Design, synthesis, and preliminary anticancer evaluation of diphenylpropan-1-one derivatives: in vitro, in vivo and in silico studies

Abstract

Breast cancer is a leading cause of cancer-associated mortality and morbidity worldwide, and there is a continued need for structurally diverse anticancer agents with improved properties. The present study reports a series of diphenylpropan-1-one (DPP) derivatives (4a–4i), rationally designed around a chalcone framework and synthesized via a three-step reaction sequence. The physicochemical and spectral characterization of the synthesized derivatives was performed using spectral techniques. Their anticancer potential was evaluated through a preliminary in vitro cytotoxicity screening against the MCF-7 mammary cancer cell line, followed by an in vivo efficacy assessment using a DMBA-induced mammary cancer animal model. To explore a possible molecular target, in silico molecular docking was performed against the epidermal growth factor receptor (EGFR) tyrosine kinase domain as an exploratory computational analysis. Amongst the synthesized derivatives, compound 4h demonstrated the most potent cytotoxic effects (IC₅₀ = 1.7 µg mL⁻¹), comparable to the standard drug gefitinib (IC₅₀ = 1.8 µg mL⁻¹). The in vivo studies revealed a significant and dose-dependent decline in cellular proliferation and mammary gland tumor progression, along with histopathological abnormalities upon treatment with compound 4h. Interestingly, the quantitative gene expression analysis demonstrated a significant reduction in the DMBA-induced overexpression of BRCA1, BRCA2, and TP53 in the 4h-treated groups in a dose-dependent manner. Molecular docking studies showed favorable binding of the DPP derivatives within the EGFR active site, with compound 4h exhibiting strong affinity (binding energy = −9.2 kcal mol⁻¹) and favorable predicted interactions in the ATP-binding region of EGFR. Computational ADMET profiling provided additional preliminary predictions of the pharmacokinetic and toxicity properties of compound 4h. The identified DPP derivatives demonstrated encouraging preliminary anticancer potential and may serve as promising lead molecules for future investigations.

A series of diphenylpropan-1-ones (DPP) was synthesized and screened for preliminary anticancer activity, leading to the identification of compound 4h as a promising anticancer lead through in vitro, in vivo, and exploratory in silico investigations.

1. Introduction

A steady surge in breast cancer incidence has been reported in several regions, including Saudi Arabia.¹ Aggressive forms include triple-negative breast cancer (TNBC), tumors characterized by epidermal growth factor receptor (EGFR) overexpression, and inflammatory breast cancer.² TNBC alone accounts for 15–30% of cases, and it is often associated with high relapse rates, poor prognosis, and inadequate treatment modalities.^3,4 Overexpression of EGFR is a characteristic of aggressive breast cancer subtypes, occurring in 13–78% of cases. It plays a central role in promoting tumor growth, facilitating metastatic progression, and contributing to treatment resistance in several malignancies. In addition, EGFR functions as a central hub in multi-target regulatory networks involving AKT, TP53, and inflammatory signaling pathways in breast cancer progression.⁵

EGFR, a member of the ErbB receptor family, functions as a transmembrane tyrosine kinase and is associated with multiple oncogenic signaling pathways. This family includes human epidermal growth factor receptor 1 (HER1), HER2, HER3, and HER4. These receptors are involved in signaling cascades mediated by EGFR.⁶ EGFR exists as an autoinhibited monomer in the resting state, which, upon binding to its growth-activating ligands, such as TGF-α and other activators, undergoes homo/heterodimerization with HER2. The formation of asymmetric dimers activates EGFR, where the C-terminal region of the kinase domain of the activator protein interacts with the N-terminal region of the kinase domain of the receiving protein, resulting in the phosphorylation of tyrosine residues of the kinase domain of the activator receptor. These signaling mechanisms are associated with the regulation of cell growth, migration, differentiation, and survival, as well as the production of pro-inflammatory cytokines and actin remodeling.⁷ In addition, processes such as epithelial–mesenchymal transition (EMT), cancer stem cell formation, tumor microenvironment remodeling, and epigenetic modulation through DNA methyltransferase, histone methyltransferase, histone acetyltransferase, and histone deacetylase also promote tumor progression and resistance, further enhancing disease aggressiveness and complicating treatment outcomes.^8–13

The EGFR inhibitors currently in use are primarily quinazoline-based compounds, which have shown clinical efficacy against various cancer types.¹⁴ Chronic administration of these agents often results in resistance, including EGFR-dependent secondary mutations such as Thr790Met and Cys797Ser, as well as EGFR-independent mutations in downstream signaling molecules.^15,16 Additionally, these inhibitors are frequently associated with moderate to severe side effects, including dermatological toxicity, gastrointestinal disturbances, and hepatotoxicity, which can compromise patient compliance.^17,18 Recent studies have shown that EGFR-TKI resistance is associated with metabolic reprogramming and the regulation of glycolysis-related non-coding RNAs, highlighting the complexity of resistance mechanisms. Moreover, genomic alterations such as copy number variations have been identified as predictive markers of EGFR-TKI response and patient prognosis.^19–21 These challenges highlight the need for novel chemical scaffolds that target EGFR and offer improved efficacy and safety profiles in breast cancer therapy.

Chalcone-based derivatives have attracted considerable interest as privileged scaffolds due to their broad spectrum of biological activities. They are frequently studied as promising pharmacophores in anticancer drug discovery because of their structural simplicity, ease of synthesis, and diverse biological properties, including inhibition of receptor tyrosine kinases such as EGFR.^22–26 Structural modification of the chalcone framework could enable the identification of new scaffolds with enhanced biological activities. Several reports have demonstrated that the α,β-unsaturated carbonyl framework of chalcones, along with appropriate substitution on the aromatic ring, facilitates intermolecular interactions with amino acid residues in the ATP-binding pocket of EGFR.^27–29 In this context, diphenylpropan-1-one (DPP) derivatives represent an underexplored class of compounds with potential anticancer efficacy. The presence of multiple aryl moieties and functional substituents in DPP derivatives is anticipated to facilitate favorable interactions within the ATP-binding site EGFR, potentially enabling effective inhibition of kinase activity.

Based on these considerations, the present study was conducted to design, synthesize, and perform preliminary anticancer screening of a novel series of DPP derivatives (4a–4i). In contrast to conventional chalcones, the DPP derivatives designed herein do not retain the α,β-unsaturated carbonyl linker and instead possess a saturated β-hydroxy ketone framework bearing an additional aryloxy substituent. Generated by epoxidation followed by nucleophilic ring-opening, this structural transformation removes the classical Michael-acceptor motif of chalcones and introduces an additional hydrogen-bonding domain along with conformational flexibility. In addition, aromatic substituents with electron-withdrawing, electron-donating, and bulky groups were varied to investigate their effects on EGFR binding and cytotoxic potential. Therefore, the present work explores a distinct DPP scaffold rather than a direct chalcone analogue.

Accordingly, the title compounds were synthesized and characterized using standard spectral techniques. Their anticancer potential was systematically assessed through in vitro cytotoxicity assays against a mammary cancer cell line (MCF-7), followed by in vivo evaluation in a DMBA-induced mammary cancer model, as well as in silico exploratory molecular docking against the EGFR tyrosine kinase domain and in silico ADME profiling to predict their pharmacokinetic and druggability properties. This integrated approach aimed to identify promising lead compounds for further development in breast cancer therapy.

2. Materials and methods

2.1. Instruments and chemicals

All chemicals, reagents, and solvents utilized in this study were of AR grade, procured from Sigma-Aldrich (Steinheim, Germany), and used without further purification. The melting points of the synthesized derivatives were recorded using the open capillary method on a digital melting point apparatus (Stuart SMP30, MS, USA). FT-IR spectra were recorded using a Shimadzu IRSpirit-T spectrophotometer (Japan). The ¹H NMR and ¹³C NMR spectra of the synthesized compounds were recorded using a nuclear magnetic resonance (NMR) spectrometer (Bruker Plus 400, MS, USA) operating at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR, with tetramethylsilane (TMS) and dimethyl sulfoxide (DMSO-d₆) as the internal standard and solvent, respectively. A high-resolution mass spectrometer (HRMS) (Thermo Scientific, MS, USA) was utilized to record mass spectra. Thin-layer chromatography (TLC) was performed to monitor the completion of the reaction on pre-coated (F-254) aluminum-supported TLC plates using benzene : methanol (9 : 1) as the mobile phase, and retention factors (R_f) were calculated for all synthesized compounds.

2.2. Cell lines

The human breast adenocarcinoma cell line (MCF-7) was used to perform in vitro cytotoxicity studies. This cell line was procured from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured using Dulbecco's modified Eagle medium (DMEM), supplemented with fetal bovine serum (FBS, heat-inactivated, 10% v/v) and penicillin–streptomycin solution (1% v/v) consisting of 100 U mL⁻¹ penicillin and 100 µg mL⁻¹ streptomycin. The cells were cultured under humidified conditions in an atmosphere containing 5% CO₂ at a constant temperature of 37 °C. The culture medium was replaced every 2–3 days, and the cancer cells were subcultured using trypsin-EDTA solution once the cell confluency reached approximately 70–80%.

