Khellin-Derived Benzofuran–Pyrazoline Hybrids as Kinase-Targeted Anticancer Agents

Abstract

Khellin, a naturally occurring furochromone, represents a valuable natural scaffold for the design of hybrid anticancer agents. In the present study, two series of khellin-derived hybridsbenzofuran–pyrazoline and benzofuran–pyrazoline–4-thiazolidinone derivativeswere rationally designed and synthesized through selective functionalization at the 5-position of the benzofuran core. The synthesized compounds were evaluated for their in vitro cytotoxic activity against human breast (MCF-7) and colon (HCT116) cancer cell lines, where several derivatives exhibited moderate antiproliferative activity in the micromolar range. Selected active compounds were further examined for their effects on EGFR and B-RAF protein expression levels as well as their kinase inhibitory activity, providing mechanistic insight into their potential mode of anticancer action. To assess therapeutic selectivity, the most active compounds were evaluated against normal human breast epithelial (MCF-12A) and normal colon (CCD-33Co) cell lines, revealing differential selectivity profiles among the tested derivatives. Molecular docking studies were performed on representative active compounds to rationalize observed biological activity and characterize key interactions within the ATP-binding sites of EGFR and B-RAF kinases. Collectively, these findings identify khellin-derived benzofuran–pyrazoline hybrid systems as promising candidates for targeting kinases and as potential anticancer agents, providing a rational basis for further structural optimization and mechanistic investigation.

1. Introduction

Benzofuran derivatives, obtained from natural sources or through chemical synthesis, constitute an important class of bioactive heterocycles with broad pharmacological significance. − A wide spectrum of biological activities has been attributed to benzofuran-containing compounds, including antimicrobial, anti-inflammatory, − antioxidant, , antihyperglycemic, neuroprotective, and anti-Alzheimer activities, − as well as notable antitumor effects. − In particular, benzofuran scaffolds have attracted sustained interest in oncology research due to their capacity to modulate key signaling pathways implicated in cancer progression and uncontrolled cellular proliferation. Both natural and synthetic benzofuran derivatives have demonstrated significant anticancer potential, establishing this scaffold as a privileged motif in anticancer drug discovery. Naturally occurring benzofurans such as Ailanthoidol, Ebenfuran, Moracin O, Angelicin, and Fruquintinib (Figure ) exhibit pronounced antitumor activity. − Among synthetic derivatives, Fruquintinib has achieved clinical approval for the treatment of metastatic colorectal cancer, underscoring the translational relevance of the benzofuran framework. , Mechanistically, benzofuran-based compounds have been shown to interact with multiple molecular targets involved in tumor development and progression, including alkaline phosphatase, B-RAF, VEGFR-2, , CDK2, GSK-3β, EGFR, ,− and topoisomerases, Additionally, they induce apoptosis and cell-cycle arrest. This multitarget interaction profile underscores the versatility of the benzofuran scaffold in rational anticancer drug design.

1.

Some natural and synthetic benzofurans with antitumor activity.

To further enhance its biological performance, hybridization strategies integrating benzofuran cores with additional pharmacologically active heterocycles have been widely explored. Benzofuran–pyrazoline and benzofuran–pyrazole hybrids (I–IV) have demonstrated cytotoxic activity across various cancer cell lines (Figure ), ,− supporting the concept that molecular hybridization can integrate complementary pharmacophoric features within a single framework. Additionally, recent studies report multikinase benzofuran–pyrazole hybrids with potent antiproliferative activity against panels of cancer cell lines and favorable kinase inhibition profiles, highlighting the potential of hybrid designs for multitarget anticancer activity. , These findings reinforce the utility of benzofuran-based hybrid systems in the development of structurally diversified anticancer candidates.

2.

Benzofuran-pyrazoline and benzofuran-pyrazole hybrids with cytotoxic activity.

Khellin (V, Figure , a) naturally occurring furochromone isolated from the fruits and seeds of Ammi visnaga, represents a particularly attractive benzofuran-related scaffold for semisynthetic modification. Khellin is traditionally used for the management of bronchial asthma, bronchitis, and renal colic due to its bronchodilatory and vasodilatory properties; it has also demonstrated cytotoxic activity against selected cancer cell lines. − Importantly, structural modification of the khellin framework has been shown to markedly enhance its antiproliferative activity. Several khellin derivatives (VI–VIII, Figure ) exhibited improved cytotoxic potency and inhibitory effects toward EGFR, while derivative IX displayed significant activity against breast and colon cancer cell lines. Additionally, khellinoflavinone X was reported to inhibit CYP4A1, an enzyme involved in procarcinogen bioactivation, suggesting possible chemopreventive relevance. Collectively, these findings indicate that khellin-derived scaffolds offer a promising foundation for further structural diversification and target-oriented optimization

3.

Semisynthetic khellin derivatives with cytotoxic activity.

Despite these advances, systematic structural and mechanistic exploration of khellin- and benzofuran-based anticancer hybridsincluding khellinoflavanones, benzofuran–heterocycle conjugates, and related hybrid scaffolds (refs ,– ,–)have predominantly focused on substitution at the 2- or 3-positions of the benzofuran or khellin cores. In most cases, hybridization strategies were limited to relatively simple heterocyclic conjugations, such as pyrazolines and structurally related motifs. Biologically, evaluation frequently relied on general cytotoxicity screening using a limited number of cancer models, often without systematic selectivity assessment or in-depth mechanistic investigation. Notably, direct interrogation of kinase-associated pathwaysparticularly clinically relevant oncogenic drivers such as EGFR and B-RAFhas remained comparatively underexplored. Consequently, structure–activity relationship (SAR) analyses in these systems have often lacked a defined mechanistic framework. In light of these limitations, the present study adopts a structurally differentiated design strategy centered on selective functionalization at the 5-position of the khellin-derived benzofuran scaffold, a substitution pattern that remains largely unexplored in previously reported systems. This positional modification was strategically integrated with rational hybridization into benzofuran–pyrazoline and benzofuran–pyrazoline–4-thiazolidinone frameworks, generating novel molecular architectures designed to enable systematic SAR exploration while maintaining pharmacophoric integrity. Importantly, the current work integrates dual-cell cytotoxic evaluation (HCT-116 and MCF-7), selectivity profiling against normal cells, direct assessment of EGFR and B-RAF expression modulation, and molecular docking analysis as supportive mechanistic tools. This integrated structural and biological validation framework positions the present compounds within a mechanistically guided dual-target exploration strategy, rather than as a simple structural extension of previously reported benzofuran-based hybrids. Guided by the documented cytotoxic properties of carbothioamide-containing pyrazolines , and the established anticancer activity of 4-thiazolidinone derivatives, ,− two structurally distinct hybrid series were rationally designed and synthesized: benzofuran–pyrazoline hybrids (XIIIa–d) and benzofuran–pyrazoline–4-thiazolidinone hybrids (XIVa–d and XVa–g). These derivatives were constructed from the khellin-derived intermediate khellinone (XI) via chalcone precursors (XIIa–d) (Figure ), enabling controlled structural diversification at the underexplored 5-position of the benzofuran scaffold. The synthesized compounds were evaluated for their in vitro cytotoxic activity against human colon (HCT-116) and breast (MCF-7) cancer cell lines to establish cross-cancer antiproliferative profiles. Selected active derivatives were further examined for their effects on EGFR and B-RAF protein expression levels, as well as their kinase inhibitory activity, providing a preliminary mechanistic assessment aligned with the proposed dual-targeting rationale. In addition, selectivity profiling against normal cells was conducted for representative highly active MCF-7 and HCT-116 derivatives to obtain an initial indication of therapeutic differentiation. To further support structure–activity relationship (SAR) interpretation, molecular docking studies were performed on representative highly active compounds as qualitative and comparative tools to rationalize observed biological trends and potential kinase interactions. Collectively, this integrated structural and biological evaluation establishes khellin-derived benzofuran–pyrazoline hybrid systems as an early stage, mechanistically guided platform for dual-target anticancer discovery and provides a rational framework for subsequent structural optimization and advanced mechanistic investigation.

4.

Target benzofuran-pyrazoline and benzofuran-pyrazoline-thiazolinone hybrids.

2. Results and Discussion

2.1. Chemistry

The synthetic pathways adopted for the preparation of the target derivatives XIIIa–d, XIVa–d and XVa–d were depicted in Schemes and . The key starting semisynthetic 1-(6-hydroxy-4,7-dimethoxybenzofuran-5-yl)­ethanone (Khellinone, XI) was obtained by alkaline hydrolysis of the natural furochromone Khellin (V) with 20% sodium hydroxide solution. Khellinone was converted to the corresponding chalcones (XIIa–d) via condensation with aromatic aldehydes, adopting the base-catalyzed Claisen-Schmidt condensation. The preparation of the benzofuran-pyrazoline carbothioamide derivatives (XIIIa–d) was achieved through a condensation of the chalcones (XIIa–d) with thiosemicarbazide in an alkaline medium. The formation of the pyrazoline ring was confirmed by spectral analysis. IR spectra revealed the disappearance of the band corresponding to the CO of chalcones and the appearance of bands attributed to NH₂ at 3280–3223 cm^–1, in addition to bands at 1296–1143 cm^–1 attributed to CS. ¹H NMR spectra of compounds (XIIIa–d) revealed three doublets of doublets at 3.04–3.10, 3.91–4.05, 5.77–5.89 ppm attributed to the two geminal protons of C4 (H_A and H_B) and one proton at C5 (H_X), respectively. This pattern confirmed the formation of the pyrazoline ring. In addition, a D₂O exchangeable signal attributed to NH₂ protons resonated at 7.80–7.88 ppm. ¹³C NMR for compound XIIIa as an example revealed the absence of signals attributed to the trans alkene carbons of the chalcones and the appearance of signals for C4 and C5 of the pyrazoline ring at 46.95 and 60.46 ppm, respectively, and signals at 176.40 and 149.59 ppm for the carbons of CS and CN, respectively. The mass spectrum for compounds XIIIa and XIIIc showed molecular ion peaks (M⁺) at 397 and 427 with relative intensities of 10.75 and 48.81, respectively, corresponding to their molecular weights [Scheme ].

