Development of Quinazolinone Derivatives as Modulators of Virulence Factors of Pseudomonas aeruginosa Cystic Fibrosis Strains
Abstract
:1. Introduction
2. Results and Discussion
2.1. Compounds’ Synthesis
2.2. Antivirulence Activity
2.2.1. Antibiofilm Activity and SAR Study
2.2.2. Activity against Pigment Production
2.3. Cytotoxic Effects
2.4. Molecular Modeling
3. Materials and Methods
3.1. General Chemistry Methods
- General procedure for the synthesis of 13–14. To a solution of anthranilic acid (12a or 12b) (500 mg, 1 eq.) in dry pyridine (5 mL), decanoyl chloride (850 µL, 1.5 eq.) was dropped. The reaction mixture was stirred at 25 °C for 24 h. Then, the reaction was warmed to room temperature (RT) and treated with 15 mL of 5% HCl. The mixture was then partitioned between H2O and EtOAc (3 × 20 mL). The combined organics were dried, filtered, and evaporated under reduced pressure.
- 2-Decanamido-4,5-difluorobenzoic acid (13). Pale brown solid, 76% yield. Spectroscopic data are in agreement with those reported [25].
- 4-Chloro-2-decanamidobenzoic acid (14). White solid, 91% yield. Spectroscopic data are in agreement with those reported [16].
- General procedure for the synthesis of compounds 15a–f. Anthranilic acids 12a–d or substituted anthranilic acids 13–14 (500 mg, 1 eq.) were heated at 140 °C in acetic anhydride (2.8 mL, 10 eq.) for 2 h. The reaction was warmed to RT and pH adjusted to 7 by adding solid NaHCO3. Then, the mixture was partitioned between H2O and DCM. The organic layer was dried, filtered, and evaporated under reduced pressure to obtain the title compounds 15a–f as solids.
- 6,7-Difluoro-2-methyl-4H-benzo[d][1,3]oxazin-4-one (15a). Yellow solid, 83% yield. Spectroscopic data are in agreement with those reported [16].
- 7-Chloro-2-methyl-4H-benzo[d][1,3]oxazin-4-one (15b). Yellow solid, 92% yield. Spectroscopic data are in agreement with those reported [16].
- 7-Bromo-2-methyl-4H-benzo[d][1,3]oxazin-4-one (15c). White solid, 94% yield. Spectroscopic data are in agreement with those reported [26].
- 6-Chloro-2-methyl-4H-benzo[d][1,3]oxazin-4-one (15d). Yellow solid, 90% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.02 (d, J = 2.3 Hz, 1H), 7.91 (dd, J = 8.6, 2.4 Hz, 1H), 7.56 (d, J = 8.6 Hz, 1H), 2.38 (s, 3H).
- 6,7-Difluoro-2-nonyl-4H-benzo[d][1,3]oxazin-4-one (15e). White solid, 52% yield. Spectroscopic data are in agreement with those reported [27].
- 7-Chloro-2-nonyl-4H-benzo[d][1,3]oxazin-4-one (15f). White solid, 50% yield. Spectroscopic data are in agreement with those reported [25].
- General procedure for the synthesis of compounds 3, 4, and 16–18. To a solution of the corresponding lactone 15a–f (356 mg, 1 eq.) in EtOH (6 mL), hydrazine hydrate (150 µL, 4 eq.) was added. The mixture was heated at 80 °C for 15 h. Then, the mixture was warmed at RT and treated with a saturated solution of NaHCO3 and EtOAc (3 × 10 mL). The combined organics were dried, filtered, and evaporated under reduced pressure. The title compounds 3, 4, and 16–18 have been purified through silica gel column chromatography, eluent petroleum ether/EtOAc (3:1).
- 3-Amino-6,7-difluoro-2-nonylquinazolin-4(3H)-one (3). White solid, 26% yield. Spectroscopic data are in agreement with those reported [16].
- 3-Amino-7-chloro-2-nonylquinazolin-4(3H)-one (4). White solid, 66% yield. Spectroscopic data are in agreement with those reported [25].
- 3-Amino-6,7-difluoro-2-methylquinazolin-4(3H)-one (16). Yellow solid, 62% yield. Spectroscopic data are in agreement with those reported [28].
