Design, synthesis, and biological evaluation of KA7, a novel quinolone-based sorafenib analogue with potent anticancer and immunomodulatory activities

Abstract

Although multi-kinase inhibitors like sorafenib remain an important treatment option for advanced malignancies, their toxicity and resistance to treatment are problems. Enhancing its structure by making changes to the scaffold offers a viable way to boost safety and effectiveness. In this study, 2-(4-chlorobenzylamino)-6-(pyridin-4-yloxy) quinolin-4(1H)-one (KA7), a novel sorafenib analogue, is synthesized and biologically evaluated. KA7 was synthesized by replacing the urea component of sorafenib with a quinolone scaffold. Then, KA7 was characterized by NMR and HRMS. MTT tests were used to calculate the IC₅₀ and assess cytotoxicity against breast (MDA-231, MCF-7), glioblastoma (U87), and lung (A549) cancer cells. Flow cytometry was used to evaluate cell death induction (apoptosis/necrosis), cell cycle analysis, and autophagic activity. The analysis of cytokine release in LPS-stimulated THP-1 macrophages allowed for the determination of KA7's immunomodulatory potential. Our results indicated that KA7 exhibited IC₅₀ values lower than those of sorafenib in both U87 and A549 cells, with similar effects observed in MCF-7 cells, demonstrating dose-dependent cytotoxicity. Flow cytometry also revealed a significant increase in apoptosis and G1 phase cell cycle arrest across all cancer cell lines tested. Autophagy induced by KA7 was confirmed through acridine orange staining. In the macrophage model, KA7 increased levels of anti-inflammatory cytokines (IL-8 and IL-10) while decreasing pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6), supporting an immunomodulatory profile akin to that of sorafenib. In conclusion, KA7 exhibits strong anticancer properties, inducing apoptosis, arresting the G1 cell cycle, stimulating autophagy, and displaying positive immunomodulatory effects. According to these results, KA7 is a promising lead chemical for additional preclinical testing as a potent sorafenib substitute.

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Introduction

Cancer remains one of the leading causes of death worldwide, with lung, stomach, liver, colon, and breast cancers accounting for the majority of cancer-related mortality. This urgent global health challenge underscores the critical need for the discovery and development of novel therapeutic agents with improved efficacy and safety profiles.

Sorafenib (Fig. 1) is an FDA-approved oral multikinase inhibitor currently used in the treatment of renal cell carcinoma (RCC), advanced hepatocellular carcinoma (HCC), and thyroid cancer.^1–6 Its therapeutic efficacy arises from its ability to inhibit multiple signaling pathways involved in tumor progression, including vascular endothelial growth factor receptors (VEGFR),^7,8 B-RAF serine/threonine kinase, Ras protein and Ras-mutation^9,10 and mitogen-activated protein kinase (MAPK) pathway.¹¹

Fig. 1 The chemical structure of sorafenib.

Despite its clinical success, sorafenib's broad kinase inhibition is also associated with severe adverse effects such as hand–foot skin reaction, diarrhea, hypertension, and fatigue.¹² Thus, there remains a pressing need for safer and more effective targeted therapies. The elucidation of sorafenib's structure and anticancer properties has spurred efforts to optimize its pharmacological profile and to identify analogues with improved bioavailability, reduced toxicity, and expanded activity against diverse tumor types. Therefore, considerable efforts in organic and medicinal chemistry are directed toward the development of sorafenib-based analogues with improved therapeutic potential against cancer progression.

Most sorafenib analogues retain the central urea moiety, which provides favorable hydrogen-bonding interactions with target kinases but also contributes to aggregation and plasma protein binding, causing cell toxicity and suboptimal pharmacokinetics.¹³ Replacing the urea group with alternative scaffolds is a promising strategy to overcome these limitations, although challenging, as the loss of hydrogen bonding must be compensated by other structural features.

In this context, we designed a novel analogue KA7 in which sorafenib's urea moiety was replaced with a quinolone core. The new structure is a 2-amino-6-phenoxyquinoline that also carries a benzylamine group (Fig. 2).

