Investigation of Quinoline-based (4-oxo-1,4-dihydroquinolin-2-yl) methyl nitrate derivatives: Synthesis, Anti-cancer activity, Nitric oxide release and Molecular docking studies

 

Venkata Sowjanya Thanneeru*, Naresh Panigrahi

Dept of Pharmaceutical Chemistry, GITAM School of Pharmacy, Vishakapatnam, India

 

ABSTRACT:

This study synthesized and evaluated substituted (4-oxo-1,4-dihydroquinolin-2-yl) methyl nitrate derivatives to identify those with strong anticancer activity and nitric oxide (NO) release. This study aimed to synthesize and evaluate a series of substituted (4-oxo-1,4-dihydroquinolin-2-yl) methyl nitrate derivatives to determine their efficacy in nitric oxide release, binding affinity to EGFR tyrosine kinase, and anticancer activity against non-small cell lung cancer (A-549) and pancreatic cancer (PANC-1) cell lines. Substituted derivatives were synthesized through a multi-step chemical process involving reactions with boron trichloride, chloroacetyl chloride, and silver nitrate. The synthesized compounds were characterized using analytical methods. Molecular docking studies were performed to assess binding affinities to EGFR tyrosine kinase (PDB ID: 4HJO). Nitric oxide release was measured using the Griess method in adherent cells. Anticancer activity was evaluated through IC50 assays in A-549 and PANC-1 cell lines, and selectivity indices were calculated to determine the compounds' preference for cancerous cells over normal cells. Molecular docking revealed compounds 6h (-4-OH), 6c (-4-Br), and 6e (-4-CH3) as having strong binding affinities to EGFR tyrosine kinase. In NO release assays, 6h showed the highest NO production (49.95 μmol/L). Anticancer evaluations identified 6e, 6h, and 6m (-3,4-CH3) as the most effective, with IC50 values of 21.46 ± 1.41 μM, 22.22 ± 1.10 μM, and 24.39 ± 2.39 μM, respectively, and high selectivity indices in both A-549 and PANC-1 cell lines. Compounds 6e, 6h, and 6m showed potent anticancer effects and strong binding to EGFR tyrosine kinase, indicating their potential as leads for further cancer therapy development.

 

 

 

 

INTRODUCTION: 

Chemotherapy remains a cornerstone in cancer management, offering both curative and palliative benefits through systemic inhibition of cell proliferation and DNA replication^1-2. Recently, nitric oxide (NO) has gained attention as a therapeutic agent in oncology. NO-releasing heterocyclic compounds are being developed to enhance anticancer efficacy by targeting multiple pathways³. The selection of NSCLC (A-549) and pancreatic cancer (PANC-1) cell lines is justified by their elevated expression of EGFR tyrosine kinase, a critical target for anticancer therapy⁴. NSCLC is characterized by high EGFR expression, while pancreatic cancer remains one of the most lethal malignancies with limited treatment options⁵. The overexpression of EGFR and associated therapeutic resistance in both cancers underscores the need for novel targeted strategies⁶.Current research focuses on developing quinoline-based NO-releasing compounds to improve treatment efficacy and reduce toxicity.

 

MATERIAL AND METHODS:

This experiment uses synthetic-grade chemicals and solvents from Sigma-Aldrich, Bangalore, India, without purification. The reaction was monitored using Merck-precoated aluminium TLC plates with silica gel 60 F254. Remi electronic melting point apparatus determined melting points. BRUKER DRX was used to collect 1H and 13C NMR spectra. Tetramethyl silane was used as the internal reference to calibrate chemical shift data in ppm. The letters ss (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet) represented the splitting patterns. Waters Xevo Q-Tof mass spectrometers collected HRMS spectra in positive ionization mode. ATCC cultures of the A-549, PANC-1, and HEK-293 cell lines were procured from Himedia Pvt Ltd, India.

 

Synthesis of substituted (4-oxo-1,4-dihydroquinolin-2-yl) methyl nitrate derivatives

The scheme of synthesis for the quinolinone nitrate derivative were displayed in Figure 1.

