Chemistry
General details: See Appendix A (Supplementary File).
Starting materials.
The key intermediates 2-((4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)methylene)-N-phenyl/allyl hydrazine carbothioamides 4a-l were prepared according to reported procedures44.
General procedure for the synthesis of compounds 6a-l
In a round-bottom flask, a mixture of 2-((4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)methylene)-N-phenyl/allylhydrazine-carbothioamides 4a–l (1 mmol) and chloroacetone 5 (1.2 mmol, 111 mg) was dissolved in absolute ethanol (50 mL) and catalyzed with two drops of dilute hydrochloric acid. The reaction mixture was heated under reflux for 6 h, with progress monitored by thin-layer chromatography (TLC) every hour. Upon completion, the resulting precipitate was filtered and washed multiple times with hot absolute ethanol to remove unreacted chloroacetone. This procedure afforded compounds 6a–l in good to excellent yields (75–90%), which were subsequently recrystallized from appropriate solvents.
4-Hydroxy-3-((4-methyl-3-phenylthiazol-2(3H)-ylidene)hydrazono)methyl) quinolin-2(1H)-one (6a)
Orang crystals (DMF) (80%), m.p. 250 –52 °C; 1H NMR (DMSO-d6, 400 Hz): δH = 14.10 (bs, 1H, OH), 11.43 (bs, 1H, NH), 8.35 (s, 1H, CH = N), 7.94–7.19 (m, 9 H, H-5,6,7,8 and Ph-CH),, 6.37 (s, 1H, thiazol-CH-5, H-5′), 2.18 ppm (s, 3 H, (Me, H-4a′); 13C NMR (DMSO-d6, 100 Hz): δC = 168.73 (CH = N), 165.72 (C-4), 161.89 (C = O), 150.42 (C-2′), 139.43 (C-3′), 136.88 (Ph-C), 136.81 (q-C-8a), 132,60, 130.57, 130.04, 129.40, 129.00, 123.79, 122,30 (Ph-CH, q-CH), 115.99, 115.15 (q-C-8,4a), 103.07 (q-C-3), 97.25 (C-5′), 15.01 ppm (Me, C-4a′). Anal. calcd for C20H16N4O2S: C, 63.81; H, 4.28; N, 14.88. Found: C, 63.75; H, 4.39; N, 15.01. MS-EI m/z calculated for C21H18N4O2S (376.43).
4-Hydroxy-6-methyl-3-((4-methyl-3-phenylthiazol-2(3H)-ylidene)hydrazono)-methyl)quinolin-2(1H)-one (6b)
Yellow crystals (DMF) (90%), m.p. 260 –62 °C; 1H NMR (DMSO-d6, 400 Hz): δH = 14.10 (bs, 1H, OH), 11.42 (bs, 1H, NH), 8.34 (s, 1H, CH = N), 7.94 (s, 1H, H-5), 7.92–7.18 (m, 7 H, H-7,8 and Ph-CH), 6.32 (s, 1H, thiazol-CH-5, H-5′), 2.79 (s, 3 H, (Me, H-6a), 2.12 ppm (s, 3 H, (Me, H-4a′); 13C NMR (DMSO-d6, 100 Hz): δC = 167.30 (CH = N), 165.15 (C-4), 161.79 (C = O), 150.41 (C-2′), 139.43 (C-3′), 137.30 (q-C-8a), 136.15 (Ph-C), 132.57, 130.11, 129.40, 129.11 (Ph-CH, q-C-5,7), 122.30 (q-c-8), 115.15 (q-C-4a), 103.07 (q-C-3), 97.15 (C-5′), 23.79 (Me, C-6a), 15.01 ppm (Me, C-4a′). Anal. calcd for C21H18N4O2S: C, 64.60; H, 4.65; N, 14.35. Found: C, 64.91; H, 4.79; N, 14.46. MS-EI m/z calculated for C21H18N4O2S (390.46).
