Harnessing molecular hybridization approach to discover novel quinoline EGFR-TK inhibitors for cancer treatment

Abstract

Aim: Using molecular hybridization approach, novel 18 quinoline derivatives (6a–11) were designed and synthesized as EGFR-TK inhibitors. Materials & methods: The antiproliferative activity was assessed against breast (MCF-7), leukemia (HL-60) and lung (A549) cancer cell lines. Moreover, the most active quinoline derivatives (6d and 8b) were further investigated for their potential as EGFR-TK inhibitors. In addition, cell cycle analysis and apoptosis induction activity were conducted. Results: A considerable cytotoxic activity was attained with IC₅₀ values spanning from 0.06 to 1.12 μM. Besides, the quinoline derivatives 6d and 8b displayed potent inhibitory activity against EFGR with IC₅₀ values of 0.18 and 0.08 μM, respectively. Conclusion: Accordingly, the afforded quinoline derivatives can be used as promising lead anticancer candidates for future optimization.

Plain language summary

Summary points.

• Tyrosine kinases (TK) play a pivotal role in cellular signaling pathways, making them key targets for therapeutic interventions.

• Molecular hybridization approach was utilized to design and synthesize novel eighteen quinoline derivatives (6a–11) as EGFR-TK inhibitors.

• The antiproliferative activity of the synthesized quinoline derivatives (6a–11) was assessed against breast (MCF-7), leukemia (HL-60) and lung (A549) cancer cell lines.

• A considerable cytotoxic activity was attained against the investigated cancer cell lines with IC₅₀ values spanning from 0.06 to 1.12 μM in comparison to 5-FU (0.18 to 0.51 μM).

• The quinoline derivatives 6d and 8b could display potent inhibitory activity against EFGR with IC₅₀ values of 0.18 and 0.08 μM, respectively in comparison to Lapatinib (0.05 μM).

• Additionally, cell cycle analysis displayed that quinoline derivatives 6d and 8b could significantly prompt S phase arrest in A549 cancer cells.

• The flow cytometry analysis revealed that the treatment of A549 cells with quinoline compounds 6d and 8b resulted in 34.88 and 31.41% cellular apoptosis, respectively, whereas non-treated cells showed 0.63%.

• The conducted in silico studies showed eligible binding pattern for the synthesized quinoline derivatives to EGFR-TK with acceptable pharmacokinetics and toxicity profiles.

• The afforded quinoline derivatives can be used as promising lead anticancer candidates for future optimization.

1. Introduction

Despite notable progress in the field of cancer treatment, it remains one of the most formidable medical challenges globally. The issue of cancer is becoming increasingly pervasive, impacting countries worldwide with a disproportionate burden on those with lower and intermediate incomes. This trend often reflects an initial socioeconomic correlation, highlighting the complex interplay between health outcomes and economic factors [1]. Cancer is recognized as the second leading cause of death worldwide following deaths attributed to heart disease. This underscores the significant impact of cancer on global mortality and emphasizes the ongoing need for effective prevention, diagnosis and treatment strategies [2]. Furthermore, due to their limited selectivity, chemotherapeutic agents may induce cytotoxic effects on normally dividing cells leading to notable side effects such as immunosuppression, nausea, anemia and hair loss. These adverse effects highlight the challenges associated with achieving a balance between targeting cancer cells and minimizing harm to healthy tissues during cancer treatment [3]. As a result, researchers worldwide are diligently working to discover novel treatments for various types of cancer. The pursuit of innovative therapeutic approaches is driven by the goal of developing more effective and targeted treatments while minimizing the adverse effects associated with traditional chemotherapy.

On the other side, the regulation of crucial biological processes like metabolism, cell proliferation, survival and death is significantly facilitated by protein kinases (PKs). Protein kinases play a pivotal role in cellular signaling pathways, making them key targets for therapeutic interventions especially in the context of cancer research [4]. These enzymes play a vital role in transferring the γ-phosphate group from ATP to specific hydroxyl groups of amino acids, such as tyrosine, serine or threonine on target protein substrates. This phosphorylation process is crucial for the modulation of various cell signaling cascades, affecting cellular functions [5]. Therefore, disruptions in cell signaling cascades caused by kinase hyper-activation or mutations can give rise to various diseases including cancer [6].

The epidermal growth factor receptor (EGFR), being one of the most prominent protein kinases, plays a crucial role in cell migration and proliferation [7]. Many human solid tumors, including hepatocellular carcinoma, non-small-cell lung cancer and breast cancer may exhibit overexpression of EGFR [4,8,9]. Clearly, recent cancer treatment strategies are centered on targeting specific molecules that can influence the regulatory mechanisms involved in controlling cancer cell proliferation [10,11]. Accordingly, EGFR represents a significant therapeutic target in this context due to its pivotal role in promoting aberrant cell growth and survival in various cancer types [12–17].

Erlotinib and gefitinib are examples of first-generation EGFR-tyrosine kinase inhibitors [18–21], which may exhibit resistance due to EGFR-TK mutation (EGFR-T790M) undermining their anticancer potential [22]. Accordingly, second generation EGFR-tyrosine kinase inhibitors were approved such as pelitinib and neratinib to conquer such mutations [23–28]. However, maximal-tolerated-dose (MTD) was exhibited by these drugs [29,30] leading to the development of third generation irreversible EGFR-tyrosine kinase inhibitors such as osimertinib and olmutinib [31–34], as depicted in Supplementary Figure S1. Lately, fourth-generation EGFR TK inhibitors has emerged as a strategy to combat EGFR tertiary mutations (C797S) and introduced for further clinical evaluations [35]. Fourth-generation EGFR TKIs function as allosteric kinase inhibitors, offering a potential complementary therapeutic approach to ATP-competitive kinase inhibitors. Notably, they target distinct binding sites with the target, presenting a promising avenue for overcoming resistance mechanisms in EGFR-mutant cancers [35]. Thus, there is an ongoing and persistent need to design new epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitors and modify existing ones to develop candidates with enhanced anticancer activity and reduced toxicity.

Quinoline is a significant heterocycle scaffold that has been extensively studied with versatile biological activity in numerous research investigations [36]. Quinoline heterocyclic derivatives exhibit a broad spectrum of biological activities, including anticancer [37–40], anti-microbial [41–43], anti-viral [44,45], anti-inflammatory [46,47] and anti-diabetic [48,49] activities. In particular, the literature revealed that many quinoline derivatives were designed and synthesized as significant EGFR-TK inhibitors [50–52], as shown in Supplementary Figure S2.

1.1. Rationale of the design

The EGFR-TK pocket, where ATP binds, consists of five main components; the adenine binding site: which includes relevant amino acids capable of forming hydrogen bonds with the adenine nucleus, the sugar region: characterized by its hydrophilic nature, hydrophobic region I: essential for inhibitor selectivity, hydrophobic region II: which may contribute to inhibitor specificity and the phosphate binding region: which can be targeted to enhance the pharmacokinetics of the inhibitors used. These components are illustrated in Supplementary Figure S3. Understanding the structural features of this pocket is vital for designing effective and selective EGFR-TK inhibitors.

Moreover, EGFR-TK inhibitors (e.g., Erlotinib) exhibit common pharmacophoric features including: a hydrophobic head that can fit into hydrophobic region I, a -NH spacer, a flat heteroaromatic system capable of fitting into the adenine region and forming hydrogen bonds with amino acids Met793, Thr854 and Thr790 and a hydrophobic tail that can fit into hydrophobic region II. These shared pharmacophoric elements contribute to the inhibitors' ability to interact with and modulate the EGFR-TK pocket effectively. In this current study, our objective was to design a variety of quinoline derivatives while preserving the key pharmacophoric features observed in EGFR-TK inhibitors, as illustrated in Figure 1. This design approach aimed to capitalize on the known structural motifs critical for effective interaction with the EGFR-TK pocket, enhancing the potential of these derivatives as kinase inhibitors. Herein, molecular hybridization strategy was utilized to attain the main pharmacophoric features of EGFR-TK inhibitors. Accordingly, by reacting isatin with 4-bromo acetophenone, the pharmacophores; planar aromatic ring system and hydrophobic tail were acquired in one compound. Besides, the -NH spacer was added using hydrazinolysis to afford hydrazide derivatives retaining the pharmacophores; planar aromatic ring system, hydrophobic tail and -NH spacer. Finally, diverse pharmacophoric hydrophobic heads were added (for structure–activity relationship studies) by substituting the -NH₂ of hydrazide moiety with variable aromatic and hetero-aromatic derivatives, as shown in Figure 2. In this current work, our objective is to design and synthesize novel quinoline derivatives intended as novel inhibitors of EGFR-TK with assessing their potential as anticancer agents through a combination of in silico and in vitro approaches.

Figure 1.

Steps for molecular hybridization of the designed compounds to attain the main pharmacophoric features of EGFR-TK inhibitors.

SAR: Structure–activity relationship.

Figure 2.

The molecular hybridization approach to attain the pharmacophoric features of the designed compounds (6a–11).

