New 6-nitro-4-substituted quinazoline derivatives targeting epidermal growth factor receptor: design, synthesis and in vitro anticancer studies

Abstract

Aim: Twenty compounds of 6-nitro-4-substituted quinazolines were synthesized.

Materials & methods: The new derivatives were evaluated for their epidermal growth factor receptor (EGFR) inhibitory activity. The most potent derivatives were assessed for their cytotoxicity against colon cancer and lung cancer cells, in addition to normal fibroblast cells.

Results & discussion: compound 6c showed a superior to nearly equal cytotoxicity in comparison to gefitinib, it also revealed a good safety profile. Compound 6c caused a cell cycle arrest at G2/M phase in addition to induction of apoptosis. A molecular docking study was conducted on the most active compounds to gain insights of their binding mode in the active site of EGFR enzyme besides ADME prediction of their physicochemical properties and drug likeness profile.

Plain language summary

Article highlights.

• Newly designed and synthesized compounds that are derived from 6-nitro-4-substituted quinazolines were biologically evaluated.

• The new compounds elicited a surpassing to good EGFR inhibition.

• The promising derivatives were tested against HCT-116, A549 cell and WI-38 lines.

• The most potent derivative 6c showed a superior enzyme inhibition against mutant EGFR T790M.

• In silico study was carried out for the most promising derivatives using molecular docking and ADME studies.

1. Introduction

Cancer is the growth of body cells in an unrestrained manner and spreads within the body regions [1–3]. According to the World Health Organization (WHO), cancer is the main cause of death in the world, accounting for nearly 10 million deaths in 2020 [1,2,4]. Chemotherapy works by stopping rapidly proliferating cells from dividing, which is a characteristic of malignant cells. Sadly, it also damages normal cells that proliferate rapidly [5]. Due to the toxicity and serious side effects of traditional chemotherapeutic medications, researchers tend to invent, synthesize and develop safe and effective anticancer drugs for this threatening disease [1,6–9]. Targeted therapy is a focused treatment that uses drugs to ‘target’ cancer cells irrespective to normal cells. They can identify and inhibit certain types of signals delivered inside a cancer cell that direct it to grow, or they can locate and target specific sites or substances in cancer cells [10]. The epidermal growth factor receptor (EGFR) participates in cell signaling pathways that regulate cell proliferation and survival in some types of cancer cells [11–14]. EGFR can occasionally be up regulated due to mutations that occur in the EGFR gene [15–19]. The developing of new molecules targeting EGFR is an innovative approach for exploration of novel anticancer agents [20–23]. FDA-approved EGFR inhibitors for the treatment of non-small cell lung cancer (NSCLC) include gefitinib I and erlotinib II. Other drugs such as lapatinib III and vandetinib IV are drugs that have been approved to treat breast cancer [24] (Figure 1).

Figure 1.

US FDA approved quinazolines as EGFR inhibitors and reported quinazolines as anticancer agents.

EGFR inhibitors have the common pharmacophoric features of a core scaffold that binds to the hinge region, the hydrophobic residue that fits into the hydrophobic pocket, the hydrophilic fragment that extends into the solvent-accessible site (Figure 2). T790M is the commonly reported mutated EGFR, Thr790 amino acid in the gatekeeper part is replaced with Met790 [25]. Our research focused on design and synthesis of new 6-nitro-4-anilinoquinazoline derivatives to find potent inhibitors against EGFR, both the wild and the mutated types.

Figure 2.

Rational design of the newly targeted quinazolines.

It was previously reported that 4-anilino quinazolines show significant anticancer activity as tyrosine kinase inhibitors [26–29]. 4,6-Disubstituted quinazoline V showed anticancer activity against ductal carcinoma cells (BT474) (IC₅₀ = 0.081 μM), compared with lapatinib (IC₅₀ = 0.064 μM) [30]. Quinazoline chalcone derivative VI is found to show significant anticancer activity against Madin-Darby canine kidney cells (MDCK II BCRP) (IC₅₀ = 0.39 μM) in comparison to Ko143 as a reference (IC₅₀ = 0.24 μM) [31]. Additionally, quinazolinyl benzylidene derivatives VIIa,b displayed a potent EGFR inhibition (IC₅₀ = 46.90 and 53.43 nM), respectively, in comparison to the dual EGFR/HER2 inhibitor lapatinib (IC₅₀ = 53.10 nM) [32]. Likewise, quinazoline-tethered hydrazones VIIIa,b is cited to exhibit significant anticancer activity against A549 lung cancer cell line, where compound VIIIa elicited a remarkable cytotoxicity (IC₅₀ = 15.77 μM), compared with gefitinib (IC₅₀ = 14.27 μM) [33] (Figure 1). Accordingly, based on the significant cytotoxicity and EGFR inhibitory activity of compounds V-VIIIa,b, the current study describes the synthesis of two series of quinazoline/chalcone 6a-f and quinazoline/benzylidene hydrazinylidene hybrids 9a-f (Figure 2).

Literature review revealed that compound IX displays a remarkable anticancer activity against A549 cells with good EGFR inhibition (IC₅₀ = 20.09 and 0.40 μM), respectively, compared with lapatinib (IC₅₀ = 11.98 and 0.11 μM), respectively [34]. Also, Compound X is reported to show anticancer activity against Michigan cancer foundation-7 (MCF-7) cells (IC₅₀ = 0.04 μM) compared with doxorubicin (IC₅₀ = 0.006 μM) [35]. Moreover, compound XI is cited to possess a good anticancer activity against lung cancer cells (A549) (IC₅₀ = 65.12 μM) [36] (Figure 1). Consequently, motivated by these findings, the authors designed to synthesize some derivatization concerning the hydrazinylidene moiety of 8 to obtain 4-chlorophenyl hydrazine-1-carboxamide 10, benzohydrazide 11 and isoindoline-1,3-dione 12 (Figure 2). Interestingly, compounds XII and XIII are declared to exhibit a remarkable anticancer activity against MCF-7 cells (IC₅₀ = 0.02 and 52.82 μM), respectively [35]. Also, compound XIV is reported to show a superior activity against KERATIN-forming tumor cells (KB), CNE2 and MCF-7 cell lines (IC₅₀ = 2.33, 1.49 and 5.95 μM), respectively, compared with 5-fluorouracil (IC₅₀ = 8.28, 15.1 and 14.2 μM), respectively [37]. Subsequently, another approach is concerned with further modification including the carbonyl functionality of 1-(4-aminophenyl)ethan-1-one of 7 to afford oxime derivative 13, phenyl hydrazinylidene 14 and hydrazine carbothioamide 15, in order to investigate their EGFR inhibitory activity besides their cytotoxicity (Figure 2).

2. Experimental

2.1. Chemistry

Spectral data are provided in Supplementary S1. Compounds 2 and 5a-f have been synthesized as reported [38–42].

2.1.1. General procedure for the preparation of compounds 6a-f

Compound 2 (1 g, 0.005 mole) was dissolved in glacial acetic acid (15 mL) then the appropriate chalcone 5a-f (0.005 mole) was added. The mixture was heated under reflux for 2 h. The formed solid was filtered, washed with water, dried and crystalized from ethanol.