 

Procedure for the synthesis of substituted 1-(2-aminophenyl) ethan-1-one (3a-3n)

A solution of boron trichloride (BCl3) (5.5 mmol) in 5 mL of carbon tetrachloride (CCl4) was stirred at room temperature. To this, a solution of aniline (1a-1n) (5 mmol) in 5 mL of dichloromethane (DCM) was added dropwise while maintaining the mixture in an ice bath. The resulting aniline-BCl3 complex was then slowly treated with acetonitrile (2) (6 mmol) and refluxed for 6 hours. Upon completion of the reflux period, the reaction mixture was allowed to cool to room temperature. Subsequently, 10 mL of 2N hydrochloric acid (HCl) was added while stirring at 80°C for 15 minutes. The mixture was then cooled, and the product was extracted with DCM to obtain the neutral fraction. The neutral fraction was treated with 2N sodium hydroxide (NaOH) and extracted with three 10 mL portions of DCM. The combined organic extracts were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting residue was the substituted 1-(2-aminophenyl) ethan-1-one (3a-3n)⁷.

 

Synthesis of substituted N-(2-acetylphenyl)-2-chloroacetamide (4a-4n)

In a 50 mL round-bottom flask, substituted aniline (6 mmol) was dissolved in tetrahydrofuran (THF) (5 mL). To this solution, DBU (1.2 mmol) was added. The reaction mixture was cooled in an ice-salt bath and stirred mechanically for 15 minutes. Chloroacetyl chloride (6.1 mmol) was then added dropwise from a dropping funnel, ensuring that the temperature did not exceed 5°C. After the addition was complete, the mixture was allowed to stir at room temperature for 3-6 hours. Reaction progress was monitored by thin-layer chromatography (TLC) using a hexane acetate (7:3) solvent system. Upon completion, the reaction mixture was poured into cold water, causing the product to precipitate. The solid was filtered, washed with water, and then dried. The crude product was recrystallized from ethanol to obtain the final product as a solid powder⁸.

 

Synthesis of substituted 2-(chloromethyl) quinolin-4(1H)-one (5a-5n)

The amide (1 equiv.) and crushed sodium hydroxide (3.4 equiv.) were dissolved in dry 1,4-dioxane (40 mL per 1 mmol) and the solution was refluxed under a nitrogen atmosphere for 2 hours. After allowing the reaction mixture to cool to room temperature, distilled water (30 mL per 1 mmol) and n-hexane (200 mL per 1 mmol) were added, causing the aqueous phase to become cloudy. The mixture was sonicated for 2 minutes, and the pH of the aqueous phase was adjusted to 6 using 1 M hydrochloric acid (HCl). The resulting precipitate was filtered, washed with n-hexane, and then dissolved in ethanol. The ethanol was evaporated, and the residual solid was suspended in ethyl acetate. The precipitate was filtered off and washed with ethyl acetate to yield the final product⁹.

 

Synthesis of 6a-6n

Substituted 2-(chloromethyl) quinolin-4(1H)-one (5a-5n) (0.54 mmol) was dissolved in 5 mL of anhydrous acetonitrile. To this solution, 0.138 g of silver nitrate (0.81 mmol) was added. The mixture was heated at 80°C for 2 hours under stirring. After the reaction was complete, the mixture was filtered through Celite and washed with dichloromethane (CH2Cl2). The combined filtrate was then evaporated to remove the solvent. The crude product was purified by column chromatography to yield the desired compound¹⁰.

 

Molecular docking

EGFR (4HJO) domain X-ray crystal structures co-crystallized with Erlotinib were obtained from Protein Data Bank¹¹. Schrödinger's Protein Preparation Wizard inserted hydrogen atoms and bond instructions to the protein's 3D structure to prepare the complex. LigPrep from Schrödinger optimized chiral ligands' 3D structures using the OPLS 2005 force field. Maestro 11.8's SITEMAP ANALYSIS TOOL analyzed 6LUD and 7SJ3 receptor sites, and the Schrödinger suite grid generation tool created receptor grids. Glide XP's extra-precision docking modes docked molecules, and the score was calculated using binding interaction energy, van der Waals energy, electrostatic potential energy, and strain energy. EGFR and CDK-4 active site ligand binding was studied using Schrödinger Maestro¹².