- 3-Amino-7-chloro-2-methylquinazolin-4(3H)-one (17). Yellow solid, 100% yield. 1H NMR (300 MHz, CDCl3) δ 8.15 (d, 1H J = 8.6 Hz), 7.63 (d, 1H, J = 2.0 Hz), 7.40 (dd, 1H, J = 8.6, 2.0 Hz), 4.88 (bs, 2H), 2.70 (s, 3H). ESI-MS m/z: 210 [M + H]+.
- 3-Amino-7-bromo-2-methylquinazolin-4(3H)-one (18). White solid, 68% yield. 1H NMR (300 MHz, Acetone-d6) δ 8.04 (s, 1H), 7.82–7.71 (m, 1H), 7.66–7.54 (m, 1H), 5.59 (s, 2H), 2.65 (s, 3H). ESI-MS m/z: 255 [M + H]+.
- General procedure for the synthesis of compounds 7a–d and 8a,b. To a mixture of 3, 16–18 (100 mg, 1 eq.) and the appropriate aldehyde (1 eq.) in EtOH (3 mL), AcOH (2 drops) was added. The mixture was heated at 80 °C for 20 h. The reaction mixture was then cooled to 25 °C and partitioned between H2O and EtOAc (3 × 10 mL). The combined organics were dried, filtered, and evaporated under reduced pressure. The title compounds were purified through silica gel column chromatography, eluent petroleum ether/EtOAc (3:1).
- (E)-6,7-Difluoro-3-((4-hydroxybenzylidene)amino)-2-nonylquinazolin-4(3H)-one (7a). White solid, Mp: 134 °C, 59% yield. 1H NMR (300 MHz, DMSO-d6) δ 10.42 (s, 1H), 8.69 (s, 1H), 8.07–7.90 (m, 1H), 7.82–7.57 (m, 3H), 6.91 (d, J = 8.6 Hz, 2H), 2.86–2.61 (m, 2H), 1.74–1.40 (m, 2H), 1.43–0.94 (m, 12H), 0.80 (t, J = 6.8 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 170.4, 162.4, 157.2 (d, J = 14.9 Hz), 152.5 (m), 150.9 (m), 145.66 (d, J = 14.3 Hz), 144.5 (d, J = 45.9 Hz), 131.4, 123.4, 118.5 (d, J = 18.3 Hz), 116.4, 115.4, 34.3, 31.7, 29.2, 29.0, 28.9, 26.2, 22.5, 14.4. ESI-MS m/z: 428 [M + H]+. LC-MS Rt: 14.036 min, purity 100%, MS: 428.2. HRMS ESI m/z [M + H]+ calcd for C24H28F2N3O2 428.2144, found 428.2126.
- (E)-6,7-Difluoro-3-((4-hydroxybenzylidene)amino)-2-methylquinazolin-4(3H)-one (7b). White solid, Mp: 245 °C, 32% yield. 1H NMR (300 MHz, DMSO-d6) δ 10.41 (s, 1H), 8.69 (s, 1H), 8.13–7.92 (m, 1H), 7.87–7.59 (m, 3H), 6.92 (d, J = 8.5 Hz, 2H), 2.46 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 170.3, 162.5, 154.9 (m), 147.0 (m), 144.7 (m), 131.5, 130.4, 123.4, 116.5, 115.2 (d, J = 17.6 Hz), 114.2, 49.0, 22.6. ESI-MS m/z: 316 [M + H]+. LC-MS Rt: 10.135 min, purity 95.5%, MS: 316.0.
- (E)-7-Chloro-3-((4-hydroxybenzylidene)amino)-2-methylquinazolin-4(3H)-one (7c). White solid, Mp: 285 °C, 47% yield. 1H NMR (300 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.70 (s, 1H), 8.11 (d, J = 8.6 Hz, 1H), 7.83–7.65 (m, 3H), 7.53 (dd, J = 8.5, 1.6 Hz, 1H), 6.92 (d, J = 8.5 Hz, 2H), 2.47 (s, 3H, under DMSO). 13C NMR (75 MHz, CDCl3) δ 169.1, 158.1, 155.2, 146.8, 134.9, 130.3, 129.8, 129.4, 129.0, 128.6, 127.8, 123.6, 120.2, 22.7. ESI-MS m/z: 314 [M + H]+. LC-MS Rt: 10.341 min, purity 98.7%, MS: 313.9.
