Direct halogenation with NXS  affords halogenated benzodiazepinones on the central aromatic ring whereas catalyst (Pd(OAc) Over the past decade, Pd(II)-catalyzed halogenation of CH bond guided by an intramolecular directing group has emerged as an efficient method in organic synthesis. Different halogenating reagents and directing groups have been employed for the direct transformation of C-H bond to C-X  bond123456781011To the best of our knowledge, no direct introduction of halogen atoms at the 2′ position of the 1,4-benzodiazepinone skeleton has been described. Designing such a route from the already synthesized benzodiazepinones would allow a quick entry to halogenated analogues or functionalized derivatives via metal catalyzed cross-coupling reactions.A direct CH activation of this ortho position could be a straightforward route to halogenated benzodiazepinones. We were encouraged in this approach based on recent reports that described the synthesis of 1,4-benzodiazepinones derived metallacycles with either zinc16171 to find the best experimental conditions to get halogenation of the C phenyl ring with minimum amount of side reaction on the A phenyl ring. Microwave heating was preferred to conventional heating to lower the reaction time and the amount of degradation compounds.The palladium-catalyzed directed CH activation/halogenation reactions on model compounds usually proceed in good yield and with complete control of regioselectivity1-I  along with some brominated compound on the central phenyl ring 2 (3CN by DMF resulted in more compound 2 (2 (2). The 7-bromo position was unequivocally identified by comparison with NMR spectra of 7-bromo-1,3-dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one obtained from 2-amino-4-bromobenzophenone. This regioselective control affords a straightforward access to 7-brominated benzodiazepinones. Replacing NBS by another brominating agent, CuBr2, gave the starting material unchanged  in hand, we decided to check the scope and limitation of this iodination process on differently substituted benzodiazepinones . Each st20123I, 125I or 131I) benzodiazepinones have been widely used in binding assays125I, the parent iodinated compound being synthesized from simple iodinated precursors such as iodobenzoic acid2223It is worth noting that radioiodinated 8-I and 8-II compounds compared to unsubstituted benzodiazepinones  was oxidized to Pd(IV). Then, ligand exchange provides dimeric intermediate C whose formation was confirmed by LC-MS. Finally, a reductive elimination occurs to afford mono-iodinated compound that can enter a new catalytic cycle to afford the diiodinated product (The putative mechanism involves a CH activation assisted by the chelation of the nitrogen atom of the imino moiety of the benzodiazepinone. The insertion of a molecule of N-iodosuccinimide product .1 was treated with Pd(OAc)2 in the absence of NIS. Once we suggested the formation of complex C we investigated whether the dimer would be formed from a reductive elimination or a simple homocoupling from the iodinated compound. For this purpose the same reaction was performed starting from compound 1-I and only non-iodinated dimer was formed strongly supporting the homocoupling process2627In this mechanism, we suggest that the oxidation of Pd(II) to Pd(IV) is prior to the ligand exchange step because the dimer formation, via a homoleptic complex, was not detected when benzodiazepine In conclusion, we have described herein an efficient protocol for regioselective halogenation of benzodiazepinones. This protocol takes advantage of CH activation with palladium acetate. Since radio-iodinated NIS is easily prepared, the CH activation described here offers a straightforward route to radioiodinated benzodiazepinones. Halogenated benzodiazepinones also represent ideal starting materials for more functionalized analogs via metal catalyzed cross-coupling reactions and this CH activation based approach opens new possibilities in this area. In this case, sequential halogenation/coupling procedures at the 7 then at the 2′ position or vice versa could gain libraries of benzodiazepinones.1H and 13C NMR spectra were recorded using a Bruker Avance 400 MHz Ultrashield spectrometer in CD3CN for all compounds except for compound 5-I whose NMR analysis were conducted in (CD3)2SO for solubility reasons. The following abbreviations are used in reporting NMR data: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; ddd, doublet of doublets of doublets; d appt, doublet of apparent triplet; m, multiplet. Infrared spectra were recorded on an FT-IR spectrophotometer. HPLC-MS analysis and purification were performed using a Waters system  with a binary gradient solvent delivery system. This system was coupled with a Waters Micromass ZQ system with a ZQ2000 quadrupole analyzer. The ionization was performed by electrospray and the other parameters were as follows: source temperature 120 °C, cone voltage 20 V, and continuous sample injection at 0.3 mL∕ min flow rate. Mass spectra were recorded in both positive and negative ion mode in the m/z 100–2,000 range and treated with the Mass Lynx 4.1 software. High-resolution mass spectrometry (HRMS) was performed using the Imagif platform , and recorded on ESI/TOF LCP premier XE mass spectrometer (Waters) using flow injection analysis mode.The products were isolated by flash silica gel column chromatography (0.040–0.063 mm). Reactions were run without exclusion of air/moisture in a microwave tube. Reactions were monitored by NMR. 1  in CD3CN (1.2 mL), were added Pd(OAc)2  and N-iodosuccinimide . The mixture was stirred at 100 °C under microwave irradiation for 15 minutes. The crude mixture was evaporated, diluted in ethyl acetate (10 mL), and washed with a 2 M aqueous solution of NaOH (5 mL). The residue was purified by flash chromatography (cyclohexane/ethylacetate 1:1) affording 30 mg (69%) of 1-I and 2 mg (3%) of 1-II. IR  3057, 2988, 2850, 1676, 1611, 1573, 1489, 1449, 1361, 1324, 1280, 1201, 1167, 1128, 1076, 1046, 1014, 984, 939, 915; 1H NMR  δ 7.83 , 7.56 , 7.51-7.45 , 7.42 , 7.15 , 7.09 , 6.94 , 4.58 , 3.76 , 3.39 ; 13C NMR  δ 173.5 (C = N), 170.1 (C = O), 145.5 (C-1′), 140.3 (C-3′), 132.5 (C-8), 131.6 (C-4′ and C-5′ or C-6′), 130.1 (C-5a), 129.6 (C-6), 129.3 (C-6′ or C-5′), 124.9 (C-7), 122.6 (C-9), 118.3 (C-9a), 96.8 (C-2′), 57.7 (C-3), 35.1 (1-CH3); HR-MS (ESI+) m/z Calcd for C16H14IN2O [M+H+] 377.0151, Found 377.0145.To a solution of 1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one 2  in CD3CN (500 μL), were added Pd(OAc)2  and N-iodosuccinimide . The mixture was stirred at 100 °C under microwave irradiation for 15 minutes. The crude mixture was evaporated, diluted in ethyl acetate (10 mL), and washed with a 2 M aqueous solution of NaOH (5 mL). The residue was purified by flash chromatography (cyclohexane/ethylacetate 1:1) affording 12 mg (54%) of 2-I and 2 mg (7%) of 2-II IR  3059, 2985, 2920, 2853, 1678, 1613, 1584, 1559, 1480, 1421, 1399, 1343, 1320, 1275, 1249, 1196, 1167, 1129, 1075, 1045, 1015, 983, 942, 915; 1H NMR  δ 7.89 , 7.56 , 7.61–7.49 , 7.38 , 7.23 , 7.08 , 4.65 , 3.76 , 3.40 ; 13C NMR  δ 172.1 (C = N), 169.7 (C = O), 144.8 (C-1′), 140.4 (C-3′), 135.2 (C-8), 131.9 (C-4′), 131.8 (C-5′ and C-6′), 129.4 (C-6), 124.8 (C-9), 118.3 (C-9a), 117.1 (C-7), 96.7 (C-2′), 57.7 (C-3), 35.0 (1-CH3) ppm; HR-MS (ESI+) m/z Calcd for C16H13BrIN2O [M+H+] 454.9256, Found 454.9265.To a solution of 7-bromo-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one 3  in CD3CN (300 μL), were added Pd(OAc)2  and N-iodosuccinimide . The mixture was stirred at 100 °C under microwave irradiation for 15 minutes. The crude mixture was evaporated, diluted in ethyl acetate (10 mL), and washed with a 2 M aqueous solution of NaOH (5 mL). NMR and LC-MS analysis indicates a 50% conversion of the starting material to the desired compound 3-I.To a solution of 7-chloro-6′-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one 4  in CD3CN (1.l mL), were added Pd(OAc)2  and N-iodosuccinimide . The mixture was stirred at 100 °C under microwave irradiation for 15 minutes. The crude mixture was evaporated, diluted in ethyl acetate (10 mL), and washed with a 2 M aqueous solution of NaOH (5 mL). The residue was purified by flash chromatography (cyclohexane/ethylacetate 1:1) affording 28 mg (54%) of 4-I and 4 mg (6%) of 4-II. IR  3058, 2983, 2919, 2853, 1677, 1612, 1598, 1560, 1478, 1447, 1418, 1398, 1342, 1318, 1275, 1253, 1197, 1168, 1129, 1089, 1075, 1039, 1014, 983, 942, 916; 1H NMR  δ 7.73 , 7.70 , 7.43-7.32 , 7.06 , 4.62 , 3.79 , 3.39 , 2.36 ; 13C NMR  δ 172.0 (C = N), 169.8 (C = O), 144.7 (C-1′), 142.6 (C-4′), 140.8 (C-3′), 135.1 (C-8), 132.1 (C-5a), 131.9 (C-5′ or C-6′), 131.6 (C-6), 130.1 (C-5′ or C-6′), 124.7 (C-9), 118.3 (C-9a), 117.0 (C-7), 96.8 (C-2′), 57.7 (C-3), 35.0 (1-CH3), 20.7 (4′-CH3); HR-MS (ESI+) m/z Calcd for C17H15BrIN2O [M+H+] 468.9412, Found 468.9427.To a solution of 7-bromo-4′-methyl-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one 5  in CD3CN (950 μL), were added Pd(OAc)2  and N-iodosuccinimide . The mixture was stirred at 100 °C under microwave irradiation for 15 minutes. The crude mixture was evaporated, diluted in ethyl acetate (10 mL), and washed with a 2 M aqueous solution of NaOH (5 mL). The residue was purified by flash chromatography (cyclohexane/ethylacetate 1:1) affording 17 mg (41%) of 5-I and 2 mg (4%) of 5-II. IR  3207, 3117, 2927, 2852, 1689, 1617, 1479, 1429, 1382, 1322, 1291, 1255, 1230, 1195, 1164, 1134, 1088, 1047, 1011, 945; 1H NMR 2SO) δ 10.82 , 7.89 , 7.71 , 7.52 , 7.45 , 7.22 , 7.18 , 6.96 , 4.19 ; 13C NMR 2SO) δ 170.7 (C = N), 169.1 (C = O), 143.7 (C-1′), 139.2 (C-5a), 139.1 (C-3′), 134.3 (C-8), 131.4 (C-6), 130.8 (C-4′ and C-6′), 128.1 (C-5′), 123.1 (C-9), 114.3 (C-7), 96.9 (C-2′), 56.8 (C-3); HR-MS (ESI+) m/z Calcd for C15H11BrIN2O [M+H+] 440.9099, Found 440.9090.To a solution of 7-bromo-1,3-dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one 6  in CD3CN (1.05 mL), were added Pd(OAc)2  and N-iodosuccinimide . The mixture was stirred at 100 °C under microwave irradiation for 15 minutes. The crude mixture was evaporated, diluted in ethyl acetate (10 mL), and washed with a 2 M aqueous solution of NaOH (5 mL). The residue was purified by flash chromatography (cyclohexane/ethylacetate 1:1) affording 26 mg (60%) of 6-I and 2 mg (3%) of 6-II. IR  3060, 2988, 2915, 2853, 1678, 1615, 1592, 1582, 1562, 1484, 1447, 1425, 1402, 1346, 1321, 1298, 1276, 1248, 1198, 1169, 1130, 1099, 1076, 1044, 1016, 983, 943, 916; 1H NMR  δ 7.89 , 7.58 , 7.57–7.49 , 7.44 , 7.22 , 6.94 , 4.65 , 3.85 , 3.40 ; 13C NMR  δ 172.1 (C = N), 169.7 (C = O), 144.6 (C-1′), 140.4 (C-3′), 132.3 (C-8), 131.9 (C-4′), 131.8 (C-5′ or C-6′), 131.5 (C-5a), 129.5 (C-7), 129.4 (C-6), 128.8 (C-5′ or C-6′), 124.5 (C-9), 118.3 (C-9a), 96.8 (C-2′), 57.6 (C-3), 35.0 (1-CH3); HR-MS (ESI+) m/z Calcd for C16H13ClIN2O [M+H+] 410.9761, Found 410.9766.To a solution of 7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one 3CN (740 μL), were added Pd(OAc)2  and N-iodosuccinimide . The mixture stirred at 100 °C under microwave irradiation for 15 minutes under microwave irradiation. The crude mixture was evaporated, diluted in ethyl acetate (10 mL), and washed with a 2M aqueous solution of NaOH (5 mL). The residue was purified by flash chromatography (cyclohexane/ethylacetate 1:1) affording 27 mg (69%) of 7-I and 3 mg (6%) of 7-II. IR  3061, 3029, 2995, 2852, 1676, 1612, 1494, 1479, 1454, 1403, 1357, 1319, 1265, 1220, 1185, 1090, 1067, 1015, 938, 882; 1H NMR  δ 7.90 , 7.62 , 7.50 , 7.40-7.24 , 7.06 , 7.09 , 5.24 , 5.07 , 4.77 , 4.01 ; 13C NMR  δ 172.0 (C = N), 168.8 (C = O), 144.5 (C-1′), 140.6 (C-3′), 138.6 (C-5a), 135.3 (C-4), 132.3 (C-6), 132.2 (C-Ph), 131.9 (C-5′ and C-6′), 132.0, 131.9, 129.7, 128.4, 128.3, 124.9 (Ph and C-9), 118.3 (C-9a), 117.6 (C-7), 96.8 (C-2′), 57.8 (C-3), 51.8 (CH2-Ph); HR-MS (ESI+) m/z Calcd for C22H19BrIN2O [M+H+] 530.9569, Found 530.9572.To a solution of 7-bromo-1,3-dihydro-1-benzyl-5-phenyl-2H-1,4-benzodiazepin-2-one  in CH8  in CD3CN (1.1 mL), were added Pd(OAc)2  and N-iodosuccinimide . The mixture was stirred at 100 °C under microwave irradiation for 15 minutes. The crude mixture