General methods
Solvents and reagents were purchased from Merck-Aldrich. UV and sulfuric–vanillin were used as revealing agents. 1H-NMR and 13C-NMR spectra were recorded with a JEOL Eclipse-300 spectrometer and a Bruker Avance III-400 model spectrometer by using CDCl3 or DMSO-d6 as solvents. NMR coupling constants are reported in Hertz (Hz), while chemical shifts (δ) are reported in ppm relative to the solvent signal. Signal splitting patterns are described as: singlet (s), doublet (d), triplet (t), quartet (q), quintet (quint), septet (sept), broad signal (br s), doublet of doublets (dd) or multiplet (m). Mass spectra were recorded with a JMST100LC AccuTOF LC with an ionSense DART SVP100 controller ionization source or a Jeol The MStation JMS-700.
Synthesis Of Butenones 9a-r
In a round-bottom flask, the corresponding benzaldehyde (1.98 mmol) was suspended in a mixture of Acetone-Water (1:1.25, 9 mL). Then, a solution of aqueous NaOH (0.05% w/v, 1.4 mL) was slowly added during 20 min. The mixture was heated to 50°C for 2 h. After completion of the reaction, the excess of acetone was removed under reduced pressure and the residue was extracted with AcOEt (2 x 10 mL). The organic layer was separated, dried with Na2SO4 and purified through silica gel column chromatography in a suitable Hex-AcOEt mixture to afford the corresponding 3-but-2-enones (9a-r) in moderate to excellent yields.
( E )-4-phenylbut-3-en-2-one (9a). Purified by column chromatography (Hex-AcOEt 9:1). White solid in 95% yield. Spectra is in accordance with the previously reported.
( E )-4-(4-methylphenyl)but-3-en-2-one (9b). Purified by column chromatography (Hex-AcOEt 9:1). Yellow solid in 86% yield. 1H-NMR (CDCl3/TMS, 400 MHz) δ: 7.49 (d, J = 16 Hz, 1H), 7.45–7.43 (AA´BB´, 2H), 7.21–7.19 (AA´BB´, 2H), 6.68 (d, J = 16 Hz, 1H), 2.374 (s, 3H), 2.367 (s, 3H); 13C-NMR (CDCl3/TMS, 100 MHz) δ: 198.6, 143.7, 141.2, 131.8, 129.8, 128.4, 126.4, 27.6, 21.6; MS (DART+) m/z: [M + H]+; 161; HRMS m/z calcd for C11H13O [M + H]+, 161.09664; found 161.09587.
( E )-4-(4-isopropylphenyl)but-3-en-2-one (9c). Purified by column chromatography (Hex-AcOEt 9:1). Yellow oil in 91% yield. 1H-NMR (400 MHz, CDCl3) δ: 7.53–7.45 (m, 3H), 7.28–7.23 (m, 2H), 6.69 (d, J = 16.3 Hz, 1H), 2.99–2.87 (m, 1H), 2.37 (s, 3H), 1.26 (d, J = 7.0 Hz, 6H); 13C-NMR (100 MHz, CDCl3) δ: 198.6, 152.0, 143.7, 132.2, 128.5, 127.2, 126.5, 34.2, 27.5, 23.9; MS (DART+) m/z: [M + H]+; 189; HRMS m/z calcd for C13H17O [M + H]+, 189.12794; found 189.12702.
( E )-4-(4-( tert -butyl)phenyl)but-3-en-2-one (9d). Purified by column chromatography (Hex-AcOEt 9:1). Yellow oil in 91 yield. 1H-NMR (400 MHz, CDCl3) δ: 7.56–7.42 (m, 5H), 6.72 (d, J = 16.3 Hz, 1H), 2.40 (s, 3H), 1.35 (s, 9H); 13C-NMR (100 MHz, CDCl3) δ: 198.6, 154.2, 143.5, 131.7, 128.2, 126.5, 126.0, 34.9, 31.2, 27.5; MS (DART+) m/z: [M + H]+; 203; HRMS m/z calcd for C14H19O [M + H]+, 203.14359; found 203.14269.
( E )-4-(4-methoxyphenyl)but-3-en-2-one (9e). Purified by column chromatography (Hex-AcOEt 9:1). Yellowish solid in 91% yield. 1H-NMR (CDCl3/TMS, 400 MHz) δ: 7.52–7.48 (AA´BB´, 2H), 7.47 (d, J = 16 Hz, 1H), 6.93–6.90 (AA´BB´, 2H), 6.60 (d, J = 16 Hz, 1H), 3.84 (s, 3H), 2.36 (s, 3H); 13C-NMR (CDCl3/TMS, 100 MHz) δ: 198.5, 161.7, 143.4, 130.1, 127.2, 125.2, 114.6, 55.5, 27.5; MS (DART+) m/z: [M + H]+; 177; HRMS m/z calcd for C11H12O2 [M + H]+, 177.09155; found 177.09121.