2.1.1.1. (E)-1-(4-((6-Nitroquinazolin-4-yl)amino)phenyl)-3-phenylprop-2-en-1-one (6a)

Yellow powder, (yield 50%), m.p. >300°C; IR (KBr, νmax/cm^-1): 3332 (NH), 1647 (C=O); ¹H NMR (DMSO-d₆) δ ppm: 7.47 (3H, m, H-4”, H-3" and H-5"), 7.74 (1H, d, CH-C=O, J = 15.55 Hz), 7.87–7.97 (4H, m, H-2", H-6" H-2’ and H-6'), 8.12 (2H, d, H-3' and H-5', J = 8.28 Hz), 8.22 (2H, m, H-2 and CH benzylic), 8.55 (1H, d, H-8, J = 9.56 Hz), 8.81 (1H, s, H-5), 9.66 (1H, d, H-7, J = 3.74 Hz), 10.561 (1H, s, NH); ¹³C NMR (DMSO-d₆) d (ppm): 188.1, 157.7, 153.4, 145.1, 143.9, 135.1, 133.3, 131.0, 130.0, 129.8, 129.4, 129.2, 127.2, 122.4, 122.0, 121.3; MS (m/z): 395.0 [M-1]⁺; Anal. Calc. % for C₂₃H₁₆N₄O₃ (396.41): C, 69.69; H, 4.07; N, 14.13; % Found C, 69.76; H, 4.27; N, 14.14.

2.1.1.2. (E)-3-(4-Fluorophenyl)-1-(4-((6-nitroquinazolin-4-yl)amino)phenyl)prop-2-en-1-one (6b)

Golden Yellow powder, (yield 65%), m.p. >300°C; IR (KBr, νmax/cm^-1): 3363 (NH), 1661 (C=O); ¹H NMR (DMSO-d₆) δ ppm: 7.06 (2H, d, H-3" and H-5", J = 8.55 Hz), 7.78 (1H, d, CH-C=O, J = 15.2 Hz), 7.68–7.74 (4H, m, H-2', H-2", H-6" and H-6'), 8.02–8.11 (4H, m, H-5', CH benzylic, H-2 and H-3'), 8.56 (1H, d, H-8, J = 8.10 Hz), 8.82 (1H, s, H-5), 9.83 (1H, d, H-7, J = 4.5 Hz), 11.07 (1H, s, NH); ¹³C NMR (DMSO-d₆) d (ppm): 188.1, 162.6, 159.4, 156.3, 145.4, 142.8, 131.4, 131.4, 130.9 (d, J_C,F = 36 Hz), 130.9, 129.5, 127.8, 127.6, 122.5, 121.8, 121.8, 121.5, 116.2, 116.0, 114.8; MS (m/z): 412.9 [M-1]⁺; Anal. Calc. % for C₂₃H₁₅FN₄O₃ (414.40): C, 66.66; H, 3.65; N, 13.52; % Found C, 66.86; H, 3.69; N, 13.56.

2.1.1.3. (E)-3-(4-Chlorophenyl)-1-(4-((6-nitroquinazolin-4-yl)amino)phenyl)prop-2-en-1-one (6c)

Yellow powder (yield, 63%), m.p. 276–278°C; IR (KBr, νmax/cm^-1): 3332 (NH), 1664 (C=O); ¹H NMR (DMSO-d₆) δ ppm: 7.36 (2H, d, H-3" and H-5", J = 7.70 Hz), 7.57–7.73 (5H, m, H-6', H-2', H-2", H-6" and CH-C=O), 8.02–8.19 (4H, m, CH benzylic and H-3', H-5' and H-2), 8.59 (1H, d, H-8, J = 8.03 Hz), 8.83 (1H, s, H-5), 9.87 (1H, d, H-7, J = 6.3 Hz), 11.31 (1H, s, NH); ¹³C NMR (DMSO-d₆) d (ppm): 188.0, 157.8, 153.6, 145.2, 142.4, 139.8, 135.4, 134.2, 133.2, 131.0, 130.1, 129.9, 129.4, 127.3, 123.2, 122.1, 121.4; MS (m/z): 429.15 [M-1]⁺; Anal. Calc. % for C₂₃H₁₅ClN₄O₃ (430.85): C, 64.12; H, 3.51; N, 13.00; O, 11.14; % Found C, 64.32; H, 3.57; N, 13.22; O, 11.15.

2.1.1.4. (E)-3-(4-Nitrophenyl)-1-(4-((6-nitroquinazolin-4-yl)amino)phenyl)prop-2-en-1-one (6d)

Golden yellow powder (yield, 64%), m.p. 296–298°C; IR (KBr, νmax/cm^-1): 3329 (NH), 1681 (C=O); ¹H NMR (DMSO-d₆) δ ppm: 7.68–8.01 (7H, m, CH-C=O, H-2', H-6', H-3', H-5', H-2" and H-6"), 8.15–8.40 (3H, m, H-5", H-3" and CH benzylic), 8.70 (1H, d, H-8, J = 8.00 Hz), 8.92 (1H, s, H-2), 10.00 (1H, d, H-7, J = 16.00 Hz), 10.25 (1H, s, H-5), 11.93 (1H, s, NH); ¹³C NMR (DMSO-d₆) d (ppm): 189.6, 159.7, 149.5, 148.3, 144.4, 140.6, 132.1, 132.1, 131.4, 131.3, 130.1, 129.8, 129.7, 129.6, 129.4, 126.2, 126.2, 124.1, 124.1, 118.8.;MS (m/z): 439.8 [M-1]⁺; Anal. Calc. % for C₂₃H₁₅N₅O₅ (441.40): C, 62.59; H, 3.43; N, 15.87; % Found C, 62.63 H, 3.52; N, 15.95.

2.1.1.5. (E)-3-(4-(Dimethylamino)phenyl)-1-(4-((6-nitroquinazolin-4-yl)amino)phenyl)prop-2-en-1-one (6e)

Orange powder (yield, 50%), m.p. 287–289°C; IR (KBr, νmax/cm^-1): 3325 (NH), 1650 (C=O); ¹H NMR (DMSO-d₆) δ ppm: 3.02 (6H, s, di 4-CH₃N), 6.76 (2H, d, H-5" and H-3", J = 7.13 Hz), 7.61–7.79 (4H, m, H-2", H-6", H-2'and H-6'), 7.99 (1H, d, CH-C=O, J = 15.17 Hz), 8.13 (2H, d, H-3' and H-5', J = 8.53 Hz), 8.21 (2H, m, H-2 and CH benzylic), 8.60 (1H, d, H-8, J = 9.16 Hz), 8.84 (1H, s, H-5), 9.74 (1H, d, H-7, J = 2.42 Hz), 10.65 (1H, s, NH); ¹³C NMR (DMSO-d₆) d (ppm): 187.7, 158.9, 157.8, 153.4, 152.3, 145.1, 145.0, 142.9, 134.2, 131.1, 129.9, 129.4, 127.1, 122.4, 122.0, 121.2, 116.2, 114.9, 112.1, 40.1; MS (m/z): 460.4 [M-2+Na]⁺; Anal. Calc. % for C₂₅H₂₁N₅O₃ (439.48): C, 68.33; H, 4.82; N, 15.94; % Found C, 68.34; H, 4.95; N, 15.99.

2.1.1.6. (E)-3-(2,4-Dimethylphenyl)-1-(4-((6-nitroquinazolin-4-yl)amino)phenyl)prop-2-en-1-one (6f)

Yellow powder (yield,60%), m.p. 270–272°C; IR (KBr, νmax/cm^-1): 3321 (NH), 1706 (C=O); ¹H NMR (DMSO-d₆) δ ppm: 2.32 (3H, s, 2-CH₃), 2.43 (3H, s, 4-CH₃), 7.12 (2H, m, H-3" and H-5"), 7.83 (1H, d, H-6", J = 15.43 Hz), 7.93–7.99 (3H, m, H-2', CH-C=O and H-6'), 8.16 (2H, d, H-3' and H-5', J = 8.55 Hz), 8.24 (2H, m, HC benzylic and H-2), 8.59 (1H, d, H-8, J = 9.18 Hz), 8.85 (1H, s, H-5), 9.73 (1H, d, H-7, J = 2,47 Hz), 10.66 (1H, s, NH); ¹³C NMR (DMSO-d₆) d (ppm): 188.2, 178.8, 172.5, 157.8, 145.1, 143,7 141.0, 140.8, 138.4, 133.5, 131.9, 130.9, 130.0, 129.8, 127.6, 127.2, 122.1, 121.9, 121.3, 117.7, 21.3, 19.6; MS (m/z): 423.6 [M-1]⁺; Anal. Calc. % for C₂₅H₂₀N₄O₃ (424.46): C, 70.74; H, 4.75; N, 13.20; % Found C, 70.814; H, 4.90; N, 13.26.

