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Developing multitarget coumarin based anti-breast cancer agents: synthesis and molecular modeling study – Scientific Reports


Chemistry

Melting points were determined on Stuart melting point apparatus and are uncorrected. Microanalyses were performed at Cairo University and performed on a Perkin-Elmer 240 elemental analyzer for C, H, and N elements, and the results were within the acceptable range of the theoretical values. 1H and 13C NMR were performed at Mansoura University and recorded on Brucker 400 MHz spectrometer and 100 MHz spectrometer, respectively. Chemical shifts are expressed in δ ppm with reference to TMS. Using a Nicolet iS10 infrared spectrometer, IR spectra were recorded at Mansoura University. Mass spectral analyses were performed on Thermo SCIENTIFIC DCQII at Azhar University. All the used chemicals and reagents were purchased from Aldrich Chemicals Co, USA.

General procedure for the preparation of (7-hydroxy-2-oxo-2H-chromen-4-yl) methyl benzoate derivatives (2–4)

To a solution of 1 (0.45 g, 2 mmol) in DMF (8 mL), the appropriate sodium salt of (un)substituted benzoic acid (2 mmol) was added. The reaction mixture was heated at 90 °C overnight, then cooled and poured over ice-water. The separated solid products were filtered off, dried and then recrystallized from EtOAc/MeOH (4:1).

(7-Hydroxy-2-oxo-2H-chromen-4-yl)methyl benzoate (2).

White solid; Yield 72%; mp 248–250 °C. IR (KBr) υmax (cm−1): 3154 (OH); 2943 and 2840 (CH aliphatic); 1720 and 1689 (C=O); 1614 and 1568 (C=C); 1136 (C–O–C). 1H NMR (400 MHz, DMSO-d6): δ 10.72 (s, 1H, OH, D2O exchangeable), 8.08 (d, 2H, J = 7.5 Hz, Phenyl-C2-H and C6-H), 7.73 (t, J = 7.2 Hz, 1H, Phenyl-C4-H), 7.68 (d, J = 8.6 Hz, 1H, C5-H), 7.59 (t, 2H, J = 7.6 Hz, Phenyl-C3-H and C5-H), 6.85 (dd, 1H, J1 = 8.6, J2 = 1.5 Hz, C6-H), 6.79 (d, 1H, J = 1.5Hz, C8-H), 6.29 (s, 1H, C3-H), 5.60 (s, 2H, OCH2). 13C NMR (100 Hz, DMSO-d6), δ (ppm): δ 165.6 (C=O), 162.0 (C2=O), 160.6 (C7), 155.6 (C4), 150.9 (C8a), 134.3 (Phenyl-C4), 130.0 (two Phenyl-C2 and C6), 129.5 (two Phenyl-C3 and -C5), 129.4 (Phenyl-C1), 126.6 (C5), 113.7 (C6), 110.0 (C4a), 108.8 (C3), 103.0 (C8), 62.5 (CH2). MS (m/z %): 296.22 (1.39, M+), 294.85 (M-H+), 105.18 (100). Elemental analysis for C17H12O5, calcd.: C, 68.92; H, 4.08; Found: C, 68.78; H, 4.26.

(7-Hydroxy-2-oxo-2H-chromen-4-yl)methyl 4-chlorobenzoate (3).

