mL of 1-butanol was added to each flask, and they were allowed to shake for 1 h. The resulting suspension was centrifuged at 6000 rpm for 10 min to separate organic and aqueous layers, the former of which was concentrated in vacuo to give a 5.35 g extract from a 2 L culture. The crude extract was successively partitioned between 60% MeOH (250 mL) and CH(E)-2-(Hept-2-en-1-yl)quinolin-4(1H)-one (1): UV (MeCN) λmax, nm (ε): 328 (29900), 322 (27600), 316 (29800), 292 (10700), 288 (10900), 260 (6600), 240 (51200); IR ν max (ATR) cm−1: 2927, 2873, 1636, 1595, 1553, 1505, 1473, 1445, 1355, 1322, 1275, 1104, 1028, 969, 841, 762, 676; 1H NMR (CD3OD) δH 8.20 , 7.68 , 7.57 , 7.39 , 6.21 , 5.71 , 5.61 , 3.42 , 2.09 , 1.38 , 1.34 , 0.90 ; 13C NMR (CD3OD) δC 180.8 (C4), 155.7 (C2), 141.6 (C8a), 136.5 (C11), 133.5 (C7), 126.0 (C5), 125.5 (C4a), 125.3 (C10), 125.1 (C6), 119.1 (C8), 108.9 (C3), 37.8 (C9), 33.2 (C12), 32.6 (C13), 23.2 (C14), 14.2 (C15); ESIMS–TOF (m/z): [M + H]+ calcd for C16H19NO, 242.1539; found, 242.1539.(E)-2-(Non-2-en-1-yl)quinolin-4(1H)-one (3): UV (MeCN) λmax, nm (ε): 328 (18600), 322 (16800), 316 (18200), 292 (8000), 288 (8400), 260 (5300), 240 ; IR ν max (ATR) cm−1: 2923, 2853, 1730, 1635, 1593, 1554, 1500, 1471, 1443, 1354, 1320, 1247, 1137, 1028, 965, 836, 759, 672; HRESIMS–TOF (m/z): [M + H]+ calcd for C18H24NO, 270.18524; found, 270.1855.1–8 was evaluated by a paper-disc agar diffusion method described in our previous study [T. maritimum.The antibacterial and antifungal activity of us study . FlexibaFile 11H and 13C NMR, COSY, HSQC, and HMBC spectra for compounds 1 and 3.


These hybrid molecules were prepared to combine the α-bromoacryloyl moiety with two series of indole-inspired chalcone analogues, possessing an indole derivative and a 3- or 4-pyridine ring, respectively, linked on either side of 2-propen-1-one system. The structure-activity relationship was also investigated by the insertion of alkyl or benzyl moieties at the N-1 position of the indole nucleus. We found that most of the newly synthesized displayed high antiproliferative activity against U-937, MOLT-3, K-562, and NALM-6 leukaemia cell lines, with one-digit to double-digit nanomolar IC50 values. The antiproliferative activities of 3-pyridinyl derivatives 3f–h revealed that N-benzyl indole analogues generally exhibited lower activity compared to N-H or N-alkyl derivatives 3a–b and 3c–e, respectively. Moreover, cellular mechanism studies elucidated that compound 4a induced apoptosis along with a decrease of mitochondrial membrane potential and activated caspase-3 in a concentration-dependent manner.The combination of two pharmacophores into a single molecule represents one of the methods that can be adopted for the synthesis of new anticancer molecules. To investigate the influence of the position of the pyridine nitrogen on biological activity, two different series of α-bromoacryloylamido indolyl pyridinyl propenones  Unfortunately, MOMIPP was characterized by a moderate selectivity for cancer compared with normal cells, being also cytotoxic against human mammary epithelial cells (HMEC). MOMIPP was characterized by the presence of an indolyl-pyridinyl-propenone scaffold, with a methoxy group at the 5-position of indole nucleus and a nitrogen at the para-position of the pyridine ring attached at the carbonyl of the enone system. The specific feature of this derivative as methuosis-inducing agent depends on the specific substitution pattern of both the indole and pyridine moieties. Modulating the substitution at the 2-position of the indole ring from small alkyl groups such as methyl or ethyl to alkoxycarbonyl moieties, it was possible to switch the mode of cell growth inhibitory activity from methuosis to microtubule depolymerizationn-butoxy, acetamido or p-methylester benzyloxy groups reduced the activity. These findings demonstrated that may be some flexibility at this position on the indole ring for future attempts to further improve potency.Among the indole-based chalcones investigated as potential anticancer agents, Maltese et al. have described a series of chalcones constituted by a α,β-unsaturated ketone linking two aromatic heterocyclic rings represented by indole and pyridine moietiesIn considering the potential mechanism through which MOMIPP induces cell death by methuosis, one possibility is that indolyl pyridinyl propanone motif, characterized by the presence of an electrophilic α,β-unsaturated carbonyl moiety, could act as a target-specific Michael acceptor, an active pharmacophore often employed in the design of anticancer drugs2a)2b)2c), a secondary metabolite originating from dibromotyrosine50>120 μM on murine leukaemia L1210 cells)2d), which was evaluated as a first-line single-agent chemotherapy in patients with advanced or metastatic soft tissue sarcomaThe pyrroloiminoquinone alkaloid Discorhabdin C , alkyl groups such as methyl (3c and 4a), ethyl (3d and 4b), n-propyl (3e and 4c) and isopropyl (4d) as well as the preparation of the bare benzyl derivative 3f, followed by the insertion of electron-withdrawing chlorine atom and the electron-donating methyl group on the benzene portion of the N-1 benzyl substitution . Compound 3b was also characterized by the presence of an electron-donating and lipophilic methyl group at the 2-position of the indole ring.The structure-activity relationship (SAR) was also investigated by the modification of the substituent at the 1H and 13C NMR spectra were recorded on a Bruker AC 200 and Varian 400 Mercury Plus spectrometer, respectively. Chemical shifts (δ) are given in ppm upfield from tetramethylsilane as an internal standard, and the spectra were recorded in appropriate deuterated solvents, as indicated. Positive-ion electrospray ionization (ESI) mass spectra were recorded on a double-focusing Finnigan MAT 95 instrument with BE geometry. Melting points (mp) were determined on a Buchi–Tottoli apparatus and are uncorrected. All products reported showed 1H and 13C NMR spectra in agreement with the assigned structures. The purity of tested compounds was determined by combustion elemental analyses conducted by the Microanalytical Laboratory of the Chemistry Department of the University of Ferrara with a Yanagimoto MT-5 CHN recorder elemental analyzer. All tested compounds yielded data consistent with a purity of at least 95% as compared with the theoretical values. All reactions were carried out under an inert atmosphere of dry nitrogen unless otherwise indicated. Standard syringe techniques were used for transferring dry solvents. Reaction courses and product mixtures were routinely monitored by TLC on silica gel (precoated F254 Merck plates), and compounds were visualized with aqueous KMnO4. Flash chromatography was performed using 230–400 mesh silica gel and the indicated solvent system. Organic solutions were dried over anhydrous Na2SO4. 5-Nitro-1H-indole-3-carbaldehyde 4 was prepared following the procedure previously describedH-indole-3-carbaldehyde (5) as well as copies of 1H-NMR and 13C-NMR spectra of compounds 3a–h and 4a–d are reported in the supplementary data.3a–h and 4a–d were carried out by the general methodology shown in N-alkylation or N-benzylation of 5-nitroindole-2-carboxaldehyde 4 with the appropriate alkyl or benzyl halide in presence of NaH as a base in DM, produced the key building blocks N-alkyl and N-benzyl indole derivatives 6a–d and 6e–g, respectively. The subsequent Claisen-Schmidt condensation between various 5-nitroindole-3-carboxaldehydes 4, 5 and 6a–g with 3- or 4-acetylpyridine and piperidine as a base in DMF at 100 °C, furnished the corresponding isomeric indole-pyridine-propenones 7a–h and 8a–d, respectively. Amino derivatives 9a–h and 10a–d were generated from the corresponding nitro analogues 7a–h and 8a–d by reduction with iron and ammonium chloride in a refluxing mixture of water and ethanol. Finally, the hybrid compounds 3a–h and 4a–d were prepared by the condensation of α-bromoacrylic acid and the 5-aminoindole chalcone derivatives 9a–h and 10a–d, respectively, using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDCI) in dimethylformamide.Synthesis of hybrid compounds H-indole-3-carbaldehyde 4  in dry N,N-dimethylformamide (5 ml) at 0 °C and under Argon atmosphere. After stirring for 30 min at room temperature, the appropriate alkyl or benzyl halide  was slowly added at +4 °C (ice-water bath) and the mixture stirred for 2 h at room temperature. After this time, the reaction mixture was quenched by addition of water (10 ml) and the product extracted with dichloromethane (3 × 10 ml). The combined organic extracts were washed with water (10 ml), brine, dried over Na2SO4 and concentrated under reduced pressure. Diethyl ether was added to the residue, the mixture stirred for 15 min. and the resulting precipitate was collected by filtration to furnish the title compound as a solid.Sodium hydride  was carefully added to a solution of 5-nitro-1H-indole-3-carbaldehyde 7a , 2-methyl-5-nitro-1H-indole-3-carbaldehyde 7 b  or the appropriate 5-nitro-N-alkyl/benzyl-indole-3-carbaldehyde 7c–h (2 mmol) in dry N,N-dimethylformamide (10 ml), 3-acetylpyridine or 4-acetylpiridine  and piperidine  were added and the mixture was heated at 100 °C for 24 h. After this time, a second addition of 3-acetylpyridine or 4-acetylpiridine  and piperidine  was made. After 24 h, the reaction mixture was cooled at room temperature and evaporated in vacuo. The residue was dissolved with CH2Cl2 (20 ml), the solution washed sequentially with water (5 ml) and brine (5 ml), dried and concentrated at reduced pressure. The resulting residue was purified by column chromatography or suspended in diethyl ether, the suspension stirred for 0.5 h, filtered and used without any further purification for the next reaction.To a stirred solution of 5-nitro-17a–h or 8a–d (1 mmol) in EtOH (10 ml) was added iron powder  and a solution of ammonium chloride  in water (3 ml). The reaction mixture was heated under reflux for 2 h, cooled to room temperature, and filtered through Celite. The filter cake was rinsed with dichloromethane (20 ml) and the filtrate washed with water (5 ml), brine (5 ml), dried over Na2SO4 and the solvent removed under reduced