The simultaneous targeting of EGFR and HER2 constitutes a legitimate anticancer strategy for the treatment of
solid tumors. In response, we developed and synthesized a new group of pyrazoline compounds that act as dualtarget
inhibitors for EGFR and HER-2. The structure of the newly synthesized compounds was validated with 1H NMR, 13C NMR, and elemental analysis. The novel compounds’ antiproliferative efficacy was evaluated against four cancer cell lines. All compounds showed GI50 values ranging from 23 to 66 nM, with the breast cancer cell line (MCF-7) showing the highest sensitivity. Compounds 7c, 7d, 7f, 7h, 7j, and 7l had the strongest antiproliferative activity, with derivatives 7d, 7h, and 7j outperforming erlotinib in all tested cancer cell lines. The study revealed that compounds 7d and 7h are the most  effective dual-target inhibitors of EGFR and HER-2, exceeding the reference EGFR inhibitor erlotinib and having comparable action to the clinically used HER-2 medication Lapatinib. We tested the apoptotic potential of 7d and 7h and found that both compounds cause apoptosis by turning on caspase-3, caspase-8, and Bax and decreasing the expression of Bcl-2, a protein that prevents apoptosis. Molecular docking studies revealed robust interactions of these compounds within the EGFR and HER-2 binding pockets, supported by molecular dynamics simulations that confirmed their stability. ADME profiling highlighted the pharmacokinetic advantages of these compounds, particularly 7h, as orally bioavailable and effective inhibitors. These findings suggest that pyrazoline-based inhibitors could serve as a foundation for future development of dual-targeted therapies to overcome resistance in cancer treatment.

Phenotypic screening, a widely utilized technique in drug research and development, has the advantage of not
relying on specific molecular targets, enabling a more comprehensive evaluation of the potential efficacy of
drugs. Our group developed and synthesized a series of novel 1H-pyrazole [3, 4-d]pyrimidine derivatives. We
used the phenotypic screening method to assess their activities. The results showed that compound 8r had
excellent inhibitory activity against the esophageal cancer cell line KYS-30 (IC50 = 3.53 ± 0.04 μM), significantly
better than the positive control compound 5-Fu. Further activity studies indicated that 8r could not only
considerably inhibit the colony formation and invasion of KYS-30 but also showed excellent safety both in vitro
and in vivo. Subsequently, we treated rat cardiac muscle cells H9C2 with high concentrations (10 μM and 20 μM)
of 8r. Investigations revealed that cardiac muscle cells maintained a survival rate exceeding 80 %, verifying the
minimal cardiotoxicity of compound 8r. The research confirmed the technical benefits of phenotypic screening in
maintaining a balance between drug effectiveness and safety, while also offering a novel approach for creating
anticancer drugs with high efficacy and low toxicity

Introduction: A novel series of quinolin-2(1H)-one derivative was rationally designed, synthesized, and characterized as potential dual inhibitors of EGFR and HER-2.
Methods: Structural elucidation was achieved through IR, NMR, mass spectrometry, elemental analysis, and single-crystal X-ray crystallography. The synthesized compounds were screened for antiproliferative activity against four
human cancer cell lines.
Results and Discussion: Compound 5a exhibited the most potent antiproliferative profile, particularly against MCF-7 breast cancer cells (IC50 = 34 nM), outperforming erlotinib (IC50 = 40 nM). Kinase inhibition assays further confirmed the dual activity of 5a, with IC50 values of 87 nM and 33 nM against EGFR and HER-2, respectively. Compound 5a induced apoptosis via activation of caspase-3, -8, and -9, along with upregulation of Bax, downregulation of Bcl-2, and increased cytochrome c release. Flow cytometry analysis demonstrated that 5a caused significant G0/G1 phase arrest in MCF-7 cells, indicating a cytostatic mechanism of action. Computational studies provided structural validation of the observed
biological activities. Molecular docking studies showed a strong binding affinity 5a within the ATP-binding pockets of EGFR and HER-2, supported by key hydrogen bonding and hydrophobic interactions. These findings were further
corroborated by 100 ns molecular dynamics simulations, which confirmed the structural stability and compactness of the 5a-HER-2 complex, as evidenced by low RMSD, consistent RMSF, and favorable radius of gyration and potential energy
profiles. Additionally, ADME predictions revealed that 5a possesses favorable physicochemical and pharmacokinetic properties. Density Functional Theory (DFT) calculations provided insights into the electronic structure of 5a, highlighting favorable HOMO–LUMO distribution and electrostatic potential surfaces that support its dual-binding behavior.