4-Hydroxy-7-methyl-3-((4-methyl-3-phenylthiazol-2(3H)-ylidene)hydrazono)-methyl)quinolin-2(1H)-one (6c)
Yellow crystals (DMF) (85%), m.p. 255 –57 °C; 1H NMR (DMSO-d6, 400 Hz): δH = 14.54 (bs, 1H, OH), 11.36 (bs, 1H, NH), 8.38 (s, 1H, CH = N), 8.34 (s, 1H, H-8), 7.81–6.96 (m, 7 H, H-5,6 and Ph-CH), 6.38 (s, 1H, thiazol-CH-5, H-5′), 2.79 (s, 3 H, (Me, H-7a), 2.38 ppm (s, 3 H, (Me, H-4a′); 13C NMR (DMSO-d6, 100 Hz): δC = 166.89 (CH = N), 165.51 (C-4), 161.92 (C = O), 150.88 (C-2′), 140.89 (C-3′), 137.98 (Ph-C), 136.89 (q-C-8a), 136.76, 130.04, 129.38, 129.12, 125.47 (Ph-CH, q-CH), 114.51 (q-C-4a), 103.02 (q-C-3), 97.20 (C-5′), 23.88 (Me, C-7a), 15.02 ppm (Me, C-4a′). Anal. calcd for C21H18N4O2S: C, 64.60; H, 4.65; N, 14.35. Found: C, 64.77; H, 4.71; N, 14.11. MS-EI m/z calculated for C21H18N4O2S (390.46).
4-Hydroxy-1-methyl-3-((4-methyl-3-phenylthiazol-2(3H)-ylidene)hydrazono)-methyl)quinolin-2(1H)-one (6d)
Yellow crystals (DMF) (77%), m.p. 230 –32 °C; 1H NMR (DMSO-d6, 400 Hz): δH = 14.12 (bs, 1H, OH), 8.42 (s, 1H, CH = N), 7.72–7.32 (m, 9 H, H-5,6,7,8 and Ph-CH), 6.41 (s, 1H, thiazol-CH-5, H-5′), 3.58 (s, 3 H, N-Me), 2.14 ppm (s, 3 H, (Me, H-4a′); 13C NMR (DMSO-d6, 100 Hz): δC = 167.32 (CH = N), 164.08 (C-4), 161.32 (C = O), 150.00 (C-2′), 140.00 (C-3′), 137.29 (Ph-C), 132.95, 130.00, 129.34, 129.06, 124.29 (Ph-CH), 116.94, 115.91, 115.54 (q-CH, C-4a, C-8), 102.89 (q-C-3), 97.18 (C-5′), 29.40 (N-Me), 14.89 ppm (Me, C-4a′). Anal. calcd for C21H18N4O2S: C, 64.60; H, 4.65; N, 14.35. Found: C, 64.55; H, 4.77; N, 14.47. MS-EI m/z calculated for C21H18N4O2S (390.46).
1-Ethyl-4-hydroxy-3-((4-methyl-3-phenylthiazol-2(3H)-ylidene)hydrazono)-methyl)quinolin-2(1H)-one (6e)
Yellow crystals (DMF) (76%), m.p. 239 –41 °C; 1H NMR (DMSO-d6, 400 Hz): δH = 14.12 (bs, 1H, OH), 8.58 (s, 1H, CH = N), 7.84–7.12 (m, 9 H, H-5,6,7,8 and Ph-CH), 6.24 (s, 1H, thiazol-CH-5, H-5′), 4.92–4.81 (q, J = 14, 11.2 Hz, 2 H, N-CH2CH3), 2.74 (s, 3 H, Me, H-4a′), 1.92–1.85 ppm (t, J = 14.8 Hz, 3 H, N-CH2CH3); 13C NMR (DMSO-d6, 100 Hz): δC = 166.21 (CH = N), 164.98 (C-4), 162.80 (C = O), 150.98 (C-2′), 140.20 (C-3′), 136.86 (q-C-8a), 133.06 (Ph-C), 130.07, 129.43, 129.11, 124.23, 122.56, 115.84 (q-CH, Ph-CH), 115.56 (q-C-4a), 102.74 (q-C-3), 97.31 (C-5′), 44.26 (N-CH2CH3), 15.01 (Me, C-4a′), 13.25 ppm (N-CH2CH3). Anal. calcd for C22H20N4O2S: C, 65.33; H, 4.98; N, 13.85. Found: C, 65.29; H, 5.10; N, 13.77. MS-EI m/z calculated for C21H18N4O2S (404.48).