- (E)-7-Bromo-3-((4-hydroxybenzylidene)amino)-2-methylquinazolin-4(3H)-one (7d). White solid, Mp: 280 °C, 65% yield. 1H NMR (300 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.70 (s, 1H), 8.03 (d, J = 8.5 Hz, 1H), 7.85 (d, J = 1.8 Hz, 1H), 7.78 (d, J = 8.6 Hz, 2H), 7.66 (dd, J = 8.5, 1.8 Hz, 1H), 6.92 (d, J = 8.6 Hz, 2H), 2.50 (m, 3H, under DMSO). 13C NMR (75 MHz, DMSO-d6) δ 171.5, 164.0, 159.2, 156.5, 148.9, 134.5, 133.0, 132.1, 129.4, 126.7, 121.3, 117.4, 115.1, 24.0 (under DMSO). ESI-MS m/z: 358 [M + H]+, 356 [M − H]−. LC-MS Rt: 10.494 min, purity 100%, MS: 357.90, 359.9.
- (E)-6,7-Difluoro-2-nonyl-3-4-phenylbuta-1,3-dien-1-yl)quinazolin-4(3H)-one (8a). White solid, Mp: 94 °C, 49% yield. 1H NMR (300 MHz, CDCl3) δ 8.64 (d, J = 8.9 Hz, 1H), 7.98 (t, J = 9.3 Hz, 1H), 7.67–6.92 (m, 8H), 2.99–2.70 (m, 2H), 1.93–1.57 (m, 2H), 1.55–1.04 (m, 12H), 0.85 (t, J = 6.0 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 169.48, 157.4, δ 156.5 (d, J = 14.9 Hz), 152.9 (d, J = 11.8 Hz), 150.99 (d, J = 14.5 Hz), 147.66 (d, J = 14.3 Hz), 144.3 (m), 134.9, 131.2, 130.3, 129.1, 128.9, 128.5, 127.8, 123.6, 118.1, 115.0 (m), 34.8, 31.9, 29.4, 29.3, 26.6, 22.7, 14.1. ESI-MS m/z: 438 [M + H]+. LC-MS Rt: 16.889 min, purity 95.8%, MS: 438.2.
- (E)-7-Bromo-2-methyl-3-(-(3-phenylallylidene)amino)quinazolin-4(3H)-one (8b). White solid, Mp: 200 °C, 52% yield. 1H NMR (300 MHz, CDCl3) δ 8.69 (d, J = 8.9 Hz, 1H), 8.10 (d, J = 8.5 Hz, 1H), 7.84 (d, J = 1.7 Hz, 1H), 7.63–7.48 (m, 3H), 7.46–7.35 (m, 3H), 7.27–7.03 (m, 2H), 2.50 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 169.1, 158.1, 155.2, 146.8, 134.9, 130.3, 129.8, 129.4, 129.0, 128.6, 127.8, 123.6, 120.2, 22.7. ESI-MS m/z: 368 [M + H]+. LC-MS Rt: 11.960 min, purity 95.5%, MS: 368.9, 370.0.
- General procedure for the synthesis of compounds 19a,b. Carboxylic acid 12b or 12d (2.0 g, 1 eq.) was diluted in formamide (4 mL). The reaction was heated at 150 °C for 20 h. Then, ice (2.0 g) was added to the reaction to favor the precipitation of the product, which was filtered and washed with H2O. Then, it was dried at 50 °C for 2 h.
- 6-Chloroquinazolin-4(3H)-one (19a). Starting from 12d, the title compound was obtained as a white solid, 93% yield. Spectroscopic data are in agreement with those reported [29].
- 7-Chloroquinazolin-4(3H)-one (19b). Starting from 12b, the title compound was obtained as a white solid, 90% yield. 1H NMR (300 MHz, DMSO-d6) δ 12.39 (s, 1H), 8.25–7.92 (m, 2H), 7.71 (d, J = 2.0 Hz, 1H), 7.54 (dd, J = 8.6, 2.1 Hz, 1H).
- General procedure for the synthesis of compounds 20a,b and 21. To a mixture of 19a,b (207 mg, 1 eq.) in acetone (13 mL), the appropriate aryl halide (178 mg, 1 eq.), K2CO3 (718 mg, 5 eq.), and NaI (155 mg, 1 eq.) were added. The mixture was stirred at 50 °C for 24 h. After this time, the reaction was partitioned between brine and EtOAc and the aqueous layer was washed again with EtOAc (3 × 10 mL). The combined organics were dried, filtered, and evaporated in vacuo. The title compound was purified through silica gel column chromatography, eluent petroleum ether/EtOAc (3:1).