pressure. The crude residue was suspended in diethyl ether, stirred for 15 min, the solid collected by filtration and used without further purification for the next reaction. Only compound 10b was purified by column chromatography on silica gel.To a stirred suspension of the appropriate nitroindole-pyridine-propenone 8a–i or 9a–i (1 mmol) in dry DMF (5 ml) was added EDCI  followed by α-bromoacrylic acid . The reaction was stirred at room temperature for 18 h, and then concentrated in vacuo. The residue was dissolved with a mixture of DCM (15 ml) and water (5 ml), and the organic phase was washed with brine (5 ml), dried over Na2SO4 and evaporated to dryness in vacuo. The resulting crude residue was purified by chromatography on silica gel.To an ice-cooled solution of amine indole–pyridine–propenone 1-Methyl-5-nitro-1H-indole-3-carbaldehyde (6a). Following general procedure A, using iodomethane as alkylating agent, compound 6a was isolated as a yellow solid. Yield 85%, mp 196–198 °C. 1H-NMR  δ: 3.97 , 7.80 , 8.18 , 8.55 , 8.93 , 9.99 . MS (ESI): [M + 1]+ = 205.4.1-Ethyl-5-nitro-1H-indole-3-carbaldehyde (6b). Following general procedure A, using iodoethane as alkylating agent, compound 6b was isolated as a yellow solid. Yield 89%, mp 180–182 °C. 1H-NMR  δ: 1.58 , 4.26 , 7.43 , 7.92 , 8.22 , 9.22 , 10.1 . MS (ESI): [M + 1]+ = 219.3.1-Propyl-5-nitro-1H-indole-3-carbaldehyde (6c). Following general procedure A, using 1-iodopropane as alkylating agent, compound 6c was isolated as a yellow solid. Yield 80%, mp 192–194 °C. 1H-NMR  δ: 0.87 , 1.83 , 4.34 , 7.89 , 8.16 , 8.63 , 8.95 , 10.0 . MS (ESI): [M + 1]+ = 233.32.1-Isopropyl-5-nitro-1H-indole-3-carbaldehyde (6d). Following general procedure A, using 2-iodopropane as alkylating agent, compound 6d was isolated as a yellow solid. Yield 85%, mp 180–182 °C. 1H-NMR  δ: 1.52 , 4.97 , 7.92 , 8.16 , 8.78 , 8.95 , 10.0 . MS (ESI): [M + 1]+ = 233.4.1-Benzyl-5-nitro-1H-indole-3-carbaldehyde (6e). Following general procedure A, using benzyl bromide as alkylating agent, compound 6e was isolated as a yellow solid. Yield 78%, mp 180–182 °C. 1H-NMR  δ: 5.65 , 7.34 , 7.84 , 8.15 , 8.75 , 8.96 , 10.0 . MS (ESI): [M + 1]+ = 281.3.1-(4-Chlorobenzyl)-5-nitro-1H-indole-3-carbaldehyde (6f). Following general procedure A, using 4-chlorobenzyl bromide as alkylating agent, compound 6f was isolated as a yellow solid. Yield 78%, mp 157–159 °C. 1H-NMR  δ: 5.66 , 7.34 , 7.42 , 7.84 , 8.16 , 8.75 , 8.96 , 10.0 . MS (ESI): [M + 1]+ = 315.7.1-(4-Methylbenzyl)-5-nitro-1H-indole-3-carbaldehyde (6g). Following general procedure A, using 4-methylbenzyl bromide as alkylating agent, compound 6g was isolated as a yellow solid. Yield 82%, mp 144–145 °C. 1H-NMR  δ: 2.25 , 5.59 , 7.15 , 7.23 , 7.84 , 8.15 , 8.73 , 8.96 , 10.0 . MS (ESI): [M + 1]+ = 295.3.(E)-3-(5-Nitro-1H-indol-3-yl)-1-(pyridin-3-yl)prop-2-en-1-one (7a). Following general procedure B, the residue purified by crystallization from ethyl ether yielded 7a as a red solid. Yield 78%, mp 165–167 °C.1H-NMR  δ: 7.53 , 7.66 , 7.71 , 8.09 , 8.46 , 8.88 , 8.97 , 9.32 , 12.2 . MS (ESI): [M + 1]+ = 294.2.(E)-3-(2-Methyl-5-nitro-1H-indol-3-yl)-1-(pyridin-3-yl)prop-2-en-1-one (7b). Following general procedure B, the residue purified by crystallization from ethyl ether yielded 7b as a brown solid. Yield 80%, mp 201–203 °C.1H-NMR  δ: 3.34 , 7.56 , 8.01 , 8.05 , 8.43 , 8.09 , 8.82 , 9.29 , 12.2 . MS (ESI): [M + 1]+ = 308.2.(E)-3-(1-Methyl-5-nitro-1H-indol-3-yl)-1-(pyridin-3-yl)prop-2-en-1-one (7c). Following general procedure B, after work-up the residue was purified by column chromatography, using ethyl acetate-MeOH 9.5:0.5 v/v as eluent, to afford 7c as a yellow solid. Yield 56%, mp 188–190 °C. 1H-NMR  δ: 3.95 , 7.59 , 7.73 , 7.78 , 8.06 , 8.16 , 8.42 , 8.81 , 8.97 , 9.31 . MS (ESI): [M + 1]+ = 308.3.(E)-3-(1-Ethyl-5-nitro-1H-indol-3-yl)-1-(pyridin-3-yl)prop-2-en-1-one (7d). Following general procedure B, the residue purified by crystallization from ethyl ether yielded 7d as a yellow solid. Yield 65%, mp 184–186 °C. 1H-NMR  δ: 1.46 , 4.34 , 7.59 , 7.74 , 7.83 , 8.06 , 8.13 , 8.42 , 8.54 , 8.81 , 8.95 , 9.31 . MS (ESI): [M + 1]+ = 322.4.(E)-3-(1-Propyl-5-nitro-1H-indol-3-yl)-1-(pyridin-3-yl)prop-2-en-1-one (7e). Following general procedure B, the residue purified by crystallization from ethyl ether yielded 7e as a yellow solid. Yield 64%, mp 167–168 °C. 1H-NMR  δ: 0.88 , 1.88 , 4.27 , 7.64 , 7.75 , 7.85 , 8.06 , 8.12 , 8.42 , 8.53 , 8.82 , 8.98 , 9.32 . MS (ESI): [M + 1]+ = 336.3.(E)-3-(1-Benzyl-5-nitro-1H-indol-3-yl)-1-(pyridin-3-yl)prop-2-en-1-one (7f). Following general procedure B, the residue purified by crystallization from ethyl ether yielded 7f as a yellow solid. Yield 55%, mp 178–180 °C. 1H-NMR  δ: 5.61 , 7.32 , 7.75 , 7.84 , 7.95 , 8.08 , 8.13 , 8.64 , 8.83 , 8.96 . MS (ESI): [M + 1]+ = 322.4.(E)-3-(1-(4-Chlorobenzyl)-5-nitro-1H-indol-3-yl)-1-(pyridin-3-yl)prop-2-en-1-one (7g). Following general procedure B, the residue purified by crystallization from ethyl ether yielded 7g as a yellow solid. Yield 65%, mp 176–178 °C. 1H-NMR  δ: 5.62 , 7.32 , 7.42 , 7.63 , 7.82 , 8.09 , 8.43 , 8.61 , 8.83 , 9.32 . MS (ESI): [M + 1]+ = 418.3.(E)-3-(1-(4-Methylbenzyl)-5-nitro-1H-indol-3-yl)-1-(pyridin-3-yl)prop-2-en-1-one (7h). Following general procedure B, the residue purified by crystallization from ethyl ether yielded 7h as a yellow solid. Yield 75%, mp 177–179 °C. 1H-NMR  δ: 2.25 , 5.50 , 7.13 , 7.20 , 7.53 , 7.76 , 7.83 , 8.07 , 8.10 , 8.15 , 8.61 , 8.78 , 8.97 , 9.32 . MS (ESI): [M + 1]+ = 398.4.(E)-3-(1-Methyl-5-nitro-1H-indol-3-yl)-1-(pyridin-4-yl)prop-2-en-1-one (8a). Following general procedure B, the residue purified by crystallization from ethyl ether yielded 8a as a red solid. Yield 88%, mp 264–266 °C. 1H-NMR  δ: 3.96 , 7.66 , 7.78 , 7.95 , 8.08 , 8.16 , 8.45 , 8.84 , 8.95 . MS (ESI): [M + 1]+ = 308.3.(E)-3-(1-Ethyl-5-nitro-1H-indol-3-yl)-1-(pyridin-4-yl)prop-2-en-1-one (8b). Following general procedure B, after work-up the residue was purified by column chromatography, using ethyl acetate-petroleum ether 9:1 v/v as eluent, to afford 8b as a yellow solid. Yield 61%, mp 220–221 °C. 1H-NMR  δ: 1.45 , 4.35 , 7.64 , 7.43 , 7.94 , 7.98 , 8.15 , 8.55 , 8.83 , 8.94 . MS (ESI): [M + 1]+ = 322.4.(E)-3-(1-Propyl-5-nitro-1H-indol-3-yl)-1-(pyridin-4-yl)prop-2-en-1-one (8c). Following general procedure B, after work-up the residue was purified by column chromatography, using ethyl acetate-petroleum ether 8:2 v/v as eluent, to afford 8c as a yellow solid. Yield 70%, mp 176–177 °C. 1H-NMR  δ: 0.85 , 1.84 , 4.28 , 7.66 , 7.85 , 7.93 , 8.07 , 8.11 , 8.52 , 8.83 , 8.92 . MS (ESI): [M + 1]+ = 336.4.(E)-3-(1-Isopropyl-5-nitro-1H-indol-3-yl)-1-(pyridin-4-yl)prop-2-en-1-one (8d). Following general procedure B, the residue purified by crystallization from ethyl ether yielded 8d as a yellow solid. Yield 69%, mp 194–195 °C.1H-NMR  δ: 1.52 , 4.96 , 7.68 , 7.88 , 7.95 , 8.09 , 8.71 , 8.84 , 8.93 . MS (ESI): [M + 1]+ = 336.4.(E)-3-(5-Amino-1H-indol-3-yl)-1-(pyridin-3-yl)prop-2-en-1-one (9a). Following general procedure C, after work-up the solid residue was stirred in diethyl ether. After filtration, the title compound 9a was isolated as a red solid. Yield 87%, mp 74–76 °C. 1H-NMR  δ: 4.86 , 6.59 , 7.16 , 7.24 , 7.39 , 7.56 , 7.92 , 7.99 , 8.35 , 8.78 , 9.24 , 11.6 . MS (ESI): [M + 1]+ = 264.3.(E)-3-(2-Methyl-5-amino-1H-indol-3-yl)-1-(pyridin-3-yl)prop-2-en-1-one (9b). Following general procedure C, after work-up the solid residue was stirred in diethyl ether. After filtration, the title compound 9b was isolated as a red solid. Yield 81%, mp 135–137 °C. 1H-NMR  δ: 3.34 , 4.82 , 6.54 , 7.08 , 7.20 , 7.34 , 7.65 , 7.99 , 8.42 , 8.78 , 9.24 , 11.2 . MS (ESI): [M + 1]+ = 278.3.(E)-3-(5-Amino-1-methyl-1H-indol-3-yl)-1-(pyridin-3-yl)prop-2-en-1-one (9c). Following general procedure C, after work-up the solid residue was stirred in diethyl ether. After filtration, the title compound 9c was isolated as a yellow solid. Yield 85%, mp 164–166 °C. 1H-NMR  δ: 3.84 , 4.49 , 6.75 , 7.31 , 7.49 , 7.58 , 7.95 , 8.16 , 8.77 , 9.22 . MS (ESI): [M + 1]+ = 279.3.(E)-3-(5-Amino-1-ethyl-1H-indol-3-yl)-1-(pyridin-3-yl)prop-2-en-1-one (9d). Following general procedure C, after work-up the solid residue was stirred in diethyl ether. After filtration, the title compound 9d was isolated as a red solid. Yield 78%, mp 156–158 °C. 1H-NMR  δ: 1.39 , 4.14 , 4.93 , 6.62 , 7.24 , 7.27 , 7.37 , 7.60 , 7.79 , 7.94 , 8.35 , 8.78 , 9.32 . MS (ESI): [M + 1]+ = 292.4.(E)-3-(5-Amino-1-propyl-1H-indol-3-yl)-1-(pyridin-3-yl)prop-2-en-1-one (9e). Following general procedure C, after work-up the solid residue was stirred in diethyl ether. After filtration, the title compound 9e was isolated as a red solid. Yield 67%, mp 146–148 °C. 1H-NMR  δ: 0.86 , 1.77 , 4.08 , 4.93 , 6.63 , 7.25 , 7.28 , 7.38 , 7.60 , 7.95 , 7.98 , 8.35 , 8.78 , 9.24 . MS (ESI): [M + 1]+ = 306.5.(E)-3-(5-Amino-1-benzyl-1H-indol-3-yl)-1-(pyridin-3-yl)prop-2-en-1-one (9f). Following general procedure C, after work-up the solid residue was stirred in diethyl ether. After filtration, the title compound 9f was isolated as a red solid. Yield 58%, mp 212–214 °C. 1H-NMR  δ: 4.92 , 5.37 , 6.78 , 7.28 , 7.41 , 7.58 , 7.96 , 8.11 , 8.35 , 8.78 , 9.32 . MS (ESI): [M + 1]+ = 354.4.(E)-3-(5-Amino-1-(4-chlorobenzyl)-1H-indol-3-yl)-1-(pyridin-4-yl)prop-2-en-1-one (9g). Following general procedure C, after work-up the solid residue was stirred in diethyl ether. After filtration, the title compound 9g was isolated as a red solid. Yield 65%, mp 127–129 °C. 1H-NMR  δ: 4.95 , 5.38 , 6.59 , 7.22 , 7.39 , 7.43 , 7.59 , 7.97 , 8.11 , 8.36 , 8.79 , 9.24 . MS (ESI): [M + 1]+ = 388.9.(E)-3-(5-amino-1-(4-methylbenzyl)-1H-indol-3-yl)-1-(pyridin-4-yl)prop-2-en-1-one (9h). Following general procedure C, after work-up the solid residue was stirred in diethyl ether. After filtration, the title compound 9h was isolated as a red solid. Yield 71%, mp 150–152 °C. 1H-NMR  δ: 2.24 , 4.92 , 5.31 , 6.57 , 7.24 , 7.39 , 7.56 , 7.84 , 7.95 , 8.09 , 8.17 , 8.35 , 8.78 , 9.24 . MS (ESI): [M + 1]+ = 368.4.