was evaporated, diluted in ethyl acetate (10 mL), and washed with a 2 M aqueous solution of NaOH (5 mL). The residue was purified by flash chromatography (cyclohexane/ethylacetate 1:1) affording 27 mg (61%) of 8-I and 2 mg (3%) of 8-II. IR  3057, 2982, 2920, 2852, 1674, 1598, 1572, 1481, 1447, 1360, 1323, 1280, 1203, 1166, 1128, 1076, 1040, 1010, 984, 939, 915; 1H NMR  δ 7.73 , 7.56 , 7.48-7.30 , 7.13 , 7.97 , 4.59 , 3.78 , 3.42 , 2.35 ; 13C NMR  δ 173.4 (C = N), 170.1 (C = O), 145.4 (C-1′), 142.2-142.5 (C-5a), 140.6 (C-3′), 132.4 (C-8), 131.3 (C-5′ or C-6′), 130.3 (C-4′), 129.8-129.6 , 124.8 (C-7), 122.5 (C-9), 118.2 (C-9a), 96.8 (C-2′), 57.7 (C-3), 34.9 (1-CH3), 20.6 (4′-CH3); HR-MS (ESI+) m/z Calcd for C17H16IN2O [M+H+] 391.0307, Found 391.0320.To a solution of 4′-methyl-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one 1  in DMF (400 μL), were added Pd(OAc)2  and N-bromosuccinimide . The mixture was stirred at 100 °C under microwave irradiation for 60 minutes. The crude mixture was diluted in ethyl acetate (10 mL), and washed with a 2 M aqueous solution of NaOH (5 mL). The residue was purified by flash chromatography (cyclohexane/ethylacetate 1:1) affording 5 mg (35%) of 1-Br and 4 mg (29%) of 3. IR  3065, 2990, 2853, 1672, 1611, 1600, 1572, 1490, 1468, 1448, 1361, 1324, 1281, 1202, 1166, 1129, 1077, 1047, 1026, 1010, 985, 939, 915; 1H NMR  δ 7.62-7.55 , 7.51 , 7.46 , 7.38 , 7.14 , 7.02 , 4.61 , 3.81 , 3.39 ; HR-MS (ESI+) m/z Calcd for C16H14BrN2O [M+H+] 329.0290, Found: 329.0286.To a solution of 1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one 1  in DMF (400 μL), was added N-bromosuccinimide . The mixture was stirred at 100 °C under microwave irradiation for 60 minutes. The crude mixture was diluted in ethyl acetate (10 mL), and washed with a 2 M aqueous solution of NaOH (5 mL). The residue was purified by flash chromatography (cyclohexane/ethylacetate 1:1) affording 3.5 mg (27%) of 2 and 2.5 mg (25%) of recovered starting material 1. IR  3061, 2922, 2852, 1679, 1608, 1480, 1446, 1397, 1339, 1321, 1269, 1198, 1179, 1129, 1090, 1072, 1017, 984, 941, 914; 1H NMR  δ 7.73 , 7.61–7.41 , 7.09–7.36 , 4.60 , 3.78 , 3.32 ; HR-MS (ESI+) m/z Calcd for C16H14BrN2O [M+H+] 329.0290, Found 329.0276.To a solution of 1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one 1-I were collected at 150(2) K on a Nonius Kappa-CCD area detector diffractometer  using graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å). The unit cell parameters were determined from ten frames, then refined on all data. The data (combination of φ- and ω-scans giving a complete data set up to θ = 30.5° and a minimum redundancy of 4 for 90% of the reflections) were processed with HKL20003).The data for compound 16H13IN2O, M = 376.18, monoclinic, space group P21/c, a = 8.1056(3), b = 12.9888(7), c = 13.9601(7) Å, β = 98.725(3)°, V = 1452.74(12) Å3, Z = 4, Dc = 1.720 g cm−3, μ = 2.201 mm−1, F(000) = 736. Refinement of 182 parameters on 4430 independent reflections out of 53320 measured reflections (Rint = 0.041) led to R1 = 0.032, wR2 = 0.085, S = 1.019, Δρmax = 0.82, Δρmin = −0.97 e Å–3.C†Electronic 1H and 13C NMR spectra and X-ray structural information of 1-I (CIF). CCDC 1014605.How to cite this article: Abdelkafi, H. and Cintrat, J.-C. Regioselective Halogenation of 1,4-Benzodiazepinones via CH Activation. Sci. Rep.5, 12131; doi: 10.1038/srep12131 (2015).


E,7'-dienyl)-8-hydroxy-2,6-dimethyl-2H-chromene (1) together with four known natural products, methylfarnesylquinone (2), isololiolide (3), pheophytin a (4), and β-carotene (5) were isolated from the brown alga Homoeostrichus formosana. The structure of 1 was determined by extensive 1D and 2D spectroscopic analyses. Acetylation of 1 yielded the monoacetylated derivative 2--8-acetyl-2,6-dimethyl-2H-chromene (6). Compounds 1–6 exhibited various levels of cytotoxic, antibacterial, and anti-inflammatory activities. Compound 2 was found to display potent in vitro anti-inflammatory activity by inhibiting the generation of superoxide anion (IC50 0.22 ± 0.03 μg/mL) and elastase release (IC50 0.48 ± 0.11 μg/mL) in FMLP/CB-induced human neutrophils.A new chromene derivative, 2-(4',8'-dimethylnona-3' Homoeostrichus formosana  + peak at m/z 349.2143, implying eight degrees of unsaturation. The UV spectrum showed absorption maxima at 206, 231, 265, 274 and 333 nm, suggesting a conjugated aromatic ring system + at 391.2249 in HRESIMS, thus a molecular formula of C24H32O3 was proposed for 6. Indeed, the resonance due to H-7 was observed at δ 6.68 (C-7/δ 122.8), showing the expected downfield shift in comparison to 1 and thus confirmed the location of acetyl group at C-8. The geometry of double bond C-3'/C-4' of geranylmethyl was assigned as E on the basis of the NOESY correlations of H2-2'/H3-11' and H-3'/H2-5'. Moreover, compound 1 did not show any significant Cotton effects and its poor optical activity indicates possibly it is a 1:1 mixture of two enantiomers +; HRESIMS m/z 349.2146 [M + Na]+ .2- was dissolved in pyridine (0.2 mL) and then Ac2O (0.1 mL) was added. After 24 h at room temperature, the reaction residue was subjected to column chromatography over silica gel using n-hexane-EtOAc (5:1) to yield the acetyl derivative 6 (4.1 mg).Compound E,7'-dienyl)-8-acetyl-2,6-dimethyl-2H-chromene (6): Colorless oil; c 0.1, CHCl3); UV (MeOH) λmax (log ε) 205 (4.5), 227 (4.4), 266 (3.6), 275 (3.5), 318 (3.4) nm; IR (neat) νmax 2922,1760, 1468, 1369, 1207, 1020, 894, and 723 cm−1; 1H-NMR : δ 5.58 , 6.27 , 6.68 , 6.55 , 2.16 , 1.38 , 2.16 , 1.71 , 2.11 , 5.14 , 1.97 , 2.05 , 5.11 ; 13C-NMR : δ 78.5 (C-2), 130.3 (C-3), 122.5 (C-4), 116.5 (C-5), 126.3 (C-6), 122.8 (C-7), 148.7 (C-8), 143.3 (C-9), 120.9 (C-10), 26.5 (C-3/CH3), 15.5 (C-6/CH3), 21.1 (C-8/OAc), 41.2 (C-1'), 22.6 (C-2'), 123.9 (C-3'), 135.3 (C-4'), 39.7 (C-5'), 26.7 (C-6'), 124.3 (C-7'), 131.3 (C-8'), 15.9 (C-4'/CH3), 17.7 (C-8'/CH3), 25.7 (C-8'/CH3); ESIMS m/z (relative intensity) 391 [M + Na]+; HRESIMS m/z 391.2249 [M + Na]+ .2--2,5-diphenyl-tetrazolium bromide] colorimetric method [Cell lines were purchased from the American Type Culture Collection (ATCC). Cytotoxicity assays of compounds c method ,29.Bacillus cereus (ATCC14579), Staphylococcus aureus (ATCC9144), Salmonella typhimurium (ATCC914028), Pseudomonas aeruginosa (ATCC27853), Serratia marcescens (ATCC25419), and Yersinia enterocolitica (ATCC23715), based on previous reports [The antibacterial activity of the isolated compounds was evaluated against  reports ,31. Bactp-nitroanilide as the elastase substrate [Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Measurements of superoxide anion generation and elastase release were carried out according to previously described procedures ,33. Brieubstrate .E,7'-dienyl)-8-hydroxy-2,6-dimethyl-2H-chromene (1) together with four known natural products 2–5 were isolated from the brown alga H. formosana. Both compounds 1 and 2 displayed marginal cytotoxicities toward HepG2, A-549, and MDA-MB-231 cancer cell lines. Compounds 1, 2, 5 and 6 showed potent inhibition against P. aeruginosa compared to that of ampicillin. Methylfarnesylquinone 2 revealed potent in vitro anti-inflammatory activity by inhibiting the generation of superoxide anion (IC50 0.22 ± 0.03 μg/mL) and the release of elastase (IC50 0.48 ± 0.11 μg/mL) in FMLP/CB-induced human neutrophils. Thus, compound 2 can be considered as a candidate for future anti-inflammatory drug development.A new chromene derivative 2-(4',8'-dimethylnona-3'


The hydrophilicity and large polar surface area of the potent S1-S1’-S2’ binding FXIa inhibitors compromised permeability. Initial work to expand the 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment towards the prime side to yield molecules with less hydrophilicity shows promise to afford potent, selective and orally bioavailable compounds.Activated factor XI (FXIa) inhibitors are anticipated to combine anticoagulant and profibrinolytic effects with a low bleeding risk. This motivated a structure aided fragment based lead generation campaign to create novel FXIa inhibitor leads. A virtual screen, based on docking experiments, was performed to generate a FXIa targeted fragment library for an NMR screen that resulted in the identification of fragments binding in the FXIa S1 binding pocket. The neutral 6-chloro-3,4-dihydro-1H-quinolin-2-one and the weakly basic quinolin-2-amine structures are novel FXIa P1 fragments. The expansion of these fragments towards the FXIa prime side binding sites was aided by solving the X-ray structures of reported FXIa inhibitors that we found to bind in the S1-S1’-S2’ FXIa binding pockets. Combining the X-ray structure information from the identified S1 binding 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment and the S1-S1’-S2’ binding reference compounds enabled structure guided linking and expansion work to achieve one of the most potent and selective FXIa inhibitors reported to date, compound 13, with a FXIa IC A well balanced haemostasis system is important to both minimize blood loss and disturbances of blood flow. Upon injury of the vessel wall, blood is exposed to tissue factor which via a cascade reaction leads to thrombin generation and a fibrin cross-linked clot to mend the injury and stop bleeding. Factor XI (FXI) has an important role in thrombin generation in the amplification phase of the coagulation process. However, over-production of thrombin may lead to excessive clots resulting in thrombosis. Also, high levels of thrombin cause activation of thrombin activated fibrinolysis inhibitor which hinders fibrinolysis. Therefore, decreased levels of thrombin will indirectly increase the rate of fibrinolysis. Inhibition of activated FXI (FXIa) should decrease thrombin generation in the amplification phase, but not in the initiation phase, and thus yield an antithrombotic and profibrinolytic effect with minimal risk of bleeding (see reviews –3). BleeThe role of FXIa in haemostasis and thrombosis in human has been extensively studied. Human haemophilia C patients who are severely deficient in FXI display reduced incidence of ischemic stroke . Unlike in vivo antithrombotic efficacy in rat models with BMS-262084, a potent and selective beta-lactam arginine that irreversibly inhibits FXIa with an IC50 of 2.8 nM formamide scaffold were screened. Compound 27 with a FXIa IC50 of 99 μM was identified, which displayed high permeability (see 27 was improved by approximately two orders of magnitude upon addition of the chloro atom to the aminoquinolinone P1 to afford compound 28 with an IC50 of 0.82 μM. The ADME properties of compound 28 with high permeability (Caco2 A to B = 19 · 10–6 cm/s), moderate rat in vivo clearance (18 mL/min/kg) and a measured oral bioavailability in rat of 27% show promise towards the goal of developing an oral drug. Potency improvements of this lead with maintained favorable ADME properties remain to be shown. However, comparing the potencies of the P1-P1’ compounds 28 and 14 , and realizing the potency boost feasible by adding a P2’ moiety  was carried out as described by Jin et al. . To impr3, and 10% D2O. FXIa CD stock solution was buffer exchanged and concentrated to approximately 100 μM (2.7 mg/mL) (UV absorption ε280 = 60390 cm-1) using 10 kDa cut-off Millipore filter centrifugation tubes. Control experiments with pMeBza and compound 2 were done on all protein preparations . Samples were prepared “just in time” using a Genesis liquid handling robot  and transferred to the NMR spectrometer via a SampleRail system . Mixtures were screened at 100 μM compound concentration in aqueous buffer with 5 μM FXIa CD added in the second step. At the third step, 10 μM inhibitor (compound 2) was added.NMR samples were prepared in aqueous buffer containing 50 mM deuterated TRIS, pH 7.4, 3 mM NaNarations . Stock soC on a Bruker Avance III 600 MHz NMR spectrometer equipped with a cryo probe head. 1D 1H T1ρ NMR spectra  denotes the substrate concentration.The ICFXIa. Human FXIa was from HTI, Essex Junction, USA. Inhibition by compounds was measured using 420 μM pyroGlu-Pro-Arg-pNA·HCl  as substrate in 50 mM Tris pH 7.4, 100 mM NaCl, 5 mM CaCl2, 0.1 mg/mL BSA, 1% DMSO (v/v).Trypsin. Recombinant