2.3. Animals

In vivo acute toxicity and anticancer activity were evaluated in adult female Sprague Dawley rats weighing 150 ± 25 g (6–8 weeks old), procured from the Central Animal House Facility of Jazan University, Jazan, Saudi Arabia. All animal-related experiments were conducted in strict accordance with international ethical standards and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.³⁰ Experimental protocols involving animals were reviewed and approved by the Standing Committee for Scientific Research Ethics, Jazan University, Jazan, Saudi Arabia (HAPO-10-Z-001) prior to conducting the experiments, with approval number REC-44/11/682. All experimental procedures were conducted in accordance with the guidelines set by the National Committee of Bioethics (NCBE), Saudi Arabia, and complied with the ARRIVE 2.0 guidelines.

2.4. Synthesis of DPP derivatives

The DPP derivatives were synthesized according to the reaction scheme depicted in Fig. 1.

Fig. 1. Synthetic route for the DPP analogues (4a–4i).

2.4.1. Synthesis of chalcone derivatives (2a–2c)

Aryl aldehydes (1a–1c) and acetophenone were mixed in equimolar quantities (0.001 mol) and dissolved in 3 mL of ethanol. To this mixture, an aqueous solution of sodium hydroxide (0.003 mol) was added slowly, and the reaction mixture was continuously stirred for 2 h at room temperature. Once the reaction was complete, the resultant solution was transferred to crushed ice and acidified using dilute HCl, if necessary. The solid thus obtained was filtered, air-dried, and recrystallized from ethanol.

2.4.2. Synthesis of chalcone epoxides (3a–3c)

The prepared chalcones (2a–2c; 5 mmol) were dissolved in methanol (10 mL) and cooled to 0 °C, after which a 30% H₂O₂ solution (1 mL) was added dropwise. The resulting reaction mixture was stirred for an additional 1.5 h at low temperature. The chalcone epoxides thus obtained were filtered under reduced pressure, dried at room temperature, and recrystallized from ethanol.

2.4.3. Synthesis of diphenylpropan-1-one derivatives (4a–4i)

Substituted phenols (2 mmol) and chalcone epoxides (3a–3c; 2 mmol) were dissolved in a 30% sodium hydroxide (NaOH) solution (2.5 eq.) and continuously stirred for 2 h at room temperature. Afterward, the resultant mixture was heated to 80 °C with continuous stirring for an additional 2 h, or until the reaction was complete, to obtain the desired products. The crude reaction mixture was concentrated under reduced pressure and purified using column chromatography on silica gel (60–120 mesh) as the stationary phase. Elution was performed using a gradient of ethyl acetate/hexane (10 : 90 to 40 : 60, v/v), and fractions containing the desired product were identified by TLC, combined, and concentrated under reduced pressure to afford the products as pure solids. The final purified DPP derivatives (4a–4i) were characterized using spectral techniques.

2.4.3.1. 3-(4-(Dimethylamino)phenyl)-2-hydroxy-3-phenoxy-1-phenylpropan-1-one (4a)

Color and state: yellow solid; yield: 48%; melting point (MP): 138–140 °C; FT-IR (KBr, ν, cm⁻¹): 3425 (–OH), 3055 (Ar–CH), 2922, 2854 (C–H), 1688 (C O), 1602, 1512 (Ar C C), 1242 (C–O–C); ¹H NMR (400 MHz, DMSO-d₆, δ ppm): 7.92–7.88 (m, 2H, Ar–H), 7.58.7.42 (m, 3H, Ar–H), 7.35–7.20 (m, 5H, Ar–H), 7.05 (d, 2H, J = 8.8 Hz, Ar–H), 6.72 (d, 2H, J = 8.8 Hz, Ar–H), 5.32 (d, 1H, J = 6.4 Hz, CH–OH), 4.68 (d, 1H, J = 6.4 Hz, CH–OAr), 4.05 (br s, 1H, OH), 2.91 (s, 6H, N(CH₃)₂); ¹³CNMR (100 MHz, DMSO-d₆, δ ppm): 198.2, 159.1, 136.2, 134.1, 132.6, 129.4, 128.8, 123.4, 116.7, 113.6, 77.2, 69.1, 40.3; HRMS (ESI⁺): m/z calcd for C₂₃H₂₄NO₃ [M + H]⁺, 362.1751; found, 362.1749; calcd for C₂₃H₂₃NO₃Na [M + Na]⁺, 384.1570; found, 384.1545; R_f value = 0.60; log P = 4.46.

2.4.3.2. 3-(4-Aminophenoxy)-3-(4-(dimethylamino)phenyl)-2-hydroxy-1-phenylpropan-1-one (4b)

Color and state: yellow-brown solid; yield: 54%; melting point (MP): 147–149 °C; FT-IR (KBr, ν, cm⁻¹): 3448, 3362 (NH₂/–OH), 3050 (Ar–CH), 2920, 2853 (C–H), 1686 (C O), 1601, 1510 (Ar C C), 1240 (C–O–C); ¹H NMR (400 MHz, DMSO-d₆, δ ppm): 7.91–7.86 (m, 2H, Ar–H), 7.55–7.40 (m, 3H, Ar–H), 7.04 (d, 2H, J = 8.8 Hz, Ar–H), 6.82 (d, 2H, J = 8.4 Hz, Ar–H), 6.70 (d, 2H, J = 8.8 Hz, Ar–H), 6.55 (d, 2H, J = 8.4 Hz, Ar–H), 5.28 (d, 1H, J = 6.4 Hz, CH–OH), 4.90 (br s, 2H, NH₂), 4.63 (d, 1H, J = 6.4 Hz, CH–OAr), 4.02 (br s, 1H, OH), 2.90 (s, 6H, N(CH₃)₂); ¹³CNMR (100 MHz, DMSO-d₆, δ ppm): 198.1, 158.6, 151.2, 146.4, 136.1, 132.5, 129.3, 128.7, 123.2, 117.1, 115.4, 113.5, 77.1, 69.0, 40.2; HRMS (ESI⁺): m/z calcd for C₂₃H₂₅N₂O₃ [M + H]⁺, 377.1860; found, 377.1901; calcd for C₂₃H₂₄N₂O₃Na [M + Na]⁺, 399.1679; found, 399.1712; R_f value = 0.57; log P = 3.66.

2.4.3.3. 3-(4-(Dimethylamino)phenyl)-2-hydroxy-3-(naphthalen-1-yloxy)-1-phenylpropan-1-one (4c)

Color and state: yellow-brown solid; yield: 48%; melting point (MP): 155–157 °C; FT-IR (KBr, ν, cm⁻¹): 3422 (–OH), 3055 (Ar–CH), 2921, 2850 (C–H), 1687 (C O), 1600, 1508 (Ar C C), 1245 (C–O–C); ¹H NMR (400 MHz, DMSO-d₆, δ ppm): 8.18–8.12 (m, 1H, naphthyl–H), 7.96–7.86 (m, 3H, Ar–H/naphthyl–H), 7.70–7.42 (m, 7H, Ar–H), 7.30–7.18 (m, 3H, Ar–H), 7.04 (d, 2H, J = 8.8 Hz, Ar–H), 6.70 (d, 2H, J = 8.8 Hz, Ar–H), 5.40 (d, 1H, J = 6.4 Hz, CH–OH), 4.78 (d, 1H, J = 6.4 Hz, CH–OAr), 4.08 (br s, 1H, OH), 2.91 (s, 6H, N(CH₃)₂); ¹³CNMR (100 MHz, DMSO-d₆, δ ppm): 198.3, 158.2, 151.4, 136.5, 134.3, 133.8, 129.9, 128.9, 128.4, 126.8, 125.9, 124.2, 122.8, 113.6, 77.5, 69.2, 40.3; HRMS (ESI⁺): m/z calcd for C₂₇H₂₆NO₃ [M + H]⁺, 412.1907; found, 412.1912; calcd for C₂₇H₂₅NO₃Na [M + Na]⁺, 434.1727; found 434.1765; R_f value = 0.68; log P = 5.46.

2.4.3.4. 2-Hydroxy-3-(4-hydroxyphenyl)-3-phenoxy-1-phenylpropan-1-one (4d)

Color and state: light-brown solid; yield: 45%; melting point (MP): 172–174 °C; FT-IR (KBr, ν, cm⁻¹): 3460–3350 (–OH), 3052 (Ar–CH), 1685 (C O), 1602, 1514 (Ar C C), 1241 (C–O–C); ¹H NMR (400 MHz, DMSO-d₆, δ ppm): 9.45 (br s, 1H, phenolic OH), 7.92–7.86 (m, 2H, Ar–H), 7.58–7.42 (m, 3H, Ar–H), 7.34–7.18 (m, 5H, Ar–H), 6.92 (d, 2H, J = 8.4 Hz, Ar–H), 6.72 (d, 2H, J = 8.4 Hz, Ar–H), 5.30 (d, 1H, J = 6.4 Hz, CH–OH), 4.65 (d, 1H, J = 6.4 Hz, CH–OAr), 4.03 (br s, 1H, alcoholic OH); ¹³CNMR (100 MHz, DMSO-d₆, δ ppm): 198.0, 158.9, 156.1, 136.2, 134.0, 132.5, 129.4, 128.8, 128.0, 123.2, 116.2, 115.5, 77.0, 68.9; HRMS (ESI⁺): m/z calcd for C₂₁H₁₉O₄ [M + H]⁺, 335.1278; found, 335.1312; calcd for C₂₁H₁₈O₄Na [M + Na]⁺, 357.1097; found, 357.1132; R_f value = 0.60; log P = 3.79.