2. Materials & methods

2.1. Chemistry

Solvents and reagents were obtained from Aldrich (MO, USA) and were used without further purification unless otherwise indicated. Melting points were determined by open capillary tube method using Stuart SMP10 melting point apparatus and were uncorrected. The elemental analysis was carried out by Thermo Scientific™ (MA, USA) FLASH 2000 CHNS/O analyzer by Thermo Fisher Scientific at The Regional Center for Mycology and Biotechnology, Al-Azhar University, Egypt. Infrared Spectra were recorded as potassium bromide discs on Bruker FT-IR spectrophotometer (MA, USA), MUST university and expressed in wave number ν_max (cm^-1). ¹H NMR spectra were performed on Bruker 400 MHz spectrophotometer using TMS as internal standard, chemical shifts(δ) were recorded in ppm on δ scale at Ain Shams University, Egypt. ¹³C NMR spectra were carried out using Bruker 100 MHz using TMS as internal standard, chemical shifts (δ) were recorded in ppm on δ scale at at Ain Shams University, Egypt. Mass spectra were run on Hewlett Packard 5988 spectrometer (CA, USA) or Shimadzu QP-2010 plus (Kyoto, Japan) at The Regional Center for Mycology & Biotechnology, Al-Azhar University, Egypt. Progress of the reactions was monitored by TLC using pre-coated aluminum sheets silica gel (Merck 60 F₂₅₄) using chloroform:methanol (9.5:0.5) as the eluting system and was visualized by UV lamp.

2.1.1. Procedure for synthesis of 2-(4-bromophenyl)quinoline-4-carboxylic acid (1)

A mixture of Isatin (10 mmol, 1.47 g), 4-bromo acetophenone (10 mmol, 1.99 g) and 33% KOH (10 ml) in ethanol (10 ml) was heated under reflux for 12 h. The reaction mixture was left to cool then acidified with HCl. The formed residue was washed with H₂O, filtered, dried and crystallized from ethanol to give compound 1 [53].

Yellowish orange powder, Yield 91%., mp 232–234°C, reported (236–237°C). IR (KBr, cm^-1): 3446 (OH), 3178 (CH aromatic), 2980 (CH aliphatic), 1708 (C=O).

2.1.2. Procedure for synthesis of ethyl 2-(4-bromophenyl)quinoline-4-carboxylate (2)

2-(4-Bromophenyl)quinoline-4-carboxylic acid (1) (10 mmol, 3.28 g) in absolute ethanol (20 ml) containing (2 ml) of conc H₂SO₄ was heated under reflux for 12 h. After cooling, the reaction mixture was rendered alkaline using aqueous solution of NaHCO₃. The formed residue was washed with H₂O, filtered, dried and crystallized from ethanol to give compound 2 [54].

Buff crystals, Yield 80%., mp 92–94°C, reported (90–92°C). IR (KBr, cm^-1): 3138 (CH aromatic), 2993 (CH aliphatic), 1716 (C=O).

2.1.3. Procedure for synthesis of 2-(4-bromophenyl)quinoline-4-carbohydrazide (3)

Ethyl 2-(4-bromophenyl)quinoline-4-carboxylate (2) (10 mmol, 3.56 g) was dissolved in absolute ethanol (20 ml) and 98% hydrazine hydrate (6 ml) was added. The reaction mixture was heated under reflux for 7 h. After cooling, the reaction mixture was poured into ice cooled water. The formed residue was, filtered, dried and crystallized from ethanol to give acid the hydrazide 3 [55].

White powder, Yield 75%., mp 245–247°C, reported (246–248°C). IR (KBr, cm^-1): 3263 (NH), 3305 (NH₂), 3055 (CH aromatic), 2983 (CH aliphatic), 1645 (C=O).

2.1.4. General procedure for synthesis of 4-hydroxy-3-methoxy-5-(substituted)methylbenzaldehyde (4a–c)

To a solution of formaldehyde 37% (15 mmol, 0.55 ml) in absolute ethanol (25 ml), the appropriate secondary amine (12 mmol) and vanillin (10 mmol, 1.5 g) were added, and the mixture was heated under reflux for 5 h, after cooling, the excess solvent was removed under pressure and the formed residue was crystallized from ethanol to obtain the desired compounds 4a–c [56,57].

2.1.4.1. 4-Hydroxy-3-methoxy-5-(morpholinomethyl)benzaldehyde (4a)

White powder, Yield 84%., mp 99–101°C, reported (99–100°C). IR (KBr, cm^-1): 3446 (OH), 3117 (CH aromatic), 2970 (CH aliphatic), 2864 (CH aldehydic), 1681 (C=O).

2.1.4.2. 4-hydroxy-3-methoxy-5-((4-methylpiperazin-1-yl)methyl)benzaldehyde (4b)

White powder, Yield 69%., mp 118–120°C. IR (KBr, cm^-1): 3419 (OH), 3008 (CH aromatic), 2943(CH aliphatic), 2891 (CH aldehydic), 1678 (C=O).

2.1.4.3. 4-hydroxy-3-methoxy-5-((4-phenylpiperazin-1-yl)methyl)benzaldehyde (4c)

White powder, Yield 87%., mp 156–158°C, reported (156–157°C). IR (KBr, cm^-1): 3460 (OH), 3125 (CH aromatic), 2951 (CH aliphatic), 2829(CH aldehydic), 1681 (C=O).

2.1.5. General procedure for synthesis of 1,3-diaryl-2-propen-1-ones (5a–c)

To a mixture of the appropriate substituted benzaldehyde (10 mmol) and the appropriate substituted acetophenone (10 mmol) in absolute ethanol (20 ml), an aqueous solution of potassium hydroxide (30%, 10 ml) was added while stirring over a period of 15 min. after complete addition, the reaction mixture was stirred at room temperature for 3 h. The separated product was collected by filtration and crystallized from methanol to give compounds 5a–c [58–60].

2.1.5.1. 1,3-Diphenylprop-2-en-1-one (5a)

Yellow crystals, Yield 99%., mp 65–67°C, reported (65–69°C). IR (KBr, cm^-1): 3084 (CH aromatic), 2931 (CH aliphatic), 1658 (C=O).

2.1.5.2. 3-(4-fluorophenyl)-1-phenylprop-2-en-1-one (5b)

Yellow powder, Yield 88%., mp 67–69°C, reported (65–66°C). IR (KBr, cm^-1): 3101 (CH aromatic), 2974 (CH aliphatic), 1660 (C=O).

2.1.5.3. 1-(4-bromophenyl)-3-(2-chlorophenyl)prop-2-en-1-one (5c)

White powder, Yield 82%., mp 102–104°C, reported (88–91°C). IR (KBr, cm^-1): 3082 (CH aromatic), 2978 (CH aliphatic), 1654 (C=O).

2.1.6. General procedure for synthesis of 2-(4-bromophenyl)-N‘-(substituted-benzylidene)quinoline-4-carbohydrazide (6a–e)

A mixture of the acid hydrazide 3 (10 mmol, 3.41 g) and the appropriate aromatic aldehyde derivatives (10 mmol) in absolute ethanol (30 ml) containing 2 drops of glacial acetic acid was heated under reflux for 3–6 h. The obtained solid was filtered and crystallized from ethanol to afford compounds (6a–e).

2.1.6.1. 2-(4-Bromophenyl)-N'-(3,4,5-trimethoxybenzylidene)quinoline-4-carbohydrazide (6a)

White powder, Yield 65% (3.38 g) mp 307–309°C. IR (KBr, cm^-1): 3446 (NH), 3184 (CH Aromatic), 2976, 2937 (CH aliphatic), 1641 (C=O), 1577 (C=N). ¹H NMR (400 MHz, DMSO-d₆), δ ppm: 3.74 (s, 3H, OCH₃), 3.87 (s, 6H, 2OCH₃), 7.09 (s, 2H, Ar-H), 7.63–8.00 (m, 4H, Ar-H), 8.14–8.19 (m, 2H, Ar-H), 8.21–8.27 (m, 4H, azomethine CH, Ar-H), 12.42 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (100 MHz, DMSO-d₆), δ ppm: 55.7, 56.7 (2C), 105.5 (2C), 122.9, 123.9, 124.9, 125.3, 126.3, 128.0 (2C), 129.3, 130.0, 130.7, 132.4 (2C), 137.5, 139.9, 141.9, 147.3, 149.7, 153.4 (2C), 154.8, 162.6. MS m/z (%): 522.68 (M+2, 44.38), 520.14 (M⁺, 45.51), 322.46 (100). Anal. Calcd. for C₂₆H₂₂BrN₃O₄ (520.38): C, 60.01; H, 4.26; N 8.08; Found: C, 60.23; H, 4.50; N, 8.27.

2.1.6.2. 2-(4-Bromophenyl)-N'-(3,4-dimethoxybenzylidene)quinoline-4-carbohydrazide (6b)

White powder, Yield 70% (3.43 g) mp 262–264°C. IR (KBr, cm^-1): 3419 (NH), 3184 (CH Aromatic), 2997 (CH aliphatic), 1651 (C=O), 1598 (C=N). ¹H NMR (400 MHz, DMSO-d₆), δ ppm: 3.67 (s, 3H, OCH₃), 3.83 (s, 3H, OCH₃), 7.06 (d, 1H, J = 7.6 Hz. Ar-H), 7.25 (d, 1H, J = 7.6 Hz. Ar-H), 7.43 (s, 1H, Ar-H), 7.61–7.91 (m, 4H, Ar-H), 8.15–8.24 (m, 2H, Ar-H), 8.29–8.35 (m, 4H, azomethine CH, Ar-H), 12.15 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (100 MHz, DMSO-d₆), δ ppm: 54.6, 56.0, 108.6, 112.0, 117.1, 122.6, 123.9, 124.6, 125.2, 126.6, 127.7, 128.0, 129.3 (2C), 130.7, 131.7 (2C), 137.2, 141.9, 145.3, 148.0, 149.1, 151.4, 154.5, 163.0. MS m/z (%): 492.33 (M+2, 26.12), 490.56 (M⁺, 25.80), 388.03 (98.88). Anal. Calcd. for C₂₅H₂₀BrN₃O₃ (490.36): C, 61.24; H, 4.11; N 8.57; Found: C, 61.08; H, 4.29; N, 8.80.