1. Reagents and Conditions: (a) NaOH, Reflux, 1 h; (b) ArCHO, Ethyl Alcohol, NaOH, 24 h; (c) NaOH, Thiosemicarbazide, Ethanol, Reflux, 8 h.

2. Reagents and Conditions: (a) Monochloroacetic Acid, Anhydrous Sodium Acetate, Glacial Acetic Acid, Reflux, 3 h; (b) Aromatic Aldehyde, Anhydrous Sodium Acetate, Glacial Acetic Acid, Reflux, 6 h.

Synthesis of (4,5-Dihydropyrazol-1-yl)­thiazol-4­(5H)-one derivatives (XIVa–d) were achieved by the reaction of 4,5-dihydropyrazole-1-carbothioamide derivatives (XIIIa–d) with monochloroacetic acid in the presence of anhydrous sodium acetate and glacial acetic acid. − Structures of (XIVa–d) were confirmed by their ¹H NMR spectra where a sharp singlet resonated at 3.94–9.96 ppm corresponding to the protons of CH₂ of the cyclized thiazolidinone ring, this is in addition to the disappearance of the exchangeable peak attributed to the protons of NH₂. ¹³C NMR spectrum of compound XIVa as an example showed a new peak in the aliphatic region at 47.91 ppm which was assigned to the CH₂ carbon of the thiazolidinone, and, a peak appeared at 187.29 ppm corresponding to the CO group of the thiazolidinone concomitantly with the disappearance of the CS peaks of the thioamide moieties. Additionally, Mass spectrum of compound XIVd showed molecular ion peak (M+) at 480 [Scheme ].

The arylidene derivatives of (4,5-dihydropyrazol-1-yl)­thiazol-4­(5H)-one (XVa–g) were obtained from the corresponding thiazolidinone derivatives (XIVa–c) by reacting them with the aromatic aldehydes in the presence of anhydrous sodium acetate in glacial acetic acid under reflux. Substitution of the CH₂ protons of the thiazolidinone was confirmed by ¹H NMR spectra which showed the disappearance of the singlets that previously appeared at 3.94–9.96 ppm. Additionally, an increase in the aromatic integration at 6.93–7.67 ppm was observed that could be assigned to the protons of the added benzylidene moieties ¹³C NMR spectrum of compound XVb, as an example, showed the disappearance of the signal corresponding to CH₂ carbon (previously appeared at 47.91 ppm) and the appearance of new signals in the aromatic region corresponding to the added benzylidene carbons. Mass spectra of compounds XVb, XVd and XVf showed molecular ion peaks (M+) at 593, 555 and 558, respectively corresponding to their molecular weights [Scheme ].

2.2. Biological Screening

2.2.1. In Vitro Cytotoxic Activity

2.2.1.1. In Vitro Cytotoxic Activity Against Colon Cancer (HCT-116) Cells

The cytotoxic activity of the synthesized compounds (XIIIa–d, XIVa–d, and XVa–g) was evaluated against the human colorectal carcinoma HCT-116 cell line using the sulforhodamine B (SRB) assay − following the protocol reported by Skehan et al. IC ₅₀ values were calculated from dose–response curves and are expressed as mean ± SD from three independent experiments.

As summarized in Table , the thioamide derivatives XIIIa–d displayed moderate cytotoxic activity, with IC ₅₀ values ranging from 39.09 to 52.33 μM. Within this series, XIIId, bearing an N,N-dimethylamino phenyl substituent at the 5-position of the pyrazoline ring, was the most active member (IC ₅₀ = 39.09 ± 0.22 μM). Compounds XIIIc (4-methoxyphenyl) and XIIIb (4-chlorophenyl) exhibited nearly comparable activity (IC ₅₀ = 45.43 ± 0.30 and 48.38 ± 0.32 μM, respectively), while the unsubstituted phenyl analogue XIIIa was the least active (IC ₅₀ = 52.33 ± 0.50 μM).

1. Cytotoxic Activity of the Synthesized Compounds Against HCT-116 Cell Line.

compound no	IC 50  ± SD (μM)
XIIIa	52.33 ± 0.50
XIIIb	48.38 ± 0.32
XIIIc	45.43 ± 0.30
XIIId	39.09 ± 0.22
XIVa	27.69 ± 0.24
XIVb	38.00 ± 0.18
XIVc	21.41 ± 0.28
XIVd	37.71 ± 0.24
XVa	36.00 ± 0.36
XVb	32.04 ± 0.40
XVc	50.77 ± 0.36
XVd	30.99 ± 0.24
XVe	31.44 ± 0.20
XVf	13.56 ± 0.24
XVg	16.24 ± 0.26
doxorubicin	9.76 ± 0.13

Transformation of the thioamide scaffold into the corresponding thiazolidinone derivatives (XIVa–d) resulted in a consistent enhancement of cytotoxic potency, yielding IC ₅₀ values between 21.41 and 38.00 μM. The most pronounced activity in this series was observed for XIVc, incorporating a 4-methoxyphenyl substituent, with an IC ₅₀ value of 21.41 ± 0.28 μM. This improvement suggests a favorable contribution of thiazolidinone ring formation to antiproliferative activity against HCT-116 cells.

Further optimization through styryl extension produced the XV series (XVa–g), which exhibited the highest overall cytotoxic activity among the tested compounds. Notably, XVf and XVg, each containing a single 4-chlorophenyl moiety positioned either on the arylidene or pyrazoline fragment, demonstrated the strongest inhibition of HCT-116 cell growth (IC ₅₀ = 13.56 ± 0.24 and 16.24 ± 0.26 μM, respectively). In contrast, compound XVb, bearing two chlorophenyl substituents, showed reduced potency (IC ₅₀ = 32.04 ± 0.40 μM), indicating that excessive aromatic substitution may not be favorable for cytotoxic activity within this scaffold.

Overall, the cytotoxicity data reveal a clear structure–activity trend, where conversion from thioamide to thiazolidinone, followed by styryl conjugation, progressively enhances antiproliferative activity against HCT-116 cells. While the reference drug doxorubicin remained more potent (IC ₅₀ = 9.76 ± 0.13 μM), several synthesized derivatives, particularly XVf and XVg, displayed low-micromolar activity, supporting the relevance of this scaffold for further optimization.

2.2.1.2. In Vitro Cytotoxic Activity against Breast Cancer (MCF-7) Cells

The cytotoxic activity of selected compounds (XIIIa–c, XIVa–d, XVb, and XVf) was evaluated against the human breast adenocarcinoma MCF-7 cell line using the sulforhodamine B (SRB) assay, following established procedures. The obtained IC ₅₀ values are summarized in Table and are reported as mean ± SD from three independent experiments.

2. In Vitro Cytotoxic Activity of Compounds XIIIa–c, XIVa–d, XVb, and XVf against MCF-7 Cells.

compound no	IC 50 μM MCF-7
XIIIa	117.75 ± 74.41
XIIIb	2.51 ± 0.700
XIIIc	62.39 ± 0.005
XIVa	201.44 ± 0.531
XIVb	352.23 ± 0.215
XIVc	10.43 ± 5.638
XIVd	245.69 ± 0.030
XVb	21.59 ± 0.009
XVf	54.87 ± 3.235
doxorubicin	37.84 ± 0.058

As shown in Table , the tested compounds exhibited a broad range of cytotoxic activity against MCF-7 cells, with IC ₅₀ values spanning from 2.51 to 352.23 μM, reflecting a marked dependence on structural features.

Among the evaluated derivatives, compound XIIIb, bearing a 4-chlorophenyl substituent, demonstrated the highest antiproliferative potency with an IC ₅₀ value of 2.51 ± 0.70 μM, surpassing the reference drug doxorubicin (IC ₅₀ = 37.84 ± 0.06 μM). In contrast, the methoxy-substituted analogue XIIIc displayed moderate activity (IC ₅₀ = 62.39 ± 0.01 μM), while the unsubstituted phenyl derivative XIIIa again was less active (IC ₅₀ = 117.75 ± 74.41 μM), indicating a clear influence of aromatic substitution on cytotoxic response.

