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

August 17, 2023

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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) υ_max (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 (C_8a), 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 (C_4a), 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) υ_max (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 (C_8a), 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 (C_4a), 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) υ_max (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-C_4-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 (C_8a), 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 (C_4a), 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, J_{2 =} 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(C_8a), 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 (C_4a), 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 (C_8a), 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 (C_4a), 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 2–4 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 C_2`-H and C_6’–H), 7.73 (t, 2H, J = 7.4 Hz, Phenyl C₄ -H and C_4’-H) 7.68 (d, 1H, J = 8.7 Hz, C₅-H), 7.59–7.57 (m, 4H, Phenyl C₃–H, C₅–H, C_3’–H and C_5’–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 (C_8a), 134.3 (two Phenyl-C_4’ and Phenyl-C_1’), 133.10 (two Phenyl-C₁ and Phenyl-C₄), 129.9 (two Phenyl-C₂ and C₆), 129.5 (two Phenyl-C_2` and C_6`), 129.3 (two Phenyl-C_3’ and C_5’), 128.4 (two Phenyl-C₃ and C₅), 126.6 (C₅), 113.7 (C₆), 109.7 (C_4a), 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 C_2’–H and C_6’–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 C_3’–H, C_5’–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 (C_8a), 136.3 (Phenyl-C_4`), 134.7 (Phenyl-C_1`), 134.4 (Phenyl-C₁), 131.8 (Phenyl-C₄), 129.7 (two Phenyl-C_2`and C_6`), 129.3 (two Phenyl-C₂ and C₆), 128.4 (two Phenyl-C_3`and C_5`), 128.3 (two Phenyl-C₃ and C₅), 126.4 (C₅), 113.4 (C₆), 111.1 (C_4a), 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, C_2’–H and C_6’–H), 7.78 (d, 1H, J = 8.9 Hz, C₅–H), 7.73 (t, 1H, J = 7.4 Hz, Phenyl C_4’–H), 7.66 (d, 2H, J = 8.5 Hz, Phenyl C₃–H and C₅–H), 7.61 (t, 2H, J = 7.6 Hz, Phenyl C_3’–H and C_5’–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 (C_8a), 139.2 (Phenyl-C₄), 134.6 (Phenyl-C_4’), 134.5 (Phenyl-C_1’), 131.8 (Phenyl-C₂), 131.8 (Phenyl-C₆), 129.7 (Phenyl-C_2’), 129.7 (Phenyl-C_6’), 129.4 (Phenyl-C_3’), 129.4 (Phenyl-C_5’), 128.4 (Phenyl-C₃), 128.4 (Phenyl-C₅), 128.2 (Phenyl-C₁), 126.4 (C₅), 113.4 (C₆), 111.1 (C_4a), 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, C_2`-H and C_6`-H), 7.78 (d, 1H, J = 8.9 Hz, C₅–H), 7.68–7.65 (m, 4H, Phenyl C₃–H, C₅–H, C_3’–H and C_5’–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 (C_8a), 139.2 (two Phenyl-C₄ and C_4’), 131.8 (two Phenyl-C₂ and C₆), 130.4 (two Phenyl- C_2’ and C_6’), 129.6 (two Phenyl-C_3’ and C_5’), 129.4 (two Phenyl-C₃ and C₅), 128.3 (two Phenyl-C₁ and C_1’), 126.5 (C₅), 113.9 (C₆), 113.4 (C_4a), 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 C_2’–H and C_6’–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 C_4’–H), 7.64–7.62 (m, 3H, Phenyl C_3’–H and C_5’–H and C_4’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 (C_8a), 134.7 (Phenyl-C₄), 134.6 (Phenyl-C_4`), 134.4 (phenyl-C_1`), 131.6 (phenyl-C₆), 129.3 (Phenyl-C_2`), 129.3 (Phenyl-C_6`), 128.4 (Phenyl-C_3`), 128.4 (Phenyl-C_5`), 126.4 (C₅), 120.8 (Phenyl-C₁), 119.4 (Phenyl-C₅), 113.2 (Phenyl-C₃), 113.1 (C₆), 111.1 (C_4a), 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 C_2’–H and C_6’–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 C_3’–H and C_5’–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-C_2`), 155.3 (C₄), 150.9 (C_8a), 139.2 (Phenyl-C_4`), 134.7 (Phenyl-C₄), 133.3 (Phenyl-C_1`), 131.7 (Phenyl-C₆), 131.6 (Phenyl-C_2`), 130.3 (Phenyl-C_6`), 129.4 (Phenyl-C₃), 126.5 (Phenyl-C_3`), 126.4 (Phenyl-C_5`), 120.8 (C₅), 119.4 (Phenyl-C₁), 113.2 (Phenyl-C₅), 113.1 (C₆), 111.1 (C_4a), 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 (C_8a), 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 (C_4a), 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 (C_8a), 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 (C_4a), 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 (C_8a), 134.7 (phenyl-C₄), 131.6 (Phenyl C₆), 126.5 (Phenyl-C₁), 126.4 (phenyl-C₅), 120.7 (C₅), 119.3 (C₆), 115.2 (C_4a), 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 method^46,47.

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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