White solid; Yield 80%; mp 173–175 °C. IR (KBr) υmax (cm−1): 3174 (OH); 2930 and 2846 (CH aliphatic); 1721 and 1690 (C=O); 1599 (C=C); 1130 (C–O–C). 1H NMR (400 MHz, DMSO-d6): δ 10.69 (s, 1H, OH, D2O exchangeable), 7.95 (d, 2H, J = 8.3 Hz, Phenyl-C2-H and C6-H), 7.58 (d, 2H, J = 8.3 Hz, Phenyl-C3-H and C5-H), 7.69 (d, 1H, J = 8.7 Hz, C5-H), 6.86 (dd, 1H, J1 = 8.7, J2 = 1.3 Hz, C6-H), 6.77 (d, 1H, J = 1.3 Hz, C8-H), 6.43 (s, 1H, C3–H), 5.50 (s, 2H, OCH2). 13C NMR (100 Hz, DMSO-d6), δ (ppm) 166.9 (C=O), 161.7 (C2=O), 160.8 (C7), 155.7 (C4), 151.5 (C8a), 138.4 (Phenyl-C4), 131.6 (Phenyl-C2), 131.6 (Phenyl-C6), 129.9 (Phenyl-C1), 129.3 (two Phenyl-C3 and C5), 127.1 (C5), 113.6 (C6), 111.5 (C4a), 109.9 (C3), 102.9 (C8), 62.5 (CH2). MS (m/z %): 332.03 (22.66, M + 2), 330.50 (24.62, M+), 252.22 (100). Elemental analysis for C17H11ClO5, calcd.: C, 61.74; H, 3.35; Found: C, 61.97; H, 3.51.

(7-Hydroxy-2-oxo-2H-chromen-4-yl)methyl 2-methoxybenzoate (4).

White solid; Yield 64%; mp 268–270 °C. IR (KBr) υmax (cm−1): 3260 (OH); 3097 (CH Aromatic), 2999, 2941 and 2836 (CH aliphatic); 1721 and 1693 (C=O); 1612 and 1567 (C=C); 1130 (C–O–C). 1H NMR (400 MHz, DMSO-d6): δ 10.69 (s, 1H, C7–OH, D2O exchangeable), 7.77 (d, 1H, J = 7.5 Hz, Phenyl-C6-H), 7.69 (d, 1H J = 8.7 Hz, C5–H), 7.62 (t, 1H, J = 7.9 Hz, Phenyl-C4-H),7.22 (d, 1H, J = 8.4 Hz, Phenyl-C3-H), 7.08 (t, 1H, J = 7.5 Hz, Phenyl-C5-H), 6.85 (dd, 1H, J1 = 8.7, J2 = 1.6 Hz, C6-H), 6.78 (d, 1H, J = 1.6 Hz, C8-H), 6.37 (s, 1H, C3-H), 5.55 (s, 2H, OCH2), 3.88 (s, 3H, CH3). 13C NMR (100 Hz, DMSO-d6), δ (ppm) δ 165.6 (C=O), 162.7 (C2=O), 161.8 (C7), 160.6 (Phenyl-C2), 158.8 (C4), 155.5 (C8a), 151.1 (Phenyl-C4), 134.7 (Phenyl-C6), 131.6 (C5), 126.5 (Phenyl-C1), 120.7 (Phenyl-C5), 119.3 (C6), 113.5 ((Phenyl-C3), 113.1 (C4a), 109.6 (C3), 108.6 (C8), 62.3 (CH2), 56.2 (OCH3). MS (m/z %): 326.24 (4.06, M+), 325.61 (10.6, M-H+), 44.22 (100). Elemental analysis for C18H14O6, calcd.: C, 66.26; H, 4.32; Found: C, 66.45; H, 4.59.

General procedure for the preparation of 4-(chloromethyl)-7-(2-oxo-2-phenylethoxy)-2H-chromen-2-one derivatives (5, 6)

To a solution of 1 (0.32 g, 1.5 mmol) in acetone (10 mL), anhydrous K2CO3 (0.29 g, 3 mmol) and the appropriate phenacyl bromide (1.5 mmol) were added. The reaction mixture was kept under stirring at room temperature for about 4 h and was monitored by TLC (petroleum ether/ethyl acetate, 7:3 v/v). After filtration, the solution was concentrated under vacuum. The obtained residue was recrystallized from a mixture of EtOAc/acetone (4:1).

4-(Chloromethyl)-7-(2-oxo-2-phenylethoxy)-2H-chromen-2-one (5).