2.1.6.3. 2-(4-Bromophenyl)-N'-(4-methoxybenzylidene)quinoline-4-carbohydrazide (6c)

White powder, Yield 68%. (3.13 g), mp 281–283°C. IR (KBr, cm^-1): 3242 (NH), 3041 (CH Aromatic), 2960 (CH aliphatic), 1654 (C=O), 1608 (C=N). ¹H NMR (400 MHz, DMSO-d₆), δ ppm: 3.83 (s, 3H, OCH₃), 7.06 (d, 2H, J = 8.8 Hz. Ar-H), 7.61–7.73 (m, 3H, Ar-H), 7.76 (d, 2H, J = 8.8 Hz. Ar-H), 7.78–7.89 (m, 1H, Ar-H), 8.15–8.24 (m, 2H, Ar-H), 8.30–8.34 (m, 4H, azomethine CH, Ar-H), 12.09 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (100 MHz, DMSO-d₆), δ ppm: 56.1, 114.9 (2C), 117.6, 122.2, 123.9, 125.6, 125.9, 126.2, 127.2, 128.3 (2C), 129.9, 130.9 (2C), 132.3 (2C), 137.6, 138.3, 142.0, 149.2, 152.3, 156.4, 162.0. MS m/z (%): 462.45 (M+2, 36.27), 460.76 (M⁺, 33.55), 166.05 (100). Anal. Calcd. for C₂₄H₁₈BrN₃O₂ (460.33): C, 62.62; H, 3.94; N, 9.13; Found: C, 62.81; H, 4.15; N, 9.36.

2.1.6.4. 2-(4-Bromophenyl)-N'-(4-methylbenzylidene)quinoline-4-carbohydrazide (6d)

White powder, Yield 70% (3.11 g), mp 246–248°C. IR (KBr, cm^-1): 3419 (NH), 3070 (CH Aromatic), 2916 (CH aliphatic), 1653 (C=O), 1606 (C=N). ¹H NMR (400 MHz, DMSO-d₆), δ ppm: 2.37 (s, 3H, CH₃), 7.06 (t, 1H, Ar-H), 7.31 (d, 2H, J = 8 Hz. Ar-H), 7. 63 (d, 2H, J = 8 Hz. Ar-H), 7. 80 (d, 2H, J = 8 Hz. Ar-H) 7.87 (t, 1H, Ar-H), 8.10–8.23 (m, 2H, Ar-H), 8.29–8.35 (m, 4H, azomethine CH, Ar-H), 12.17 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (100 MHz, DMSO-d₆), δ ppm: 21.4, 117.5, 123.9, 124.2, 127.8, 128.0 (2C), 129.7, 130.1 (2C), 131.0, 131.8 (2C), 132.4 (2C), 137.7, 140.9, 141.9, 145.5, 148.5, 149.4, 149.7, 155.1, 163.2. MS m/z (%): 446.24 (M+2, 22.24), 444.97 (M⁺, 24.52), 307.03 (100). Anal. Calcd. for C₂₄H₁₈BrN₃O (444.33): C, 64.88; H, 4.08; N, 9.46; Found: C, 64.72; H, 4.25; N, 9.73.

2.1.6.5. 2-(4-Bromophenyl)-N'-(4-hydroxy-3-methoxybenzylidene)quinoline-4-carbohydrazide (6e)

White powder, Yield 69%. (3.28 g) mp 280–282°C. IR (KBr, cm^-1): 3207 (NH), 3045 (CH Aromatic), 2972 (CH aliphatic), 1647 (C=O), 1589 (C=N). ¹H NMR (400 MHz, DMSO-d₆), δ ppm: 3.86 (s, 3H, OCH₃), 6.88 (d, 1H, J = 8.4 Hz. Ar-H), 7.14 (d, 1H, J = 8.4 Hz. Ar-H), 7.41 (s, 1H, Ar-H), 7.62 (t, 1H, Ar-H), 7.77 (d, 2H, J = 8.4 Hz. Ar-H), 7. 85 (t, 1H, Ar-H), 8.16 (d, 1H, J = 8 Hz. Ar-H), 8.22 (d, 1H, J = 8 Hz. Ar-H), 8.27–8.33 (m, 4H, azomethine CH, Ar-H), 9.66 (s, 1H, OH, D₂O exchangeable), 12.09 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (100 MHz, DMSO-d₆), δ ppm: 56.1, 116.0, 117.4, 123.0, 124.3, 125.7, 125.8, 128.1, 128.8 (2C), 129.8, 130.1, 131.0, 132.4 (2C), 132.6, 135.2, 137.7, 142.2, 148.3, 148.6, 149.9, 155.1, 163.0. MS m/z (%): 478.19 (M+2, 38.67), 476.78 (M⁺, 39.66), 408.52 (100). Anal. Calcd. for C₂₄H₁₈BrN₃O₃ (476.33): C, 60.52; H, 3.81; N,8.82; Found: C, 60.68; H, 4.02; N, 9.06

2.1.7. General procedure for synthesis of 2-(4-bromophenyl)-N'-(1-( substituted)ethylidene)quinoline-4-carbohydrazide (7a–d)

A mixture of the acid hydrazide 3 (10 mmol, 3.41 g) and the appropriate acetophenone derivatives (10 mmol) in absolute ethanol (30 ml) containing 2 drops of glacial acetic acid was heated under reflux for 6–18 h. The obtained solid was filtered and crystallized from ethanol to afford compounds (7a–d).

2.1.7.1. 2-(4-Bromophenyl)-N'-(1-(3,4,5-trimethoxyphenyl)ethylidene)quinoline-4-carbohydrazide (7a)

White powder, Yield 69%. (3.69 g) mp 243–245°C. IR (KBr, cm^-1): 3460 (NH), 3192 (CH Aromatic), 2933 (CH aliphatic), 1654 (C=O), 1587 (C=N). ¹H NMR (400 MHz, DMSO-d₆), δ ppm: 2.37 (s, 3H, CH₃), 3.73 (s, 3H, OCH₃), 3.87 (s, 6H, 2OCH₃), 7.19 (s, 2H, Ar-H), 7.63–7.88 (m, 4H, Ar-H), 8.12–8.18 (m, 2H, Ar-H), 8.23 (s, 1H, Ar-H), 8.92–8.32 (m, 2H, Ar-H), 12.42 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (100 MHz, DMSO-d₆), δ ppm: 14.2, 54.9 (2C), 60.1, 103.2 (2C), 122.8, 123.9, 125.6, 127.3, 129.0 (2C), 130.4, 131.7 (2C), 133.8, 137.2, 138.8, 142.7, 145.7, 147.7, 148.7, 152.4, 154.8 (2C), 156.6, 163.7. MS m/z (%): 536.60 (M+2, 33.04), 534.09 (M⁺, 32.63), 218.87 (100). Anal. Calcd. for C₂₇H₂₄BrN₃O₄ (534.41): C, 60.68; H, 4.53; N 7.86; Found: C, 60.85; H, 4.70; N, 8.09.

2.1.7.2. 2-(4-Bromophenyl)-N'-(1-(3,4-dimethoxyphenyl)ethylidene)quinoline-4-carbohydrazide (7b)

White powder, Yield 70%. (3.53 g), mp 215–217°C. IR (KBr, cm^-1): 3226 (NH), 3182 (CH Aromatic), 2964 (CH aliphatic), 1654 (C=O), 1608 (C=N), 1540, 1490, 1436, (NH, C=C). ¹H NMR (400 MHz, DMSO-d₆), δ ppm: 2.35 (s, 3H, CH₃), 3.63 (s, 3H, OCH₃), 3.83 (s, 3H, OCH₃), 6.37 (s, 1H, Ar-H), 6.74–7.04 (m, 2H, Ar-H), 7.43–7.85 (m, 5H, Ar-H), 8.16–8.36 (m, 4H, Ar-H), 11.62 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (100 MHz, DMSO-d₆), δ ppm: 13.5, 54.6, 56.4, 108.0, 109.7, 111.3, 116.5, 119.9, 124.2, 125.3, 127.3, 129.0 (2C), 130.7, 132.7 (2C), 137.4, 142.6, 142.9, 145.3, 147.3, 148.4, 148.7, 150.0, 154.5, 163.7. MS m/z (%): 504.56 (M+2, 14.64), 506.26 (M⁺, 17.80), 166.12 (100). Anal. Calcd. for C₂₆H₂₂BrN₃O₃ (504.38): C, 61.91; H, 4.40; N,8.33; Found: C, 62.08; H, 4.54; N, 8.60.

2.1.7.3. 2-(4-Bromophenyl)-N'-(1-(4-methoxyphenyl)ethylidene)quinoline-4-carbohydrazide (7c)

White powder, Yield 68%. (3.23 g), mp 258–260°C. IR (KBr, cm^-1): 3385 (NH), 3099 (CH Aromatic), 2956 (CH aliphatic), 1662 (C=O), 1610 (C=N). ¹H NMR (400 MHz, DMSO-d₆), δ ppm: 2.34 (s, 3H, CH₃), 3.63 (s, 3H, OCH₃), 7.03 (d, 2H, J = 8.8 Hz. Ar-H), 7. 10 (d, 2H, J = 8.8 Hz. Ar-H), 7.71–7.86 (m, 3H, Ar-H), 7.88 (t, 1H, Ar-H), 8.17 (d, 1H, J = 8.4 Hz. Ar-H), 8.29–8.35 (m, 4H, Ar-H), 11.15 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (100 MHz, DMSO-d₆), δ ppm: 15.2, 55.7, 114.7 (2C), 123.2, 123.9, 124.6, 125.6, 127.6, 128.3 (2C), 130.0 (2C), 130.7, 132.0 (2C), 137.8, 142.6, 145.7, 148.7, 150.0, 154.5, 155.8, 161.2, 164.0. MS m/z (%): 473.93 (M+2, 74.84), 476.85 (M⁺, 79.68), 323.78 (100). Anal. Calcd. for C₂₅H₂₀BrN₃O₂ (474.36): C, 63.30; H, 4.25; N, 8.86; Found: C, 63.54; H, 4.33; N, 8.98.

