2.1. Experimental
Materials: The different reagents used in the synthesis (e.g., triethylamine 99.5%, thionyl chloride (SOCl2) 98% and amino acids 99%, sodium hydroxide pellets (NaOH) 99.99%) and analytical solvents (e.g., tetrahydrofuran 99.5%, anhydrous dimethylformamide 99.8%, ethyl acetate (HPLC-grade) and hexane (HPLC-grade)) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
General procedure for the preparation of 2-oxo-1,2-dihydroquinoline-4-carboxamide esters, 1a–2e: A mixture of 1.2 mol of thionyl chloride (SOCl2) and 1 mol of 2-oxo-1,2-dihydroquinoline-4-carboxylic acid was refluxed for 2 h. The excess of thionyl chloride was evaporated under reduced pressure, then 1.5 mol of protected amino acid solubilized in 20 mL of DMF and 3.3 mol of triethylamine (TEA) were added in small amounts to the mixture. After 20 min, the reaction was abandoned at room temperature for 12 h.
General synthetic procedure for the preparation of 2-oxo-1,2-dihydroquinoline-4-carboxamide acids, 3a–e and 4a–e: A mixture of quinoline carboxamide esters 1a–e and 2a–e (1 mol) and 1.2 mol of sodium hydroxide in 20 mL of THF/water (1/2: v/v) was stirred at room temperature for 24 h. After the THF was evaporated, the aqueous solution was extracted 3 times by dichloromethane (DCM) to remove the amount of ester that did not react. Half of the aqueous phase was evaporated and acidified by HCl (3 M) to obtain precipitate, and the crude product was isolated through simple filtration.
Chromatographic analyses: Column liquid chromatography was performed on 60 Merck silica gel (230–400 mesh ASTM). Thin layer chromatography (TLC) was performed on Merck aluminium plates coated with 60 F254 Merck silica gel (thickness 0.2 mm). The synthesized compounds were revealed by an ultra-violet lamp set at 254 nm and their melting points were determined by Electrothermal IA 9000 Series digital fusiometer using capillary tubes.
Spectroscopic analyses: NMR spectra and electrospray ionization time-of-flight (ESI-TOF) mass spectra were recorded at the Department of Chemistry, University of Helsinki, Finland. Fluorescence spectra were generated at the Department of Chemistry, University of Mulhouse France. NMR spectra were performed on Bruker Ascend 400 MHz-Avance III HD NMR spectrometer (Bruker Corporation, Billerica, MA, USA). 1H NMR spectra were recorded at 400 MHz, and the 13C NMR spectra at 100 MHz using DMSO-d6 or CDCl3 as solvent. Recorded spectra were calibrated by solvent signals when applicable and processed with MesterNova software. The chemical shift (δ) of different peaks was expressed in ppm and the coupling constants (nJ) in Hz. In describing the multiplicity of signals, the following abbreviations have been used: singlet (s), doublet (d), doublet of doublet (dd), multiplet (m), triplet (t) and quadruplet (q). High-resolution mass spectra were measured by Bruker micro TOF-MS.
2-(2-oxo-1,2-dihydroquinoline-4-carboxamido)propanoic acid: 3a. White solid, mp = 246–248 °C. 1H NMR δ (ppm) 400 MHz, DMSO-d6:12.22 (s, 1 H, NHquinoline), 9.1 (s, 1H, 3JH-H = 6.9 Hz, NH), 7.75–7.21 (m, 4H, Har)), 6.57 (s, 1H, CHethylenic), 4.44 (qd, 1H, 3JH-H = 7.2 Hz, 3JH-H = 6.9 Hz, *CH-N), 4–3(m, OHacid and water), 1.38 (d, 3H, CH3). 13C NMR δ (ppm) 100 MHz, DMSO-d6: 174.2 (C=Oacid), 166.22 (C=Oamide), 161.7 (C=Oamide quinoline), 146.5 (C4a-C8a), 139.6 (C4a-C8a), 131.3 (Ct5), 126.5 (Ct7), 122.4 (=C3), 120.2 (Ct6), 116.7 (Ct4), 116.0 (Ct8), 48.4 (*CH-N), 17.0 (CH3). Mass Spectrometry: m/z = 260.1086 [M]+, m/z = 519.1848 [2M+H]+.