- 6-Chloro-3-(4-nitrobenzyl)quinazolin-4(3H)-one (20a). Yellow solid, 66% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.63 (s, 1H), 8.18 (d, J = 8.6 Hz, 2H), 8.05 (d, J = 2.3 Hz, 1H), 7.86 (dd, J = 8.7, 2.4 Hz, 1H), 7.73 (d, J = 8.7 Hz, 1H), 7.59 (d, J = 8.6 Hz, 2H), 5.32 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 159.7, 148.9, 147.4, 147.1, 144.5, 135.1, 132.0, 130.0, 129.2, 125.5, 124.2, 123.3, 49.3. ESI-MS m/z: 316 [M + H]+.
- 7-Chloro-3-(4-nitrobenzyl)quinazolin-4(3H)-one (20b). Yellow solid, 60% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.65 (s, 1H), 8.14 (dd, J = 23.0, 8.6 Hz, 3H), 7.76 (d, J = 1.9 Hz, 1H), 7.57 (dd, J = 12.5, 5.4 Hz, 3H), 5.30 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 160.0, 149.8, 149.4, 147.3, 144.6, 139.6, 129.1, 128.6, 128.0, 126.9, 124.1, 120.8, 49.2. ESI-MS m/z: 316 [M + H]+.
- 3-(4-Bromobenzyl)-7-chloroquinazolin-4(3H)-one (21). Yellow solid, 67% yield. 1H NMR (300 MHz, CDCl3) δ 8.19 (t, J = 15.5 Hz, 1H), 8.08 (s, 1H), 7.69 (s, 1H), 7.47 (d, J = 7.4 Hz, 3H), 7.22 (d, J = 7.7 Hz, 2H), 5.11 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 160.0, 148.9, 146.9, 134.5, 131.8, 129.7, 128.2, 127.0, 122.5, 120.1, 49.2. ESI-MS m/z: 350 [M + H]+.
- General procedure for the synthesis of compounds 22a,b. To a mixture of 20a,b (182 mg, 1 eq.) in EtOH (6 mL), heated at 55 °C, saturated NH4Cl (6 mL) was added. After that, iron powder (330 mg, 10 eq.) was added and the mixture was heated at 70 °C for 2 h. Solvent was removed, the mixture was diluted in EtOAc and filtered on Celite®, then pH was adjusted to 8 by adding saturated NaHCO3. The mixture was then partitioned between H2O and EtOAc. The organic layer was dried, filtered, and evaporated to afford the final product.
- 3-(4-Aminobenzyl)-6-chloroquinazolin-4(3H)-one (22a). White solid, Mp: 194 °C, 54% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.53 (s, 1H), 8.07 (d, J = 2.4 Hz, 1H), 7.82 (dd, J = 8.7, 2.5 Hz, 1H), 7.68 (d, J = 8.7 Hz, 1H), 7.07 (d, J = 8.3 Hz, 2H), 6.48 (d, J = 8.3 Hz, 2H), 5.09 (s, 2H), 4.97 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 159.5, 148.9, 148.8, 147.0, 131.8, 129.9, 129.6, 125.5, 123.7, 123.3, 114.1, 49.2. ESI-MS m/z: 286 [M + H]+. LC-MS Rt: 8.251 min, purity 99.4%, MS: 286.0.
- 3-(4-Aminobenzyl)-7-chloroquinazolin-4(3H)-one (22b). Yellow solid, Mp: 168 °C, 95% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.55 (s, 1H), 8.12 (d, J = 8.6 Hz, 1H), 7.73 (d, J = 1.9 Hz, 1H), 7.53 (dt, J = 30.1, 15.1 Hz, 1H), 7.03 (t, J = 19.0 Hz, 2H), 6.48 (d, J = 8.4 Hz, 2H), 5.11 (s, 2H), 4.96 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 159.8, 149.8, 149.2, 148.9, 148.7, 139.3, 129.7, 128.5, 127.9, 126.4, 123.4, 114.1, 48.9. ESI-MS m/z: 286 [M + H]+. LC-MS Rt: 8.233 min, purity 97.7%, MS: 286.0.