Fig. 2 Sorafenib and its quinolone analogue KA7.

Quinolines are privileged scaffolds in drug discovery, characterized by a fused benzene–pyridine bicyclic structure that imparts favorable electronic and steric properties. Quinoline derivatives have been widely studied for their diverse pharmacological activities, including antibacterial, antifungal, antiviral, anti-inflammatory, and anticancer effects.^14–16 Importantly, their ability to interfere with key cellular processes relevant to tumorigenesis has made them attractive candidates for anticancer drug development.^17,18

Herein, we report the synthesis of 2-(4-chlorobenzylamino)-6-(pyridin-4-yloxy) quinolin-4(1H)-one (KA7), a sorafenib analogue featuring a quinoline core. We evaluate its cytotoxic effects across a panel of human cancer cell lines, including breast (MDA-231, MCF-7), glioblastoma (U87), and lung (A549), along with normal human dermal fibroblasts (HDF) for selectivity. Furthermore, we investigate its mechanism of action through apoptosis, cell cycle and autophagy assays, as well as its immunomodulatory profile in macrophage models.

Materials and methods

Chemistry

In our present research 2-(4-chlorobenzylamino)-6-(pyridin-4-yloxy)quinolin-4(1H)-one (KA7) was synthesized via the nucleophilic displacement of SMe groups in the starting compound 5-[bis(methylsulfanyl)-methylidene]-2,2-dimethyl-1,3-dioxane-4,6-dione 1 with 4-(pyridin-4-yloxy)benzenamine and (4-chlorophenyl)methanamine, respectively to get intermediate 2. Intermediate 2 is converted to quinoline by heating at 230 °C to give the final product KA7 (Fig. 3).

Fig. 3 General synthesis method of 2-(4-chlorobenzylamino)-6-(pyridin-4-yloxy) quinolin-4(1H)-one (KA7).

Experimental part

Chemicals and reagents used were purchased from Combi-Blocks, Acros Organics and Sigma-Aldrich. All reagents and chemicals used without any further purification.

All NMR spectra were acquired in deuterated DMSO using a Bruker spectrometer at 500 MHz. High-resolution mass spectra (HRMS) were obtained using an electrospray ionization technique on a Bruker Impact II mass spectrometer in positive-ion mode at 2500 V Fig. S1.

High-performance liquid chromatography (HPLC) was performed using a Thermo Fisher Scientific P1000 pump integrated into AS 3000 system, and UV 2000 detector. Separation was achieved on a C18 column (150 mm × 4.6 mm1), mobile phase 80% CH₃OH and 20% Deionized water. The detection wavelength 254 nm, pump flow rate 1 mL min⁻¹ flow rate Fig. S2.

Procedure

2-(4-Chlorobenzylamino)-6-(pyridin-4-yloxy) quinolin-4(1H)-one (KA7). 1 (0.40 g, 1.60 mmol) and 4-(pyridin-4-yloxy) benzenamine (0.33 g, 1.76 mmol) in 10 ml THF was heated at reflux for 1.5 h and then (4-chlorophenyl) methanamine (0.22 ml, 1.76 mmol) was added. The reaction was stirred for an additional 2 h. The THF was evaporated and the crude solid was washed with diethyl ether to yield the intermediate 2. The intermediate 2 was heated in diphenyl ether at 230 °C for 15 minutes. The reaction mixture was cooled to room temperature then the diethyl ether was added. The resulting precipitate was filtered off to give 0.2 g (33%) of KA7 as off-white solid.

¹H NMR (500 MHz, DMSO) δ: 10.92 (s, 1H), 8.43–6.89 (m, 11H), 5.25 (s, 1H), 4.46 (s, 2H)·¹³C NMR (126 MHz, DMSO) δ: 164.97, 151.93, 148.64, 147.92, 131.76 129.41, 128.80, 124.29, 124.21, 124.06, 123.77, 112.37, 110.64, 44.33. HRMS ESI⁺, ([M + H]⁺)m/z: calculated for C₂₁H₁₇ClN₃O₂ 378.1009; found, 378.0990. Fig. S3

Cell culture

MDA-231, MCF-7, U87, A549, and THP-1 cells. All cells were donated from the cell therapy center at the University of Jordan.

