Hybrid chalcone-pyrazoline derivatives: synthesis and in silico evaluation for anticancer potential

ABSTRACT

Aim

This study aimed to design, synthesize, and evaluate a hybrid chalcone-pyrazoline derivatives as potential anticancer agents targeting B-Raf kinase in lung cancer.

Material & Methods

Chalcone–pyrazoline derivatives (PY1–PY10) were synthesized via Claisen–Schmidt condensation followed by cyclization, characterized using FT-IR, NMR, and LC-MS. In vitro cytotoxic activity was assessed against A549 human lung cancer cells using the MTT assay. Molecular docking studies were performed with B-Raf kinase (PDB ID: 2FB8) using Schrödinger software. ADME properties were predicted using SwissADME.

Result

Compound PY7 exhibited the most potent cytotoxicity (IC₅₀ = 6.45 µM) and the highest docking score (−8.89 kcal/mol), showing strong binding interactions with GLN530 in B-Raf kinase. Structure Activity Relationship analysis revealed that electron-withdrawing para-nitro substituents enhanced potency, while electron-donating groups generally reduced activity. ADME profiling confirmed all compounds complied with Lipinski’s Rule of Five, had high gastrointestinal absorption, and displayed favorable drug-likeness.

Conclusion

The findings identify PY7 as a promising lead candidate with potent anticancer activity, strong B-Raf binding affinity, and favorable pharmacokinetics. This work supports chalcone–pyrazole scaffolds as viable templates for the development of novel targeted lung cancer therapeutics.

GRAPHICAL ABSTRACT

1. Introduction

Cancer is a heterogeneous group of diseases in which the cells that grow abnormally are out of control and have the potential of invading the neighboring tissues and, in many instances, invade other tissues in other body organs through a process called metastasis [1]. It is the consequence of genetic and epigenetic changes that interrupt ordinary cellular regulatory networks, such as cell growth, apoptosis, and differentiation. These changes are usually caused by an interaction between environmental exposures, lifestyle, and hereditary genetic disorders [2]. Cancer is still among the predominant morbidity and mortality causes in the whole world, and thus this presents a considerable burden to care systems [3]. The International Agency for Research on Cancer (IARC) estimates released in GLOBOCAN 2024 show that the world experienced around 20.6 million new cancer cases and 9.9 million cancer deaths [4]. The incidence and mortality rates continue to rise, primarily due to the aging population, lifestyle changes, and exposure to carcinogenic factors [4,5]. The highest prevalence of detected cancers projected for the near future, specifically in 2024, includes lung cancer (12.4%), breast cancer (11.7%), colorectal cancer (9.6%), prostate cancer (7.3%), and stomach cancer (5.6%). Lung cancer is the leading cause of cancer deaths, accounting for approximately 18.7% of total cancer fatalities, followed by colorectal cancer at 9.3% and liver cancer at 7.7% (Figure S1) [6]. It is estimated that by 2040, the number of new cancer cases worldwide will reach 28.4 million annually, representing a 37% increase compared to 2024 [7].

Lung cancer is one of the most aggressive malignancies and is considered the top cause of cancer-related deaths all over the world [8,9]. Lung cancer primarily originates from the epithelial cells of the respiratory tract and can be classified into two main categories: non-small cell lung cancer and small cell lung cancer (SCLC), which is characterized by rapid growth and early metastasis (Figure S2) [10]. Tobacco smoking is significantly associated with the development of lung cancer; however, other factors such as environmental exposures, genetic predispositions, and occupational carcinogens also play important roles in its development [11]. Regardless of the progress made in diagnosis methods and treatment measures, which are the targeted therapies and the immunotherapies, lung cancer is commonly diagnosed in its aggressive outfit, leading to a poor overall prognosis [12,13]. The identification of the disease at the early stage, the individualized approach to treatment and the continuous study of the molecular mechanisms will help increase the chances of survival and quality of life of people with diseases [14,15].

BRAF stands for v-Raf murine sarcoma viral oncogene homolog B. The BRAF gene, which is related to the serine/threonine kinase protein, regulates cell growth, differentiation, and survival through regulating the MAPK/ERK (Mitogen-Activated Protein Kinase/Extracellular Signal Regulated Kinase) pathway [16,17]. In BRAF, the acquisition of specific mutations contributes to activation of the pathway in the absence of ligand, and this is the V600E mutation that becomes active, leading to unregulated cell growth and oncogenesis [18]. The mutations are predominant in various cancers, including melanoma, colorectal cancer, thyroid cancer, and non-small cell lung cancer, making these cancers prime molecular targets for treatment. The design of selective BRAF inhibitors (e.g., Vemurafenib and Dabrafenib) has resulted in great benefits for the outcomes of patients with BRAF-mutant tumors [19–21]. Nonetheless, acquired resistance is one of the most pressing clinical challenges that may require a combination treatment, including BRAF and MEK (Mitogen-Activated Protein Kinase) inhibitors, to increase its efficacy and evade resistance. Subsequently, BRAF continues to be a key focus in targeted cancer treatment and precision cancer medicine [22]. Various BRAF inhibitors were reported and studied during the last decade, and they are mostly used to treat cancers that contain activating BRAF gene mutations, and in particular, the V600E mutation and the recently developed BRAF inhibitors as mentioned in Figure 1. Vemurafenib is the first BRAF selective inhibitor to be used clinically on patients with metastatic melanoma [23]. This is followed by dabrafenib, which debases BRAF V600E as well as BRAF V600K mutations and has been approved to be used in melanoma, non-small cell lung cancer (NSCLC), and anaplastic thyroid cancer, especially when used together with the MEK inhibitor trametinib to enhance the outcome of clinical conditions and reduce resistance. Encorafenib is yet another important breakthrough, and it is granted in combination with binimetinib in patients with metastatic melanoma [24]. These are first-generation inhibitors that can actively suppress the monomeric version of the mutant BRAF protein. Second-generation and experimental BRAF inhibitors have been reported to overcome the constraints imposed by the development of acquired resistance and other side effects like paradoxical activation of the MAPK pathway in wild-type BRAF cells [25]. They encompass PLX8394, a so-called paradigm shifter that inhibits mutated BRAF dimers but not wild-type BRAF, and LXH254, a pan-RAF (pan–Rapidly Accelerated Fibrosarcoma) with potent BRAF and CRAF (Cellular Rapidly Accelerated Fibrosarcoma) isoform inhibition and more general applicability in RAF-driven tumors [26]. In other related compounds, TAK-580 (MLN2480) and RAF709 are being tested in clinical trials to overcome resistance and have an effect on cancers with BRAF mutations or the related RAS (Rat Sarcoma) pathway mutations [27]. Additional novel agent CCT196969 has been studied to be more selective, possess dual inhibition of RAF and MEK or improve pharmacodynamic properties [28].