White solid; Yield 63%; mp 180–182 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.06 (d, 2H, J = 7.2 Hz, Phenyl-C2-H and C6-H), 7.79 (d, 1H, J = 8.9 Hz, C5-H), 7.73 (t, 1H, J = 7.4 Hz, Phenyl-C4-H), 7.60 (t, 2H, J = 7.6 Hz, Phenyl-C3-H and C5-H), 7.16 (d, 1H, J = 2.5 Hz, C8-H), 7.10 (dd, 1H, J1 = 8.9, J2 = 2.5 Hz, C6-H), 6.52 (s, 1H, C3-H), 5.79 (s, 2H, OCH2), 5.02 (s, 2H, CH2Cl). 13C NMR (100 Hz, DMSO-d6), δ (ppm) 194.8 (C=O), 161.8 (C2=O), 160.4 (C7), 155.5(C8a), 151.2 (C4), 134.6 (Phenyl-C4), 134.4 (Phenyl-C1), 129.3 (two Phenyl-C2 and-C6), 128.3 (two Phenyl-C3 and -C5), 126.8 (C5), 113.2 (C6), 112.6 (C3), 111.2 (C4a), 102.45 (C8), 71.07 (CH2), 41.8 (CH2). MS (m/z %): 328.88 (19.45, M+), 330.20 (15.51, M+ + 2), 329.80 (10.36, M+ + 1), 125.26 (100). Elemental analysis for C18H13ClO4, calcd.: C, 65.76; H, 3.99; Found: C, 66.02; H, 4.13.

4-(Chloromethyl)-7-(2-(4-chlorophenyl)-2-oxoethoxy)-2H-chromen-2-one (6)

Buff solid; Yield 66%; mp 195–197 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.07 (d, 2H, J = 8.4 Hz, Phenyl-C2-H and C6–H), 7.79 (d, 1H, J = 8.8 Hz, C5–H), 7.69 (d, 2H, J = 8.4 Hz, Phenyl-C3-H and C5-H), 7.17 (d, 1H, J = 2.2 Hz, C8–H), 7.11 (dd, 1H, J1 = 8.8 Hz, J2 = 2.2 Hz, C6-H), 6.53 (s, 1H, C3-H), 5.77 (s, 2H, OCH2), 5.02 (s, 2H, CH2Cl). 13C NMR (100 Hz, DMSO-d6): δ (ppm) 193.3 (C=O), 161.7 (C2=O), 160.4 (C7), 155.5 (C8a), 151.2 (C4), 139.2 (Phenyl-C4), 133.3 (Phenyl-C1), 130.3 (two phenyl-C2 and-C6), 129.4 (two phenyl-C3 and -C5), 126.8 (C5), 113.2 (C6), 112.6 (C3), 111.2 (C4a), 102.4 (C8), 71.1 (CH2), 41.8 (CH2). MS (m/z %): 362.08 (2.37, M+), 363.81 (4.51, M+ + 2), 139.18 (100). Elemental analysis for C18H12Cl2O4, calcd.: C, 59.53; H, 3.33; Found: C, 59.74.; H, 3.58.

General Procedure for the Preparation of 7-[(2-oxo-2-phenylethoxy)-2H-chromen-4-yl] methyl benzoate derivatives (7–12)

Pathway (A)

The same procedure used for the synthesis of 5 and 6 was adopted, except that 7-hydroxycoumarin derivatives 2–4 were used instead of 1.

Pathway (B)

The same procedure used for the synthesis of derivatives 24 was adopted, except that coumarin derivatives 5 and 6 were used instead of 1.

Pathway (C)

To a solution of 1 (0.32 g, 1.5 mmol) in DMF (10 mL), the appropriate sodium salt of (un)substituted benzoic acid (1.5 mmol), anhydrous K2CO3 (0.29 g, 3 mmol) and the appropriate phenacyl bromide (1.5 mmol) were added. The reaction mixture was kept under stirring at room temperature for about 4 h, heated at 90 °C overnight, then cooled and poured over ice-water. The separated solid products were found to be an impure mixture of different products with very low yields.