2.1.1.7. (E)-1-(4-((6-Nitroquinazolin-4-yl)amino)phenyl)ethan-1-one (7)

Equimolar amounts of formimidamide 2 (0.32 g, 0.0015 mole) and 4-aminoacetophenone (2 g, 0.0015 mole) in glacial acetic acid (8 mL) were heated under reflux at 120°C for 2 h, the reaction mixture was allowed to cool. The formed precipitate was filtered, washed with water, dried and crystallized from ethanol.

Yellow powder (yield, 61%), m.p. 268–270°C; IR (KBr, νmax/cm^-1): 3231 (NH), 1662 (C=O); ¹H NMR (DMSO-d₆) δ ppm: 2.56 (3H, s, CH₃C=O), 7.89–8.03 (5H, m, H-2', H-3', H-4', H-2 and H-6'), 8.50 (1H, d, H-8, J = 9.16 Hz), 8.74 (1H, s, H-5), 9.59 (1H, d, H-7, J = 2.46 Hz), 10.50 (1H, s, NH); ¹³C NMR (DMSO-d₆) d (ppm): 197.0, 158.8, 157.6, 153.3, 145.0, 143.5, 132.6, 129.9, 129.3, 127.0, 121.8, 121.2, 114.9, 26.9; MS (m/z): 309.1 [M+1]⁺; Anal. Calc. % for C₁₆H₁₂N₄O₃ (308.30): C, 62.33; H, 3.92; N, 18.17; % Found C, 62.30; H, 3.99; N, 18.22.

2.1.1.8. (E)-N-(4-(1-Hydrazinylideneethyl)phenyl)-6-nitroquinazolin-4-amine (8)

A mixture of 1-(4-aminophenyl)ethan-1-one 7 (3 g, 0.01 mole) and hydrazine hydrate (6 ml, 0.1 mole) in ethanol (40 ml) was heated under reflux for 6 h. The reaction mixture was cooled. The formed solid was filtered, washed with ethanol, dried and crystallized from ethanol.

Orange powder, (yield 50%), m.p. 266–268°C; IR (KBr, νmax/cm^-1): 3367 (NH), 3309 and 3275 (NH₂); ¹H NMR (DMSO-d₆) δ ppm: 2.92 (3H, s, CH₃C=N), 6.35 (2H, s, NH₂), 7.86 (2H, d, H-2' and H-6', J = 8.18 Hz), 7.82 (2H, d, H-3' and H-5', J = 7.62 Hz), 7.92 (1H, d, H-8, J = 8.80 Hz), 8.54 (1H, d, H-7, J = 9.27 Hz), 8.71 (1H, s, H-2), 9.66 (1H, 2, H-5), 10.44 (1H, s, NH); ¹³C NMR (DMSO-d₆) d (ppm): 159.0, 158.1, 153.5, 144.9, 142.3, 137.8, 136.4, 129.9, 127.0, 125.3, 122.7, 121.3, 114.9, 11.7; MS (m/z): 320.9 [M-1]⁺; Anal. Calc. % for C₁₆H₁₄N₆O₂ (322.33): C, 59.62; H, 4.38; N, 26.07; % Found C, 59.69; H, 4.43; N, 26.37.

2.1.2. General procedure for the preparation of compounds (9a-f)

A mixture of hydrazinylidene 8 (0.32 g, 0.001 mole), appropriate aldehyde (0.001 mole), anhydrous potassium carbonate (0.28 g, 0.002 mole) in absolute ethanol (20 mL) was heated under reflux for 3–18 h. The solution was filtered while hot and the filtrate was concentrated, cooled, where a solid product was obtained, filtered, washed with petroleum ether 60–80°C, dried and crystallized from absolute ethanol to obtain the desired products 9a-f.

2.1.2.1. (E)-N-(4-((E)-1-(((E)-Benzylidene)hydrazinylidene)ethyl)phenyl)-6-nitroquinazolin-4-amine (9a)

Yellow powder (yield, 50%), m.p. 270–272°C; Reaction time: 3 h; IR (KBr, νmax/cm^-1): 3306 (NH); ¹H NMR (DMSO-d₆) δ ppm: 4.35 (3H, s, CH₃C=N), 7.50 (2H, d, H-2' and H-6', J = 6.37 Hz), 7.76–7.98 (7H, m, H-3', H-5', H-2", H-3", H-4", H-5" and H-6"), 8.43–8.52 (4H, m, H-2, H-8, CH benzylic and H-7), 9.53 (1H, s, H-5), 10.6 (1H, s, NH); ¹³C NMR (DMSO-d₆) d (ppm): 164.3, 158.9, 158.8, 157.7, 143.4, 135.0, 131.2, 129.3, 128.5, 128.0, 127.4, 125.9, 123.1, 121.9, 56.5, 15.0; MS (m/z): 409.1 [M-1]⁺; Anal. Calc. % for C₂₃H₁₈N₆O₂ (410.44): C, 67.31; H, 4.42; N, 20.48; % Found C, 67.35; H, 4.55; N, 20.6

2.1.2.2. N-(4-((E)-1-(((E)-4-Fluorobenzylidene)hydrazinylidene)ethyl)phenyl)-6-nitroquinazolin-4-amine (9b)

Golden Yellow powder, (yield 50%), m.p. 262–264°C; Reaction time: 3 h; IR (KBr, νmax/cm^-1): 3282 (NH); ¹H NMR (DMSO-d₆) δ ppm: 4.35 (3H, s, CH₃C=N), 7.36 (2H, d, H-2' and H-6', J = 8.62 Hz), 7.62 (1H, s, CH benzylic), 7.68 (2H, d, H-3" and H-5", J = 8.07 Hz), 7.87–7.99 (6H, m, H-3', H-5', H-2", H-6", H-7 and H-8), 8.37 (1H, s, H-2), 8.54 (1H, s, H-5), 9.37(1H, s, NH); ¹³C NMR (DMSO-d₆) d (ppm): 159.6, 158.7, 156.4, 155,8, 142.6, 131.8, 130.7, 130.6, 130.1(d, J_C,F = 32 Hz), 127.3, 125.3, 123.5, 122.2, 116.5, 116.2, 115,9, 14.9; MS (m/z): 427.2 [M-1]⁺; Anal. Calc. % for C₂₃H₁₇FN₆O₂ (428.43): C, 64.48; H, 4.00; N, 19.62; % Found C, 64.55; H, 4.11; N, 19.77.

2.1.2.3. N-(4-((E)-1-(((E)-4-Chlorobenzylidene)hydrazinylidene)ethyl)phenyl)-6-nitroquinazolin-4-amine (9c)

Yellow powder (yield 52%), m.p. 260–262°C; Reaction time: 3 h; IR (KBr, νmax/cm^-1):3282 (NH); ¹H NMR (DMSO-d₆) δ ppm: 4.37 (3H, s, CH₃C=N), 7.58 (2H, d, H-2' and H-6', J = 8.50 Hz), 7.92–8.03 (7H, m, H-3", H-5", H-3', H-5', H-2", H-6" and CH benzylic), 8.52 (1H, s, H-2), 8.59 (1H, d, H-8, J = 9.17 Hz), 8.79 (1H, s, H-5), 9.72 (1H, d, H-7, J = 2.45 Hz), 10.59 (1H, s, NH); ¹³C NMR (DMSO-d₆) d (ppm): 164.1, 163.5, 158.0, 156.9, 149.2, 145.0, 135.9, 133.8, 133.5, 130.2, 130.0, 129.4, 127.6, 122.4, 121.3, 115.6, 15.1; MS (m/z): 443.3 [M-1]⁺; Anal. Calc. % for C₂₃H₁₇ClN₆O₂ (444.88): C, 62.10; H, 3.85; N, 18.89; % Found C, 62.18; H, 3.95; N, 18.97.