( E )-4-(4-(methylthio)phenyl)but-3-en-2-one (9f). Purified by column chromatography (Hex-AcOEt 9:1). Yellow solid in 89% yield. 1H-NMR (CDCl3/TMS, 400 MHz) δ: 7.48–7.44 (m, 3H), 7.24–7.21 (AA´BB´), 6.67 (d, J = 16 Hz, 1H), 2.50 (s, 3H), 2.36 (s, 3H); 13C-NMR (CDCl3/TMS, 100 MHz) δ: 198.4, 143.0, 142.5, 131.0, 128.7, 126.3, 126.1, 27.6, 15.2; MS (DART+) m/z: [M + H]+;193 HRMS m/z calcd for C11H13OS [M + H]+, 193.06871; found 193.06864.
( E )-4-([1,1'-biphenyl]-4-yl)but-3-en-2-one (9g). Purified by column chromatography (Hex-AcOEt 95:5). White solid in 94% yield. 1H-NMR (400 MHz, CDCl3) δ: 7.66–7.60 (m, 6H), 7.56 (d, J = 16.2 Hz, 1H), 7.49–7.44 (m, 2H), 7.41–7.36 (m, 1H), 6.77 (d, J = 16.2 Hz, 1H), 2.41 (s, 3H). 13C-NMR (100 MHz, CDCl3) δ: 198.4, 143.3, 143.0, 140.1, 133.4, 128.9, 128.8, 127.9, 127.6, 127.1, 27.6; MS (DART+) m/z: [M + H]+; 223; HRMS m/z calcd for C16H15O [M + H]+, 223.11229; found 223.11250.
( E )-4-(4-chlorophenyl)but-3-en-2-one (9h). Purified by column chromatography (Hex-AcOEt :1). Off-white solid in 87% yield. 1H-NMR (CDCl3/TMS, 400 MHz) δ: 7.46 (d, J = 16 Hz, 1H), 7.48–7.46 (AA´BB´, 2H), 7.38–7.35 (AA´BB´, 2H), 6.68 (d, J = 16 Hz, 1H), 2.38 (s, 3H); 13C-NMR (CDCl3/TMS, 100 MHz) δ: 198.2, 142.0, 136.6, 133.1, 129.5, 129.4, 127.6, 27.8; MS (DART+) m/z: [M + H]+; 181; HRMS m/z calcd for C10H10ClO [M + H]+, 181.04202; found 181.04282.
( E )-4-(4-fluorophenyl)but-3-en-2-one (9i). Purified by column chromatography (Hex-AcOEt 95:5). Yellow oil in 75% yield. 1H-NMR (400 MHz, CDCl3) δ: 7.56–7.52 (m, 2H), 7.48 (d, J = 16.2 Hz, 1H), 7.13–7.05 (m, 2H), 6.65 (d, J = 16.3 Hz, 1H), 2.38 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ: 198.1, 165.3, 162.8, 142.0, 130.2, 130.1, 126.9, 116.3, 116.0, 27.6; 19F-NMR (376 MHz, CDCl3) δ: -109.2; MS (DART+) m/z: [M + H]+; 165; HRMS m/z calcd for C10H10FO [M + H]+, 165.07157; found 165.07120.
( E )-4-(4-(trifluoromethyl)phenyl)but-3-en-2-one (9j). Purified by column chromatography (Hex-AcOEt 9:1). White solidl in 78% yield. 1H-NMR (400 MHz, CDCl3) δ: 7.65 (s, 4H), 7.52 (d, J = 16.3 Hz, 1H), 6.77 (d, J = 16.3 Hz, 1H), 2.40 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ: 198.0, 141.4, 138.0, 132.6, 132.2, 131.9, 131.6, 129.2, 128.5, 126.1, 126.1, 126.0, 126.0, 28.0; 19F-NMR (376 MHz, CDCl3) δ: -62.91; MS (DART+) m/z: [M + H]+; 215; HRMS m/z calcd for C11H10F3O [M + H]+, 215.06837; found 215.06858.
( E )-4-(3-methoxyphenyl)but-3-en-2-one (9k). Purified by column chromatography (Hex-AcOEt 9:1). Yellow oil in 40% yield. 1H-NMR (400 MHz, CDCl3) δ: 7.47 (d, J = 16.3 Hz, 1H), 7.31 (t, J = 7.9 Hz, 1H), 7.15–7.11 (m, 1H), 7.06 (dd, J = 2.6, 1.6 Hz, 1H), 6.94 (ddd, J = 8.3, 2.6, 0.9 Hz, 1H), 6.69 (d, J = 16.3 Hz, 1H), 3.83 (s, 3H), 2.38 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ: 198.4, 160.0, 143.4, 135.8, 130.0, 127.4, 121.0, 116.4, 113.0, 55.3, 27.5; MS (DART+) m/z: [M + H]+; 177; HRMS m/z calcd for C13H13O2 [M + H]+, 177.09155; found 177.09148.