Figure 1.

Structures of reported BRAF inhibitors.

The BRAF pathway is a critical part of a signaling pathway known as the Mitogen-Activated Protein Kinase signaling pathway, whose key role is to control the proliferation, differentiation, metabolism, and survival of cells [29]. The route is prompted when growth factors in the extracellular space attach to the tyrosine kinase receptors (e.g., the epidermal growth factor receptor, EGFR), which activate the small GTPase RAS. RAS, on activation, acts on the RAF family kinase that consists of ARAF (v-Raf murine sarcoma 3611 viral oncogene homolog A), CRAF, and most prominently BRAF [30,31]. Of these, one that is often mutated in a variety of human cancers is BRAF, especially the V600E mutation, which leads to a constitutively active kinase without dependence on upstream stimulus [32]. Phosphorylated and activated BRAF causes the phosphorylation of MEK, leading to the phosphorylation of ERK (Extracellular Signal-Regulated Kinase) [33]. The activated ERK translocates to the nucleus and controls the expression of genes controlling cell cycle events, cell survival, apoptosis, and drug resistance [34,35]. Simultaneously, EGFR may also stimulate the PI3K/AKT/mTOR pathway, which also makes an important contribution to metabolism and survival of cells, as shown in Figure S3 [36]. Dysregulation of the BRAF-MEK-ERK signaling pathway, particularly by oncogenic BRAF mutations, results in unregulated cell growth, which contributes to the pathogenesis of many malignancies such as melanoma, colorectal cancer, and non-small cell lung cancer [37].

Pyrazole derivatives have emerged as an important class of heterocyclic compounds in medicinal chemistry due to their broad spectrum of pharmacological activities, particularly their promising anticancer potential [38]. A flexible pyrazole core, a five-membered nitrogen-containing heteroaromatic ring with two adjacent nitrogen atoms, provides a platform to use in structural modification, whereby physicochemical and biological properties can be fine-tuned. It has been extensively shown that pyrazole-based compounds can be effective in blocking important molecular pathways found in tumor progression such as kinases, tubulin polymerization, and other enzymes in the regulation of cell cycle and apoptosis. They are also structurally versatile, which makes them able to accommodate various sets of substituents to increase modifications to the target selectivity and binding affinity [39]. Also, validating this scaffold as a privileged structure in anticancer drug design are crizotinib and celecoxib analogs that have demonstrated high efficacy in a variety of malignancies and are clinically relevant pyrazole derivatives. In addition to that, pyrazoles exhibit favorable pharmacokinetic characteristics that entail good oral absorption, bioavailability, and metabolic stability essential in therapeutic use. This property to activate various signaling pathways that include MAPK/ERK, PI3K/AKT, and apoptotic cascades highlights the prospects of pyrazole derivatives as multi-target anticancer drugs. As a result, pyrazole-derived molecule discovery and optimization has remained a strategic direction about new, efficacious, and selective anticancer therapeutics [40].

1.1. Rationale

The design of the novel N-acetyl substituted and unsubstituted chalcone–pyrazole derivatives in this study was driven by the need to develop potent and selective anticancer agents targeting B-Raf kinase, a key oncogenic protein in the MAPK/ERK signaling pathway as shown in Figure 2. Structure–Activity Relationship (SAR) insights guided the incorporation of specific substituents to enhance binding affinity and pharmacological activity. Electron-withdrawing groups, such as p–NO₂ at the R position, were introduced to improve potency by facilitating polar interactions and stabilizing the ligand–protein complex. Conversely, electron-donating groups (–NH₂, –OCH₃) were found to reduce activity in most cases, likely due to increased electron density that disrupts optimal binding conformation and reduces membrane permeability. Small, lipophilic groups such as cyclopropyl at R₁ enhanced hydrophobic interactions, improving cellular uptake and potency. N-acetylation was employed to modulate electronic effects, potentially increasing ring stability and bioactivity, as observed in compound PY5. The most potent compound, PY7, combined a para-nitro substitution with an unsubstituted pyrazole nitrogen, allowing greater basicity and electron richness, which may have contributed to stronger B-Raf binding. This rational design approach integrates physicochemical optimization, SAR findings, and target-based docking insights to produce molecules with high anticancer potential and favorable drug-like properties.

Figure 2.

Rationale behind the synthetic proposed compounds.

1.2. Experimental

The experiments were performed with oven dried glassware and dry, purified solvents. Most of the chemicals were sourced from CDH, LOBA, Avra, and Sigma Aldrich. All the chemicals used in the synthesis were laboratory and analytical reagent grades. The reaction progress was tracked through TLC on pre coated silica plates (Merck Silica gel F254) using n-hexane and ethyl acetate, chloroform-methanol solvent system. Compounds were recrystallized using ethanol, acetone, and isopropyl alcohol. The melting points of the compounds were determined using melting point apparatus. IR spectra were recorded using Thermo-scientific FT-IR spectrophotometer with potassium bromide pellets. ¹H-NMR and ¹³C-NMR spectra were recorded using 500-MHz: Bruker Ascend 500 from DST-FIST 500 MHz NMR Facility, Department of Chemistry, Indian Institute of Technology, Indore, with CDCl₃ solvents. Chemical shifts were measured in parts per million (ppm) relative to tetra methyl silane (TMS) used as an internal standard and coupling constant (J) values were reported in Hertz (Hz). Mass spectrometry data were obtained from LC-MS (APCI) spectrometry from SAIF, Panjab University, Chandigarh, Punjab. India. SAIF, Panjab University, Chandigarh, Punjab, India conducted the elemental analysis of the synthesized compounds.