2.1.7.4. 2-(4-Bromophenyl)-N'-(1-(p-tolyl)ethylidene)quinoline-4-carbohydrazide (7d)

White powder, Yield 73%. (3.35 g), mp 238–240°C. IR (KBr, cm^-1): 3226 (NH), 3030 (CH Aromatic), 2918 (CH aliphatic), 1660 (C=O), 1614 (C=N). ¹H NMR (400 MHz, DMSO-d₆), δ ppm: 2.13 (s, 3H, CH₃), 2.34 (s, 3H, CH₃), 7.03 (d, 2H, J = 8 Hz. Ar-H), 7. 28 (d, 2H, J = 8 Hz. Ar-H), 7.67 (t, 1H, Ar-H), 7.76–7.87 (m, 3H, Ar-H), 8.14–8.19 (m, 2H, Ar-H), 8.27–8.35 (m, 3H, Ar-H), 11.20 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (100 MHz, DMSO-d₆), δ ppm: 15.2, 21.0, 123.9, 124.6, 125.3, 125.9, 126.3 (2C), 127.7, 128.6 (2C), 129.0, 130.0 (2C), 131.0, 132.7 (2C), 134.7, 137.5, 139.5, 142.6, 147.3, 149.7, 156.5, 163.7. MS m/z (%): 460.07 (M+2, 34.32), 458.96 (M⁺, 32.05), 291.71 (100). Anal. Calcd. for C₂₅H₂₀BrN₃O (458.36): C, 65.51; H, 4.40; N, 9.17; Found: C, 65.32; H, 4.56; N, 9.41.

2.1.8. General procedure for synthesis 2-(4-bromophenyl)-N'-(4-hydroxy-3-methoxy-5-(substituted)benzylidene)quinoline-4-carbohydrazide (8a–c)

A mixture of equimolar amount of the acid hydrazide 3 (10 mmol, 3.41 g) and the appropriate aldehydes 5a–c in absolute ethanol (20 ml) containing 2 drops of glacial acetic acid was heated under reflux for 4–8 h. The obtained solid was filtered and crystallized from ethanol to afford compounds (8a–c).

2.1.8.1. 2-(4-Bromophenyl)-N'-(4-hydroxy-3-methoxy-5-(morpholinomethyl)benzylidene)quinoline-4-carbohydrazide (8a)

Buff crystals, Yield 78% (4.5 g), mp 192–194°C. IR (KBr, cm^-1): 3460 (OH), 3419 (NH), 3055 (CH Aromatic), 2962 (CH aliphatic), 1651 (C=O), 1589 (C=N). ¹H NMR (400 MHz, DMSO-d₆), δ ppm: ¹H NMR (400 MHz, DMSO-d₆), δ ppm: 2.46–2.50 (m, 4H, CH₂-N-CH₂), 3.34 (s, 2H, CH₂), 3.39 (s, 1H, OH, D₂O exchangeable), 3.62–3.65 (m, 4H, CH₂-O-CH₂), 3.86 (s, 3H, OCH₃), 7.15 (s, 1H, Ar-H), 7.31 (s, 1H, Ar-H), 7.69 (t, 1H, Ar-H), 7.79 (d, 2H, J = 8 Hz. Ar-H), 7.91(t, 1H, Ar-H), 8.10–8.18 (m, 3H, Ar-H), 8.21–8.25 (m, 3H, azomethine CH, Ar-H), 12.08 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (100 MHz, DMSO-d₆), δ ppm: 52.6 (2C), 56.0, 58.4, 66.5 (2C), 109.0, 117.4, 122.9, 123.5, 123.9, 124.6, 124.9, 125.9, 127.7, 129.7 (2C), 130.7, (2C), 132.4, 137.2, 141.5, 142.6, 148.4, 148.7, 149.7, 155.2, 157.2, 162.6. MS m/z (%): 577.48 (M+2, 24.24), 575.55 (M⁺, 23.38), 245.22 (100). Anal. Calcd. for C₂₉H₂₇BrN₄O₄ (575.46): C, 60.53; H, 4.73; N 9.74; Found: C, 60.79; H, 4.88; N, 10.02.

2.1.8.2. 2-(4-Bromophenyl)-N'-(4-hydroxy-3-methoxy-5-((4-methylpiperazin-1-yl)methyl)benzylidene)quinoline-4-carbohydrazide (8b)

Buff powder, Yield 76.4%. (4.5 g), mp 180–182°C. IR (KBr, cm^-1): 3460 (OH), 3419 (NH), 3010 (CH Aromatic), 2939 (CH aliphatic), 1649 (C=O), 1589 (C=N). ¹H NMR (400 MHz, DMSO-d₆), δ ppm: 2.18 (s, 3H, CH₃), 2.37 (s, 4H, piperazine), 3.43 (s, 5H, piperazine +OH, D₂O exchangeable), 3.70 (s, 2H, CH₂), 3.85 (s, 3H, OCH₃), 7.10 (s, 1H, Ar-H), 7.29 (s, 1H, Ar-H), 7.65–7.72 (m, 1H, Ar-H), 7.75–7.97 (m, 3H, Ar-H), 8.14–8.24 (m, 3H, Ar-H), 8.28–8.35 (m, 3H, azomethine CH, Ar-H), 12.07 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (100 MHz, DMSO-d₆), δ ppm: 45.8, 51.9 (2C), 54.6, 55.7 (2C), 58.7, 108.6, 117.4, 122.6, 123.5, 124.2, 124.6, 124.9, 125.3, 125.9, 128.3 (2C), 129.0, 130.7, 131.7 (2C), 137.1, 142.2, 144.3, 145.0, 148.7, 149.7, 155.2, 168.6. MS m/z (%): 588.19 (M+2, 19.99), 590.45 (M⁺, 23.38), 529.24 (100). Anal. Calcd. for C₃₀H₃₀BrN₅O₃ (588.51): C, 61.23; H, 5.14; N 11.90; Found: C, 62.52; H, 5.12; N, 10.98.

2.1.8.3. 2-(4-Bromophenyl)-N'-(4-hydroxy-3-methoxy-5-((4-phenylpiperazin-1-yl)methyl)benzylidene)quinoline-4-carbohydrazide (8c)

White powder, Yield 89%. (5.8 g), mp 166–168°C. IR (KBr, cm^-1): 3446 (OH), 3421 (NH), 3035 (CH Aromatic), 2941 (CH aliphatic), 1662 (C=O), 1597 (C=N). ¹H NMR (400 MHz, DMSO-d₆), δ ppm: 2.63 (s, 4H, piperazine), 3.18 (s, 4H, piperazine), 3.46 (s, 1H, OH, D₂O exchangeable), 3.73 (s, 2H, CH₂), 3.87 (s, 3H, OCH₃), 6.78 (t, 1H, Ar-H), 6.94 (d, 2H, J = 7.6 Hz. Ar-H), 7.17–7.23 (m, 3H, Ar-H), 7.32 (s, 1H, Ar-H), 7.65–7.75 (m, 1H, Ar-H), 7.79 (d, 2H, J = 8 Hz. Ar-H), 7.89 (t, 1H, Ar-H), 8.11–8.23 (m, 3H, Ar-H), 8.24–8.28 (m, 3H, azomethine CH, Ar-H), 12.18 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (100 MHz, DMSO-d₆), δ ppm: 48.5 (2C), 52.6 (2C), 56.0, 58.4, 109.0, 117.4 (2C), 118.1, 120.1, 123.5, 124.2, 124.9, 125.9, 126.6, 127.3, 128.0, 129.3 (2C), 129.7, 130.4, 130.7 (2C), 132.4, 137.8, 141.9, 147.3, 148.3, 149.4, 150.0, 151.4, 154.4, 154.5, 163. MS m/z (%): 652.12 (M+2, 7.54), 650.72 (M⁺, 8.28), 529.24 (100). Anal. Calcd. for C₃₅H₃₂BrN₅O₃ (650.58): C, 64.62; H, 4.96; N 10.77; Found: C, 64.51; H, 5.12; N, 10.98.

2.1.9. General procedure for synthesis of 2-(4-bromophenyl)-N'-(2-substituted oxoindolin-3-ylidene)quinoline-4-carbohydrazide (9a,b)

A mixture of the acid hydrazide 3 (10 mmol, 3.41 g) and substituted isatin (10 mmol) in absolute ethanol (30 ml) containing 2 drops of glacial acetic acid was heated under reflux for 8–10 h. The obtained solid was filtered and crystallized from methanol.

2.1.9.1. 2-(4-Bromophenyl)-N'-(2-oxoindolin-3-ylidene)quinoline-4-carbohydrazide (9a)

Yellow powder, Yield 83% (3.91 g). mp 294–296°C. IR (KBr, cm^-1): 3292 (NH), 3099 (CH Aromatic), 2993 (CH aliphatic), 1691, 1624 (C=O), 1587 (C=N). ¹H NMR (400 MHz, DMSO-d₆), δ ppm: 6.85–7.44 (m, 4H, Ar-H), 7.70–7.88 (m, 4H, Ar-H), 8.20 (d, 1H, J = 8.8 Hz. Ar-H), 8.29–8.40 (m, 3H, Ar-H), 8.52 (s, 1H, Ar-H), 11.31 (s, 1H, NH, D₂O exchangeable), 13.67 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (100 MHz, DMSO-d₆), δ ppm: 115.8, 117.4, 120.5, 122.2, 122.5, 122.8, 123.5, 123.9, 124.2, 125.6, 126.6, 128.3 (2C), 130.0, 130.7 (2C), 131.4, 132.0, 137.2, 143.3, 148.7, 155.2, 163.3, 169.8. MS m/z (%): 473.65 (M+2, 9.83), 471.85 (M⁺, 10.76), 407.56 (100). Anal. Calcd. for C₂₄H₁₅BrN₄O₂ (471.31): C, 61.16; H, 3.21; N 11.89; Found: C, 61.40; H, 3.37; N, 12.16.