human trypsin was from Polymun, Vienna, Austria. Inhibition by compounds was measured using 300 μM Bz-Val-Gly-Arg-pNA  as substrate in 50 mM Tris pH 7.4, 40 mM NaCl, 20 mM CaCl2, 0.1% Tween 20 (v/v), 1% DMSO (v/v).Thrombin. Human recombinant thrombin was prepared in house. Inhibition by compounds was measured using 300 μM pyroGlu-Pro-Arg-pNA·HCl (Km = 280 μM) as substrate in 50 mM Tris/HCl pH 7.4, 100 mM NaCl, 0.2% BSA (w/v), 1% DMSO (v/v).Plasmin. Human plasmin was from Enzyme Research Laboratories, Swansea, UK. Inhibition by compounds was measured using 300 μM pyroGlu-Pro-Arg-pNA·HCl (Km = 275 μM) as substrate in 50 mM Tris pH 7.4, 100 mM NaCl, 0.1% Tween 80 (v/v), 1% DMSO (v/v).FIXa. Human FIXa was from Enzyme Research Laboratories, Swansea, UK. Inhibition by compounds was measured using 500 μM CH3SO2-(D)-CHG-Gly-Arg-pNA·AcOH  as substrate in 33 mM Tris/HCl pH 7.4, 66 mM NaCl, 3.3 mM CaCl2, 33% Ethylene glycol (v/v), 0.3% BSA (w/v), 1% DMSO (v/v).FXa. Human FXa was from HTI, Essex Junction, USA. Inhibition by compounds was measured with 100 μM Z-D-Arg-Gly-Arg-pNA  as substrate in 50 mM Tris pH 7.4, 100 mM NaCl, 5 mM CaCl2, 0.2% BSA (w/v), 1% DMSO (v/v), 1% DMSO (v/v).tPa. Human tPA was from Biopool, Umeå, Sweden. Inhibition by compounds was measured with 250 μM CH3SO2-D-HHT-Gly-Arg-pNA  in 50 mM Tris/HCl pH 7.4, 100 mM NaCl, 0.032% Tween 80 (v/v), 1% DMSO (v/v).PKK. Human PKK was from Calbiochem, Darmstadt, Germany. Inhibition by compounds was measured with 400 μM D-Pro-Phe-Arg-pNA·HCl  in 50 mM Tris/HCl pH 7.4, 100 mM NaCl, 5 mM CaCl2, 0.2% BSA (v/w), 1% DMSO (v/v).The chromatographic data was obtained utilizing a Waters Aquity UPLC chromatographic system running a gradient from 5–95% acetonitrile at pH7.4. The stationary phase was a Waters BEH C18 2.1x50 mm. Standardized retention data were obtained by normalization against a set of compounds with precisely determined (octanol/water) logD-values. Lipophilicity values for samples were derived from a corresponding calibration curve.μmol/l in HBSS buffer pH 6.5) to the apical side of the monolayer. Samples for quantitative analysis by LC/MS/MS were withdrawn before the addition of the test substrate and at 45 and 120 min post addition of the test substrate. During incubation the transwell plates were placed in a shaking incubator at 37°C between sampling. The peak areas were exported to Excel where Papp-values and recoveries were calculated. Twenty-two reference compounds with known human oral bioavailability were used to establish a correlation curve with Papp versus the fraction of the oral dose absorbed (fa).A monolayer of Caco2 cells, cultured on semi-permeable polycarbonate surfaces, was used to study the permeability in the apical to basolateral direction. The process was atomized by a robotic Tecan EVO platform  and 24-transwell plates from Costar . HBSS buffer pH 7.4 was dispensed to the basal side of the monolayer. The assay was initiated by adding the test substrate (10 app AB) as well as in the basolateral to apical direction and (Papp BA). Efflux ratios were derived from the following equation: Papp BA/Papp AB.Efflux was studied in the same Caco2 cells with the modification that HBSS buffer pH 7.4 was used on both sides of the monolayer. The permeability was studied in the apical to basolateral direction  of parent compound was calculated from the slope in the regression analysis of the natural logarithm of parent concentration vs. time curve.Rat hepatocyte metabolic stability was determined in accordance with the method described by Jacobson et al. . Cryo prTwo days prior to dosing, female Sprague Dawley rats were prepared by cannulation of the left carotid artery for blood sampling and by cannulation of the right jugular vein for intravenous administration. The catheters were filled with heparin (100 IU/mL), exteriorized at the nape of the neck and sealed. The surgery was performed under isoflurane  anesthesia. After surgery the rats were housed individually and had free access to food and water. About 16 h prior to dosing the animals were deprived of food, and fasted until 4 h after dosing. The rats had free access to drinking water during the experiment. On the experiment day, the test item formulation was administered orally by gavage or intravenously in the jugular vein. At pre-defined time points, blood samples of about 0.150 mL were withdrawn from the carotid artery up to 24h after dosing. A total of 10 samples were withdrawn. The blood samples were collected in heparinized plastic tubes and centrifuged, within 30 min, for five min at 10 000 g and +4°C. The plasma was transferred to a 96 well plate and stored at -20°C until analysis by LC/MS/MS. The studies were approved by the Göteborg Animal Research Ethical Board.1H NMR spectra were recorded on a Bruker biospin GmbH 400, 500 or 600 MHz spectrometer. Chemical shifts are reported in parts-per-million (δ) relative to DMSO-d6 at 2.50 ppm, CDCl3 at 7.26 ppm or CD3OD at 3.31 ppm as an internal standard. High-resolution mass spectrometry (HRMS) analysis was performed using a Waters XEVO-qTOF instrument  with MassLynx 4.1 software. If necessary, the purity was determined by ultra high performance liquid chromatography (U(H)PLC). Purity of all final compounds was 95% or higher at 210 nm. The instrument was a Waters U(H)PLC Acquity system . The column was a Waters Acquity BEH C18, 1.7 μm particle size (100 mm × 2.1 mm).Chemicals and solvents from commercially available sources was purchased and used without further purification. 1, 2, 4 and 27 were obtained from the AstraZeneca corporate compound collection, compounds 3, 5, 6, 7, 12, 17, and 19 were commercially available and compounds 9 1, 2, 4 a + C29H25N5O4 508.1985, found 508.1988.N-(4-Chlorophenyl)-3-oxobutanamide (13A). A mixture of methyl 3-oxobutanoate , 4-chloroaniline , DMAP  and pyridine (4 mL) was refluxed in xylenes (20 mL) for 8 h. The reaction mixture was partitioned between 2M HCl (20 mL) and EtOAc (20 mL), the aqueous phase was extracted with EtOAc (2x10 mL) and the combined organic fractions was dried (Na2SO4) and then concentrated to produce compound 13A as a pale brown solid . 1H NMR  δ 2.20 , 3.55 , 7.36 , 7.60 , 10.22 .4-Bromo-N-(4-chlorophenyl)-3-oxobutanamide (13B). To a stirred solution of N-(4-chlorophenyl)-3-oxobutanamide (13A)  in AcOH (80 mL) at room temperature was dropwise added a solution of bromine  in AcOH (20 mL) over 3 h. Resulting mixture was stirred for 3 h, producing a brown precipitate. The reaction mixture was poured into a 1L beaker filled to 2/3 with crushed ice. A crude brown precipitate containing a mixture of various brominated species was collected by filtration. The product was purified by silica column chromatography . Relevant fractions was pooled and concentrated under reduced pressure to give compound 13B as a pale brown solid . 1H NMR  δ 2.19 , 3.53 , 7.31–7.47 , 7.54–7.61 , 10.18 .4-(Bromomethyl)-6-chloroquinolin-2(1H)-one (13C). 4-Bromo-N-(4-chlorophenyl)-3-oxobutanamide (13B)  was heated at 45°C in sulfuric acid (60 mL) for 24 h. Due to slow conversion of the starting material another portion of sulfuric acid (60 mL) was added and the reaction was continued until complete conversion was observed (LCMS), a total of 46 h. The reaction was allowed to reach ambient temperature and then poured into a 2L beaker filled to 2/3 with crushed ice, producing a pink precipitate. The precipitate was collected by filtration, washed with ice-cold water and then dried on a freeze-drier giving compound 13C as a pink solid . 1H NMR  δ 4.93 , 6.80 , 7.35 , 7.59 , 7.89 , 11.95 .Diethyl 2-{methyl}malonate (13D). To a stirred solution of diethyl malonate  in THF (15 mL) at room temperature was added NaH  in portions. Resulting mixture was stirred at room temperature for 10 min before a slurry of 4-(bromomethyl)-6-chloroquinolin-2(1H)-one (13C)  in THF (15 mL) was added dropwise. The resulting brown suspension was refluxed for 1 h and then concentrated under reduced pressure to give a brown oil. Water was then added until a precipitate formed. The precipitate was filtered and washed with cold water and then dried on a freeze-drier, giving compound 13D as a white solid . 1H NMR  δ 1.12 , 3.30 , 3.93 , 4.10 , 6.38 , 7.32 , 7.56 , 7.87 , 11.83 .3-propanoic acid (24A). A suspension of diethyl 2-{methyl}malonate (13D)  in 20% HCl (aq) (30 mL) was refluxed for 12 h. The reaction was then cooled under stirring on an ice bath to form a precipitate. The precipitate was filtered, washed with cold water and dried on a freeze-drier to give compound 24A as a white solid . 1H NMR  δ 2.62 , 3.03 , 6.40 , 7.32 , 7.55 , 7.80 , 11.77 , 12.30 .4-(Aminomethyl)-6-chloroquinolin-2(1H)-one (13E). A mixture of 4-(bromomethyl)-6-chloroquinolin-2(1H)-one (13C)  and hexamethylene tetraamine  in dichloroethane (20 mL) was refluxed for 5 h. A white precipitate formed and was collected by filtration. The intermediate solid was then refluxed in concentrated hydrochloric acid (10 mL) for 2 h. The reaction was stirred on an ice-bath and quenched with water, forming a new precipitate. The precipitate was filtered, washed with cold water and then dried on a freeze-drier to give compound 13E as a white solid . 1H NMR  δ 4.10 , 6.66 , 6.97 , 7.36 , 7.57 , 7.81 .(S)-N-methylamino}-1-oxo-3-phenylpropan-2-yl)-4-hydroxy-2-oxo-1,2-dihydroquinoline-6-carboxamide (13). A mixture of (S)-2--3-phenylpropanoic acid (15B) , T3P , 4-(aminomethyl)-6-chloroquinolin-2(1H)-one (13E)  and TEA  was heated at 120°C for 20 min using a microwave reactor. Excess DMF was removed under reduced pressure to produce a crude brown oily residue, containing the desired product and an inseparable phosphorous ester of the product and the coupling reagent. The crude mixture was treated with 6M HCl at room temperature for 1h to cleave phosphorous ester byproducts. The reaction was concentrated and then purified by preparative HPLC to give compound 13 as a pale pink solid . 1H NMR  δ 2.97–3.08 , 3.13 , 4.41–4.56 , 4.67–4.76 , 6.46 , 6.72 , 7.11 , 7.19 , 7.24–7.31 , 7.34 , 7.47 , 7.53 , 7.78 , 7.88 , 8.29 , 8.71 , 8.77 . 2 protons were not observed. HRMS: calcd. for [M+H]+ C29H23ClN4O5 543.1435, found 543.1447.(S)-Tert-butyl 1-{methylamino}-1-oxo-3-phenylpropan-2-ylcarbamate (14A). 1H-Benzo[d][(S)-2-Amino-N-{methyl}-3-phenylpropanamide (14B). A solution of (S)-tert-butyl methyl}amino]-1-oxo-3-phenylpropan-2-yl)carbamate (14A)  in DCM (8 mL) and TFA (3 mL) was stirred at room temperature for 16 h. Excess solvents was removed under reduced pressure to give compound 14B as a white solid . 1H NMR  δ 3.05 , 4.11 , 4.44–4.56 , 6.43 , 7.22 , 7.34 , 7.57 , 7.81 , 8.32 , 9.01 , 11.84 .(S)-2-Acetamido-N-{methyl}-3-phenylpropanamide (14). Acetic acid  and 2-(1H-benzo[d]+ C21H20ClN3O3 398.1271, found 398.1270.4-Hydroxy-2-oxo-1,2-dihydroquinoline-6-carboxylic acid (15A). To a solution of methyl 4-aminobenzoate  in DCM (40 mL) was dropwise added ethyl 3-chloro-3-oxopropanoate  and resulting mixture was stirred at room temperature for 16 h. The reaction mixture was washed with 1M NaOH (2 x 40 mL) and brine (40 mL). The organic layer was dried (Na2SO4), filtered and then concentrated under reduced pressure. The residue was hydrolyzed directly by stirring in a mixture of THF (10 mL), water (10 mL) and lithium hydroxide  at room temperature for 16 h. Excess THF was removed under reduced pressure before the pH was adjusted to 3 with 2M HCl. The product precipitated and was collected by filtration. The solid material was then heated in polyphosphoric acid  at 120°C for 2 h. The reaction mixture was cooled on an ice bath and water (100 mL) was added under stirring. A white solid was formed and collected by filtration and washed with ice-cold water giving compound 15A as a white solid . 1H NMR  δ 5.77 , 7.32 , 7.96–8.07 , 8.38 , 11.50 , 11.61 .(S)-2--3-phenylpropanoic acid (15B). A mixture of 4-hydroxy-2-oxo-1,2-dihydroquinoline-6-carboxylic acid (15A) , 2-(1H-benzo[d]+ C20H19N3O4 366.1454, found 366.1462.