1.3. Synthesis of substituted chalcone derivatives (3)

The synthesis of substituted chalcones is commonly carried out through the Claisen–Schmidt condensation reaction, which involves the base-catalyzed condensation of a substituted aromatic aldehyde (1) with a substituted aromatic ketone (2) (Figure 3A,B). In a typical procedure, equimolar amounts of a substituted acetophenone (1 mmol) and a substituted benzaldehyde (1 mmol) are dissolved in ethanol. To this mixture, an aqueous solution of sodium hydroxide (20%) is added dropwise with continuous stirring. The reaction mixture is stirred at room temperature for 12–24 h. The progress of the reaction is monitored using TLC. Upon completion, the reaction mixture is poured into ice-cold water to precipitate the chalcone product. The resulting solid is filtered, washed with cold water, and dried. The crude chalcone is further purified by recrystallization using solvents like ethanol or ethyl acetate.

Figure 3.

Synthetic scheme for the synthesis of: (A) N-acetyl substituted pyrazoline derivatives PY(1–5); (B) unsubstituted pyrazoline derivatives PY(6–10).

1.4. Synthesis of N-acetyl pyrazole derivatives ((PY(1–5))

Hydrazine hydrate (5 mmol) was added to a solution of chalcone (3) (1 mmol) in acetic acid (8–10 ml) in a dry round-bottom flask (Figure 3A). The reaction mixture was refluxed for approximately 6–10 h. The progress of the reaction was monitored using TLC (n-hexane: ethyl acetate, 6:4). After the completion of the reaction, the reaction mixture was cooled, poured into crushed ice, and allowed to stand overnight at room temperature.

1.5. Synthesis of unsubstituted pyrazole derivatives ((PY(6–10))

Hydrazine hydrate (5 mmol) was added to a solution of chalcone (1 mmol) in ethanol (10 ml) in a dry round-bottom flask (Figure 3B). The reaction mixture was refluxed for approximately 6 h. The progress of the reaction was monitored using TLC (n-hexane: ethyl acetate, 6:4). After the completion of the reaction, the reaction mixture was cooled, poured into crushed ice, and allowed to stand overnight at room temperature. All synthesized compounds are given in Table 1.

Table 1.

Synthesized new chalcone derived pyrazole derivatives.

Compound	R	R1
PY1
PY2
PY3
PY4
PY5
PY6
PY7
PY8
PY9
PY10

1.6. Physicochemical properties

The physicochemical properties of synthesized compounds are shown in Table 2. All compounds were obtained in good percent yield that indicate the suitability of synthetic procedures. Compounds such as PY3, PY4, PY6, and PY7 were obtained in high yield (70–80%) compared to that of other compounds in the series.

Table 2.

Physicochemical characteristics of the final compound PY(1–10).

Comp.	Mol. formula	Color	M.P. (°C)	Rf	% yield
PY1	C19H21N3O3	Yellow	133–135	0.6	62
PY2	C19H19N3O5	Pale yellow	131–133	0.3	67
PY3	C20H23N3O4	Light yellow	141–143	0.5	73
PY4	C19H17N3O3	Brown	138–140	0.4	75
PY5	C19H21N3O3	Pale yellow	135–137	0.4	63
PY6	C18H21N3O3	Yellow	110–112	0.6	83
PY7	C17H15N3O2	Light yellow	107–109	0.4	79
PY8	C17H17N3	Brown	105–107	0.6	65
PY9	C17H17N3	Pale yellow	110–112	0.4	61
PY10	C18H20N2O3	Yellow	108–110	0.4	52

1.6.1. 1-(3-(4-aminophenyl)-5-(2,5-dimethoxyphenyl)-4,5-dihydro-1 H-pyrazol-1-yl)ethan-1-one (PY1)

Yield: 62%, Yellow solid, m.p.: 133–135°C; FT-IR (KBr, ѵ cm⁻¹) 3458 (NH), 1623 (C = N), 1351 (C-N), 1279, 1220 (C-O), 1730 (C = O). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 7.67 (d, J = 5 Hz, 1H), 7.64 (d, J = 10 Hz, 1H), 7.29 (CDCl₃), 7.00 (d, J = 5 Hz, 1H), 6.96 (d, J = 25 Hz, 1H), 6.95 (s, 1H), 6.90 (d, J = 10 Hz, 1H), 6.80 (d, J = 5 Hz, 1H), 5.49 (s, 2H), 4.85 (s, 1H), 3.94 (s, J = 120 Hz, 2H), 3.71 (s, 3H), 3.69 (s, 3H), 1.97 (s, 3H). ¹³C NMR (126 MHz) δ (ppm): 168.64, 152.97, 151.56, 150.68, 148.36, 130.53, 130.35, 130.07, 126.21, 114.48, 114.25, 113.27, 112.49, 111.34, 77.31, 77.07, 76.79, 60.61, 56.19, 55.58, 40.19, 23.70. LC/MS calculated for C₁₉H₂₁N₃O₃ is 339.16, Found: 340.35. Elem. Anal. Calcd.: C, 67.24; H, 6.24; N, 12.38. Found: C, 67.99; H, 6.02; N, 12.56.

1.6.2. 1-(5-(2,5-dimethoxyphenyl)-3-(4-nitrophenyl)-4,5-dihydro-1 H-pyrazol-1-yl)ethan-1-one (PY2)

Yield: 67%, Pale Yellow solid, m.p.: 131–133°C; FT-IR (KBr, ѵ cm⁻¹) 3441 (NH), 1493, 1463 (N = O), 1369 (C-N), 1597 (C = N), 1294, 1263 (C-O), 1707 (C = O). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 8.38 (d, J = 25 Hz,1H), 8.30 (d, J = 20 Hz, 1H), 8.16 (d, J = 50 Hz, 1H), 8.06 (d, J = 15 Hz, 1H), 7.30 (CDCl₃), 6.86 (s, 1H), 6.77 (d, J = 5 Hz, 1H), 6.71 (d, J = 10 Hz, 1H), 5.28 (t, J = 25 Hz, 1H), 3.83 (d, J = 80 Hz, 2H), 3.73 (s, 3H), 3.71 (s, 3H), 1.97 (s, 3H). ¹³C NMR (126 MHz) δ (ppm): 168.94, 152.32, 151.40, 150.50, 145.10, 142.15, 130.52, 127.97, 127.90, 127.86, 127.84, 113.65, 112.72, 111.41, 77.30, 77.05, 76.79, 60.48, 56.02, 55.79, 40.42, 23.42. LC/MS calculated for C₁₉H₁₉N₃O₅ is 369.38, Found: 370.45. Elem. Anal. Calcd.: C, 61.78; H, 5.18; N, 11.38. Found: C, 62.02; H, 5.76; N, 11.66.