Conversion of the thioamide scaffold into the corresponding thiazolidinone derivatives (XIVa–d) resulted in divergent effects on cytotoxic potency, depending on the nature of the aryl substituent. Specifically, transformation of XIIIb into XIVb led to a pronounced reduction in activity (IC ₅₀ = 352.23 ± 0.22 μM), and a similar decrease was observed for XIIIa/XIVa (IC ₅₀ = 117.75 vs 201.44 μM). Conversely, thiazolidinone formation markedly enhanced activity in the methoxy-substituted series, where XIVc exhibited a significantly improved IC ₅₀ value of 10.43 ± 5.64 μM, representing a ∼6-fold increase relative to its thioamide precursor and greater potency than doxorubicin.

Evaluation of the styryl-containing derivatives revealed that XVb, bearing two chlorophenyl moieties, retained notable cytotoxic activity (IC ₅₀ = 21.59 ± 0.01 μM), whereas XVf, containing a single chlorophenyl group, showed reduced potency (IC ₅₀ = 54.87 ± 3.24 μM). These results indicate that both substitution pattern and scaffold rigidity critically modulate antiproliferative activity in this series.

2.2.1.3. In Vitro Cytotoxic Activity against Normal Breast Cell Line (MCF-12A) and Normal Colon Cell Line (CCD-33Co)

To gain preliminary insight into the selectivity of the most active compounds toward malignant cells, selected derivatives were evaluated against normal human cell lines corresponding to their respective cancer models. Compounds XIIIb and XIVc, which demonstrated the highest cytotoxic potency against MCF-7 breast cancer cells, were assessed against the normal human breast epithelial cell line MCF-12A, while compounds XVf and XVg, the most active agents against HCT116 colon cancer cells, were evaluated against the normal human colon cell line CCD-33Co. The results are summarized in Table .

3. In Vitro Cytotoxic Activity of Compounds XIIIb and XIVc Against Normal Breast Cell Line (MCF-12A) and Compounds XVf and XVg Against Normal Colon Cell Line (CCD-33Co).

compound no	IC 50 μM MCF-7	IC 50 μM MCF-12A	IC 50 μM HCT-116	IC 50 μM CCD-33o
XIIIb	2.51 ± 0.700	88.73 ± 0.002	.···.···.	.···.···
XIVc	10.43 ± 5.638	38.44 ± 0.002	.···.···	.···.···.
XVf	.···...	.···.···	13.56 ± 0.24	10.34 ± 0.002
XVg	.···.···	.···..	16.24 ± 0.26	21.65 ± 0.002
doxorubicin	37.84 ± 0.058		9.76 ± 0.13

Compound XIIIb exhibited weak cytotoxicity toward MCF-12A cells (IC ₅₀ = 88.73 ± 0.002 μM), corresponding to a high selectivity index (SI = 35.4) relative to MCF-7 cells and indicating a favorable therapeutic window. In contrast, compound XIVc showed moderate cytotoxicity toward normal breast cells (IC ₅₀ = 38.44 ± 0.002 μM) and a lower selectivity index (SI = 3.7), reflecting reduced discrimination between malignant and nonmalignant breast cells.

Evaluation against the normal colon cell line revealed that compound XVf displayed cytotoxicity toward CCD-33Co cells (IC ₅₀ = 10.34 ± 0.002 μM) comparable to its activity against HCT116 cells (IC ₅₀ = 13.56 ± 0.24 μM), yielding a selectivity index of 0.76 and indicating limited selectivity. Conversely, compound XVg demonstrated lower cytotoxicity toward CCD-33Co cells (IC ₅₀ = 21.65 ± 0.002 μM) relative to HCT116 cells (IC ₅₀ = 16.24 ± 0.26 μM), corresponding to a selectivity index of 1.33 and suggesting modest preferential activity toward colon cancer cells.

Overall, these findings identify compound XIIIb as exhibiting the most favorable selectivity profile among the tested derivatives, whereas selectivity toward colon cancer cells was more limited for compounds XVf and XVg.

2.2.1.4. Future-Work and Selectivity Framing

While the present study establishes clear antiproliferative trends and identifies multiple low-micromolar inhibitors across two cancer models, evaluation of cytotoxicity against normal cells was intentionally focused on the most potent MCF-7 and HCT-116 active compounds to provide an initial assessment of selectivity. Future studies will extend normal-cell profiling to additional highly active derivatives, and will incorporate a broader panel of nonmalignant cell lines to more comprehensively define therapeutic windows. Moreover, comparative selectivity analyses against clinically established kinase-targeting agents will be pursued to contextualize the safety and translational potential of this scaffold class. Such investigations will support further lead optimization and guide the rational development of cancer-type–selective analogues.

2.2.1.5. Cross-Cancer Structure–Activity Relationship (SAR)

Across both colorectal (HCT-116) and breast (MCF-7) cancer models, the cytotoxic activity of the synthesized compounds reveals scaffold-dependent and physicochemically driven structure–activity relationships. In the thioamide series (XIII), moderate antiproliferative activity is observed, with potency strongly influenced by the electronic nature and lipophilicity of aromatic substituents. Electron-donating groups (e.g., OCH₃, N­(CH₃)₂) increase molecular polarity and hydrogen-bonding capacity, which in several cases correlates with reduced membrane permeability and diminished cellular potency, whereas halogen substitution (e.g., Cl) enhances lipophilicity and hydrophobic surface area, contributing to improved cell penetration and moderately enhanced activity in selected analogues.

Transformation of the thioamide scaffold into the thiazolidinone core (XIV series) introduces increased molecular rigidity and polarity, leading to context-dependent biological responses. While this cyclization enhances binding preorganization in some derivatives (e.g., XIVc), it also increases steric bulk and polar surface area, which in other cases likely impairs passive diffusion across cellular membranes, resulting in pronounced activity loss. These observations suggest that subtle changes in molecular polarity, steric demand, and conformational flexibility critically influence intracellular bioavailability and target engagement.

Introduction of styryl conjugation in the (XV series) significantly increases molecular planarity, π-conjugation, and lipophilicity, favoring enhanced hydrophobic interactions and improved membrane permeability. This structural evolution correlates with improved cytotoxic activity in HCT-116 cells, particularly for XVf and XVg, which display low-micromolar IC₅₀ values. In contrast, activity in MCF-7 cells remains more variable, indicating that cell-type-specific uptake, polarity tolerance, and intracellular target accessibility play dominant roles in determining potency. The differential performance of compounds such as IXVc and XVf across both models further supports that balanced lipophilicity, controlled polarity, and optimal molecular size are critical determinants of dual anticancer activity rather than scaffold class alone.

Collectively, the SAR analysis indicates that cytotoxic potency in this series is governed by a multifactorial interplay between electronic effects, lipophilicity, polarity, steric bulk, molecular planarity, and conjugation length, rather than by single structural modifications. Structural changes that disrupt this balance can lead to sharp activity losses due to reduced membrane permeability, altered intracellular accumulation, or suboptimal target engagement, providing a mechanistic rationale for the observed activity variations. These findings highlight the importance of physicochemical property optimization, rather than purely structural modification, as a guiding principle for further scaffold refinement and cancer-type-specific optimization.

The experimentally observed activity trends are further supported by molecular docking studies, which qualitatively demonstrate favorable binding orientations and interaction patterns for the more active compounds within the EGFR and B-RAF active sites.

Based on the observed cytotoxic and selectivity trends across both cancer models, selected compounds were further evaluated for their enzyme expression inhibition and inhibitory activity to explore potential mechanistic contributors to the antiproliferative effects.

2.2.2. In Vitro Expression Inhibitory Assay

2.2.2.1. In Vitro Evaluation of B-RAF Expression Inhibition in HCT116 Cells

B-RAF is a serine/threonine kinase that functions within the RAS/MEK/ERK signaling pathway regulating cell proliferation and survival. , Activating B-RAF mutations, particularly V600E, occur in approximately 10–15% of colorectal cancers and contribute to tumor progression. Accordingly, modulation of B-RAF expression represents a relevant therapeutic strategy for targeting colorectal malignancies.

Based on their antiproliferative activity against HCT116 cells, Compounds XIVc, XVf, and XVg were evaluated for their ability to inhibit B-RAF expression relative to the reference inhibitor sorafenib. As summarized in Table , compound XIVc exhibited pronounced B-RAF expression inhibition (68.74%), approaching the activity of sorafenib (71.25%). In comparison, compounds XVf and XVg produced moderate inhibition levels of 58.19 and 59.09%, respectively, while doxorubicin showed 66.98% inhibition under identical experimental conditions.

4. In Vitro B-RAF Expression Inhibition (%) of Selected Compounds in HCT116 Cells.

compound no	B-Raf % inhibition
XIVc	68.74
XVf	58.19
XVg	59.09
sorafinib	71.25
doxorubicin	66.98

These findings indicate that compound XIVc exerts a strong suppressive effect on B-RAF expression in HCT116 cells, whereas XVf and XVg display moderate activity. The observed inhibition profile supports the selection of these compounds for further biological evaluation.

2.2.2.2. In Vitro Evaluation of EGFR Expression Inhibition in MCF-7 Cells

Epidermal growth factor receptor (EGFR/ErbB1/HER1) is a receptor tyrosine kinase that regulates critical signaling pathways involved in cell proliferation and survival. EGFR overexpression is frequently observed in breast cancer.