( E )-4-(3-fluorophenyl)but-3-en-2-one(9l). Purified by column chromatography (Hex-AcOEt 9:1). Yellow oil in 72% yield. 1H-NMR (400 MHz, CDCl3) δ: 7.45 (d, J = 16.2 Hz, 1H), 7.36 (td, J = 7.9, 5.6 Hz, 1H), 7.30 (dt, J = 7.7, 1.4 Hz, 1H), 7.22 (ddd, J = 9.7, 2.5, 1.6 Hz, 1H), 7.08 (tdd, J = 8.2, 2.6, 1.2 Hz, 1H), 6.69 (d, J = 16.3 Hz, 1H), 2.37 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ: 198.0, 164.3, 161.8, 141.9, 141.8, 130.6, 130.5, 128.2, 124.3, 124.3, 117.5, 117.4, 117.2, 114.5, 114.3, 27.7; 19F-NMR (376 MHz, CDCl3) δ: -112.4; MS (DART+) m/z: [M + H]+; 165; HRMS m/z calcd for C10H10FO [M + H]+, 165.07157; found 165.07140.
( E )-4-([1,1'-biphenyl]-3-yl)but-3-en-2-one (9m). Purified by column chromatography (Hex-AcOEt 9:1). White oil in 79% yield. 1H-NMR (400 MHz, CDCl3) δ: 7.78 (t, J = 1.8 Hz, 1H), 7.66–7.58 (m, 4H), 7.56 (dt, J = 7.7, 1.5 Hz, 1H), 7.49 (tt, J = 8.7, 2.1 Hz, 3H), 7.44–7.37 (m, 1H), 6.82 (d, J = 16.3 Hz, 1H), 2.43 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ: 198.4, 143.4, 142.1, 140.4, 135.0, 129.4, 129.4, 128.9, 127.8, 127.5, 127.2, 127.1, 127.0, 27.6; MS (DART+) m/z: [M + H]+; 223; HRMS m/z calcd for C16H15O [M + H]+, 223.11229; found 223.11250.
( E )-4-(thiophen-2-yl)but-3-en-2-one (9n). Purified by column chromatography (Hex-AcOEt 95:5). Brown oil in 80% yield. 1H-NMR (CDCl3/TMS, 400 MHz) δ: 7.66 (d, J = 16 Hz, 1H), 7.4 (d, J = 8 Hz, 1H), 7.29 (d, J = 4 Hz, 1H), 7.07(dd, J = 8 and 4 Hz, 1H), 6.53 (d, J = 16 Hz, 1H), 2.33 (s, 3H); 13C-NMR (CDCl3/TMS, 100 MHz) δ: 197.9, 139.9, 135.8, 131.6, 129.0, 128.4, 125.9, 27.8; MS (DART+) m/z: [M + H]+; 153; HRMS m/z calcd for C8H9OS [M + H]+, 153.03741; found 153.03726.
( E )-4-(1-tosyl-1H-indol-2-yl)but-3-en-2-one (9o). Purified by column chromatography (Hex-AcOEt 75:25). Brown solid in 45% yield. 1H-NMR (400 MHz, CDCl3) δ: 8.02 (d, J = 8.1 Hz, 1H), 7.90 (s, 1H), 7.82 (dd, J = 8.1, 3.7 Hz, 3H), 7.66 (d, J = 16.1 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.35 (t, J = 7.4 Hz, 1H), 7.28 (s, 1H), 7.26 (s, 1H), 6.84 (d, J = 15.9 Hz, 1H), 2.41 (s, 3H), 2.37 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ: 198.18, 145.65, 135.67, 134.72, 134.51, 130.17, 128.88, 128.07, 127.23, 127.05, 125.63, 124.19, 120.74, 118.16, 113.88, 77.40, 77.08, 76.76, 27.68, 21.66; MS (DART+) m/z: [M + H]+; 340; HRMS m/z calcd for C19H18NO3S [M + H]+, 340.10074; found 340.09970.
(3 E,5E)-6-phenylhexa-3,5-dien-2-one (9p). Purified by column chromatography (Hex-AcOEt 9:1). Yellow solid in 89% yield. 1H-NMR (CDCl3/TMS, 400 MHz) δ: 7.49–7.46 (m, 2H), 7.39–7.29 (complex, 4H), 6.96 (d, J = 16 Hz, 1H), 6.88 (ddd, J = 16, 10.4 and 0.4 Hz, 1H), 6.26 (d, J = 16 Hz, 1H), 2.32 (s, 3H); 13C-NMR (CDCl3/TMS, 100 MHz) δ: 198.6, 143.6, 141.4, 136.1, 130.6, 129.4, 129.0, 127.4, 126.8, 27.5; MS (DART+) m/z: [M + H]+; 173; HRMS m/z calcd for C11H12OS [M + H]+, 173.09664; found 173.09675.