2.4.3.5. 3-(4-Aminophenoxy)-2-hydroxy-3-(4-hydroxyphenyl)-1-phenylpropan-1-one (4e)

Color and state: light-brown solid; yield: 47%; melting point (MP): 180–182 °C; FT-IR (KBr, ν, cm⁻¹): 3455, 3360 (NH₂/–OH), 3050 (Ar–CH), 1684 (C O), 1601, 1512 (Ar C C), 1240 (C–O–C); ¹H NMR (400 MHz,DMSO-d₆, δ ppm): 9.43 (br s, 1H, phenolic OH), 7.90–7.84 (m, 2H, Ar–H), 7.56–7.40 (m, 3H, Ar–H), 6.90 (d, 2H, J = 8.4 Hz, Ar–H), 6.80 (d, 2H, J = 8.4 Hz, Ar–H), 6.70 (d, 2H, J = 8.4 Hz, Ar–H), 6.54 (d, 2H, J = 8.4 Hz, Ar–H), 5.27 (d, 1H, J = 6.4 Hz, CH–OH), 4.88 (br s, 2H, NH₂), 4.60 (d, 1H, J = 6.4 Hz, CH–OAr), 4.01 (br s, 1H, alcoholic OH); ¹³CNMR (100 MHz, DMSO-d₆, δ ppm): 198.0, 158.5, 156.0, 146.3, 136.1, 132.4, 129.3, 128.7, 123.0, 117.0, 115.5, 115.2, 76.9, 68.8; HRMS (ESI⁺): m/z calcd for C₂₁H₂₀NO₄ [M + H]⁺, 350.1387; found, 350.1422; calcd for C₂₁H₁₉NO₄Na [M + Na]⁺, 372.1206; found, 372.1223; R_f value = 0.51; log P = 2.98.

2.4.3.6. 2-Hydroxy-3-(4-hydroxyphenyl)-3-(naphthalen-1-yloxy)-1-phenylpropan-1-one (4f)

Color and state: brown solid; yield: 43%; melting point (MP): 200–202 °C; FT-IR (KBr, ν, cm⁻¹): 3460–3350 (-OH), 3055 (Ar–CH), 1686 (C O), 1600, 1508 (Ar C C), 1244 (C–O–C); ¹H NMR (400 MHz, DMSO-d₆, δ ppm): 9.46 (br s, 1H, phenolic OH), 8.16–8.10 (m, 1H, naphthyl–H), 7.95–7.84 (m, 3H, Ar–H/naphthyl–H), 7.68–7.40 (m, 7H, Ar–H), 6.92 (d, 2H, J = 8.4 Hz, Ar–H), 6.72 (d, 2H, J = 8.4 Hz, Ar–H), 5.36 (d, 1H, J = 6.4 Hz, CH–OH), 4.74 (d, 1H, J = 6.4 Hz, CH–OAr), 4.06 (br s, 1H, alcoholic OH); ¹³CNMR (100 MHz, DMSO-d₆, δ ppm): 198.2, 158.2, 156.1, 136.4, 134.1, 133.7, 129.5, 128.8, 128.3, 127.5, 126.7, 125.8, 124.1, 122.6, 115.5, 77.3, 69.0; HRMS (ESI⁺): m/z calcd for C₂₅H₂₁O₄ [M + H]⁺, 385.1434; found, 385.1476; calcd for C₂₅H₂₀O₄Na [M + Na]⁺, 407.1254; found, 407.1304; R_f value = 0.70; log P = 4.78.

2.4.3.7. 3-(4-Chlorophenyl)-2-hydroxy-3-phenoxy-1-phenylpropan-1-one (4g)

Color and state: yellow-brown solid; yield: 46%; melting point (MP): 204–206 °C; FT-IR (KBr, ν, cm⁻¹): 3424 (–OH), 3052 (Ar–CH), 1687 (C O), 1601, 1510 (Ar C C), 1240 (C–O–C), 805 (C–Cl); ¹H NMR (400 MHz, DMSO-d₆, δ ppm): 7.92–7.86 (m, 2H, Ar–H), 7.58–7.42 (m, 3H, Ar–H), 7.38 (d, 2H, J = 8.4 Hz, Ar–H), 7.28–7.18 (m, 5H, Ar–H), 7.08 (d, 2H, J = 8.4 Hz, Ar–H), 5.33 (d, 1H, J = 6.4 Hz, CH–OH), 4.67 (d, 1H, J = 6.4 Hz, CH–OAr), 4.05 (br s, 1H, –OH); ¹³CNMR (100 MHz, DMSO-d₆, δ ppm): 198.2, 158.8, 137.2, 136.3, 134.1, 132.7, 129.4, 128.9, 128.3, 127.8, 123.4, 123.4, 116.7, 77.1, 69.0; HRMS (ESI⁺): m/z calcd for C₂₁H₁₈ClO₃ [M + H]⁺, 353.0939; found, 353.0941; calcd for C₂₁H₁₇ClO₃Na [M + Na]⁺, 375.0758; found, 375.0811; R_f value = 0.50; log P = 4.73.

2.4.3.8. 3-(4-Aminophenoxy)-3-(4-chlorophenyl)-2-hydroxy-1-phenylpropan-1-one (4h)

Color and state: brown solid; yield: 44%; melting point (MP): 197–199 °C; FT-IR (KBr, ν, cm⁻¹): 3450, 3355(–NH₂/OH), 3052 (Ar–CH), 2892 (C–H), 1694 (C O), 1514 (Ar C C), 1242 (C–O–C), 800 (C–Cl); ¹H NMR (400 MHz, DMSO-d₆, δ ppm): 7.91–7.85 (m, 2H, Ar–H), 7.53–7.38 (m, 3H, Ar–H), 7.36 (d, 2H, J = 8.4 Hz, Ar–H), 6.81 (d, 2H, J = 8.4 Hz, Ar–H), 6.71 (d, 2H, J = 8.4 Hz, Ar–H), 6.54 (d, 2H, J = 8.4 Hz, Ar–H), 5.32 (d, 1H, J = 6.4 Hz, CH–OH), 4.86 (br s, 2H, NH₂), 4.62 (d, 1H, J = 6.4 Hz, CH-OAr), 4.01 (br s, 1H, OH); ¹³CNMR (100 MHz, DMSO-d₆, δ ppm): 198.1, 157.4, 147.4, 137.7, 136.2, 132.7, 131.9, 129.1, 128.2, 127.6, 116.2, 115.2, 77.8, 68.4; HRMS (ESI⁺): m/z calcd for C₂₁H₁₉ClNO₃ [M + H]⁺, 368.1048; found, 368.1064; calculated for C₂₁H₁₈ClNNaO₃ [M + Na]⁺, 390.0867; found, 390.0901; R_f value = 0.76; log P = 3.93.

2.4.3.9. 3-(4-Chlorophenyl)-2-hydroxy-3-(naphthalen-1-yloxy)-1-phenylpropan-1-one (4i)

Color and state: deep brown solid; yield: 48%; melting point (MP): 186–188 °C; FT-IR (KBr, ν, cm⁻¹): 3424 (OH), 3055 (Ar–CH), 1688 (C O), 1600, 1508 (Ar C C), 1245 (C–O–C), 804 (C–Cl); ¹H NMR (400 MHz, DMSO-d₆, δ ppm): 8.17–8.10 (m, 1H, naphthyl–H), 7.96–7.84 (m, 3H, Ar–H/naphthyl–H), 7.70–7.42 (m, 7H, Ar–H), 7.38 (d, 2H, J = 8.4 Hz, Ar–H), 7.08 (d, 2H, J = 8.4 Hz, Ar–H), 5.38 (d, 1H, J = 6.4 Hz, CH–OH), 4.76 (d, 1H, J = 6.4 Hz, CH–OAr), 4.07 (br s, 1H, OH); ¹³CNMR (100 MHz, DMSO-d₆, δ ppm): 198.3, 158.2, 137.3, 136.4, 134.2, 133.8, 132.7, 129.6, 128.9, 128.4, 127.8, 127.5, 126.8, 125.9, 124.2, 77.4, 69.1; HRMS (ESI⁺): m/z calcd for C₂₅H₂₀ClO₃ [M + H]⁺, 403.1095; found, 403.1103; calculated for C₂₅H₁₉ClO₃Na [M + Na]⁺, 425.0915; found, 425.0934; R_f value = 0.65; log P = 5.73.