1.6.3. 1-(3-(4-aminophenyl)-5-(2,3,4-trimethoxyphenyl)-4,5-dihydro-1 H-pyrazol-1-yl)ethan-1-one (PY3)

Yield: 73%, Light Yellow solid, m.p.: 141–143°C; FT-IR (KBr, ѵ cm⁻¹) 3364 (NH), 1731 (C = O), 1628 (C = N), 1361, 1291 (C-N), 1229, 1200, 1141 (C-O). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 7.72 (d, J = 20 Hz, 1H), 7.65 (d, J = 5 Hz, 1H), 7.29 (CDCl₃), 7.01 (d, J = 15 Hz, 1H), 6.89 (d, J = 15 Hz, 1H), 6.82 (d, J = 35 Hz,1H), 6.56 (d, J = 10 Hz, 1H), 5.49 (s, 2H), 4.95 (t, J = 45 Hz,1H), 3.88 (s, 3H), 3.83 (s, 3H), 3.80 (d, J = 55 Hz, 2H) 3.68 (s, 3H), 1.98 (s, 3H). ¹³C NMR (126 MHz) δ (ppm): 168.56, 153.15, 152.14, 151.28, 150.66, 142.27, 130.24, 128.18, 127.89, 122.39, 121.65, 115.28, 114.64, 107.33, 77.32, 77.06, 76.81, 60.96, 60.79, 60.75, 56.12, 42.00, 23.40. LC/MS calculated for C₂₀H₂₃N₃O₄ is 369.42, Found: 370.48. Elem. Anal. Calcd.: C, 65.03; H, 6.28; N, 11.37. Found: C, 65.99; H, 6.07; N, 11.98.

1.6.4. (E)-1-(3-(4-nitrophenyl)-5-styryl-4,5-dihydro-1 H-pyrazol-1-yl)ethan-1-one (PY4)

Yield: 75%, Brown solid, m.p.: 138–140°C; FT-IR (KBr, ѵ cm⁻¹) 3359 (NH), 1347, 1176, 1146, 1107 (C-N), 1604 (C = N), 1516, 1347 (NO), 1713 (C = O). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 8.45 (d, J = 5 Hz, 1H), 8.34 (d, J = 5 Hz, 1H), 8.24 (d, J = 45 Hz, 1H), 8.08 (d, J = 15 Hz, 1H), 7.38 (d, J = 10 Hz, 1H), 7.29 (CDCl₃), 7.28 (d, J = 10 Hz, 1H), 7.27 (d, J = 10 Hz, 1H), 7.24 (d, J = 5 Hz, 1H), 7.14 (d, J = 15 Hz, 1H), 6.57 (d, J = 10 Hz, 1H), 6.45 (d, J = 30 Hz,1H), 4.39 (t, J = 20 Hz,1H), 2.46 (d, J = 90 Hz, 2H), 1.97 (s, 3H). ¹³C NMR (126 MHz) δ (ppm): 168.94, 151.40, 150.50, 142.15, 136.50, 134.58, 128.80, 128.74, 128.68, 128.60, 128.14, 127.90, 127.86, 127.84, 127.82, 127.40, 77.30, 77.05, 76.79, 51.44, 39.78, 23.42. LC/MS calculated for C₁₉H₁₇N₃O₃ is 335.36, Found: 336.49. Elem. Anal. Calcd.: C, 68.05; H, 5.11; N, 12.53. Found: C, 67.87; H, 5.75; N, 12.59.

1.6.5. 1-(3-(2-aminophenyl)-5-(3,4-dimethoxyphenyl)-4,5-dihydro-1 H-pyrazol-1-yl)ethan-1-one (PY5)

Yield: 63%, Pale Yellow solid, m.p.: 135–137°C; FT-IR (KBr, ѵ cm⁻¹) 3351 (NH), 1326, 1262 (C-N), 1608 (C = N), 1139, 1026 (CO), 1723 (C = O). ¹H NMR (500 MHz CDCl₃) δ (ppm): 7.67 (d, J = 10 Hz, 1H), 7.38 (t, J = 10 Hz, 1H), 7.29 (CDCl₃), 7.00 (d, J = 5 Hz, 1H), 6.97 (t, J = 30 Hz, 1H), 6.97 (s, 1H), 6.92 (d, J = 10 Hz, 1H), 6.90 (d, J = 10 Hz, 1H), 4.98 (s, 2H), 4.96 (t, J = 15 Hz, 1H), 3.94 (d, J = 125 Hz, 2H), 3.83 (s, 3H), 3.71 (s, 3H), 1.97 (s, 3H). ¹³C NMR (126 MHz) δ (ppm): 168.78, 151.40, 150.50, 149.24, 147.33, 134.58, 131.14, 121.59, 121.50, 118.32, 118.08, 117.94, 116.54, 109.19, 77.30, 77.05, 76.79, 65.78, 56.32, 56.17, 39.78, 23.42. LC/MS calculated for C₁₉H₂₁N₃O₃ is 339.40, Found: 340.51. Elem. Anal. Calcd.: C, 67.24; H, 6.24; N, 12.38. Found: C, 67.72; H, 6.56; N, 12.52.

1.6.6. 2-(5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1 H-pyrazol-3-yl)aniline (PY6)

Yield: 83%, Yellow solid, m.p.: 110–112°C; FT-IR (KBr, ѵ cm⁻¹) 3455 (NH), 1592 (C = N), 1343, 1318 (C-N), 1266, 1234, 1182 (C-O). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 9.50 (s, 1H), 7.67 (d, J = 10 Hz, 1H), 7.40 (t, J = 25 Hz,1H), 7.28 (CDCl₃), 6.98 (d, J = 15 Hz,1H), 6.68 (d, J = 5 Hz, 1H), 6.67 (s, 1H), 6.66 (s, 1H), 4.81 (s, 2H), 3.95 (d, J = 5 Hz, 2H), 3.90 (d, J = 20 Hz,1H), 3.73 (s, 3H), 3.71 (s, 3H), 3.65 (s, 3H); ¹³C NMR (126 MHz) δ (ppm): 152.83, 152.06, 151.61, 150.46, 137.94, 137.40, 131.66, 121.50, 118.51, 117.23, 116.46, 102.16, 102.00, 60.88, 77.31, 77.07, 76.79, 56.37, 56.19, 51.56, 42.59. LC/MS calculated for C₁₈H₂₁N₃O₃ is 327.38, Found: 328.56. Elem. Anal. Calcd.: C, 66.04; H, 6.47; N, 12.84. Found: C, 66.72; H, 6.02; N, 12.23.