A novel series of pyrano-quinoline compounds 5a–l was designed, synthesized, and investigated for
antiproliferative efficacy as multi-EGFR/HER-2/BRAFV600E inhibitors. This work addresses the reaction
between 4-hydroxy-2-oxo-1,2-dihydroquinolines and 2-benzylidenemalononitriles, which produces
a new series of 2-amino-5-oxo-4-phenyl-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile
derivatives 5a–l, giving good yields. The suggested mechanism was considered. The structures of 5a–l
were elucidated using NMR spectroscopy, mass spectrometry, and elemental analysis. The cell viability
assay of 5a–l against a normal cell line showed that none of the studied compounds exhibited
cytotoxicity, and all hybrids retained above 90% cell viability at a dose of 50 mM. The antiproliferative
activity of 5a–l was assessed against a panel of four cancer cell lines using the MTT assay. Compounds
5e and 5h had the most antiproliferative activity, with GI50 values of 26 and 28 nM, respectively, making
them more efficient than erlotinib (GI50 = 33 nM). Inhibitory assays on EGFR, HER-2, and BRAFV600E
indicated that compounds 5e and 5h were the most efficacious derivatives, with IC50 values of 71 nM
(EGFR), 62 nM (BRAFV600E), and 21 nM (HER-2) for compound 5e, whereas compound 5h displayed IC50
values of 75 nM (EGFR), 67 nM (BRAFV600E), and 23 nM (HER-2). Molecular docking studies were
conducted on a series of quinoline-based compounds to evaluate their binding affinity with EGFR and
HER-2 kinases. Compound 5e showed promising interactions, forming stable complexes with key
residues like Met769 (EGFR) and Asp863 (HER-2). The docking simulations revealed critical hydrogen
bonding, p–p stacking, and hydrophobic interactions, supporting its potential as a kinase inhibitor for
cancer treatment.

A novel series of benzimidazole‐based derivatives (5a–g), (6a–b), and (7a–b) were designed, synthesized, and evaluated for their potential as dual inhibitors of EGFR and HER‐2. The synthesized compounds were subjected to in vitro screening against a panel of selected human cancer cell lines. Additionally, their cytotoxicity was assessed using normal human mammary epithelial cells (MCF‐10A) to evaluate their safety profile. Among the tested derivatives, compounds 5b, 5f, and 6a demonstrated the most pronounced antiproliferative activity, exhibiting IC₅₀ values of 6, 8, and 5 μM, respectively. These values reflect a potency at least fourfold greater than that of the reference drug doxorubicin (IC₅₀ = 33 μM). EGFR and HER‐2 enzyme inhibition assays were conducted to explore the potential molecular targets responsible for the observed anticancer effects. Notably, compound 6a (R₁ = phenyl, thiosemicarbazide) exhibited superior efficacy against the MCF‐7 breast cancer cell line, with an IC₅₀ of 5 μM, approximately six times more potent than doxorubicin. Conversely, compound 7b, with an IC₅₀ value of 85 μM against MCF‐7 cells, was the least active, underscoring the critical role of the phenyl moiety in antiproliferative activity. Furthermore, a molecular docking study was conducted to investigate the binding interactions of 6a within the active sites of EGFR and HER‐2, providing insight into its potential mechanism of action.