2.5. In vitro cytotoxicity assay

To assess the cytotoxic potential of the DPP derivatives (4a–4i), the MTT assay was conducted on the human breast cancer cell line MCF-7 using the procedure described earlier.³¹ MCF-7 cells were merely selected as an established human breast adenocarcinoma model to perform preliminary cytotoxicity screening of the tested compounds and were not selected on the basis of experimentally confirmed EGFR overexpression. EGFR expression was not independently quantified in the cells during the present investigation. Briefly, exponentially growing cells were selected for the study, and approximately 1 × 10⁴ cells were seeded into the MTT microplate wells. Cells were suspended in 100 µL of RPMI medium, left to adhere overnight to the bottom of the plate, and then incubated in a CO₂ incubator. Meanwhile, the DPP derivatives were thoroughly dissolved in the culture medium containing 10% DMSO, ensuring that the final DMSO concentration in the wells did not exceed 0.05%. The following day, the culture medium was replaced with fresh medium consisting of various concentrations of DPP derivatives, and the cultures were incubated for 24–48 h for treatment. One set of wells was kept as a control, which did not receive the DPP derivatives but contained 0.05% DMSO. A commercial drug, gefitinib, was used as a positive control in a separate set of wells. After 48 h, once the reaction was complete, the plates were removed from the incubator, and cytotoxicity was observed under a microscope, with a clear reduction in viable cells. Subsequently, MTT solution (10 mg mL⁻¹, 100 µL) was added to each well. The plates were covered with aluminium foil to prevent light exposure and returned to the incubator for 4 h. At the end of the incubation period, the plates were taken out of the incubator, and the supernatant was gently removed without disturbing the formazan crystals. DMSO (100 µL) was added to each well to dissolve the formazan crystals, and the absorbance of the purple color formed was measured at 570 nm using a microplate reader. The cell viability and growth inhibition percentages were calculated using eqn (1) and (2), respectively.

1

2

where A_{treated cells} = absorbance of treated cells and A_{control cells} = absorbance of control cells.

Experiments were carried out in triplicate, and the findings are presented as the mean ± standard deviation (SD). The statistical comparisons were made between the treated and untreated control groups at different concentrations, and the IC₅₀ values were calculated using non-linear regression analysis of the concentration–response curves.

2.6. In vivo acute toxicity study

The acute toxicity study of the most active compound, 4h, was performed in adult female Sprague-Dawley rats in accordance with OECD Guideline 423, with minor modifications.³² The animals were first acclimatized to standard laboratory conditions with free access to feed and water. Prior to dosing, the animals were fasted overnight and given only water ad libitum. Compound 4h was suspended in 0.1% carboxymethylcellulose (CMC) and administered by gavage, with a stepwise dose escalation. The control group of animals only received normal saline. All animal groups were observed for 48 h and subsequently monitored daily for 14 days, or until signs of toxicity or mortality became evident. The maximum safe dose was defined as the highest dose administered without mortality or severe toxicity.

2.7. In vivo anticancer activity against the DMBA-induced cancer model

The in vivo anticancer activity of the most active compound, 4h, was evaluated using a 7,12-dimethylbenz-(a)-anthracene (DMBA)-induced breast cancer model in Sprague-Dawley rats. A total of 55 adult female rats, aged 6 to 8 weeks, with weights in the range of 150 ± 25 g, were used in the study and were divided into five groups. The animals were housed under standard environmental conditions (25 °C ± 2 °C, 50–60% relative humidity (RH), 12 h light/dark cycle) with free access to food and water. Group 1 (n = 10) served as the normal control, where the rats were administered 0.1% carboxymethyl cellulose (CMC) via oral gavage. A total of 45 female Sprague-Dawley rats received a subcutaneous injection of DMBA (80 mg kg⁻¹), previously dissolved in corn oil (0.5 mL), into the mammary region to induce breast tumors.³³ The animals were monitored weekly for tumor development, and after approximately 4 months, those exhibiting palpable mammary tumors were screened for the study and assigned to treatment groups. The breast cancer-induced rat models were categorized into four groups: the DMBA control group, which received no treatment (group 2, n = 15); the reference drug group, where animals were administered tamoxifen p.o. at a dose of 20 mg per kg per day (group 3, n = 10), the 4h-25 group (group 4, low-dose treated, n = 10), and the 4h-50 group (group 5, high-dose treated, n = 10) groups, in which the animals in the latter two groups were orally administered with compound 4h at doses of 25 mg per kg per day and 50 mg per kg per day, respectively. The test and standard compounds were dissolved in 0.1% CMC before administration, and the treatment was continued for 28 days.

Upon completion of the experiment, the animals were anesthetized with a mixture of ketamine (100 mg kg⁻¹) and xylazine (10 mg kg⁻¹), administered intraperitoneally. An adequate depth of anesthesia was confirmed by the absence of the pedal withdrawal reflexes before the collection of samples. Blood samples were then collected from the inferior vena cava into heparinized tubes and centrifuged at 5000 rpm for 10 min to extract plasma. The obtained plasma samples were stored as aliquots at −80 °C until further analysis. Following sample collection, the animals were euthanized by exsanguination under deep anesthesia, and both mammary tumors and normal mammary tissues were carefully excised from all experimental groups for subsequent analyses. For histopathological assessment, inguinal mammary gland tissues from all animals of each experimental group were fixed in 10% neutral-buffered formalin solution for 72 h. The tissues were subsequently processed through a series of ascending concentrations of alcohol for dehydration, cleared using xylene, and embedded in paraffin wax. The paraffinized tissue blocks were then sectioned into 5 µm-thick slices using a microtome, followed by hematoxylin and eosin (H & E) staining for microscopic examination.³⁴ Morphological changes, including epithelial proliferation, nuclear atypia, stromal alterations, and the presence of mitotic figures, were examined under a light microscope. To reduce observer bias and improve the robustness of the pathological evaluation, histopathological evaluation was performed by a qualified pathologist blinded to the treatment groups during microscopic examination.

2.8. RNA extraction and cDNA synthesis

Total RNA was isolated from the mammary tumor tissues collected from the normal control, DMBA-control, tamoxifen-treated, low-dose 4h-treated, and high-dose 4h-treated groups using TRIzol reagent under RNase-free conditions following the manufacturer's protocol (Solarbio Science, Beijing).³⁵ cDNA was synthesized from the total RNA using a reverse transcription kit (high-capacity cDNA kit) in the presence of an RNase inhibitor (applied biosystems, 4 368 814). The oligomer primer sequences of the BRCA1, BRCA2, and TP53 target genes are displayed in Table 1. Power Up™ SYBR™ Green Master Mix (applied biosystems, A25742) was used for qRT-PCR amplification on an applied biosystems 7700 real-time sequence detection system. The value of each sample was normalized to GAPDH expression, which served as the reference gene. The qRT-PCR assay was carried out using an amplification program including an initial denaturation, followed by several cycles of denaturation, primer annealing, and extension. Melt-curve analysis was subsequently performed to verify the specificity of the amplified products. The comparative mRNA expression levels were estimated using the 2^−ΔΔCT method and expressed as fold changes relative to the normal control or the DMBA-treated control group, where applicable. Groups were compared statistically, and significant changes in gene expression following treatment with compound 4h and standard tamoxifen were observed.

Table 1. List of primer sequences.

Oligo	Primer sequences	bp
BRCA1	5′-TGAAGACTGCTCGCAGAGTGATA-3′ forward	23
	5′-AGCTTCCAGGTGAGCCATTTC-3′ reverse	21
BRCA2	5′-TTGAGGACCCCAAGACCTGT-3′ forward	20
	5′-CCGGAGAGACAAAGGTGCA-3′ reverse	19
TP53	5′-GCAGAGTTGTTAGAAGGC-3′ forward	18
	5′-TTGAGAAGGGACGGAAGA-3′ reverse	18
GAPDH	5′-AACTTTGGCATTGTGGAAGG-3′ forward	20
	5′-ACACATTGGGGGTAGGAACA-3′ reverse	20

2.9. Exploratory in silico molecular docking analysis

Molecular docking analysis of the synthesized DPP derivatives (4a–4i) was performed as an exploratory computational analysis to investigate their potential interactions and predicted binding compatibility within the ATP-binding region of the EGFR-tyrosine kinase domain. The crystal structure of EGFR complexed with gefitinib (PDB ID: 4WKQ) was downloaded from the RCSB Protein Data Bank website and employed for the docking study. Gefitinib served as the native reference ligand to identify the active binding site and validate the docking protocol. Docking simulations were performed using UCSF Chimera 1.13.1 integrated with AutoDock Vina software (version 1.2.0, The Scripps Research Institute, USA).^36,37 The three-dimensional structures of the synthesized compounds were imported to the Chimera interface for ligand preparation. Prior to docking, the EGFR protein structure was processed using the Chimera Dock Prep module. For protein preparation, only chain A was retained, while crystallographic water and other solvent molecules were removed. Hydrogens atoms and partial charges were assigned to obtain the final protein structure, which was then saved as a.mol2 file for the next step. The structures of the test compounds were optimized for ligand geometry, atomic charges, and energies using the AMBER force field, followed by hydrogen addition and assignment of Gasteiger charges.