(E)-3-(5-Amino-1-methyl-1H-indol-3-yl)-1-(pyridin-4-yl)prop-2-en-1-one (10a). Following general procedure C, after work-up the solid residue was stirred in diethyl ether. After filtration, the title compound 10a was isolated as a red solid. Yield 80%, mp 196–198 °C. 1H-NMR  δ: 3.76 , 4.95 , 6.64 , 7.23 , 7.29 , 7.87 , 7.92 , 8.90 . MS (ESI): [M + 1]+ = 279.3.(E)-3-(5-Amino-1-ethyl-1H-indol-3-yl)-1-(pyridin-4-yl)prop-2-en-1-one (10b). Following general procedure C, after work-up the residue was purified by column chromatography, using ethyl acetate:petroleum ether 4:6 v/v as eluent, to afford 10b as a yellow solid. Yield 65%, mp 180–182 °C. 1H-NMR  δ: 1.39 , 4.13 , 4.92 , 6.63 , 7.22 , 7.27 , 7.37 , 7.87 , 7.93 , 8.00 , 8.79 . MS (ESI): [M + 1]+ = 292.3.(E)-3-(5-Amino-1-propyl-1H-indol-3-yl)-1-(pyridin-4-yl)prop-2-en-1-one (10c). Following general procedure C, after work-up the solid residue was stirred in diethyl ether. After filtration, the title compound 10c was isolated as a red solid. Yield 76%, mp 154–155 °C. 1H-NMR  δ: 0.85 , 1.77 , 4.05 , 4.93 , 6.62 , 7.22 , 7.28 , 7.37 , 7.94 , 8.80 . MS (ESI): [M + 1]+ = 306.4.(E)-3-(5-Amino-1-isopropyl-1H-indol-3-yl)-1-(pyridin-4-yl)prop-2-en-1-one (10d). Following general procedure C, after work-up the solid residue was stirred in diethyl ether. After filtration, the title compound 10d was isolated as a red solid. Yield 78%, mp 146–148 °C. 1H-NMR  δ: 1.45 , 4.66 , 4.93 , 6.63 , 7.20 , 7.31 , 7.87 , 7.94 , 8.13(s. 1H), 8.88 . MS (ESI): [M + 1]+ = 306.4.(E)-2-Bromo-N-(3-(3-oxo-3-(pyridin-3-yl)prop-1-en-1-yl)-1H-indol-5-yl)acrylamide (3a). Following general procedure D, after workup as described previously, the residue was purified by flash chromatography on silica gel using light petroleum ether-EtOAc 1–9 (v/v) as eluent, affording compound 3a as a yellow solid. Yield: 59%, mp 181–183 °C. 1H-NMR  δ: 6.31 , 6.78 , 7.46 , 7.53 , 7.64 , 8.04 , 8.17 , 8.31 , 8.37 , 8.81 , 9.24 , 10.3 , 12.0 . 13C NMR  δ: 111.89, 112.37, 112.64, 114.92, 116.88, 117.56, 123.89, 124.92, 125.48, 132.51, 133.69, 134.48, 135.46, 138.32, 139.76, 148.99, 152.63, 160.81, 187.83. MS (ESI): [M]+ = 396.2, [M + 2]+ = 398.2. Anal. calcd for C19H14BrN3O2. C, 57.59; H, 3.56; N, 10.60; found: C, 57.38; H, 3.44; N, 10.39.(E)-2-Bromo-N-(2-methyl-3-(3-oxo-3-(pyridin-3-yl)prop-1-en-1-yl)-1H-indol-5-yl)acrylamide (3b). Following general procedure D, after workup as described previously, the residue was purified by flash chromatography on silica gel using EtOAc as eluent, affording compound 3b as a yellow solid. Yield: 63%, mp 172–174 °C. 1H-NMR  δ: 2.59 , 6.30 , 6.77 , 7.36 , 7.41 , 7.60 , 8.05 , 8.26 , 8.36 , 8.80 , 9.24 , 10.2 , 11.9 . 13 C NMR  δ: 11.90, 109.18, 109.56, 111.38, 111.75, 113.39, 115.97, 123.88, 125.36, 125.59, 132.56, 133.12, 133.90, 135.35, 138.59, 145.64, 148.85, 152.52, 160.77, 187.47. MS (ESI): [M]+ = 410.1, [M + 2]+ = 412.1. Anal. calcd for C20H16BrN3O2. C, 58.55; H, 3.93; N, 10.24; found: C, C, 58.39; H, 3.77; N, 10.08.(E)-2-Bromo-N-(1-methyl-3-(3-oxo-3-(pyridin-3-yl)prop-1-en-1-yl)-1H-indol-5-yl)acrylamide (3c). Following general procedure D, after workup as described previously, the residue was purified by flash chromatography on silica gel using light petroleum ether: EtOAc 2:8 as eluent, affording compound 3c as a yellow solid. Yield: 45%, mp 179–180 °C. 1H-NMR  δ: 3.86 , 6.32 , 6.79 , 7.55 , 7.63 , 7.72 , 7.99 , 8.16 , 8.32 , 8.41 , 8.80 , 9.24 , 10.3 . 13C NMR  δ: 33.71, 111.45, 112.09, 112.49, 115.41, 117.35, 124.42, 125.89, 126.11, 133.37, 134.22, 134.78, 135.62, 135.98, 138.20, 139.59, 149.51, 153.17, 161.37, 188.26. MS (ESI): [M]+ = 410.4, [M + 2]+ = 412.4. Anal. calcd for C20H16BrN3O2. C, 58.55; H, 3.93; N, 10.24; found: C, 58.36; H, 3.71; N, 10.02.(E)-2-Bromo-N-(1-ethyl-3-(3-oxo-3-(pyridin-3-yl)prop-1-en-1-yl)-1H-indol-5-yl)acrylamide (3d). Following general procedure D, after workup as previously described, the residue was purified by flash chromatography on silica gel using light petroleum ether-EtOAc 1–9 (v/v) as eluent, affording compound 3d as a yellow solid. Yield: 64%, mp 172–173 °C. 1H-NMR  δ: 1.43 , 4.26 , 6.32 , 6.79 , 7.53 , 7.63 , 8.00 , 8.25 , 8.32 , 8.36 , 8.80 , 9.25 , 10.3 . 13C NMR  δ: 15.52, 41.55, 110.16, 111.44, 112.29, 112.52, 115.48, 117.34, 124.41, 125.79, 126.11, 133.32, 134.21, 134.65, 135.97, 136.44, 139.56, 149.53, 153.17, 161.36, 188.28. MS (ESI): [M]+ = 424.3, [M + 2]+ = 426.3. Anal. calcd for C21H18BrN3O2. C, 59.45; H, 4.28; N, 9.90; found: C, 59.19; H, 4.08; N, 9.78.(E)-2-Bromo-N-(1-propyl-3-(3-oxo-3-(pyridin-3-yl)prop-1-en-1-yl)-1H-indol-5-yl)acrylamide (3e). Following general procedure D, after workup as described previously, the residue was purified by flash chromatography on silica gel using light petroleum ether: EtOAc 2:8 as eluent, affording compound 3e as a yellow solid. Yield: 66%, mp 178–180 °C. 1H-NMR  δ: 0.85 , 1.80 , 4.18 , 6.30 , 6.77 , 7.51 , 7.60 , 7.98 , 8.21 , 8.30 , 8.34 , 8.79 , 9.23 , 10.3 . 13C NMR  δ: 10.96, 22.72, 47.61, 111.01, 111.63, 111.97, 114.99, 116.80, 123.85, 125.24, 125.56, 130.82, 132.71, 133.64, 134.44, 135.41, 136.58, 138.99, 148.97, 152.61, 160.79, 187.71. [M]+ = 438.2, [M + 2]+ = 440.2. Anal. calcd for C22H20BrN3O2. C, 60.28; H, 4.60; N, 9.59; found: C, 60.03; H, 4.40; N, 9.38.(E)-2-Bromo-N-(1-benzyl-3-(3-oxo-3-(pyridin-3-yl)prop-1-en-1-yl)-1H-indol-5-yl)acrylamide (3f). Following general procedure D, after workup as described previously, the residue was purified by flash chromatography on silica gel using light petroleum ether: EtOAc 2:8 as eluent, affording compound 3f as a yellow solid. Yield: 74%, mp 193–195 °C. 1H-NMR  δ: 5.49 , 6.31 , 6.78 , 7.28 , 7.59 (m 4H), 8.05 , 8.33 , 8.81 , 9.25 , 10.3 . 13C NMR  δ: 30.61, 49.60, 111.30, 112.05, 112.12, 115.53, 117.01, 123.87, 125.00, 125.60, 127.10 (2 C), 127.63, 128.62 (2 C), 132.83, 133.59, 134.30, 135.47, 136.73, 136.96, 138.81, 149.02, 152.66, 160.81, 187.81. MS (ESI): [M]+ = 410.4, [M + 2]+ = 412.4. Anal. calcd for C26H20BrN3O2. C, 64.21; H, 4.14; N, 8.64; found: C, 64.01; H, 3.98; N, 8.44.(E)-2-Bromo-N-(1-(4-chlorobenzyl)-3–(3-oxo-3-(pyridin-3-yl)prop-1-en-1-yl)-1H-indol-5-yl)acrylamide (3g). Following general procedure D, after workup as described previously, the residue was purified by flash chromatography on silica gel using light petroleum ether-EtOAc 2-8 (v/v) as eluent, affording compound 3g as a yellow solid. Yield: 61%, mp 198–200 °C. 1H-NMR  δ: 5.50 , 6.32 , 6.79 , 7.29 , 7.40 , 7.56 (m 4H), 8.00 , 8.34 , 8.82 , 9.25 , 10.3 . 13 C NMR  δ: 48.84, 111.27, 112.10, 112.26, 115.63, 117.07, 123.90, 125.34, 125.66, 128.63 (2C), 129.02 (2C), 132.26, 132.92, 133.56, 134.20, 135.50, 136.01, 136.68, 136.84, 138.77, 149.04, 152.71, 160.83, 187.82. MS (ESI): [M]+ = 520.1, [M + 2]+ = 522.1. Anal. calcd for C26H19BrClN3O2. C, 59.96; H, 3.68; N, 8.07; found: C, 59.78; H, 3.56; N, 7.89.(E)-2-Bromo-N-(1-(4-methylbenzyl)-3–(3-oxo-3-(pyridin-3-yl)prop-1-en-1-yl)-1H-indol-5-yl)acrylamide (3h). Following general procedure D, after workup as described previously, the residue was purified by flash chromatography on silica gel using light petroleum ether: EtOAc 3:7 as eluent, affording compound 3h as a yellow solid. Yield: 63%, mp 154–156 °C. 1H-NMR  δ: 2.23 , 5.41 , 6.29 , 6.76 , 7.13 , 7.16 , 7.58 (m 4H), 7.98 , 8.29 , 8.31 , 8.35 , 8.79 , 9.23 , 10.3 . 13C NMR  δ: 21.12, 45.96, 111.89, 112.60, 115.96, 117.51, 124.42, 125.45, 126.21, 127.73 (2 C), 129.70 (2 C), 132.95, 133.36, 134.14, 134.45, 134.80, 136.02, 136.70. 137.27, 137.45, 139.40, 149.57, 153.21, 161.35, 188.33. MS (ESI): [M]+ = 500.1, [M + 2]+ = 502.2. Anal. calcd for C27H22BrN3O2. C, 64.81; H, 4.43; N, 8.40; found: C, 64.67; H, 4.31; N, 8.22.(E)-2-Bromo-N-(1-methyl-3-(3-oxo-3-(pyridin-4-yl)prop-1-en-1-yl)-1H-indol-5-yl)acrylamide (4a). Following general procedure D, after workup as described previously, the residue was purified by flash chromatography on silica gel using light petroleum ether:EtOAc 1:9 as eluent, affording compound 4a as a yellow solid. Yield: 89%, mp 226–228 °C. 1H-NMR  δ: 3.86 , 6.32 , 6.80 , 7.42 , 7.58 , 7.66 , 7.88 , 7.98 , 8.16 , 8.31 , 8.83 , 10.3 . 13C NMR  δ: 33.20, 110.99, 111.56, 111.96, 114.70, 116.94, 120.80, 121.27 (2 C), 125.32, 125.62, 132.90, 135.11, 137.98, 140.05, 144.80, 150.56 (2C), 160.82, 188.52. MS (ESI): [M]+ = 410.3, [M + 2]+ = 412.3. Anal. calcd for C20H16BrN3O2. C, 58.55; H, 3.93; N, 10.24; found: C, 58.31; H, 3.77; N, 10.03.(E)-2-Bromo-N-(1-ethyl-3-(3-oxo-3-(pyridin-4-yl)prop-1-en-1-yl)-1H-indol-5-yl)acrylamide (4b). Following general procedure D, the residue was purified by flash chromatography on silica gel using light petroleum ether: EtOAc 2:8 as eluent, affording compound 4b as a yellow solid. Yield: 64%, mp 165–167 °C. 1H-NMR  δ: 1.43 , 4.26 , 6.32 , 6.79 , 7.43 , 7.65 , 7.88 , 7.99 , 8.26 , 8.30 , 8.83 , 10.3 . 13C NMR  δ: 14.96, 41.05, 110.99, 111.75, 111.99, 114.78, 116.92, 120.76, 121.28 (2C), 125.24, 125.62, 132.86, 134.14, 136.26, 140.06, 144.80, 150.55 (2 C), 160.81, 188.56. MS (ESI): [M]+ = 424.3, [M + 2]+ = 426.3. Anal. calcd for C21H18BrN3O2. C, 59.45; H, 4.28; N, 9,90; found: C, 59.27; H, 4.15; N, 9,77.(E)-2-Bromo-N-(1-propyl-3-(3-oxo-3-(pyridin-4-yl)prop-1-en-1-yl)-1H-indol-5-yl)acrylamide (4c). Following general procedure D, after workup as described previously, the residue was purified by flash chromatography on silica gel using light petroleum ether:EtOAc 2:8 as eluent, affording compound 4c as a yellow solid. Yield: 58%, mp 193–195 °C. 1H-NMR  δ: 0.86 , 1.82 , 4.20 , 6.32 , 6.79 , 7.44 , 7.63 , 7.88 , 7.98 , 8.24 , 8.30 , 8.83 , 10.3 . 13C NMR  δ: 10.96, 22.73, 47.66, 111.10, 111.65, 112.01, 114.86, 116.94, 120.70, 121.28 (2C), 125.46, 125.62, 132.80, 134.48, 136.93, 140.04, 144.80, 150.55 (2C), 160.81, 188.56. MS (ESI): [M]+ = 438.3, [M + 2]+ = 440.3. Anal. calcd for C22H20BrN3O2. C, 60.28; H, 4.60; N, 9.59; found: C, 60.03; H, 4.46; N, 9.38.(E)-2-Bromo-N-(1-isopropyl-3-(3-oxo-3-(pyridin-4-yl)prop-1-en-1-yl)-1H-indol-5-yl)acrylamide (4d). Following general procedure D, after workup as described previously, the residue was purified by flash chromatography on silica gel using light petroleum ether:EtOAc 2:8 as eluent, affording compound 4d as a yellow solid. Yield: 62%, mp 202–204 °C. 1H-NMR  δ: 1.50 , 4.81 , 6.32 , 6.79 , 7.45 , 7.65 , 7.88 , 7.99 , 8.29 , 8.40 , 8.83 , 10.3 . 