- Synthesis of 7-Chloro-3-((4′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)methyl)quinazolin-4(3H)-one (9). To a solution of 21 (100 mg, 1 eq.) in anhydrous EtOH (2.5 mL) and anhydrous 1,2-diethoxyethane (2.5 mL), Pd0 (1.3 mg, 0.04 eq.) was added. The mixture was refluxed and after 30 min, Na2CO3 (189 mg, 6 eq.) in H2O (1 mL) and (4-(trifluoromethyl)phenyl)boronic acid (80 mg, 1.4 eq.) in EtOH (0.5 mL) were added. The mixture was refluxed for 12 h. After this time, H2O was added (1 mL) and the mixture was extracted with EtOAc (10 mL). The aqueous layer was extracted twice with EtOAc (2 × 5 mL). The combined organic layers were dried, filtered, and evaporated under reduced pressure. The title compound 9 was purified through silica gel column chromatography, eluent petroleum ether/EtOAc (3:1). Yellow solid, Mp: 167 °C, 63% yield. 1H NMR (300 MHz, CDCl3) δ 8.30–7.92 (m, 2H), 7.90–7.06 (m, 10H), 5.17 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 160.4, 148.9, 147.4, 147.3 (dd, J = 14.3, 3.6 Hz), 143.7, 140.6, 138.9, 135.4, 132.1, 129.6, 128.6, 128.3 (d, J = 2.0 Hz), 128.1 (d, J = 2.9 Hz), 127.9, 127.3, 127.1 (d, J = 2.7 Hz), 127.0, 125.9, 125.8 (d, J = 3.8 Hz), 125.7, 120.6, 49.5. ESI-MS m/z: 437 [M + Na]+. LC-MS Rt: 12.716 min, purity 95.0%, MS: 415.1.
- Synthesis of 3-(4-(1H-pyrrol-1-yl)benzyl)-7-chloroquinazolin-4(3H)-one (10). According to published procedures [30,31], a mixture of 4-chloropyridine hydrochloride (79.5 mg, 5 eq.) in 1,4-dioxane was heated at 150 °C for 30 min. Then, 22b (125 mg, 1 eq.) and 2,5-dimethoxy tetrahydrofuran (57 µL, 1 eq.) were added and the mixture was heated at 150 °C for 2 h. The reaction mixture was then filtered on Celite® and the product was purified through silica gel column chromatography, eluent petroleum ether/EtOAc (3:1). White solid, Mp: 195 °C, 92% yield. 1H NMR (300 MHz, CDCl3) δ 8.24 (d, J = 8.6 Hz, 1H), 8.14 (s, 1H), 7.70 (d, J = 1.7 Hz, 1H), 7.56–7.30 (m, 5H), 7.05 (d, J = 1.9 Hz, 2H), 6.33 (d, J = 1.9 Hz, 2H), 5.18 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 160.4, 148.9, 147.3, 147.2, 140.7, 140.6, 132.6, 129.4, 128.3, 128.1, 127.1, 120.8, 120.6, 119.1, 110.7, 49.3. ESI-MS m/z: 336 [M + H]+. LC-MS Rt: 11.546 min, purity 97.5%, MS: 336.0.
- General procedure for the synthesis of compounds 11a–f. To a well-stirred solution of 22a,b (180 mg, 1 eq.) in MeOH (5 mL), the appropriate aldehyde (1 eq.) and AcOH (5 drops) were added. The mixture was heated at 50 °C for 12 h. After this time, the reaction was cooled to 25 °C and NaBH3CN (60 mg, 1.5 eq.) was added. The mixture was stirred for an additional 2 h. Then, pH was adjusted to 8 by adding a saturated NaHCO3 aqueous solution. The crude was partitioned between H2O and EtOAc; the aqueous layer was extracted with EtOAc (3 × 10 mL). The title compound was purified through silica gel column chromatography, eluent petroleum ether/EtOAc (3:1).