Docking simulations of the DPP derivatives within the EGFR active site were run in the AutoDock Vina module integrated into UCSF Chimera. The ATP-binding cavity was defined based on the location of the native co-crystallized ligand, gefitinib. An imaginary grid box was generated with center coordinates of x = 3.5, y = 194, z = 25 Å and dimensions of 25 × 25 × 25 Å, which adequately encompassed the active binding region of EGFR. The docking search parameters were adjusted to allow an energy difference of 3 kcal mol⁻¹ between conformers, with a low exhaustiveness value of 8 and the generation of 10 binding conformations for each ligand. Validation of the docking protocol was performed through redocking of the reference inhibitor/native co-crystallized ligand into the EGFR binding pocket, and a root–mean–square deviation (RMSD) value of ≤2 Å was considered acceptable for reliable prediction of binding poses. The experimentally obtained redocking RMSD value was found to be 1.64 Å. The binding conformations of the synthesized compounds were ranked according to their docking energies (ΔG, kcal mol⁻¹), RMSD values, and intermolecular interactions, including hydrogen bonds. The optimal binding orientations and ligand–receptor interactions were further examined using Discovery Studio Visualizer 2016. Gefitinib was used as the reference standard for the comparative evaluation of the binding affinities and interaction patterns of the synthesized DPP derivatives with EGFR.

2.10. In silico ADME, pharmacokinetic, and drug-likeness prediction

The pharmacokinetic, drug-likeness, and toxicity profiles of the most active compound, 4h, were evaluated using two widely used computational platforms: SwissADME³⁸ and ADMETlab 3.0.³⁹ The SMILES notation for compound 4h was generated and submitted to both platforms for analysis. SwissADME was used to predict the physicochemical properties, aqueous solubility, lipophilicity, gastrointestinal (GI) absorption, blood–brain barrier (BBB) permeability, P-glycoprotein (P-gp) substrate/inhibitor status, medicinal chemistry filters (PAINS and Brenk alerts), synthetic accessibility, and compliance with the major drug-likeness rules, such as the Lipinski, Ghose, Veber, Egan, and Muegge criteria. Further, the BOILED-Egg (Brain Or IntestinaL EstimateD permeation) predictive model built into SwissADME was employed to evaluate passive gastrointestinal absorption and BBB permeation based on the relationship between lipophilicity (WLOGP) and topological polar surface area (TPSA).⁴⁰

To predict a broader pharmacokinetic and toxicity profile, compound 4h was further analyzed using ADMETlab 3.0. Parameters related to absorption, distribution, metabolism, excretion, and toxicity (ADMET) were predicted, including human intestinal absorption, plasma protein binding, cytochrome P450 interactions, P-gp transport, BBB penetration, drug-induced liver injury (DILI), mutagenicity, carcinogenicity, and organ-specific toxicities. Moreover, the ADMETlab radar plot was used to visualize the overall drug-likeness and pharmacokinetic characteristics of compound 4h relative to the recommended ranges for orally active drug candidates.

2.11. Statistical analysis

All experiments were carried out in triplicate, and the data were expressed as the mean ± standard deviation (SD). Statistical evaluation of the results was conducted using GraphPad Prism software (version 10.6.0; GraphPad Software, San Diego, CA, USA). Comparisons among different experimental groups were made using one-way analysis of variance (ANOVA), followed by Tukey's multiple-comparison post hoc test. Statistical significance was defined as p < 0.05.

3. Results and discussion

3.1. Synthesis and characterization of title compounds (4a–4i)

A series of novel 3-(4-substituted phenyl)-2-hydroxy-3-aryloxy-1-phenylpropan-1-ones (4a–4i) was designed from a chalcone scaffold and successfully synthesized via a simple three-step synthetic route. The reaction scheme involved the Claisen–Schmidt condensation of aryl aldehydes with aromatic ketones to obtain chalcones (2a–2c), followed by epoxidation using hydrogen peroxide to yield chalcone epoxides (3a–3c), and eventually, the nucleophilic ring-opening reaction with substituted phenols to afford the targeted DPP derivatives (4a–4i). The final compounds were obtained in moderate yields ranging from 43% to 54%. All the synthesized compounds were characterized using standard spectroscopic techniques, confirming their structural integrity and purity. As the epoxide ring-opening reactions were conducted under achiral conditions, no stereochemical induction or resolution was performed. Therefore, the stereochemical configurations of the synthesized β-hydroxy ketone derivatives were not assigned, and the compounds were evaluated as obtained. The resolution and stereochemical characterization of individual stereoisomers will be addressed in future investigations.

The FT-IR spectra exhibited characteristic absorption peaks corresponding to important functional groups. The carbonyl group (C O) of the propanone moiety was confirmed by a strong absorption band at 1680–1700 cm⁻¹, whereas a broad absorption peak at around 3400–3450 cm⁻¹ was observed, corresponding to the hydroxyl (–OH) stretching vibration. Compounds with amino groups showed an additional band in the region of 3300–3350 cm⁻¹, corresponding to the –NH₂ groups. Aromatic C C stretching vibrations in all compounds were distinct in the region of 1500–1600 cm⁻¹. The ¹H NMR spectra also showed characteristic signals corresponding to the synthesized compounds, where the methine protons (CH–OH and CH–OAr) were observed as doublets in the regions of δ 5.2–5.4 and δ 4.6–4.8 ppm, with J = 6.4 Hz, confirming the formation of a β-hydroxy ketone framework. Aromatic protons were observed downfield as multiplets in the range of δ 6.5–8.73 ppm, depending on the substitution pattern. The –OH and –NH₂ protons were observed as broad singlets in the downfield region, owing to the presence of hydrogen bonding.

The ¹³C NMR spectra further confirmed the structure, as the carbonyl carbon appeared in the region of δ 197–199 ppm, aromatic carbons appeared in the range of δ 113–160 ppm, and the methine carbons of the propanone moiety were observed in the region of δ 65–78 ppm, which is consistent with the proposed structures. The mass spectral analysis (HRMS-ESI⁺) also showed the corresponding molecular ion peaks, confirming the respective molecular weights of the synthesized compounds.

3.2. In vitro cytotoxicity assay

The cytotoxicity of the title derivatives (4a–4i) was assessed against the human breast cancer cell line MCF-7 using the MTT assay, and the results are shown in Table 2. As evident from the table, a wide variation in anticancer activity was observed, with IC₅₀ values ranging from 1.7 to >30 µg mL⁻¹, depending on the substituents present on the aromatic rings. Interestingly, among the tested compounds, 4h exhibited the highest cytotoxic activity, with an IC₅₀ of 1.7 µg mL⁻¹, comparable to the standard reference drug, gefitinib (IC₅₀ = 1.8 µg mL⁻¹). Compound 4g also showed marked cytotoxic activity (IC₅₀ = 2.8 µg mL⁻¹), followed by compound 4a (IC₅₀ = 6.25 µg mL⁻¹) and compound 4i (IC₅₀ = 12 µg mL⁻¹). In contrast, compounds 4d (IC₅₀ = 23 µg mL⁻¹), 4e (IC₅₀ = 31 µg mL⁻¹), and 4b (IC₅₀ = 35 µg mL⁻¹) exhibited relatively weaker cytotoxic effects, whereas compounds 4c and 4f showed no significant inhibition within the tested concentration range. Compounds 4g and 4h produced significant concentration-dependent reductions in MCF-7 cell viability compared with the control (p < 0.001). The concentration–response curves obtained during the in vitro cytotoxicity assay for derivatives 4g and 4h and the standard gefitinib are presented in Fig. 2, whereas the curves for the other compounds are included in the SI (Fig. S1).

Table 2. In vitro cytotoxicity assay results for DPP derivatives (4a–4i).

Compound no.	IC50 (µg mL−1)
Control	—
4a	6.3
4b	35
4c	No inhibition
4d	23
4e	31
4f	No inhibition
4g	2.8
4h	1.7
4i	12
Gefitinib (standard)	1.8

Fig. 2. Concentration-response curves obtained during in vitro cytotoxicity assay of DPP derivatives 4g and 4h and the standard drug gefitinib. Data are presented as the mean of triplicate measurements ± S.D.

The synthesized compounds were designed as a systematic substitution matrix to investigate the effects of both electronic and steric factors on biological activity. The p-dimethylamino and p-hydroxy groups were selected as polar electron-donating substituents, whereas the p-chloro group was chosen as a relatively lipophilic electron-withdrawing substituent. In addition, the aryloxy component was also varied from phenoxy to 4-aminophenoxy to introduce additional hydrogen-bond donor/acceptor functionality, as well as to the naphthalen-1-yloxy ring to examine the effects of increased aromatic surface area and steric bulk. This design strategy helped evaluate the contributions of electronic character, hydrogen-bonding capability, lipophilicity, and steric factors across the compound series.