3-hydroxy-2-(2-oxo-1,2-dihydroquinoline-4-carboxamido)propanoic acid: 3b. White solid, mp = 200–202 °C. 1H NMR δ (ppm) 400 MHz, DMSO-d6: 12.1 (s, 1 H, NHquinoline), 9.17 (s, 1H, 3JH-H 6.9 Hz, NH), 7.75–7.21 (m, 4H, Har), 6.57 (s, 1H, CHethylenic), 4.5 (dd, 1H, 3JH-H = 9 Hz, 3JH-H = 6.9 Hz, *CH-N), 4.01 (d, 2H, 3JH-H = 9 Hz, CH2-OH). 13C NMR δ (ppm) 100 MHz, DMSO-d6: 173.1 (C=Oacide), 166.0 (C=Oamide), 161.6 (C=Oamide quinoline), 146.4 (C4a-C8a) 139.5 (C4a-C8a), 131.3 (Ct5), 126.4 (Ct7), 122.3 (=Ctethylenic), 119.8 (C6), 116.4 (Ct4), 115.9 (Ct8), 61.7 (CH2-O), 51.1 (*CH-N). Mass Spectrometry: m/z = 275.0662 [M]+, m/z = 551.1409 [2M+H] +.
2-(2-oxo-1,2-dihydroquinoline-4-carboxamido)-2-phenylacetic acid: 3c. White solide mp = 218–220 °C. 1H NMR δ (ppm) 400 MHz, DMSO-d6: 13.04 (s, 1H, OHacid), 11.93 (s, 1 H, NHquinoline), 9.52 (s, 1H, 3JH-H = 7.3 Hz, NH), 7.8–7 (m, 9H, Har), 6.50 (s, 1H, CHethylenic), 4.58 (d, 1H, 3JH-H = 7.2 Hz,*CH-N). 13C NMR δ (ppm) 100 MHz, DMSO-d6: 172 (C=Oacid), 166.3 (C=Oamide), 161.6 (C=Oamide quinoline), 146.1 (C4a-C8a), 139.6 (C4a-C8a), 1386.7 (Cqar phenyl), 131.2 (Ct5), 129.0 (Ct7), 128.6 (=Ctethylenic), 128.5 (Ctar phenyl), 1276.3 (Ct4), 122.4 (=Ctethylenic), 120.5 (C6), 116.7 (Cq phenyl), 116.0 (Ct8), 57.2 (*CH-N). Mass Spectrometry: m/z = 321,0875 [M]+, m/z = 643,1801 [2M+H] +.
2-(2-oxo-1,2-dihydroquinoline-4-carboxamido)-3-phenylpropanoic acid: 3d. White solid, mp = 216–217 °C. 1H NMR δ (ppm) 400 MHz, DMSO-d6: 11.91 (s, 1 H, NHquinoline), 9.02 (s, 1H, 3JH-H = 8.4 Hz, NH), 7.53–7 (m, 9H, Har), 6.24 (s, 1H, CHethylenic), 4.69 (ddd, 1H, 3JH-H = 10.9Hz,3JH-H 8.3, 3JH-H 4.3 Hz *CH-N), 3.25 (dd, 1H, 3JH-H = 4.8 Hz, 2JH-H = 13.9 Hz, CH2-Ar), 2.94 (dd, 1H, 3JH-H = 11.1 Hz, 2JH-H 13.9 Hz, CH2-Ar). 13C NMR δ (ppm) 100 MHz, DMSO-d6: 173.2 (C=Oacid), 166.0 (C=Oamide), 161.6 (C=Oamide quinoline), 146.6 (C4a-C8a), 139.5 (C4a-C8a), 138.4 (Cqar phenyl),131.3 (Ct5), 129.6 (Ct7),128.6 (=Ctethylenic), 126.9 (Ctar phenyl), 126.4 (Ct4), 122.3 (=Ctethylenic), 119.8 (C6), 116.4 (Cq phenyl), 115.9 (Ct8), 54.1 (*CH-N), 36.8 (CH2). Mass Spectrometry: m/z = 335,1034 [M]+, m/z = 671,2136 [2M+H] +.
2-(2-oxo-1,2-dihydroquinoline-4-carboxamido)-3-phenylpropanoic acid: 3e. Grey solid, mp = 203–205 °C. 1H NMR δ (ppm) 400 MHz, DMSO-d6: 13.00(s, 1H, OHacid), 11.94 (s, 1 H, NHquinoline), 10.9 (s, 1H, 3JH-H = 7.5 Hz, NHindol), 9.06 (s, 1H, 3JH-H = 7.9 Hz, NH), 7.61–6.98 (m, 9H, Har+ Hindol), 6.30(s, 1H, CHethylenic), 4.72 (ddd, 1H, 3JH-H = 10.2 Hz, 3JH-H = 7.9 Hz, 3JH-H = 4.4 Hz *CH-N), 3.31 (dd, 1H, 3JH-H = 4.4 Hz, 2JH-H = 14.4 Hz, CH2), 3.13 (dd, 1H, 3JH-H = 10.3 Hz, 2JH-H = 14.4 Hz, CH2-Indol). 13C NMR δ (ppm) 100 MHz, DMSO-d6: 173.4 (C=Oacid), 166.3 (C=Oamide), 161.6 (C=Oamide quinoline), 146.6 (C4a-C8a), 139.5 (C4a-C8a), 136.6 (Cqar tryptophan),131.2 (Ct5),127.5 (Cqtryptophan), 126.4 (Ct7), 124.1 (Ctar tryptophan), 122.3 (Ctar tryptophan), 121.4 (=Ctethylenic), 120 (C4), 118.9 (Ct6), 118.6 (Ctar tryptophan), 116.5 (Ctar tryptophan), 116.5 (Cq ehtylenic) 115.9 (Ct8), 111.9 (=Ctethylenic tryptophan), 110.5 (=Cqethylenic tryptophan), 53.6 (*CH-N), 27 (CH2-Indol). Mass Spectrometry: m/z = 374,1137 [M]+, m/z = 749,2292 [2M+H] +.