MDA-231 and U87 cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) (EuroClone, Pero, Italy), while MCF-7, A549, and THP-1 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (EuroClone, Italy). Both media were supplemented with 10% fetal bovine serum (FBS) (Cytiva, Marlborough, MA, USA), 1% penicillin-streptomycin, 1% l-glutamine, and 0.1% amphotericin B (EuroClone S.p.A., Pero, Italy). Cells were incubated at 37 °C in a humidified incubator with 5% CO₂ (Esco Lifesciences Group, Singapore) and subcultured upon reaching 70–90% confluence using trypsin-EDTA 1× in PBS (EuroClone, Pero, Italy).

MTT assay-IC₅₀

5000 cells per well of the cell lines, MDA-231, MCF-7, U87, and A549, were cultured in a 96-well plate, and were treated with a serial dilution ranging from 50 to 1.5 µM to determine the IC₅₀. Following 72 hours of treatment, 15 µL of MTT reagent was added to each well. The plate was incubated at 37 °C for 3 hours. Later, 50 µL of the stop solution was added to each well. Absorbance was measured at 570 nm using a microplate reader (Thermo Fisher Scientific, USA).

For the calculation of IC₅₀ values, nonlinear regression analysis was performed using GraphPad Prism version 8.4, with logarithmic concentration plotted against viability percentage values.

Cell death modality (apoptosis/necrosis assay)

To evaluate the impact of KA7 on cell death, apoptosis, and necrosis were assessed in MDA-231, MCF-7, U87, and A549 cells, with Sorafenib used as a positive control. Initially, cells were seeded at a density of 250 000 cells per well in 6-well plates (SPL Life Sciences, Pocheon, Korea) and treated with KA7 and sorafenib at the IC₅₀ and double the IC₅₀ concentrations for 72 hours. Following treatment, cells were harvested using 1× trypsin (EuroClone S.p.A., Pero, Italy), collected by centrifugation. Following, cells were stained with Annexin V and propidium iodide (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol and analyzed using BD Accuri™ C6 Plus flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) using FL1 filter for Annexin V signal detection and FL2 filter for PI signal detection.

Cell cycle assay

To evaluate the impact of KA7 on cell cycle progression, initially, MDA-231, MCF-7, U87, and A549 cells were seeded at a density of 250 000 cells per well in 6-well plates (SPL Life Sciences, Pocheon, Korea) and treated with KA7 and sorafenib at the IC₅₀ and double the IC₅₀ concentrations for 72 hours. Following treatment, cells were harvested using 1× trypsin (EuroClone S.p.A., Pero, Italy), collected by centrifugation. The collected samples for the cell cycle assay were then fixed in 500 µL of cold methanol and stored at −20 °C until processing the samples. Next, samples were washed twice with PBS, resuspended in 50 µL PBS, and incubated with 150 µL of an RNase A/propidium iodide (PI) solution (NZYTech, Lisbon, Portugal; Tocris Bioscience, Bristol, UK) at final concentrations of 0.2 mg mL⁻¹ and 0.04 mg mL⁻¹, respectively. Samples were incubated in the dark for 30 min, followed by the addition of 100 µL PBS. Flow cytometry analysis was performed using BD Accuri™ C6 Plus using FL2 filter, and data were processed using FlowJo software version 10 (BD Biosciences, Franklin Lakes, NJ, USA).

Acridine orange (AO) staining

Based on fluorescence emission, AO staining was performed to assess cellular changes following treatment MDA-231, MCF-7, U87, and A549 cells were seeded at a density of 120 000 cells per well in a 12-well plate and allowed to adhere overnight. Cells were then treated with IC₅₀ concentrations of KA7 for 72 hours. After incubation, cells were washed with PBS, harvested, and collected by centrifugation, and the supernatant was discarded. The cell pellet was resuspended in 10 µM AO and incubated for 15 minutes in a dark place. Following staining, excess AO was removed by washing with PBS, and the cells were centrifuged and resuspended in 250 µL of PBS. Fluorescence analysis was performed using BD Accuri™ C6 Plus flow cytometry, with FITC and PerCP filters. Data analysis was conducted using BD Accuri™ software.