3-(3-Allyl-4-methylthiazol-2(3H)-ylidene)hydrazono)methyl)-4-hydroxyquinolin-2(1H)-one (6f)
Yellow crystals (DMF) (82%), m.p. 220 –22 °C; 1H NMR (DMSO-d6, 400 Hz): δH = 14.17 (bs, 1H, OH), 11.47 (bs, 1H, NH), 8.55 (s, 1H, CH = N), 7.95–7.93 (d, J = 8 Hz, 1H, H-5), 7.58–7.54 (m, 1H, H-7), 7.31–7.29 (d, J = 8 Hz, 1H, H-8), 7.24–7.20 (t, J = 8 Hz, 1H, H-6), 6.24 (s, 1H, thiazol-CH-5, H-5′), 6.01–5.94 (m, 1H, allyl-CH=), 5.21–503 (m, 2 H, allyl-CH2=), 4.56–4.55 (m, 2 H, allyl-CH2), 2.17 ppm (s, 3 H, Me, H-4a′); 13C NMR (DMSO-d6, 100 Hz): δC = 167.18 (CH = N), 165.59 (C-4), 161.97 (C = O), 149.79 (C-2′), 139.38 (C-3′) 137.23 (allyl-CH=), 123.75, 132.50 (q-CH), 123.75 (q-C-8), 115.98 (allyl-CH2=), 115.23 (q-C-4a), 103.15 (q-C-3), 96.40 (C-5′), 46.72 (allyl-CH2), 13.87 ppm (Me, C-4a′). Anal. calcd for C17H16N4O2S: C, 59.98; H, 4.74; N, 16.46. Found: C, 59.98; H, 4.74; N, 16.46. MS-EI m/z calculated for C21H18N4O2S (340.40).
3-((3-Allyl-4-methylthiazol-2(3H)-ylidene)hydrazono)methyl)-4-hydroxy-6-methylquinolin-2(1H)-one (6Â g)
Yellow crystals (DMF) (90%), m.p. 228–230 °C; 1H NMR (DMSO-d6, 400 Hz): δH = 14.13 (bs, 1H, OH), 11.39 (bs, 1H, NH), 8.54 (s, 1H, CH = N), 7.73 (s, 1H, H-5), 7.32–7.30 (d, J = 8 Hz, 1H, H-7), 7.20–7.19 (d, J = 4 Hz, 1H, H-8), 6.24 (s, 1H, thiazol-CH-5, H-5′), 5.98–5.96 (m, 1H, allyl-CH=), 5.20–5.03 (m, 2 H, allyl-CH2=), 4.55–4.54 (m, 2 H, allyl-CH2), 2.37 (s, 3 H, Me, H-6a), 2.17 ppm (s, 3 H, Me, H-4a′); 13C NMR (DMSO-d6, 100 Hz): δC = 167.14 (CH = N), 165.32 (C-4), 161.85 (C = O), 149.91 (C-2′), 137.40 (C-3′) 137.21 (allyl-CH=), 133,77 (q-C-6), 132.96, 131.34 (q-C-7,6), 123.75 (q-C-8), 116.94 (allyl-CH2=), 1115.94 (q-C-4a), 115.02 (q-C-8a), 103.14 (q-C-3), 96.36 (C-5′), 46.71 (allyl-CH2), 20.96 (Me-C-6a), 13.88 ppm (Me, C-4a′). Anal. calcd for C18H18N4O2S: C, 61.00; H, 5.12; N, 15.81. Found: C, 61.13; H, 4.97; N, 15.77. MS-EI m/z calculated for C21H18N4O2S (354.43).