2-(6-bromo-2-oxo-1,2-dihydroquinoline-4-carboxamido)propanoic acid: 4a. White solid, mp = 250–252 °C. 1H NMR δ (ppm) 400 MHz, DMSO-d6: 12.10 (s, 1 H, NHquinoline), 9.24 (d, 1H, 3JH-H = 6.9 Hz, NH), 7.7–7.4 (m, 3H, Har), 6.59 (s, 1H, CHethylenic), 4.50 (qd, 1H, 3JH-H = 7.2 Hz, 3JH-H = 6.9 Hz, *CH-N), 1.4 (d, 3H, CH3). 13C NMR δ (ppm) 100 MHz, DMSO-d6: 174.1 (C=Oacid), 166.4 (C=Oamide), 161.3 (C=Oamide quinoline), 144.8 (C4a-C8a), 139.6 (C4a-C8a), 133.9 (Ct5), 128.4 (Ct7), 121.6 (=Ctethylenic), 118.3 (Ct4), 118.2 (Ct8), 114.3 (Cq), 48.5 (*CH-N), 16.9 (CH3). Mass Spectrometry: m/z = 336. 9785 [M]+, m/z = 674.9670 [2M+H]+.
3-hydroxy-2-(6-bromo-2-oxo-1,2-dihydroquinoline-4-carboxamido)propanoic acid: 4b. White solid, mp = 213–215 °C. 1H NMR δ (ppm) 400 MHz, DMSO-d6: 11.97 (s, 1 H, NHquinoline), 9.17 (s, 1H, 3JH-H = 6.9 Hz, NH), 7.75–7.21 (m, 4H, Har), 6.57 (s, 1H, CHethylenic), 4.5 (dd, 1H, 3JH-H = 9 Hz, 3JH-H = 6.9Hz, *CH-N), 3.8 (d, 2H, 3JH-H = 9 Hz, CH2-OH). 13C NMR δ (ppm) 100 MHz, DMSO-d6: 173 (C=Oacide), 166.6 (C=Oamide), 161.7 (C=Oamide quinoline), 146.2 (C4a-C8a) 139.6 (C4a-C8a), 131.3 (Ct5), 126.4 (Ct7), 120.4 (=Ctethylenic), 116.6 (C6), 116 (Ct4), 114.4 (Ct8), 61.3 (CH2-O), 52.6 (*CH-N). Mass Spectrometry: m/z = 352,9761 [M]+.
2-(6-bromo-2-oxo-1,2-dihydroquinoline-4-carboxamido)-2-phenylacetic acid: 4c. White solid, mp = 230–232 °C. 1H NMR δ (ppm) 400 MHz, DMSO-d6: 13.04 (s, 1H, OHacid), 11.93 (s, 1 H, NHquinoline), 9.53 (s, 1H, 3JH-H =5.61 Hz, NH), 7.9–7 (m, 9H, Har), 6.5 (s, 1H, CHethylenic), 5.56 (d, 1H, 3JH-H 5.61 Hz,*CH-N). 13C NMR δ (ppm) 100 MHz, DMSO-d6: 172 (C=Oacid), 166.7 (C=Oamide), 161.2 (C=Oamide quinoline), 145.1 (C4a-C8a), 139.6 (C4a-C8a), 139 (Cqar phenyl), 138.1 (Ctar), 3.8 (Ct5), 129.5 (Ct7), 128.7 (Ctar phenyl), 128.6 (Ctar phenyl), 127 (Ct7), 121.30 (=Ctethylenic), 116.2 (Cq4), 116 (Ct8), 57.6 (*CH-N). Mass Spectrometry: m/z = 398,9975 [M]+, m/z = 799,0034 [2M+H] +.