( E )-4-(3',5'-difluoro-[1,1'-biphenyl]-4-yl)but-3-en-2-one (9r). White solid in 56% yield. 1H-NMR (400 MHz, CDCl3) δ: 7.55–7.47 (m, 4H), 7.45 (d, J = 16.6 Hz, 1H), 7.06–6.99 (m, 2H), 6.73 (dt, J = 8.8, 2.3 Hz, 1H), 6.67 (d, J = 16.3 Hz, 1H), 2.31 (s, 3H); 13C-NMR (75 MHz, CDCl3) δ: 198.3, 165.2, 165.0, 161.9, 161.7, 143.6, 143.4, 143.3, 142.4, 140.8, 134.7, 129.0, 127.7, 127.6, 110.1, 110.0, 109.9, 109.8, 103.5, 103.2, 102.8, 27.7; 19F NMR (375 MHz, CDCl3) δ: -109.23; MS (DART+) m/z: [M + H]+; 259; HRMS m/z calcd for C16H13F2O [M + H]+, 259.09345; found 259.09249.
Synthesis of imidazo[1,2- a ]pyrimidines 4a-r
To a solution of the corresponding butenone 4a–r (0.5 mmol) in EtOH (9.8 mL), was added CuBr2 (084 mmol) in one portion. The mixture was allowed to react at room temperature for 12–24 h. After consumption of the methylketone, the mixture was evaporated and passed through a short silica gel pad eluted with AcOEt. After remotion of the organic solvent, the residue was redissolved in EtOH (4.9 mL) and 2-aminopyrimidine (0.5 mmol) was added. The Hanztsch reaction was allowed to reflux for 6–8 h until the starting material disappeared. The presence of a very polar fluorescent compound (blue fluorescence at 365 nm), which correspond to the desired imidazo[1,2-a]pyrimidine, indicated the completion of the reaction. The ethanol was evaporated under reduced pressure and the residue was redissolved in water. The mixture was basified to pH = 8 with an aqueous solution of NaOH 1.0 M and then extracted with DCM (2 x 15 mL). The bicycles 4a–r were isolated after a flash column chromatography using AcOEt as eluent.
( E )-2-styrylimidazo[1,2- a ]pyrimidine (4a). Brown solid in 59% yield. 1H-NMR (300 MHz, CDCl3) δ: 8.47 (dd, J = 4.2, 2.0 Hz, 1H), 8.34 (dd, J = 6.6, 2.1 Hz, 1H), 7.67 (d, J = 16.1 Hz, 1H), 7.54 (d, J = 1.6 Hz, 1H), 7.51 (s, 2H), 7.38–7.24 (m, 3H), 7.09 (d, J = 16.2 Hz, 1H), 6.77 (dd, J = 6.8, 4.1 Hz, 1H); 13C-NMR (75 MHz, CDCl3) δ: 150.3, 148.8, 145.9, 137.0, 133.0, 132.5, 128.8, 128.1, 126.9, 119.5, 108.7, 108.6; MS (DART+) m/z: [M + H]+; 222; HRMS m/z calcd for C14H12N3 [M + H]+, 222.10312; found 222.10229; IR (ATR cm− 1): 3024, 2923, 2853, 1607, 1499, 1223,
( E )-2-(4-methylstyryl)imidazo[1,2- a ]pyrimidine (4b). White off solid in 52% yield. 1H-NMR (300 MHz, CDCl3) δ: 8.51 (dd, J = 4.2, 2.0 Hz, 1H), 8.35 (dd, J = 6.7, 2.0 Hz, 1H), 7.67 (d, J = 16.0 Hz, 1H), 7.51 (s, 1H), 7.46 (d, J = 7.9 Hz, 2H), 7.17 (d, J = 7.9 Hz, 2H), 7.08 (d, J = 16.1 Hz, 1H), 6.81 (dd, J = 6.7, 4.1 Hz, 1H), 2.36 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ: 150.2, 148.8, 146.3, 138.2, 134.3, 132.8, 132.7, 129.6, 126.9, 118.5, 108.6, 108.3, 21.5; MS (DART+) m/z: [M + H]+; 236; HRMS m/z calcd for C15H14N3 [M + H]+, 236.11877; found 236.11891; IR (ATR cm− 1): 3023, 2917, 2851, 1607, 1490, 1224.
( E )-2-(4-isopropylstyryl)imidazo[1,2- a ]pyrimidine (4c). White solid in 46% yield. 1H-NMR (300 MHz, CDCl3) δ: 8.45 (dd, J = 4.2, 2.0 Hz, 1H), 8.33 (dd, J = 6.7, 2.1 Hz, 1H), 7.65 (d, J = 16.0 Hz, 1H), 7.49 (s, 1H), 7.46 (d, J = 8.2 Hz, 2H), 7.21 (d, J = 8.2 Hz, 2H), 7.05 (d, J = 16.1 Hz, 1H), 6.75 (dd, J = 6.7, 4.1 Hz, 1H), 2.90 (hept, J = 6.9 Hz, 1H), 1.25 (d, J = 6.9 Hz, 6H); 13C-NMR (100 MHz, CDCl3) δ: 150.1, 149.1, 148.8, 146.1, 134.6, 132.9, 132.5, 126.9, 126.9, 108.5, 108.5, 34.0, 24.0; MS (IE+) m/z: [M+]+; 263; HRMS m/z calcd for C17H17N3 [M+]+, 263.1422; found 263.1428. IR (ATR cm− 1): 3081, 3024, 2958, 1610,1512, 1221.