The most cytotoxic compounds against MCF-7 cells, XIIIb and XIVc (IC ₅₀ = 2.5 and 10.43 μM, respectively), were tested for their ability to inhibit EGFR expression, with Erlotinib as a reference. As reported in Table , EGFR expression was inhibited by 80.94% (XIIIb) and 87.88% (XIVc), compared to 95.68% inhibition by Erlotinib. These results demonstrate that the selected compounds effectively reduce EGFR expression, with XIVc showing the highest activity.

5. In Vitro EGFR Expression Inhibition (%) of the Most Active Compounds in MCF-7 Cells.

compound no	EGFR % inhibition
XIIIb	80.94
XIVc	87.88
erlotinib	95.68
doxorubicin	86.71

2.2.2.3. In Vitro Inhibitory Activity of Compounds XIIIb, XIVc, and XVf against EGFR and B-RAF Enzymes

Recent studies have underscored the therapeutic advantage of dual inhibition of EGFR and B-RAF in cancers driven by aberrant MAPK signaling, particularly colorectal and certain breast tumors. Furthermore, the presence of B-RAF mutations in subsets of breast cancer cell lines suggests a potential therapeutic role for combined EGFR/B-RAF targeting strategies. − Guided by these findings, compounds XIIIb, XIVc, and XVf, previously identified as the most cytotoxic derivatives against MCF-7 and HCT-116 cell lines, were selected for enzymatic evaluation to further elucidate their kinase inhibitory potential.

EGFR and B-RAF inhibitory activities were determined using recombinant human kinases following established assay protocols. Test compounds were evaluated over a concentration range of up to 10 μM to generate dose–response curves, and IC ₅₀ values were calculated by nonlinear regression analysis from three independent experiments (n = 3). Reactions were conducted at 37 °C with a 1 h incubation period. Statistical significance was assessed using one-way ANOVA followed by Tukey’s post hoc test, with p < 0.05 considered statistically significant.

As shown in Table , compound XIIIb exhibited potent EGFR inhibition with an IC ₅₀ value of 0.12 μM, demonstrating approximately 3-fold greater potency than the reference inhibitor Erlotinib (IC ₅₀ = 0.38 μM). In contrast, XIVc showed moderate EGFR inhibition (IC ₅₀ = 3.59 μM), consistent with its broader inhibitory profile. Regarding B-RAF inhibition, compounds XIVc and XVf exhibited IC ₅₀ values of 2.7 and 5.8 μM, respectively, compared with Sorafenib (IC ₅₀ = 1.9 μM). Notably, XIVc retained measurable activity against both EGFR and B-RAF (EGFR: 3.59 μM; B-RAF: 2.7 μM), highlighting its potential as a dual inhibitor.

6. In Vitro EGFR and B-raf Inhibitory Activity of the Most Active Compounds.

compound no	EGFR IC 50 μM	B-raf IC 50 μM
XIIIb	0.12	.···
XIVc	3.59	2.7
XVf	.···.	5.8
erlotinib	0.38	.···.
sorafinib	.···.	1.9

From a structure–activity relationship (SAR) perspective, the superior EGFR inhibition observed for XIIIb may be attributed to favorable positioning of the heteroaromatic scaffold within the ATP-binding pocket, potentially strengthening hinge-region interactions while maintaining an optimal lipophilic balance that enhances binding affinity. In contrast, the dual inhibitory profile of XIVc suggests structural flexibility that permits accommodation within both kinase active sites, albeit with moderate potency. The observed activity trends align with reported selectivity patterns of known EGFR and B-RAF inhibitors, where precise hinge-binding interactions primarily govern EGFR potency, whereas extended hydrophobic interactions and solvent-exposed substituents frequently contribute to B-RAF inhibition.

The rationale for prioritizing XIIIb and XIVc for further investigation is supported by their pronounced cytotoxic profiles combined with significant enzymatic inhibition, indicating that kinase targeting likely contributes to their antiproliferative effects. Collectively, these findings validate the designed scaffold as a promising framework for further optimization toward dual EGFR/B-RAF inhibitory agents.

These findings further validate the rational design strategy outlined in the Introduction, confirming that 5-position functionalization and hybrid scaffold engineering represent an effective approach for dual-target anticancer agent development.

2.3. Molecular Docking Methodology

Molecular docking studies were conducted as a supportive structural approach to provide mechanistic insight into the experimentally observed inhibitory activities against EGFR and B-RAF kinases, rather than as a quantitative predictive tool. Docking simulations were performed using a validated molecular modeling platform (MOE 2019). The X-ray crystallographic structures of EGFR and B-RAF were retrieved from the Protein Data Bank (PDB ID: 1XKK for EGFR − and 2FB8 for B-RAF), , which are cocrystallized with the reference inhibitors lapatinib and SB-590885, respectively.

Protein structures were prepared by removal of crystallographic water molecules and native ligands, followed by the addition of hydrogen atoms and assignment of appropriate protonation states at physiological pH. Energy minimization was carried out using the default force field implemented in the docking software to relieve steric clashes and optimize protein geometry.

The chemical structures of the selected compounds were initially drawn using ChemDraw and subsequently converted into three-dimensional structures. Ligand preparation was performed using the MOE ligand preparation module, where protonation states were assigned at physiological pH, hydrogen atoms were added, geometry optimization was performed using the default force field, partial charges were assigned, and multiple low-energy conformations were generated to ensure adequate conformational flexibility during docking simulations. The optimized ligand structures were subsequently used as input for docking calculations.

Docking simulations employed the software’s standard scoring function, and multiple ligand conformations were generated for each compound. The top-ranked poses were selected based on docking score, binding orientation, pocket complementarity, and consistency of key molecular interactions within the active sites.

Docking protocol validation was performed by redocking the native cocrystallized ligands into their respective binding sites. The resulting root-mean-square deviation (RMSD) values (<2.0 Å) confirmed the reliability and accuracy of the docking protocol. Docking results are presented as supportive qualitative interpretations intended to rationalize experimental biological findings and observed IC₅₀ trends, rather than as quantitative predictors of binding affinity.

2.3.1. Molecular Docking Studies (Supportive Analysis)

To provide structural interpretation of the observed biological activity trends, comparative molecular docking studies were performed for the most active compounds against EGFR and B-RAF kinases. Compounds XIIIb and XIVc were docked into the EGFR kinase domain, while compounds XIVc and XVf were evaluated against the B-RAF kinase. This comparative design was employed to support structure–activity relationship (SAR) analysis and to rationalize differences in inhibitory potency observed experimentally. The selected compounds represent highly active and moderately active analogues, enabling qualitative correlation between binding interactions and experimental inhibitory activity. The docking results are presented as supportive mechanistic insights rather than definitive evidence of binding affinity.

2.3.1.1. Binding Mode of Compounds XIVc and XIIIb in the EGFR Active Site

To rationalize the EGFR inhibitory activity of the selected compounds, molecular docking studies were performed within the ATP-binding site of the kinase domain. Within the EGFR active site, compounds XIIIb (IC ₅₀ = 0.12 μM) and XIVc (IC ₅₀ = 3.59 μM) adopted stable binding conformations comparable to that of the reference inhibitor. Both compounds established a conserved hinge-region interaction through hydrogen bonding with the backbone NH of Met793, a characteristic feature of ATP-competitive EGFR inhibitors. Notably, compound XIIIb exhibited an expanded interaction network, forming an additional hydrogen bond with Leu792 and a water-mediated hydrogen bond with Thr854, which further stabilized its binding orientation within the catalytic pocket. Moreover, The aromatic system of XIIIb engaged in favorable hydrophobic contacts with the Leu718 side chain, contributing to the stabilization of the ligand–protein complex. These interactions were complemented by hydrophobic contacts with residues including Leu718, Val726, Leu792, Met1002 Ala743, Ile744, Asp855, and Gln791, enhancing ligand accommodation within the binding cleft (Figure A–D).

5.

2D diagram (A) and 3D representation (B) of compound XIVC, 2D diagram (C) and 3D representation (D) of compound XIIIb in the EGFR binding site.

This extensive interaction network is consistent with the highly favorable docking score (−20.762 kcal/mol) and low RMSD value (1.2 Å), supporting the superior inhibitory potency of XIIIb. In contrast, compound XIVc exhibited a reduced interaction density within the binding pocket, lacking the cooperative hydrogen-bonding network and electrostatic stabilization observed for XIIIb. This diminished interaction profile is consistent with its weaker docking score (−18.128 kcal/mol) and higher RMSD value (1.7 Å), reflecting reduced binding stability and lower enzymatic inhibition.

Consistent with its superior enzymatic potency relative to XIVc, compound XIIIb displayed improved pocket complementarity and greater binding stability. This binding pattern is consistent with a type-I ATP-competitive inhibition mode, characterized by ligand accommodation within the adenine-binding region, formation of the canonical hinge interaction, and complementary hydrophobic packing within the catalytic cleft. The observed agreement between docking stability and experimental IC₅₀ values supports a coherent structure–activity relationship, suggesting that strengthened hinge anchoring, cooperative hydrogen bonding, and enhanced hydrophobic packing collectively contribute to improved EGFR inhibition

2.3.1.2. Binding Mode of Compounds XIVc and XVf in the B-RAF Active Site

To rationalize the B-RAF inhibitory activity of the selected compounds, molecular docking was performed within the ATP-binding site of the kinase domain. Both XIVc and XVf were accommodated within the catalytic pocket; however, their interaction networks differed significantly, correlating with their enzymatic potencies. Docking of compound XIVc (IC ₅₀ = 2.7 μM) within the B-RAF ATP-binding pocket revealed deep accommodation of the ligand in a conformation consistent with established B-RAF inhibitor binding modes. The ligand formed a conserved hinge-region hydrogen bond with the backbone of Cys532, a key anchoring interaction for effective B-RAF inhibition. Additional stabilizing hydrogen bonds were established with Asp594 and Gly466, reinforcing ligand positioning within the catalytic cleft.