3-((3-Allyl-4-methylthiazol-2(3H)-ylidene)hydrazono)methyl)-6-chloro-4-hydroxy quinolin-2(1H)-one (6Â h)
Yellow crystals (DMF) (75%), m.p. 245 –47 °C; 1H NMR (DMSO-d6, 400 Hz): δH = 14.19 (bs, 1H, OH), 11.30 (bs, 1H, NH), 8.54 (s, 1H, CH = N), 8.03 (s, 1H, H-5), 7.32–7.30 (d, J = 8 Hz, 1H, H-7), 7.21–7.19 (d, J = 8 Hz, 1H, H-8), 6.21 (s, 1H, thiazol-CH-5, H-5′), 5.98–5.96 (m, 1H, allyl-CH=), 5.21–5.03 (m, 2 H, allyl-CH2=), 4.55–4.54 (m, 2 H, allyl-CH2), 2.16 ppm (s, 3 H, Me, H-4a′); 13C NMR (DMSO-d6, 100 Hz): δC = 167.06 (CH = N), 165.55 (C-4), 162.01 (C = O), 151.79 (C-2′), 141.72 (q-C-6), 137.40 (C-3′), 138.46 (allyl-CH=), 132.70, 129.05 (q-C-5,6), 123.11 (q-C-8), 117.56 (allyl-CH2=), 117.30 (q-C-4a), 117.04 (q-C-8a), 103.10 (q-C-3), 95.89 (C-5′), 47.04 (allyl-CH2), 13.46 ppm (Me, C-4a′). Anal. calcd for C17H15ClN4O2S: C, 54.47; H, 4.03; N, 14.95. Found: C, 54.33; H, 3.97; N, 15.10. MS-EI m/z calculated for C17H15ClN4O2S (374.84).
3-((3-Allyl-4-methylthiazol-2(3H)-ylidene)hydrazono)methyl)-4-hydroxy-7-methyl quinolin-2(1H)-one (6i)
Yellow crystals (DMF) (88%), m.p. 225 –27 °C; 1H NMR (DMSO-d6, 400 Hz): δH = 14.56 (bs, 1H, OH), 11.39 (bs, 1H, NH), 8.53 (s, 1H, CH = N), 7.82 (s, 1H, H-8), 7.81–6.97 (m, 2H, H-5,6), 6.95 (s, 1H, thiazol-CH-5, H-5′), 6.01–5.93 (m, 1H, allyl-CH=), 5.21–5.03 (m, 2 H, allyl-CH2=), 4.54–4.53 (m, 2 H, allyl-CH2), 2.78 (s, 3 H, Me, H-7a), 2.16 ppm (s, 3 H, Me, H-4a′); 13C NMR (DMSO-d6, 100 Hz): δC = 166.96 (CH = N), 166.75 (C-4), 162.63 (C = O), 150.25 (C-2′), 140.82 (C-3′) 139.55 (q-C-7), 137.94 (allyl-CH=), 131.81, 125.77 (q-C-5,6), 123.77 (q-C-8), 116.91 (allyl-CH2=), 115.69 (q-C-4a), 114.49 (q-C-8a), 103.10 (q-C-3), 96.31 (C-5′), 46.67 (allyl-CH2), 21.95 (Me-C-6a), 13.87 ppm (Me, C-4a′). Anal. calcd for C18H18N4O2S: C, 61.00; H, 5.12; N, 15.81. Found: C, 61.17; H, 5.25; N, 15.99. MS-EI m/z calculated for C18H18N4O2S (354.43).