2.1.2.4. 6-Nitro-N-(4-((E)-1-(((E)-4-nitrobenzylidene)hydrazinylidene)ethyl)phenyl)quinazolin-4-amine (9d)

Orange powder (yield, 55%), m.p. 296–298°C; Reaction time: 6 h; IR (KBr, νmax/cm^-1): 3282 (NH); ¹H NMR (DMSO-d₆) δ ppm: 4.35 (3H, s, CH₃C=N), 7.97–8.05 (5H, m, H-2' and H-6', H-3',H-5' and CH benzylic), 8.16 (2H, d, H-2" and H-6", J = 8.89 Hz), 8.35 (2H, d, H-3" and H-5", J = 8.8 Hz), 8.59 (1H, d, H-8, J = 9.2 Hz), 8.64 (1H, s, H-2), 8.80 (1H, s, H-5), 9.73 (1H, d, H-7, J = 2.46 Hz), 10.60 (1H, s, NH); ¹³C NMR (DMSO-d₆) d (ppm): 164.6, 158.9, 157.8, 155.8, 153.4, 148.7, 144.9, 140.8, 133.0, 129.9, 129.4, 127.7, 127.0, 124.3, 122.2, 121.2, 115.1, 15.2; MS (m/z): 454.2 [M-1]⁺; Anal. Calc. % for C₂₃H₁₇N₇O₄ (455.43): C, 60.66; H, 3.76; N, 21.53; % Found C, 60.75; H, 3.82; N, 21.59.

2.1.2.5. N-(4-((E)-1-(((E)-2,4-Dimethylbenzylidene)hydrazinylidene)ethyl)phenyl)-6-nitroquinazolin-4-amine (9e)

Orange powder, (yield 55%), m.p. 251–253°C; Reaction time: 3 h; IR (KBr, νmax/cm^-1): 3390 (NH); ¹H NMR (DMSO-d₆) δ ppm: 2.34 (3H, s, 2-CH₃), 2.52 (3H, s, 4-CH₃), 4.35 (3H, s, CH₃C=N), 7.14 (2H, d, H-2' and H-6', J = 6.03 Hz), 7.78–8.05 (7H, m, H-3", H-5", H-3', H-5', H-8, H-6" and CH benzylic), 8.55 (1H, d, H-7, J = 9.13 Hz), 8.71 (1H, s, H-2), 9.67 (1H, s, H-5), 10.59 (1H, s, NH); ¹³C NMR (DMSO-d₆) d (ppm): 163.7, 158.9, 157.9, 157.4, 153.5, 144.8, 140.8, 138.3, 133.5, 132.1, 130.1, 129.8, 128.6, 127.5, 127.3, 126.9, 122.3, 121.2, 115.1, 21.4, 20.1, 15.2; MS (m/z): C437.2 [M-1]⁺; Anal. Calc. % for C₂₅H₂₂N₆O₂ (438.49): C, 68.48; H, 5.06; N, 19.17; % Found C, 68.55; H, 5.13; N, 19.27.

2.1.2.6. N-(4-((E)-1-(((E)-3,4-Dimethoxybenzylidene)hydrazinylidene)ethyl)phenyl)-6-nitroquinazolin-4-amine (9f)

Orange powder, (yield 50%), m.p. >300°C; Reaction time: 18 h; IR (KBr, νmax/cm^-1): 3363 (NH); ¹H NMR (DMSO-d₆) δ ppm: 3.84 (3H, s, 4-OCH₃), 3.85 (3H, s, 3-OCH₃), 4.35 (3H, s, CH₃C=N), 7.08 (1H, d, H-5", J = 8.35 Hz), 7.40 (1H, d, H-6", J = 8.32 Hz), 7.95–8.01 (6H, m, H-2", H-2', H-5', H-8, H-6' and H-3'), 8.46 (1H, s, CH benzylic), 8.57 (1H, d, H-7, J = 6.85 Hz), 8.78 (1H, s, H-2), 9.70 (1H, s, H-5), 10.50 (1H, s, NH); ¹³C NMR (DMSO-d₆) d (ppm): 163.3, 159.0, 158.3, 157.9, 153.5, 151.8, 149.4, 145.0, 140.7, 133.8, 129.9, 127.6, 127.4, 127.0, 123.4, 122.3, 121.3, 114.9, 111.9, 109.9, 56.0, 55.9, 15.0; MS (m/z): 469.2 [M-1]⁺; Anal. Calc. % for C₂₅H₂₂N₆O₄ (470.49): C, 63.82; H, 4.71; N, 17.86; % Found C, 63.92; H, 4.81; N, 17.94.

2.1.2.7. (E)-N-(4-Chlorophenyl)-2-(1-(4-((6-nitroquinazolin-4-yl)amino)phenyl)ethylidene)hydrazine-1-carboxamide (10)

A mixture of hydrazinylidene 8 (0.32 g, 0.001 mole) and 4-chlorophenyl isocyanate (0.17 g, 0.001 mole) in dry benzene (10 mL) was heated under reflux for 6 h. The product obtained on cooling was filtered off, washed with benzene, dried and crystallized from ethanol.

Orange powder, (yield 53%), m.p. 297–300°C; IR (KBr, νmax/cm^-1): 3406 (NH), 3363 (NH), 3201 (NH), 1689 (C=O); ¹H NMR (DMSO-d₆) δ ppm: 2.30 (3H, s, CH₃C=N), 7.36 (2H, d, H-2' and H-6', J = 8.09 Hz), 7.73 (2H, d, H-3" and H-5", J = 8.45 Hz), 7.97–8.03 (5H, m, H-3' and H-5', H-2", H-6" and H-8), 8.58 (1H, d, H-7, J = 9.25 Hz), 8.77 (1H, s, H-2), 9.01 (1H, s, H-5), 9.71 (1H, s, NH), 9.85 (1H, s, NH-C=O), 10.55 (1H, s, NH-N=C); ¹³C NMR (DMSO-d₆) d (ppm): 159.0, 158.0, 153.9, 153.5, 146.4, 145.0, 139.5, 138.5, 134.1, 129.9, 128.7, 127.0, 126.5, 122.5, 121.8, 121.3, 114.9, 14.1; MS (m/z): 477.8 [M+2]⁺; Anal. Calc. % for C₂₃H₁₈ClN₇O₃ (475.89): C, 58.05; H, 3.81; N, 20.60; % Found C, 58.25; H, 3.84; N, 20.64.

2.1.2.8. (E)-N'-(1-(4-((6-Nitroquinazolin-4-yl)amino)phenyl)ethylidene)benzohydrazide (11)

Benzoyl chloride (0.42 g, 0.003 mole) was added to a solution of compound 8 (0.32 g, 0.001 mole) in methylene chloride (10 mL) and in the presence of triethylamine (0.2 g, 0.002 mole). The reaction mixture was warmed at 30°C for 1 h then stirred overnight at R.T. The obtained solid was filtered, washed with methylene chloride, dried and crystallized from ethanol.

