Design of 2-thiopyrimidinones derivatives as new aldose reductase inhibitors via alkyne-azide click reaction

Design of 2-thiopyrimidinones derivatives as new aldose reductase inhibitors via alkyne-azide click reaction

• Author
• Ilaria Martina Silva Lins
• Co-authors
• Larissa Gonçalves Maciel , Janaína Versiani dos Anjos
• Abstract

• INTRODUCTION

  Aldose reductase (AR, EC 1.1.1.21) is a widely distributed enzyme member of the aldo-keto reductase family1. Its primary role is to catalyze the conversion of noxious aldehydes generated by reactive oxygen species into inert alcohols2. In individuals with high blood sugar levels, AR activity is frequently linked to complications arising from diabetes.3To perform its enzymatic function, AR heavily relies on nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor, which can lead to reduced NADPH availability for other enzymes like glutathione reductase (GR). This, in turn, can trigger oxidative stress within cells3. Another consequence of increased AR activity is the accumulation of sorbitol, the primary product of AR reactions. Because sorbitol is highly hydrophilic, it doesn't readily pass through cell membranes. Instead, it can build up in tissues, disrupting ion balance and causing cellular osmotic stress1,3. This accumulation of sorbitol is associated with diabetic complications such as retinopathy, neuropathy, and nephropathy3.Pyrimidines and their oxo-derivatives are extensively recognized for their diverse range of biological activities. These compounds have demonstrated noteworthy effects in various fields, including such as anti-inflammatory,4 antioxidant,5 anticancer agent, 6 antiviral,7 antimicrobial,5,8 and AR inhibition.9,10. Thiopyrimidinones are a subclass of pyrimidinones that exhibit a distinctive tautomeric equilibrium due to conjugated double bonds both within and outside the heterocycle11. Among them, thiouracil stands out as a bioactive thiopyrimidinone closely related to uracil. Thiouracil derivatives have garnered significant attention in the literature due to their pharmacological properties12–14. This study focuses on the synthesis of new thiopyrimidinone derivatives utilizing the alkyne-azide click chemistry approach. Copper-catalyzed alkyne-azide cycloaddition (CuAAC) is a powerful and widely employed chemical reaction in modern organic synthesis. This versatile click chemistry reaction enables the efficient and selective formation of 1,2,3-triazole rings by coupling an alkyne and an azide in the presence of a copper catalyst. This can provide a powerful tool for the efficient assembly of diverse molecular architectures, making it an attractive choice for designing novel derivatives15,16. In addition to the synthesis of these compounds, this research also encompasses their evaluation as potential inhibitors of aldose reductase (ARIs). In order to assess their potential as aldose reductase inhibitors, molecular docking simulations17 were employed. By using this approach, we aim to gain insights into the binding modes and affinities of the newly synthesized 2-thiopyrimidinones derivatives with aldose reductase, shedding light on their potential as therapeutic agents. 

  AIMS

  Design of novel aldose reductase enzyme inhibitors derived from 2-thiopyrimidinones through alkyne-azide click reaction.·       alkyne-azide click reaction (CuAAc)viaSynthesis of 2-thiopyrimidinones ·       Molecular docking simulations to assess the binding modes of new candidates against AR·       Evaluation of compounds through AR screening inhibition assay kit 

  METHODS·       Chemistry In this study, five new derivatives of 2-thiopyrimidinones containing a triazole ring were synthesized through an alkyne-azide click reaction. The first step began with a multicomponent synthesis to form the 2-thiopyrimidinone ring (4a-e). This involved a reaction between aromatic aldehydes (1a-e), ethyl cyanoacetate (2) and thiourea (3). These three components reacted in the presence of potassium carbonate as a base and in an ethanolic medium, under reflux conditions, as described previously18,19. Step two involves adding the 'alkyne' moiety to these derivatives of 2-thiopyrimidinones (4a-e) through an S-alkylation process. The reaction takes place by mixing the derivatives 4a-e with propargyl bromide in acetone, using triethylamine as a base, and stirring them at room temperature for 24 hours resulting in alkynes 5a-e. This procedure was similar to the one described by Lins and coworkers11. The third and final step involves the alkyne-azide click reaction, where the alkynes (5a-e) react with 4-azidobenzoic acid (6) in dimethylformamide, using CuI as a catalyst and triethylamine as a base, resulting in the final compounds 7a-e16. ·       Molecular docking simulations To assess the binding modes of the described compounds (7a-e) at the aldose reductase active site, molecular docking simulations were conducted using AutoGrid v.4.020 and AutoDock21 v.4.022,23, as detailed in our previous publications11.To prepare the enzyme (PDB ID: 2FZD) for simulations, polar hydrogens were added, and partial charges for protein atoms were assigned based on AMBER86 force field parameters24. All atoms were assigned charges accordingly. Ligands, whether cocrystallized or target compounds, had charges assigned using the Gasteiger method to ensure that all residues exhibited integer charges.To assess the reproducibility of our simulation procedure, a validation method (commonly referred to as redocking) was employed to demonstrate that the procedure can accurately reproduce the structural conformation of the cocrystallized ligand at the active site of the respective enzymes tested. At the end of each simulation step, ligand conformations with the most favorable binding energies were selected, yielding atomic coordinates for the 200 conformers that best fit the binding site. All obtained conformers were structurally compared based on their RMSDs and clustered into groups of similar conformations with an RMSD tolerance of 2 Å for each cluster. The resulting structures and figures were examined and generated using PyMOL25 and Discovery Studio26. 