3-((3-Allyl-4-methylthiazol-2(3H)-ylidene)hydrazono)methyl)-4-hydroxy-8-methyl quinolin-2(1H)-one (6j)
Yellow crystals (DMF) (87%), m.p. 241 –43 °C; 1H NMR (DMSO-d6, 400 Hz): δH = 14.12 (bs, 1H, OH), 10.60 (bs, 1H, NH), 8.58 (s, 1H, CH = N), 7.84–7.82 (d, J = 7.6 Hz, 1H, H-5), 7.43–7.41 (d, J = 7.2 Hz, 1H, H-7), 7.16–7.12 (t, J = 7.6 Hz, 1H, H-6), 6.24 (s, 1H, thiazol-CH-5, H-5′), 5.98–5.96 (m, 1H, allyl-CH=), 5.22–5.04 (m, 2 H, allyl-CH2=), 4.56–4.55 (m, 2 H, allyl-CH2), 2.42 (s, 3 H, Me, H-8a), 2.17 ppm (s, 3 H, Me, H-4a′); 13C NMR (DMSO-d6, 100 Hz): δC = 167.25 (CH = N), 165.75 (C-4), 162.14 (C = O), 150.25 (C-2′), 140.44 (C-3′) 139.55 (q-C-8), 137.94 (allyl-CH=), 132.97, 131.81 (q-C-5,6), 123.77 (q-C-8), 116.81 (allyl-CH2=), 115.25 (q-C-4a), 114.10 (q-C-8a), 102.49 (q-C-3), 96.14 (C-5′), 46.49 (allyl-CH2), 22.13 (Me-C-6a), 13.37 ppm (Me, C-4a′). Anal. calcd for C18H18N4O2S: C, 61.00; H, 5.12; N, 15.81. Found: C, 61.14; H, 5.18; N, 15.77. MS-EI m/z calculated for C18H18N4O2S (354.43).
3-((3-Allyl-4-methylthiazol-2(3H)-ylidene)hydrazono)methyl)-4-hydroxy-1-methyl quinolin-2(1H)-one (6k)
Yellow crystals (DMF) (75%), m.p. 210 –12 °C; 1H NMR (DMSO-d6, 400 Hz): δH = 14.16 (bs, 1H, OH), 8.61 (s, 1H, CH = N), 8.06–7.33 (m, 3 H, H-5,6,7,8), 6.23 (s, 1H, thiazol-CH-5, H-5′), 5.97 (m, 1H, allyl-CH=), 5.21–5.03 (m, 2 H, allyl-CH2=), 4.56-4 (bs, 2 H, allyl-CH2), 3.61 (s, 3 H, N-Me), 2.17 ppm (s, 3 H, Me, H-4a′); 13C NMR (DMSO-d6, 100 Hz): δC = 167.37 (CH = N), 164.08 (C-4), 161.32 (C = O), 150.33 (C-2′), 137.25 (allyl-CH=), 132.98 (q-C-8a), 124.19, 122.52 (q-C-5,6), 116.93 (allyl-CH2=), 115.94 (q-C-8), 115.54 (q-C-4a), 102.83 (q-C-3), 96.48 (C-5′), 46.73 (allyl-CH2), 29.44 (N-Me), 13.88 ppm (Me, C-4a′). Anal. calcd for C18H18N4O2S: C, 61.00; H, 5.12; N, 15.81. Found: C, 60.97; H, 4.99; N, 15.97. MS-EI m/z calculated for C18H18N4O2S (354.43).