Inflammatory response

Differentiation and stimulation of THP-1 cells. THP-1 cells were seeded at a density of 1 × 10⁶ cells per well in a 6-well plate and maintained in RPMI medium (EuroClone, Italy) supplemented with 150 nM phorbol 12-myristate 13-acetate (PMA) (ChemCruz, Santa Cruz Biotechnology, Dallas, TX, USA) to promote differentiation into macrophage-like cells. Following a 24-hour incubation, the differentiated macrophages were further stimulated with 1 µg per mL lipopolysaccharide (LPS) (ChemCruz, Santa Cruz Biotechnology, Dallas, TX, USA) for an additional 24 hours to induce an inflammatory response. Cells were subsequently treated with 10 µM KA7 and 10 µM sorafenib, then incubated at 37 °C in a CO₂ incubator for 24 hours.

Cytokine detection using cytometric bead array (CBA). At the end of the treatment period, culture supernatants were collected and stored at −20 °C for cytokine profiling. The levels of IL-1β, IL-6, IL-8, IL-10, IL-12p70, and TGF-β were evaluated using the cytometric bead array (CBA) kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. Flow cytometric analysis was conducted using the BD Accuri™ C6 Plus flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) using PE filter. Cytokine concentrations were determined by interruption from a standard curve made with a freshly prepared cytokine bead mixture. The results were expressed as cytokine concentration (pg mL⁻¹) relative to the untreated control.

Statistical analysis. All statistical analyses were done using GraphPad Prism software. All experiments were done in triplicate in three independent experiments (n = 3). Data are presented as mean ± standard deviation (SD). Two-way ANOVA was used to evaluate apoptosis and cell cycle assays, while one-way ANOVA was used for Acridine orange and CBA assays, and the Dunnett Test for multiple comparisons between treated groups and controls for all assays.

Statistical significance was stated as *p < 0.05.

Results

Comparative cytotoxicity of KA7 and sorafenib across cancer cell lines

The MTT assay was employed to assess the inhibitory effect of the KA7 compound on various cancer cell lines. After 72 hours of treatment, the IC₅₀ values for both KA7 and sorafenib (SOR) were determined using the MTT assay. The results are presented in Fig. 4. As shown in Fig. 4, the IC₅₀ of MDA-231, U87, and A549 cells treated with KA7 was lower compared to their treatment with sorafenib. The IC₅₀ results were (MDA-231:6.70 ± 0.78, U87:5.40 ± 0.75, and A549:4.70 ± 0.66) cells treated with KA7 compound, while the IC₅₀ for cells treated with sorafenib were as follows: (MDA-231:9.40 ± 1.03, U87:15 ± 1.10, and A549:11 ± 1.30). Though MCF-7 treatment showed comparable IC₅₀ values, as IC₅₀ was 9.30 ± 0.97 for cells treated with KA7 and 8.50 ± 1.30 for cells treated with sorafenib.

Fig. 4 IC₅₀ values for the cell viability after 72 hours of treatment with the KA7 compound and sorafenib (SOR). Nonlinear regression analysis was performed to calculate IC₅₀ values, using GraphPad Prism version 8.4, with logarithmic concentration plotted against viability percentage values.

Cell death modality (apoptosis/necrosis assay)

To investigate the impact of KA7 on cell death modality, MDA-231, MCF-7, U87, and A549 were treated with IC₅₀ and double IC₅₀ concentrations of KA7 or sorafenib, then assessed using an Annexin V/PI staining assay after 72 hours of treatment.

Our results demonstrated that, across all cell lines, there was a significant increase in the percentage of Annexin V-positive cells compared to the control untreated groups (p < 0.05) in all treated cell lines. The IC₅₀ results for the percentage of Annexin V-positive cells were as follows: for MDA-231:39.06 ± 263, MCF-7: 6.14 ± 2.14, U87:3.30 ± 0.30, and A549:16.90 ± 1.89. For the double IC₅₀, the results were MDA-231:36.23 ± 1.60, MCF-7: 11.98 ± 0.98, U87:7.70 ± 3.30, and A549 47.16 ± 3.80.