Orange powder, (yield 40%), m.p. 280–282°C; IR (KBr, νmax/cm^-1): 3414 (NH), 1674 (C=O); ¹H NMR (DMSO-d₆) δ ppm: 2.62 (3H, s, CH₃C=N), 7.58 (3H, m, H-3", H-4" and H-5"), 8.03 (2H, d, H-2' and H-6', J = 9.26 Hz), 8.01–8.19 (5H, m, H-3' and H-5', H-2", H-6" and H-2), 8.49 (2H, m, H-8 and H-5), 8.59 (1H, d, H-7, J = 2.39 Hz), 9.72 (1H, s, NH), 10.69 (1H, s, NH-C=O); ¹³C NMR (DMSO-d₆) d (ppm): 197.1, 162.3, 159.0, 154.3, 144.5, 143.6, 137.5, 132.6, 131.7, 130.0, 129.4, 129.0, 128.8, 127.2, 121.7, 121.2, 113.6, 26.9; MS (m/z): 427.92 [M+1]⁺; Anal. Calc. % for C₂₃H₁₈N₆O₃ (426.44): C, 64.48; H, 4.71; N, 19.62; % Found C, 64.56; H, 4.71; N, 19.69.

2.1.2.9. (E)-2-((1-(4-((6-Nitroquinazolin-4-yl)amino)phenyl)ethylidene)amino)isoindoline-1,3-dione (12)

A mixture of hydrazinylidene 8 (0.32 g, 0.001 mole) and phthalic anhydride (0.3 g, 0.002 mole) in glacial acetic acid (10 mL) was heated under reflux for 3 h. The product obtained on cooling was filtered off, washed with water, dried and crystallized from ethanol.

Yellow powder, (yield 50%), m.p. 268–270°C; IR (KBr, νmax/cm^-1): 3329 (NH), 1751 (C=O); ¹H NMR (DMSO-d₆) δ ppm: 2.59 (3H, s, CH₃C=N), 7.98–8.25 (8H, m, H-3', H-2', H-6', H-5', H-3", H-4", H-5" and H-6"), 8.59 (2H, m, H-8 and H-7), 8.83 (1H, s, H-2), 9. 72 (1H, s, H-5), 10.63(1H, s, NH); ¹³C NMR (DMSO-d₆) d (ppm): 197.1, 159.0, 157.8, 153.4, 145.1, 143.5, 143.5, 136,7, 132.7, 130.1, 129.4, 127.2, 125.2, 121.9, 121.3, 115.0, 26.9; MS (m/z): 452.82; Anal. Calc. % for C₂₄H₁₆N₆O₄ (452.43): C, 63.71; H, 3.56; N, 18.58; % Found C, 63.79;H, 3.66; N, 18.69.

2.1.2.10. (E)-1-(4-((6-Nitroquinazolin-4-yl)amino)phenyl)ethan-1-one oxime (13)

1-(4-aminophenyl)ethan-1-one 7 (0.32 g, 0.001 mole) was dissolved in hot methanol (10 mL), then a mixture of hydroxylamine HCl (0.13 g, 0.002 mole) and anhydrous sodium acetate (0.16 g, 0.002 mole) in water (2 mL) was added. The reaction mixture was heated under reflux for 6 h, then allowed to cool where a solid precipitate was obtained. The formed solid was filtered, washed with methanol, dried and crystallized from absolute ethanol.

Yellow powder, (yield 50%), m.p. >300°C; IR (KBr, νmax/cm^-1): 3282 (NH), 3120(OH); ¹H NMR (DMSO-d₆) δ ppm: 2.19 (3H, s, CH₃C=N), 7.72 (2H, d, H-2' and H-6', J = 8.37 Hz), 7.95 (2H, d, H-3' and H-5', J = 8.38 Hz), 7.91 (1H, d, H-8, J = 9.16 Hz), 8.57 (1H, d, H-7, J = 9.30 Hz), 8.75 (1H, s, H-2), 9.65 (1H, s, H-5), 10.51 (1H, s, NH), 11.16 (1H, s, OH); ¹³C NMR (DMSO-d₆) d (ppm): 159.1, 158.0, 153.5, 152.9, 144.9, 139.2, 133.3, 129.9, 129.4, 127.0, 126.1, 122.0, 121.2, 114.9, 114.8, 11.8; MS (m/z):323.67; Anal. Calc. % for C₁₆H₁₃N₅O₃ (323.31): C, 59.44; H, 4.05; N, 21.66; % Found C, 59.46; H, 4.25; N, 21.76.

2.1.2.11. (E)-6-Nitro-N-(4-(1-(2-phenylhydrazinylidene) ethyl)phenyl)quinazolin-4-amine (14)

A mixture of 1-(4-aminophenyl)ethan-1-one 7 (0.32 g, 0.001 mole), phenyl hydrazine (0.1 g, 0.001 mole) in ethanol (10 ml) in presence of 3 drops of glacial acetic acid was heated under reflux for 18 h. The obtained solid was filtered, washed with ethanol, dried and crystallized from ethanol.

Dark red powder, (yield 55%), m.p. 226–228°C; IR (KBr, νmax/cm^-1): 3383 (NH), 3251 (NH); ¹H NMR (DMSO-d₆) δ ppm: 2.30 (3H, s, CH₃C=N), 7.22–7.29 (5H, m, H-2", H-3", H-5", H-6" and H-4"), 7.82 (2H, d, H-2' and H-6', J = 8.77 Hz), 7.92 (2H, d, H-5' and H-3', J = 8.76 Hz), 8.12 (1H, d, H-8, J = 9.16 Hz), 8.75 (1H, d, H-7, J = 9.14 Hz), 8.97 (1H, s, H-2), 9.37 (1H, s, H-5), 9.85 (1H, s, NH), 11.75 (1H, s, NH-N=C); ¹³C NMR (DMSO-d₆) d (ppm): 160.0, 154.1, 146.4, 146.0, 144.7, 140.1, 137.9, 136.2, 129.6, 129.3, 125.8, 124.4, 123.4, 122.2, 119.4, 114.2, 113.3, 19.0; MS (m/z): 398.72; Anal. Calc. % for: C₂₂H₁₈N₆O₂ (398.43): C, 66.32; H, 4.55; N, 21.09; % Found C, 66.33; H, 4.64; N, 21.29.

2.1.2.12. (E)-2-(1-(4-((6-Nitroquinazolin-4-yl)amino)phenyl)ethylidene)hydrazine-1-carbothioamide (15)

A mixture of 1-(4-aminophenyl)ethan-1-one 7 (0.32 g, 0.001 mole), thiosemicarbazide (0.27 g, 0.003 mole) in ethanol (10 ml) and in presence of glacial acetic acid was heated under reflux for 30 h. The reaction mixture was cooled, and the formed precipitate was filtered, washed with ethanol, dried and crystallized from absolute ethanol.

Orange powder, (yield 50%), m.p. 220–222°C; IR (KBr, νmax/cm^-1): 3410 and 3367 (NH₂), 3263 (NH), 3174 (NH); ¹H NMR (DMSO-d₆) δ ppm: 2.33 (3H, s, CH₃C=N), 7.91–8.04 (6H, m, H-2', H-3', H-5', H-6' and NH₂), 8.29 (1H, s, H-2), 8.56 (1H, d, H-8, J = 9.16 Hz), 8.75 (1H, s, H-5), 9.67 (1H, d, H-7, J = 2.45 Hz), 10.22 (1H, s, NH), 10.50 (1H, s, NH-C=S); ¹³C NMR (DMSO-d₆) d (ppm): 179.2, 158.9, 157.9, 153.4, 147.9, 144.9, 139.9, 133.7, 129.9, 127.3, 127.0, 122.3, 121.2, 114.8, 14.2.; MS (m/z): 381.75; Anal. Calc. % for C₁₇H₁₅N₇O₂S (381.41): C, 53.53; H, 3.96; N, 25.71; % Found C, 53.64; H, 4.00; N, 25.86.