 

MTT assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cytotoxic effects and cell viability of Quinoline Nitrate derivatives 6a-6n. Two human cancer cell lines, A-549 (Non-Small Cell Lung Cancer) and PANC-1, along with one normal Human Embryonic Kidney Cell line (HEK-293), were cultivated in 96-well plates and allowed to adhere overnight. The cells were exposed to various concentrations (0.1 μM, 10 μM, 50 μM, and 100 μM) of the produced 1H-1,2,4-triazole-3-carboxamide derivatives 4a-4n for 72 hours. Following the specified incubation period, MTT solution was added to each well, and the plates were then incubated to promote the development of formazan crystals. The formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured at 540nm using a microplate reader (PerkinElmer EnVision Multilabel Plate Reader). Decreased absorbance values correlated with higher cytotoxicity and reduced cell viability. The IC50 values of the quinoline nitrate derivatives for each cell line were obtained using MTT assay data collected at different concentrations and time points. The IC50 values were calculated using GraphPad Prism version 9.0. The tests were conducted in triplicates using control groups (Cells treated with erlotinib served as a positive control and Untreated cells served as a negative control) to guarantee the correctness and dependability of the assay¹³.

 

NO-Releasing assay

The nitric oxide (NO) release from adherent cells was evaluated using the Griess reagent assay, following standard protocols¹⁴. Cells were cultured under standard conditions and treated with a nitric oxide donor, sodium nitroprusside (SNP), at various concentrations and time intervals, while untreated cells served as the control. Following incubation, culture supernatants were collected and centrifuged to remove cellular debris. The clear supernatants were transferred to labeled tubes for further analysis. The Griess reagent was prepared according to the manufacturer's instructions (Promega), and a series of sodium nitrite standards were prepared to generate a standard curve. Equal volumes of each sample and Griess reagent were added to a 96-well microplate, gently mixed, and incubated at room temperature for the specified duration. Absorbance was measured at 540 nm using a microplate reader. Nitrite concentrations were quantified by extrapolation from the standard curve, and the levels of NO release were compared between control and treated groups. In this study, a single concentration (100 μM) of each test compound was used throughout the assay. All procedures were conducted under sterile conditions, and safety and waste disposal protocols were strictly followed. Where necessary, the protocol was adapted to accommodate experimental requirements in line with the reagent kit guidelines.

 

 

 

Figure 1: Scheme of synthesis for the quinolinone nitrate derivative

 

RESULTS AND DISCUSSION:

Substituted (4-oxo-1,4-dihydroquinolin-2-yl) methyl nitrate derivatives (6a–6n) were synthesized via a multi-step procedure. Initially, substituted 1-(2-amino phenyl) ethan-1-one derivatives (3a–3n) were prepared from substituted anilines using boron trichloride and acetonitrile. These intermediates were converted to N-(2-acetylphenyl)-2-chloroacetamide derivatives (4a–4n) via reaction with chloroacetyl chloride in THF. Subsequent cyclization with NaOH in 1,4-dioxane yielded substituted 2-(chloro methyl) quinolin-4(1H)-one derivatives (5a–5n), which were then treated with silver nitrate in acetonitrile to obtain the target nitrate derivatives (6a–6n). The final products, isolated as pale-yellow crystals, were obtained in good yields (78–87%). Structural and physicochemical details are presented in Table 1, with analytical data discussed in the following section.

 

Analytical characterization of synthesized Compounds

6a: (4-oxo-1,4-dihydroquinolin-2-yl) methyl nitrate

¹H NMR (500 MHz, Chloroform-d) δ 9.05 (s, 1H), 8.32 (dd, J = 8.2, 1.6 Hz, 1H), 7.59 (dd, J = 8.0, 1.6 Hz, 1H), 7.54 (td, J = 7.8, 1.5 Hz, 1H), 7.31 (td, J = 8.0, 1.6 Hz, 1H), 6.44 (t, J = 1.5 Hz, 1H), 5.29 (d, J = 1.4 Hz, 2H). ¹³C NMR (125 MHz, Chloroform-d) δ 69.12, 108.90, 117.82, 122.96, 123.66, 125.66, 131.59, 139.71, 147.10, 176.31. HRMS: m/z: For C₁₀H₈N₂O₄ ([M + H]+): 221.0598, found 221.0592.