13C NMR  δ: 22.20 (2 C), 47.44, 111.06, 111.81, 111.97, 114.83, 116.82, 120.68, 121.29 (2 C), 125.23, 125.60, 132.83, 133.28, 133.92, 140.08, 144.81, 150.53 (2 C), 160.79, 188.62. MS (ESI): [M]+ = 438.2, [M + 2]+ = 440.2. Anal. calcd for C22H20BrN3O2. C, 60.28; H, 4.60; N, 9.59; found: C, 60.06; H, 4.44; N, 9.32.N, N, N′, N′-tetramethylethylenediamine were from Bio-Rad . All other chemicals were obtained from Sigma .Stock solutions of 100 mM of compounds were made in dimethyl sulfoxide (DMSO) and aliquots were frozen at −20 °C. Poly(vinylidene difluoride) (PVDF) membranes were purchased from Millipore . Acrylamide, bisacrylamide, ammonium persulfate and 2 as describedH-tetrazolium bromide (MTT) assay as previously described50) were determined graphically for each experiment by a non-linear regression using the curve-fitting routine of the computer software Prism™ 4.0 (GraphPad) and the equation derived by De Lean and co-workersU-937 (human myeloid leukaemia), MOLT-3 (acute lymphoblastic leukaemia), K-562 (chronic myeloid leukaemia), NALM-6 (B cell precursor leukaemia) and SK-MEL-1 (melanoma) cells were from DSMZ . The Burkitt’s lymphoma RAJI cells were from the American Type Culture Collection . Cells were cultured in RPMI 1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum, 100 μg/mL streptomycin and 100 U/mL penicillin, incubated at 37 °C in a humidified atmosphere containing 5% CO−1 bis-benzimide trihydrochloride (Hoechst 33258) and incubated at room temperature for 15 min. Stained nuclei were visualized using Zeiss fluorescent microscopy.Cells were harvested and fixed in 3% paraformaldehyde and incubated at room temperature for 10 min. The fixative was removed and the cells were washed with PBS, resuspended in 30–50 μL of PBS containing 20 μg mLg, washed with PBS and resuspended in 50 μL of PBS. Following dropwise addition of 1 ml of ice-cold 75% ethanol, fixed cells were stored at −20 °C for 1 h. Samples were then centrifuged at 500 × g and washed with PBS before resuspension in 1 mL of PBS containing 50 μg mL−1 propidium iodide and 100 μg mL−1 RNase A and incubation for 1 h at 37 °C in the dark. The percentage of cells with decreased DNA staining, composed of apoptotic cells resulting from either fragmentation or decreased chromatin, was determined of a minimum of 10,000 cells per experimental condition. Cell debris was excluded from analysis by selective gating based on anterior and right angle scattering. Apoptosis was also determined by translocation of phosphatidylserine to the cell surface using the annexin V-fluoresceine isothiocyanate (FITC) apoptosis detection kit (BD Pharmingen) according to the manufacturer’s protocol.To study changes in the cell DNA content, histogram measurements of hypodiploid DNA formation was performed by flow cytometry using a BD FACSVerse™ cytometer . Histograms were analysed with the Flowing Software™. Cells were collected and centrifuged at 500 × 5) were collected by centrifugation, washed with PBS and incubated in 30 μL of lysis buffer , containing 1 μg μL−1 RNase A at 37 °C for 1 h. Then, 3 μL of proteinase K (10 μg μL−1) was added and the mixture was incubated at 50 °C for an additional 2 h. DNA was extracted with 100 μL of phenol-chloroform-isoamyl alcohol (24:24:1) and mixed with 5 μL of loading solution . Samples were separated by electrophoresis in 2% agarose gels in TAE buffer , visualized by ultraviolet illumination after ethidium bromide (0.5 μg mL−1) staining and the images were captured by a digital camera .A late biochemical hallmark of apoptosis is the fragmentation of the genomic DNA. It is an irreversible event and occurs before changes in plasma membrane permeability. DNA isolation and gel electrophoresis were performed as described previously6 mL−1) were treated in the absence or presence of compounds 3a–e and 4a–d (0.3 μM) for 6 h and harvested by centrifugation at 500 × g for 10 min. Cell pellets were then resuspended in ice-cold buffer  containing 250 mM sucrose. After 15 min incubation on ice, cells were lysed by pushing them several times through a 29-gauge needle and the lysate spun down at 1000 × g for 5 min at 4 °C. Protein concentration in the nuclear fraction was measured by the Bradford method and samples containing equal amounts of proteins were boiled in sodium dodecyl sulfate sample buffer for 5 min before loading on a 7.5% polyacrylamide gel containing 0.1% sodium dodecyl sulphate. Proteins were electrotransferred to poly(vinylidene difluoride) (PVDF) membranes, blocked with 5% fat-free dry milk in Tris-buffered saline  with 0.1% Tween 20 and then incubated with anti-poly(ADP-ribose)polymerase monoclonal antibody  overnight at 4 °C. After washing and incubation with anti-mouse antibody conjugated to horseradish peroxidase , the antigen-antibody complexes were visualized by enhanced chemiluminescence (Millipore) using the manufacturer's protocol.Cells (1 × 10pNA (for caspase-3 like protease activity), VEID-pNA (for caspase-6 activity), IETD-pNA (for caspase-8 activity) and LEHD-pNA (for caspase-9 activity) as described previouslyCaspase activity was evaluated by measuring proteolytic cleavage of the chromogenic substrates DEVD-The membrane potential was measured by flow cytometry using the fluorochrome 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). Flow cytometric analysis was carried out using a BD FACSVerse™ cytometer . All of the methods have been described in detail elsewheret-test. p values of <.05 were considered significant.Statistical significance of differences between means of control and treated samples were calculated using Student’s 3a–h and 4a–d along with some selected amino precursors 9a, 9c, 9e, and 10c against a panel of six human tumour cell lines such as leukaemia , Burkitt’s lymphoma (RAJI) and melanoma (SK-MEL-1) cells.In 50 values. Among them, derivatives 3a, 3b, 3c, and 4a exhibited the most potent activity, with IC50 values of 10–64, 9–47, 6–47, and 7–59 nM, respectively, against U-937, MOLT-3, K-562, and NALM-6 leukaemia cell lines. In general, the two leukaemia cell lines MOLT-3 and NALM-6 were the most sensitive to the influence of conjugates 3a–e and 4a–c, with compound 3c which was active in the single-digit nanomolar range against both cancer cell lines.We found that most of the new hybrid compounds are characterized by selective effects on cancer cell lines, displaying high antiproliferative activity towards leukaemia cells, with one-digit to double-digit nanomolar IC3a, 3c, 3e, and 4c with those of the amino derivative precursors 9a, 9c, 9e, and 10c, respectively. These latter compounds exhibited tumour cell growth inhibitory activity from two- to four-order of magnitude lower than their corresponding conjugates, demonstrating that the presence of the α-bromoacryloyl group was essential for antiproliferative effects. The cell growth inhibitory activities of the hybrid molecules and their selectivity against the different cancer cell lines were influenced by the substituent at the N-1 position of the indole ring as well as the position of the nitrogen (meta or para) on the pyridine of the indolyl–pyridinyl–chalcone motif.The validity of the hybridization approach was confirmed comparing the activity of hybrid derivatives 3a, the alkylation of the indole nitrogen with small alkyl groups such as methyl or ethyl was tolerated, while the extension of the alkyl chain from ethyl to n-propyl, and in particular the introduction of benzyl and p-substituted benzyl, it was instead unfavourable for the antiproliferative activity. This effect resulted more evident against the four human leukaemia  cell lines.Compared with the 3-pyridinyl derivative 3a, the insertion of a methyl group at the 2-position of indole ring, to furnish 3b, had varying effects on antiproliferative activity. Derivatives 3a and 3b are equipotent against MOLT-3, NALM-6, and RAJI cells, while 3a was 1.5-fold less potent than 3b against U-937 and K-562 leukaemia cells. The opposite effect was observed on SK-MEL-1 cells, with the 2-methyl indole derivative 3b which was 1.5-fold less active than 3a.Relative to the activity of N-methylation of indole ring nitrogen of 3-pyridinyl derivative 3a, to furnish compound 3c, caused a 2-fold increased activity with respect to 3a against U-937 and MOLT-3 cells and a 1.5-fold in K562 cells, while an opposite effect was observed against SK-MEL-1 cells, with a 2-fold reduced potency. The two molecules 3a and 3c showed similar cell growth inhibitory activity against NALM-6 and RAJI cells.The 4a–d, increasing the length of the alkyl chain from methyl to ethyl  caused a 1.5- to 2-fold reduction in cytostatic activity against MOLT-3, NALM-6, K-562, and SK-MEL-1, with the two derivatives which were equipotent against U-937 and RAJI cells. A similar effect was observed in the series of 3-pyridine analogues between N-methyl and N-ethyl derivatives 3c and 3d, respectively, with the N-ethyl derivative 3d which was 1.5- to 2-fold less active than N-methyl counterpart 3c against U-937, MOLT-3, NALM-6, K-562, and RAJI, while there were minor differences in IC50 values against SK-MEL-1 cells.In the series of 4-pyridine analogues N-1 position of the indole ring , changing the position of the nitrogen atom in the pyridine ring from the meta-position in 3-pyridine to the para-position in 4-pyridine had contrasting effects on the activity against the panel of human cancer cell lines.By the comparison of compounds with the same substituent at the N-methyl indole derivatives 3c and 4a, there was little difference in activity between these two isomeric pyridine derivatives against U-937, MOLT-3, K-562, and RAJI cells. This latter compound halved the activity of the former against NALM-6.For the N-ethyl indole derivatives 3d and 4b, the 3-pyridinyl derivative 3d was from 1.3- to 2-fold more potent than the 4-pyridinyl counterpart 4b against NALM-6, K-562 and SK-MEL-1 cells, while 4b exerted a 2-fold more pronounced antiproliferative activity in comparison with 3d toward U-937 cells. The differences between the two compounds were minimal in MOLT-3 and RAJI cell lines.For the N-n-propyl indole derivatives 3e and 4c were similar against MOLT-3, K-562, RAJI and SK-MEL-1 cells, while the 3-pyridinyl analogue 3e had 1.7- and 2.8-fold reduced activity as compared with 4-pyridinyl