2.1.9.2. N'-(5-Bromo-2-oxoindolin-3-ylidene)-2-(4-bromophenyl)quinoline-4-carbohydrazide (9b)

Orange powder, Yield 78% (4.31 g). mp 298–300°C. IR (KBr, cm^-1): 3273 (NH), 3095 (CH Aromatic), 2948 (CH aliphatic), 1689, 1620 (C=O), 1587 (C=N). ¹H NMR (400 MHz, DMSO-d₆), δ ppm: 7.12 (d, 1H, J = 6.4 Hz. Ar-H), 7.55 (d, 1H, J = 6.4 Hz. Ar-H), 7.66 (t, 1H, Ar-H), 7.75 (d, 2H, J = 8.8 Hz. Ar-H), 7.84 (m, 1H, Ar-H), 8.07–8.15 (m, 3H, Ar-H), 8.24–8.31 (m, 3H, Ar-H), 10.66 (s, 1H, NH, D₂O exchangeable), 13.32 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (100 MHz, DMSO-d₆), δ ppm: 119.9, 123.5, 123.9, 124.2, 124.9, 127.0, 127.7, 129.0 (2C), 130.4, 132.0 (2C), 132.7, 133.1, 133.4, 135.4, 136.5, 137.8, 141.9, 148.4, 150.0, 154.8, 164.4, 168.8. MS m/z (%): 552.92 (M+2, 26.31), 550.15 (M⁺, 22.76), 230.08 (100). Anal. Calcd. for C₂₄H₁₄Br₂N₄O₂ (550.21): C, 52.39; H, 2.56; N 10.18; Found: C, 52.51; H, 2.80; N, 10.43.

2.1.10. General procedure for synthesis of 2-(4-bromophenyl)-N'-((1,2)-1,3-substituted allylidene)quinoline-4-carbohydrazide (10a–c)

A mixture of the acid hydrazide 3 (10 mmol, 3.41 g) and the corresponding chalcones (4a–c) (10 mmol) in absolute ethanol (30 ml) containing 5 drops of glacial acetic acid was heated under reflux for 24 h. The obtained solid was filtered and crystallized from acetonitrile to afford compounds (10a–c).

2.1.10.1. 2-(4-Bromophenyl)-N'-((1Z,2Z)-1,3-diphenylallylidene)quinoline-4-carbohydrazide (10a)

White powder, Yield 74% (3.9 g). mp 222–224°C. IR (KBr, cm^-1): 3419 (NH), 3061 (CH Aromatic), 2927 (CH aliphatic), 1678 (C=O), 1589 (C=N). ¹H NMR (400 MHz, DMSO-d₆), δ ppm: 6.84 (d, 1H, J = 8 Hz. =CH), 7.11–7.36 (m, 2H, =CH, Ar-H), 7.38–7.46 (m, 4H, Ar-H), 7.54–7.61 (m, 2H, Ar-H), 7.62–7.70 (m, 3H, Ar-H), 7.74–7.79 (m, 2H, Ar-H), 8.15–8.22 (m, 3H, Ar-H), 8.27–8.30 (m, 1H, Ar-H), 8.36 (d, 2H, J = 7.6 Hz. Ar-H), 8.45 (s, 1H, Ar-H), 11.79 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (100 MHz, DMSO-d₆), δ ppm: 119.0, 123.8, 125.1, 127.2, 127.6, 127.9, 128.0, 128.1 (2C), 128.4 (2C), 128.6 (2C), 128.7 (2C), 129.2, 129.4, 129.5, 129.6 (2C), 130.5 (2C), 131.9, 135.6, 137.1, 137.3, 140.2, 142.2, 155.6, 156.0, 163.2. MS m/z (%): 534.91 (M+2, 17.42), 532.84 (M⁺, 15.83), 295.71 (100). Calcd. for C₃₁H₂₂BrN₃O (532.44): C, 69.93; H, 4.16; N 7.89; Found: C, 69.70; H, 4.37; N, 8.15.

2.1.10.2. 2-(4-Bromophenyl)-N'-((1Z,2Z)-3-(4-fluorophenyl)-1-phenylallylidene)quinoline-4-carbohydrazide (10b)

White powder, Yield 69% (3.8 g). mp 198–200°C. IR (KBr, cm^-1): 3419 (NH), 3062 (CH Aromatic), 2931 (CH aliphatic), 1681 (C=O), 1593 (C=N), 1540. ¹H NMR (400 MHz, DMSO-d₆), δ ppm: 6.83 (d, 1H, J = 8 Hz, =CH), 7.15 (d, 1H, J = 8 Hz, =CH), 7.22–7.28 (m, 2H, Ar-H), 7.30–7.34 (m, 1H, Ar-H), 7.54–7.62 (m, 2H, Ar-H), 7.66–7.74 (m, 3H, Ar-H), 7.76–7.80 (m, 2H, Ar-H), 7.85–7.91 (m, 1H, Ar-H), 8.12–8.19 (m, 3H, Ar-H), 8.21–8.35 (m, 3H, Ar-H), 8.43 (s, 1H, Ar-H), 12.34 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (100 MHz, DMSO-d₆), δ ppm:115.8 (2C), 119.2, 124.2, 125.3, 128.7 (2C), 129.0 (2C), 129.7, 130.0 (2C), 130.7, 131.0, 131.4 (2C), 131.7 (2C), 132.0, 134.4, 136.5, 137.8, 138.5, 139.2, 142.6, 147.3, 148.0, 159.2, 161.2, 164.0, 166.0. MS m/z (%): 552.07 (M+2, 10.76), 550.73 (M⁺, 12.98), 469.79 (100). Anal. Calcd. for C₃₁H₂₁BrFN₃O (550.43): C, 67.65; H, 3.85; N, 7.63. Found: C, 67.84; H, 4.06; N, 7.81.

2.1.10.3. 2-(4-Bromophenyl)-N'-((1Z,2Z)-1-(4-bromophenyl)-3-(2-chlorophenyl)allylidene)quinoline-4-carbohydrazide (10c)

White powder, Yield 64% (4.14 g). mp 228–230°C. IR (KBr, cm^-1): 3444 (NH), 3062 (CH Aromatic), 2976 (CH aliphatic), 1664 (C=O), 1589 (C=N). ¹H NMR (400 MHz, DMSO-d₆), δ ppm: 7.07 (d, 1H, J = 8 Hz, =CH), 7.10–7.35 (m, 2H, =CH, Ar-H), 7.38–7.60 (m, 5H, Ar-H), 7.63–7.77 (m, 2H, Ar-H), 7.83–7.98 (m, 4H, Ar-H), 8.10–8.15 (m, 1H, Ar-H), 8.16–8.34 (m, 3H, Ar-H), 8.43 (s, 1H, Ar-H), 11.96 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (100 MHz, DMSO-d₆), δ ppm: 118.1, 123.9, 124.9, 126.3, 127.0, 127.3, 128.0, 128.4, 128.6 (2C), 129.0 (2C), 129.3, 129.7, 130.0 (2C), 131.7 (2C), 132.4, 132.7, 133.4, 135.4, 137.8, 138.4, 140.2, 141.5, 144.3, 148.0, 154.5, 155.8, 164.0. MS m/z (%): 547.51 (M+2, 15.79), 645.43 (M⁺, 19.41), 508.90 (100). Anal. Calcd. for C₃₁H₂₀Br₂ClN₃O (645.78): C, 57.66; H, 3.12; N, 6.51. Found: C, 57.89; H, 3.39; N, 6.65.

2.1.11. Procedure for synthesis of 2-(4-bromophenyl)-N‘-(3,4-dihydronaphthalen-2(1H)-ylidene)quinoline-4-carbohydrazide (11)

A mixture of the acid hydrazide 3 (10 mmol, 3.41 g) and tetralone (10 mmol, 1.32 ml) in absolute ethanol (20 ml) containing 5 drops of glacial acetic acid was heated under reflux for 6 h. The obtained solid was filtered and crystallized from ethanol to afford compounds (11).

White powder, Yield 84% (3.95 g). mp 271–273°C. IR (KBr, cm^-1): 3446 (NH), 3055 (CH Aromatic), 2937 (CH aliphatic), 1647 (C=O), 1608 (C=N). ¹H NMR (400 MHz, DMSO-d₆), δ ppm: 1.80–1.86 (m, 2H, CH₂, tetralone ring), 2.66 (s, 2H, CH₂, tetralone ring), 2.68–2.80 (m, 2H, CH₂, tetralone ring), 6.78–7.35 (m, 4H, Ar-H), 7.69–7.77 (m, 4H, Ar-H), 7.80–8.35(m, 5H, Ar-H), 11.19 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (100 MHz, DMSO-d₆), δ ppm: 21.4, 27.1, 29.5, 123.5, 124.6, 124.9, 125.3, 125.6, 126.3, 127.0, 128.0, 128.6, 129.0 (2C), 130.0, 130.4 (2C), 131.1, 132.0, 137.2, 139.9, 142.2, 148.0, 155.1, 155.8, 164.0. MS m/z (%): 472.43 (M+2, 17.22), 470.92 (M⁺, 17.83), 370.91 (100). Anal. Calcd. for C₂₆H₂₀BrN₃O (470.37): C, 66.39; H, 4.29; N, 8.93. Found: C, 66.54; H, 4.37; N, 9.12.