The structure–activity relationship (SAR) analysis revealed a clear dependence of biological activity on the nature and position of substituents attached to the aromatic rings of the DPP scaffold. Both electronic and steric factors markedly influenced the biological activity of the tested derivatives. In general, it was observed that the derivatives containing electron-withdrawing functionalities exhibited greater cytotoxic potential than those containing electron-donating groups. An electron-withdrawing functional group, such as a chloro substituent, improved the cytotoxic activity of the compounds, possibly by enhancing the hydrophobic character of the molecule. The chlorine substituent is also known to improve van der Waals interactions and enhance the occupancy of hydrophobic pockets within the protein binding sites, contributing to stronger overall interactions within the ATP-binding region of EGFR. Furthermore, substituents such as halogens can improve metabolic stability and membrane permeability and may therefore further increase the cytotoxicity of compounds possessing them. Compound 4h demonstrated the highest cytotoxic activity, which might be attributed to the presence of both a chloro and an aminophenoxy moiety, which may facilitate electronic interactions and hydrogen bonding within the target binding site and thereby improve ligand orientation within the active site.

In contrast, compounds containing only electron-donating groups, such as the dimethylamino group in compounds 4a–4c and the hydroxyl group in compounds 4d–4f, showed low to moderate anticancer potential. Although these functionalities may contribute to hydrogen-bond formation, their increased electron density and polarity might have reduced favorable hydrophobic interactions within the receptor-binding pocket, leading to reduced potency compared to compound 4h.

The nature of the aryloxy substituent was also observed to influence cytotoxic activity. Among the chloro-substituted series (4g–4i), replacing the phenoxy group in 4g with a 4-aminophenoxy group in 4h improved the activity from 2.8 to 1.7 µg mL⁻¹, suggesting that the amino group might have contributed to additional hydrogen-bonding interactions and improved the ligand orientation within the target binding region. However, replacing the phenoxy group with a bulkier naphthalene-1-yloxy group in 4i reduced the cytotoxic activity to 12 µg mL⁻¹, indicating that an excessive aromatic bulk might have introduced steric constraints and hindered the optimal fit of the molecule within the binding pocket.

The influence of steric effects was also evident in the tested derivatives. It was also noted that derivatives bearing the naphthyl moiety (4c, 4f, and 4i) generally showed reduced anticancer activity, possibly due to increased steric hindrance, leading to a poor fit within the binding site and thereby limiting optimal binding interactions. Although the naphthyl group increases the hydrophobic surface area and theoretically supports π–π or hydrophobic interactions, its bulkiness might disrupt the preferred orientation of the DPP derivatives, limiting optimal receptor interactions. Therefore, the preliminary SAR study indicated the important roles of both electronic and steric factors in modulating the anticancer activity of the DPP derivatives.

In summary, the overall comparison of the tested series of compounds indicated that the biological activity was favoured by the chloro-substituted aromatic ring, while the aryloxy functionality was required for an appropriate balance between hydrogen-bonding capacity and steric demand. The superior efficacy of compound 4h compared to compound 4g suggested that the introduction of a 4-aminophenoxy substituent could enhance the activity within the chloro series. However, its replacement with a bulkier naphthalen-1-yloxy group, as in compound 4i, reduced the potency. The lack of activity observed for the naphthyl-containing compounds 4c and 4f further indicated that the increasing aromatic surface area alone was not sufficient and might have been detrimental to the activity, as the favourable molecular interactions were compromised by the increased steric demand.

The present DPP derivatives (4a–4i) were structurally distinct from conventional chalcone derivatives because they lack the α,β-unsaturated carbonyl group. Since this electrophilic enone system can undergo Michael addition with cellular nucleophiles, the absence of this group in the synthesized compounds might have reduced the potential for non-specific covalent reactivity. However, to establish the effects of this structural modification in the improvement of target selectivity, direct experimental evaluation and off-target reactivity studies are warranted.

3.3. In vivo acute toxicity study

The in vivo acute oral toxicity study of the most active compound, 4h, was performed in female rats in accordance with the OECD guidelines. Compound 4h did not show any mortality or severe toxic manifestations up to the highest tested dose (2000 mg kg⁻¹), indicating a favorable safety profile. No visible changes in the general behavior, locomotor activity, feeding behavior, or other physiological parameters were noticeable in the treated group of animals compared with the control group. Based on the findings of this study, the median lethal dose (LD₅₀) of compound 4h was considered to be greater than 2000 mg kg⁻¹, and therefore, the selected doses (1/40 and 1/80 of this maximum safe dosage) for in vivo anticancer activity were calculated to be 50 and 25 mg kg⁻¹ body weight, respectively.

3.4. In vivo anticancer activity against the DMBA-induced cancer model

The histopathological examination of H & E-stained mammary gland sections from the normal control, DMBA-induced control, and treatment groups revealed marked differences (Fig. 3). The mammary gland tissue of the normal control group exhibited well-preserved histoarchitecture (Fig. 3A). The mammary gland revealed a tubular, branched, ductal, and glandular alveolar arrangement, all of which were lined by a bilayer of epithelial and myoepithelial cells. The glandular structures possessed well-developed lumina and were surrounded by a connective tissue matrix interspersed with adipose stroma. No evidence of cellular atypia, inflammatory infiltration, or abnormal proliferation was noted.

Fig. 3. Histopathological assessment of mammary gland tissues in the DMBA-induced breast cancer model. (A) Normal control mammary gland showing normal histoarchitecture with intact tubular, branched ducts and alveolar structures lined by epithelial and myoepithelial cells; (B and C) DMBA-induced tumors demonstrating malignant proliferation, hyperchromatic pleomorphic cells, myoepithelial loss, and desmoplastic stroma; (D) low-dose 4h-treated group (25 mg kg⁻¹) showing a marked reduction in the epithelial proliferation and partial restoration of mammary tissue architecture; (E) high-dose 4h-treated group (50 mg kg⁻¹) showing substantial reduction in the proliferation and improved histological organization; and (F) tamoxifen-treated group showing reduced papillary formation and infiltration with residual mild brisk proliferation. H & E staining; representative photomicrographs are shown at low and high magnifications.

In contrast, the mammary glands of female rats pretreated with DMBA exhibited malignant transformations characterized by abnormal proliferation of ductal epithelial cells and glandular structures (Fig. 3B and C). These alterations were characterized by the presence of hyperchromatic, pleomorphic malignant cells with enlarged nuclei, loss of the myoepithelial layer, and increased mitotic activity. In addition, a desmoplastic stromal response was noticed, confirming successful induction of mammary carcinogenesis.

Interestingly, treatment with compound 4h led to a dose-dependent improvement in mammary gland histology. A marked reduction in mammary gland tumor progression was observed in rats treated with a low dose of compound 4h (25 mg kg⁻¹). This was manifested by decreased cellular proliferation and the absence of papillary formations (Fig. 3D). Although mild desmoplastic stromal changes were noted, the extent of tumor-associated cellular abnormalities was remarkably reduced compared with that in the untreated DMBA group.

A more pronounced protective effect was observed in the high-dose-treated group of animals (4h, dose = 50 mg kg⁻¹), with a marked reduction in epithelial cell proliferation (Fig. 3E). The histological examination showed significant suppression of nuclear pleomorphism, decreased mitotic activity, and improved preservation of mammary gland architecture, suggesting enhanced anticancer efficacy of compound 4h at the higher dose.

Correspondingly, treatment with the reference drug (tamoxifen) also resulted in a significant improvement in the histology of mammary gland tissues compared with the DMBA control group (Fig. 3F). The absence of papillary formations, reduced ductal wall infiltration, and diminished desmoplastic changes were evident. However, occasional brisk mitotic activity and residual proliferative changes still persisted.

Although compound 4h demonstrated promising antitumor activity, comprehensive in vivo efficacy and safety assessments were not performed. Future studies will include longitudinal measurement of tumor growth, which is required for the calculation of tumor growth inhibition, body weight monitoring, tumor multiplicity assessment, and biochemical evaluation of hepatic and renal function. Similarly, the systemic safety profile and overall therapeutic index of compound 4h need to be established. These parameters will be calculated and incorporated with detailed pharmacokinetic and mechanistic investigations to further assess the preclinical potential of the compound. Although blinded histopathological evaluation was performed to minimize observer bias, quantitative evaluation of tumor necrosis and proliferative indices was not performed in the present study. The evaluation was qualitative and intended to describe treatment-related morphological changes.

3.5. RNA extraction and cDNA synthesis

The effects of compound 4h on the expression levels of target genes BRCA1, BRCA2, and TP53 were quantified by qRT-PCR in the DMBA-induced breast tumor tissues of treated rats, and the results are presented in Fig. 4. Significant alterations in the expression levels of the tested genes were observed among the experimental groups, showing the impact of DMBA-induced carcinogenesis and treatment with compound 4h. The animals in the DMBA-induced positive control group showed significantly higher relative mRNA expression levels of the BRCA1, BRCA2, and TP53 genes than those in the normal control group (p < 0.001), indicating successful induction of DMBA-induced mammary carcinogenesis. Treatment with 4h-25 (low dose) and 4h-50 (high dose) showed a significant, dose-dependent decline in the DMBA-induced overexpression of BRCA1, BRCA2, and TP53 (p < 0.01 to p < 0.001) compared with the untreated DMBA control group.

Fig. 4. Dose-dependent effects of compound 4h (low dose = 25 mg kg⁻¹ and high dose = 50 mg kg⁻¹) and the standard drug tamoxifen on the amplification of target genes BRCA1, BRCA2, and TP53 in the DMBA-induced mammary tumor tissues. Data are presented as mean ± SD, and the statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test; ***p < 0.001 versus the normal control; ^##p < 0.01 and ^###p < 0.001 versus the DMBA-induced positive control.