compound 4c against NALM-6 and U-937 cell lines, respectively.The cell growth inhibitory activities of 3f–h, compound 3f with no substituent on the benzene ring resulted in the most active derivative against the six cancer cell lines. For the p-chlorobenzyl derivative 3g, its activity was lower than that of 3f against MOLT-3, NALM-6, and SK-MEL-1 cells, while it resulted in 2-fold more potent than 3f against RAJI cells. The two compounds were equipotent against U-937 and K-562 cells. By comparing the effect of substituents with opposite electronic properties at the para-position on the phenyl of the benzyl moiety, the presence of the electron-donating methyl group was less favourable for the activity with respect to the electron-withdrawing chlorine atom, with the p-chlorobenzyl derivative 3g which was from 1.5- to 5-fold more active than p-methyl benzyl counterpart 3h against five of the six cancer cell lines, while the two compounds were equipotent against NALM-6 cell line.In the series of benzyl derivatives n-propyl and the branched iso-propyl derivatives 4c and 4d, respectively, were very similar against the NALM-6, K-562, and SK-MEL-1 cells, while 4d exhibited reduced activity (from 2- to 13-fold) as compared with 4c against U-937, MOLT-3, and RAJI cell lines.The cell growth inhibitory activities of 4a for 24 h and cell viability was determined by the MTT assay. The human tumour leukaemia U-937 cells were included in the experiment as a positive control. The results indicate no cytotoxicity (up 0.1 µM) to either fresh or proliferating PBMC growth, while there was an 80% reduction in cell viability in U-937 cells , quiescent and phytohemagglutinine (PHA)-activated healthy human PBMC were incubated in presence of increasing concentrations of the 4-pyridine indole derivative 37 cells , suggest,3a–h and 4a–d to induce apoptosis, using human myeloid leukaemia U-937 cell lines as the experimental system  is characteristic of apoptosis and reflects fragmented DNA.Degradation of DNA into a specific fragmentation pattern was considered the end-point of the apoptotic pathway and results from activation of caspase-activated endonuclease0-G1 peak. These experiments showed that the percentage of hypodiploid cells increased approximately 16-, 14-, 22-, 20-, and 20-fold after 6 h of treatment at a concentration of 0.3 μM of compounds 3a, 3b, 3d, 4a, and 4b in U-937 cells, respectively , a family of intracellular cysteine proteases expressed as inactive zymogens in living cells that, once activated in response to proapoptotic signals, lead to irreversible cell death3a–e and 4a–d in U-937 cells, cell lysates were assayed for cleavage of the tetrapeptides DEVD-pNA, VEID-pNA, IETD-pNA and LEHD-pNA as specific substrates for caspase-3/7, caspase-6, caspase-8, and caspase-9, respectively. As shown in 3b and 4a in U-937 cells, indicating that apoptotic cell death takes place in a caspase-dependent pathway. There was also caspase activation after 6 h of treatment with the same compounds that increased the percentage of hypodiploid cells . These findings indicated that these derivatives induce a fast activation of caspases in U-937 cells.To determine whether caspases were involved in the apoptotic cell death induced by hybrid derivatives 3a, 3b, 3d, 4a, and 4b, in accordance with the experiments of caspase activation. Etoposide (10 μM) was included in both experiments (caspase activation and PARP cleavage) as a positive control.Poly(ADP-ribose)polymerase (PARP) is involved in DNA repair predominantly in response to environmental stress and is important for the maintenance of cell viability4a was one of the most cytotoxic compounds against U-937 cells as well a potent apoptotic inducer, additional experiments were done with this molecule. The quantification of apoptosis obtained by measurement of the number of hypodiploid cells by flow cytometry showed that the percentage of hypodiploid cells increased approximately 20-fold in 4a-treated cells, after 6 h exposure at a concentration as low as 0.3 μM and this effect was dose-dependent. Maximal levels of apoptotic cells (approximately 50-fold increase with respect to control) were observed at 6 h with a 1 μM concentration of 4a  as detected by annexin V-FITC staining in U-937 cells  of compound 4a for 6 h, showed an accumulation of early apoptotic cells  in comparison with the untreated cells (2.4%).To characterize the mode of cell death induced by 37 cells . As show4a at the concentration of 0.3 μM. Control non-treated U-937 cells exhibited normal features, with the nuclei round and homogeneousApoptosis is regulated by genetic mechanisms and characterized by morphological and biochemical changes in the cell nucleus, including chromatin condensation and nuclear shrinking,4a. Dose-response experiments show that a low concentration (0.1 μM) of compound 4a was sufficient to induce caspase-3/7 activation  created by the proteins of the respiratory chain located on the inner mitochondrial membrane is also a key event in mitochondria-controlled apoptotic pathwaysm) is involved in the mechanism of cell death, U-937 cells were treated with increasing concentrations of 4a, stained using the dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine (JC-1) to monitor changes in ΔΨm induced by 4a and analysed by flow cytometry. m dropped after 4 h of treatment at different concentrations , which suggests that the dissipation of the mitochondrial membrane potential contributed to apoptosis induced by the hybrid compound 4a. After a 4 h treatment of U-937 cells at two different concentrations (0.3 and 1 μM) of 4a, the results showed in m-chlorophenyl hydrazone  that depolarizes mitochondrial membranes, was used as a positive control.Increasing evidence indicated that mitochondria play a vital role in the progression of apoptosis3 and 4 that incorporate these two moieties within their structures. These two series of compounds have been synthesized to further explore the influence of the pyridinyl moiety attached at the carbonyl of the propenone system by switching the position of the pyridine nitrogen from the meta- to the para-position, to obtain the 3- and 4-pyridinyl derivatives 3a–h and 4a–d, respectively. The results revealed that substituent at the N-1 position of the indole ring was a crucial determinant for the biological activity of these two classes of compounds. We found that most of the α-bromoacryloylamido indole–pyridine–propenone derivatives selectively inhibit the growth of U-937, MOLT-3, K-562, and NALM-6 leukaemia cell lines, with single- to double-digit nanomolar IC50 values. With the only two exceptions (3g and 4d), the tested compounds resulted more active against MOLT-3 and NALM-6 cell lines. Notably, derivatives 3a–e and 4a–c showed potent growth inhibitory activities against MOLT-3 and NALM-6 cells with IC50 values in the range 6–48 and 9–87 nM, respectively. The considerable differences in activity between the hybrid compounds and their corresponding amino indolyl pyridinyl propanone precursors, with these later that showed weak or no antiproliferative activity against the panel of cancer cell lines, revealed that the insertion of the α-bromoacryloyl moiety was an important molecular change that influences the bioactivity, leading to a significant increase in potency. Comparing derivatives with the same substituent at the N-1 position of the indole ring , there is no great difference in activity between 3- and 4-pyridine analogues against MOLT-3, K-562, RAJI and SK-MEL-1 cells.Because of their ability to interact with cellular nucleophiles, Michael acceptors are often employed as a powerful tool in the design of anticancer agents. The observation that the α,β-unsaturated carbonyl system of indolyl pyridinyl propenone and the α-bromoacryloyl group are capable of undergoing Michael addition and thus can act as trapping agents of cellular nucleophiles led us to prepare and evaluate two series of α-bromoacryloylamido indolyl pyridinyl propenone derivatives with general structures 3f–h revealed that while the N-alkylation of indole nucleus was generally well-tolerated (derivatives 3c–e), the N-benzylation was detrimental for the potency. In general, the presence of either electron-withdrawing chlorine or electron-releasing methyl substituents on the phenyl of the benzyl moiety  reduced the cytostatic activity as compared with the unsubstituted benzyl derivative 3f.The antiproliferative activities of 3-pyridinyl derivatives 4a showed typical morphological features of apoptosis such as chromatin condensation and fragmented nuclei. Apoptosis was accompanied by cleavage of the nuclear protein PARP, a recognized caspase-3 substrate which is involved in DNA repair and it is important for maintaining cell viability. These results lead us to conclude that derivative 4a induced apoptosis by activation of the extrinsic and the intrinsic pathways of cell death. The induction of apoptosis was confirmed by the annexin V test. The increase in annexin V-positive cells demonstrated activation of the apoptotic pathway by this compound. Finally, our results indicated that compound 4a induced apoptotic cell death through a mechanism that involved activation of multiple caspases and by important changes in mitochondrial function, such as dissipation of the transmembrane potential. Finally, the low cytotoxicity of compound 4a toward normal human cells (PBMC), revealed its potential to be clinically developed for anticancer chemotherapy.Moreover, all hybrid molecules were also evaluated for their effects on induction of apoptosis in U-937 cells. These molecules caused an increase in the number of hypodiploid cells and cell death was found to be associated with the disruption of the mitochondrial potential and activation of caspases. U-937 cells treated with compound