[2-Oxo-7-(2-oxo-2-phenylethoxy)-2H-chromen-4-yl]methyl benzoate (7)

Buff solid; Yield 66%; mp 242–244 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.07 (d, 2H, J = 7.3 Hz, Phenyl C2–H and C6–H), 7.96 (s, 2H, Phenyl C2`-H and C6’–H), 7.73 (t, 2H, J = 7.4 Hz, Phenyl C4 -H and C4’-H) 7.68 (d, 1H, J = 8.7 Hz, C5-H), 7.59–7.57 (m, 4H, Phenyl C3–H, C5–H, C3’–H and C5’–H), 6.85 (dd, 1H, J1 = 8.7 Hz, J2 = 2.0 Hz, C6-H), 6.78 (d, 1H, J = 2.0 Hz, C8–H), 6.28 (s, 1H, C3–H), 5.79 (s, 2H, COCH2), 5.60 (s, 2H, OCH2). 13C NMR (100 Hz, DMSO-d6), δ (ppm) 196.4 (C=O), 165.6 (C=O), 162.9 (C2=O), 160,6 (C7), 155.6 (C4), 150.9 (C8a), 134.3 (two Phenyl-C4’ and Phenyl-C1’), 133.10 (two Phenyl-C1 and Phenyl-C4), 129.9 (two Phenyl-C2 and C6), 129.5 (two Phenyl-C2` and C6`), 129.3 (two Phenyl-C3’ and C5’), 128.4 (two Phenyl-C3 and C5), 126.6 (C5), 113.7 (C6), 109.7 (C4a), 108.8 (C3), 102.9 (C8), 70.0 (COCH2), 62.5 (OCH2). MS (m/z %): 414.14 (23.61, M+), 413.24 (6.94, M-H+), 105.12 (100). Elemental analysis for C25H18O6, calcd.: C, 72.46; H, 4.38; Found: C, 72.23; H, 4.57.

{7-[2-(4-Chlorophenyl)-2-oxoethoxy]-2-oxo-2H-chromen-4-yl}methyl benzoate (8)

White solid; Yield 51%; mp 245–247 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.08 (d, 2H, J = 8.7 Hz, Phenyl C2–H and C6–H), 8.05 (d, 2H, J = 8.9 Hz, Phenyl C2’–H and C6’–H), 7.78 (d, 1H, J = 8.9 Hz, C5–H), 7.73 (t, 1H, J = 7.4 Hz, Phenyl C4–H), 7.66 (d, 2H, J = 8.6 Hz, Phenyl C3’–H, C5’–H), 7.61 (t, 2H, J = 7.7 Hz, Phenyl C3–H, C5–H,), 7.18 (d, 1H, J = 2.3 Hz, C8–H), 7.09 (dd, 1H, J1 = 8.6 Hz, J2 = 2.3 Hz, C6-H), 6.41 (s, 1H, C3-H), 5.81 (s, 2H, COCH2), 5.64 (s, 2H, OCH2). 13C NMR (100 Hz, DMSO-d6): δ (ppm): δ 194.2 (C=O), 164.8 (C=O), 161.8 (C2=O), 160.4 (C7), 155.4 (C4), 150.5 (C8a), 136.3 (Phenyl-C4`), 134.7 (Phenyl-C1`), 134.4 (Phenyl-C1), 131.8 (Phenyl-C4), 129.7 (two Phenyl-C2`and C6`), 129.3 (two Phenyl-C2 and C6), 128.4 (two Phenyl-C3`and C5`), 128.3 (two Phenyl-C3 and C5), 126.4 (C5), 113.4 (C6), 111.1 (C4a), 110.1 (C3), 102.4 (C8), 71.1 (COCH2), 62.8 (OCH2). MS (m/z %): 447.97 (12.78, M+), 450.63 (32.38, M+ + 2), 43.19 (100). Elemental analysis for C25H17ClO6, calcd.: C, 66.90; H, 3.82; Found: C, 67.12; H, 3.97.