Among the treated groups, animals treated with the high dose (50 mg kg⁻¹) of 4h showed gene expression levels similar to those observed in the standard drug, tamoxifen-treated group, indicating comparable anticancer activity at the molecular level. These findings were consistent with histological observations and further supported the potential protective properties of compound 4h against DMBA-associated mammary carcinogenesis. Therefore, the observed normalization of BRCA1, BRCA2, and TP53 expression by compound 4h may be an indirect consequence of reduced DMBA-induced DNA damage and cellular stress rather than a direct transcriptional regulatory effect.⁴¹ The outcomes of the present study were consistent with previous investigations in rats, showing that DMBA induces oxidative stress, genomic instability, and compensatory activation of DNA repair-associated genes during the development of breast cancer.^41,42 Compound 4h and tamoxifen effectively attenuated the transcriptional response associated with DMBA-induced molecular changes in mammary tissues, thereby contributing to the observed reduction in tumor progression in a dose-dependent manner.

DMBA is a potent genotoxic carcinogen that acts by inducing oxidative stress, DNA damage, and genomic instability. Consequently, activation of the DNA damage response results in increased transcription of DNA repair- and cell cycle-associated genes, including BRCA1, BRCA2, and TP53. Therefore, the increased expression of these genes, as observed in the DMBA-treated group, was due to a compensatory response to carcinogen-induced genomic stress, injury, and DNA damage, leading to activation of DNA repair mechanisms and cell-cycle checkpoint pathways. Although BRCA1, BRCA2, and TP53 function as tumor suppressor genes, their increased transcription under genotoxic stress reflects an adaptive response to extensive DNA damage rather than enhanced tumor–suppressive activity.

Expression of these genes following carcinogen exposure may vary depending on the stage of tumor development and the extent of DNA damage. In chemically induced breast cancer models, transient upregulation of these genes has been attributed to the cellular attempt to repair DNA lesions and preserve genomic integrity. However, despite this compensatory response, persistent DNA damage may overwhelm these protective effects and eventually result in malignant transformation. BRCA1, BRCA2, and TP53 were selected for the study as representative molecular markers, as they are key regulators of genomic stability and are frequently implicated in the development of breast cancer. BRCA1 and BRCA2 play central roles in homologous recombination-mediated DNA repair, while TP53 functions as a key regulator of cell-cycle arrest, DNA repair, and apoptosis in response to genotoxic stress.

3.6. Exploratory in silico molecular docking analysis

The binding compatibilities of the synthesized compounds (4a–4i) within the ATP-binding pocket of the EGFR-tyrosine kinase domain were assessed by molecular docking studies using AutoDock Vina. The docking scores, expressed as binding energies (kcal mol⁻¹), and the corresponding RMSD values for all compounds are summarized in Table 3. All synthesized compounds demonstrated favorable binding affinities toward EGFR, with the calculated binding energies ranging from −7.6 to −9.6 kcal mol⁻¹, suggesting moderate to strong ligand–receptor interactions within the active site of the protein. Among the tested analogues, compound 4g showed the highest binding affinity with a docking score of −9.6 kcal mol⁻¹, closely followed by compound 4h (−9.2 kcal mol⁻¹) and compound 4i (−9.0 kcal mol⁻¹). Notably, these three compounds exhibited stronger predicted binding affinities than the reference EGFR inhibitor, gefitinib, which had a binding affinity of −8.4 kcal mol⁻¹. The calculated RMSD values for all the investigated compounds were <2 Å, demonstrating the acceptable reliability and stability of the predicted docking conformations.

Table 3. Results of in silico molecular docking studies of DPP derivatives 4a–4i with the EGFR binding site.

Compound no.	Binding energy (kcal mol−1)	RMSD (Å)
4a	−8.1	0.135
4b	−7.8	0.935
4c	−8.3	1.614
4d	−8.2	1.297
4e	−8.1	1.382
4f	−7.6	1.513
4g	−9.6	1.784
4h	−9.2	1.352
4i	−9.0	1.091
Gefitinib (reference inhibitor)	−8.4	1.74

Although compound 4g showed the most favorable docking score, compound 4h was considered the lead candidate based on its superior overall biological profile, including in vitro cytotoxicity and in vivo anticancer activity. The analysis of the docking results revealed that compound 4h occupied the same ATP-binding pocket in EGFR and exhibited a binding orientation similar to that of gefitinib (Fig. 5 and 6). The docked ligand adopted an extended conformation within the tyrosine kinase domain and was stabilized through multiple hydrophobic and hydrogen-bonding interactions. Two hydrogen bonds with Thr854, one π–sulfur interaction with Met762, and one π-anion interaction with Asp800 are observed for 4h in its interaction with EGFR. Also, a total of six π-alkyl interactions were predicted for 4h: two with Val726, one with Ala743, one with Lys745, one with Leu844, and one with Leu788.

Fig. 5. Binding interactions of compound 4h within the EGFR tyrosine kinase active site.

Fig. 6. Binding interactions of gefitinib within the EGFR tyrosine kinase active site.

These predicted interactions suggested that compound 4h is geometrically and energetically compatible with the ATP-binding pocket of EGFR; however, docking alone cannot confirm the biochemical inhibition mechanism or demonstrate that the observed cytotoxicity is EGFR-mediated. The diphenylpropan-1-one moiety of compound 4h significantly influenced binding and contributed to its stabilization within the hydrophobic region of the binding site, as observed in Fig. 5. Additionally, the chloro and aminophenoxy substituents contributed to ligand–receptor interactions, thereby explaining the superior activity of this compound observed experimentally.

Fig. 6 shows the binding of the reference standard, gefitinib, to EGFR. The molecular docking results were in agreement with the results from the in vitro cytotoxicity assay using the MCF7 breast cancer cells. A preliminary structure–activity relationship could also be derived from the docking results, as the compounds possessing electron-withdrawing chloro-substituted aromatic rings (4g and 4h) showed stronger binding affinities than the derivatives containing electron-releasing hydroxyl or dimethylamino groups toward the EGFR binding site. Compounds with weaker docking scores, including 4b and 4f, also exhibited comparatively lower cytotoxic effects, demonstrating a correlation between molecular docking and biological evaluation. Docking protocol validation by redocking the reference inhibitor/native co-crystallized ligand indicated that the protocol was acceptable because the RMSD between the native and redocked poses was 1.64 Å. The overlay of the native and redocked gefitinib poses is shown in Fig. 7.

Fig. 7. Docking validation (RMSD = 1.64 Å) overlay poses of native gefitinib (pink) and redocked gefitinib (green).

Although compound 4h was found to be geometrically compatible with the EGFR ATP-binding pocket and showed favorable predicted ligand–receptor interactions, molecular docking can only estimate binding compatibility, not biochemical inhibition. Similarly, in vitro cytotoxicity assays using MCF-7 cells were not target-specific and could have resulted from EGFR-independent mechanisms. Therefore, the findings presented in the study support the anticancer activity of compound 4h, with predicted EGFR-binding potential; however, direct EGFR kinase inhibition was not established. Biochemical kinase assays assessing EGFR phosphorylation and downstream signaling are required to confirm the proposed mechanism.

Additionally, the selectivity of compound 4h for EGFR over other subtypes of the ErbB family of receptors was not established. The structural versatility of the DPP scaffold might have permitted interactions with other cancer-associated kinases. ATP-binding pockets within the ErbB kinase family share significant structural similarity, and cross-reactivity with HER2 or other kinases is possible; therefore, future biochemical kinase-panel studies are required to fully establish the potency and selectivity of the scaffold. Future optimization of this scaffold should consider a multi-target strategy rather than exclusive EGFR selectivity.

3.7. In silico ADME pharmacokinetic and drug-likeness prediction

The pharmacokinetic and drug–likeness properties of the most active compound, 4h, were evaluated using SwissADME and ADMETlab 3.0 platforms to complement the biological and molecular docking studies. The predicted parameters collectively provided crucial insights into the developability of compound 4h as a potential anticancer lead molecule.

The SwissADME analysis revealed that compound 4h had favorable physicochemical properties, including a molecular weight of approximately 367 g mol⁻¹, a TPSA of 72.55 Ų, and a moderate lipophilicity profile, which is required for good oral absorption. Interestingly, the compound met all the lipinski, ghose, veber, egan, and muegge drug-likeness criteria without major violations and exhibited good compatibility with the physicochemical requirements typically associated with orally active drugs. The predicted parameters for compound 4h from the SwissADME and ADMETlab tools are summarized in Table 4.

Table 4. Summary of SwissADME and AMDETlab parameters predicted for compound 4h.