2.2. Biological evaluation

2.2.1. Anti-proliferative activity against different cancer cell lines

The anti-proliferative activity of the synthesized 2-(4-bromophenyl)quinoline derivatives 6a–11 was assessed against breast MCF-7, leukemia HL-60 and lung A549 cancer cell lines. The results were reported as half maximal inhibitory concentration values (IC₅₀). See details in the Supplementary Material.

2.2.2. EGFR kinase inhibitory activity

The most active quinoline compounds against A549 cell line, compounds 6d and 8b, were selected to investigate for the potential EGFR kinase inhibitory activity. Lapatinib was used as reference EFGR inhibitor. The results were reported as half maximal inhibitory concentration values, as determined from triplicate measurements. See details in the Supplementary Material.

2.2.3. Cell cycle analysis

The cellular cycle analysis was performed to investigate the prevention of proliferation in cancer cells (A549 lung cancer cell line) by the most potent quinoline derivatives 6d and 8b. A549 cells were treated with compounds 6d and 8b at the IC₅₀ concentration for 48 h and the treated A549 cells were stained with propidium iodide (PI) and analyzed by FACS analysis. The obtained results were compared with non-treated MCF-7 cells as control. See details in the Supplementary Material.

2.2.4. Apoptosis inducing activity

Flow cytometric analysis was performed to determine the mechanism of cellular the mechanism of cellular death which observed by the most active quinoline compounds in A549 lung cancer cells by Annexin V-FITC/PI double staining analysis. In this regard, non-treated cells were used as negative control. The cells were treated with compounds 6d and 8b at the IC₅₀ concentration dose for 48 h. After that, the cells were stained with FITC Annexin V and PI solution, respectively and the percentage of cells were determined by FACS analysis.

2.3. In silico studies

2.3.1. Molecular docking

The binding interactions of the newly synthesized quinoline derivatives (6a–11) at the EGFR-TK active site were analyzed using molecular docking with the MOE 2019 suite [61]. The co-crystallized ligand (erlotinib) at the target site served as a reference control for comparison and assessment of the binding interactions of the newly designed compounds.

2.3.1.1. Preparation of the investigated derivatives

To prepare for the molecular docking process, the assessed compounds were chemically drawn using PerkinElmer ChemOffice Suite 2017. Subsequently, these chemical structures were imported into a database file along with erlotinib (MDB file), as previously described. This preparation is essential for setting up the molecular docking simulations and analyzing the interactions between the compounds and the target protein at the EGFR-TK active site [62–70].

2.3.1.2. Preparation of the EGFR-TK target protein

The Protein Data Bank (PDB) file of the EGFR-TK target protein was obtained from the PDB (PDB entry: 1m17) [71]. Following this, the target protein underwent preparation and energetic minimization to ensure it was thoroughly ready for the subsequent docking process, as detailed in previous discussions [63,70]. This preparation step is crucial to create an optimized and reliable structure of the target protein for accurate molecular docking simulations.

2.3.1.3. Docking of the afforded compounds (6a–11) to EGFR-TK target protein

The general docking protocol was employed according to the default procedures discussed previously in detail [65,72–75] and the docking process was executed for the selected compounds. For each docked compound, the pose with the best affinity energy scores, root-mean-square deviation (RMSD) values and notable amino acid and nucleobase interactions were chosen and saved for subsequent visualization. This step ensures that the most favorable binding conformations and interactions of the compounds with the EGFR-TK target protein are retained for further analysis and interpretation.

2.3.2. Physicochemical, absorption, distribution, metabolism, excretion & toxicity, & pharmacokinetic properties prediction

The Swiss Institute of Bioinformatics (SIB) provides the free Swiss ADME web tool, a resource used for evaluating the physicochemical characteristics predicting pharmacokinetic features and anticipating the absorption, distribution, metabolism and excretion (ADME) parameters of the synthesized compounds. To perform these calculations, SMILES notations representing the chemical structures of the synthesized compounds were inputted into the online server of the Swiss ADME web tool for further processing and analysis. This approach aids in understanding the compounds’ potential pharmacokinetic behavior and provides insights into their suitability for drug development [76]. Besides, the toxicity features of the investigated compounds were examined using the pkCS web platform [77].

3. Results & discussion

3.1. Chemistry

In this study, 18 compounds containing quinoline moiety were synthesized as depicted in Figures 3 & 4. The synthesis of the desired hydrazone derivatives 6a–e in a good yield (65–70%) was accomplished by condensing the acid hydrazide 3 with substituted benzaldehyde derivatives in boiling ethanol containing catalytic amount of glacial acetic acid. The structure of the target compounds was confirmed by IR spectra, which revealed the disappearance of the NH₂ forked peak. ¹H NMR spectra showed singlet signal at δ 8.21–8.35 ppm corresponding to the proton of azomethine group. ¹³C NMR spectra revealed signal at δ 148.3–149.2 ppm referring to the carbon of azomethine group. 2-(4-Bromophenyl)-N'-(1-(substituted)ethylidene)quinoline-4-carbohydrazide derivatives 7a–d were prepared in good yield (68–73%) via reaction of acid hydrazide 3 with substituted acetophenone derivative under reflux in the presence glacial acetic acid. IR spectra of 7a–d showed absence of the characteristic -NH₂ forked band. ¹H NMR spectra revealed singlet signal at δ 2.13–2.37 ppm corresponding to the proton of CH₃. In the ¹³C NMR spectra, CH₃ resonating at 13.5–15.2 ppm and carbon atom of imine (C=N) resonating at δ 148.7–149.7 ppm. Subsequent reaction of acid hydrazide 3 with compounds 4a–c in absolute ethanol containing catalytic amount of glacial acetic acid afforded 2-(4-bromophenyl)-N'-(4-hydroxy-3-methoxy-5-(substituted)benzylidene)quinoline-4-carbohydrazide derivatives 8a–c in high yield (76–89%). IR spectra of compounds 8a–c showed stretching bands of OH and NH at 3446–3460 and 3419–3421 cm^-1 respectively, and absence of NH₂ stretching bands. ¹H NMR spectra revealed characteristic signals at δ 2.37–3.65 ppm assigned for aliphatic protons of morpholinyl, N-methylpiperazinyl and N-phethylpiperazinyl, in addition to singlet signal of N-CH₂ and OCH₃ at δ 3.34–3.73 ppm and δ 3.85–3.87 ppm respectively. Moreover, singlet signal of N=CH appeared at δ 8.21–8.35 ppm. Besides, the presence of only one singlet signal of D₂O-exchangeable proton at δ 12.07–12.18 ppm corresponding to NH. The D₂O-exchangeable proton of phenolic OH noticed at δ 3.39–3.46 ppm. ¹³C NMR spectra of 8a–c showed characteristic signals at δ 45.8–66.5 ppm assigned for carbons of morpholinyl, N-methylpiperazinyl and N-phenylpiperazinyl, along with signals of OCH₃ and N-CH₂ at δ 54.6–56.0 and 58.4–58.7, respectively. In addition, characteristic signals of N=CH at δ 149.7–150.0 ppm was noticed. Interestingly, 2-(4-bromophenyl)-N'-(2-substituted oxoindolin-3-ylidene)quinoline-4-carbohydrazide 9a,b were prepared in a good yield (78–83%) through the reaction of acid hydrazide 3 with isatin derivatives in absolute ethanol containing few drops of glacial acetic acid. IR spectra of 9a,b showed stretching bands of two NH at 3273–3292 cm^-1 and absence of NH₂ stretching bands along with presence of two major sharp absorption bands at 1689–1691 and 1620–1624 cm^-1 characterizing two carbonyl functional groups. ¹H NMR spectra of compounds 9a,b revealed characteristic signals at δ 10.66–11.31 and 13.32–13.67 ppm corresponding to D₂O-exchangeable protons of two NH and absence of NH₂ signal. The other aromatic protons of 2-oxoindoline moiety appeared at the expected chemical shift. ¹³C NMR spectra of compounds 9a,b showed, two signals at δ 163.3–164.4 and 168.8–169.8 ppm corresponding to two carbonyl groups. On the other hand, quinoline–chalcone hybrids 10a–c were prepared in a good yield (64–74%) via straightforward reaction of chalcone derivatives 5a–c with acid hydrazide 3 in absolute ethanol containing glacial acetic acid in a catalytic amount. The IR spectra of 10a–c showed absence of absorption band of NH₂ and presence of absorption band at 1664–1678 cm^-1 referring to carbonyl functional groups accompanied by the presence of stretching band of NH at 3419–3444 cm^-1. ¹H NMR spectra of compounds 10a–c revealed characteristic signals at δ 11.79–12.34 ppm corresponding to D₂O-exchangeable NH proton and absence of signal of NH₂. The appearance of two signals of olefinic protons of chalcone at δ 6.83–7.07 and 7.10–7.36 ppm confirmed the open hydrazone structure. The coupling constant of these protons were around 8 Hz so that, the formed compounds were in cisoid form. The other aromatic protons of chalcone moiety appeared at the expected chemical shift. ¹³C NMR spectra showed numerous signals referring to carbons of chalcone moiety. Condensation of tetralone as a cyclic ketone with acid hydrazide 3 in ethanol containing few drops of glacial acetic acid provided compound 11 in high yield (84%). IR spectra of 2-(4-bromophenyl)-N‘-(3,4-dihydronaphthalen-2(1H)-ylidene)quinoline-4-carbohydrazide (11) showed absence of absorption band of NH₂ and presence of absorption band at 3446 and 1647 cm^-1 referring to NH and carbonyl functional group respectively. ¹H NMR spectrum showed absence of signal of NH₂ protons and appearance of three signals at δ 1.80–1.86, 2.66 and 2.68–2.80 ppm corresponding to aliphatic protons of tetralone ring as long as, the drastic increase in the number of aromatic protons and their integration were all doubtless evidence the preparation of the target compound. ¹³C NMR spectrum tracked the appearance of signals referring to aliphatic carbon of tetralone moiety at δ 21.4, 27.1, 29.5 ppm besides, the elevated number of aromatic carbons of tetralone moiety. Electron ionization mass spectrometry of the synthesized compounds showed molecular ion peaks corresponding to their molecular weight in the ratio (1:1).