In the control untreated cells, the percentages of Annexin V-positive cells were as follows: MDA-231:21.11 ± 1.61, MCF-7: 3.18 ± 0.56, U87:1.06 ± 0.09, and A549:6.24 ± 0.42. For the PI-positive cells, only MDA-231 and MCF-7 cells treated with KA7 exhibited a significant increase in the percentage of PI-positive cells compared to the control untreated cells (p < 0.05). Consequently, these results indicate that KA7 induced apoptotic cell death in the treated cells.

Similarly, the sorafenib-treated cells showed a highly significant increase in both Annexin V-positive and PI-positive cells among all cell lines treated. The IC₅₀ results for Annexin V-positive cells were MDA-231:86.30 ± 0.63, MCF-7: 2.40 ± 0.40, U87:3.30 ± 0.37, and A549:77.76 ± 2.16. For the PI-positive cells, the results were MDA-231:5.80 ± 0.70, MCF-7: 12.13 ± 2.51, U87:1.96 ± 0.40, and A549:20.40 ± 2.10.

Similar trends were observed in cells treated with double IC₅₀ for Annexin V-positive cells (MDA-231:80.30 ± 0.15, MCF-7: 13.00 ± 2.17, U87:7.3 ± 0.30, and A549:66.09 ± 2.06) and PI-positive cells (MDA-231:12.30 ± 0.37, MCF-7: 32.70 ± 2.52, U87:9.80 ± 1.10, and A549 29.10 ± 1.87), as shown in Fig. 5.

Fig. 5 (A) Flow cytometric analysis and (B) Flow cytometric dot plots of the percentages of healthy and dead cells (apoptotic/necrotic cells) of MDA-231, MCF-7, U87, and A549 cells following 72 hours of treatment with KA7 and sorafenib (SOR) at IC₅₀ and double the IC₅₀ concentrations (µM) compared to untreated controls. Data are presented as mean ± SD. Statistical significance was determined using two-way ANOVA (*p < 0.05).

Cell cycle assay

To investigate the effect of KA7 on cell cycle progression, MDA-23, MCF-7, U87, and A549 cells were treated with IC₅₀ and double IC₅₀ concentrations of KA7 and sorafenib for 72 hours.

Our results demonstrated that both KA7 and sorafenib caused an arrest in the G1 phase of treated cells, resulting in a significant reduction in the percentage of cells progressing to the G2 phase across all cell lines.

The results for KA7 at IC₅₀ were as follows: for MDA-231 cells, the percentage was 76.29 ± 1.33; for MCF-7 cells, it was 79.50 ± 0.93; for U87 cells, it was 85.5 ± 0.50; and for A549, it was 58.70 ± 1.93. For cells treated with double KA7 IC₅₀, the percentages were: MDA-231:83.30 ± 3.0, MCF-7: 87.58 ± 0.58, U87:85.50 ± 0.58, and A549:90.40 ± 0.40. In contrast, the control group of untreated cells showed the following percentages: MDA-231 at 36.51 ± 3.30, MCF-7 at 41.12 ± 1.49, U87 at 46.30 ± 3.50, and A549 at 67.70 ± 2.29.

Similarly, G1 arrest was observed in cells treated with sorafenib at IC₅₀, with results as follows: MDA-231 at 89.20 ± 7.87, MCF-7 at 87.90 ± 1.53, and U87 at 66.17 ± 1.10. For double IC₅₀ doses of sorafenib, the results were: MDA-231 at 92.30 ± 1.36, MCF-7 at 85.40 ± 4.14, and U87 at 69.02 ± 1.02. However, in the case of A549 cells treated with IC₅₀ and double IC₅₀ of sorafenib, the cells were arrested in the G2 phase, and the percentages of cells in the G1 and S phases decreased significantly, as illustrated in Fig. 6.