Euphorbia prolifera. Their structures were elucidated as 2α-O-isobutyryl-3β,5α,7β,10,15β-penta-O-acetyl-14α-O-benzoyl-10,18-dihydromyrsinol (1), 2α-O-isobutyryl-3β-O-propion-yl-5α,7β,10,15β-tetra-O-acetyl-10,18-dihydromyrsinol (2), and 2α,14α-di-O-benzoyl-3β,5α,7β,10,15β-penta-O-acetyl-10,18-dihydromyrsinol (3) on the basis of spectroscopic data analyses . Their neuroprotective activities were evaluated and compounds 1 and 2 showed neuroprotective effects against MPP+-induced neuronal cell death in SH-SY5Y cells.Three new myrsinol diterpenes were isolated from the roots of  Euphorbia prolifera Buch-Ham, belonging to the family Euphorbiaceae, is a perennial herbaceous plant distributed in southwest China , four oxygenated methines , and one oxygenated methylene [δH 4.09 and 3.49 ]. In addition, 11 methyls and one monosubstituted benzene ring were also revealed in the 1H-NMR spectrum. The 13C-NMR spectrum of 1 exihibited 41 carbon resonances. From the 1H- and 13C-NMR spectra of 1, five acetyl groups , and one benzoyl group   , two methylenes (C-1 and C-17), nine methines , and five quaternary carbons . By comparing the chemical shifts of C-1-C-20 of compound 1 with those of myrsinol diterpenes reported in the literature  −34.5 ; IR (KBr) νmax cm−1: 2982, 1739, 1602, 1452, 1372, 1246; ESI-MS: m/z 807 [M + Na]+; HR-ESI-MS m/z 807.3195 [M + Na]+, calcd. for C41H52O15Na 807.3204; 1H-NMR  data see 13C-NMR  data see 2α-O-isobutyryl-3β-O-propionyl-5α,7β,10,15β-tetra-O-acetyl-10,18-dihydromyrsinol (2). Colorless needles (MeOH); m.p. 91-93 °C; [α] −56.0 ; IR (KBr) νmax cm−1: 3524, 2982, 1735, 1462, 1371, 1244; ESI-MS: m/z 717 [M + Na]+; HR-ESI-MS m/z 717.3101 [M + Na]+, calcd. for C35H50O14Na 717.3098; 1H-NMR  data see 13C-NMR  data see 2α,14α-di-O-benzoyl-3β,5α,7β,10,15β-penta-O-acetyl-10,18-dihydromyrsinol (3). White powder; [α] −73.2 ; IR (KBr) νmax cm−1: 2955, 1742, 1452, 1373, 1246; ESI-MS: m/z 841 [M + Na]+; HR-ESI-MS m/z 841.3040 [M + Na]+, calcd. for C44H50O15Na 841.3047; 1H-NMR  data see 13C-NMR  data see 2. The cells were disassociated by incubation with 1 mM of ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) in phosphate-buffered saline (PBS) for 15 min and then seeded in 96-well culture plates (1 × 104 cells/well). The cells were incubated at 37 °C in a 5% CO2 humidified air incubator for 24 h. The cells were pre-treated for 2 h with various concentrations  of the compounds before incubation in a medium containing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPP+). MTT dissolved in phosphate-buffered saline was added at the end of incubation to a final concentration of 0.5 mg/mL. After incubation for 4 h at 37 °C and 5% CO2, the supernatant was removed and the formed formazan crystals in the viable cells were measured at 490 nm by using a microplate reader . Experiments were carried out in triplicate. A statistical analysis was performed by a one-way analysis of variance (ANOVA) followed by post hoc multiple comparisons using the Student-Newman-Keuls method. Data are expressed as the mean ± SD of three assays. Human dopaminergic neuroblastoma SH-SY5Y cells were cultured at 37 °C in DMEM supplemented with 10% (v/v) inactivated fetal bovine serum and 100 U/mL of penicillin/streptomycin in a water-saturated atmosphere of 95% air and 5% COE. prolifera. Their structures were elucidated on the basis of extensive spectroscopic data analyses. Biological evaluation disclosed that two of them exhibited strong neuroprotective effects against MPP+-induced neuronal cell death in human dopaminergic neuroblastoma SH-SY5Y cells. The results of our chemical investigation further revealed the chemical constituents of E. prolifera. While, the current biological data suggest that myrsinol diterpenes with strong neuroprotective activities may probably be useful for the treatment of nervous system diseases, such as Parkinson's disease [In summary, three new myrsinol diterpenes were successfully isolated from the roots of  disease . Further


N-trans-coumaroyltyramine (1), N-trans-feruloyltyramine (2), N-trans-feruloyloctopamine (3), 5,7-dihydroxy-8-methoxyflavone (4) and (3S)3,5,4′-trihydroxy-7-methoxy-6-methylhomoisoflavanone (5), were isolated from the fibrous roots of Liriope muscari (Liliaceae). Compounds 2–5 were isolated for the first time from the Liriope genus. Their in vitro antioxidant activities were assessed by the DPPH and ABTS scavenging methods with microplate assays. The structure-activity relationships of compounds 1–3 are discussed.Five phenolic compounds, namely  However, some chemically synthesized antioxidants like BHA and BHT are now being restricted by legislation because of doubts over their possible toxic and carcinogenic effects , one flavone  and one homoisoflavanone  were isolated from L. muscari. Compounds 2–5 were isolated for the first time from the Liriope genus. Chinese) , especiadiseases . Previouycosides ,12,13. IL. muscari have been studied. Their in vitro antioxidant activities were assessed by the DPPH and ABTS scavenging method. N-trans-feruloyltyramine  and N-trans-feruloyloctopamine  showed potential antioxidant activities.To our knowledge, this is the first time that the phenolic components of L. muscari. The structures of compounds 1–4 were characterized by examination of their ESI-MS, NMR (1H- and 13C-) data and comparison with literature reports.The compounds were isolated using silica gel and Sephadex LH-20 gel column chromatography from 80% ethanol extract of 5 was first isolated in 1985 from Ophiopogonis +. C17H17NO3. 1H-NMR  δ: 9.36 (-OH), 8.11 , 7.39 , 7.30 , 7.01 , 6.78 , 6.67 , 6.38 , 3.31 , 2.64 . The 1H- and 13C-NMR . Colourless oil (petroleum/acetic ether). Rf 0.62 (acetic ether) ESI-MS: m/z 314 [M+H]+. C18H19NO4. 1H-NMR  δ: 9.46, 9.21 , 8.01 , 7.31 , 7.12 , 7.01 , 6.98 , 6.78 , 6.68 , 6.43 , 3.80 , 3.33 , 2.64 . The 1H- and 13C-NMR . Colourless oil (petroleum/acetic ether). Rf 0.47 (acetic ether) ESI-MS: m/z 330 [M+H]+. C18H19NO5. 1H-NMR  δ: 9.47, 9.32 , 7.96 , 7.31 , 7.15 , 7.12 , 6.98 , 6.79 , 6.72 , 6.55 , 4.54 , 3.80 , 3.38, 3.18 . The 1H- and 13C-NMR . Yellow needle crystal (petroleum/acetic ether). Rf 0.40 (P/E 2:1) ESI-MS: m/z 283 [M−H]−. C16H12O5. 1H-NMR  δ:12.52 , 10.87 , 8.08 , 7.62 , 7.02 , 6.32 , 3.86 . 13C-NMR  δ: 182.5 (C-4), 163.5 (C-2), 157.8 (C-7), 156.7 (C-5), 150.1 (C-8a), 132.6 (C-4′), 131.3 (C-1′), 129.8 , 128.2 (C-8), 126.8 , 105.5 (C-3), 104.2 (C-4a), 99.6 (C-6), 61.5 (-OCH3). The 1H- and 13C-NMR spectral data are consistent with published data −. C18H18O6. 1H-NMR  δ: 7.09 , 6.78 , 6.10 , 4.23 , 4.06 , 3.91 , 2.96 , 2.06 . 13C-NMR  δ: 198.3 (C-4), 166.5 (C-7), 161.1 (C-8a), 160.1 (C-5), 154.9 (C-4′), 131.8 , 126.2 (C-1′), 115.3 , 106.5 (C-6), 100.2 (C-4a), 91.0 (C-8), 72.3 (C-3), 71.9 (C-2), 56.0 (-OCH3), 40.8 (C-9), 6.9 (-CH3). The 1H-NMR spectral data are consistent with published data [r2: 0.9985): In this method, a microplate reader and 96 well plate were used to carry out the determination of the spectral absorption values. This assay is based on the classic method developed by Blois in 1958 ,37. Unli517-C is absorbance of a control with no radical scavenger and λ517-S is absorbance of the remaining DPPH in the presence of scavenger.The scavenging capability of test compounds was calculated using the following equation:2S2O8), and then storing in the dark for 6 h at room temperature, as described by Gülçin [·+ solutions (50 μL) were added to samples of different concentrations . These solutions were gently mixed and incubated in the dark for 30 min at room temperature. Then the absorbances of the resulting solutions were measured at 734 nm. Different concentrations of ABTSradical solutions (55–220 μg/mL) were used to prepare the standard curve. The ABTS radical concentration (μg/mL) in the reaction medium was calculated from the following calibration curve, determined by linear regression (r2: 0.9985):The ABTS assay was carried out using a method based on the original and classic method developed by Miller in 1993  with som734-C is absorbance of a control with no radical scavenger and λ734-S is absorbance of the remaining ABTS in the presence of scavenger.The scavenging capability of test compounds was calculated using the following equation:L. muscari were studied for the first time. Three amides, one flavone and one homoisoflavonone were isolated and their antioxidant activities were evaluated using two microplate assay methods. N-trans-feruloyltyramine (2) and N-trans-feruloyloctopamine (3) showed effective activity and the structure-activity relation investigation indicates that the -OCH3 group at C-3 affects the antioxidant activity.Phenolic components of


Docking and molecular dynamics studies revealed that the three inhibitors made interactions with residues at the secondary binding site to phosphate, exclusive for PTP1B. The data reported here support these compounds as hits for the design more potent and selective inhibitors against PTP1B in the search of new antidiabetic treatment.The Atlas of Diabetes reports 415 million diabetics in the world, a number that has surpassed in half the expected time the twenty year projection. Type 2 diabetes is the most frequent form of the disease; it is characterized by a defect in the secretion of insulin and a resistance in its target organs. In the search for new antidiabetic drugs, one of the principal strategies consists in promoting the action of insulin. In this sense, attention has been centered in the protein tyrosine phosphatase 1B (PTP1B), a protein whose overexpression or increase of its activity has been related in many studies with insulin resistance. In the present work, a chemical library of 250 compounds was evaluated to determine their inhibition capability on the protein PTP1B. Ten molecules inhibited over the 50% of the activity of the PTP1B, the three most potent molecules were selected for its characterization, reporting Ki values of 5.2, 4.2 and 41.3 µM, for compounds  In its sixth edition, the Atlas of Diabetes reported 415 million diabetics worldwide, surpassing in half the time the twenty year projection . Type 2 To this end, one of the main strategies consists in promoting the action of insulin , and theS)-isomer of isothiazolidinone ((S)-IZD) sulfanyl}-1-ethanone (4); SP 00892 for 4-{5-[5--2-furyl]-1,2,4-oxadiazol-3-yl}phenyl-N,N-dimethylsulfamate (5); RJF 01991 for ′N′1-{2-[(2-oxo-4-propyl-2H-chromen-7-yl)oxy]propanoyl}-3-(trifluoromethyl)benzene-1-sulfonohydrazide (6); HTS 02534 for ′N-(3-chloro-4-fluorophenyl)-2-[sulfanyl]acetamide (7); RH 02067 for ′N-{3-[oxy]pyridin-2-yl}-4-pentylbenzenesulfonamide (8). The general synthesis method for compounds 1, 2, 3, 9 and 10 is outlined below.The tested chemical library was composed of an in-house set of 100 compounds and 150 small molecules of the Fragment Library and HitFinder1–3 and 9 were prepared from the appropriate benzimidazole-2-amine and the adequate acid or anhydride under the guidelines of our synthetic procedure previously reported for similar benzimidazole derivatives -1-methyl-2-(methylthio)-1H-benz-imidazole-6-carboxamide (1). Recrystallized from DMF/MeOH white solid (89%); m.p. 269-270 °C. 1H-NMR : δ 2.75 ; 3.72 ; 6.69 ; 7.27 ; 7.33 ; 7.37 ; 7.68 ; 7.71 ; 7.91 ; 12.40 . 