The aromatic framework of XIVc occupied a hydrophobic subpocket, engaging residues including Val471, Ala481, Lys483, Leu505, Leu514, Ile527, Gly534, and Phe583, thereby promoting favorable pocket complementarity and structural stabilization. This cooperative interaction network is consistent with the favorable docking score (−21.49 kcal/mol) and low RMSD value (1.5 Å), supporting the observed B-RAF inhibitory potency of XIVc.

In contrast, compound XVf (IC ₅₀ = 5.8 μM) adopted a less optimized orientation within the ATP-binding site. Although XVf maintained hinge recognition through interaction with Cys532, the overall hydrogen-bonding network was less extensive than that observed for XIVc. The ligand established fewer cooperative stabilizing contacts within the catalytic pocket and exhibited reduced hydrophobic enclosure, consistent with weaker pocket complementarity. This diminished stabilization is reflected in its less favorable docking score (−17.49 kcal/mol) and higher RMSD value (2.0 Å), in agreement with its lower enzymatic inhibition potency (Figure A–D).

6.

2D diagram (A) and 3D representation (B) of compound XIVc, 2D diagram (C) and 3D representation (D) of compound XVf in the B-RAF binding site.

Collectively, the comparative docking analysis reveals a structure–activity relationship in which enhanced hinge anchoring combined with improved hydrophobic packing promotes stronger B-RAF inhibition. The observed agreement between docking stability and experimental IC ₅₀ values provides structural support for the rational design of dual EGFR/B-RAF-targeting inhibitors.

Overall, the comparative docking analysis demonstrates that enhanced biological activity correlates with the ability of active compounds to establish conserved hinge-region hydrogen bonding and extensive hydrophobic stabilization within the ATP-binding sites of EGFR and B-RAF kinases. In the EGFR model, the superior inhibitory potency of XIIIb is associated with an expanded interaction network and improved pocket complementarity relative to XIVc. In the B-RAF model, the stronger inhibitory activity of XIVc compared with XVf is consistent with a more cooperative hydrogen-bonding network and enhanced hydrophobic packing within the catalytic cleft.

Although molecular docking does not provide quantitative binding affinity measurements, the observed interaction patterns offer structural support for the experimental findings and reinforce the proposed structure–activity relationship model. Collectively, these results provide mechanistic insight into kinase inhibition by the synthesized compounds and support their continued investigation as EGFR- and B-RAF-targeting anticancer agents.

3. Conclusion

In this study, structurally differentiated khellin-derived benzofuran–pyrazoline and benzofuran–pyrazoline–4-thiazolidinone hybrids were rationally designed and synthesized through targeted functionalization at the 5-position of the benzofuran scaffold. Biological evaluation demonstrated that several derivatives exhibited moderate antiproliferative activity against human colon (HCT116) and breast (MCF-7) cancer cell lines, indicating that positional modification and heterocyclic hybridization significantly influence cytotoxic behavior.

Among the evaluated compounds, XVf and XVg exhibited the highest cytotoxic activity against HCT116 cells and were associated with notable reduction of B-RAF expression, while XIIIb and XIVc showed the most pronounced activity against MCF-7 cells and displayed favorable selectivity profiles relative to normal human breast epithelial (MCF-12A). Selected active derivatives were further shown to modulate EGFR and B-RAF expression levels and to exhibit measurable inhibitory activity in in vitro kinase assays. Notably, compound XIVc demonstrated activity across both cancer models and showed pronounced inhibitory effects on EGFR and B-RAF signaling.

Molecular docking studies provided structural insight into ligand–target interactions and supported observed structure–activity relationships by revealing conserved hinge-region interactions and hydrophobic stabilization within the ATP-binding sites of both kinases. Although docking does not provide quantitative binding affinity predictions, the observed interaction patterns offer structural support for the experimental findings.

Collectively, these results identify khellin-derived benzofuran–pyrazoline hybrid systems as promising kinase-targeting anticancer candidates and provide a rational basis for further structural optimization, expanded biological evaluation, and in vivo investigation.

4. Experimental Section

4.1. Chemistry

All reagents were purchased from Sigma-Aldrich (St. Louis, MO) or Alfa Aesar and used without further purification. Thin-layer chromatography was performed on Sorbtech TLC plates (silica gel w/UV254), visualizing with UV-light 254 nm. ¹H NMR and ¹³C NMR spectra were recorded at 400 and 100 MHz on a Bruker spectrometer using solvent peak as the internal standard. The multiplicities are reported as follows: singlet (s), doublet (d), doublet of doublets (dd), multiplet (m). Coupling constants are reported in hertz. mass microanalysis spectra were recorded on an Agilent 6220 using electrospray ionization (ESI) time-of-flight.

The reported compounds XI and XIIa–d were prepared according to the reported procedure and confirmed by their melting points XI [110–111 °C] and XIIa–d [125–126, 152, 94, and 158 °C respectively].

4.1.1. General Procedure for the Synthesis of 3-(6-hydroxy-4,7-dimethoxybenzofuran-5–yl)-5-(substituted)­phenyl-4,5-dihydropyrazole-1-carbothioamide (XIIIa–d)

A mixture of chalcones (XIIa–d) (10 mmol), thiosemicarbazide (0.91g, 10 mmol) and NaOH (0.4 g, 25 mmol) was refluxed in ethanol (25 mL) for 6–8 h (TLC). After cooling, the solution was poured onto ice water; the precipitate was collected by filtration, left to dry, and crystallized from methanol.

4.1.1.1. 3-(6-Hydroxy-4,7-dimethoxybenzofuran-5-yl)-5-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (XIIIa)

C₂₀H₁₉N₃O₄S (397.45), Yield: 0.38 g (90%), mp 190–192 °C; IR (KBr, cm^–1): 3483 (OH), 3323, 1620 (NH₂), 1571 (CN), 1296 (CS). MS (m/z) 397 rel. intensity: [M⁺, 10.75]. ¹H NMR (DMSO-d ₆) δ ppm: 3.07 (dd, 1H, C4–H_A pyrazoline, J _AB = 18.28 Hz, J _AX = 3.08 Hz), 3.94 (s, 3H, OCH₃), 3.97 (s, 3H, OCH₃), 3.99 (dd, 1H, C4–H_B pyrazoline, J _BA = 18.28 Hz, J _BX = 11.32 Hz), 5.90 (dd, 1H, C5-Hx pyrazoline, J _XB = 11.32 Hz, J _XA = 3.08 Hz), 7.12 (d, 1H, furan H, J = 2.28 Hz), 7.24 (t, 3H, Ar–H, J = 7.68 Hz), 7.34 (t, 2H, Ar–H, J = 7.68 Hz), 7.80 (s, 2H, NH₂, exch. with D₂O), 7.88 (d, 1H, furan H, J = 2.28 Hz), 9.93 (br. s, 1H, OH, exch. with D₂O). ¹³C NMR (DMSO-d ₆) δ ppm: 176.4 (CS), 155.6, 149.6, 147.9, 146.7, 144.4, 143.4, 129.1, 128.1, 127.3, 126.0, 112.4, 107.3, 105.8 (aromatic carbons), 62.8 (CHCH₂), 61.2 (OCH₃), 60.5 (OCH₃), 46.9 (CHCH₂).

4.1.1.2. 5-(4-Chlorophenyl)-3-(6-hydroxy-4,7-dimethoxybenzofuran-5-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (XIIIb)

C₂₀H₁₈N₃O₄SCl (431.98), Yield: 0.36 g (83%), mp 240–242 °C; ¹H NMR (DMSO-d ₆) δ ppm: 3.05 (dd, 1H, C4–H_A pyrazoline, J _AB = 18.32 Hz, J _AX = 3.08 Hz), 3.89 (s, 3H, OCH₃), 3.92 (s, 3H, OCH₃), 3.96 (dd, 1H, C4–H_B pyrazoline, J _BA = 18.32 Hz, J _BX = 11.40 Hz), 5.89 (dd, 1H, C5-HX pyrazoline, J _XB = 11.40 Hz, J _XA = 3.08 Hz), 7.13 (d, 1H, furan H, J = 2.2 Hz), 7.24 (d, 2H, Ar–H, J = 8.4 Hz), 7.40 (d, 2H, Ar–H, J = 8.4 Hz), 7.80 (br. s, 2H, NH₂, exch. With D₂O), 7.88 (d, 1H, furan H, J = 2.2 Hz), 9.86 (s, 1H, OH, exch. with D₂O).