( E )-2-(4-(tert-butyl)styryl)imidazo[1,2- a ]pyrimidine (4d). Brown solid in 44% yield. 1H-NMR (400 MHz, CDCl3) δ: 8.48 (dd, J = 4.2, 2.0 Hz, 1H), 8.34 (dd, J = 6.7, 2.1 Hz, 1H), 7.67 (d, J = 16.1 Hz, 1H), 7.51–7.47 (m, 3H), 7.38 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 16.0 Hz, 1H), 6.78 (dd, J = 6.7, 4.1 Hz, 1H), 1.33 (s, 9H); 13C-NMR (100 MHz, CDCl3) δ 151.4, 150.1, 148.8, 146.1, 134.2, 132.9, 132.4, 126.7, 125.7, 118.7, 108.5, 108.4, 34.8, 31.4; MS (DART+) m/z: [M + H]+; 278; HRMS m/z calcd for C18H20N3 [M + H]+, 278.16572; found 278.16475. IR (ATR cm− 1): 3025, 2956, 2865, 1609, 1489, 1225.
( E )-2-(4-methoxystyryl)imidazo[1,2- a ]pyrimidine (4e). Brown solid in 39% yield. 1H-NMR (300 MHz, CDCl3) δ: 8.51 (dd, J = 4.1, 2.0 Hz, 1H), 8.35 (dd, J = 6.7, 2.0 Hz, 1H), 7.66 (d, J = 16.0 Hz, 1H), 7.53–7.47 (m, 3H), 7.00 (d, J = 16.0 Hz, 1H), 6.94–6.88 (m, 2H), 6.81 (dd, J = 6.7, 4.1 Hz, 1H), 3.83 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ: 159.8, 150.1, 146.5, 132.7, 132.3, 130.1, 129.9, 128.3, 117.4, 114.3, 108.5, 108.1, 55.5; MS (DART+) m/z: [M + H]+; 252; HRMS m/z calcd for C15H14N3O [M + H]+, 252.11369; found 252.11283. IR (ATR cm− 1): 3141, 3034, 2922, 1601, 1509, 1243.
( E )-2-(4-(methylthio)styryl)imidazo[1,2- a] pyrimidine (4f). Brown solid in 18% yield. 1H-NMR (300 MHz, CDCl3) δ: 8.50 (dd, J = 4.1, 2.0 Hz, 1H), 8.34 (dd, J = 6.7, 2.0 Hz, 1H), 7.64 (d, J = 16.0 Hz, 1H), 7.50 (s, 1H), 7.49–7.43 (m, 2H), 7.25–7.20 (m, 2H), 7.07 (d, J = 16.0 Hz, 1H), 6.80 (dd, J = 6.7, 4.1 Hz, 1H), 2.49 (s, 3H); 13C-NMR (75 MHz, CDCl3) δ: 150.3, 148.9, 146.1, 138.6, 134.0, 132.8, 132.0, 127.3, 126.7, 118.8, 108.6, 108.5, 15.8; MS (DART+) m/z: [M + H]+; 268; HRMS m/z calcd for C15H14N3S [M + H]+, 268.09084; found 268.09158. IR (ATR cm− 1): 3085, 3022, 2921, 1606, 1497, 1224
( E )-2-(2-([1,1'-biphenyl]-4-yl)vinyl)imidazo[1,2- a ]pyrimidine (4g). Brown solid in 43% yield. 1H-NMR (400 MHz, CDCl3) δ: 8.53 (dd, J = 4.2, 2.0 Hz, 1H), 8.37 (dd, J = 6.7, 2.0 Hz, 1H), 7.75 (d, J = 16.0 Hz, 1H), 7.66–7.60 (m, 6H), 7.56 (s, 1H), 7.47–7.43 (m, 2H), 7.38–7.33 (m, 1H), 7.18 (d, J = 16.0 Hz, 1H), 6.83 (dd, J = 6.7, 4.2 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ_ 150.2, 148.8, 146.0, 140.8, 140.7, 136.0, 132.7, 132.1, 128.8, 127.4, 127.3, 127.0, 119.4, 108.6, 108.5; MS (DART+) m/z: [M + H]+; 298; HRMS m/z calcd for C20H16N3 [M + H]+, 298.13442; found 298.13359. IR (ATR cm− 1): 3028, 2923, 2852, 1610, 1494, 1229.