3-((3-Allyl-4-methylthiazol-2(3H)-ylidene)hydrazono)methyl)-1-ethyl-4-hydroxy quinolin-2(1H)-one (6Â L)
Yellow crystals (DMF) (76%), m.p. 216 –18 °C; 1H NMR (DMSO-d6, 400 Hz): δH = 14.18 (bs, 1H, OH), 8.62 (s, 1H, CH = N), 8.06–7.32 (m, 3 H, H-5,6,7,8), 6.25 (s, 1H, thiazol-CH-5, H-5′), 5.98–5.94 (m, 1H, allyl-CH=), 5.20–5.04 (m, 2 H, allyl-CH2=), 4.56–4.25 (m, 4 H, allyl-CH2, N-CH2CH3), 2.18 (s, 3 H, Me, H-4a′), 1.06 (t, 3 H, N-CH2CH3); 13C NMR (DMSO-d6, 100 Hz): δC = 167.11 (CH = N), 165.50 (C-4), 161.97 (C = O), 149.38 (C-2′), 137.23 (allyl-CH=), 132.96 (q-C-8a), 132.50, 124.19, 122.52, 123.75 (q-C-5,6,7,8), 117.23 (allyl-CH2=), 115.98 (q-C-4a), 102.96 (q-C-3), 96.15 (C-5′), 46.72 (allyl-CH2), 46.15 (N-CH2CH3), 14.23 (N-CH2CH3), 13.87 ppm (Me, C-4a′). Anal. calcd for C19H20N4O2S: C, 61.94; H, 5.47; N, 15.21. Found: C, 62.09; H, 5.55; N, 15.13. MS-EI m/z calculated for C18H18N4O2S (368.45).
Biology
Cell viability assay
The effects of compounds 6a–l on MCF-10 A normal cell viability were assessed using an MTT colorimetric assay45,46 after 4 days of incubation. See Appendix A for detailed experimental procedures.
Antiproliferative assay
The antiproliferative activity of the novel compounds 6a–l was evaluated against three human cancer cell lines—MCF-7 (breast), HepG2 (liver), and HCT-116 (colon), alongside the WI-38 normal human fibroblast cell line. All cell lines were purchased from ATCC (American Type Cell Culture) via the Holding Company for Biological Products and Vaccines (VACSERA) in Cairo, Egypt. Growth inhibition was measured using the MTT assay47, and IC50 values were calculated from dose-response curves. Data represent the mean of at least two independent experiments performed in triplicate (see Appendix A for full details).
EGFR inhibitory assay
The inhibitory efficacy of compounds 6a-c against EGFR kinase was evaluated using a commercial EGFR Kinase Assay Kit (Catalog #40321, San Diego, CA)48,49. Their IC50 values were determined and compared directly to Erlotinib as the reference standard. Detailed experimental protocols are outlined in Appendix A.
HER-2 inhibitory assay
The inhibitory efficacy of compounds 6a-c against HER-2 was evaluated using a commercial HER-2 TK Assay Kit27,50. Their IC50 values were determined and compared to Lapatinib as the reference standard. Detailed experimental protocols are outlined in Appendix A.
VEGFR-2 inhibitory assay
The in vitro inhibitory efficacy of compounds 6a–c against VEGFR-2 was evaluated using a VEGFR-2 Kinase Assay Kit, with Sorafenib as the reference compound51,52. The resulting IC50 values were determined to quantify potency. Full experimental protocols are available in Appendix A.
Apoptotic markers assay
Compound 6b was evaluated for its ability to activate caspase-3/7 using the Caspase-Glo 3/7 luminescent assay60 in the HepG-2 hepatocellular carcinoma cell line, with staurosporine as the reference standard. Detailed experimental protocols are outlined in Appendix A.
Docking study
To investigate potential binding modes, the three-dimensional crystal structures of EGFR (PDB ID: 1M17)64,65, HER-2 (PDB ID: 3PPO)67, and VEGFR-2 (PDB ID: 3WZE)68 were retrieved from the RCSB Protein Data Bank. The structure of compound 6b was sketched and geometrically optimized using the Avogadro molecular editor. Target proteins were prepared using AutoDock Tools by removing co-crystallized ligands and water molecules, then adding polar hydrogen atoms and Kollman charges. Molecular docking simulations were executed via AutoDock Vina, and the top-scoring binding conformations were visualized and analyzed using the Discovery Studio Visualizer.
