Development of Pyrazole and Pyrazolo[4,5-b]pyridine Analogues with Potent Fungal Biofilm Inhibition Properties.

Abstract

Introduction: Fungal infections continue to pose significant health risks because of two factors, which include drug resistance and the restricted availability of treatment options. Molecular hybridization has been an effective method for developing new drugs with improved therapeutic effects. Fused heterocyclic systems provide researchers with useful frameworks for synthesizing compounds that more effectively engage biological targets. The pyrazole and pyrazolo[ 4,5-b]pyridine derivatives have attracted significant scientific interest due to their multiple biological functions and their ability to be modified in various ways.

Methods: The target compounds were prepared through cyclization of naphthalene hydrazide 1 with 2-cyano-N-aryl-acetamide 2a-d to give the key 5-amino-pyrazoles 3a-d. These compounds were then converted into pyrazolo[4,5-b]pyridine derivatives (5a, 5b, 5d, 7a-c) using various chemical routes. Spectroscopic methods (FT-IR, ¹H NMR, and ¹³C NMR) were used for structural identification. An ADMET analysis was conducted to assess the drug likeness and pharmacokinetics of each compound. Finally, the selected compounds were evaluated for antifungal activity against Candida albicans by assessing their ability to inhibit biofilm formation and filamentation. SEM and gene expression studies were conducted to investigate the mechanisms of action, while molecular docking analysis was performed to identify binding interactions with biofilm-associated proteins.

Results: Compound 5a showed the highest inhibitory activity (IC50 = 16 μg/mL), with high biofilm inhibition (69%) and filamentation suppression compared to Fluconazole (IC50 = 8 μg/mL, 76.7%). SEM and gene expression data confirmed disruption of the biofilm matrix and the suppression of virulence-related genes. Docking analysis also provided evidence of high-affinity binding to several biofilm-associated proteins.

Discussion: The significant biological activity of compound 5a can be explained by the chloro group present on the pyrazolo[4,5-b]pyridine system. On the other hand, compound 7a, despite containing similar groups, exhibited low activity, possibly due to bulky substituents that cause steric hindrance, preventing its interaction with the target molecules involved in fungal virulence. Thus, both biological and molecular docking studies confirm the suitability of compound 5a for use as an antifungal drug.

Conclusion: This research has shown that the synthesized pyrazolo[4,5-b]pyridine derivatives showed high antifungal activity. Specifically, compound 5a has been identified as a lead candidate due to its high capacity to disrupt biofilm formation, suppress virulence factor production, and exhibit strong binding affinity for these proteins, thereby making it suitable for the development of future antifungal drugs.