2.2. Biological evaluation

EGFR inhibitory assay for the newly synthesized compounds, cytotoxicity of the most active derivatives, cell cycle analysis and apoptotic assay were established. According to the approved protocol number REC0723.

2.2.1. EGFR-TK inhibitory assay

The materials of the in vitro assessment of the newly targeted compounds for their EGFR inhibitory activities are described in the Supplementary S2 [43,44].

2.2.2. In vitro cytotoxic activity

Three candidates 6c, 8 and 9f eliciting a superior EGFR inhibition were selected to be tested for their in vitro cytotoxicity versus two cell lines; lung cancer cells (A549) and colorectal cancer cells (HCT-116) in addition to normal fibroblast cells (WI-38) [45]. The methods are presented in Supplementary S2.

2.2.3. Cell cycle analysis

Cell cycle examination was performed to demonstrate the effect of 6c on cell cycle progression in HCT-116 cells [46], the experimental assay is elucidated in Supplementary S2.

2.2.4. Apoptotic assay

An annexin V-FITC/propidium iodide dual staining assay was utilized to examine the apoptotic induction impact and apoptosis percentage produced by 6c in HCT-116 cells [47]. The additional data offering more information is discussed in Supplementary S2.

2.3. In silico studies

2.3.1. Molecular docking study

A molecular docking study was carried out for compounds 6c, 8 and 9f on EGFR using the pdb file (ID: 1M17) which is downloaded from protein data bank [48] through using AutoDock Vina 1.1.2. Software [49,50] MGL tools 1.5.7. was implemented for preparation of the protein and the ligand, then should be saved as pdbqt format which is a pre-requisite to carry out the docking procedure by AutoDock Vina. The results are visualized by Discovery Studio Visualizer v21.1.0.20298 [51].

2.3.2. ADME prediction

ADME prediction was carried out using the free web server SwissADME [52] (http://www.swissadme.ch/index.php) which evaluates several physicochemical parameters and determine the possibility of drug-likeness of the tested derivatives. Three derivatives 6c, 8 and 9f were tested on the server. The additional data offered more information which is discussed in Supplementary S3.

3. Results

3.1. Chemistry

The methodology followed for the preparation of the designed quinazolines are illustrated in Figures 3–6. Reaction of 2-amino-4-nitrobenzonitrile with DMF/DMA under reflux condition gave the (E)-N'-(2-cyano-4-nitrophenyl)-N,N-dimethyl formimidamide intermediate 2 [53], which upon reaction with the appropriate chalcone gives compounds 6a-f. The desired chalcones 5a-f were synthesized as reported [54–58] via reaction of 4-aminoacetophenone 3 with appropriate aromatic aldehydes 4a-f in ethanol, as depicted in Figure 3.

Figure 3.

Synthesis of the reported intermediates 2 & 5a-f and the targeted compounds 6a-f: Reagents and conditions: (i) DMF-DMA, reflux, 2 h; (ii) Aqueous NaOH 60%, ethanol, ice bath, 30 min, R.T, overnight; (iii) Glacial acetic acid, reflux, 2 h.

Figure 4.

Synthesis of compounds 7, 8 and 9a-f: Reagents and conditions: (i) Glacial acetic acid, reflux, 2 h; (ii) Hydrazine hydrate, ethanol, reflux, 6 h; (iii) Appropriate aromatic aldehyde, anhydrous potassium carbonate, ethanol, reflux, 3-18 h.

Figure 5.

Synthesis of the targeted compounds 10, 11 and 12: Reagents and conditions: (i) 4-Chlorophenyl isocyanate, dry benzene, reflux, 6 h; (ii) Benzoyl chloride, triethylamine, methylene chloride, warming at 30°C for 1 h, R.T overnight; (iii) Phthalic anhydride, glacial acetic acid, reflux, 3 h.

Figure 6.

Synthesis of compounds 13, 14 and 15: Reagents and conditions: (i) Hydroxylamine HCl, anhydrous sodium acetate, methanol, reflux, 6 h; (ii) Phenyl hydrazine, ethanol, reflux, 18 h; (iii) Thiosemicarbazide, ethanol, reflux, 30 h.

Reaction of the formimidamide intermediate 2 with 4-aminoacetophenone 3 in refluxing glacial acetic acid afforded 1-(4-aminophenyl)ethan-1-one 7, as described in Figure 4.

Furthermore, reaction of intermediate 7 with hydrazine hydrate in ethanol under reflux afforded the hydrazinylidene 8. Moreover, heating compound 8 with appropriate aromatic aldehyde in ethanol and in presence of anhydrous potassium carbonate gave the benzylidene derivatives 9a-f.

As presented in Figure 5, the hydrazinylidene 8 was subsequently reacted with 4-chlorophenyl isocyanate by heating under reflux in benzene to afford the targeted hydrazino carboxamide intermediate 10. Additionally, benzohydrazide 11 was produced by stirring compound 8 with benzoyl chloride in methylene chloride in presence of triethylamine at room temperature. Also, isoindoline-1,3-dione 12 was produced by heating under reflux of compound 8 with phthalic anhydride in glacial acetic acid.

Reaction of 1-(4-aminophenyl)ethan-1-one 7 with hydroxylamine HCl in refluxing methanol and in presence of sodium acetate afforded ethanone oxime 13, as shown in Figure 6. Additionally, phenylhydrazinylidene 14 was obtained by reflux of compound 7 with phenyl hydrazine in ethanol (Figure 6). Finally, heating of compound 7 with thiosemicarbazide in ethanol afforded carbothioamide 15 (Figure 6).

3.2. Biological evaluation

3.2.1. EGFR-TK inhibitory assay

The whole set of compounds were appraised for evaluating their EGFR inhibitory activity to estimate their inhibition on the enzyme. The results are presented in Table 1.

Table 1.

IC₅₀ values of the tested compounds and gefitinib in in vitro epidermal growth factor receptor kinases assay.

Compound	EGFR, (IC50 μM)	Compound	EGFR, (IC50 μM)
6a	0.0319 ± 0.001	9d	0.0576 ± 0.0014
6b	0.0342 ± 0.0013	9e	0.0267 ± 0.0009
6c	0.0131 ± 0.0008	9f	0.0185 ± 0.001
6d	0.0497 ± 0.0031	10	0.941 ± 0.042
6e	0.0553 ± 0.0025	11	0.136 ± 0.007
6f	0.0401 ± 0.0021	12	0.82 ± 0.035
7	0.596 ± 0.026	13	5.882 ± 0.259
8	0.0095 ± 0.0008	14	2.088 ± 0.092
9a	0.0414 ± 0.0017	15	0.61 ± 0.026
9b	0.0424 ± 0.0023	Gefitinib	0.199 ± 0.009
9c	0.0712 ± 0.0029

3.2.2. In vitro cytotoxic activity

Three compounds 6c, 8 and 9f that elicit a superior EGFR inhibition were selected to be evaluated for their in vitro cytotoxicity against two human cancer cell lines, namely: A549 (lung cancer cells) and HCT-116 (colorectal cancer cells), in addition to WI-38 (normal fibroblast cells) using the sulforhodamine method [59–61]. Gefitinib was used as a reference standard. The in vitro cytotoxic data are presented in Supplementary S2 & Table 2 & Figures 1–3.

3.2.3. Cell cycle analysis

Compound 6c showing an outstanding cytotoxicity against HCT-116 was subjected to cell cycle examination by flow cytometry analysis to study its effect in cell cycle progression [62–65]. The results are shown in Supplementary S2 & Table 3 & Figure 4.