4.1.1.3. 3-(6-Hydroxy-4,7-dimethoxybenzofuran-5-yl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (XIIIc)

C₂₁H₂₁O₅N₃S (427.47), Yield: 0.36 g (85%), mp 200–202 °C; MS (m/z): 427 rel. intensity: [M⁺, 48,81]. ¹H NMR (DMSO-d ₆) δ ppm: 3.06 (dd, 1H, C4–H_A pyrazoline, J _AB = 18.28 Hz, J _AX = 2.88 Hz), 3.73 (s, 3H, Ar-OCH₃), 3.90 (s, 3H, OCH₃), 3.93 (s, 3H, OCH₃), 3.95 (dd, 1H, C4–H_B pyrazoline, J _BA = 18.28 Hz, J _BX = 11.18 Hz), 5.85 (dd, 1H, C5-Hx pyrazoline, J _XB = 11.18 Hz, J _XA = 2.88 Hz), 6.89 (d, 2H, Ar–H, J = 8.64 Hz), 7.13 (d, 1H, furan H, J = 2.2 Hz), 7.17 (d, 2H, Ar–H, J = 8.64 Hz), 7.80 (br. s, 2H, NH₂, exch. With D₂O), 7.89 (d, 1H, furan H, J = 2.2 Hz), 9.95 (s, 1H, OH, exch. with D₂O). ¹³C NMR (DMSO-d ₆) δ ppm: 176.3 (CS), 158.7, 155.7, 149.6, 147.9, 146.7, 144.9, 135.4, 129.2, 127.4, 114.2, 112.4, 107.4, 105.8 (aromatic carbons), 61.8 (CHCH₂), 61.2 (OCH₃), 60.8 (OCH₃), 55.5 (OCH₃), 46.9 (CHCH₂).

4.1.1.4. 5-(4-Dimethylaminophenyl)-3-(6-hydroxy-4,7-dimethoxybenzofuran-5-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (XIIId)

C₂₂H₂₄O₄N₄S (440.52), Yield: 0.37 g (85%), mp 168–170 °C; IR (KBr, cm^–1): 3479 (OH), 3367 (NH₂), 1616 (CN), 1261 (CS). ¹H NMR (DMSO-d ₆) δ ppm: 2.85 (s, 6H, N­(CH₃)₂), 3.10 (dd, 1H, C4–H_A pyrazoline, J _AB = 18.3 Hz, J _AX = 3.9 Hz), 3.89 (s, 3H, OCH₃), 3.91 (dd, 1H, C4–H_B pyrazoline, J _BA = 15.7 Hz, J _BX = 10.9 Hz), 3.95 (s, 3H,OCH₃), 5.77 (d, 1H, C5–H Pyrazoline, J _XB = 10.9 Hz, J _XA= 3.3 Hz), 6.68 (d, 2H, Ar–H, J = 8.8 Hz), 7.13 (d, 1H, furan H, J = 2.04 Hz), 7.52 (d, 2H, Ar–H, J = 8.7 Hz), 7.88 (s, 2H, NH₂, exch. With D₂O), 7.90 (d, 1H, furan H, J = 2.12 Hz), 10.23 (s, 1H, OH, exch. with D₂O).

4.1.2. General Procedure for the Synthesis of 2-(3-(6-Hydroxy-4,7-dimethoxybenzofuran-5-yl)-5-(substituted)­phenyl-4,5-dihydropyrazol-1-yl)­thiazol-4­(5H)-one (XIVa–d)

A mixture of the thiocarbomoyl pyrazoline derivatives (XIIIa–d) (10 mmol), monochloroacetic acid (0.94g, 10 mmol) and anhydrous sodium acetate (0.82g, 10 mmol) were heated under reflux in glacial acetic acid (10 mL) for 3 h (TLC). After cooling, the solution was poured onto ice water; the precipitate was collected by filtration, washed, dried and crystallized from ethanol.

4.1.2.1. 2-(3-(6-Hydroxy-4,7-dimethoxybenzofuran-5-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl) thiazol-4­(5H)-one (XIVa)

C₂₂H₁₉O₅N₃S (437.47), Yield: 0.37 g (85%), mp 220–222 °C; IR (KBr, cm^–1): 3122 (OH), 1620–1521 (CN), 1406 (C–N), 1695 (CO thiazolidinone). ¹H NMR (DMSO-d ₆) δ ppm: 3.31 (dd, 1H, C4–H_A pyrazoline, J _AB= 18.58 Hz, J _AX= 3.72 Hz), 3.91 (s, 3H, OCH₃), 3.94 (s, 2H, CH₂ thiazolidinone), 3.98 (s, 3H, OCH₃), 4.14 (dd, 1H, C4-Hb pyrazoline, J _BA= 18.58 Hz, J _BX= 11.28 Hz), 5.75 (dd, 1H, C5-Hx pyrazoline, J _XB = 11.24 Hz, J _XA = 3.68 Hz), 7.16 (d, 1H, furan H, J = 2.16 Hz), 7.32 (t, 3H, Ar–H, J = 6.64 Hz), 7.39 (d, 2H, Ar–H, J = 6.80 Hz), 7.92 (d, 1H, furan H, J = 2.24 Hz), 9.97 (s, 1H, OH, exch. with D₂O). ¹³C NMR (DMSO-d ₆) δ ppm: 187.3 (CO), 177.6, 160.2, 149.8, 147.7, 146.6, 145.1, 141.0, 129.3, 129.2, 128.4, 126.4, 112.5, 106.9, 105.8 (aromatic carbons), 63.1­(CHCH₂), 61.5 (OCH₃), 61.3 (OCH₃), 60.9 (CH₂ thiazolidinone), 47.9 (CHCH₂).

4.1.2.2. 2-(5-(4-Chlorophenyl)-3-(6-hydroxy-4,7-dimethoxybenzofuran-5-yl)-4,5-dihydro-1H-pyrazol-1-yl)­thiazol-4­(5H)-one (XIVb)

C₂₂H₁₈O₅N₃SCl (471.91), Yield: 0.38 g (80%), mp 268–270 °C; ¹H NMR (DMSO-d ₆) δ ppm: 3.17 (dd, 1H, C4–H_A pyrazoline, J _AB = 18.72 Hz, J _AX = 3.76 Hz), 3.91 (s, 3H, OCH₃), 3.97 (s, 2H, CH₂), 4.06 (s, 3H, OCH₃), 4.11 (dd, 1H, C4–H_B pyrazoline, J _BA = 18.72 Hz, J _BX = 11.32 Hz), 5.78 (dd, 1H, C5–H_X pyrazoline, J _XB = 11.24 Hz, J _XA = 3.76 Hz), 7.14 (d, 1H, furan H, J = 2.12 Hz), 7.35 (d, 2H, Ar–H, J = 8.44 Hz), 7.47 (d, 2H, Ar–H, J = 8.40 Hz), 7.92 (d, 1H, furan H, J = 2.20 Hz), 9.96 (s, 1H, OH, exch. with D₂O).

4.1.2.3. 2-(3-(6-Hydroxy-4,7-dimethoxybenzofuran-5-yl)-5-(4-methoxyphenyl)-4,5- dihydro-1H-pyrazol-1-yl)­thiazol-4­(5H)-one (XIVc)

C₂₃H₂₁O₆N₃S (467.49), Yield: 0.40 g (85%), mp 256–258 °C; ¹H NMR (DMSO-d ₆) δ ppm: 3.30 (dd, 1H, C4–H_A pyrazoline, J _AB= 18.68 Hz, J _AX= 3.42 Hz), 3.75 (s, 3H, OCH₃), 3.92 (br. s, 5H, OCH₃ and CH₂), 4.00 (s, 3H, OCH₃), 4.06 (dd, 1H, C4–H_B pyrazoline, J _BA= 18.68 Hz, J _BX= 11.28 Hz), 5.70 (dd, 1H, C5–H_X pyrazoline, J _XB= 11.10 Hz, J _XA= 3.42 Hz), 6.95 (d, 2H, Ar–H, J = 8.56 Hz), 7.17 (d, 1H, furan H, J = 2.04 Hz), 7.27 (d, 2H, Ar–H, J = 8.56 Hz), 7.93 (d, 1H, furan H, J = 2.08 Hz), 9.97 (s, 1H, OH, exch. with D₂O).

4.1.2.4. 2-(5-(4-(Dimethylamino)­phenyl)-3-(6-hydroxy-4,7-dimethoxybenzofuran-5-yl)-4,5-dihydro-1H-pyrazol-1-yl)­thiazol-4­(5H)-one (XIVd)

C₂₄H₂₄O₅N₄S (480.54), Yield: 0.39 g (82%), mp 200–202 °C; MS (m/z) 480 rel. intensity: [M+, 3.75], 53 (100). ¹H NMR (DMSO-d ₆) δ ppm: 2.88 (s, 6H, N­(CH₃)₂), 3.17 (dd, 1H, C4–H_A pyrazoline, J _AB = 15.6 Hz, J _AX = 4.24 Hz), 3.90 (s, 3H, OCH₃), 3.96 (s, 2H, CH₂), 4.02 (s, 3H, OCH₃), 4.07 (dd, 1H, C4–H_B pyrazoline, J _BA = 17.9 Hz, J _BX = 11.30 Hz), 5.63 (dd, 1H, C5–H_X pyrazoline, J _XB = 11.12 Hz, J _XA = 3.56 Hz), 6.71 (d, 2H, Ar–H, J = 8.70 Hz), 7.17 (d, 1H, furan H, J = 2.12 Hz), 7.53 (d, 2H, Ar–H, J = 8.40 Hz), 7.94 (d, 1H, furan H, J = 2.20 Hz), 10.19 (s, 1H, OH, exch. with D₂O). ¹³C NMR (DMSO-d ₆) δ ppm: 187.3 (CO), 177.2, 160.4, 150.5, 149.8, 147.7, 146.6, 145.1, 129.2, 128.2, 127.5, 112.7, 112.5, 107.0, 105.9 (aromatic carbons), 63.0­(CHCH₂), 61.6 (OCH₃), 61.3 (OCH₃), 60.9 (CH₂ thiazoldinone), 47.7 (CHCH₂), 40.5­(N­(CH₃)₂).