1.6.7. (E)-3-(4-nitrophenyl)-5-styryl-4,5-dihydro-1 H-pyrazole (PY7)

Yield: 79%, Light Yellow solid, m.p.: 107–108°C; FT-IR (KBr, ѵ cm⁻¹) 3299 (NH), 1597 (C = N), 1340, 1261, 1108 (C-N), 1563, 1340 (NO). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 9.14 (s, 1H), 8.41 (d, J = 5 Hz, 1H), 8.36 (d, J = 5 Hz,1H), 8.08 (d, J = 5 Hz, 1H), 8.03 (d, J = 5 Hz, 1H), 7.39 (t, J = 15 Hz, 1H), 7.35 (t, J = 15 Hz, 1H), 7.29 (CDCl₃), 7.29 (t, J = 0, 1H), 7.24 (d, J = 10 Hz, 1H), 7.18 (d, J = 5 Hz, 1H) 6.56 (d, J = 5 Hz, 1H), 6.21 (d, J = 10 Hz, 1H), 3.35 (t, J = 20 Hz, 1H), 2.53 (t, J = 125 Hz, 2H); ¹³C NMR (126 MHz), δ (ppm): 151.82, 150.15, 145.46, 136.09, 134.71, 128.90, 128.72, 128.64, 128.50, 128.33, 127.96, 127.75, 127.26, 127.16, 127.02, 77.31, 77.05, 76.80, 50.58, 41.96. LC/MS calculated for C₁₇H₁₅N₃O₂ is 293.33, Found: 294.45. Elem. Anal. Calcd.: C, 69.61; H, 5.15; N, 14.33. Found: C, 69.02; H, 5.35; N, 14.57.

1.6.8. (E)-3-(5-styryl-4,5-dihydro-1 H-pyrazol-3-yl)aniline (PY8)

Yield: 65%, Brown solid, m.p.: 105–107°C; FT-IR (KBr, ѵ cm⁻¹) 3373 (NH), 1598 (C = N), 1351, 1071, 1028 (C-N). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 9.13 (s,1H), 7.45 (d, 15 Hz, 1H), 7.39 (t, J = 15 Hz, 1H), 7.35 (t, J = 10 Hz, 1H), 7.28 (CDCl₃), 7.27 (d, J = 10 Hz, 1H), 7.24 (d, J = 5 Hz,1H), 7.18 (t, J = 10 Hz, 1H), 7.12 (t, J = 5 Hz, 1H), 7.02 (s, 1H), 6.89 (d, J = 5 Hz, 1H), 6.56 (d, J = 5 Hz, 1H), 6.19 (d, 10 Hz, 1H), 5.48 (s, 2H), 3.35 (t, J = 20 Hz, 1H), 2.53 (d, J = 125 Hz, 2H); ¹³C NMR (126 MHz) δ (ppm): 151.41, 148.86, 136.21, 134.84, 134.58, 133.57, 128.90, 128.79, 128.57, 128.43, 128.27, 127.70, 123.38, 117.22, 113.23, 77.30, 77.07, 76.80, 51.56, 42.59. LC/MS calculated for C₁₇H₁₇N₃ is 263.34, Found: 264.85. Elem. Anal. Calcd.: C, 77.54; H, 6.51; N, 15.96. Found: C, 77.99; H, 6.32; N, 15.76.

1.6.9. (E)-4-(5-styryl-4,5-dihydro-1 H-pyrazol-3-yl)aniline (PY9)

Yield: 61%, Pale Yellow solid, m.p.: 110–111°C; FT-IR (KBr, ѵ cm⁻¹) 3384 (NH), 1594 (C = N), 1351, 1176, 1129, 1071 (C-N). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 9.13 (s, 1H), 7.67 (d, J = 5 Hz, 1H), 7.64 (d, J = 15 Hz, 1H), 7.38 (t, J = 10 Hz, 1H), 7.31 (t, J = 30 Hz, 1H), 7.29 (CDCl₃), 7.25 (t, J = 20 Hz, 1H), 7.21 (d, J = 15 Hz, 1H), 7.11 (d, J = 10 Hz, 1H), 6.91 (d, J = 5 Hz, 1H), 6.88 (d, J = 15 Hz,1H), 6.57 (d, J = 15 Hz,1H), 6.21 (d, J = 5 Hz, 1H), 5.48 (s, 2H), 3.30 (t, J = 10 Hz, 1H) 2.53 (d, J = 90 Hz, 2H); ¹³C NMR (126 MHz) δ (ppm): 151.50, 150.77, 136.31, 134.56, 130.84, 130.43, 129.01, 128.82, 128.62, 128.42, 128.25, 127.23, 126.20, 114.63, 114.49, 77.31, 77.06, 76.81, 63.06, 47.49, 41.93. LC/MS calculated for C₁₇H₁₇N₃ is 263.14, Found: 264.36. Elem. Anal. Calcd.: C, 67.24; H, 6.24; N, 12.38. Found: C, 67.99; H, 6.02; N, 12.56. Elem. Anal. Calcd.: C, 77.54; H, 6.51; N, 15.96. Found: C, 77.95; H, 6.07; N, 15.67.