3.2.4. Apoptotic assay

According to a previously published method [66–68], an Annexin V-FITC/propidium iodide dual staining experiment was performed in order to confirm and quantify the proportion of apoptosis caused by compound 6c in HCT-116 cells. The Annexin V assay allows assessments of the kinetics of apoptotic death in relation to the cell cycle and the possibility of detecting early stages of apoptosis before the loss of membrane structural stability [63,69]. The outcome of compound 6c on apoptotic induction in HCT-116 cells is shown in Supplementary S2 & Table 4 & Figure 5.

3.3. In silico studies

3.3.1. Molecular docking study

Molecular docking study was performed for the most active derivatives 6c, 8 and 9f on EGFR (pdb ID: 1M17). Docking setup was validated firstly through docking of the co-crystallized ligand “erlotinib” into the vicinity of the EGFR active site, the co-crystallized ligand showed a docking score of -9.35 kcal/mol. with accepted root mean square deviation (rmsd) value, 1.42 Supplementary S3 & Figure 1.

The docking procedure was then carried out on the three selected compounds 6c, 8 and 9f, in addition to the reference drug gefitinib, the score in kcal/mol. is presented in Supplementary S3 & Table 1.

Binding of the co-crystallized ligand “erlotinib” to the EGFR active site is through hydrogen bond interactions with the key amino acid Met769 with quinazoline nitrogen (Supplementary S3 & Figure 1) [70]. The docked compounds show a satisfactory binding energy score Supplementary S3 & Table 1. that is comparable to that of the co-crystallized ligand. Figure 7 represents the 2D interaction of compound 6c with EGFR active site. Other compounds are provided in Supplementary S3 & Figures 3–6. The 3D overlay of gefitinib (blue) and 6c (purple) is present in Supplementary S3 & Figure 2.

Figure 7.

2D interaction of compound 6c with EGFR active site.

EGFR T790M mutant enzyme in complex with naquotinib (pdb ID: 5Y9T) was used to carry out the docking for the most promising compound 6c. Through analyzing its binding affinity & interactions, it shows a good binding energy score (-7.56 kcal./mol.) compared with the co-crystallized ligand “naquotinib” with a score of (-7.91 kcal/mol.) and to the 6c binding score to the normal EGFR active site (-7.86 kcal/mol.). It exhibits hydrogen bond interaction through the carbonyl oxygen with MET793 in a similar pattern to naquotinib in addition to hydrophobic bonding with the mutant MET790 amino acid residue (Supplementary S3 & Figure 7).

3.3.2. ADME prediction

Swiss ADME web tool of bioinformatics (SIB) [52,71–75] is used for the calculation of the physicochemical descriptors and to predict ADME parameters for the most potent compounds 6c, 8 and 9f.

Polarity, lipophilicity, size, insolubility, flexibility and unsaturation were studied (Supplementary S3 & Figure 8) as a bioavailability radar chart in which the pink colored area indicates suitable parameters for good oral bioavailability, the presence of several aromatic unsaturated rings makes the unsaturation parameter has violation in all of the tested derivatives, other parameters were satisfactory.

The pink-colored zone is the suitable space for oral bioavailability Lipinski's rule of five [] is also applied to assess the oral bioavailability and drug-likeness of the tested compounds, the results are summarized in (Supplementary S3 & Table 2).

Finally, a boiled egg chart is also constructed by Swiss ADME server, where human intestinal absorption (HIA) area is in the white area, while the blood-brain barrier (BBB) penetration area is in the yellow yolk area. Only two of the tested derivatives are predicted to have a good intestinal absorption, which are 6c and 8. Besides, through observing the color given to each compound either being red or blue, we deduct the possibility of each compound to act as a substrate for permeability glycoprotein (PGP) as the blue color indicates that it may act as a substrate while the red color not. PGP is an efflux pump [76] for many drugs that causes them to be out of the target cells and this is responsible of drug resistance, all the tested compounds are in red and so are not predicted to be PGP substrates (Supplementary S3 & Figure 9).

4. Discussion

4.1. Chemistry

IR spectra of compounds 6a-f displayed the existence of the characteristic stretching vibration of NH group in the range of 3321–3363 cm^-1 along with the appearance of new band at 1647–1706 cm^-1 related to the new carbonyl group. ¹H NMR spectrum of 6e showed a singlet signal at 3.02 ppm assigned to dimethyl amino protons. The 2,4-di-CH₃ protons of 6f appeared as two singlet signals at 3.32 and 3.43 ppm. Additionally, compounds 6a-f revealed the appearance of a singlet signal around 10.61–11.90 ppm corresponding to the NH protons, Also, Compound 6b showed J coupling = 8.55 Hz in the aromatic protons compared with literature value J = 8.8 Hz [31]. ¹³C NMR spectra of 6a, b, c, d, e and f demonstrated a signal at 188.1, 188.1, 188.0, 189.6, 187.7 and 188.2 ppm attributed to the carbonyl carbons, respectively. Moreover, compound 6e showed aliphatic signal at 40.0 ppm for the carbons of N(CH₃)₂ and compound 6f revealed two aliphatic signals at 21.3 and 19.6 ppm attributed to the carbons of 2,4-di-CH₃. Compound 6b showed J_C,F = (130.9, d, 36 Hz) compared with literature value J_C,F = (133.45, d, 30 Hz) [77].

IR spectrum of 7 showed the appearance of new band at 1662 cm^-1 related to the carbonyl group, whereas ¹H NMR spectrum showed singlet signals at 2.56 ppm corresponding to the methyl protons, along with the appearance of an exchangeable singlet signal at 10.50 ppm assigned to the NH proton. ¹³C NMR spectrum of 7 exhibited aliphatic signals at 26.9 and 197.8 ppm attributed to methyl and carbonyl carbons, respectively.

IR spectrum of 8 lacked the carbonyl band and revealed a forked band at 3367 and 3309 cm^-1 corresponding to the NH₂ group, as well as the appearance of the characteristic stretching vibration of NH at 3275 cm^-1. ¹H NMR spectrum showed a singlet signal at 2.05 ppm corresponding to methyl protons and exchangeable singlet signals at 10.64 and 6.35 ppm assigned to the NH and NH₂ protons, respectively. ¹³C NMR spectrum of 8 supported the carbon skeleton of the obtained structure by lacking the signal of carbonyl carbon and showing a signal at 11.7 ppm assigned to methyl carbon. Moreover, heating compound 8 with appropriate aromatic aldehyde in ethanol and in presence of anhydrous potassium carbonate gave the benzylidene derivatives 9a-f. IR spectra of compounds 9a-f revealed the presence of a sharp band corresponding to NH groups at 3282–3390 cm^-1, along with the disappearance of bi-forked NH₂ band. ¹H NMR spectra of 9a-f displayed an increase in the integration of the aromatic protons indicating the presence of additional aromatic ring, in addition, Compound 9b displayed J coupling = 8.07 Hz in the aromatic protons compared with literature value J = 8.2 Hz [32]. Also, compound 9e showed two singlet signals at 2.34 and 2.52 ppm corresponding to dimethyl protons. The di-OCH₃ protons of 9f appeared as two singlet signals at 3.84 and 3.85 ppm, ¹³C NMR spectra of compounds 9a-f supported the carbon skeleton of the obtained structures. Compound 9e exhibited aliphatic signals at 20.1 and 21.4 ppm, assigned to methyl carbons. However, compound 9f demonstrated signals at 55.9 and 56.0 ppm due to 3, 4-dimethoxy carbons. Compound 9b shows J_C,F = (130.7, d, 32 Hz) compared with literature value J_C,F = (133.45, d, 30 Hz) [77].

