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Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay

Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay

Published on: February 9, 2021

New Nitrogen-, Oxygen-, and Sulfur-Containing Heterocyclic Compounds as Anti-Colon Cancer Agents: Synthesis,

Nahed Nasser Eid El-Sayed¹, Najeh Krayem², Hamed Ahmed Derbala³

¹Egyptian Drug Authority, 51 Wezaret El-Zerra St., Giza 35521, Egypt.

Pharmaceuticals (Basel, Switzerland)

|June 27, 2025

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.

Insights

Novel thiophene compounds show promise as multi-target agents against colorectal cancer by inhibiting key pathways like oxidative stress and the Warburg effect. Further optimization is needed to improve solubility and bioavailability for potential drug development.

Area of Science:

Medicinal Chemistry
Oncology
Biochemistry

Background:

Colorectal cancer hallmarks include oxidative stress, the Warburg effect, and apoptosis resistance.
Developing multi-target compounds is crucial for effective cancer therapy.

Purpose of the Study:

To synthesize and evaluate novel compounds targeting key pathways in colorectal tumorigenesis.
To identify lead compounds with potent anticancer activity and favorable safety profiles.

Main Methods:

Synthesis of 24 novel compounds with diverse heterocyclic scaffolds.
Evaluation of antioxidant activity, PDK-1/LDHA inhibition, and cytotoxicity against colon cancer cells (LoVo, HCT-116) and normal cells (HUVECs).
In-depth mechanistic studies including cell cycle analysis, apoptosis induction, ROS levels, mitochondrial potential, gene expression, molecular docking, and in silico ADMET predictions.

Main Results:

Thiophenes 3b and 3d demonstrated potent inhibition of PDK-1/LDHA and antioxidant activity, with significant cytotoxicity against colon cancer cells (IC50 values in µM range) and minimal toxicity to normal cells.
Molecular docking confirmed favorable interactions with LDHA and PDK-1 active sites.
Compound 3d induced cell cycle arrest (G2/M or G1), promoted apoptosis via ROS generation, modulated Bax/Bcl-2 expression, disrupted mitochondrial potential, and activated caspase-3. In silico analysis suggested good drug-likeness but highlighted limitations in solubility and potential toxicity.

Conclusions:

Thiophenes 3b and 3d are promising multi-target candidates for anti-colon cancer therapy.
Structural optimization is necessary to enhance solubility, bioavailability, and safety for further preclinical development.

Read full analysis

Keywords:

ADMET Warburg effect apoptosis colorectal cancer heterocyclic compounds multitarget-directed ligands oxidative stress

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