  RESULTS AND DISCUSSION

  Synthesis of ARI CandidatesIn this study, five new compounds derived from 2-thiopyrimidinones containing a triazole ring were synthesized. The compounds were tested against AR through molecular docking simulations.Synthesis of 2-thiopyrimidinones (4a-e)The formation of the 2-thiopyrimidinone ring involves a multicomponent reaction14, in which three or more reagents come together to form a single product. In this step, five derivatives containing the heterocyclic ring were obtained with yields ranging from 45-82%, and their respective melting points were compared with those from the literature. The mechanism of formation of this pyrimidine ring is described by Andrade and colleagues14. The synthesis begins with a Knoevenagel reaction between ethyl cyanoacetate and the respective aromatic aldehydes, leading to the formation of a Michael intermediate. Subsequently, the intermediate reacts with thiourea, completing the cyclization of the ring and oxidation of the ring promoted by atmospheric oxygen. As these compounds are well-described in the literature, structural characterizations were not necessary, and we proceeded to the next step.Synthesis of alkynes (5a-e)The functionalization of the ring with the alkyne moiety derived from propargyl bromide followed the procedure described by Lins and colleagues. Since these compounds were not previously described in the literature, nuclear magnetic resonance (NMR) and high-resolution mass spectra (HRMS) analyses were conducted before proceeding to the final step.Synthesis of compounds 7a-e via CuAAC.The alkynes 5a-e reacted with 4-azidobenzoic acid to form derivatives containing the triazole ring. These compounds required repurification through column chromatography in ethyl acetate/methanol, with great caution after synthesis, as the residual copper from the click reaction tends to interfere with the appearance of some signals in the hydrogen and carbon NMR spectra. This issue has already been reported by Fakhrutdinov and colleagues27. The five products are novel and are on the verge of being published.Molecular docking evaluation.The molecular docking simulations followed the methodology previously established by our research group11. Through the results from molecular docking simulations, it was observed that the compounds interact with key residues such as His110 and Tyr48. Furthermore, the theoretical inhibition constant (theoretical KI) obtained after molecular docking for the synthesized candidates ranged from 2.9 to 1.13 nM. The theoretical KI value for the co-crystallized ligand was 87.36 nM. Although this is theoretical data, it raises the possibility that the newly synthesized compounds in this study show promising results in terms of aldose reductase inhibition, which will be further evaluated experimentally in in vitro assays. 

  CONCLUSION

  In this study, the search for new inhibitors for the aldose reductase enzyme was proposed with the aim of treating diabetic complications caused by high blood sugar levels. As a result, eight novel compounds were synthesized in this work, of which five showed promising results in terms of binding modes and binding energy against AR enzyme. Comparing the binding modes and energy to the co-crystallized ligand and the commercial drug Tolrestat, the molecular docking results indicate that the compounds synthesized here exhibit better interactions with the enzyme's active site, resulting in higher score values and theoretical inhibition constants (theoretical KI). The next step to complete this work involves the in vitro evaluation of the compounds to determine their inhibition percentage and IC50 values against aldose reductase. 

  ACKNOWLEDGMENT 

  We thank North Dakota State University and Federal University of Alagoas for NMR and HMRS analysis. IMSL acknowledges CAPES for the graduate fellowship. 

  REFERENCES 

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• Keywords
• aldose reductase, CuAAC, thyopirimidinones, inhibition, molecular docking
• Modality
• Pôster
• Subject Area
• Drug Design and Discovery, Synthesis and Natural Products