Fig. 6 (A) Cell cycle analysis and (B) cell cycle flow cytometric histograms of MDA-231, MCF-7, U87, and A549 cells following 72 hours of treatment with KA7 and sorafenib (SOR) at IC₅₀ and double the IC₅₀ concentrations (µM) compared to untreated controls. Data are presented as mean ± SD. Statistical significance was determined using two-way ANOVA (*p < 0.05).

Acridine orange (AO) staining

To assess autophagic activity, cells were treated with IC₅₀ values of the KA7 compound for 72 hours. Following the treatment, the cells were stained with acridine orange to evaluate autophagic activity.

The results indicated a significant increase in the percentage of acridine orange-positive cells across all cell lines. Specifically, the percentages were: MDA-231:25.70% ± 4.04, MCF-7: 23.03% ± 3.60, U87:13.40% ± 2.20, and A549:33.32% ± 1.15. These values were notably higher when compared to the control untreated groups, which showed the following percentages: MDA-231:3.30% ± 0.40, MCF-7: 1.963% ± 0.55, U87:1.90% ± 0.43, and A549:4.96% ± 1.41. The findings are illustrated in Fig. 7.

Fig. 7 (A) Acridine orange analysis and (B) acridine orange flow cytometric results of treated MDA-231, MCF-7, U87, and A549 cells for 72 hours with KA7 and sorafenib (SOR) at IC₅₀ concentrations (µM) compared to untreated controls. Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA (*p < 0.05).

Inflammatory response

To evaluate the inflammatory responses of the KA7 compound, THP-1 cells were infected with lipopolysaccharide (LPS) and treated with 10 µM of KA7 or sorafenib. The secretion levels of cytokines were measured using multiplex flow cytometry.

The results indicated a decrease in the levels of pro-inflammatory cytokines, including IL-1β, TNF-α, and IL-6, in cells treated with KA7 and sorafenib (SOR). The results were as follows: IL-1β: Control: 235.99 ± 36.50 KA7: 14.77 ± 1.04, SOR: 2.76 ± 2.77 pg ml⁻¹. IL-6: Control: 936.35 ± 117, KA7: 176.32 ± 12.80, SOR: 28.4 ± 4.16 pg ml⁻¹. TNF-α: Control: 268.05 ± 38.04 pg ml⁻¹, KA7: 4.06 ± 0.05 pg ml⁻¹, SOR: 4.70 ± 0.46 pg ml⁻¹.

Additionally, the secretion levels of anti-inflammatory cytokines IL-10 and IL-8 showed a significant increase (p < 0.05). The levels were as follows: IL-10:Control: 0.70 ± 0.11, KA7: 4.26 ± 0.12, SOR: 5.39 ± 0.14 pg ml⁻¹. IL-8:Control: 680.14 ± 121.40, KA7: 755.48 ± 25.40 pg ml⁻¹, SOR: 1363.03 ± 62.60 pg ml⁻¹. Whereas for IL-12p70 no statistical difference was observed among all treatment groups: IL-12p70: Control: 7.42 ± 1.94, KA7: 7.69 ± 0.06 pg ml⁻¹, SOR: 7.84 ± 0.064 pg ml⁻¹.

Consequently, these results indicate the anti-inflammatory potential of the KA7 compound, which is comparable to the effects of sorafenib, as illustrated in Fig. 8.

Fig. 8 Inflammatory response of macrophage cells, activated with LPS and treated with 10 µM KA7 and 10 µM sorafenib (SOR) for 72 hours, and compared to the control untreated. Measurement of the secretion level of cytokines at the protein level, measured by cytometric bead array (CBA)-flow cytometry. A One-way ANOVA test was used for statistical analysis. Bars represent means ± SD (*p < 0.05).

Discussion

In this study, a novel sorafenib analogue KA7 was synthesized and characterized, in which the urea moiety was replaced with a quinolone scaffold. KA7 demonstrated potent cytotoxic effects across multiple cancer cell lines, including breast (MDA-231, MCF-7), glioblastoma (U87), and lung (A549). Notably, KA7 was approximately twice as potent as sorafenib against U87 cells and three times more potent against A549 cells. These findings suggest that KA7 retains broad anticancer activity similar to sorafenib, while showing enhanced potency in selected cancer models.