( E )-2-(4-chlorostyryl)imidazo[1,2- a ]pyrimidine 4h). Yellow solid in 62% yield. 1H-NMR (300 MHz, CDCl3) δ: 8.53 (dd, J = 4.1, 2.0 Hz, 1H), 8.36 (dd, J = 6.7, 2.0 Hz, 1H), 7.66 (d, J = 16.0 Hz, 1H), 7.53 (s, 1H), 7.50–7.44 (m, 2H), 7.36–7.30 (m, 2H), 7.10 (d, J = 16.0 Hz, 1H), 6.83 (dd, J = 6.7, 4.1 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ: 150.5, 148.9, 145.7, 135.6, 133.8, 132.9, 131.4, 129.0, 128.1, 120.0, 108.8, 108.8; MS (DART+) m/z: [M + H]+; 256; HRMS m/z calcd for C14H11ClN3 [M + H]+, 256.06415; found 256.06310. IR (ATR cm− 1): 3085, 3028, 1606, 1496, 1223, 763.
( E )-2-(4-fluorostyryl)imidazo[1,2- a ]pyrimidine (4i). White off solid in 43% yield. 1H-NMR (400 MHz, CDCl3) δ: 8.45 (dd, J = 4.2, 2.0 Hz, 1H), 8.30 (dd, J = 6.7, 2.1 Hz, 1H), 7.60 (d, J = 16.0 Hz, 1H), 7.49–7.42 (m, 3H), 7.01–6.95 (m, 3H), 6.76 (dd, J = 6.7, 4.1 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ: 163.8, 161.4, 150.3, 148.7, 145.8, 133.2, 133.1, 132.8, 131.3, 128.4, 128.3, 119.1, 115.8, 115.6, 108.6, 108.5; MS (DART+) m/z: [M + H]+; 240; HRMS m/z calcd for C14H11FN3 [M + H]+, 240.09370; found 240.09430. IR (ATR cm− 1): 3071, 3034, 1608, 1506, 1225.
( E) -2-(4-(trifluoromethyl)styryl)imidazo[1,2- a ]pyrimidine (4j). Brown solid in 41% yield. 1H-NMR (300 MHz, Acetone-d6) δ: 8.87 (dd, J = 6.8, 2.0 Hz, 1H), 8.53 (dd, J = 4.1, 2.1 Hz, 1H), 7.97 (s, 1H), 7.85 (d, J = 8.1 Hz, 2H), 7.71 (d, J = 8.6 Hz, 3H), 7.46 (d, J = 16.2 Hz, 1H), 6.99 (dd, J = 6.8, 4.1 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ: 150.7, 148.8, 145.2, 140.4, 132.9, 130.9, 126.9, 125.7, 125.7, 125.7, 125.6, 121.7, 109.2, 108.7; 19F-NMR (375 MHz, CDCl3) δ: -62.51; MS (DART+) m/z: [M + H]+; 290; HRMS m/z calcd for C15H11F3N3 [M + H]+, 290.09051; found 290.09128. IR (ATR cm− 1): 3070, 3033, 2924, 1610, 1322, 1103.
( E )-2-(3-methoxystyryl)imidazo[1,2- a ]pyrimidine (4k). Brown solid in 56% yield. 1H-NMR (300 MHz, CDCl3) δ: 8.48 (dd, J = 4.1, 2.0 Hz, 1H), 8.34 (dd, J = 6.7, 2.0 Hz, 1H), 7.64 (d, J = 16.0 Hz, 1H), 7.51 (s, 1H), 7.26 (t, J = 7.9 Hz, 1H), 7.15–7.05 (m, 3H), 6.84–6.80 (m, 1H), 6.78 (dd, J = 6.7, 4.1 Hz, 1H), 3.83 (s, 3H); 13C-NMR (75 MHz, CDCl3) δ: 160.0, 150.3, 148.8, 145.9, 138.5, 132.9, 132.5, 129.8, 119.9, 119.6, 113.9, 112.1, 108.7, 108.6, 55.4; MS (IE+) m/z: [M+]+; 251; HRMS m/z calcd for C15H13N3O [M+]+, 251.1059; found 251.1060. IR (ATR cm− 1): 3039, 2921, 1611, 1473, 1212.
( E )-2-(3-fluorostyryl)imidazo[1,2- a ]pyrimidine (4l). Yellow solid in 42% yield. 1H-NMR (300 MHz, CDCl3) δ: 8.48 (dd, J = 4.3, 2.0 Hz, 1H), 8.36 (dd, J = 6.8, 2.0 Hz, 1H), 7.63 (d, J = 15.9 Hz, 1H), 7.53 (s, 1H), 7.31–7.18 (m, 3H), 7.08 (d, J = 16.0 Hz, 1H), 6.94 (ddt, J = 8.7, 6.2, 2.8 Hz, 1H), 6.79 (dd, J = 6.7, 4.1 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ: 164.4, 162.0, 158.3, 150.5, 148.7, 145.2, 139.4, 139.3, 133.1, 131.1, 131.1, 130.3, 130.2, 122.9, 122.9, 120.8, 114.9, 114.7, 113.0, 112.8, 109.2, 108.7; MS (DART+) m/z: [M + H]+; 240; HRMS m/z calcd for C14H11FN3 [M + H]+, 240.09370; found 240.09427. IR (ATR cm− 1): 3039, 1609, 1476, 1230. 963.