(S)-N-(1-(Dimethylamino)-1-oxo-3-phenylpropan-2-yl)-4-hydroxy-2-oxo-1,2-dihydroquinoline-6-carboxamide (16). A suspension of 4-hydroxy-2-oxo-1,2-dihydroquinoline-6-carboxylic acid (15A)  and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate  was stirred in DMF (5 mL) at room temperature for 10 min before (S)-2-amino-N,N-dimethyl-3-phenylpropanamide hydrochloride  and TEA  was added, resulting mixture was stirred at room temperature for 16 h. DMSO (5 mL) was added and excess DMF was then removed under reduced pressure. The remaining DMSO solution was purified by preparative HPLC to give compound 16 as a white solid . 1H NMR  δ 2.79 , 2.94 , 3.01 , 5.07 , 5.73 , 7.17 , 7.2–7.32 , 7.93 , 8.34 , 8.82 , 11.34 , 11.43–11.78 . HRMS: calcd. for [M+H]+ C21H21N3O4 380.1610, found 380.1601.2-(Trimethylsilyl)ethyl methylcarbamate (18A). A mixture of 2-acetic acid (20E) , diphenyl phosphorazidate , TEA  in DMF (5 mL) was stirred at room temperature for 1 h before 2-(trimethylsilyl)ethanol  was added. The resulting mixture was heated at 100°C under microwave irradiation for 10 min. Excess solvent was removed under reduced pressure and the residual crude material was purified by preparative HPLC. Fractions containing the product were pooled and freeze-dried to give compound 18A as a colorless oil , that solidified over time. 1H NMR  δ -0.03–0.1 , 0.88–1 , 2.46 , 2.68 , 3.11 , 3.16–3.23 , 4.04–4.13 , 5.59 , 6.83 , 7.16–7.23 , 8.34 .4-(Aminomethyl)-6-chloro-3,4-dihydroquinolin-2(1H)-one (18B). To a solution of 2-(trimethylsilyl)ethyl carbamate (18A)  in acetonitrile (25 mL) was added tetrabutylammonium fluoride . Resulting mixture was stirred at 60°C for 16 h. The conversion was slow and additional tetrabutylammonium fluoride  was added and the stirring continued at 60°C for 1 h. Excess solvents was removed under reduced pressure, the crude material was retaken in DMSO and purified by preparative HPLC. Fractions containing the product were pooled and freeze-dried to give compound 18B as a colorless oil. Tetrabutylammonium salt was still present in the NMR and additional purification was needed. The oil was dissolved in EtOAc/THF/MeOH and hydrochloric acid in dioxane  was added. Resulting mixture was stirred at room temperature for 16 h, forming a precipitate. The precipitate was collected by filtration and dried under reduced pressure to give compound 18B as a white solid . The material was used without further purifications. 1H NMR  δ 2.60 , 2.90 , 3.02–3.17 , 3.2–3.27 , 6.94 , 7.27–7.37 . 3 protons were not observed.N--1-oxo-3-phenylpropan-2-yl)-4-hydroxy-2-oxo-1,2-dihydroquinoline-6-carboxamide (18). A mixture of 4-(aminomethyl)-6-chloro-3,4-dihydroquinolin-2(1H)-one x HCl (18B) , (S)-2--3-phenylpropanoic acid , 2-(1H-benzo[d]+ C27H24ClN7O3 530.1707, found 530.1704.6-Chloro-3,4-dihydroquinolin-2(1H)-one (21A). To a mixture of 4-chloro-2-iodoaniline  in DMSO (10 mL) was added ethyl acrylate , tributyltin hydride  and (E)-2,2'-bis(2-methylpropanenitrile) in toluene . The resulting mixture was heated at 100°C for 16 h. The reaction was diluted with DCM and washed with brine. The organic layer was dried (Na2SO4), filtered and concentrated under reduced pressure. The residue was filtered through a silica plug, using 40% EtOAc in Heptane as the eluent. Fractions containing the desired product was pooled and concentrated under reduced pressure to give compound 21A as a crude solid . The crude material was used in the next reaction step without further purifications.6-Chloro-1-(4-methoxybenzyl)-3,4-dihydroquinolin-2(1H)-one (21B). Sodium hydride in mineral oil  was carefully added to a mixture of 6-chloro-3,4-dihydroquinolin-2(1H)-one (21A)  in DMF (3 mL). After stirring at room temperature for 30 min, 1-(chloromethyl)-4-methoxybenzene  was added in one portion and resulting mixture stirred for 2 h at room temperature. The reaction mixture was partitioned between DCM and water and the organic layer was collected, dried (Na2SO4) and concentrated under reduced pressure. The crude residue containing compound 21B  was used directly in the next reaction step without further purifications.Tert-butyl 2-(6-chloro-1-(4-methoxybenzyl)-2-oxo-1,2,3,4-tetrahydroquinolin-3-yl)acetate (21C). To a solution of 6-chloro-1-(4-methoxybenzyl)-3,4-dihydroquinolin-2(1H)-one (21B)  in dry THF (3 mL), at -78°C under an atmosphere of nitrogen, was dropwise added lithium diisopropylamide . After complete addition resulting mixture was stirred at -78°C for 10 min and then allowed to slowly reach room temperature. The reaction was again cooled to -78°C and tert-butyl 2-bromoacetate  was added by dropwise addition. Resulting mixture was stirred at -78°C for 1 h, followed by 1 h at 0°C. The reaction mixture was concentrated under reduced pressure, purified by silica column chromatography, using 0–100% EtOAc in heptane as the eluent. Fractions containing the desired product was pooled and concentrated under reduced pressure to give compound 21C as a pale yellow solid . 1H NMR  δ 1.48 , 2.41 , 2.81–3.01 , 3.10 , 3.77 , 5.08 , 6.83 , 7.05–7.15 .2-acetic acid (21D). A neat solution of tert-butyl 2-(6-chloro-1-(4-methoxybenzyl)-2-oxo-1,2,3,4-tetrahydroquinolin-3-yl)acetate (21C)  in 2,2,2-trifluoroacetic acid  was heated to 80°C for 2 h. The reaction mixture was concentrated under reduced pressure to give compound 21D as a crude pale yellow solid . The crude material was used directly without further purifications in the next reaction step.N-(4-(1H-Tetrazol-5-yl)phenyl)-2-acetamido)-3-phenylpropanamide (21). A mixture of 2-acetic acid (21D) , 2-acetic acid (22A). A suspension of 4-chloro-2-iodoaniline , dimethyl 2-methylenesuccinate , diacetoxypalladium , triphenyl phosphine  and sodium acetate  in dry DMF (50 mL) was heated under an inert atmosphere at 110°C for 16 h. Excess solvent was removed under reduced pressure to give the crude ester as a brown solid. The ester was hydrolyzed directly by stirring at room temperature in 4M NaOH for 1 h. The reaction mixture was washed with EtOAc (4 x 100 mL) and then acidified to pH 1 with concentrated HCl, forming a precipitation. The precipitate was collected by filtration and washed with cold water and then dried under reduced pressure. The residual solid was retaken in THF and passed through a celite plug using EtOAc as the eluent. The filtered solution was dried (MgSO4) and concentrated under reduced pressure to give compound 22A  as a pale brown solid. 1H NMR  δ 3.09 , 7.16 , 7.45 , 7.64 , 7.75 , 10.45 , 12.11–13.4 .N-(4-(1H-Tetrazol-5-yl)phenyl)-2-acetamido)-3-phenylpropanamide (22). A mixture of 2-acetic acid (22A) , TEA  and 2-(1H-benzo[d]+ C27H22ClN7O3 528.1551, found 528.1534.Ethyl 6-chloro-2-oxo-1,2-dihydroquinoline-3-carboxylate (23A). A mixture of 2-amino-5-chlorobenzaldehyde , diethyl malonate  and piperidine  in EtOH (60 mL) was refluxed for 6 h. Upon cooling to room temperature a precipitation was formed. The precipitation was collected by filtration, washed with cold EtOH and dried under reduced pressure to give compound 23A  as a crude solid. The crude material was used without further purifications. 1H NMR  δ 1.29 , 4.27 , 7.32 , 7.63 , 7.95 , 8.45 , 12.16 .6-Chloro-3-(hydroxymethyl)quinolin-2(1H)-one (23B). To a slurry of ethyl 6-chloro-2-oxo-1,2-dihydroquinoline-3-carboxylate (23A)  Et2O (30 mL), under an atmosphere of nitrogen at 0°C, was dropwise added DIBAL-H . Resulting yellow-orange solution was allowed to slowly reach room temperature upon stirring over night. The reaction mixture was cooled to 0°C MeOH and 4M HCl was then carefully added. Resulting mixture was extracted with EtOAc (3 x 100 mL), the combined organic fractions was washed with brine, dried (Na2SO4), filtered and concentrated under reduced pressure to give compound 23B as a pale brown oil . The crude material contained traces of byproducts with a reduced ring system, but was used without further purifications. 1H NMR  δ 4.40 , 5.28 , 7.31 , 7.48 , 7.78–7.93 , 11.91 .6-Chloro-3-(chloromethyl)quinolin-2(1H)-one (23C). A neat mixture of 6-chloro-3-(hydroxymethyl)quinolin-2(1H)-one (23B)  and SOCl2 (10 mL) was refluxed for 6 h. Excess SOCl2 was removed under reduced pressure and the residue carefully quenched with saturated NaHCO3. The mixture was diluted with EtOAc and the layers were separated. The aqueous phase was extracted with EtOAc (3 x 20 mL). The combined organic fractions was dried (Na2SO4), filtered and concentrated under reduced pressure to give compound 23C as a crude pale yellow solid . The crude material was used directly in the next reaction step without further purifications.3-propanoic acid (23D). A mixture of diethyl malonate , sodium hydride  in THF (15 mL) was stirred at room temperature under an atmosphere of nitrogen for 10 min. A slurry of 6-chloro-3-(chloromethyl)quinolin-2(1H)-one (23C)  in THF (15 mL) was dropwise added to the reaction mixture and the resulting pale yellow suspension was refluxed for 2 h. Excess solvent was removed under reduced pressure and the residue poured on crushed ice, forming a white precipitate. The precipitate was collected by filtration, washed with cold water and decarboxylated immediately by adding concentrated HCl (20 mL) and refluxing the resulting suspension for 16 h. The yellow solution was allowed to reach room temperature and then stirred on an ice bath to form a new precipitate. The precipitate was filtered, washed with cold water and dried under reduced pressure to give compound 23D as a pale yellow solid . 1H NMR  δ 2.55 , 2.71 , 7.30 , 7.43–7.52 , 7.73 , 11.90 . 1 proton was not observed.(S)-N-(4-(1H-Tetrazol-5-yl)phenyl)-2-propanamido)-3-phenylpropanamide (23). A mixture of 3-propanoic acid (23D) , 2-(1H-benzo[d]+ C28H24ClN7O3 542.1707, found 542.1732.(S)-N-(4-(1H-Tetrazol-5-yl)phenyl)-2-propanamido)-3-phenylpropanamide (24). A mixture of 3-propanoic acid (24A) , 2-(1H-benzo[d]+ C28H24ClN7O3 542.1707, found 542.1701.6-Chloro-4-({methylamino}methyl)quinolin-2(1H)-one (25A). A mixture of 4-(bromomethyl)-6-chloroquinolin-2(1H)-one (13C) , 2M methanamine in THF  and TEA  was stirred at 50°C for 1 h. A solid precipitate formed upon cooling to room temperature, the precipitate was collected by filtration and washed with cold THF. The crude solid of compound 25A  was used directly in the next reaction step without further purifications.(S)-Tert-butyl 1-[{methyl}(methyl)amino]-1-oxo-3-phenylpropan-2-ylcarbamate (25B). A mixture of 6-chloro-4-({methylamino}methyl)quinolin-2(1H)-one (25A) , (S)-2-(tert-butoxycarbonylamino)-3-phenylpropanoic acid , 1H-benzo[d]-1-oxo-3-phenylpropan-2-ylcarbamate (25B)  in excess of 4M HCl in dioxane (6 mL) was stirred at room temperature for 2 h. LCMS indicated full conversion to the desired product. The solvent was evaporated to give compound 25C as a crude solid. The crude product used without further purifications in the next reaction step.(S)-N-methyl}(methyl)amino]-1-oxo-3-phenylpropan-2-yl)-4-hydroxy-2-oxo-1,2-dihydroquinoline-6-carboxamide (25). A mixture of (S)-2-amino-N-{methyl}-N-methyl-3-phenylpropanamide (25C) , 4-hydroxy-2-oxo-1,2-dihydroquinoline-6-carboxylic acid (15A) , 1H-benzo[d]+ C30H25ClN4O5 557.1592, found 557.1571.(S)-tert-butyl 1-[{methyl}(methyl)amino]-1-oxo-3-phenylpropan-2-yl(methyl)carbamate (26A). A mixture of 6-chloro-4-({methylamino}methyl)quinolin-2(1H)-one (25A) , (S)-2-(tert-butoxycarbonyl(methyl)amino)-3-phenylpropanoic acid , 1H-benzo[d][(S)-N--N-methyl-2-(methylamino)-3-phenylpropanamide (26B). A solution of (S)-tert-butyl 1-[{amino)-1-oxo-3-phenylpropan-2-yl(methyl)carbamate (26A)  in excess of 4M hydrogen chloride in dioxane (6 mL) was stirred at room temperature for 3 h. The reaction was concentrated under reduced pressure to give the crude compound 26B (0.300 g 95%). The crude material was used without further purifications in the next reaction step.(S)-N-methyl}(methyl)amino]-1-oxo-3-phenylpropan-2-yl)-4-hydroxy-N-methyl-2-oxo-1,2-dihydroquinoline-6-carboxamide (26). A mixture of (S)-N-{methyl}-N-methyl-2-(methylamino)-3-phenylpropanamide (26B) , 4-hydroxy-2-oxo-1,2-dihydroquinoline-6-carboxylic acid (15A) , 1H-benzo[d]+ C31H27ClN4O5 571.1748, found 571.1756.N-{methyl}-N,4-dimethyl-3-phenyl-1H-pyrazole-5-carboxamide (28). A mixture of 6-chloro-4-{(methylamino)methyl}quinolin-2(1H)-one (25A) , 4-methyl-3-phenyl-1H-pyrazole-5-carboxylic acid , 1H-benzo[d]+ C22H19ClN4O2 407.1275, found 407.1288.S1 File(DOCX)Click here for additional data file.