4.1.3. General Procedure for the Synthesis of 5-Arylidene-2-(3-(6-hydroxy-4,7-dimethoxybenzofuran-5-yl)-5-aryl-4,5-dihydropyrazol-1-yl)­thiazol-4­(5H)-one (XVa–g)

A mixture of (XIVa–d) (10 mmol), the appropriate aromatic aldehyde (13 mmol) and anhydrous sodium acetate (0.82g, 10 mmol) was refluxed in glacial acetic acid (20 mL) for 6 h TLC. After cooling the mixture was poured onto ice cold water then the precipitate was filtered, dried and crystallized from ethanol.

4.1.3.1. 5-Benzylidene-2-(3-(6-hydroxy-4,7-dimethoxybenzofuran-5-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)­thiazol-4­(5H)-one (XVa)

C₂₉H₂₃N₃O₅S (525.58), Yield: 0.42 g (80%), mp 276–278 °C; ¹H NMR (DMSO-d ₆) δ ppm: 3.31 (dd, 1H, C4–H_A pyrazoline J _AB= 18.7 Hz, J _AX= 4.4 Hz), 3.95 (s, 3H, OCH₃), 4.01 (dd, 1H, C4–H_B pyrazoline, J _BA= 18.48 Hz, J _BX= 12.8 Hz), 4.12 (s, 3H, OCH₃), 5.78 (dd, 1H, C5–H_X pyrazoline, J _XB= 12.4 Hz, J _XA= 4.4 Hz), 7.14 (d, 1H, furan H, J = 2.4 Hz), 7.26–7.55 (m, 11H, 10 Ar–H and 1 benzylidene H) 7.93 (d, 1H, furan H, J = 2.4 Hz), 9.96 (s, 1H, OH, exch. with D₂O).

4.1.3.2. 5-(4-Chlorobenzylidene)-2-(5-(4-chlorophenyl)-3-(6-hydroxy-4,7-dimethoxybenzofuran-5-yl)-4,5-dihydro-1H-pyrazol-1-yl)­thiazol-4­(5H)-one (XVb)

C₂₉H₂₁Cl₂N₃O₅S (594.47), Yield: 0.51 g (85%), m.p.240–242 °C; MS (m/z) 593, rel. intensity: [M+, 6.79], 64 (100). ¹H NMR (DMSO-d ₆) δ ppm: 3.32 (dd, 1H, C4–H_A pyrazoline, J _AB= 18.4 Hz, J _AX= 4.18 Hz), 3.95 (s, 3H, OCH₃), 4.02 (s, 3H, OCH₃), 4.16 (dd, 1H, C4–H_B pyrazoline, J _BA= 18.5 Hz, J _BX= 11.20 Hz), 5.76 (dd, 1H, C5–H_X pyrazoline, J _XB= 11.08 Hz, J _XA= 3.40 Hz), 7.16 (d, 1H, furan H, J = 1.40 HZ), 7.35–7.64 (m, 9H, 8 Ar–H and 1 benzylidene H) 7.92 (d, 1H, furan H, J = 1.60 Hz), 9.95 (s, 1H, OH, exch. with D₂O). ¹³C NMR (DMSO-d ₆) δ ppm: 187.24 (CO), 177.68, 170.53, 161.23, 160.14, 149.78, 147.62, 146.51, 145.12, 139.91, 134.89, 133.18, 132.99, 131.80, 130.18, 128.75, 128.14, 112.48, 112.31, 106.76, 105.84 (aromatic carbons), 62.67 (CHCH₂), 61.49 (OCH₃), 56.51 (OCH₃), 47.89 (CHCH₂).

4.1.3.3. 2-(3-(6-Hydroxy-4,7-dimethoxybenzofuran-5-yl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-5-(4-methoxybenzylidene)­thiazol-4­(5H)-one (XVc)

C₃₁H₂₇N₃O₇S (585.63),Yield: 0.53 g (90%), mp 238–240 °C; ¹H NMR (DMSO-d ₆) δ ppm: 3.31 (dd, 1H, C4–H_A pyrazoline, J _AB= 18.6 Hz, J _AX= 2.96 Hz), 3.77 (s, 3H, OCH₃), 3.83 (s, 3H, OCH₃), 3.94 (s, 3H, OCH₃), 4.08 (s, 3H, OCH₃), 4.13 (dd, 1H, C4–H_B pyrazoline, J _BA= 14.72 Hz, J _BX= 11.2 Hz), 5.72 (dd, 1H, C5–H_X pyrazoline, J _XB= 10.96 Hz, J _XA= 3.48 Hz), 7.16 (d, 1H, furan H, J = 2 Hz), 6.93–7.61 (m, 9H, 8 Ar–H and 1 benzylidene H) 7.94 (d, 1H, furan H, J = 2 Hz), 9.96 (s, 1H, OH, exch. with D₂O).

4.1.3.4. 2-(3-(6-Hydroxy-4,7-dimethoxybenzofuran-5-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-5-(4-methoxybenzylidene)­thiazol-4­(5H)-one (XVd)

C₃₀H₂₅N₃O₆S (555.60), Yield: 0.44 g (80%), mp 150–152 °C; MS (m/z) 555 rel. intensity: [M+, 56.95], 164 (100). ¹H NMR (DMSO-d ₆) δ ppm: 3.40 (dd, 1H, C4–H_A pyrazoline, J _AB= 19.2 Hz, J _AX= 3.48 Hz), 3.82 (s, 3H, OCH₃), 3.94 (s, 3H, OCH₃), 4.01 (s, 3H, OCH₃), 4.16 (dd, 1H, C4–H_B pyrazoline, J _BA= 18.62 Hz, J _BX= 11.22 Hz), 5.88 (dd, 1H, C5–H_X pyrazoline, J _XB= 11.10 Hz, J _XA= 3.66 Hz), 7.09 (d, 2H, Ar–H, J = 8.76 Hz), 7.19 (d, 1H, furan H, J = 2.24 Hz), 7.33–7.44 (m, 5H, 5 Ar–H), 7.60 (d, 2H, Ar–H, J = 8.84 Hz), 7.62 (s, 1H, benzylidene H), 7.94 (d, 1H, furan H, J = 2.24 Hz), 9.92 (s, 1H, OH, exch. with D₂O). ¹³C NMR (DMSO-d ₆) δ ppm: 179.8 (CO), 170.6, 161.1, 160.8, 149.8, 147.6, 146.4, 145.1, 140.8, 132.1, 131.5, 129.4, 129.2, 128.5, 126.7, 126.5, 125.4, 115.3, 112.4, 106.7, 105.9 (aromatic carbons), 63.2 (CHCH₂), 61.4 (OCH₃), 61.3 (OCH₃), 55.9 (OCH₃), 48.1 (CHCH₂).

4.1.3.5. 5-(4-(Dimethylamino)­benzylidene)-2-(3-(6-hydroxy-4,7 dimethoxybenzofuran-5-yl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)­thiazol-4­(5H)-one (XVe)

C₃₂H₃₀N₄O₆S (598.67), Yield: 0.48 g (80%), mp 250–252 °C. ¹H NMR (DMSO-d ₆) δ ppm: 2.99 (s, 6H, N­(CH₃)₂), 3.28 (dd, 1H, C4–H_A pyrazoline, J _AB= 18.04 Hz, J _AX= 3.44 Hz), 3.92 (s, 3H, OCH₃), 3.93 (s, 3H, OCH₃), 3.99 (s, 3H, OCH₃), 4.07 (dd, 1H, C4–H_B pyrazoline, J _BA= 18.6 Hz, J _BX= 11.4 Hz), 5.67 (dd, 1H, C5–H_X pyrazoline, J _XB= 11.12 Hz, J _XA= 3.2 Hz), 7.16 (d, 1H, furan H, J = 1.7 Hz), 6.79–7.53 (m, 9H, 8H Ar–H and 1 benzylidene H) 7.92 (d, 1H, furan H, J = 1.8 Hz), 9.96 (s, 1H, OH, exch. with D₂O).