[2-Oxo-7-(2-oxo-2-phenylethoxy)-2H-chromen-4-yl]methyl 4-chlorobenzoate (9)

White crystals; Yield 77%; mp 217–219 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.10–8.07 (m, 4H, Phenyl C2–H, C6–H, C2’–H and C6’–H), 7.78 (d, 1H, J = 8.9 Hz, C5–H), 7.73 (t, 1H, J = 7.4 Hz, Phenyl C4’–H), 7.66 (d, 2H, J = 8.5 Hz, Phenyl C3–H and C5–H), 7.61 (t, 2H, J = 7.6 Hz, Phenyl C3’–H and C5’–H), 7.18 (d, 1H, J = 2.4 Hz, C8-H), 7.09 (dd, 1H, J1 = 8.9 Hz, J2 = 2.4 Hz, C6-H), 6.41 (s, 1H, C3–H), 5.81 (s, 2H, COCH2), 5.64 (s, 2H, OCH2). 13C NMR (100 Hz, DMSO-d6): δ 194.2 (C=O), 164.8 (C=O), 161.8 (C2=O), 160.4 (C7), 155.3 (C4), 150.5 (C8a), 139.2 (Phenyl-C4), 134.6 (Phenyl-C4’), 134.5 (Phenyl-C1’), 131.8 (Phenyl-C2), 131.8 (Phenyl-C6), 129.7 (Phenyl-C2’), 129.7 (Phenyl-C6’), 129.4 (Phenyl-C3’), 129.4 (Phenyl-C5’), 128.4 (Phenyl-C3), 128.4 (Phenyl-C5), 128.2 (Phenyl-C1), 126.4 (C5), 113.4 (C6), 111.1 (C4a), 110.1 (C3), 102.4 (C8), 71.1 (CH2), 62.8 (CH2). MS (m/z %): 448.41 (38.92, M+), 450.14 (22.31, M+ + 2), 447.68 (28.44, M+-1), 384.02 (100). Elemental analysis for C25H17ClO6, calcd.: C, 66.90; H, 3.82; Found: C, 66.83; H, 4.05.

{7-[2-(4-Chlorophenyl)-2-oxoethoxy]-2-oxo-2H-chromen-4-yl}methyl 4-chlorobenzoate (10)

Buff crystals; Yield 51%; mp 228–230 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.09–8.07 (m, 4H, Phenyl C2–H, C6–H, C2`-H and C6`-H), 7.78 (d, 1H, J = 8.9 Hz, C5–H), 7.68–7.65 (m, 4H, Phenyl C3–H, C5–H, C3’–H and C5’–H), 7.20 (d, 1H, J = 2 Hz, C8–H), 7.09 (dd, 1H, J1 = 8.9, J2 = 2.0 Hz, C6–H), 6.41 (s, 1H, C3–H), 5.78 (s, 2H, COCH2), 5.64 (s, 2H, OCH2). 13C NMR (100 Hz, DMSO-d6): δ 193.3 (C=O), 164.8 (C=O), 161.8 (C2=O), 160.4 (C7), 155.6 (C4), 150.7 (C8a), 139.2 (two Phenyl-C4 and C4’), 131.8 (two Phenyl-C2 and C6), 130.4 (two Phenyl- C2’ and C6’), 129.6 (two Phenyl-C3’ and C5’), 129.4 (two Phenyl-C3 and C5), 128.3 (two Phenyl-C1 and C1’), 126.5 (C5), 113.9 (C6), 113.4 (C4a), 103.1 (C8), 102.4 (C3), 71.56 (CH2), 62.8 (CH2). MS (m/z %): 483.13 (35.54, M+), 485.01 (15.28, M+ + 2), 141.69 (100). Elemental analysis for C25H16Cl2O6, calcd.: C, 62.13; H, 3.34; Found: C, 61.96; H, 3.56.