1.6.10. 5-(3,4-dimethoxyphenyl)-3-(2-methoxyphenyl)-4,5-dihydro-1 H-pyrazole (PY10)

Yield: 52%, yellow solid, m.p.: 108–109°C; FT-IR (KBr, ѵ cm⁻¹) 3342 (NH), 1659, 1632 (C = N), 1350 (C-N), 1259, 1161 (C-O). ¹H NMR (500 MHz) δ (ppm): 9.95 (d, J = 5 Hz, 1H), 7.69 (t, J = 30 Hz, 1H), 7.53 (t, J = 5 Hz,1H), 7.45 (d, J = 5 Hz,1H), 7.29 (CDCl₃), 7.10 (d, J = 10 Hz,1H), 7.06 (d, J = 5 Hz,1H), 6.98 (d, J = 5 Hz,1H), 6.89 (s, 1H), 4.00 (s, 3H), 3.88 (d, J = 40 Hz, 1H), 3.79 (s, 2H), 3.75 (s, 3H), 3.62 (s, 3H); ¹³C NMR (126 MHz) δ (ppm): 160.29, 151.40, 149.65, 147.57, 136.82, 132.29, 131.63, 121.74, 121.66, 118.74, 117.29, 111.41, 109.85, 77.30, 77.05, 76.79, 56.18, 56.02, 55.79, 51.66, 41.17. LC/MS calculated for C₁₈H₂₀N₂O₃ is 312.37, Found: 313.65. Elem. Anal. Calcd.: C, 69.21; H, 6.45; N, 8.97. Found: C, 69.43; H, 6.02; N, 8.53.

2. Result

2.1. Biological evaluation of synthesized compounds

2.1.1. In vitro anticancer activity

The test compounds were assessed for their anticancer efficacy against the A549 lung cancer cell line via the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay method. The percentage of cell viability of the synthesized compounds was assessed using the A549 cell line. The cell line was inoculated in 96-well microculture plates with suitable DMEM (Dulbecco’s Modified Eagle Medium) and RPMI-1640 (Roswell Park Memorial Institute 1640) medium, enriched with 10% heated fetal bovine serum, and thereafter incubated for 24 h at 37°C in a 5% CO₂ incubator.

Cells were seeded in 96-well plates at a density of 5000 cells per well in 100 μl of complete media and incubated for 24 h. The test chemical was solubilized in DMSO (Dimethyl Sulfoxide) and formulated at different concentrations (0–10 µg/mL). Subsequent to the incubation, the cells were subjected to these concentrations for a duration of 72 h, followed by the addition of 0.5 mg/mL of MTT reagent to the medium and incubated at 37°C for 4 h. The formazan crystals were dissolved by adding 200 µL of DMSO and incubated for further 30 min at 37°C. The concentration of formazan crystals was quantified using a spectrophotometer microtiter plate reader at a wavelength of 570 nm. IC₅₀ values of all the synthesized compounds were mentioned in Table 3.

Table 3.

In vitro anticancer activity of the synthesized compounds (PY1–10) by MTT assay against (A-549) cell line.

S.No.	Compound code	IC50 (µM) at 100 µM
1	PY1	18.5
2	PY2	12.4
3	PY3	17.2
4	PY4	13.6
5	PY5	7.42
6	PY6	14.2
7	PY7	6.45
8	PY8	22.8
9	PY9	24.2
10	PY10	16.3
11	Dabrafenib	8.56

2.1.2. In silico studies

2.1.2.1. Molecular docking studies

A series of chalcone pyrazole derivatives were synthesized, and their molecular structures were shown using ChemDraw Professional 16.0. The compounds were further analyzed through computational methods and Schrodinger’s software to assess their possible characteristics and interactions.

2.1.2.2. Protein preparation

The crystal structure of B-Raf kinase (PDB ID: 2FB8) was obtained from the Protein Data Bank. The structure was subsequently built via the Protein Preparation Wizard in Schrodinger. This method entailed the extraction of water molecules exceeding 5 Å from heteroatom groups, the incorporation of hydrogen atoms, the designation of bond ordering, and the optimization of the hydrogen bonding network, as illustrated in Figure 4A. Protein underwent controlled minimization utilizing the OPLS3e force field to mitigate steric conflicts.

Figure 4.

In silico studies: (A) 3D xray structure of minimised B-Raf kinase. The 3D structure was obtained from Protein data bank (www.Rscb.org) with a resolution of 2.90 Å (PDB ID: 2FB8); (B) Docking interactions of co-crystallised ligand (PDB ID-2FB8) showed docking score −7.24 kcal/mol with interactions of amino acids PHE583 and HIS539; (C) Docking interactions of compound PY7 showed a docking score of −8.89 kcal/mol with interactions of amino acids GLN530.

2.1.2.3. Grid generation

A receptor grid was created with the Receptor Grid Generation feature in Glide. The centroid of the bound ligand in the crystal structure was used to establish the center of the grid box. The grid box was configured at a dimension that included the active site, facilitating sufficient sampling of the ligand conformations.

2.1.2.4. Ligand preparation

The ligands were synthesized via the LigPrep module in Schrödinger Maestro 13.5. The structures were ionized at physiological pH (7.0 ± 2.0) utilizing Epik, and potential tautomers and stereoisomers were produced. Subsequently, the energy of the ligands was minimized with the OPLS-3E force field.

2.1.2.5. Docking

The synthesized ligands were docked into the constructed receptor grid utilizing the Glide docking tool. Both standard precision (SP) and additional precision (XP) docking modes were utilized. During docking, the ligands were permitted flexibility, whilst the receptor remained stiff. The Glide score was employed to rate the docked conformations according to their binding affinities.

2.1.2.6. Post-docking analysis

The highest-ranked poses from the docking results were examined to assess their binding interactions with the active site residues of B-Raf kinase. The interactions, including hydrogen bonds, hydrophobic contacts, and π-π stacking interactions, were analyzed to determine the binding mode of the ligands. The binding poses were visualized via the Maestro tool in Schrödinger. Docking scores of all synthesized compounds mentioned in Table 4 and docking interactions of the co-crystallized ligand and the most potent compound PY7, were shown in Figure 4B,C.

Table 4.

Docking score of the synthesized derivatives.