Parameter	Predicted value
Molecular weight	367.1 g mol−1
TPSA	72.55 Å2
LogP	3.21
LogS	−4.576
pKa (acid)	9.11
pKa (base)	4.793
Rotatable bonds	6
Hydrogen-bond acceptors	3
Hydrogen-bond donors	2
Lipinski/ghose/veber/egan/muegge violations	0
Human intestinal absorption	High
BBB permeability	Positive (0.838)
P-gp substrate	No
P-gp inhibitor	0.741
Plasma protein binding	96.56%
Volume of distribution	0.389
Oral bioavailability score	0.55
CYP3A4 inhibitor	Yes (0.998)
CYP3A4 substrate	Yes (0.886)
CYP2C9 inhibitor	Yes (0.951)
CYP2C19 inhibitor	Yes (0.755)
Ames toxicity	Low risk (0.149)
Carcinogenicity	Low risk (0.277)
DILI	Moderate risk (0.694)
Synthetic accessibility	3.23

The BOILED-egg model further predicted the permeability characteristics of compound 4h (Fig. 8). It was observed that compound 4h was located in the yolk region of the BOILED-egg plot, suggesting a high probability of passive permeation across the BBB while remaining within the physicochemical space associated with efficient GI absorption. Although BBB permeability is not a prerequisite for breast cancer treatment, the predicted ability of compound 4h to cross biological barriers may reflect favorable membrane permeability characteristics, which need to be confirmed through further experimental pharmacokinetic investigation. Moreover, the molecule was represented by a red marker, indicating that it is predicted to be a non-substrate of P-gp, thereby reducing the probability of transporter-mediated efflux and potentially favouring cellular uptake.

Fig. 8. BOILED-egg model obtained for compound 4h, predicting passive GI absorption and BBB permeability based on lipophilicity (WLOGP) and TPSA. The white region represents molecules with a high probability of GI absorption, whereas the yellow region (yolk) corresponds to molecules predicted to permeate the BBB. The red marker denotes that the molecule is predicted to be a non-substrate of P-gp.

The ADMETlab 3.0 analysis provided additional pharmacokinetic and safety-related information. It predicted potentially favorable intestinal absorption and distribution properties for compound 4h, together with adequate physicochemical characteristics for oral administration; however, these predictions require experimental confirmation. The compound demonstrated a high predicted plasma protein binding value, suggesting strong binding to circulating plasma proteins and potentially influencing systemic distribution and half-life. Metabolic predictions using the tool indicated potential interactions with several cytochrome P450 isoforms, particularly CYP3A4, CYP2C9, and CYP2C19, warranting future experimental metabolism studies to confirm potential drug–drug interactions.

The predicted toxicity profile of 4h was generally acceptable, with a low probability of mutagenic or carcinogenic effects, suggesting a computationally predicted toxicity profile with no major alerts for several endpoints, which requires experimental confirmation. However, moderate risks were predicted for certain toxicity endpoints, including DILI. These findings are commonly observed during early-stage lead identification and warrant further experimental validation.

The overall ADMET characteristics of compound 4h were further visualized and compared using the SwissADME and ADMETlab radar plots (Fig. 9A and B). These radar plots demonstrated that most predicted properties of compound 4h were within the recommended drug-like region, indicating a balanced combination of physicochemical, pharmacokinetic, and medicinal chemistry attributes. The close alignment of the compound-specific profile with the optimal property space suggested that compound 4h possessed a favorable balance between biological activity and developability. As evident from the figures, the majority of the predicted parameters for compound 4h were located within the optimal ADMET space, and the compound demonstrated acceptable values for molecular size, lipophilicity, polarity, flexibility, and hydrogen-bonding capacity, all of which contributed to its favorable drug-like profile.

Fig. 9. Drug-likeness and ADMET radar plots of compound 4h. (A) SwissADME bioavailability radar. The pink region represents the optimal range of six key parameters, including lipophilicity (LIPO), molecular size (SIZE), polarity (POLAR), solubility (INSOLU), flexibility (FLEX), and saturation (INSATU), while the red polygon corresponds to the predicted properties of compound 4h. (B) ADMETlab 3.0 radar plot illustrating the predicted physicochemical and pharmacokinetic properties of compound 4h. The yellow polygon represents the predicted properties of compound 4h, the blue shaded region represents the recommended range for drug-like molecules, and the green region represents the lower acceptable limits. A close alignment between the profile of compound 4h and the optimal ADMET space indicated favorable drug-likeness and promising predicted pharmacokinetic characteristics.

Collectively, the SwissADME and ADMETlab predictions supported the suitability of compound 4h as a promising lead candidate for further optimization. The combination of favorable predicted oral absorption characteristics, compliance with major drug-likeness criteria, acceptable toxicity predictions, and balanced predicted pharmacokinetic properties strengthens the overall therapeutic potential of this DPP derivative and complements the encouraging in vitro, in vivo, and molecular docking results obtained in the present study. However, it is important to emphasize that the pharmacokinetic and toxicity properties reported in this study are based exclusively on computational prediction models and therefore need to be confirmed through experimental investigations. Although SwissADME and ADMETlab are valuable tools for early-stage lead optimization, their predictions should be regarded as preliminary, requiring comprehensive experimental validation through in vitro ADME assays, pharmacokinetic investigations, and in vivo toxicological studies before drawing definitive conclusions about the potential of compound 4h for drug development.

4. Conclusions and future perspectives

A series of novel diphenylpropan-1-one (DPP) derivatives was successfully designed, synthesized, and biologically evaluated as potential anticancer agents for breast cancer therapy. The findings from in vitro, in vivo, and in silico studies were in good agreement, leading to the identification of the most active compound, 4h. The anticancer activity of 4h was comparable to that of the standard drugs, gefitinib and tamoxifen. The exploratory molecular docking studies indicated that the DPP derivatives may interact with the ATP-binding pocket of EGFR in a manner comparable to that of the reference inhibitor/native ligand, gefitinib, an FDA-approved drug. The ADMET profiling of compound 4h using SwissADME and ADMETlab tools suggested favorable drug-like properties, high GI absorption, acceptable pharmacokinetic characteristics, and an overall good development profile consistent with that of orally active small molecules. Compound 4h exhibited promising efficacy in the DMBA-induced breast cancer model, which was supported by favorable histopathological findings.

However, additional in vivo studies, including quantitative tumor burden measurements and comprehensive assessments of efficacy and safety profiles, are required to fully establish the therapeutic potential of compound 4h. The interaction of compound 4h with EGFR was established using exploratory molecular docking studies; however, further confirmation requires experimental validation using direct biochemical kinase inhibition assays, assessment of EGFR phosphorylation and downstream signaling, and other target-validation studies. Also, the docking analysis merely represents static predicted binding poses and cannot assess the dynamic stability of the ligand–protein complexes. Future investigations should include molecular dynamics simulations, followed by appropriate trajectory analyses including RMSD, RMSF, hydrogen-bond persistence, and binding energy calculations, which will provide crucial insights into the stability of compound 4h within the EGFR binding pocket. Similarly, the pharmacokinetics and safety profile of the newly synthesized derivative were predicted using computational tools and warrant experimental ADME and toxicological investigations to fully establish these claims. Moreover, anticancer evaluation against additional breast cancer cell lines representing other molecular subtypes could be conducted to better understand the therapeutic potential of this newly identified scaffold.

Based on the favorable in vitro cytotoxicity, in vivo antitumor activity, and molecular docking results, compound 4h may have shown its action by attenuating EGFR-associated proliferative signaling, thereby reducing genomic stress and the compensatory activation of DNA damage response pathways. However, direct regulation of these genes by compound 4h was not investigated in the present study and requires further mechanistic validation. Another limitation of the study is that the cytotoxicity of compound 4h was evaluated only against the MCF-7 breast cancer cell line, and the selectivity of the compound toward malignant cells relative to the normal mammary epithelial cells could not be established. Future studies should evaluate cytotoxicity in non-tumorigenic normal mammary epithelial cells, such as MCF-10 A, to determine the selectivity index. Furthermore, MCF-7 cells generally express lower levels of EGFR than some triple-negative breast cancer cell lines, such as MDA-MB-468 and MDA-MB-231. Therefore, the confirmation of EGFR expression in EGFR-overexpressing breast cancer cell lines is warranted. In conclusion, the DPP scaffold may represent a promising structural framework for further anticancer investigations, with compound 4h emerging as the most active derivative. However, advanced mechanistic studies, target-validation assays, stereochemical characterization, quantitative in vivo efficacy assessment, and experimental pharmacokinetic and toxicological evaluation are necessary to determine its selectivity, mechanism of action, and potential for future preclinical development.

Author contributions

A. N.: conceptualization, methodology, project administration, resources, validation, writing – review & editing. M. S. A.: formal analysis, methodology, software, writing – original draft. W. A.: methodology, writing – original draft. K. Z.: methodology, supervision, writing – review & editing. S. M.: data curation, methodology, writing – original draft. N. T.: data curation, methodology, writing – original draft. M. M. E. T.: data curation, methodology, writing – original draft. M. A.: data curation, methodology, writing – original draft. M. A. B.: investigation, methodology, software. A. M.: project administration, software, writing – review & editing. M. A.: project administration, software, writing – review & editing.

Conflicts of interest

None declared.

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information (SI). Should any raw data files be needed in another format, they are available from the corresponding author upon reasonable request. Supplementary information: concentration response curves for derivatives 4a, 4b, 4c, 4d, 4e, 4f, 4i, and gefitinib. See DOI: https://doi.org/10.1039/d6ra06656a.