Achillea tenuifolia Lam. (Asteraceae) afforded a methanolic extract from which after fractionation in solvents with different polarities, two known flavones 3’, 5- dihydroxy- 4’, 6, 7- trimethoxy flavone , 5- hydroxy- 3’,4’, 6, 7- tetramethoxyflavone (compound 4), besides stearic acid (compound 1), lupeol (compound 2), daucosterol , 2, 4- dihydroxy methyl benzoate (compound 6) were isolated for the first time. The structure of isolated compounds was elucidated by means of different spectroscopic methods such as UV, IR, Mass and 1H- NMR (1D and 2D) and 13C-NMR. For further confirming the structures of isolated compounds, comparison of the spectral data of them with those reported in the litratures have been done.  Achillea(Asteraceae) comprises 115 species in the world which nineteen are present in Iran  instruments using the UNIX data system at 300, 400 and 500 MHz, respectively. The 13C-NMR spectrum was recorded at 75, 100 and 125 MHz, respectively using CDCL3, CD3OD and C5D5N as solvent. 1H-13C HMBC and HMQC were recorded as mentioned above. EI-MS spectra were recorded on a Finnigan MAT 312. HR-EIMS were carried out on Jeol JMS 600 mass spectrometer. Column chromatography was carried out on silica gel (M&N), 70-230 and 230-400 meshes. All solvents and chemical reagents were purchased from Merk . Compounds on the TLC were detected at 254 and 366 nm and by ceric sulphate as spraying reagent.The FT-IR spectra were recorded on a vector 22 instrument. The Plant materialAchillea tenuifolia (Asteraceae) was collected in May 2008, from populations growing in Zanjan province, Iran. The plant was identified in Department of Pharmacognosy, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran. A voucher specimen (NO.487) was deposited in the herbarium of the above mentioned college.The aerial flowering part of Extraction and isolationA.tenuifolia(4 Kg) were extracted by maceration with methanol (3×15 L) at room temperature, three times, each time three days. The methanolic extract was evaporated under reduced pressure to give a dark residue (300 g), which was suspended in water and defatted with petroleum ether. The defatted aqueous extract successively fractionated with dichloromethane, n-butanole (3 times each). The dichloromethane fraction (50 g) was subjected on a silica gel column chromatography using hexane with increasing gradient of EtOAC up to 100% and followed by methanol to give ten fractions.The dried aerial parts of 3) to yield 4 sub fractions. Sub fraction B purified by preparative TLC with the system of hexane: EtOAC (8.5:1.5) to give compound 1.Fraction 1 (hexane eluate) was subjected on silica gel column (Hex: CHCl3 to give three sub fractions (M- O). Sub fraction N (hexane: CHCl3 = 6: 4 eluate) was further purified by recrystallization from MeOH to yield compound 2 .Fraction 2 (hexane:EtOAC = 9.5 : 0.5 eluate )was subjected to silica gel column chromatography, using hexane: CHCl2CO, hexane: EtOAC as mobile phase, respectively.Fraction 6 (hex: EtOAC=8:2) was rechromatographed on silica gel column (hexane: aceton=6:4) to render 8 sub fractions (a-h).Subfractions 6c, 6f was further separated on preparative TLC to yield compounds 3, 4 using hexane: Me3: MeOH (2.5:7.5:0.5) as mobile phase and afforded compound 5.Fraction 9 (EtOAC: MeOH= 9.5:0.5 eluate) was loaded on silica gel column using hexane: CHCl2CO: MeOH as mobile phase. From fraction eluted with Me2CO: MeOH (9.5: 0.5) obtained compound 6.The structure of all compounds have been shown in Finally fraction eluted with EtOAc: MeOH (9:1) was subjected on silica gel column using Me18H36O2);EI/MS: m/z(rel.%): 284(4.7), 256(25.95), 213(11.49), 185(14.45),171(14.84), 157(17.15), 143(11.18), 129(43.89), 115(17.54), 101(11.75), 87(26.50), 73(100);1H- NMR:δ=2.17, 1.49, 1.16, 0.76.Stearic acid (compound 1): White powder(10 mg); m.p. 70 °C;HR EI/MS: 284.2722 ;EI/MS: m/z(rel.%): 426(24.77), 411(5.73), 393(2.02), 357(2.10), 302(4.30), 257(6.61), 229(6.83), 218(9.66), 207(43.76), 203(37.62), 189(57.00), 161(22.52), 121(51.33);1H-NMR: δ= 0.70, 0.8, 0.83, 0.94, 1.00, 1.01, 1.65, 3.15, 4.53 , 4.63.Lupeol (compound 2): Colorless crystals (20 mg); m.p. 214°- 217 °C; IR (KBr)υmax(MeOH): 273, 340; IRυmax (KBr)cm -1: 3448, 1650, 1603, 1457,1270,1120,1013,840;HR EI/MS m/z: 344.0896;FAB MS [M+1]+:345,[M-1]+:343;EI-MSm/z(rel.int.): 343.8(35.13), 328(30.38), 313(100.00), 300(11.27), 196 (9.44), 180.8(12.45), 152.8(52.66), 148(14.45), 133(13.85);1H&13 C-NMR: see 3’, 5- dihydroxy- 4’, 6, 7- trimethoxyflavone : yellow solid (10 mg); m. p. 192 °C; Uv: λmax(MeOH):270,340;IRυmax (KBr)cm-1:3530,1660,1605,1460,1273,1130,840,800; HR EI/MS m/z: 358.1047 ;FAB MS [M+1]+:359,[M-1]+:357; EIMSm/z(rel.int.): 358(57.04), 343(50.2), 328(100.00), 313(98.6), 299(19.75), 285(32.8), 196(20), 181(30.5), 162(25.25), 153(80);1H- NMR: see 5- hydroxy- 3’, 4’, 6, 7- tetramethoxyflavone (compound 4):pale yellow crystals (10.5 mg); m. p. 195 °C; Uv:λmax: 3460, 3050, 1650 cm-1;HR EI/MS m/z : 576.4386 ; FAB MS[M-1]+: 575; EI/MS m/z(rel.%): 414(8.4), 399(8.1), 396(100.0), 381 (14.6), 329(4.5), 303(6.5), 275(9.9), 273(4.9), 255(20.2);1H-NMR: δ= 0.64, 0.83, 0.86, 0.92, 0.96, 3.97, 4.27-4.58, 5.04 .Daucosterol: White powder (25 mg); m. p. 278°- 282 °C;[α]: -14.5°; IR(KBr)υ8H8O4); EI/MS m/z(rel.%): 168.0 (100.00), 152.9 (73.29), 136.0 (25.90), 125.0(4.76), 107.9 (16.28), 97.0 (48.67), 84.9 (48.37);1H-NMR : δ= 3.87, 6.77 , 7.50 , 7.57.2,4-dihydroxy-methyl benzoate (compound 6):Brown solid (12 mg); m. p. 88°- 95 °C; HR EI/MS m/z 168.0417  to Band Іa (in AlCl3/HCl) was 18 nm, which indicated a hydroxyl group at position 5 and one methoxyl group at position 6 , which is typical for the proton of flavone (H-3).The 1H-NMR spectrum displayed three resonances in the aromatic region at δ7.44,δ7.38,δ6.91. J=2.4, 8.8 HZ showed meta and ortho couplings, respectively, which showed that there were substitution in position 3´,4´,thus H- 2´, 6´,5´ have meta and ortho couplings with each other. Also three singlets (each integrating for three protons) at δ 3.97, 3.89, 3.94 referred to three methoxyl moieties at 4´, 6, 7 which the placement of them have been confirmed by the HMBC spectrum. The singlet at δ6.52 integrating for one proton was assigned to H- 8. The 13 C-NMR spectra also showed the presence of 18 carbons, which were resolved through DEPT experiment as 3 methyl, 5 methine and 10 quaternary carbons. Considering all the above evidences, the structure of the compound 3 confirmed as 3´,5-dihydroxy- 4´, 6,7-trimethoxyflavone. The structural similarities between compound 3& 4 were revealed by comparison of the 1H-NMR spectra of these two compounds ,H-8(δ 6.67),3´,4´-substituted B ring  were observed. Additional singlet at δ3.95 (3H integration) was observed, which was not present in compound 3. This was further supported by differences between HREI- MS spectra of compound 3,[M]+ at m/z 344.0896 and compound 4,[M]+ at m/z 358.1053. In the EI- MS spectrum of compound 4 besides 358 as base ion, there were two fragment ions in m/z 181,162, characteristic retro-Diels- Alder fragment ions for three oxygenated substituents in the Ring A and two methoxyl moieties in the Ring B. All these data approved the structure of compound 4 to be, 5-hydroxy- 3´, 4´, 6, 7-tetramethoxyflavone.Compound 3 was found to be 3´, 5-dihydroxy-4´ ,6 ,7-trimethoxyflavone. The HREI- MS of compound 3 showed the [M]sition 6 . This daompounds . In the A.tenuifolia contains compounds present in the other species of the genus. The isolation of these flavones was in accordance with the results of our previous work (Achillea which reported that the genus Achillea are characterized by the predomination of flavonoids, methylated aglycones, 6-hydroxyflavones, and their O- Me ethers (ous work  and withe ethers -18.


Those complexes share the same supramolecular interaction leading to similar thermal off-rates in the range of 0.09 and 0.36 s−1, compared to 1.7 s−1 for the monovalent complex. On the other hand, the backbones exhibit different flexibility, and we determined a broad range of rupture lengths between 0.3 and 1.1 nm, with higher most-probable rupture forces for the stiffer backbones. Interestingly, the medium-flexible connection has the highest rupture forces, whereas the ligands with highest and lowest rigidity seem to be prone to consecutive bond rupture. The presented approach allows separating bond and backbone effects in multivalent model systems.Multivalent biomolecular interactions allow for a balanced interplay of mechanical stability and malleability, and nature makes widely use of it. For instance, systems of similar thermal stability may have very different rupture forces. Thus it is of paramount interest to study and understand the mechanical properties of multivalent systems through well-characterized model systems. We analyzed the rupture behavior of three different bivalent pyridine coordination complexes with Cu In a multivalent molecular system, two partners interact with each other through two or more non-covalent equivalent interaction centers. This principle is important in biochemistry  and suprrb pyridin-3-yl}butan-1-ol (4): A suspension of 4-{[2-(pyridin-3-yl)ethynyl]pyridin-5-yl}but-3-yn-1-ol (3) + calcd for C16H21N2O, 257.1648, found, 257.1634.1-ol (3)    in anhydrous CH2Cl2 (0.6 mL) was added EDAC  and the resulting mixture was stirred at room temperature under an atmosphere of argon for 5 days. The solvent was evaporated and the residue was purified by dialysis (MW cut-off: 1000 g/mol) against MeOH to provide the pyridine-PEG conjugate 5  as a colorless solid. Mp 127 °C; 1H NMR  δ 1.54–1.62, 1.64–1.72 , 2.62 , 3.06 , 3.64 , 6.94 , 7.19 , 7.34–7.41 , 7.50 , 8.30 , 8.38–8.44  ppm.Synthesis of 2-methyl-3-(pyridin-4-yloxy)-2-[(pyridin-4-yloxy)methyl]propan-1-ol (8) and 1,1,1-tris[pyridine-4-yloxy)methyl]ethane (9): To a solution of 2-(hydroxymethyl)-2-methylpropane-1,3-diol  in dry DMF (25 mL) was added NaOH . After 15 min stirring at room temperature, 4-fluoropyridine  was added. The mixture was heated to reflux for 4 days and after cooling to room temperature diluted with CH2Cl2/water. The organic phase was washed several times with water and then dried (Na2SO4). Column chromatography on aluminum oxide (CH2Cl2/MeOH 94:6) afforded 338 mg of a yellowish solid (mixture of 8 and 9) and 21 mg (3%) of compound 8 as colorless solid. By further purification steps (second chromatography on silica gel followed by HPLC) additional 8  and 9  were isolated.Data of compound 8: Mp 161–162 °C; 1H NMR  δ 1.19 , 3.68 , 4.09 , 7.00 , 8.33  ppm; 13C NMR  δ 17.1 , 42.0 , 64.7 , 71.0 , 111.9 , 155.7 , 159.7  ppm; IR (ATR) ν: 3135 (OH), 3100–3025 (=C-H), 2960–2865 (C-H), 1590–1460 , 1055–1025 (C-O) cm−1; HRMS (pos. ESI-TOF) m/z: [M + H]+ calcd for C15H19N2O3, 275.1396; found, 275.1403; anal. calcd for C15H18N2O3: C, 65.68; H, 6.61; N, 10.21; found: C, 65.17; H, 6.45; N, 10.13.Data of compound 9: Mp 143–144 °C; 1H NMR  δ 1.33 , 4.10 , 6.80 , 8.41  ppm; 13C NMR  δ 17.3 , 40.2 , 69.4 , 110.3 , 151.3 , 164.6  ppm; IR (ATR) ν: 3050–3035 (=C-H), 2950–2870 (C-H), 1685–1455 , 1110 (C-O) cm−1; HRMS (pos. ESI–TOF) m/z: [M + H]+ calcd for C20H22N3O3, 352.1656; found, 352.1659; anal. calcd for C20H21N3O3: C, 68.36; H, 6.02; N, 11.96; found: C, 68.02; H, 6.00; N, 11.93.Synthesis of pyridine-PEG conjugate 10, ligand of 2c: To a solution of the bifunctional poly(ethylene glycol)  in anhydrous CH2Cl2 (0.6 mL) was added EDAC  and the resulting mixture was stirred at room temperature under an atmosphere of argon for 9 days. The solvent was evaporated and the residue was purified by dialysis (MW cut-off: 1000 g/mol) against MeOH to provide the pyridine-PEG conjugate 10  as a colorless solid (mp. 55–58 °C). 1H NMR  δ 6.81 , 8.43  ppm.3N4 cantilevers  were cleaned by UV/O3 treatment [Surface films of the polymers were prepared in a similar manner as described before . A droplreatment  (Penray 4 (2b), 30 mM CuSO4 (3c) or DI water (blank). SFM cantilevers were calibrated using the thermal noise method [SMF measurements were performed as in , i.e., oe method . In DFS,4 (blank) showed a different force-loading rate behavior, proving specific interactions with the Cu2+ ligand. In 3 mM CuSO4, ligand 2c showed the same behavior as the monovalent system 1, but a different in 30 mM CuSO4. The latter was used for the analysis presented here.Force–distance curves were processed as described in . In shor