13C-NMR : δ 14.48 (S-CH3); 30.56 (N-CH3); 110.93 (C-7 benzimidazole); 115.64 (C-6 dichlorophenoxy); 118.45 (C-4 benzimidazole); 118.59 (C-7′a benz-imidazole); 121.36 (C-2 dichlorophenoxy); 123.73 (C-5 benzimidazole); 124.69 (C-4 dichlorophenoxy); 127.79 C-6 benzimidazole); 129.11 (C-5 dichlorophenoxy); 133.23 (C-3 dichlorophenoxy); 135.63 (C-7a benzimidazole); 144.89 (C-3a benzimidazole); 145.04 (C-6′ benzimidazole); 148.58 (C-2′ benzimidazole); 155.10 (C-1 dichlorophenoxy); 157.27 (C-2 benzimidazole); 166.85 (C=O amide). EI-MS: m/z 565 (M+); HRMS (FAB+): 565.9752 [M + H]+ .2-[6-Chloro-5--1H-benzimidazol-2-yl]thio-N-(thiazol-2-yl)acetamide (2). Recrystallized from methanol to give a beige solid (20% yield); m.p. 155-157 °C. 1H-NMR : δ 4.37 . 6.64 , 7.23 , 7.27 , 7.37 , 7.48 , 7.61–7.57 , 7.64 , 7.72 , 7.99–7.95 , 8.27–8.22 , δ 12.70 . 13C-NMR : δ 34.79 (SCH2), 109.88 (C-2 naphtyloxy), 113.78 (C-3 naphtyloxy), 119.12 (C-5 thiazolyl), 121.36 (C-8 naphtyloxy), 122.45 (C-4 naphtyloxy), 124.86 (C-5 or C-6), 126.02 (C-6 or C-7 naphtyloxy), 126.13 (C-6 or C-7 naphtyloxy), 126.85 (C-4a or C-8a naphtyloxy), 127.77 (C-5 naphtyloxy), 134.45 (C-4a or C-8a naphtyloxy), 137.76 (C-4 thiazolyl), 146.06 (C-3a or C-7a), 151.96 (C-3a or C-7a), 153.37 (C-2), 157.81 (C-2 thiazolyl), 166.22 (CONH). MS (DART): m/z (%): 467 . HRMS (DART): Calcd for C22H15ClN4O2S2 [M + H]+: 467.04032, found: 467.04177.N-[6-Chloro-5--1H-benzimidazol-2-yl]-2,2,2-trifluoroacetamide (3). Purified by washing with cold water. Beige solid; m.p. > 200 °C (d). 1H-NMR  δ: 6.84 ; 7.21 ; 7.32 ; 7.44 ; 7.66 ; 13.07 . 13C-NMR : δ 105.22 (C-4), 114.10 (C-7), 116.97 (C-6′), 117.52 , 120.37 (C-6), 122.15 (C-2′), 125.65 (C-4′), 127.03, 129.22, 129.36 (C-5′), 133.41 (C-3′), 147.07 (C-5), 153.98 (C-2), 154.17 (C-1’), 162.99 .N-[6-Chloro-5--1-methyl-1H-benzimidazol-2-yl]acetamide (9). Recrystallized from ethanol, white crystals (84% yield); m.p. 237.5–238.9 °C. 1H-NMR  δ: 2.16 ; 3.64 ; 6.65 ; 7.24 ; 7.36 ; 7.42 ; 7.88 ; 10.97 . 13C-NMR : δ 23.43 (CO-CH3), 30.97 (N-CH3), 111.95 (C-4), 112.27 (C-7), 115.51 (C-6′), 119.52 (C-6), 121.28 (C-2′), 124.70 (C-4′), 129.16 (C-5′), 133.25, 140.41 (C-3′), 145.39 (C-4), 148.37 (C-2), 155.14 (C-1′), 170.57 (CO). EA. Calc.: C16H12Cl3N3O2: C, 49.96; H, 3.14; N, 10.9. Found: C, 49.82; H, 2.57; N, 10.6. HMRS (ESI) Calcd for C16H12Cl3N3O2 [M + Na]: 406.0073; found 406.E. coli BLR strains were transformed for the overexpression of the protein. With this purpose, 500 mL of LB liquid culture medium was grown supplemented with Kanamycin (50 µg/mL) at 37 °C, once it reached an optical density of 0.9 at 600 nm, 1mM of IPTG was added to induce the overexpression, incubating four more hours. Right away, cells were cultured by centrifugation and lysed by sonication. The supernatant was passed through a Ni-agarose column and the enzyme was purified by an imidazole gradient. The fractions were analyzed by SDS-PAGE electrophoresis and those with the presence of the protein were pooled and concentrated with a Plus-70 centricon, immediately the enzyme was precipitated with ammonium sulfate (80% saturation).The region of the gene PTPN1 that encodes for PTP1B (residues 1–321) was synthetized by Integrated DNA Technologies and inserted in the pIDTSmart plasmid. Then, the gene was liberated by restriction reactions using Ndel and BamH1 enzymes and inserted into the overexpression vector pET28A. Afterwards, −1 cm−1).The PTP1B activity was measured based on the Goldstein method . The ass50) of the PTP1B activity was determined through curves at different concentration of each compound, adjusting the data to the equation reported elsewhere [1 the concentrations used were 3, 6, 10 and 20 µM; in the case of compound 2 were 2, 4, 6, 8, and 10 µM; and for compound 3 were 20, 50, 70 and 90 µM. The experimental data were analyzed through the Lineweaver-Burk plot using the software Sigma Plot V12.3 .Inhibition assays were performed under the above described conditions, adding to the reaction each one of the molecules at a final concentration of 200 µM. The concentration that inhibits 50% (IClsewhere . The inhwww.schrodinger.com) and prepared in Ligprep 2.3 [The molecules were built in Maestro 10.4  were calculated and obtained using PRODRG [The initial structures for the simulations were those with the lowest binding energy of each complex obtained by docking. The necessary topology files for each ligand (compounds g PRODRG . The sysg PRODRG  using thg PRODRG . The FAF-Drugs4 server  and the With respect to toxicological parameters, PROTOX  server wIn the present work three new mixed type inhibitors for PTP1B are reported, which based on their inhibition capability and mechanism, potential selectivity against TCPTP, and predicted drug-like properties, could represent a good starting point for the development of more potent molecules that can guide the design of a new drug to treat type 2 diabetes.


Geometry optimization of these compounds was executed by PM3, PM5 and Minimize Energy methods to describe them via molecular descriptors. The antimicrobial activity of the synthesized compounds was evaluated against different microbial strains using two different methods: the diffusion method and the micro-dilution method. All data indicated that the products possess antimicrobial activity which depends on the nature of substituent attached to the benzopyran moiety. In general, after 24 h the MIC values of most tested coumarins was 0.13 mg/mL, but compounds 1 and 6b displayed the strongest antimicrobial activity on the tested cultures of bacteria after 48 h. Compound 13b has the strongest growth inhibitory potential on fungus C. albicans, tested by diffusion method, with an inhibition zone of 30-37 mm at a concentration of 150 µg/mL. The conclusion of this experiment is that the synthesized compounds have varied and different influence on different classes of bacteria and the fungus C. albicans.Several novel 4-hydroxy-chromene-2-one derivatives  Coumarins are benzopyran derivatives. Naturally occurring coumarins have been isolated from over 800 species of plants and microorganisms , and manThe first reports of the application of commercial microwave ovens to the synthesis of small organic molecules appeared in 1986. ,12. MicrThe essence of this work was synthesis of coumarin derivatives using microwave irradiation, in comparison with conventional methods. Coumarin derivatives have previously been studied in our laboratory ,24,25,26C. albicans.The use of solvent-free conditions for the fast synthesis of novel coumarin derivatives by the Knoevenagel condensation under microwave irradiation offers several advantages : solvent2b-8b by the conventional condensation method were characterized by lower yields of the desired compounds (no more than 32%), followed by time consuming purifications by column chromatography. Thus, we report herein substantial improvements to the synthesis of these compounds using a solvent free microwave promoted reaction. The obtained results indicated significant yield increases (up to 87%), decreases in reaction times and, as far as purification of products is concerned, requiring only recrystallization from appropriate solvents.Preparations of coumarin derivatives 1) microwave heating provided several advantages compared to the conventional method of preparation by azeotropic water removal. Due to homogenous heating achievable under microwaves side reactions were almost eliminated, which resulted in increased yields of the desired compounds 9b-16b (up to 90%). Additionally purifications were limited to only recrystallization from methanol, without requiring time and solvent consuming column chromatography.Also, in a case of synthesis of imino derivatives of 4-hydroxychromene-2-one  analysis data of all compounds were in full agreement with the suggested molecular structures. The IR spectra of pure products 2b-8b indicated the presence of a broadened OH band in the 3437-3422 cm-1 region, corresponding to the hydroxyl fragment of the coumarin moiety. A strong coumarin moiety lactone C=O band at 1706-1699 cm-1 was also observed. Synthesized compounds 2b, 3b, 4b and 5b possess additional strong C=O bands from ester COOMe or COOEt groups at 1731-1729 cm-1. Additional acetyl group C=O bands were observed at 1700-1686 cm-1 for compounds 3b, 6b and 8b. The IR spectra of compounds 7b and 8b showed broadened OH bands at 3111 and 3100 cm-1 and C=O bands at 1699-1689 cm-1 from COOH groups. Strong absorptions in the 2231-2229 cm-1 region indicated the presence of C≡N groups in the prepared compounds 5b and 7b. The 1H-NMR spectra of compounds 2b-8b showed the characteristic coumarin aromatic proton signals in the 7.83-7.29 ppm range . All these spectra also contain resonances from methyl fragments  and hydroxyl groups . The protons of the methyl group from the COOMe fragment of compounds 3b, 4b and 5b were observed as singlets at 3.86-3.81 ppm, while the compound 2b showed ethyl proton resonances at 4.21 ppm  and 1.23 ppm . In the vinyl proton region compound 4b showed an absorption at 5.76 ppm . Since the compounds 7b and 8b were prepared from acetic acid derivatives, broadened OH proton peaks were observed at low field . The methyl protons from the acetyl fragment of compounds 3b, 6b and 8b were identified in the range of 2.29-2.26 ppm.Both elemental and spectral . In the 1H-NMR spectrum of compound 16b a multiplet at 1.82 ppm for a –CH2-CH2- group, two triplets at 2.46 ppm and 3.60 ppm for a methylene protons from C’-4 and C’-1, respectively, and a broadened singlet from the hydroxyl group of COOH fragment