[2-Oxo-7-(2-oxo-2-phenylethoxy)-2H-chromen-4-yl]methyl 2-methoxybenzoate (11)

White solid; Yield 45%; mp 220–222 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.06 (d, 2H, J = 7.7 Hz, Phenyl C2’–H and C6’–H), 7.78 (d, 1H, J = 6.4 Hz, C5–H), 7.76 (d, 1H, J = 8.7 Hz, Phenyl C6–H), 7.73 (m, 1H, Phenyl C4’–H), 7.64–7.62 (m, 3H, Phenyl C3’–H and C5’–H and C4’H), 7.23 (d, 1H, J = 7.2 Hz, C6–H), 7.18 (s, 1H, C8-H), 7.09 (dd, 2H, J1 = 13.7, J2 = 6.5 Hz, Phenyl C3-H and C5-H), 6.46 (s, 1H, C3-H), 5.81 (s, 2H, COCH2), 5.60 (s, 2H, OCH2), 3.88 (s, 3H, OCH3). 13C NMR (100 Hz, DMSO-d6): δ (ppm) 194.2 (C=O), 165.6 (C=O), 161.7 (C2=O), 160.4 (C7), 158.9 (Phenyl-C2), 155.3 (C4), 151.0 (C8a), 134.7 (Phenyl-C4), 134.6 (Phenyl-C4`), 134.4 (phenyl-C1`), 131.6 (phenyl-C6), 129.3 (Phenyl-C2`), 129.3 (Phenyl-C6`), 128.4 (Phenyl-C3`), 128.4 (Phenyl-C5`), 126.4 (C5), 120.8 (Phenyl-C1), 119.4 (Phenyl-C5), 113.2 (Phenyl-C3), 113.1 (C6), 111.1 (C4a), 109.8 (C3), 102.4 (C8), 71 (COCH2), 62.4 (OCH2), 56.28 (OCH3). MS (m/z %): 444.46 (9.60, M+), 77.32 (100). Elemental analysis for C26H20O7, calcd.: C, 70.27; H, 4.54; Found: C, 70.44; H, 4.63.

{7-[2-(4-Chlorophenyl)-2-oxoethoxy]-2-oxo-2H-chromen-4-yl}methyl 2-methoxybenzoate (12)

White solid; Yield 51%; mp 218–220 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.07 (d, 2H, J = 8.2 Hz, Phenyl C2’–H and C6’–H), 7.78 (d, 1H, J = 6.8 Hz, C5–H), 7.74 (d, 1H, J = 7.7 Hz, Phenyl C6–H), 7.69 (d, 2H, J = 8.3 Hz, Phenyl C3’–H and C5’–H), 7.62 (t, 1H, J = 8.0 Hz, Phenyl C4–H), 7.22 (d, 1H, J = 7.7 Hz, C6–H), 7.19 (s, 1H, C8–H), 7.09 (dd, 2H, J1 = 11.3 Hz, J2 = 7.7 Hz, Phenyl C3–H and C5–H), 6.46 (s, 1H, C3–H), 5.78 (s, 2H, COCH2), 5.60 (s, 2H, OCH2), 3.87 (s, 3H, OCH3). 13C NMR (100 Hz, DMSO-d6): δ (ppm) 193.3 (C=O), 165.6 (C=O), 161.6 (C2=O), 160.4 (C7), 158.9 (Phenyl-C2`), 155.3 (C4), 150.9 (C8a), 139.2 (Phenyl-C4`), 134.7 (Phenyl-C4), 133.3 (Phenyl-C1`), 131.7 (Phenyl-C6), 131.6 (Phenyl-C2`), 130.3 (Phenyl-C6`), 129.4 (Phenyl-C3), 126.5 (Phenyl-C3`), 126.4 (Phenyl-C5`), 120.8 (C5), 119.4 (Phenyl-C1), 113.2 (Phenyl-C5), 113.1 (C6), 111.1 (C4a), 109.8 (C3), 102.3 (C8), 71.1 (COCH2), 62.3 (OCH2), 56.2 (OCH3). MS (m/z %): 478.24 (8.62, M+), 479.23 (18.46, M+ + 1), 480.20 (12.17, M+ + 2), 235.15 (100). Elemental analysis for C26H19ClO7, calcd.: C, 65.21; H, 4.00; Found: C, 65.03; H, 4.19.