N-bromosuccinimide (NBS), and Suzuki-Miyaura cross-coupling of the pyrazolyl bromide with 4-carbamoylphenylboronic acid to give the target 4-arylpyrazole in 35% overall yield, suitable for study in WS cells.Microwave-assisted Suzuki-Miyaura cross-coupling reactions have been employed towards the synthesis of three different MAPKAPK2 (MK2) inhibitors to study accelerated aging in Werner syndrome (WS) cells, including the cross-coupling of a 2-chloroquinoline with a 3-pyridinylboronic acid, the coupling of an aryl bromide with an indolylboronic acid and the reaction of a 3-amino-4-bromopyrazole with 4-carbamoylphenylboronic acid. In all of these processes, the Suzuki-Miyaura reaction was fast and relatively efficient using a palladium catalyst under microwave irradiation. The process was incorporated into a rapid 3-step microwave-assisted method for the synthesis of a MK2 inhibitor involving 3-aminopyrazole formation, pyrazole C-4 bromination using  WRN that give rise to a pathology attributed for the most part to the key role of RecQ3 in cellular responses to specific types of DNA damage. Lack of RecQ3 leads to frequent DNA replication fork stalling and subsequently genomic instability diazepinone PF-3644022 pyridin-4-one inhibitor 6, the stressed cellular morphology and shortened replicative lifespan were both corrected, although the extension in life span was much smaller than the one seen upon treatment with SB203580 pyridin-4-one inhibitor 6 quinoline ring (see H)-one scaffold  requires 274.1222); IR (KBr) νmax/cm−1 3411 (NH), 2922 (CH), 1626, 1604, 1586, 1479; 1H-NMR  δH/ppm 11.18 , 8.26 , 7.96 , 7.69 , 7.63 , 7.58 , 7.46–7.41 , 7.35 , 6.47 , 5.76 , 5.17 ; 13C-NMR  δC/ppm 157.5 (C), 134.3 (C), 141.0 (C), 137.0 (C) 133.5 (C), 130.2 (C), 128.5 (CH), 127.9 (CH), 126.8 (CH), 122.7 (CH), 120.9 (CH), 118.8 (CH), 115.2 (CH), 114.9 (CH), 110.0 (CH), 101.5 (CH), 96.7 (CH); m/z (EI) 275 , 274 , 273 (19), 246 (7), 205 (6), 191 (9), 133 (14), 84 (21).A mixture of 3-amino-1-(3-bromophenyl)-1compound    and NBS  in THF (10 mL) was stirred at room temperature for 16 h and then the solvent was vaporated in vacuo. The residue was dissolved in EtOAc, filtered through Celite and evaporated in vacuo. Purification by column chromatography on silica gel, eluting with hexane–EtOAC (3:1 v/v), gave the title compound  as a brown solid, mp 93–95 °C ; IR (KBr) νmax/cm−1 3415, 3312, 3213, 1629, 1594, 1556; 1H-NMR  δH/ppm 8.55 , 7.88 , 7.67 , 7.37–7.35 , 5.38 ; 13C-NMR  δC/ppm 159.9 (C), 142.5 (C), 131.8 (CH), 128.6 (CH), 127.5 (CH), 122.7 (C), 119.3 (CH), 115.1 (CH), 85.2 (C); m/z (EI) 275 ; 319  317 , 315 , 220 (42), 205 (100), 189 (8), 177 (14), 145 (15), 105 (12).A solution of 3-amino-1-(3-bromophenyl)-1title compound was prepared as a yellow solid, exactly in accordance with our previously published microwave-assisted procedure thiophene moiety could be envisaged and thus incorporated with this transformation [Suzuki-Miyaura cross-coupling reactions have been investigated for the synthesis of three different MK2 inhibitors for study in WS cells. Towards the synthesis of PF-3644022, cross-coupling of a 2-chloroquinoline with a 3-pyridinylboronic acid provided the desired biaryl target but the pyridine moiety and the chloroquinoline precursor both proved incompatible with the subsequent Yamazaki cyanation of a 6-nitroquinoline, en route to the benzothiophene ring. Thus, although a relatively early-stage Suzuki-Miyaura coupling would appear incompatible with this cyanation method, alternative strategies for the incorporation of the 3-aminobenzo[ormation . Towards


The brominated indoles 1–3 along with the sesquiterpene debilone (4) have been isolated from Laurenciacomplanata. For the first time, debilone (4) has been obtained from a marine plant. From the methanol extract of Calloseris sp., we have achieved the second isolation of the unusual A-ring contracted steroids (−)-2-ethoxycarbonyl-2β-hydroxy-A-nor-cholest-5-en-4-one (9) and phorbasterone B (10). The crude extracts of Laurenciacomplanata exhibited antimicrobial activity against Bacillus cereus, Staphylococcus aureus, Streptococcus pneumoniae, and Candida albicans.Several species of red algae (Rhodophyta) from the coastal regions of Madagascar have been investigated for their natural products. The most abundant compound was cholesterol ( Gelidiummadagascariense, Mollion identified an agar polymer in which the methylated fraction contains predominantly 6-O-methyl galactose and traces of 4-O-methyl galactose  [11) were isolated from the diethyl ether fraction of Calloseris sp. (After extraction of a sample of -one (9)  and (−)-9) [10)] , and choeris sp. .9 was isolated as colorless oil. The molecular mass of 458 was deduced from the ESI-MS by the masses of the protonated molecule and the ammonium adduct (m/z = 459 [M + H]+ and 476 [M + NH4]+). Infrared absorptions at ν = 3440 and 1740 cm−1 revealed the presence of hydroxyl and ester groups, respectively. A second absorption for a carbonyl group at 1721 cm−1 was assigned to the ketone moiety. The absorption at 1648 cm−1 results from a C=C double bond. The 1H NMR and 13C NMR spectra displayed a close resemblance to cholestane steroids. Obvious differences are the two carbonyl signals at δ = 172.90 (C-3) and 201.05 (C-4) ppm and the signals for an ethoxy group in the 1H NMR spectrum at δ = 4.25 ppm  and δ = 1.27 ppm . The separated 1H NMR signals for the geminal protons at δ = 2.10 ppm  and 2.18  indicated the presence of a contracted ring system. The olefinic proton at δ = 6.74 ppm  displays a downfield shift as compared to cholesterol (5), which can be explained by the conjugated electron-withdrawing group. Extensive 2D NMR measurements  allowed the assignment of all 1H NMR and 13C NMR signals for (−)-2-ethoxycarbonyl-2β-hydroxy-A-nor-cholest-5-en-4-one (9). With respect to the stereochemistry at C-2, the presence of a NOESY correlation between the hydroxyl proton and the protons of the methyl group (C-19) was in agreement with the β-orientation of the hydroxyl group. Compound 9 has been isolated only once before by Lin and coworkers from the soft coral Dendronephthya sp.  −; Anal. calcd for C8H3Br4N: C 22.20, H 0.70, N 3.24; found: C 23.10, H 0.62, N 3.22%.2,3,5,6-Tetrabromoindole (3–154 °C ); UV -3-Hydroxy-1,1,7,7a-tetramethyl-1,1a,2,3,6,7,7a,7b-octahydro-5H-cyclopropa[a]naphthalen-5-one (9-hydroxyaristol-1(10)-en-2-one, debilone) (4) +, 469 [2M + H]+.(1aone) (4) ,43,44: C8): Colorless solid; 1H NMR : δ (ppm) = 0.69 , 0.82 , 0.84 , 0.87 , 0.92–0.95 , 0.93 , 0.96–1.00 , 1.00–1.02 , 1.02 , 1.03–1.20 , 1.24–1.40 , 1.44–1.62 , 1.63–1.72 , 1.80–1.89 , 1.95–1.99 , 1.99–2.04 , 2.23–2.27 , 2.28–2.33 , 3.50–3.57 , 5.36 ; 13C NMR and DEPT : δ (ppm) = 11.84 (C-18), 12.31 (C-24′), 18.81 (C-21), 18.95 (C-27), 19.39 (C-28), 19.58 (C-19), 21.07 (C-11), 22.99 (C-23), 24.30 (C-15), 26.34 (C-25), 28.23 (C-16), 28.91 (C-26), 31.66 (C-2), 31.89 , 33.89 (C-22), 36.26 (C-20), 36.49 (C-10), 37.24 (C-1), 39.76 (C-12), 42.31 (C-4), 42.59 (C-13), 46.04 (C-24), 50.11 (C-9), 56.01 (C-17), 56.75 (C-14), 71.81 (C-3), 121.72 (C-6), 140.75 (C-5); GC-MS : m/z (%) = 414 , 399 (35), 396 (52), 381 (34), 329 (51), 303 (48), 273 (22), 255 (29), 213 (40), 145 (41), 107 (38), 105 (40), 55 (44), 43 (67).24-Methyl-25-homocholesterol (9): Colorless oil; 3) +, 476 [M + NH4]+.(−)-2-Ethoxycarbonyl-2β-hydroxy-A-nor-cholest-5-en-4-one  ); CD Δε 10): Colorless oil; CD Δε MeOH (λ nm): −0.24 (239), +0.29 (269); UV (MeOH): λ (nm) = 246; fluorescence (MeOH): λmax (246 nm) = 298 nm; IR (ATR): ν (cm−1) = 3456 (br), 2924, 2852, 1743, 1718, 1649, 1459, 1376, 1267, 1232, 1175, 1102, 1054, 960, 886, 767, 716; 1H NMR : δ (ppm) = 0.73 , 0.87 , 0.88 , 0.94 , 0.99–1.12 , 1.12–1.20 , 1.19–1.24 , 1.21 , 1.32–1.42 , 1.48–1.55 , 1.58–1.64 , 1.66–1.74 , 1.82–1.92 , 2.07 , 2.11 , 2.19 , 2.41 , 3.79 , 3.80 , 6.75 ; 13C NMR and DEPT : see m/z = 445 [M + H] +, 462 [M + NH4] +, 911 [2M + Na] +.Phorbasterone B : δ (ppm) = 0.83–0.93 , 1.23–1.35 , 1.43–1.58 , 1.59–1.66 , 2.03 , 2.26–2.33 , 3.68 , 4.04 , 4.13 , 5.45 , 5.59–5.67 ; 13C NMR and DEPT  : δ (ppm) = 14.1 (CH3), 14.3 (CH3), 22.6 (CH2), 24.9 (CH2), 25.4 (CH2), 28.7–29.7 , 31.8 (CH2), 32.1 (CH2), 34.4 (CH2), 37.3 (CH2), 60.2 (CH2), 73.2 (CH), 132.3 (CH), 133.2 (CH), 174.0 (C); ESI-MS (10 V): m/z = 281 [M – H2O + H]+ (12), 295 [M − H2O + H]+ (13), 309 [M − H2O + H]+ (14), 316 [M + NH4]+ (12), 330 [M + NH4]+ (13), 344 [M + NH4]+ (14), 561 [2M − 2H2O + H]+ (12 + 12), 575 [2M − 2H2O + H]+ (12 + 13), 589 [2M − 2H2O + H]+ (13 + 13) (12 + 14), 603 [2M − 2H2O + H]+ (13 + 14), 617 [2M − 2H2O + H]+ (14 + 14).Mixture of allylic alcohols 1, 2, and 3 were tested for their antimicrobial activity against four Gram-negative bacteria: Enterobacter cloacae (ATCC 700323), Klebsiella oxytoca (ATCC 8724), Escherichia coli, Salmonella enteridis; three Gram-positive bacteria: Bacillus cereus (ATCC 13061), Staphylococcus aureus (ATCC 11632), Streptococcus pneumoniae (ATCC 6301); and against one yeast strain: Candida albicans. The pathogens were supplied by the Laboratoire de Microbiologie de l’Environnement (LME), Centre National de Recherche sur l’Environnement (CNRE), Antananarivo, Madagascar. The crude extracts and the compounds 1–3 were dissolved in methanol at a concentration of 1 mg/mL. A sample (10 μL) of each solution was added via a pipette onto a sterile antibiotic filter disc of 6 mm diameter and oven dried at 40–50 °C. The discs were placed on Müller-Hinton agar plates that had been inoculated with the microorganisms mentioned above. The plates were incubated for 24 h at 37 °C for the bacteria and for 48 h at 25 °C for the yeast. The diameters of the inhibition zones generated around the discs were measured (Ø in mm). The tests were performed in triplicate and the mean values are given in Antimicrobial assay: The antimicrobial activities were determined using the agar diffusion technique in Petri dishes. The crude algal extracts and the three compounds Plasmodium falciparum was determined by using the microfluorimetric assay previously reported [50 value in µg/mL.Antimalaria test: The antiplasmodial activity against the FCM29 strain of reported . The res5) is the major sterol in most of the red algae (Rhodophyta) [5) and oxygenated cholesterol derivatives have been identified as the major components of Grateloupia sp., Gracilaria corticata, Halymenia sp., Metamastophora sp., and Spyridia sp. From the extracts of Spyridia sp. and Calloseris sp. we obtained 24-methyl-25-homocholesterol (8) and we achieved the first full assignment of its 1H and 13C NMR data. The three known brominated indoles 1–3 have been isolated from Laurencia complanata along with the sesquiterpene debilone (4). For the first time, we have isolated debilone (4) from a marine organism. From the extracts of Calloseris sp. we achieved the second isolation of the two A-ring contracted steroids (−)-2-ethoxycarbonyl-2β-hydroxy-A-nor-cholest-5-en-4-one (9) and phorbasterone B (10). A mixture of the three fatty acid esters 12–14 with an internal allylic alcohol function has been obtained from Neurymenia fraxinifolia. These compounds probably derive from autoxidation of palmitoleates and oleates. We have screened the crude plant extracts for their antimicrobial activity. The extract of Laurenciacomplanata showed the highest antimicrobial activities against Bacillus cereus, Staphylococcus aureus, Streptococcus pneumoniae, and Candida albicans.In his early work on the sterols of marine algae, Patterson reported that cholesterol (dophyta) . This fi