4.1.3.6. 5-(4-Chlorobenzylidene)-2-(3-(6-hydroxy-4,7-dimethoxybenzofuran-5-yl)-5- phenyl-4,5-dihydro-1H-pyrazol-1-yl) thiazol-4­(5H)-one (XVf)

C₂₉H₂₂ClN₃O₅S (560.02), Yield: 0.45 g (80%), mp 228–230 °C; MS (m/z) 558 rel. intensity: [M+, 43.94], 438 (100). ¹H NMR (DMSO-d ₆) δ ppm: 3.29 (dd, 1H, C4–H_A pyrazoline, J _AB= 18.8 Hz, J _AX= 3.56 Hz), 3.94 (s, 3H, OCH₃), 4.01 (s, 3H, OCH₃), 4.06 (dd, 1H, C4–H_B pyrazoline, J _BA= 18.6 Hz, J _BX= 11.36 Hz), 5.75 (dd, 1H, C5–H_X pyrazoline, J _XB= 11.36 Hz, J _XA= 3.68 Hz), 7.12 (d, 1H, furan H, J = 1.8 Hz), 7.23–7.67 (m, 10H, 9 Ar–H and 1 benzylidene H), 7.92 (d, 1H, furan H, J = 1.8 Hz), 9.95 (s, 1H, OH, exch. with D₂O).

4.1.3.7. 5-Benzylidene-2-(5-(4-chlorophenyl)-3-(6-hydroxy-4,7-dimethoxybenzofuran-5-yl)-4,5-dihydro-1H-pyrazol-1-yl)­thiazol-4­(5H)-one (XVg)

C₂₉H₂₂ClN₃O₅S (560.02), Yield: 0.45 g (80%), mp 198–200 °C; ¹H NMR (DMSO-d ₆) δ ppm: 3.29 (dd, 1H, C4–H_A pyrazoline, J _AB= 18.60 Hz, J _AX= 3.66 Hz), 3.93 (s, 3H, OCH₃), 4.08 (s, 3H, OCH₃), 4.16 (dd, 1H, C4–H_B pyrazoline, J _BA= 18.60 Hz, J _BX= 11.08 Hz), 5.90 (dd, 1H, C5-HX pyrazoline, J _XB= 11.06, Hz, J _XA= 3.66 Hz), 7.19 (d, 1H, furan H, J = 2.24 Hz), 7.33–7.45 (m, 5H, 5 Ar–H), 7.58 (d, 2H, Ar–H, J = 8.52 Hz), 7.66–7.68 (m, 3H, 2 Ar–H and 1 benzylidene H), 7.94 (d, 1H, furan H, J = 2.24 Hz), 9.88 (s, 1H, OH, exch. with D₂O). ¹³C NMR (DMSO-d ₆) δ ppm: 179.5 (CO), 170.4, 161.3, 149.8, 147.6, 146.3, 145.1, 140.6, 134.9, 133.2, 131.8, 130.0, 129.8, 129.4, 129.2, 129.1, 128.6, 126.5, 112.4, 106.7, 105.9 (aromatic carbons), 63.3 (CHCH₂), 61.4 (OCH₃), 61.3 (OCH₃), 48.1 (CHCH₂).

4.2. Biological Screening

The biological screening was carried out at the laboratory of the National Cancer Institute (Cairo, Egypt) and Vacsera Laboratories (Cairo, Egypt).

4.2.1. In Vitro Cytotoxic Activity Against Colon Cancer (HCT-116) Cell Line

All the newly synthesized derivatives XIIIa–d, XIVa–d, and XVa–g were tested against the colon cancer HCT-116 cell line using sulfo-Rhodamine B (SRB) assay as described by Skehan et al. The assay was carried out at the National Cancer Institute, Cairo, Egypt, according to the manufacturer’s instructions. The procedure is explained in detail in the Supporting Information and Doxorubicin was used as a reference drug.

4.2.2. In Vitro Cytotoxic Activity Against Breast Cancer Cell Line MCF-7

The test was carried out using the same procedure mentioned above for compounds XIIIa–c, XIVa–d, XVb, and XVf against the MCF-7 cell line using sulfo-Rhodamine B (SRB) assay and according to manufacturer’s instructions. Doxorubicin was used as a reference drug. The procedure is illustrated in detail in the Supporting Information.

4.2.3. In Vitro Cytotoxic Activity Against Normal Cell Lines (MCF-12A and CCD-33Co)

Normal human breast epithelial cells (MCF-12A) and normal human colon fibroblast cells (CCD-33Co) were used to evaluate the cytotoxic selectivity and safety profile of the selected compounds. Cells were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen/Life Technologies) according to the manufacturer’s instructions. Cytotoxicity evaluation was performed under standard culture conditions, and cell viability was assessed using the same protocol applied for cancer cell lines. The detailed experimental procedure is provided in the Supporting Information.

4.2.4. In Vitro EGFR Expression Inhibition Assay (MCF-7 Cells)

Compounds XIIIb and XIVc were evaluated for their inhibitory activity against EGFR expression in MCF-7 cells using a human epidermal growth factor receptor enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s protocol (Enzyme-Linked Immunosorbent Assay Kit for EGFR; Assay Biotech, Fremont, CA). Erlotinib was used as the reference inhibitor. The detailed experimental procedure is explained in the Supporting Information.

4.2.5. In Vitro B-RAF Expression Inhibition Assay (HCT-116 Cells)

Compounds XVf and XVg were evaluated for their inhibitory activity against B-RAF expression in HCT116 cells using a colorimetric cell-based ELISA kit according to the manufacturer’s protocol (Colorimetric Cell-Based ELISA Kit – B-RAF Cell-Based ELISA; Assay Biotech, Fremont, CA). Sorafenib was used as the reference inhibitor. The detailed experimental procedure is illustrated in the Supporting Information.

4.2.6. In Vitro Determination of IC ₅₀ Against EGFR Kinase (Enzymatic Inhibitory Assay)

Compounds XIIIb and XIVc were evaluated for their inhibitory activity against epidermal growth factor receptor (EGFR) kinase using a luminescence-based EGFR kinase assay kit according to the manufacturer’s instructions (BPS Bioscience Inc., EGFR Kinase Assay Kit, Cat. No. 40321). The assay is based on the measurement of ATP consumption using the Kinase-Glo MAX detection system. Erlotinib was employed as the reference standard inhibitor. The detailed experimental procedure is provided in the Supporting Information.

4.2.7. In Vitro Determination of IC ₅₀ Against B-RAF Kinase (Enzymatic Inhibitory Assay)

Compounds XVf and XVg were investigated for their inhibitory effect against B-RAF kinase using a luminescence-based BRAF (WT) kinase assay kit following the manufacturer’s protocol (BPS Bioscience Inc., BRAF (WT) Kinase Assay Kit, Cat. No. 78316). The assay relies on ATP depletion measurement using the Kinase-Glo MAX luminescent detection reagent. Sorafenib was used as the reference standard inhibitor. The experimental procedure is described in detail in the Supporting Information.

4.3. Molecular Docking

Molecular docking studies were performed as a supportive structural tool to rationalize the experimentally observed EGFR and B-RAF inhibitory activities rather than as a quantitative predictive method. Docking simulations were carried out using the Molecular Operating Environment (MOE) software package (MOE 2019).

The X-ray crystal structures of the target proteins were retrieved from the Protein Data Bank, including EGFR cocrystallized with lapatinib (PDB ID: 1XKK) and B-RAF cocrystallized with SB-590885 (PDB ID: 2FB8). These cocrystallized ligands were used as structural reference controls for binding-site definition and protocol validation. Protein structures were prepared by removing crystallographic water molecules and native ligands, followed by the addition of hydrogen atoms and assignment of appropriate protonation states at physiological pH. Energy minimization was performed using the MMFF94X force field with an RMSD gradient cutoff of 0.001 kcal·mol^–1, and partial charges were automatically assigned.

The chemical structures of the selected compounds were drawn using ChemDraw and converted into their corresponding three-dimensional conformations. Ligand preparation was performed using the MOE ligand preparation module, where protonation states were assigned at physiological pH, hydrogen atoms were added, partial charges were automatically calculated, and geometry optimization was carried out using the MMFF94X force field with an RMSD gradient cutoff of 0.001 kcal·mol^–1. Multiple low-energy conformations were generated for each ligand to ensure adequate conformational flexibility during docking simulations.

Docking calculations were conducted using the standard MOE scoring function, and multiple binding poses were generated for each compound. The optimal docking poses were selected based on docking score, binding orientation, pocket complementarity, and the formation of key interactions within the ATP-binding sites of EGFR and B-RAF kinases.

Protocol validation was performed by redocking the native cocrystallized ligands (lapatinib and SB-590885) into their respective binding sites. The resulting root-mean-square deviation (RMSD) values were below 2.0 Å, confirming the reliability and reproducibility of the docking protocol and its ability to reproduce experimentally observed binding conformations and key molecular interactions.

Docking results are presented as qualitative structural interpretations to support and rationalize the experimental enzyme inhibition data and observed IC ₅₀ trends, rather than as quantitative predictors of binding affinity.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c12128.

• Figures S1–S4: Structures of natural and synthetic benzofurans and target benzofuran–pyrazoline and benzofuran–pyrazoline–thiazolidinone hybrids; Schemes S1 and S2: Synthesis of benzofuran–pyrazoline derivatives; biological assays: in vitro cytotoxicity (HCT-116, MCF-7, MCF-12A, CCD-33Co) and EGFR/B-RAF enzyme expression and inhibition studies; Figures S5–S40: ¹H NMR, ¹³C NMR, and mass spectra of synthesized compounds XIIIa–XIIIc, XIVa–XIVd, XVb, XVc, XVd, XVe, XVf, and XVg; experimental details (PDF)

The authors declare no competing financial interest.