- 6-Chloro-3-(4-((furan-2-ylmethyl)amino)benzyl)quinazolin-4(3H)-one (11a). Yellow solid, Mp: 161 °C, 73% yield. 1H NMR (300 MHz, CDCl3) δ 8.26 (d, J = 1.8 Hz, 1H), 8.06 (s, 1H), 7.63 (q, J = 8.7 Hz, 2H), 7.33 (s, 1H), 7.19 (d, J = 8.4 Hz, 2H), 6.62 (d, J = 8.4 Hz, 2H), 6.30 (s, 1H), 6.20 (d, J = 2.8 Hz, 1H), 5.04 (s, 2H), 4.37–4.08 (m, 3H). 13C NMR (75 MHz, CDCl3) δ 160.0, 152.2, 147.7, 146.5, 142.0, 134.5, 133.0, 129.7, 129.1, 126.2, 124.3, 123.2, 113.2, 110.4, 110.4, 107.1, 49.5, 41.1. ESI-MS m/z: 366 [M + H]+, 388 [M + Na]+. LC-MS Rt: 11.122 min, purity 100%, MS: 365.9. HRMS ESI m/z [M + Na]+ calcd for C20H16ClN3O2Na 388.0823, found 388.0809.
- 7-Chloro-3-(4-((furan-2-ylmethyl)amino)benzyl)quinazolin-4(3H)-one (11b). White solid, Mp: 126 °C, 70% yield. 1H NMR (300 MHz, Acetone-d6) δ 8.41 (s, 1H), 8.19 (d, J = 8.6 Hz, 1H), 7.64 (d, J = 1.9 Hz, 1H), 7.50 (dd, J = 8.6, 2.0 Hz, 1H), 7.42 (s, 1H), 7.25 (d, J = 8.5 Hz, 3H), 6.69 (d, J = 8.5 Hz, 3H), 6.27 (dd, J = 17.7, 2.4 Hz, 2H), 5.47 (t, J = 5.3 Hz, 1H), 5.10 (s, 3H), 4.30 (d, J = 5.9 Hz, 3H). 13C NMR (75 MHz, Acetone-d6) δ 159.7, 153.3, 149.4, 148.7, 148.3, 141.7, 139.3, 129.4, 128.2, 127.1, 126.6, 124.5, 121.0, 112.6, 110.2, 106.6, 48.8, 40.3. ESI-MS m/z: 366 [M + H]+. LC-MS Rt: 11.154 min, purity 99.1%, MS: 366.1. HRMS ESI m/z [M + H]+ calcd for C20H17ClN3O2 366.1004, found 366.0993.
- 6-Chloro-3-(4-(((2,3-dihydrobenzofuran-5-yl)methyl)amino)benzyl)quinazolin-4(3H)-one (11c). White solid, Mp: 153 °C, 75% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.53 (s, 1H), 8.05 (s, 1H), 7.82 (dd, J = 8.7, 2.5 Hz, 1H), 7.67 (d, J = 8.7 Hz, 1H), 7.23–7.05 (m, 3H), 7.00 (d, J = 7.9 Hz, 1H), 6.64 (d, J = 8.1 Hz, 1H), 6.49 (d, J = 8.5 Hz, 2H), 6.23 (t, J = 5.9 Hz, 1H), 4.97 (s, 2H), 4.44 (t, J = 8.7 Hz, 2H), 4.09 (d, J = 5.8 Hz, 2H), 3.08 (t, J = 8.6 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 159.6, 159.1, 148.8, 147.1, 134.9, 132.2, 131.9, 129.9, 129.6, 127.6, 127.1, 125.5, 124.4, 123.7, 123.3, 112.5, 108.9, 71.2, 49.2, 46.3, 29.6. ESI-MS m/z: 418 [M + H]+. LC-MS Rt: 11.383 min, purity 95.1%, MS: 418.0. HRMS ESI m/z [M + H]+ calcd for C24H21ClN3O2 418.1317, found 418.1302.
- 7-Chloro-3-(4-(((2,3-dihydrobenzofuran-5-yl)methyl)amino)benzyl)quinazolin-4(3H)-one (11d). White solid, 181 °C, 72% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.54 (s, 1H), 8.11 (d, J = 8.6 Hz, 1H), 7.72 (d, J = 1.9 Hz, 1H), 7.55 (dd, J = 8.6, 2.0 Hz, 1H), 7.24–6.90 (m, 4H), 6.64 (d, J = 8.1 Hz, 1H), 6.50 (d, J = 8.5 Hz, 2H), 6.20 (t, J = 5.9 Hz, 1H), 4.96 (s, 2H), 4.44 (t, J = 8.7 Hz, 2H), 4.10 (d, J = 5.9 Hz, 2H), 3.09 (t, J = 8.7 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 159.9, 159.0, 149.8, 148.7, 132.2, 129.6, 128.9, 127.8, 127.2, 126.8, 124.7, 123.6, 120.7, 112.7, 108.8, 71.2, 49.3, 46.7, 29.5. ESI-MS m/z: 418 [M + H]+. LC-MS Rt: 11.418 min, purity 99.1%, MS: 418.0. HRMS ESI m/z [M + H]+ calcd forC24H21ClN3O2 418.1317, found 418.1304.