In line with our strategy of replacing sorafenib's aryl urea moiety, other studies have explored alternative scaffolds such as 1,2,3-triazoles. For example, a series of triazole-containing sorafenib analogues, in which the urea group was substituted by a 1,2,3-triazole ring linking a phenoxy fragment, was synthesized via Huisgen 1,3-dipolar cycloaddition and nucleophilic aromatic substitution.¹⁹ Cytotoxicity studies against hepatocellular carcinoma cell lines revealed that most analogues showed only moderate to weak activity against HepG2 cells (best IC₅₀ = 61.6 ± 5.2 µM). However, several compounds displayed notable inhibitory activity against Huh7 cells, with the p-tert-butylphenoxy analogue achieving significant potency (IC₅₀ = 5.67 ± 0.57 µM). Importantly, this compound demonstrated a high selectivity index (SI > 17.6), which was markedly superior to that of sorafenib (SI = 6.73), underscoring the value of aryl urea replacement strategies in generating analogues with improved potency and safety profiles.

Similar efforts have also focused on replacing sorafenib's aryl urea moiety with pyrazole scaffolds. In one study, four series of pyrazole-containing sorafenib derivatives were synthesized and tested for cytotoxicity against A549, HepG2, MCF-7, and PC-3 cancer cell lines. Among them, one compound, bearing a 3-bromo substitution at the C-3 position of the pyrazole ring, emerged as the most potent. It exhibited IC₅₀ values of 2.84 ± 0.78 µM (A549), 1.85 ± 0.03 µM (HepG2), and 1.96 ± 0.28 µM (MCF-7), which were comparable to or better than sorafenib.²⁰ The superior cytotoxicity of this compound compared to unsubstituted derivatives suggests that C-3 substitutions play a critical role in the structural optimization of sorafenib analogues. In addition to triazole- and pyrazole-based analogues, chalcone derivatives of sorafenib have also attracted significant attention due to their broad biological activities.^21–23 Wang et al. reported that replacing the aryl urea group with a chalcone moiety produced derivatives with equal or superior potency to sorafenib against A549, HepG2, MCF-7, and PC-3 cell lines, including one compound with sub-micromolar activity against HepG2 (IC₅₀ = 0.56 ± 0.83 µM).²⁴ Collectively, these findings highlight that replacing the aryl urea moiety with alternative scaffolds, such as pyrazole, triazole, or chalcone rings, can enhance antitumor potency. Our quinolone-based analogue KA7 follows this rationale, demonstrating improved activity against selected cancer cell lines and reinforcing scaffold engineering as a promising strategy for optimizing sorafenib analogues. Substituting sorafenib's urea moiety with a quinolone framework represents a rational approach to address the pharmacokinetic and toxicity limitations of urea-containing kinase inhibitors, while preserving potency and, in some cases, enhancing activity against specific cancer types.

Flow cytometry analysis demonstrated that KA7 induced significant apoptosis across all tested cancer cell lines, as evidenced by increased Annexin V-positive populations. This aligns with sorafenib's established mechanism as a multikinase inhibitor, which promotes apoptosis through inhibition of the Raf–MEK–ERK and mTOR pathways, downregulation of anti-apoptotic proteins such as Mcl-1 and survivin, and upregulation of pro-apoptotic mediators like Redd1.^25–27 Sorafenib has also been reported to trigger mitochondrial fragmentation, which facilitates cytochrome c release and activates caspase-dependent apoptosis.^28,29 Several sorafenib analogues similarly promote apoptotic cell death through related mechanisms.^30–33 In addition, KA7 induced a marked arrest in the G1 phase of the cell cycle, thereby limiting progression to the G2/M phase. Notably, sorafenib has been shown to cause G2 arrest in A549 cells, whereas KA7 predominantly induced G1 arrest, suggesting subtle mechanistic distinctions between the two compounds. Future studies should investigate the involvement of key cell cycle regulatory proteins such as cyclin D1, CDK4/6, p21, and p27, which could help clarify the molecular basis of KA7-induced G1 arrest.