( E )-2-(2-([1,1'-biphenyl]-3-yl)vinyl)imidazo[1,2- a ]pyrimidine (4m). Brown solid in 44% yield. 1H-NMR (400 MHz, CDCl3) δ: 8.51 (dd, J = 4.1, 2.0 Hz, 1H), 8.36 (dd, J = 6.7, 2.0 Hz, 1H), 7.80–7.73 (m, 2H), 7.65–7.59 (m, 2H), 7.55–7.51 (m, 2H), 7.50–7.47 (m, 1H), 7.47–7.40 (m, 2H), 7.39–7.34 (m, 1H), 7.19 (d, J = 16.0 Hz, 1H), 6.80 (dd, J = 6.7, 4.1 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ: 150.4, 148.8, 145.9, 141.8, 141.2, 137.5, 132.9, 132.5, 129.3, 128.9, 127.6, 127.3, 127.0, 125.9, 125.7, 119.9, 108.7, 108.7; MS (DART+) m/z: [M + H]+; 298; HRMS m/z calcd for C20H16N3 [M + H]+, 298.13442; found 298.13424. IR (ATR cm− 1): 3058. 2922, 1611, 1494, 1221.
( E )-2-(2-(thiophen-2-yl)vinyl)imidazo[1,2- a ]pyrimidine (4n). Green solid in 37% yield. 1H-NMR (400 MHz, CDCl3) δ: 8.47 (dd, J = 4.1, 2.0 Hz, 1H), 8.34 (dd, J = 6.7, 2.0 Hz, 1H), 7.80 (d, J = 15.7 Hz, 1H), 7.47 (s, 1H), 7.21 (dt, J = 5.1, 1.0 Hz, 1H), 7.12 (dt, J = 3.6, 0.9 Hz, 1H), 7.00 (dd, J = 5.1, 3.5 Hz, 1H), 6.89 (d, J = 15.7 Hz, 1H), 6.78 (dd, J = 6.7, 4.1 Hz, 1H); 13C-NMR (75 MHz, CDCl3) δ: 158.4, 150.3, 145.5, 142.5, 132.9, 127.8, 127.3, 125.7, 125.1, 118.9, 111.7, 108.6; MS (IE+) m/z: [M+]+; 227; HRMS m/z calcd for C12H9N3S [M+]+, 227.0517; found 227.0524. IR (ATR cm− 1): 3127, 3064, 2922, 1608, 1485, 1228.
( E )-2-(2-(1-tosyl-1H-indol-2-yl)vinyl)imidazo[1,2- a ]pyrimidine (4o). Brown solid in 22% yield. 1H-NMR (300 MHz, CDCl3) δ: 8.51 (bs, 1H), 8.36 (d, J = 6.6 Hz, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.87 (d, J = 7.4 Hz, 1H), 7.81–7.72 (m, 4H), 7.53 (s, 1H), 7.39–7.26 (m, 2H), 7.24–7.12 (m, 3H), 6.81 (dd, J = 6.7, 4.0 Hz, 1H), 2.32 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ: 158.2, 150.4, 145.2, 135.6, 135.0, 132.9, 130.0, 129.0, 126.9, 125.1, 124.7, 123.7, 123.2, 120.6, 120.6, 120.1, 113.8, 108.7, 29.7; MS (IE+) m/z: [M+]+; 414; HRMS m/z calcd for C23H18N3O2S [M+]+, 414.1150; found 414.1151. IR (ATR cm− 1): 3121, 2921, 2852,1612, 1445, 1172.
2-((1 E,3E)-4-phenylbuta-1,3-dien-1-yl)imidazo[1,2-a]pyrimidine (4p). Brown solid in 32% yield. 1H-NMR (400 MHz, CDCl3) δ: 8.50 (dt, J = 4.9, 2.3 Hz, 1H), 8.34 (dd, J = 6.8, 2.0 Hz, 1H), 7.54–7.43 (m, 4H), 7.36–7.30 (m, 2H), 7.26–7.21 (m, 1H), 6.98 (dd, J = 15.5, 10.9 Hz, 1H), 6.82–6.79 (m, 1H), 6.76 (d, J = 15.6 Hz, 1H), 6.70 (d, J = 15.3 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ: 150.1, 148.8, 146.0, 137.2, 134.4, 133.2, 132.6, 128.7, 127.8, 126.6, 123.3, 108.5, 108.4; MS (IE+) m/z: [M+]+; 247; HRMS m/z calcd for C16H13N3 [M+]+, 247.1109; found 247.1109. IR (ATR cm− 1) 3127, 3024, 2923, 1607, 1482, 1235, 990.