Figure 3.

Synthesis of starting materials 1–5a–c. Reagents and conditions: (A) 33%KOH, 96% EtOH, reflux 12h. (B) Absolute EtOH, conc H₂SO₄, reflux 12 h. (C) NH₂NH₂.H₂O, EtOH, reflux 7 h. (D) HCHO,appropriate secondary amine, EtOH , reflux 5 h. (E) 30% KOH, EtOH, stirring room temperature, 2 h.

Figure 4.

Synthesis of compounds 6a–11. Reagents and conditions: (A) Aromatic aldehydes, EtOH, HOAc, reflux 3–6 h. (B) Substituted acetophenone, EtOH, HOAc, reflux, 6–18 h. (C) Aldehydes 5a–c, EtOH, HOAc, reflux, 4–8 h. (D) Isatin derivative EtOH, HOAc, reflux 8 h. (E) Chalcones 4a–c, EtOH, HOAc, reflux, 24 h. (F) 2-tetralone EtOH, HOAc, reflux 6 h.

3.2. Biological evaluation

3.2.1. Antiproliferative activity against different cancer cell lines

The antiproliferative activity of the synthesized 2-(4-bromophenyl)quinoline derivatives 6a–11 was assessed against breast (MCF-7), leukemia (HL-60) and lung (A549) cancer cell lines. Selection of these cancer cell types depended on the documented high expression of the EGFR in such tumor cells [78–80]. The obtained results are summarized in Table 1. From the obtained results, it was found that the assessed quinoline derivatives 6a–11 showed considerable cytotoxic activity against breast (MCF-7), leukemia (HL-60) and lung (A549) cancer cell lines with IC₅₀ values 0.14–0.82, 0.10–1.12 and 0.06–0.96 μM, respectively in comparison to 5-Fluorouracil (5-FU), a positive control, which showed IC₅₀ values of 0.51, 0.18 and 0.41 μM, respectively. Regarding activity against breast MCF-7 breast cancer cells, compounds 10a and 11 were the least active molecule with IC₅₀ value of 0.82, 0.76 μM, respectively compared with other tested quinoline derivatives (IC₅₀ ranges: 0.14–0.60 μM). The 4-(4-methoxybenzylidenehydraziny) bearing quinoline in 6c was equipotent to 4-(4-methylbenzylidenehydrazinyl) quinoline in 6d. The presence of 4-(3,4-dimethoxybenzylidenehydrazinyl) quinoline 6b resulted in higher activity than 4-(3,4,5-trimethoxybenzylidenehydrazinyl) quinoline 6a and 4-(4-hydroxy-3-methoxybenzylidenehydrazinyl)quinoline 6e. In addition, the N-(arylethylidene)quinoline-4-carbohydrazides 7a–d showed best activity in case of 4-methoxyphenyl derivative 7c (IC₅₀ = 0.16 μM) followed by 4-methylphenyl 7d (IC₅₀ = 0.34 μM), the 3,4,5-trimethoxyphenyl 7b (IC₅₀ = 0.46 μM) and the least cytotoxic activity showed by 3,4-dimethoxyphenyl 7a (IC₅₀ = 0.54 μM). Additionally, the N-methylpiperazinyl derivative 8b (IC₅₀ = 0.23 μM) demonstrated equipotent activity to the N-phenylpiperazinyl congener 8c (IC₅₀ = 0.22 μM). Moreover, the N-(5-bromo-2-oxindolin-3-ylidene)quinoline-4-carbohydrazide derivative 9b (IC₅₀ = 0.34 μM) exerted higher activity than the unsubstituted derivative 9a (IC₅₀ = 0.47 μM). Furthermore, the presence of electron withdrawing group enhances the cytotoxic activity of compounds 10b and 10c as compared with with unsubstituted phenyl bearing congener 10a.

Table 1. The IC₅₀ values (μM) of all target (4-bromophenyl)quinoline derivatives 6–11 against MCF-7, HL-60 and A549 cell lines.

Compounds	IC50 (μM)
	MCF-7	HL-60	A549	HEL-299
6a	0.49	0.69	0.96	NT
6b	0.14	0.32	0.22	NT
6c	0.31	0.97	0.21	NT
6d	0.30	0.41	0.06	24.96
6e	0.50	0.46	0.24	NT
7a	0.46	1.12	0.20	NT
7b	0.54	0.32	0.37	NT
7c	0.16	0.31	0.22	NT
7d	0.34	0.23	0.26	NT
8a	0.60	0.12	0.31	NT
8b	0.23	0.39	0.07	32.09
8c	0.22	0.64	0.29	NT
9a	0.47	0.65	0.44	NT
9b	0.34	0.20	0.21	NT
10a	0.82	0.79	0.28	NT
10b	0.20	0.10	0.27	NT
10c	0.44	0.30	0.16	NT
11	0.74	0.49	0.09	NT
5-FU	0.51	0.18	0.41	22.51

5-FU: 5-Fluorouracil; NT: Not tested.

The antiproliferative screening against leukemia HL-60 cancer cell line revealed that compounds 8a and 10b emerged as the most potent counterpart demonstrating IC₅₀ values of 0.12 and 0.10 μM, respectively exceeding the activity of 5-FU (IC₅₀ = 0.18 μM) by 1.50- and 1.80-fold, respectively. In addition, compounds 7d and 9b exhibited equipotent cytotoxic activity to 5-FU with IC₅₀ values of 0.23 and 0.20 μM, respectively. The remaining molecules possessed weak to moderate activity compared to 5-FU.

Concerning A549 lung cancer cell line, it was found that compounds 6d, 8b and 11 were the most potent showing IC₅₀ values of 0.06, 0.07 and 0.09 μM, respectively surpassing the cytotoxic activity of 5-FU by nearly 5.86-fold which possessed IC₅₀ value of 0.41 μM. Thereafter, quinoline compounds 6b, 6c, 6e, 7a, 7c, 9b and 10c displayed 1.80-fold superior activity compared to 5-FU with IC₅₀ values of 0.22, 0.21, 0.24, 0.20, 0.22, 0.21 and 0.16 μM, respectively. In addition, compounds 7b, 7d, 8a, 8c, 10a,b exerted better cytotoxic activity than 5-FU with IC₅₀ ranges: 0.24–0.37 μM. It is noteworthy that the tested quinoline compounds 6d and 8b showed lower cytotoxicity against normal lung HEL-299 cell line as denoted from their IC₅₀ values compared with 5-FU. Thus the tested quinoline compounds 6d and 8b proved to be safe and selective toward the lung cancer cell line.

3.2.2. EGFR kinase inhibitory activity

The most active quinoline compounds against A549 cell line, 4-(4-methylbenzylidenehydrazinyl)quinoline 6d and N-phenyl piperazinyl-bearing 4-(4-hydroxy-3-methoxybenzylidenehydraziny)quinoline 8b were selected to investigate for the potential EGFR kinase inhibitory activity. Lapatinib was used as reference EFGR inhibitor. The results were reported as IC₅₀ values, as determined from triplicate measurements and are presented in Figure 5. It was revealed that the examined quinoline derivatives 6d and 8b displayed potent inhibitory activity against EFGR with IC₅₀ values of 0.18 and 0.08 μM, respectively. The results concluded that quinoline molecules 6d and 8b emerged as not only the most efficient antiproliferative agents toward the examined cell lines (MCF-7, HL-60 and A549 cell lines) but also showed potent EGFR kinase inhibitory activity in the current study.

Figure 5.

The IC₅₀ (μM) of the target quinoline molecules 6d and 8b against EGFR kinase activity compared to Lapatinib.

3.2.3. Cell cycle arrest

The cellular cycle analysis was performed to investigate the prevention of proliferation in cancer cells (A549 lung cancer cell line) by the most potent quinoline derivatives 6d and 8b. A549 cells were treated with compounds 6d and 8b at the IC₅₀ concentration for 48 h and the treated A549 cells were stained with PI and analyzed by FACS analysis. The obtained results were compared with non-treated A549 cells as control. As shown in Figure 6, the treatment of A549 cells with 4-(4-methylbenzylidenehydrazinyl)quinoline derivative 6d and N-phenyl piperazinyl-bearing 4-(4-hydroxy-3-methoxybenzylidenehydraziny)quinoline derivative 8b at the IC₅₀ concentration (μM) increased the percentage of S phase cells from 25.84 to 33.71 and 38.22%, respectively. These results confirmed that 4-(4-methylbenzylidenehydrazinyl)quinoline 6d and N-phenyl piperazinyl-bearing 4-(4-hydroxy-3-methoxybenzylidenehydraziny)quinoline 8b significantly cause S phase arrest in A549 cancer cells.

Figure 6.

Influence of quinoline compounds 6d and 8b on the cellular cycle distribution in A549 cancer cell line compared to untreated control cells.

