New Nitrogen-, Oxygen-, and Sulfur-Containing Heterocyclic Compounds as Anti-Colon Cancer Agents: Synthesis, Multitargeted Evaluations, Molecular Docking Simulations and ADMET Predictions

Abstract

Background/Objectives: Oxidative stress, the Warburg effect, and resistance to apoptosis are key hallmarks driving colorectal tumorigenesis. This study aimed to develop novel multi-target compounds capable of modulating these pathways. Methods: A library of 24 newly synthesized compounds-incorporating annulated thiophene, thiazole, quinazolinone, 2-oxoindoline, and 1,2,3-oxadiazole scaffolds, as well as N-(1-(4-hydroxy-3-methoxyphenyl)-3-oxo-3-(2-(phenylcarbamothioyl)hydrazineyl) prop-1-en-2-yl)benzamide-was evaluated for antioxidant activity (DPPH assay), PDK-1 and LDHA inhibition, cytotoxic effects against LoVo and HCT-116 colon carcinoma cells, with parallel assessment of safety profiles on normal HUVECs. The underlying anticancer mechanism of the most active compound was investigated through analysis of cell cycle distribution, apoptosis induction, intracellular reactive oxygen species levels, mitochondrial membrane potential disruption, and expression levels of apoptosis-related genes. Molecular docking assessed binding interactions within LDHA and PDK-1 active sites. The physicochemical, drug-likeness, and ADMET properties of the multi-bioactive candidates were predicted in silico. Results: Among the synthesized compounds, thiophenes 3b and 3d exhibited potent PDK-1/LDHA and DPPH/LDHA inhibitions, along with significant cytotoxic effects on LoVo/HCT-116 cells (IC₅₀ in µM: 190.30/170.21 and 156.60/160.96, respectively), while showing minimal cytotoxicity toward HUVECs. Molecular docking revealed favorable interactions with key amino acid residues within the LDHA and/or PDK-1 active sites. Compound 3d notably induced G2/M (LoVo) and G1 (HCT-116) arrest and promoted apoptosis via enhancing ROS generation, modulating Bax/Bcl-2 expressions, disrupting mitochondrial membrane potential, and ultimately activating caspses-3. In silico predictions indicated their promising drug-likeness and pharmacokinetics, though high lipophilicity, poor solubility (especially for 3b), and potential toxicity risks were identified as limitations. Conclusions: Thiophenes 3b and 3d emerged as promising multi-target candidates; however, structural optimization is warranted to enhance their solubility, bioavailability, and safety to support further development as lead anti-colon cancer agents.

Keywords: ADMET; Warburg effect; apoptosis; colorectal cancer; heterocyclic compounds; multitarget-directed ligands; oxidative stress.

Scheme 1

Synthesis of compounds 3a–e, 8, and 9a–c.

Scheme 2

Synthesis of compounds 13, 14, 17, and 18a–h.

Scheme 3

Synthesis of compounds 20, 22, 23, and 25.

Figure 1

Effects of compound 3d on the cell cycle distribution of LoVo and HCT-116 cells: (A) negative control LoVo cells, (B) 3d-treated LoVo cells, (C) negative control HCT-116 cells, (D) 3d-treated HCT-116 cells, (E) bar chart depicting mean cell cycle distribution (% ± SD, n = 2).

Figure 2

qRT-PCR quantification of Bax, Bcl-2 and Casp-3 genes expression in LoVo and HCT-116 cells treated with compound 3d (30 µg/mL, 48 h). Data are expressed as fold changes relative to untreated controls and normalized to GAPDH.

Figure 3

Flow cytometric analysis of apoptosis in LoVo and HCT-116 cells treated with compound 3d (30 µg/mL, 48 h): (A) untreated LoVo cells (control), (B) 3d-treated LoVo cells, (C) untreated HCT-116 cells (control), (D) 3d-treated HCT-116 cells. (E) bar chart quantifying early apoptosis, late apoptosis, total apoptosis, and necrosis expressed as mean percentages ± SD of two independent experiments.

Figure 4

Mechanistic flowchart summarizing the sequential events through which compound 3d exerts anticancer effects in colorectal carcinoma cells. Arrows indicate; pathway flow and potential crosstalk with extrinsic apoptosis. Abbreviations: ROS, reactive oxygen species; ΔΨm, mitochondrial membrane potential.

Figure 5

Validation of the docking process against LDHA and PDK-1 enzymes. Superimposition of the co-crystallized ligands (purple) and re-docked ligands (cyan).

Figure 6

A 2D representation of the interactions with amino acid residues in the active binding site of LDHA: (a) Compound 3b, (b) compound 3d.

Figure 7

A 2D representation of the interactions between compound 3b and amino acid residues in the active binding site of PDK-1.

Figure 8

Radar chart representations of compounds 3b and 3d, exhibiting their physicochemical properties.