( E )-2-(2-(3',5'-difluoro-[1,1'-biphenyl]-4-yl)vinyl)imidazo[1,2-a]pyrimidine (4r). White solid in 50% yield. 1H-NMR (400 MHz, DMSO-d6) δ: 8.95 (dd, J = 6.7, 2.0 Hz, 1H), 8.52 (dd, J = 4.1, 2.0 Hz, 1H), 8.02 (s, 1H), 7.85–7.73 (m, 4H), 7.63–7.37 (m, 4H), 7.22 (tt, J = 9.3, 2.3 Hz, 1H), 7.02 (dd, J = 6.7, 4.1 Hz, 1H); MS (DART+) m/z: [M + H]+; 259; HRMS m/z calcd for C20H14F2N3 [M + H]+, 334.11558; found 334.11504. IR (ATR cm− 1): 3138, 3082, 3030, 1609, 1589, 1112, 968, 820, 763.
Anti-proliferative Test
Human cancer cell lines were supplied by the National Cancer Institute, USA. The anti-proliferative effect was determined by using sulforhodamine B (SRB) in a microculture assay to measure cell growth, according to the protocols established by the NCI. The cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10,000 units/ mL of penicillin G sodium, 10 µg/m streptomycin sulfate and 25 µg/mL amphotericin B (Gibco) and 1% non-essential amino acids (Gibco). The cultures were maintained at 37°C in a 5% CO2 humidified atmosphere. As determined with trypan blue, the viability of the cell used in the experiments exceeded 95%. The cells were removed from the tissue culture flasks by treatment with trypsin and diluted with fresh media. 100 µL of cell suspension aliquots, containing 5000–10,000 cell per well, were transferred into a 96 well microtiter plates (Costar) and incubated at 37°C for 24 h in a 5% CO2 atmosphere. Stock solutions of test compounds 4a-r initially dissolved in DMSO (20 mM) were prepared and further diluted in medium to produce the desired concentrations. 100 µL aliquots of diluted solutions of the imidazopyrimidines 4a-r were added to each well. The cultures were exposed for 48 h to the compounds at concentrations of 20 µM. After the incubation period, cells were fixed to the plastic substratum by the addition of 50 µL of cold 50% aqueous trichloroacetic acid. The plates were incubated at 4°C for 1 h, washed with tap H2O, and air-dried. The trichloroacetic-acid-fixed cells were stained by the addition of 0.4% SRB. Free SRB solution was the removed by washing with 1% aqueous acetic acid. The plates were the air-dried, and the bound dye was solubilized by the addition of 100 µL 10 mM-unbuffered Tris base. The plates were placed on a shaker for 5 min, prior analysis. Optical densities were determined in an Ultra Microplated Reader (Elx 808, BIO-TEX Instruments, Inc,) λ = 515 nm. A dose–response curve was plotted for each compound and the concentration (IC50), and the 50% of inhibition was estimated through non-linear regression analysis. Statistical analysis Data were expressed as the mean ± standard error of three independent experiments and statistically analyzed using GraphPad Prism V6 software (Graph- Pad Software Inc., La Jolla, CA). One-way analysis of variance (ANOVA) followed by Dunnett’s test was performed to determine significant difference between the test group and the solvent control. A p value of < 0.05 was considered statistically significant.
Microdilution method for antimicrobial screening.
Antibacterial assays were performed following the methodology described by the CLSI M07-A10 using a microdilution test [32]. Briefly, all tested microorganisms (Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, multiresistant clinical isolates) were grown in Müeller-Hinton broth overnight. Subsequently, the bacterial suspension was adjusted to an optical density (OD600nm) of 0.08–0.13 equivalent to 1.5 x 108 UFC/mL, and then diluted 1/20 in the same media. Tested compounds 4a-p were dissolved in DMSO at 2.5 mM. Microdilution assays were carried out in a 96-well plate at a final volume of 100 µL. To each well, 10 µL of diluted bacterial suspension, 2 µL of molecules (4a-4r) to a final concentration of 50 µM, and 88 µL of Müeller-Hinton broth were added, and the OD600nm was measured (t0). Afterward, the plates were incubated at 37°C for 24 h and measured again (t24, OD600nm). Data are reported as the percentage of inhibition. Gentamicin was used as positive control. The minimum inhibitory concentration (MIC) experiments were performed following the same methodology at final concentrations ranging from 0.0025 to 250 µM. MIC was calculated by non-linear regression using a modified Gompertz function33 with GraphPad Prism (Eq. 1).
\(y =A+ {Ce}^{{-c}^{B(x-m)}}\) Eq. 1
where is the lower asymptote of y (approximately zero), B is a slope parameter, C is the distance between the upper and lower asymptote (approximately 1) and M is the log concentration of the inflexion point.