 

6b: (6-chloro-4-oxo-1,4-dihydroquinolin-2-yl) methyl nitrate

¹H NMR (500 MHz, Chloroform-d) δ 9.28 (s, 1H), 8.15 (d, J = 2.4 Hz, 1H), 7.40 (dd, J = 8.2, 2.3 Hz, 1H), 7.13 (d, J = 8.2 Hz, 1H), 6.45 (t, J = 1.5 Hz, 1H), 5.29 (d, J = 1.4 Hz, 2H). ¹³C NMR (125 MHz, Chloroform-d) δ 177.33, 146.81, 137.75, 129.63, 127.17, 125.14, 122.46, 118.04, 108.90, 69.12. HRMS: m/z: For C₁₀H₇ClN₂O₄ ([M + H]+): 256.6217, found 256.6217.

 

6c: (6-bromo-4-oxo-1,4-dihydroquinolin-2-yl) methyl nitrate

¹H NMR (500 MHz, Chloroform-d) δ 9.37 (s, 1H), 8.04 (d, J = 2.5 Hz, 1H), 7.44 (dd, J = 8.3, 2.4 Hz, 1H), 7.26 (d, J = 8.3 Hz, 1H), 6.47 (t, J = 1.5 Hz, 1H), 5.27 (d, J = 1.4 Hz, 2H). ¹³C NMR (125 MHz, Chloroform-d) δ 178.05, 147.83, 138.48, 133.99, 128.84, 121.95, 118.54, 116.59, 109.41, 69.84. HRMS: m/z: For C₁₀H₇BrN₂O₄ ([M + H]+): 299.9543, found 299.9539.

6d: (6-fluoro-4-oxo-1,4-dihydroquinolin-2-yl) methyl nitrate

¹H NMR (500 MHz, Chloroform-d) δ 9.34 (s, 1H), 7.69 (dd, J = 12.1, 2.7 Hz, 1H), 7.31 (dd, J = 8.2, 4.6 Hz, 1H), 7.12 (ddd, J = 10.5, 8.2, 2.7 Hz, 1H), 6.50 (d, J = 1.4 Hz, 1H), 5.29 (d, J = 1.4 Hz, 2H). ¹³C NMR (125 MHz, Chloroform-d) δ 70.06, 109.19, 111.78, 111.97, 117.63, 117.81, 119.41, 119.48, 122.20, 122.28, 136.94, 136.97, 147.10, 154.45, 156.42, 179.78, 179.81. HRMS: m/z: For C₁₀H₇FN₂O₄ ([M + H]+): 239.1709, found 239.1705.

 

6e: (6-methyl-4-oxo-1,4-dihydroquinolin-2-yl) methyl nitrate

¹H NMR (500 MHz, Chloroform-d) δ 9.17 (s, 1H), 7.83 (d, J = 2.1 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.20 (dd, J = 8.2, 2.2 Hz, 1H), 6.58 (t, J = 1.6 Hz, 1H), 5.27 (d, J = 1.4 Hz, 2H), 2.59 (s, 3H). ¹³C NMR (125 MHz, Chloroform-d) δ 21.34, 69.83, 110.64, 117.18, 122.61, 125.22, 130.53, 131.88, 137.75, 147.32, 177.11. HRMS: m/z: For C₁₁H₁₀N₂O₄ ([M + H]+): 235.2087, found 235.2081.