IR spectrum of 10 displayed the presence of the stretching vibration of NH groups in the range of 3406–3201 cm^-1 along with the appearance of a strong band at 1689 cm^-1 due to carbonyl group stretching. ¹H NMR spectrum of compound 10 revealed the appearance of three singlet signals around 9.71–10.55 ppm corresponding to the NH protons. ¹³C NMR spectrum of 10 demonstrated a signal at 159.0 ppm attributed to carbonyl carbon. IR spectrum of 11 displayed the disappearance of the characteristic stretching vibrations of the NH₂ moiety along with the appearance of a new band at 1674 cm^-1 related to the new carbonyl group. ¹H NMR spectrum of compound 11 revealed the appearance of two singlet signals 9.72–10.69 ppm corresponding to the NH protons. ¹³C NMR spectrum of 11 showed a signal at 197.1 ppm attributed to the carbonyl carbon. IR spectrum of 12 showed a band at 1751 cm^-1, attributed to the carbonyl group. ¹H NMR spectrum of compound 12 showed a singlet signal at 10.63 ppm corresponding to the NH proton. ¹³C NMR spectrum of 12 showed a signal at 197.1 ppm attributed to carbonyl carbons.

IR spectrum of 13 showed NH stretching bands at 3282 cm^-1 and OH stretching bands 3120 cm^-1. ¹H NMR spectrum showed two singlet signals at 10.51 and 11.16 ppm corresponding to the NH and OH protons, respectively. ¹³C NMR spectrum of 13 supported the carbon skeleton of the obtained structure. IR spectrum of 14 showed two NH stretching bands at 3383 and 3251 cm^-1. ¹H NMR spectrum of compound 14 showed two exchangeable singlet signals at 9.85 and 11.75 ppm NH protons. ¹³C NMR spectrum of 14 supported the carbon skeleton of the obtained structure. IR spectrum of 15 lacked the C=O band and showed two NH stretching bands at 3263 and 3174 cm^-1 in addition to NH₂ stretching bands at 3410 and 3367 cm^-1. ¹H NMR spectrum of compound 15 revealed three singlet signals at 8.04, 10.22 and 10.50 ppm due to NH₂ and NH protons, respectively. ¹³C NMR spectrum of 15 showed a signal at 179.2 ppm attributed to the C=S carbon.

4.2. Biological evaluation

4.2.1. EGFR-TK inhibitory assay

The majority of the screened derivatives display a superior to a significant EGFR-TK inhibitory activity in a sub-micromolar level (IC₅₀ = 0.0095–0.941 μM). Unsubstituted chalcone derivative 6a and 4-fluorophenyl analogue 6b exhibited a superior enzyme inhibition (IC₅₀ = 0.032 and 0.034 μM), respectively, compared with gefitinib (IC₅₀ = 0.199 μM). 4-Chlorophenyl chalcone 6c elicited the surpassing enzyme inhibition among this series with (IC₅₀ = 0.0131 μM). 4-Nitrophenyl 6d, 4-dimethylamino phenyl 6e and 2,4-dimethyl phenyl 6f analogues demonstrated a remarkable enzyme inhibition (IC₅₀ = 0.049, 0.055 and 0.040 μM), respectively compared with gefitinib (IC₅₀ = 0.199 μM). Unsubstituted hydrazinylidene derivative 8 displayed a surpassing inhibitory activity (IC₅₀ = 0.0095 μM). On the other hand, the benzylidene derivatives 9a-f demonstrated a significant inhibitory activity as donated by their IC₅₀ values = 0.0185–0.0712 μM but less than the precursor 8. However, the unsubstituted benzylidene 9a and those bearing electron withdrawing groups 4-fluoro 9b, 4-chloro 9c and 4-nitro 9d congeners displayed a potent enzyme inhibition (IC₅₀ = 0.041, 0.042, 0.071 and 0.057 μM). Moreover, the substituted benzylidene derivatives having electron donating groups: 2,4-dimethyl 9e and 3,4-dimethoxy 9f elicited a superior enzyme inhibition (IC₅₀ = 0.027 and 0.018 μM), respectively. 4-Chlorophenyl hydrazine-1-carboxamide 10, benzohydrazide 11, isoindoline-1,3-dione 12 and hydrazine-1-carbothiamide 15 showed a promising enzyme inhibition (IC₅₀ = 0.136–0.941 μM), where compound 11 elicited a superior enzyme activity (IC₅₀ = 0.136 μM). Oxime 13 and phenyl hydrazinylidene 14 displayed the least activity among the tested compounds (IC₅₀ = 5.88 and 2.08 μM), respectively. Compound 6c was further tested against mutant EGFR kinase (T790M) and its result is shown in Supplementary S2 & Table 1. It was noticed that compound 6c has a outstanding inhibition (IC₅₀ = 0.119 μM) relative to geftinib (IC₅₀ = 0.185 μM) versus the mutated type.

4.2.2. In vitro cytotoxic activity

The tested compounds 6c, 8 and 9f showed a preferential activity against HCT-116 cell line. These compounds displayed a superior anticancer activity against HCT-116 in a submicromolar level (IC₅₀ = 0.007–0.376 μM) compared with gefitinib (IC₅₀ = 1.69 μM). 4-Chlorophenyl chalcone 6c elicited a superior anticancer activity against HCT-116 and A549 (IC₅₀ = 0.007 and 0.017 μM), respectively relative to gefitinib (IC₅₀ = 1.69 and 0.0103 μM), respectively. Also, it showed a nearly equipotent cytotoxicity against A549 (IC₅₀ = 0.017 μM) to that expressed by gefitinib (IC₅₀ = 0.0103 μM). Furthermore, compounds 6c, 8 and 9f displayed a good safety profile in HCT-116 more than A549. Although hydrazinylidene 8 exhibited a superior EGFR enzyme inhibition, it did not show the most potent cytotoxicity against HCT-116 and A549 relative to 4-chlorophenyl chalcone 6c and 3,4-dimethoxy benzylidene 9f.

4.2.3. Cell cycle analysis

Compound 6c showing an outstanding cytotoxicity against HCT-116 was subjected to cell cycle examination by flow cytometry analysis to study its effect in cell cycle progression [62–65]. The results are shown in Supplementary S2 & Table 3 & Figure 4.

4.2.4. Apoptotic assay

The results revealed that the percentage of the total apoptotic cells in HCT-116 cell line is amplified after addition of 6c (29.55%) relative to control cells (0.54%) which indicates a distinguished sign of apoptosis.

4.3. In silico studies

4.3.1. Molecular docking study

All the docked compounds exhibit the key binding interaction with Met769 through the quinazoline nitrogen atom in a similar pattern to the co-crystallized ligand “erlotinib”. For EGFR T790M mutant enzyme, the promising derivative 6c shows a similar binding mode to that of the ligand “naquotinib”.

4.3.2. ADME prediction

All the tested compounds show satisfactory ADME properties with no violations in the radar chart, the boiled egg chart revealed that the tested derivatives are non-PGB substrates.

5. Conclusion

Twenty novel compounds were synthesized and their ability to inhibit EGFR was assessed. Fourteen compounds out of twenty demonstrated a superior enzyme inhibition (IC₅₀ = 0.0095–0.0712 μM) in comparison to gefitinib (IC₅₀ = 0.199 μM). Three compounds 6c, 8 and 9f that cause an outstanding enzyme inhibition were further tested for their anticancer activity against HCT-116 and A549 cell lines, in addition to WI-38 normal fibroblast cell line. The most active compound 6c was screened for its inhibition on mutant EGFR (EGFR^T790M) where it showed a superior activity in comparison to gefitinib. Also, it caused a cell cycle arrest at G2/M phase in HCT-116 in addition to induction of apoptosis. The binding affinity of the synthesized drugs is compatible with their EGFR inhibitory activity, as demonstrated by the results of the molecular docking investigation. In physicochemical studies, compounds 6c and 8 are predicted to have a good intestinal absorption. Moreover, all the tested compounds are not substrates for permeability glycoprotein.

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17568919.2024.2389772