- 6-Chloro-3-(4-((ferrocene-2-ylmethyl)amino)benzyl)quinazolin-4(3H)-one (11e). Red solid, Mp: 112 °C, 70% yield. 1H NMR (300 MHz, CDCl3) δ 8.29 (d, J = 2.6 Hz, 1H), 8.09 (d, J = 6.1 Hz, 1H), 7.71–7.55 (m, 3H), 7.18 (dd, J = 17.4, 7.8 Hz, 2H), 6.61 (d, J = 8.5 Hz, 2H), 5.07 (s, 3H), 4.22 (s, 3H), 4.17 (s, 6H), 4.14 (s, 2H), 3.93 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 160.1, 148.3, 146.5, 135.3, 134.6, 133.0, 129.8, 129.1, 126.2, 123.8, 123.3, 113.0, 86.0, 70.3, 69.5, 69.1, 68.3, 49.6, 43.2. ESI-MS m/z: 484 [M + H]+. LC-MS Rt: 12.287 min, purity 97.9%, MS: 483.0.
- 7-Chloro-3-(4-((ferrocene-2-ylmethyl)amino)benzyl)quinazolin-4(3H)-one (11f). Red solid, Mp: 155 °C, 75% yield. 1H NMR (300 MHz, CDCl3) δ 8.22 (d, J = 8.5 Hz, 1H), 8.07 (s, 1H), 7.66 (s, 1H), 7.40 (d, J = 8.5 Hz, 1H), 7.20 (d, J = 8.2 Hz, 2H), 6.60 (d, J = 8.2 Hz, 2H), 5.03 (s, 2H), 4.31–4.01 (m, 9H), 3.92 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 160.5, 149.0, 148.3, 147.6, 140.2, 129.8, 128.3, 127.7, 126.9, 123.8, 120.7, 112.9, 86.0, 68.5, 68.1, 67.9, 49.5, 43.2. ESI-MS m/z: 484 [M + H]+. LC-MS Rt: 12.302 min, purity 99.4%, MS: 483.1.
3.2. Biological Methods
3.2.1. Bacterial Strains
3.2.2. “CF-like” Experimental Conditions
3.2.3. Drug Susceptibility Assays of Planktonic Cells: MIC and MBC Measurements
3.2.4. Biofilm Formation Assay
3.2.5. Pyocyanin and Pyoverdine Formation Assays
3.2.6. Cytotoxicity Evaluation
3.3. Statistical Analysis
3.4. Molecular Modeling
3.4.1. Protein and Ligand Preparation
3.4.2. Molecular Docking
3.4.3. Molecular Dynamics Simulation
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Sample Availability
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Carullo, G.; Di Bonaventura, G.; Rossi, S.; Lupetti, V.; Tudino, V.; Brogi, S.; Butini, S.; Campiani, G.; Gemma, S.; Pompilio, A. Development of Quinazolinone Derivatives as Modulators of Virulence Factors of Pseudomonas aeruginosa Cystic Fibrosis Strains. Molecules 2023, 28, 6535. https://doi.org/10.3390/molecules28186535
Carullo G, Di Bonaventura G, Rossi S, Lupetti V, Tudino V, Brogi S, Butini S, Campiani G, Gemma S, Pompilio A. Development of Quinazolinone Derivatives as Modulators of Virulence Factors of Pseudomonas aeruginosa Cystic Fibrosis Strains. Molecules. 2023; 28(18):6535. https://doi.org/10.3390/molecules28186535
Chicago/Turabian StyleCarullo, Gabriele, Giovanni Di Bonaventura, Sara Rossi, Veronica Lupetti, Valeria Tudino, Simone Brogi, Stefania Butini, Giuseppe Campiani, Sandra Gemma, and Arianna Pompilio. 2023. "Development of Quinazolinone Derivatives as Modulators of Virulence Factors of Pseudomonas aeruginosa Cystic Fibrosis Strains" Molecules 28, no. 18: 6535. https://doi.org/10.3390/molecules28186535