 

Molecular docking

Molecular docking of substituted (4-oxo-1,4-dihydroquinolin-2-yl) methyl nitrate derivatives with EGFR tyrosine kinase (PDB ID: 4HJO) revealed varying binding affinities (Table 1). Compounds 6h (-4-OH, -6.801), 6c (-4-Br, -6.72), and 6e (-4-CH₃, -6.553) showed the strongest binding, likely due to favorable hydrogen bonding and hydrophobic interactions at the para position (Figure 1). Moderately active compounds such as 6i, 6k, 6m, and 6n displayed slightly less negative docking scores (−6.29 to −6.48), with functional groups contributing to electrostatic and van der Waals interactions. In contrast, derivatives like 6b, 6l, 6d, 6g, and 6f exhibited lower binding affinities (−4.91 to −6.25), possibly due to electron-withdrawing effects or steric hindrance from bulky substituents. 6j (-4-OCH₃, -4.869) showed the weakest interaction, suggesting limited contribution of the methoxy group to binding. Although none of the derivatives outperformed erlotinib (−7.040), compounds 6h and 6c demonstrated comparable binding scores, indicating potential as lead candidates. The results emphasize the critical role of substituent type and position on the quinoline scaffold, where para-substituted electron-donating or moderately withdrawing groups enhance binding, while bulky or inert groups reduce affinity.

 

Table 1: Summary of experimental results of quinoline nitrate derivatives (6a-6n)

Compound	R			IC50 Values (μM)			Selectivity Index
		Docking Score	Concentration of released NO (μmol/L)	Non-Small Cell Lung Cancer line (A-549)	Pancreatic cancer cell line (PANC-1)	Human Embryonic Kidney Cell line (HEK-293)	(A549)	(PANC1)
6a	-H	-5.854	37.63	34.52 ± 2.41	35.35 ± 2.23	50.03 ± 2.26	1.45	1.42
6b	-4-Cl	-6.248	35.98	32.71 ± 1.34	35.39 ± 1.20	48.31 ± 2.04	1.48	1.37
6c	-4-Br	-6.72	44.52	26.55 ± 2.30	25.00 ± 2.59	54.08 ± 1.84	2.04	2.16
6d	-4-F	-5.294	29.47	32.50 ± 1.88	33.34 ± 1.11	51.10 ± 4.12	1.57	1.53
6e	-4-CH3	-6.553	45.81	21.46 ± 1.41	22.69 ± 1.18	54.53 ± 1.06	2.54	2.4
6f	-4-C2H5	-4.918	31.35	31.97 ± 1.39	29.33 ± 4.24	43.22 ± 3.77	1.35	1.47
6g	-4-CF3	-5.32	30.48	27.67 ± 2.25	28.99 ± 1.62	50.32 ± 1.88	1.82	1.74
6h	-4-OH	-6.801	49.95	22.22 ± 1.10	28.18 ± 1.13	46.08 ± 1.47	2.07	1.63
6i	-4-NO2	-6.482	40.07	41.11 ± 2.45	43.55 ± 2.79	48.47 ± 1.84	1.18	1.11
6j	-4-OCH3	-4.869	32.25	31.13 ± 1.37	32.52 ± 1.26	46.39 ± 2.86	1.49	1.43
6k	-4-(NCH3)2	-6.29	41.24	29.73 ± 3.41	28.23 ± 2.22	54.52 ± 1.24	1.83	1.93
6l	-3-CH3	-6.269	35.74	36.54 ± 1.38	34.28 ± 2.17	53.38 ± 1.04	1.46	1.56
6m	-3,4-CH3	-6.316	42.61	24.39 ± 2.39	26.82 ± 2.73	48.63 ± 1.80	1.99	1.81
6n	-3-OCH3	-6.417	34.87	33.76 ± 1.33	38.72 ± 1.23	50.64 ± 2.80	1.5	1.31
Erlotinib	-	-7.040	-	21.85±3.25	24.15±2.70	34.97±1.94	1.6	1.45

 

 