General procedure for the preparation of (7-acetoxy-2-oxo-2H-chromen-4-yl)methyl benzoate derivatives (13–15)

The appropriate 2–4 derivative (1.5 mmol) was added to a mixture of acetic anhydride and acetic acid (1:1, 10 mL). The reaction mixture was refluxed for 12 h, cooled and poured onto ice. The crude off-white solid was then filtered, washed with water, and dried. The obtained solid was recrystallized from ethanol.

(7-Acetoxy-2-oxo-2H-chromen-4-yl)methyl benzoate (13)

White solid; Yield 71%; mp 192–194 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.09 (d, 2H, J = 7.3 Hz, Phenyl C2–H and C6–H), 7.92 (d, 1H, J = 8.6 Hz, C5–H), 7.73 (t, 1H, J = 7.1 Hz, Phenyl C4–H), 7.59 (t, 2H, J = 7.4 Hz, Phenyl C3–H and C5–H), 7.37 (s, 1H, C8–H), 7.25 (d, 1H, J = 8.5 Hz, C6–H), 6.56 (s, 1H, C3–H), 5.66 (s, 2H, OCH2), 2.33 (s, 3H, COCH3). 13C NMR (100 Hz, DMSO-d6): δ (ppm) 169.2 (C=O), 165.5 (C=O), 159.8 (C2=O), 154.2 (C7), 153.6 (C4), 150.2 (C8a), 134.3 (phenyl-C4), 129.9 (two Phenyl-C2 and C6), 129.4 (two Phenyl-C3 and C5), 129.3 (phenyl-C1), 126.4 (C5), 119.2 (C6), 115.3 (C4a), 112.7 (C8), 110.9 (C3), 62.5 (OCH2), 21.3 (COCH3). MS (m/z %): 338.55 (24.52, M+), 337.37 (18.27, M+-1), 280.45 (100). Elemental analysis for C19H14O6, calcd.: C, 67.45; H, 4.17; Found: C, 67.63; H, 4.39.

(7-Acetoxy-2-oxo-2H-chromen-4-yl)methyl 4-chlorobenzoate (14).

Off-White solid; Yield 88%; mp 216–218 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.09 (d, 2H, J = 8.2 Hz, Phenyl C2–H and C6-H), 7.92 (d, 1H, J = 7.0 Hz, C5–H), 7.67 (d, 2H, J = 6.6 Hz, Phenyl C3–H and C5–H), 7.37 (s, 1H, C8–H), 7.25 (d, 1H, J = 5.2 Hz, C6–H), 6.60 (s, 1H, C3–H), 5.67 (s, 2H, OCH2), 2.34 (s, 3H, COCH3). 13C NMR (100 Hz, DMSO-d6): δ (ppm) 169.4 (C=O), 165.4 (C=O), 159.4 (C2=O), 154.3 (C7), 153.6 (C4), 149.9 (C8a), 134.6 (phenyl-C4), 131.8 (two Phenyl-C2 and C6), 129.6 (two Phenyl-C3 and C5), 128.2 (phenyl-C1), 126.4 (C5), 119.2 (C6), 115.5 (C4a), 112.8 (C8), 110.9 (C3), 62.7 (OCH2), 21.4 (COCH3). MS (m/z %): 372.14 (43.76, M+), 374.07 (63.76, M+ + 2), 312.09 (100). Elemental analysis for C19H13ClO6, calcd C, 61.22; H, 3.52; Found C, 61.39; H, 3.68.