H-benzo[d]imidazol-3-ium chloride (1-Cl), 1,3-bis-1H-benzo[d]imidazol-3-ium chloride (2-Cl), 1,3-diphenyl-1H-benzo[d]imidazol-3-ium chloride (3-Cl), and 1,3-di(pyridin-2-yl)-1H-benzo[d]imidazol-3-ium chloride (4-Cl) were prepared in a two-step synthesis avoiding chromatographic work-up. In the key step triethyl orthoformate is reacted with the corresponding N1,N2-diarylbenzene-1,2-diamines and then further transformed in situ, by alkoxy abstraction using trimethylsilyl chloride (TMSCl), and concomitant imidazole ring closure.An atom-economic synthetic route to benzimidazolium salts is presented. The annulated polycyclic systems: 1,3-bis-1 Imidazole-based N-heterocyclic carbenes (NHCs) are stable systems serving as ancillary ligands mainly to construct organometallic complexes. These NHCs are sterically and electronically tunable, strongly binding ligand units in complexes in the search for new materials –5 and foAfter the discovery of NHCs in 1968 by Wanzlick and Öfele, who isolated stable diamino-substituted carbenes, around 20 years later Arduengo further stabilized these potential ligand groups by embedding them into imidazole rings and increased in addition the steric congestion –22 or tw1,N2-substituents. As mentioned before the synthetic pre-stages of NHCs are always imidazolium salts, but benzimidazolium salts, which possess two aromatic N1,N2-substituents are rare. More common are benzimidazolium salts having different N1,N2-substituents, where one of the N-substituent is an aromatic or a benzylic group and the other substituent an alkyl imidazol-3-ium chloride (2-Cl), 1,3-diphenyl-1H-benzo[d]imidazol-3-ium chloride (3-Cl) [H-benzo[d]imidazol-3-ium chloride (4-Cl) ,,2 by emp 5 imidazol-3-ium chloride (1-Cl). In a 125 mL two neck round-bottomed flask was weighed N1,N2-bis-1,2-benzenediamine  and triethyl orthoformate (60 mL) was added and a fractional distillation apparatus equipped with a Vigreux column and a thermometer on the head of the latter was connected with the central neck of the flask. The green solution was stirred at 145 °C until the color switched to orange (15 min) and then a small flux of nitrogen was passed through the solution until 30 mL of a mixture of EtOH and HC(OEt)3 distilled out (1 hour). Then the temperature was lowered to 90 °C and trimethylsilyl chloride  was added at once. A white-grey solid started to form after 10 minutes. Then the solution was cooled down to room temperature and the precipitate was filtered off and washed with Et2O (3 × 15 mL) then dried to afford in 455 mg of 1-Cl as an off-white powder . Yield 67%. 1H NMR  δ 10.09 , 7.84 , 7.56 , 7.30 , 2.45 , 2.11 ; 13C{1H} δ 143.55 (N-C1-N), 136.54 (N-C2=C7-N), 132.94 (C4=C5), 131.27 , 130.24 , 129.34 , 114.93 , 21.34 (p-CH3), 17.45 (o-CH3); HRMS–ESI (MeOH): calcd for C25H27N2+, 355.21688; found, 355.21644.Synthesis of 1,3-bis-1H-benzo[d]imidazol-3-ium chloride (2-Cl). In a 50 mL two neck round-bottomed flask was weighed N1,N2-bis-1,2-benzenediamine  and triethyl orthoformate (20 mL) was added and a fractional distillation apparatus equipped with a Vigreux column and a thermometer on the head of the latter was connected with the central neck of the flask. The colourless suspension was stirred at 145 °C then after 20 minutes the colour switched to green. A small flux of nitrogen was passed through the solution until 15 mL of a mixture of EtOH and HC(OEt)3 distilled out (40 min). Then fresh triethyl orthoformate (3 mL) was added to the solution, followed by the trimethylsilyl chloride  added at once. The solution, that switched from dark green to dark red, was stirred at 50 °C for 3 hours, then the solvent was removed and the red solid was triturated with Et2O (20 mL) and the precipitate was filtered off, washed with further Et2O (3 × 10 mL), then triturated with acetone (3 mL) and finally dried to afford 31 mg of 2-Cl as a grey powder . Yield 28%. 1H NMR  δ 12.95 , 7.74 , 7.52 , 7.39 , 2.29 , 1.33 , 1.17 ; 13C NMR  δ 146.80 (N-C1-N), 146.02 (N-C8), 132.21 (N-C2=C7-N), 128.5 , 127.45 , 124.97 , 113.42 (C4=C5), 29.42 , 24.73 , 23.10 ; HRMS–ESI (MeOH): calcd for C31H39N2+, 439.31078; found, 439.31042.Synthesis of 1,3-diphenylbenzimidazolium chloride (3-Cl). In a 50 mL two neck round-bottomed flask was weighed N,N’-diphenylbenzene-1,2-diamine  and triethyl orthoformate (15 mL) was added and a fractional distillation apparatus equipped with a Vigreux column and a thermometer on the head of it, was connected with the central neck of the flask. The light blue suspension was stirred at 145 °C until the suspension became a light green solution (20 min), then a small flux of nitrogen was passed through the solution until 10 mL of a mixture of EtOH and HC(OEt)3 distilled out (ca. 45 min). Then the trimethylsilyl chloride  was added at once and a light blue precipitate formed immediately. The solution was cooled down to room temperature and the precipitate was filtered off, washed with Et2O (3 × 10 mL) and then dried to afford 48 mg of 3-Cl as a slightly green powder . Yield 83%. 1H NMR  δ 11.51 , 8.19 , 7.79 , 7.68 , 7.61 ; 13C{1H} δ 139.90 (N-C1-N), 130.68 (N-C2=C7-N), 129.39 (C8), 128.80 (C4=C5), 128.53 (C3), 126.08 (C10), 123.32 (C11), 111.96 (C9); HRMS–ESI (MeOH/H2O 1:1): calcd for C19H15N2, 271.12297; found, 271.12281.Synthesis of 1,3-di(pyridin-2-yl)-1H-benzo[d]imidazol-3-ium chloride (4-Cl). In a 125 mL two neck round-bottomed flask was weighed N1,N2-di(pyridin-2-yl)benzene-1,2-diamine  and triethyl orthoformate (30 mL) was added and a fractional distillation apparatus equipped with a Vigreux column and a thermometer on the head of the latter was connected with the central neck of the flask. The pink suspension was stirred at 145 °C until the suspension became a red solution (10 min), then a small flux of nitrogen was passed through the solution until 20 mL of a mixture of EtOH and HC(OEt)3 distilled out (50 min). Then trimethylsilyl chloride  was added at once and the solution switched from deep red to blue and a blue precipitate formed immediately. The solution was cooled down to room temperature and the precipitate was filtered off, washed with Et2O (3 × 10 mL) and then dried to afford 344 mg of 4-Cl as a blue powder . Yield 98%. 1H NMR  δ 10.07 , 8.84 , 8.48 , 8.28 , 8.03 , 7.89 , 7.77 ; 13C{1H} δ 150.0 , 147.52 , 140.73 , 130.96 (N-C2=C7-N), 128.52 (C4=C5), 125.89 , 117.81 , 116.10 ; HRMS–ESI (MeOH/H2O 1:1): calcd for C17H13N4, 273.11347; found, 273.11308.Synthesis of N1,N2-diphenylbenzene-1,2-diamine (5). In a glove-box a 250 mL Schlenk flask was charged with Pd(dba)2  and P(t-Bu)3 . Subsequently was added toluene (10 mL) and the solution was stirred for 10 min at room temperature. Then 1,2-dichlorobenzene  was added followed by aniline addition  and the t-BuONa reagent . An additional amount of toluene (40 mL) was used to rinse the walls of the Schlenk vessel and the solution was then heated at 92 °C. The reaction was monitored by GC–MS and after completion (4 h) to the solution were added AcOEt (100 mL) and water (100 mL). The organic layer was washed with brine and afterwards dried over MgSO4 and filtered. After the evaporation of the solvent under reduced pressure we obtained 1547 mg of a deep blue solid . The product has a sky-blue color when in solution, which is due to a small amount of impurities. The 1H NMR analysis confirmed that 5 was pure enough to be used for the next synthetic step. Yield 89.1%; GC–MS (EI+): 11.074 min [260.3–261.2]; (98%). 1H NMR  δ 7.28 , 6.97 , 5.65 ; 13C{1H} NMR  δ 144.8 , 135.0 , 129.3 , 122.35 , 120.3 , 119.4 , 116.56 (C8).Synthesis of N1,N2-bisbenzene-1,2-diamine (6). Inside a glove box a 250 mL Schlenk flask was charged with Pd(dba)2 , the P(t-Bu)3  and subsequently toluene (10 mL). The solution was stirred for 10 minutes at room temperature. Then 1,2-dichlorobenzene  was added followed by 2,4,6-trimethylaniline  and t-BuONa  and an additional amount of toluene (40 mL) was used to rinse the walls of the Schlenk tube. The solution was then heated at 115 °C for 15 hours and the solid material that precipitated out of the solution was isolated by suction filtration. The so obtained cake was washed with water and ether and then dried to afford 941 mg of 6 as a grayish powder . Yield 82%. The 1H NMR analysis confirmed that 6 was pure enough to be used for the next step without further purification. MS–EI+: 12.485 min [344.5–345.3]; 1H NMR  δ 7.025 , 6.72 , 6.37 , 5.169 , 2.385 , 2.26 ; 13C{1H} δ 136.99 , 135.31 , 134.07 , 133.45 , 129.36 , 120.04 , 114.31 , 20.90 (p-CH3), 18.18 (o-CH3).Synthesis of N1,N2-bisbenzene-1,2-diamine (7). Inside a glove box a 250 mL Schlenk flask was charged with Pd(dba)2 , P(t-Bu)3 , then toluene (20 mL) was added, and the solution was stirred for 5 minutes at room temperature. Addition of 2,6-diisopropylaniline , t-BuONa  and 1,2-dibromobenzene , was followed by additional toluene (80 mL). The solution was then heated at 115 °C for 15 hours until a white solid separated from the solution and depositing on the walls of the Schlenk tube. The volume of toluene was reduced to one half and the suspension was filtered through a glass-frit and washed with water (50 mL) and with a mixture of water–methanol , then the solid was dried under vacuum to afford 8.45 g of 7 as a grey product . The 1H NMR analysis established that the product obtained was pure enough to be used for the next step. Yield 58%. MS–EI+: 9.97 min [430–431]; 1H NMR  δ 7.31 , 6.66 , 6.29 , 5.30 , 3.24 , 1.24 , 1.18 ; 13C{1H}  δ 145.77 (HN-C1=C2-NH), 137.25 (NH-C7), 137.12 , 126.49 (C10), 124.14 , 120.20 , 114.69 (C6), 28.61 , 24.848 , 23.29 .File 15.CIF file of compound File 2NMR and HRMS–ESI analyses.


Penicillium purpurogenum G59 resulted in activation of silent biosynthetic pathways for the secondary metabolite production. Upon treatment of G59 spores with neomycin and dimethyl sulfoxide (DMSO), a total of 56 mutants were obtained by single colony isolation. The acquired resistance of mutants to neomycin was testified by the resistance test. In contrast to the G59 strain, the EtOAc extracts of 28 mutants inhibited the human cancer K562 cells, indicating that the 28 mutants have acquired the capability to produce bioactive metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses further indicated that diverse secondary metabolites have been newly produced in the bioactive mutant extracts. Followed isolation and characterization demonstrated that five bioactive secondary metabolites, curvularin (1), citrinin (2), penicitrinone A (3), erythro-23-O-methylneocyclocitrinol (4) and 22E-7α-methoxy-5α,6α-epoxyergosta-8(14),22-dien-3β-ol (5), were newly produced by a mutant, 4-30, compared to the G59 strain. All 1–5 were also not yet found in the secondary metabolites of other wild type P. purpurogenum strains. Compounds 1–5 inhibited human cancer K562, HL-60, HeLa and BGC-823 cells to varying extents. Both present bioassays and chemical investigations demonstrated that the introduction of neomycin-resistance into the marine-derived fungal G59 strain could activate silent secondary metabolite production. The present work not only extended the previous DMSO-mediated method for introducing drug-resistance in fungi both in DMSO concentrations and antibiotics, but also additionally exemplified effectiveness of this method for activating silent fungal secondary metabolites. This method could be applied to other fungal isolates to elicit their metabolic potentials to investigate secondary metabolites from silent biosynthetic pathways.Introduction of neomycin-resistance into a marine-derived, wild-type  Fungi from marine habitats have come to be a rich source of new compounds with biological and pharmaceutical properties. A number of structurally novel and bioactive compounds, including a lot of drug leads and other health care ingredients ,2,3,4, hPenicillium purpurogenum G59, a marine-derived fungal strain initially isolated by our group + in ESI-MS, was identified as curvularin + in ESI-MS, and compound 3,c 0.2, MeOH), m/z 381 [M + H]+ in ESI-MS, were identified as citrinin + in ESI-MS, and compound 5,c 0.4, CHCl3), m/z 465 [M + Na]+ in ESI-MS, were identified as erythro-23-O-methyl neocyclocitrinol A/B ring system, and a plausible biosynthetic route has been proposed for the C25 steroids to explain the origin of their bicyclic system and side chain +, 907 [2M + Na]+. 1H NMR  δ: 5.26 , 5.22 , 4.20 , 3.75 , 3.43 , 3.25 , 2.53–2.41 , 2.40–2.31 , 2.29–2.18 , 2.14 , 2.16–2.07 , 2.00–1.82 , 1.77–1.64 , 1.60–1.16 , 1.04 , 0.95 , 0.91 , 0.89 , 0.87 , 0.85 . 13C NMR  δ: 154.5 (C-14), 136.8 (C-22), 133.4 (C-23), 124.1 (C-8), 74.3 (C-7), 69.3 (C-3), 67.0 (C-5), 59.3 (C-6), 58.2 (C-17), 55.1 (CH3O-7), 44.4 , 41.5 (C-9), 40.7 (C-4), 40.4 (C-20), 37.9 (C-12), 37.1 (C-10), 34.4 (C-25), 33.4 (C-1), 31.9 (C-2), 28.4 (C-16), 25.8 (C-15), 21.8 (C-21), 20.5 (C-26), 20.2 (C-27), 20.1 (C-11), 18.6 (C-18), 18.2 (C-28), 16.9 (C-19). The1H NMR data of 5 in CDCl3 in the 221–5 in the EtOAc extracts of mutant 4-30 and strain G59 was carried out by HPLC-PDAD-UV and HPLC-ESI-MS analyses. A sample solution of crude 1–5 in MeOH at 10 mg/mL was used as reference standard in the HPLC-PDAD-UV analysis. HPLC-PDAD-UV and HPLC-ESI-MS were performed at the conditions given in 1–5 were elucidated as peaks with retention times (tR), 40.88 min for 1, 19.02 min for 2, 54.30 min for 3, 51.65 min for 4, and 69.53 min for 5. Compounds 1–5 were all detected in the mutant 4-30 extract but not in the G59 extract both by retention times and UV spectra  monitoring with both the extracted ion chromatograms and related MS spectra, 1–5 were all detected in the mutant 4-30 extract, but none of these metabolites were detected in the G59 extract (Examination of  spectra . In the  extract .P. purpurogenum G59 to neomycin could activate silent secondary metabolite production. The acquired resistance could be introduced by treatment of the G59 spores with neomycin and DMSO at 4 °C. The present work not only extended the previous DMSO-mediated method for introducing drug-resistance in fungi both in DMSO concentrations and antibiotics, but also additionally exemplified the effectiveness of this method for activating silent fungal secondary metabolites. This method could be applied to other fungal isolates to elicit their metabolic potentials to investigate bioactive secondary metabolites from silent biosynthetic pathways.The introduction of acquired resistance of