Nitric oxide (NO) release study

The nitric oxide (NO) release profile of substituted (4-oxo-1,4-dihydroquinolin-2-yl) methyl nitrate derivatives, assessed using the Griess method in adherent cells (Table 1), demonstrated considerable variation depending on the nature and position of the substituents. Compounds 6h (-4-OH, 49.95 μmol/L), 6e (-4-CH₃, 45.81 μmol/L), and 6c (-4-Br, 44.52 μmol/L) showed the highest NO release, suggesting that electron-donating or moderately withdrawing groups at the para position enhance NO liberation, possibly by increasing solubility or reactivity of the nitrate group. Moderate NO release was observed with 6m, 6k, and 6i (40–42 μmol/L), where electronic and steric effects of dimethyl, dimethylamino, and nitro groups may influence NO stability and release dynamics. Lower NO release was recorded for 6b, 6l, and 6n (34–36 μmol/L), likely due to intermediate electronic effects or suboptimal positioning of functional groups. 6j and 6f (≈31–32 μmol/L) released less NO, possibly due to steric hindrance from bulky ethyl or ineffective electronic contribution from methoxy groups. The weakest NO-releasing derivatives were 6g (-CF₃, 30.48 μmol/L) and 6d (-F, 29.47 μmol/L), where strong electron-withdrawing effects likely over-stabilize the nitrate moiety, hindering its decomposition into NO. Overall, NO release correlated with both electronic properties and steric bulk of the substituents. Electron-donating groups enhanced NO release, while strongly withdrawing or bulky groups reduced it, underscoring the importance of rational substituent selection in designing NO-releasing anticancer agents.

 

Anticancer activity

Non-small cell lung cancer line (A-549)

In the A-549 cell line (Table 1), compounds 6e (−4-CH₃), 6h (−4-OH), and 6m (−3,4-CH₃) demonstrated the highest anticancer activity with low IC₅₀ values of 21.46 ± 1.41 μM, 22.22 ± 1.10 μM, and 24.39 ± 2.39 μM, respectively. These compounds also exhibited favorable selectivity indices (SI), indicating preferential cytotoxicity toward cancer cells over normal HEK-293 cells. Notably, 6e showed an SI of 2.54 (A-549/HEK-293) and 2.40 (PANC-1/HEK-293), followed by 6h (2.07 and 1.63) and 6m (1.99 and 1.81), highlighting their therapeutic potential with minimal off-target effects. Moderately active compounds included 6c, 6d, 6b, 6f, 6g, 6n, and 6l, with IC₅₀ values ranging from 26.55 ± 2.30 μM to 36.54 ± 1.38 μM. Compounds like 6c and 6g showed moderate selectivity (SI ≈ 2.04–2.16 and 1.74–1.82, respectively), suggesting potential for further structural optimization to enhance selectivity. Lower activity was observed for 6i (−4-NO₂) and 6k (−4-(NCH₃)₂), with IC₅₀ values of 41.11 ± 2.45 μM and 29.73 ± 3.41 μM, respectively. Their lower SI values (e.g., 6i: 1.18, 6k: 1.83) indicate limited selectivity, reflecting reduced cancer cell specificity and making them less favorable candidates for targeted therapy.

 

Pancreatic cancer cell line (PANC-1)

In the PANC-1 cell line (Table 1), compounds 6e (−4-CH₃), 6m (−3,4-CH₃), and 6h (−4-OH) exhibited the highest anticancer activity with IC₅₀ values of 22.69 ± 1.18 μM, 26.82 ± 2.73 μM, and 28.18 ± 1.13 μM, respectively. These compounds also showed favorable selectivity indices (SI), with 6e at 2.40, 6m at 1.81, and 6h at 1.63 (PANC-1/HEK-293), indicating strong therapeutic potential and selective cytotoxicity toward cancer cells. Moderate activity was observed in compounds 6c, 6d, 6b, 6f, 6g, 6n, and 6l, with IC₅₀ values ranging from 25.00 ± 2.59 μM to 34.28 ± 2.17 μM. Selectivity indices for these compounds, such as 6c (SI: 2.16) and 6g (SI: 1.74), suggest a moderate preference for targeting cancer cells over normal cells. Compounds with the lowest activity included 6i (−4-NO₂) and 6k (−4-(NCH₃)₂), having IC₅₀ values of 43.55 ± 2.79 μM and 28.23 ± 2.22 μM, respectively. Their lower SI values (6i: 1.11, 6k: 1.93) reflect limited selectivity and reduced potency, making them less promising for targeted pancreatic cancer therapy.