(7-Acetoxy-2-oxo-2H-chromen-4-yl)methyl 2-methoxybenzoate (15).

White solid; Yield 74%. mp 186–188 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.89 (d, 1H, J = 8.5 Hz, Phenyl C6–H), 7.79 (d, 1H, J = 6.7 Hz, C5–H), 7.62 (t, 1H, J = 7.5 Hz, Phenyl C4–H), 7.35 (s, 1H, C8–H), 7.25 (d, 1H, J = 7.9 Hz, C6–H), 7.22 (d, 1H, J = 8.8 Hz, Phenyl C3–H), 7.08 (t, 1H, J = 7.4 Hz, Phenyl C5–H), 6.63 (s, 1H, C3–H), 5.62 (s, 2H, OCH2), 3.88 (s, 3H, OCH3), 2.33 (s, 3H, COCH3). 13C NMR (100 Hz, DMSO-d6): δ (ppm) 169.3 (C=O), 165.6 (C=O), 158.9 (C2=O), 155.5 (phenyl-C2), 154.2 (C7), 153.5 (C4), 150.2 (C8a), 134.7 (phenyl-C4), 131.6 (Phenyl C6), 126.5 (Phenyl-C1), 126.4 (phenyl-C5), 120.7 (C5), 119.3 (C6), 115.2 (C4a), 113.5 (phenyl-C3), 113.1 (C8), 109.6 (C3), 62.5 (OCH2), 56.2 (OCH3), 21.3 (COCH3). MS (m/z %): 368.36 (6.80, M+), 44.13 (100). Elemental analysis for C20H16O7, calcd.: C, 65.22; H, 4.38; Found C, 65.41; H, 4.45.

Biological tests

In vitro cytotoxic activity using MTT assay

In this study, all synthesized compounds were initially screened for their in vitro antitumor activities against two human BC cell lines, namely, MCF-7 (estrogen positive) and MDA-MB-231(triple negative), to select the most promising ones for further screenings via MTT assay using Doxorubicin, as a reference drug as described in the reported method46,47.

In vitro (EGFR) enzyme inhibition assay

Fourteen compounds that showed good cytotoxic activity against the TNBC cell line (MDA-MB-231) were examined for in vitro EGFR inhibition assay relative to erlotinib as a reference drug according to the reported protocol48.

In vitro (ARO) enzyme inhibition assay

Eleven compounds that showed good cytotoxic activity against the ER + cell line (MCF-7) were examined for in vitro ARO inhibition assay relative to EXM as a reference drug according to the reported method49.

Cell cycle arrest

DNA flow cytometric cell cycle analysis was performed on treated MCF-7 cells with IC50 concentration of compound 8 and treated MDA-MB-231 cells with IC50 concentration of compounds 10, 12 and 14 according to the reported procedure50.

Apoptosis assay

Annexin V-FITC/PI dual staining assay was performed according to the reported method51 to further study the ability of compounds 8, 10, 12 and 14 to induce apoptosis by introducing flow cytometry-based analysis.

Measurement of the level of Bax Bcl-2 and Caspase-9

MCF-7 cells were treated with compound 8, while MDA-MB-231 cells were exposed to compounds 10, 12 and 14 according to the manufacturer’s instructions52.

Molecular docking study with both EGFR and human ARO enzymes

Crystal structures of EGFR (PDB ID: 1M17)18 and ARO (PDB ID: 3EQM)53 were obtained from the PDB. Regarding enzyme inhibition assay, the most potent compounds were selected for docking study using Molecular Operating Environment (MOE) software.



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