Compound 1 possesses an unusual oxazo ring system at C-10 while compound 2 displays an unprecedented C–C bond cleavage between C-10 and C-11 with an N-ethylphenyl group at C-10. Biogenetic pathways of 1 and 2 are proposed. The absolute configuration of 1 was confirmed by Mosher’s method and molecular mechanics calculations (MM2). The cytotoxicities of compounds 1–10 were evaluated against a small panel of human cancer cell lines.Two new nitrogen-containing verticillene diterpenoids, cespilamides A and B ( Bellonella albiflora and eleutherobins obtained from Eleutherobia aurea showed significant cytotoxicities , a tetrasubstituted olefinic moity , and an exomethylene group . The DEPT NMR spectrum indicated an oxygenated quarternary carbon (δC 102.8), an oxygenated methine carbon (δC 68.5 d), eight methylene carbons , and three methyl groups . The 1H–1H COSY experiment (C 33.0), C-4 (δC 146.2), and C-5 (δC 43.8). Furthermore, the HMBC correlations of CH3-16, CH3-17/C-15 (δC 37.9), C-1 (δC 43.5), C-11 (δC 166.2) and H-13/C-12 (δC 133.5), C-20 (δC 177.1), C-11 indicated that compound 1 possesses a 2′,2′-dimethylcyclohexene moiety. The HMBC correlations of H-9/C-7 (δC 134.8), C-8 (δC 132.3), C-10 (δC 102.8), C-11 and CH3-19/C-7, C-8, C-9 (δC 46.6) were used to establish the planar structure of compound 1, except for the C1′-C2′ moiety. Comparison of the 1H- and 13C-NMR data of 1 with those of cespitulactam D revealed that they have similar verticillene skeletons , a 1,1-disubstituted olefin (δC 145.7) with an exomethylene group , an oxygenated methine carbon (δC 65.8), and a phenyl group . Thus, eight degrees of unsaturation were counted, leaving one further ring to be elucidated. The 1H–1H COSY /H-1′/H-2′ and H-4′/H-5′/H-6′/H-7′/H-8′ revealed the sequences of three fragments including H-5 to H-7, H-3 to H-12 and a benzylethyl amine side chain. The HMBC correlations  and indicated eleven degrees of unsaturation. The IR absorption of 1676 cm−1 suggested the presence of a conjugated amide group. The 1H, 13C (C 170.0), a trisubstituted olefin , an exomethylene group , a tetrasubstituted olefin , a phenyl group , an aliphatic CH group , and four aliphatic CH2 group . The above findings accounted for five of the eight degrees of unsaturation, indicating that compound 3 is a tricyclic sesquiterpene with a phenyl group. 1H–1H COSY spectrum of 3 showed four sets of correlations, H-1/H-2/H-3, H-5/H-6, H-1′/H-2′, and H-4′/ H-5′/ H-6′/ H-7′/ H-8′. The HMBC correlations  by HRESIMS data . The IR spectrum revealed the presence of hydroxy (3421 cm−1) and α,β-unsaturated γ-lactam (1695 cm−1) moieties. The 1H and 13C NMR spectra , 6.74, d, J = 8.4 Hz (2H); δC 154.6 (s), 130.8 (s), 130.0 (d), 115.4 (d), 41.0 (t), 34.3 (t)] on the nitrogen atom, rather than a phenylethyl group as found in compound 3. Interpretation of 1H–1H COSY and HMBC spectra of compound 4 also indicated the presence of a hydroxy group at C-6′. The relative configuration of compound 4 was determined by comparison with the NMR and the optical rotation of compound 3.The molecular formula of  spectra  of compo5 was shown to be C25H28ON2 (Δ = 13), as deduced from HRESIMS at m/z 395.2099 . Spectroscopic data of compound 5 were found to be similar to those of 3 and 4 except for the evidence of an ethylindole moiety. The LRMS of compound 5 exhibited a peak at m/z 229 [M + H − C10H10N]+, also consistent with the presence of an ethylindole group. In the 1H and 13C NMR spectra ; δC 39.9 (t), 24.8 (t), 113.3 (s), 121.9 (d), 124.7 (s), 111.1 (d), 121.9 (d), 119.3 (d), 118.6 (d), and 127.6 (s)] were also observed. The 3-ethylindole group on the tertiary nitrogen in 5 was revealed by detailed analysis of 2D NMR spectra (C 170.2), C-8 (δC 137.2) as well as correlations of H-2′/C-3′, C-4′ and C-11′ indicated that the phenylethyl side chain at the nitrogen in compound 3 was replaced by the 3-ethylindole group in compound 5. Assignment of the 1H and 13C-NMR spectroscopic data of 5 were accomplished by application of 1H–1H COSY, HMQC, and HMBC correlations. The relative configuration of compound 5 was assigned the same as those of compounds 3 and 4.The molecular formula of compound  spectra , signals spectra . The HMB6) was isolated as a colorless, amorphous solid. The molecular formula, C21H30O3, was established by the HRESIMS at m/z 353.2096 [M + Na]+ . The IR bands at 1720 and 1706 cm−1 were attributed to an ester and a carbonyl group, which were confirmed by the presence of the acetate (δC 170.1) and ketocarbonyl (δC 202.1). The 13C-NMR , six aliphatic methylene carbons , a methine carbon (δC 43.1), an oxygenated methine carbon (δC 72.3), an aliphatic quaternary carbon (δC 35.4), two olefinic methine carbons (δC 129.0 and 135.4), an olefinic methylene carbon (δC 113.5), three olefinic quaternary carbons , and two additional carbonyl signals. The 1H–1H COSY spectrum showed the connectivities of H-7/H-6/H-5 and H-3/H-2/H-1/H-14/H-13/H-12. Resonances at δC 133.3 (C-8) and 129.0 (C-7) were correlated in the HMBC spectrum with proton signals at δH 5.15 , and with the vinylic methyl protons at δH 1.76 (Me-19), and suggested that compound 6 contains an E-trisubstituted double bond bearing a methyl group  and a 1,1-disubstituted olefin (δC 144.7) with an exomethylene group  were also implied by interpretation of the HMBC data of compound 6. Moreover, HMBC correlations of δH 5.38  with δC 170.1 indicated that C-6 (δC 72.3) is attached to an acetoxy group (δC 21.3). HMBC correlations of H-12/C-11, C-10, C-15, H-9/C-10, C-11, Me-16/C-11, C-15, C-1 and Me-17/C-11, C-15, C-1, H-18/C-3, C-5 established the final structure of 6. The relative configuration of 6 was determined by NOESY analysis and comparison of the coupling constants of 6 with the data reported +. Its IR bands showed the presence of a hydroxy (3397 cm−1) and conjugated carbonyl (1697 cm−1) groups. The 1H and 13C-NMR spectroscopic (C 214.5 and 208.1), a trisubstituted olefin , and an exocyclic double bond , a trisubstituted double bond +) in the HRESIMS. There were very few differences between the 1H-NMR spectroscopic data  and C-20 . Furthermore, the COSY and HMBC correlations were closely comparable (Supporting Information). The NOESY correlations of H-20/Me-17 in 10 confirmed the β-orientation of H-20. The only difference between 9 and 10 is the configuration of the ethoxy group at C-20. The optical rotations of 10 [9 [2Cl2)] supported the conclusion to be made that compound 10 is the 20-epimer of cespitaenin D.Cespitaenin E , medulloblastoma (Daoy), and cervical epitheloid carcinoma (Hela) cancer cells with IC50 of 17.5, 22.3, and 24.7 μM, respectively. Compound 6 showed significant cytotoxicity against human breast adenocarcinoma (MCF-7) cancer cells with the IC50 at 21.2 μM.Four human cancer cell lines were chosen to test the nds 1–10 . Compoun1H and 13C-NMR spectra as well as 2D NMR spectra  were recorded in CDCl3 (or CD3OD) using Bruker DRX NMR spectrometers operating at 300 or 500 MHz for 1H and 75 or 125 MHz for 13C using the CDCl3 solvent peak as internal standard (δH 7.26 for 1H and δC 77.0 for 13C). Low-resolution ESIMS and HRESIMS were run on a JEOL JMS-HX 110 mass spectrometer. Silica gel 60  was used for column chromatography (CC). Precoated silica gel plate  was used for preparative TLC. Sephadex LH-20  was used for separation. LiChrospher Si 60  and LiChrospher 100 RP-18e  were used for NP-HPLC and RP-HPLC , respectively.Optical rotations were obtained on a JASCO DIP-1000 polarimeter. IR spectra were recorded using a Horiba FT-720 spectrophotometer. The Cespitularia taeniata was collected in Green Island, Taiwan, in March 2004. This soft coral was identified by one of the authors (Y.-C.S.). A voucher specimen (GSC-1) has been deposited in the School of Pharmacy, National Taiwan University, Taipei, Taiwan.C. taeniata  were extracted with EtOAc and CH2Cl2  at room temperature and concentrated under reduced pressure to yield a crude extract. The crude extract was partitioned between H2O and EtOAc to yield an EtOAc-soluble fraction (100 g), which was chromatographed on a Si gel column (1 kg) and initially eluted with n-hexane , n-hexane/EtOAc , and finally MeOH  to give 12 fractions. Fractions six (3.1 g) and eight (1.7 g) were further separated on a Sephadex LH-20 column using CH2Cl2-MeOH (4:1) to furnish nine and five fractions , respectively. Separation of fraction 6-5 (390 mg) was performed by a Si gel column (1.2 g) using a solvent mixture of n-hexane-CH2Cl2-MeOH (100:100:1~5:5:1) to yield six fractions (6-2-1~6-2-6). Fraction 6-5-3 (34 mg) was further purified with a NP-HPLC column  to give cespitaenin A . Fraction 6-5-4 (121 mg) and fraction 6-5-5 (68 mg) were separated with a NP-HPLC column  and then a RP-HPLC column was used  to yield cespitaenin C , cespitaenin D , cespilamide C  and cespitaenin E . Fraction 6-6 (310 mg) was purified with a NP-HPLC column  and with preparative TLC  to give cespilamide D . Fraction 6-8 (16 mg) was further purified with a RP-HPLC column  to yield cespilamide E . Fraction 8-4 (779 mg) and 8-5 (68 mg) were further separated with a NP-HPLC column  and with a RP-HPLC column  to yield cespilamide A , cespitaenin B  and cespilamide B .The whole animals of 1): colorless, amorphous solid;c 0.2, CH2Cl2); IR (neat) νmax 3421, 2936, 1695 cm−1; 1H-NMR  and 13C-NMR  data, see m/z 358.2380 .Cespilamide A (2): colorless, amorphous solid; c 0.2, CH2Cl2); IR (neat) νmax 3371, 2929, 2360, 1701, 1647 cm−1; 1H-NMR  and 13C-NMR  data, see m/z 448.2825 .Cespilamide B (3): colorless, amorphous solid; c 0.2, CH2Cl2); IR (neat) νmax 2926, 1676 cm−1; 1H-NMR  and 13C-NMR  data, see m/z 356.1992 .Cespilamide C (4): colorless, amorphous solid; c 0.2, CH2Cl2); IR (neat) νmax 3312, 2927, 1649 cm−1; 1H-NMR  and 13C-NMR  data, see m/z 372.1937 .Cespilamide D (5): colorless, amorphous solid; c 0.2, CH2Cl2); IR (neat) νmax 2929, 1659, 1340 cm−1; 1H-NMR  and 13C-NMR  data, see m/z 395.2099 .Cespilamide E (6): colorless, amorphous solid; c 0.2, CH2Cl2); IR (neat) νmax 1720, 1706 cm−1; 1H-NMR  and 13C-NMR  data, see m/z 353.2096 .Cespitaenin A (7): colorless, amorphous solid; c 0.2, CH2Cl2); IR (neat) νmax 3397, 2359, 2339, 1697, 1276 cm−1; 1H-NMR  and 13C-NMR  data, see m/z 359.1837 .Cespitaenin B (8): colorless, amorphous solid; c 0.2, CH2Cl2); IR (neat) νmax 3385, 2924, 1738 cm−1; 1H-NMR  and 13C-NMR  data, see m/z 361.2378 .Cespitaenin C (9): colorless, amorphous solid; c 0.2, CH2Cl2); IR (neat) νmax 3444, 2986, 2950, 1731 cm−1; 1H-NMR  and 13C-NMR  data, see m/z 401.2306 .Cespitaenin D (10): colorless, amorphous solid; c 0.2, CH2Cl2); IR (neat) νmax 3390, 2930, 1757 cm−1; 1H-NMR  and 13C-NMR  data, see m/z 401.2305 .Cespitaenin E (R-(−)- or S-(+)-MPTA chloride (one drop) was added to a solution of 1 (3 mg in 2 mL pyridine) and the solution was allowed to stand at room temperature for 12 h. After purification using preparative LC, the resultant ester  was analyzed by 1H NMR spectroscopic measurement, and ∆ = δS − δR was calculated. 1a: Compound 1H-NMR  δH 5.578 , 5.542 , 1.199, 1.466 , 4.788 , 4.770 , 1.597 , 4.12 , 3.92 , 3.89 , 3.25 ; 1b:Compound 1H-NMR  δH 5.525 , 5.402 , 1.189, 1.444 , 4.871 , 4.835 , 1.586 , 4.12 , 3.92 , 3.87 , 3.25 .50 value was defined by a comparison with the untreated cells as the concentration of test sample resulting in 50% reduction of absorbance. Mitomycin C was used as the positive control.Cytotoxicity was tested against the MCF-7 (breast carcinoma), Daoy (medulloblastoma), DLD-1 (colon adenocarcinoma), and Hela  human tumor cell lines. The assay procedure using MTT [3--2,5-diphenyltetrazolium bromide was carried out as previously described. The cellCespitularia taeniata.This paper describes the first isolation of five novel nitrogen-containing diterpenoids and sesquiterpenoids, and five bicyclic verticillenes and nor-verticillenes from Taiwanese soft coral