 

Impact of substituent groups on anticancer activity

The anticancer activity of substituted (4-oxo-1,4-dihydroquinolin-2-yl)methyl nitrate derivatives (6a–6n) is strongly influenced by the electronic and steric nature of the substituents on the quinolinone core. Electron-donating groups (EDGs) significantly enhance activity. Notably, 6e (−4-CH₃) and 6m (−3,4-CH₃) showed the lowest IC₅₀ values in both A-549 (21.46 μM and 24.39 μM) and PANC-1 (22.69 μM and 26.82 μM) cells, indicating high potency. These methyl groups likely increase electron density, enhancing interactions with the target proteins.

 

 

6c

 

6e

Figure 2: Interactions of compound 6c, and 6e at the active site of EGFR tyrosine kinase

 

Similarly, 6n (−3-OCH₃) exhibited moderate activity, further supporting the favorable role of EDGs. In contrast, electron-withdrawing groups (EWGs) generally decreased activity. 6i (−4-NO₂) showed the highest IC₅₀ values (41.11 μM in A-549, 43.55 μM in PANC-1), likely due to reduced electron density hindering effective binding. 6d (−4-F) displayed intermediate activity, suggesting some contribution from halogen interactions. Halogen-substituted compounds such as 6b (−4-Cl) and 6c (−4-Br) showed variable activity, with 6c being more active, likely due to better size and electronic compatibility with the binding site. 6h (−4-OH) also exhibited strong activity (22.22 μM in A-549, 28.18 μM in PANC-1), possibly due to hydrogen bonding potential. The dimethylamino group in 6k led to intermediate activity, indicating a balanced electronic effect. Overall, EDGs (especially methyl and hydroxyl) enhanced anticancer activity by increasing electron density and promoting target interaction, while strong EWGs (e.g., nitro, trifluoromethyl) reduced it. Halogens and dimethylamino groups had moderate or variable effects depending on size and polarity. Correlating NO release, docking affinity, and cytotoxicity, compounds such as 6e and 6m showed high NO release, strong EGFR binding affinity, and potent anticancer activity, supporting a positive correlation between these parameters. However, exceptions like 6i, which had relatively high NO release but poor activity and binding, highlight that multiple molecular factors—beyond NO donation and docking—govern therapeutic outcomes. These findings underscore the importance of strategic substituent modification to optimize efficacy in quinoline-based NO-releasing anticancer agents. The correlation between NO release, docking studies data, and anticancer activity suggests that compounds exhibiting high NO release and strong binding affinity to target proteins generally show enhanced anticancer efficacy. High NO release contributes to oxidative stress and apoptosis in cancer cells, while effective binding to target proteins aids in inhibiting cancer cell growth. However, inconsistencies in some compounds indicate that while NO release and binding affinity are crucial, other factors also play a role in determining anticancer activity. Thus, a multifaceted approach considering NO release, target binding, and other biological interactions is essential for optimizing anticancer agents.

 

CONCLUSION:

The synthesized (4-oxo-1,4-dihydroquinolin-2-yl) methyl nitrate derivatives revealed that substituents play a critical role in determining their biological activities. Among the tested compounds, 6e (-4-CH3), 6h (-4-OH), and 6m (-3,4-CH3) were identified as the most effective, showing superior anticancer activity with the lowest IC50 values and high selectivity indices in A-549 and PANC-1 cell lines. These compounds also exhibited high NO release, correlating with their potent anticancer effects. Molecular docking results confirmed their strong binding affinities to EGFR tyrosine kinase. The study highlights that enhancing NO release and optimizing target binding through strategic substituent modifications can significantly improve anticancer activity. Compounds 6e, 6h, and 6m are thus considered promising leads for further development, emphasizing the importance of substituent choice in maximizing therapeutic potential.

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Received on 16.03.2025      Revised on 11.04.2025 Accepted on 22.07.2025      Published on 02.08.2025 Available online from August 08, 2025 Research J. Pharmacy and Technology. 2025;18(8):4038-4044.. 10.52711/0974-360X.2025.00580 © RJPT All right reserved
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