Evaluation of Novel Spiro-pyrrolopyridazine Derivatives as Anticancer Compounds: In Vitro Selective Cytotoxicity, Induction of Apoptosis, EGFR Inhibitory Activity, and Molecular Docking Analysis

Abstract

Cancer, characterized by uncontrolled cell proliferation, remains a global health challenge. Despite advancements in cancer treatment, drug resistance and adverse effects on normal cells remain challenging. The epidermal growth factor receptor (EGFR), a transmembrane tyrosine kinase protein, is crucial in controlling cell proliferation and is implicated in various cancers. Here, the cytotoxic and apoptotic potential of 21 newly synthesized spiro-pyrrolopyridazine (SPP) derivatives was investigated on breast (MCF-7), lung (H69AR), and prostate (PC-3) cancer cells. XTT assay was used for cytotoxicity assessment. Flow cytometry and western blot (WB) analyses were conducted for apoptosis detection. Additionally, the EGFR inhibitory potential of these derivatives was evaluated via a homogeneous timeresolved fluorescence (HTRF) assay, and WB and molecular docking studies were conducted to analyze the binding affinities of SPP10 with EGFR. SPPs, especially SPP10, exhibit significant cytotoxicity across MCF-7, H69AR, and PC-3 cancer cells with IC 50 values of 2.31 ± 0.3, 3.16 ± 0.8, and 4.2 ± 0.2 μM, respectively. Notably, SPP10 demonstrates selective cytotoxicity against cancer cells with a low impact on nontumorigenic cells (IC 50 value: 26.8 ± 0.4 μM). Flow cytometric analysis demonstrated the potent induction of apoptotic cell death by SPP10 in all of the tested cancer cells. Western blot analysis revealed the involvement of key apoptotic proteins, with SPP10 notably inhibiting antiapoptotic Bcl-2 while inducing pro-apoptotic Bax and cytochrome c. SPP10 exhibited significant EGFR kinase inhibitory activity, surpassing the efficacy of the reference drug erlotinib. Molecular docking studies support these findings, revealing strong binding affinities of SPP10 with both wild-type and mutated EGFR. The study underscores the significance of heterocyclic compounds, particularly spiro-class heterocyclic molecules, in advancing cancer research. Overall, SPP10 emerges as a promising candidate for further investigations in cancer treatment, combining potent cytotoxicity, apoptotic induction, and targeted EGFR inhibition.

InCl3-Catalyzed One-Pot Synthesis of Pyrrolo/Indolo- and Benzooxazepino-Fused Quinoxalines

“…Recently, hybrid molecules, in which two or more pharmacophores are combined together in one molecule, have garnered significant interest since they provide enhanced or unusual activities as compared to their individual counterparts. , Notably, heterocyclic hybrid molecules have exhibited better specificity, patient compliance, and aptitude to overcome drug resistance and reduced side effects . An ever-continuing aspect of these studies is to find novel hybrid molecules that will provide a new mode of action for the treatment of a specific disease.…”