In addition to apoptosis and cell cycle arrest, our findings revealed that KA7 also triggered autophagy, as demonstrated by acridine orange staining. Autophagy is a double-edged process in cancer: while it can function as a pro-survival mechanism under stress, excessive activation may lead to autophagic cell death.³⁴ Consistent with our observations, sorafenib has been widely reported to induce autophagy in cancer cells.^35–38 For example, in prostate cancer, sorafenib-induced formation of Atg5-deficient autophagosomes promoted the interaction of p62 with RIPK, leading to necroptotic cell death,³⁶ whereas in renal cancer cells, sorafenib activated a cytoprotective form of autophagy that interfered with the effectiveness of sorafenib-mediated apoptosis.³⁵ These contrasting reports underscore the context-dependent role of autophagy in cancer therapy. Our data suggest that KA7 shares this autophagy-inducing property, but further studies are needed to determine whether KA7-mediated autophagy enhances cell death or contributes to adaptive resistance. Future investigations should examine the expression of key autophagy regulators such as LC3-II, Beclin-1, and p62/SQSTM1 to elucidate the molecular mechanisms underlying KA7-induced autophagy.

An important observation of this study was the immunomodulatory activity of KA7. In LPS-stimulated THP-1 macrophages, both KA7 and sorafenib significantly reduced the secretion of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) while enhancing anti-inflammatory mediators (IL-8 and IL-10). These effects are consistent with previous reports describing sorafenib's ability to modulate the tumor microenvironment and immune signaling.^39–41 Elevated TNF-α levels have been implicated in driving epithelial–mesenchymal transition and promoting sorafenib resistance in hepatocellular carcinoma cells.⁴² The ability of KA7 to downregulate TNF-α may therefore represent an important mechanism for overcoming resistance and improving therapeutic outcome. By combining this anti-inflammatory activity with direct cytotoxic effects, KA7 could reduce tumor-promoting inflammation while reinforcing antitumor immunity. This dual mechanism underscores KA7 as a promising candidate for incorporation into combination strategies with immunotherapeutic agents.

All of these findings point to KA7 as a viable structural scaffold inspired by sorafenib that needs more refinement. Although KA7 showed increased potency in some cell lines, these findings should be considered model-dependent and preliminary rather than a sign of widespread therapeutic superiority. As a result, KA7 represents a lead molecule for further mechanistic characterization, in vivo validation, and structure–activity refinement.

Furthermore, given its immunomodulatory profile, KA7 should be investigated in tumor-immune co-culture systems and in vivo models that better reflect the complexity of the tumor microenvironment.

In conclusion

The effective synthesis of KA7, a quinolone-based analogue of sorafenib, is highlighted in this paper. It showed improved cytotoxicity against a number of cancer cell lines, including breast cancer, lung cancer, and glioblastoma cell models. Its capacity to trigger autophagy, induce apoptosis, and cause G1 phase cell cycle arrest was validated by mechanistic investigations. Crucially, KA7 had an immunomodulatory profile, lowering pro-inflammatory cytokines and increasing anti-inflammatory mediators, which may have both immunoregulatory and anticancer effects. All things considered, these results indicate that replacing the urea moiety with a quinolone scaffold yields KA7 as a promising structural analogue rather than a confirmed superior therapeutic alternative to sorafenib. KA7 should therefore be viewed as a lead candidate for further optimization and preclinical evaluation, with particular focus on improving selectivity, clarifying target engagement, and determining its translational potential.

Author contributions

D. A. designed and planned the experiments. A. A. worked out the chemistry part. M. M., H. K., and N. A. worked out the biological part. A. H. took the lead in writing the manuscript. All authors provided critical feedback and helped shape the research, analysis, and manuscript.

Conflicts of interest

The authors declare no conflict of interest.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d5ra07478a.

Acknowledgements

The authors would like to express their gratitude to the University of Petra for support and funding (research no. 20/4/2024).

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