2-(6-bromo2-oxo-1,2-dihydroquinoline-4-carboxamido)-3-phenylpropanoic acid: 4d. White solid, mp = 240–242 °C. 1H NMR δ (ppm) 400 MHz, DMSO-d6: 13.03 (s, 1H, OHacid), 12.08 (s, 1 H, NHquinoline), 9.17 (s, 1H, 3JH-H = 5.61 Hz, NH), 7.9–7 (m, 9H, Har), 6.32 (s, 1H, CHethylenic), 4.69 (d, 1H, 3JH-H = 5.61 Hz,*CH-N), 3.25 (dd, 1H, 3JH-H = 4.8 Hz, 2JH-H = 13.8Hz, CH2-Ar), 2.95 (dd, 1H, 3JH-H = 11.1Hz, 2JH-H =13.8 Hz, CH2-Ar). 13C NMR δ (ppm) 100 MHz, DMSO-d6: 173 (C=Oacid), 165.7 (C=Oamide), 161.2 (C=Oamide quinoline), 145 (C4a-C8a), 138.7 (C4a-C8a), 138.1 (Cqar phenyl), 134 (Ct5), 129.5 (Ct7), 128.6 (=Ctethylenic), 128.3 (Ctar phenyl), 127 (Ct4), 121.3 (=Ctethylenic), 118.2 (C6), 118.0 (Cq phenyl), 114.2 (Ct8), 53.9 (CH2-Ar), 54 (*CH-N), 36.7 (CH2). Mass Spectrometry: m/z = 413,0124 [M]+, m/z = 827,0356 [2M+H] +.
2-(6-bromo-2-oxo-1,2-dihydroquinoline-4-carboxamido)-3-phenylpropanoic acid: 4e. White solid. Mp = 260–262 °C. 1H NMR δ (ppm) 400 MHz, DMSO-d6: 12.98 (s, 1H, OHacid), 12.22 (s, 1 H, NHquinoline), 10.88 (s, 1H, 3JH-H = 7.5 Hz, NHindol), 9.15 (s, 1H, 3JH-H = 7.9 Hz, NH), 8.43(d, 1H, 4JH-H = 2.2 Hz, Har), 7.61–6.98 (m, 8H, Har + Hindol), 6.37(s, 1H, CHethylenic), 4.67 (ddd, 1H, 3JH-H = 10.2 Hz, 3JH-H = 7.9 Hz, 3JH-H = 4.4 Hz *CH-N), 3.31(dd, 1H, 3JH-H =4.4 Hz, 2JH-H = 14.4 Hz, CH2-Indo), 3.14 (dd, 1H, 3JH-H = 10.3 Hz, 2JH-H = 14.4 Hz, CH2-Indol). 13C NMR δ (ppm) 100 MHz, DMSO-d6: 173.4 (C=Oacid), 166.7 (C=Oamide), 161.6 (C=Oamide quinoline), 146.3 (C4a-C8a), 138.6 (C4a-C8a), 136.6 (Cqar tryptophan), 133.8 (Ct5), 127.56 (Cqtryptophan), 125.99 (Ct7), 124.1 (Ctar tryptophan), 121.4 (=Ctethylenic), 118.8 (Ctar tryptophan), 118.5 (C4), 118.3 (Ctar tryptophan), 118.1 (Ctar tryptophan), 117.9 (Cq ehtylenic) 114.5 (Ct8), 111.9 (=Ctethylenic tryptophan), 110.5 (=Cqethylenic tryptophan), 53.9 (*CH-N), 26.9 (CH2-Indol). Mass Spectrometry: m/z = 452,0241 [M]+, m/z = 905,0624 [2M+H]+.
Antibacterial assay-minimum inhibitory concentration (MIC). The MICs of the amino ester derivatives
1a–2e and the amino acid derivatives
3a–4e were determined by the broth microdilution method [
31]. A 96-well polypropylene microliter plate was filled with 50 µL of Mueller–Hinton broth (MHB). Then, 50 µL of each sample at a final concentration of 10 mg/mL was added into the first well. Serial ½ dilutions were realized by pipetting 50 µL from the first well and transferred to the next one. This operation was repeated until the 12th well and the last 50 µL mixture was discarded. Finally, a volume of 50 µL of bacterial suspension was added into each well at a final concentration of approximately 106 CFU/mL. The 96-well plate was then covered and was incubated at 37 °C for 24 h. After that, 5 µL of resazurin was pipetted into all the wells and incubated another time at 37 °C for 2 h. The MIC was presented as the lowest concentration that showed a negative bacterial growth translated by a non-change in resazurin color. A positive one is detected by reduction of the blue dye resazurin to pink resorufin [
32].
Fluorometry: The fluorescence spectra of all the solutions were measured at room temperature using a Shimadzu RF-5001-Pc spectrofluorometer and were investigated in DMSO at concentration of 10 mg/mL (DMSO).