3.2.4. Apoptosis inducing activity

Flow cytometric analysis was performed to determine the mechanism of cellular the mechanism of cell death which observed by the most active quinoline compounds in A549 lung cancer cells by Annexin V-FITC/PI double staining analysis [81]. In this regard, non-treated cells were used as negative control. The cells were treated with 4-(4-methylbenzylidenehydrazinyl)quinoline 6d and N-phenyl piperazinyl-bearing 4-(4-hydroxy-3-methoxybenzylidenehydraziny)quinoline 8b at the IC₅₀ concentration dose for 48 h. After that, the cells were stained with FITC Annexin V and PI solution, respectively and the percentage of cells were determined by FACS analysis. As shown in Figure 7, the treatment of A549 cells with quinoline compounds 6d and 8b resulted in 34.88 and 31.41%, respectively of cellular apoptosis, whereas control non-treated cells showed 0.63%. These results confirmed that the cytotoxicity of quinoline compounds 6d and 8b is associated with cellular apoptosis of A549 cell line.

Figure 7.

Influence of quinoline compounds 6d and 8b on the percentage of apoptosis after staining with Annexin V/PI in A549 cancerous cell line compared to untreated control cells.

PI: Propidium iodide.

3.2.5. Caspase 3/7 assay

It was reported that, several EGFR inhibitors can alter the mitochondrial membrane potential causing the release of cytochrome-c and other pro-apoptotic substances, which subsequently leads to activation of caspase 3/7 and eventually apoptotic induction [82]. To evaluate the effect of 4-(4-methylbenzylidenehydrazinyl)quinoline 6d and N-phenyl piperazinyl-bearing 4-(4-hydroxy-3-methoxybenzylidenehydraziny)quinoline 8b on the level of active caspase 3/7, A549 cells were treated with these compounds at their IC₅₀ concentration for 48 h. As illustrated in Supplementary Figure S4, 4-(4-methylbenzylidenehydrazinyl)quinoline 6d and N-phenyl piperazinyl-bearing 4-(4-hydroxy-3-methoxybenzylidenehydraziny)quinoline 8b increased the levels of active caspase 3/7 by 28.43- and 22.50-fold, respectively compared with control non-treated A549 cells.

3.3. In silico studies

3.3.1. Molecular docking studies

In the molecular docking study, the co-crystallized inhibitor (erlotinib) served as a reference standard to evaluate the binding modes of the newly synthesized anticancer candidates against the target protein of EGFR. This provided insights into how the newly synthesized compounds interacted with the target protein. To ensure the accuracy of the MOE program used for docking, pre-screening validation was conducted. This validation involved re-docking the native inhibitor (erlotinib), and a reasonably low RMSD value of 1.41 Å was attained. This low RMSD value underscores the validity and reliability of the MOE program in predicting binding conformations, confirming its accuracy in the molecular docking analysis [62,83–85], as illustrated in Supplementary Figure S5. The re-docked co-crystallized and the native co-crystallized erlotinib 2D overlay is illustrated in Supplementary Figure S6. Subsequently, erlotinib along with the newly synthesized compounds (6a–11) underwent molecular docking analysis. Supplementary Table S1 presents the binding free energy values as well as the amino acid binding interactions of the assessed compounds.

By docking studies analysis, it was shown that co-crystalized erlotinib could compose two hydrogen bonds with Asp831 via the -NH spacer and the phenyl ring of quinazoline nucleus at distances of 3.51 and 3.44 Å, respectively. Besides, the methoxy group of the erlotinib hydrophobic tail could form hydrogen bond with Met769 at a distance of 2.98 Å. Moreover, the quinazoline nucleus of erlotinib could form pi–H bond with Cys773 at a distance of 3.78 Å, as depicted in Supplementary Figure S7. Considering their prominent anticancer activities, it was revealed that compound 6d could form two pi–H bonds with Leu694 via the quinoline scaffold at distances of 4.36 and 4.63 Å, as shown Supplementary Figure S8. Additionally, it was shown that compound 8b could form pi–H bond with Arg817 via the phenyl ring of the hydrophobic head at a distance of 3.61 Å, as illustrated in Supplementary Figure S9. The 2D/3D binding interactions along with 3D protein positioning for all investigated compounds are depicted in Supplementary Figure S10.

3.3.2. In silico physicochemical & absorption, distribution, metabolism, excretion & toxicity properties

The pharmacokinetic properties and ADME parameters of the newly synthesized compounds along with the estimation of their physical and chemical properties were predicted using the free Swiss ADME web application from the SIB. To perform these calculations, the SMILES notations of the chemical structures of the synthesized compounds were submitted to the online server for further analysis. This approach provides valuable insights into the potential pharmacokinetic behavior and characteristics of the investigated compounds, aiding in the assessment of their suitability for further drug development [76] Moreover, the pkCSM descriptors algorithm protocol was utilized to predict the toxicity parameters of the synthesized compounds. This computational approach is designed to provide insights into the potential toxicity of compounds based on their structural features [77].

Accordingly, regarding their physicochemical features, all the assessed compounds displayed poor water solubility and high gastrointestinal tract absorption owing to their eligible lipophilicity. Hence, we can anticipate feasible bio-availabilities through oral route [86,87]. Besides, not all investigated compounds could pass through the blood brain barrier assuring lower CNS side effects. Interestingly, except for compounds 8a and 8b, the all synthesized compounds are not good substrates for P-glycoprotein assuring better bioavailability, as shown in Supplementary Figure S11. Moreover, all the afforded compounds could notably display inhibition for all or most of the common hepatic metabolizing enzymes (CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4). Moreover, all of the synthesized derivatives obey Lipinski's rule of five [88], hence they could be employed in oral preparations. Additionally, the bioavailability snapshot radars for the investigated compounds were subject to intuitive examination. These unique snapshots from SwissADME provide drug similarity graphs expressed in a hexagonal shape, where each vertex represents a parameter reflecting a product's bioavailability and the colored zone indicates the suitable physicochemical space for oral bioavailability. These radar plots were illustrated in Supplementary Figure S12, offering a visual representation of the compounds’ bioavailability characteristics.

Furthermore, to anticipate the potential toxicity based on the structural characteristics, the pkCSM descriptors algorithm protocol were employed. It was revealed that compounds 6b, 6c, 6e, 9a and 9b could display Ames toxicity, opening eyes on their possible mutagenicity [89]. In addition, all the synthesized compounds are non-inhibitors of hERG I, so they do not exhibit a cardiotoxic effect on the human heart's electrical activity [90]. However, along with eroltinib, all the synthesized compounds are hERG II inhibitors and thus may provoke a cardiac arrhythmia threat [91]. Notably, all the synthesized compounds as well as erlotinib are hepatotoxic. Finally, owing to smaller oral rat chronic toxicity values, compounds 6c, 7d, 10a, 10b and 10c could manifest appropriate tolerability as depicted in Supplementary Tables S2 & S3.

3.4. Structure–activity relationship study

Structure–activity relationship (SAR) is a fundamental concept in medicinal chemistry and drug design since variable substituents play a crucial role in modifying the properties of a molecule and its activity. Hence, SAR study was conducted based on the average IC₅₀ values of the synthesized compounds on the investigated cancer cell lines. Herein, the hydrophobic tail, planar aromatic ring system and -NH spacer were maintained. However, diverse aryl and hetero–aryl hydrophobic heads were employed for the conducted SAR studies.

By substituting the hydrazone moiety with variable small-sized substituted phenyl ring (compounds 6a–e, 7a–d), it was interestingly revealed that 4-methoxy phenyl derivative (compound 7c), 3,4-dimethoxy phenyl derivative (compound 6b), and 4-methyl phenyl derivative (compound 6d) exhibited the best anticancer activity. However, by substituting the hydrazone moiety with variable more bulky substituted phenyl ring (compounds 8a–c), it was shown that 4-hydroxy-3-methoxy-5-((4-methylpiperazin-1-yl)methyl) phenyl derivative (compound 8b) displayed prominent antineoplastic activity. In addition, by substituting the hydrazone moiety with hetero-aryl scaffold (isatin) (compounds 9a,b), it was shown that 5-bromo-isatin (compound 9b) could exhibit better anticancer activity than unsubstituted one (compound 9a). Finally, by substituting the hydrazone moiety with substituted 1,3-diphenylallylidene derivatives (compounds 10a–c), or with 3,4-dihydro, 1H-naphthalene (compound 11), it was revealed that the fluorinated diphenylallylidene derivative (compound 10b) could display better cytotoxic activity, as depicted in Supplementary Figure S13.

4. Conclusion

Intriguingly, by retaining the main EGFR-TK pharmacophores using molecular hybridization, the synthesized quinoline derivatives could show promising anti-proliferative activity, in particular for compounds 8a and 10b for leukemia treatment, surpassing 5-FU by 1.50 and 1.80 fold, respectively, and for compounds 6d, 8b and 11 for lung cancer treatment, surpassing 5-FU by nearly 5.86-fold. Besides, compounds 6d and 8b emerged as not only the most efficient antiproliferative agents toward the examined cell lines (MCF-7, HL-60 and A549 cell lines) but also with potent EGFR kinase inhibitory activity. Moreover, flow cytometry and cell cycle analysis assured the apoptotic potential of quinoline compounds 6d and 8b at A549 cell line with S phase arrest. Additionally, the conducted in silico studies, assured the eligibility of the synthesized quinoline derivatives as EGFR-TK inhibitors by pursuing their binding pattern with acceptable pharmacokinetics and toxicity profiles. The established SAR shed light on that substituting the hydrazone moiety with 4-methyl phenyl derivative (compound 6d) and 4-hydroxy-3-methoxy-5-((4-methylpiperazin-1-yl)methyl) phenyl derivative (compound 8b) could display prominent antineoplastic activity, paving the way for affording more effective compounds through lead optimization.

Supplemental Material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17568919.2024.2342201

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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