Ligand name	Binding score (Kcal/mol)	Interactions
PY1	−4.88	ILE463, GLY466
PY2	−5.14	ASH594, LYS483
PY3	−4.69	SER536
PY4	−7.39	GLU501, PHE595, ASH594, LYS483, ALA481
PY5	−7.64	CYS532, THR529
PY6	−7.57	PHE583, GLN530, GLY534, SER536
PY7	−8.89	GLN530
PY8	−6.56	GLN530
PY9	−7.62	GLN530
PY10	−7.07	PHE583, GLY534
Dabrafenib	−7.24	PHE583, HIS539

2.1.2.7. Drug likeness, ADME, and toxicity predictions

The various physicochemical and structural parameters of the synthesized compounds contribute to the drug-likeness properties of the molecule. It helps to predict the oral bioavailability of the substances. For the compound to function as a therapeutic molecule, it must adhere to Lipinski’s Rule of Five. Lipinski outlines criteria for evaluating the drug-likeness of chemical compounds based on their physicochemical characteristics and potential for oral bioavailability. The essential aspects of Lipinski’s Rule of Five are as follows: (a) Molecular Weight: The molecular weight of the chemical must be below 500 Daltons, (b) Lipophilicity: The compound’s LogP (octanol-water partition coefficient) must be below 5. This signifies intermediate lipophilicity, striking a balance between solubility in lipids and aqueous conditions, (c) Hydrogen bond donors: The compound should have no more than five hydrogen bond donor groups (e.g., OH or NH groups), (d) Hydrogen Bond Acceptors: The compound must possess no more than 10 hydrogen bond acceptor atoms (e.g., oxygen or nitrogen), and (e) The polar surface area (PSA): The compound’s polar surface area must be below 140 Å. This quantifies the molecule surface area occupied by polar atoms and functional groups. The current study assessed the drug-likeness properties of the compounds using the Swiss ADME online tool. The lipophilicity (LogP) values of all synthesized compounds range from 2.4 to 3.06. Compound PY7 possesses a logP value of 2.44, adhering to Lipinski’s Rule of Five. Gastrointestinal absorption has been elevated for PY7, indicating oral bioavailability. Qikprop predictions indicated that pyrazole derivatives exhibit superior biological activity metrics and may serve as lead molecules for subsequent modifications. ADME (Absorption, Distribution, Metabolism, and Excretion) predictions of all synthesized derivatives were mentioned in Table 5.

Table 5.

ADME prediction of synthesized derivatives.

Molecule	Formula	MW	HBA	HBD	TPSA	iLoGP	GI absorption	Lipinski violations
PY1	C19H21N3O3	339.40	4	1	77.15	2.94	High	0
PY2	C19H19N3O5	369.38	6	0	96.95	2.95	High	0
PY3	C20H23N3O4	369.42	5	1	86.38	3.06	High	0
PY4	C19H17N3O3	335.36	4	0	78.49	2.8	High	0
PY5	C19H21N3O3	339.40	4	1	77.15	2.95	High	0
PY6	C18H21N3O3	327.38	4	2	78.1	2.64	High	0
PY7	C17H15N3O2	293.33	3	1	70.21	2.44	High	0
PY8	C17H17N3	263.34	1	2	50.41	2.47	High	0
PY9	C17H17N3	263.34	1	2	50.41	2.4	High	0
PY10	C18H20N2O3	312.37	4	1	52.08	2.8	High	0

MW= Molecular weight, TPSA= Topological polar surface area, HBA= Hydrogen bond acceptor, HBD= Hydrogen bond donor.

2.1.2.8. Structure-activity relationship (SAR) (Figure 5)

Figure 5.

Structure-activity Relationship (SAR) studies of synthesized derivatives.

             SAR studies of synthesized compounds showed that the incorporation of p-NO₂ substituents at position R significantly enhances cytotoxic activity, with PY7 demonstrating the highest potency (IC₅₀ = 6.45 µM), followed by PY2 (12.4 µM) and PY4 (13.6 µM). The strong electron-withdrawing properties facilitate polar interactions within the B-Raf catalytic site, where PY7 shows superior binding affinity to GLN530 and achieves the most favorable docking energy (−8.89 kcal/mol). Electron-releasing amino (–NH₂) substituents generally reduce biological activity, as seen in compounds PY1 (18.5 µM), PY3 (17.2 µM), PY6 (14.2 µM), PY8 (22.8 µM), and PY9 (24.2 µM). This is likely due to increased electron density, which alters binding geometry and diminishes lipophilicity. However, PY5 (7.42 µM) is a notable exception, where the incorporation of dimethoxyphenyl and N-acetylation mitigates adverse electronic effects through enhanced hydrophobic interactions. Methoxy-functionalized phenyl moieties at positions R or R₁ influences biological potency via spatial and electronic mechanisms. PY3 (17.2 µM), which contains the sterically bulky trimethoxy phenyl group, exhibited reduced efficacy, while PY6 (14.2 µM), which has unmodified pyrazole nitrogen, displayed improved binding characteristics. This suggests that minimal steric hindrance can partially restore biological activity. Modification at position R₁ with a styryl group increases potency only when paired with electron-deficient substituents. The combination of styryl and –NO₂ in PY7 yielded maximal activity, whereas the styryl–NH₂ analogues PY8 and PY9 showed minimal efficacy, indicating potential spatial obstruction and reduced hydrophobic complementarity. N-acetylation influences binding interactions and molecular stability, proving beneficial for PY5 (7.42 µM) through increased lipophilicity and ring stabilization. However, it demonstrates diminished effectiveness in NO₂-bearing PY2 (12.4 µM) when compared to the unmodified pyrazole nitrogen in PY7, which preserved optimal hydrogen-bonding potential.

3. Discussion

The given research reports the effective designing, synthesis, and biological testing of the new series of hybrid chalcone-pyrazoline derivatives that may serve as the potential anticancer agents working against B-Raf kinase. Lung cancer continues to be a predominant cause of cancer related mortality across the world and the MAPK/ERK pathway, notably B-Raf kinase, has been observed to take center stage in tumor cell propagation, survival, and metastatic process. Activating mutations of this pathway, like that of BRAF V600E, are constitutively activated and thus the inhibition of B-Raf is an established treatment method.

Structure-Activity Relationship (SAR) analysis was used to emphasize the role of electronic and steric factors in the potency of cytotoxicity. Substituents with electron-withdrawing groups, p-nitro in particular, had greater anticancer activity, probably because they were favored in electrophilic interactions with the target molecule. On the other side, the electron-donating groups exhibited lower effectiveness, indicating potentially undesirable changes in the orientation of binding and in lipophilicity. It seemed that N-acetyl group brought stability to the ring and binding affinity in some of the derivatives, whereas the unsubstituted nitrogen at R2 position as in PY7 increased the basicity and electronic interactions, pairing with better potency.