at 11.3 ppm were observed.Formation of imine derivatives In 1, as a base for synthetic research, with compounds 6b and 13b, were subjected to geometry examination since molecule planarity and voluminous side chain groups play important roles in the bioactivity of the compounds . This apparatus is well suited for stringent reaction conditions, namely, anhydrous atmosphere, controlled temperature  and attachment of reflux condenser with constant stirring.All reagents, solvents and catalyst were of analytical grade and used directly. All the melting points were recorded on a Kofler-hot stage apparatus  and uncorrected. The purity of compounds was checked routinely by TLC using a Merck Kieselgel 60 PF1, 2.5 mmol) and toluene (50 mL) containing a catalytic amount of ammonium acetate was taken and carbonyl compound 2a-8a was added. The reaction mixture was stirred for a period of 8 h at the boiling point of the solvent with azeotropic removal of water formed. The excess solvent was distilled off and solid product was filtered, dried, purified via column chromatography (benzene-acetone=8:2) to give products 2b-8b.A solution of 3-acetyl-4-hydroxychromene-2-one , carbonyl compound 2a-8a (2.8 mmol) and ammonium acetate  was irradiated and heated in the microwave reactor at the power setting and for the time indicated in 2b-8b , amine 9a-16a (0.01 mol) and a catalytic amount of p-toluenesulfonic acid in toluene (50 mL) was heated with azeotropic removal of water for a period of 10-12 h. Progres od reaction was monitored by TLC (toluene-acetone = 7:3). At the end of the reaction, the solvent was removed under reduced pressure. The solid products were filtered, dried and recrystallized from methanol to give compounds 9b-16b.A mixture of 3-acetyl-4-hydroxychromene-2-one (p-toluenesulfonic acid was added to a toluene solution (50 mL) of equimolar amounts (0.01 mol) of 3-acetyl-4-hydroxychromene-2-one (1) and amine 9a-16a. The mixture was heated under microwaves for 3 minutes. After cooling, the solvent was removed, and the obtained solid was filtered and recrystallized from methanol ethylidene)malonate (2b): Yield: 96%; m..p. 224-226 ºC; IR : ν(OH) 3434, ν(CH3 and CH2) 2949 and 2841, ν(C=O)1731 (lactone and ester), ν(C=C)ar, 1611, 1546 and 1496, ν(C-O-C) 1368 and 1032; 1H-NMR (δ ppm): 1.23 , 4.21 , 7.31-7.83 , 16.73 ; MS: m/z (%): 346 (M+); Anal. for C18H18O7 (346.33) (%): C: 62.42, H: 5.24; found C: 62.44, H: 5.23.Methyl 2-acetyl-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)but-2-enoate (3b): Yield: 97%; m.p. 237-239 ºC; IR : νOH 3433, ν(CH3), 2948, 2930, 2856, ν(C=O) 1730 (lactone and ester), ν(C=O) 1700, ν(C=C)ar 1610, 1545, 1495 1369, ν(C-O-C) 1032 and 1024; 1H-NMR (δ ppm): 2.20 , 2.26 , 3.81 , 7.29-7.83 , 17.01 ; MS: m/z (%): 302 (M+); Anal. for C16H14O6 (302.28) (%): C: 63.57, H: 4.67; found C: 63.55, H: 4.71.Methyl 3-(4-hydroxy-2-oxo-2H-chromen-3-yl)but-2-enoate (4b): Yield: 96%; m.p. 202-203 ºC, m.p. 222-223 ºC; IR : ν(OH) 3422, ν(CH3) 2949, 2930, ν(C=O) 2841, 1729 (lactone and ester), ν(C=C)ar 1609, 1542, 1491, ν(C-O-C) 1364, 1033, 1023; 1H-NMR (δ ppm): 2.22 , 3.86 , 5.76 , 7.29-7.82 , 17.11 ; MS: m/z (%): 260 (M+); Anal. for C14H12O5 (260.24) (%): C: 64.61, H: 4.65; found C: 64.63, H: 4.69.Methyl 2-cyano-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)but-2-enoate (5b): Yield: 94%; m.p. 247-249 ºC; IR : ν(OH) 3434, ν(CH3) 2949, 2929, 2854, ν(C≡N) 2231, ν(C=O) 1731 (lactone and ester), ν(C=C)ar 1611, 1546, 1497, ν(C-O-C) 1368, 1032, 1025; 1H-NMR (δ ppm): 2.23 , 3.80 , 7.29-7.83 , 17.06 ; MS: m/z (%): 285 (M+); Anal. for C15H11NO5 (285.06) (%): C: 63.16, H: 3.89, N: 4.91; found C: 63.18, H: 3.84, N: 4.95.3-(1-(4-Hydroxy-2-oxo-2H-chromen-3-yl)-ethylidene)-pentane-2,4-dione (6b): Yield: 94%; m.p. 241-243 ºC; IR : ν(OH) 3434, ν(CH3) 2949, 2929, ν(C=O) 1731 (lactone), ν(C=O) 1697, 1686, ν(C=C)ar 1610, 1544, 1496, ν(C-O-C) 1371, 1032; 1H-NMR (δ ppm): 2.23 , 2.29 , 7.31-7.83 , 17.01 ; MS: m/z (%): 286 (M+); Anal. for C16H14O5 (286.28) (%): C: 67.13, H: 4.93; found C: 67.15, H: 4.87.2-Cyano-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)but-2-enoic acid (7b): Yield: 84%; m.p. 256-257 ºC; IR : ν(OH) 3433 (coumarin), ν(OH) 3100 (COOH), ν(CH3) 2947, 2927, ν(C=O) 1731, ν(C=O) 1699 (COOH), ν(C=C)ar 1611, 1543’ 1495, ν(C-O-C) 1371, 1032; 1H NMR (δ ppm): 2.24 , 7.31-7.83 , 12.37 , 17.13 ; MS: m/z (%): 271 (M+); Anal. for C14H9NO5 (271.22) (%): C: 62.00, H: 3.34, N: 5.16; found C: 62.05, H: 5.11, N: 5.21.2-Acetyl-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)but-2-enoic acid (8b): Yield: 87%; m.p. 229-231 ºC; IR : ν(OH) 3437 (coumarin), ν(OH) 3111 (COOH), ν(CH3) 2991, ν(C=O) 1727 (lactone), ν(C=O) 1699, 1689 (COOH and keto group), ν(C=C)ar 1601, 1541, 1491, ν(C-O-C) 1369, 1031; 1H-NMR (δ ppm): 2.21 , 2.28 , 7.31-7.83 , 12.65 , 17.17 ; MS: m/z (%): 288 (M+); Anal. for C15H12O6 (271.22) (%): C: 62.50, H: 4.20; found C: 62.55, H: 4.21.4-Hydroxy-3-(1-(phenylimino)ethyl)-2H-chromen-2-one (9b): Yield: 95%; m.p. 169-171 ºC; IR : ν(OH) 3415, ν 3073, 3037, ν(CH3) 2929, 2853, ν(C=O) 1704 (lactone), ν(C=N) 1609 (imino), ν(C=C)ar 1592, 1561, 1480; 1H-NMR (δ ppm): 2.72 , 7.3 , 7.4 , 7.6 , 7.7 , 6.9-7.21 , 16.15 ; MS: m/z (%): 279 , 278 (100), 264 (17), 262 (10), 250 (5), 236 (2), 234 (3), 222 (2), 188 (19), 187 (28), 158 (12), 144 (4), 131 (9), 130 (14), 121 (19), 118 (10), 93 (12), 92 (6), 77 (25), 65 (6), 51 (8); Anal. for C17H13NO3 (279.29) (%): C: 73.11, H: 4.69, N: 5.02; found C: 73.09, H: 4.71, N: 4.97.4-Hydroxy-3-(1-(p-tolylimino)ethyl)-2H-chromen-2-one (10b): Yield: 97%; m.p. 147-149 ºC. IR : ν(OH) 3421, ν 3073, ν(CH3) 2985, 2922, 2852, ν(C=O) 1709 (lactone), ν(C=N) 1611 (imino), ν(C=C)ar 1597, 1569, 1513, 1483; 1H-NMR (δ ppm): 2.41 , 2.69 , 7.09-7.65 , 7.4 , 7.6 , 7.7 , 16.07 ; MS: m/z (%): 293 , 292 (100), 278 (32), 276 (20), 264 (5), 236 (2), 188 (21), 187 (12), 172 (9), 158 (5), 145 (5), 144 (12), 132 (10), 121 (23), 107 (12), 106 (11), 91 (23), 77 (8), 65 (16), 51 (3); Anal. for C18H15NO3 (293.32) (%): C: 73.71, H: 5.15, N: 4.78: found C: 73.72, H: 5.12, N: 4.79.4-Hydroxy-3-(1-(m-tolylimino)ethyl)-2H-chromen-2-one (11b): Yield 94%; m.p. 109-110 ºC. IR : ν(OH) 3417, ν(=CH)ar 3067, ν(CH3) 2982, 2929, 2853, ν(C=O) 1697 (lactone), ν(C=N) 1606 (imino), ν(C=C)ar 1600, 1566, 1484; 1H-NMR (δ ppm): 2.42 , 2.70 , 7.01 , 7.04 , 7.12 , 7.25 , 7.3 , 7.4 , 7.6 , 7.7 , 15.9 ; MS: m/z (%): 293 , 292 (100), 278 (30), 276 (12), 264 (3), 236 (3), 188 (14), 187 (27), 172 (11), 158 (7), 145 (13), 144 (12), 132 (10), 121 (19), 107 (12), 106 (6), 91 (20), 77 (9), 65 (15), 51 (4); Anal. for C18H15NO3 (293.32) (%): C: 73.71, H: 5.15, N: 4.78; found C: 73.72, H: 5.14, N: 4.79.4-Hydroxy-3-(1-(o-tolylimino)ethyl)-2H-chromen-2-one (12b): Yield 94%; m.p. 138-139 ºC. IR : ν(OH) 3466.94, ν(=CH)ar 3072.51, ν(CH3) 2935.08, 2856.34, ν(C=O) 1711.85 (lactone), ν(C=N) 1610.82 (imino), ν(C=C)ar 1594.26, 1562.86, 1486.38; 1H-NMR (δ ppm): 2.30 , 2.81 (CH3-C=N), 7.23 , 7.28 , 7.3 , 7.4 , 7.37 , 7.6 , 7.7 , 15.8 ; MS: m/z (%): 293 , 292 (34), 278 (100), 188 (7), 187 (11), 172 (8), 158 (15), 145 (4), 144 (9), 132 (9), 121 (16), 107 (2), 106 (3), 91 (17), 77 (6), 65 (13), 51 (6); Anal. for C18H15NO3 (293.32) (%): C: 73.71, H: 5.15, N: 4.78: found C: 73.75, H: 5.21, N: 4.73.4-Hydroxy-3-(1-(4-nitrophenylimino)ethyl)-2H-chromen-2-one (13b): Yield 92%; m.p. 212-215 ºC. IR : ν(OH) 3414, ν(=CH)ar 3081, 3046, ν(CH3) 2992, 2947, 2849, ν(C=O) 1707 (lactone), ν(C=N) 1608 (imino), ν(C=C)ar 1580, 1519, 1483, νar(NO2) 1557, 1340; 1H-NMR (δ ppm): 2.76 , 7.3 , 7.4 , 7.43 , 7.6 , 7.7 , 8.39 , 15.95 ; MS: m/z (%): 324 , 323 (100), 309 (8), 307 (10), 294 (14), 277 (18), 263 (8), 221 (1), 207 (7), 203 (6), 188 (26), 187 (47), 176 (6), 163 (9), 157 (9), 121 (48), 117 (12), 108 (7), 92 (14), 77 (9), 76 (13), 67 (14), 65 (12); Anal. for C17H12N2O5 (324.29) (%): C: 62.96, H: 3.73, N: 8.64: found; C: 62.97, H: 3.77, N: 8.69.4-Hydroxy-3-(1-(3-nitrophenylimino)ethyl)-2H-chromen-2-one (14b): Yield: 97%; m.p. 209-210 ºC. IR : ν(OH) 3416, ν(=CH)ar 3089, 3062, ν(CH3) 2980, 2936, 2853, ν(C=O) 1705 (lactone), ν(C=N) 1609 (imino), ν(C=C)ar 1590, 1538, 1491, νar(NO2) 1562, 1353; 1H-NMR (δ ppm): 2.61 , 7.26 , 7.3 , 7.34 , 7.4 , 7.54 , 7.6 , 7.7 , 8.09 , 16.02 ; MS: m/z (%): 324 (M+ 69), 323 (83), 307 (100), 294 (14), 263 (61), 249 (3), 234 (3), 220 (3), 207 (7), 188 (26), 187 (67), 176 (6), 163 (15), 157 (12), 145 (5), 138 (5), 130 (8), 121 (78), 117 (19), 103 (9), 92 (27), 77 (15), 76 (29), 67 (25), 65 (18), 51 (6); Anal. for C17H12N2O5 (324.29) (%): C: 62.96, H: 3.73, N: 8.64: found: C: 62.96; H: 3.73; N: 8.64.3-(1-(Benzylimino)ethyl)-4-hydroxy-2H-chromen-2-one (15b): Yield 97%; m.p. 151-152 ºC. IR : ν(OH) 3406, ν(=CH)ar 3032, 3012, ν(CH2) 2930, ν(C=O) 1698 (lactone), ν(C=N) 1612 (imino), ν(C=C)ar 1586, 1572, 1485; 1H-NMR (δ ppm): 2.64 , 3.95 , 7.29 , 7.26 , 7.39 , 7.3 , 7.4 , 7.6 , 7.7 , 16.01 ; MS: m/z (%): 293 , 292 (14), 276 (15), 262 (2), 236 (3), 202 (66), 189 (6), 173 (10), 156 (19), 144 (14), 131 (9), 121 (16), 107 (12), 106 (6), 91 (20), 77 (9), 65 (15), 51 (4); Anal. for C18H15NO3 (293.32) (%): C: 73.71, H: 5.15, N: 4.78; found C: 73.73; H: 5.13; N: 4.73.5-(1-(4-Hydroxy-2-oxo-2H-chromen-3-yl)ethylideneamino)pentanoic acid (16b): Yield 87%; m.p. 169-171 ºC; IR : ν(OH) 3418 (coumarin), ν(OH) 3602-2811 (COOH), ν 2947, 2930, 2875, ν(C=O) 1721 (COOH), ν(C=O) 1703 (lactone), ν(C=N) 1614 (imino), ν(C=C)ar 1600, 1560, 1487; 1H-NMR (δ ppm): 1.82 , 2.46 , 2.70 , 3.60 , 7.3 , 7.4 , 7.6 =303 (M+); Anal. for C16H17NO5 (303.11) (%): C: 63.36; H: 5.65; N: 4.62; found C: 63.27; H: 5.68; N: 4.63.C. albicans.New coumarin derivatives have been synthesized using conventional and microwave heating methodology and characterized. The advantages in the use of microwave methodology are shorter reaction times, higher yields and simplified work up procedures for the point of purification of the prepared compounds. All tested compounds are in principal very good potential microorganism growth inhibitors, but their structural diversity resulted in great differences in inhibitory potential. Finally, a nice result of this experiment is that we have synthesized some compounds that have varied and different influence on different classes of bacteria and the fungus