The results of the in vitro MTT assay demonstrated PY7 as the most active derivative toward the A549 lung cancer cell line (IC₅₀ = 6.45 µM) and substantially better than other synthesized derivatives. The higher binding energy of PY7 to B-Raf kinase (docking score: −8.89 Kcal/mol) was also observed by molecular docking with important hydrogen bonding residues being GLN530. This docking pattern was more superior to that of a common drug termed as Dabrafenib, and thus, indicating a vast opportunity to be transformed into a therapeutic agent.

ADME predictions showed that all of the synthesized compounds met the parameters of Lipinski Rule of Five that their gastrointestinal absorption was high, and there were signs of violation, which suggests good oral availability and drug-likeness. The versatile cytotoxicity profile, effective binding interaction, and good pharmacokinetic reasoning make these derivatives, especially PY7, potentially useful as a prospective source of new leads. The chalcone-pyrazole scaffold is a versatile platform to produce new B-Raf kinase inhibitors in lung cancer therapy but also makes this approach a useful platform in general, which could be merged with other suitable ones. Our future work will center about the in vivo testing, mechanism-of-action studies, and structural optimization to increase potency and selectivity with the view of minimizing possible off-target effects.

4. Conclusion

In this study, a novel series of hybrid chalcone-pyrazoline derivatives was efficiently designed, synthesized, and evaluated for its anticancer activity against the A549 lung cancer cell line. The synthetic strategy involved the Claisen–Schmidt condensation reaction followed by pyrazole ring formation, leading to the formation of 10 new compounds featuring structurally diverse substituents. Characterization by spectroscopic and analytical techniques (FTIR, NMR, and LC-MS) established the purity and structural integrity of the synthesized derivatives. Biological screening by the MTT assay identified that the majority of the synthesized derivatives were found to possess exceptional cytotoxic activity, with compound PY7 being the most potent (IC₅₀ = 6.45 µM) against the A549 cell line. This highlights its potential as a promising lead compound for further development in lung cancer therapy. To gain insights into the mechanism of action, molecular docking studies were performed using the crystal structure of B-Raf kinase (PDB ID: 2FB8), a key target in the MAPK signaling pathway. The docking results showed strong binding affinities for several derivatives, with PY7 again emerging as the most promising candidate (docking score: −8.89 kcal/mol), demonstrating favorable interactions with active site residues. These findings suggest that the designed compounds can act as B-Raf inhibitors, potentially disrupting dysregulated cancer signaling. In addition, in silico ADME profiling of all the synthesized compounds identified favorable pharmacokinetic profiles, high gastrointestinal absorption, and complete compliance with Lipinski’s Rule of Five, suggesting excellent drug-like features and prospects for oral bioavailability. Overall, the study presents a robust integrated approach of synthetic chemistry, in vitro cytotoxic evaluation, molecular docking, and ADME prediction, collectively identifying PY7 as a potent candidate for lung cancer therapy.

Funding Statement

This work was not funded.

Article highlights

• A novel series of 10 hybrid chalcone-pyrazoline derivatives (PY(1–10)) containing a range of electron-withdrawing and electron-donating substituents were synthesized using the Claisen–Schmidt condensation followed by cyclization as a systematic structure-activity relationship (SAR) study.

• Targeting MAPK/ERK Signaling Pathway in NSCLC, the design strategy specifically was directed against the mutant B-Raf kinase (V600E) in the MAPK/ERK signaling pathway, a proven oncogenic driver in non-small cell lung cancer (NSCLC) with the aim of disrupting deregulated cell proliferation and cell survival functions.

• Influence of substituents on the anticancer potency of the SAR analysis indicated that the relative anticancer potency was greatly enhanced by the para-substitution of a -NO₂ group on the aromatic ring (R) with PY7 (-NO₂ + styryl, unsubstituted pyrazole nitrogen) the most active compound (IC₅₀ = 6.45 µM). Electron-donor amino (-NH₂) groups tended to reduce potency, however, in PY5 (IC 50 = 7.42 µM) hydrophobic interactions and stability were increased by dimethoxyphenyl and N-acetylation.

• Cytotoxicity in vitro by MTT assay in A549 lung carcinoma cells as seen in Table 2, most derivatives had a moderate to strong inhibitory effect on the growth of A549 cells with PY7 being the most effective, even surpassing the standard B-Raf inhibitor dabrafenib (IC₅₀ = 8.56 µM), which presents it as a good lead compound.

• Molecular docking was used to verify the best in silico docking against B-Raf kinase (PDB ID: 2FB8) which was found to have a high attractive energy (PY7 = −8.89 kcal/mol) due to ideal hydrogen bonds with GLN530. The presence of other active compounds with positive interactions with essential residues was also observed, as in PY5 and PY9, which confirms the importance of the presence of specific substituents in the engagement of the target.

• Drug-Likeness and ADME Profiling of all the synthesized compounds fulfilled Lipinski Rule of Five, they presented good absorption in the gastrointestinal tract, and their physicochemical properties Metric (LogP 2.4–3.06) suggested that none of their statements fail the serious drug-likeness rules.

• Structure Property in the synthesized compounds correlation in Styryl-substitution effect was much more pronounced with electron-withdrawing group as in PY7 but anti-active bulky trimethoxy (PY3) showed reduced activity, reiterating the importance of balancing steric and electronic factors in making potent compounds

Disclosure statement

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

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Writing assistance

No writing assistance was utilized in the production of this manuscript

Data availability statement

The data supporting the findings of this study are provided in the manuscript and the Supplementary information files.

Ethical approval statement

This study did not require ethical board approval as it did not contain human or animal trials.

Authors contribution

Purabi Saha: Conducted synthesis, data analysis and wrote the main manuscript; Azra Yasmin: Prepared figures S1–S3; Ritesh Jha: Conceived of the presented idea and prepared Figures 1–3; Aarti Passi: Prepared tables; Vikramdeep Monga: Molecular Docking Studies and prepared Figure 4; Shammy Jindal: Supervision and data analysis of prepared manuscript; Kamya Goyal: Supervision, data analysis and editing of prepared manuscript. All authors reviewed the manuscript.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/17568919.2025.2561464