Synthesis and Antibacterial Activity of 3′- and 5′-Alkylamino Derivatives of Thymidine, Uridine, and 5-Methyluridine

Objective: This study aims to design, synthesize, and evaluate a novel series of pyrimidine nucleoside derivatives modified with long-chain 3′- and 5′-alkylamino substituents as a strategy to overcome evolving bacterial resistance by targeting unconventional pathways in Gram-positive pathogens. Methods: Novel 3′- and 5′-modified thymidine, uridine, and 5-methyluridine analogs were synthesized via site-specific tosylation, nucleophilic substitution with azide or fatty amines, and subsequent reductive amination. The chemical structures of all synthesized compounds were verified using UV, 1H, and 13C NMR spectroscopy, and high-resolution mass spectrometry (HRMS). In vitro cytotoxicity was assessed against the A549 cell line using the standard MTT assay, while antibacterial efficacy was determined via minimum inhibitory concentration (MIC) evaluations against a panel of six Gram-positive bacterial strains, including Staphylococcus aureus (MRSA/MSSA) and Mycobacterium smegmatis. Results and Discussion: Target derivatives IIIa–IIIb, VII, IXa–IXc, and XI exhibited moderate cytotoxicity with CD50 values ranging from 20 to 50 µM. In antibacterial screenings, the ribothymidine and thymidine derivatives VII, IXc, and XI, bearing an extended C13 aminotridecyl chain, demonstrated exceptional potency. Their inhibitory profiles were comparable to clinically established reference antibiotics and significantly outperformed previously reported N4-alkylnucleosides. Conversely, shortening the alkyl chain component (IXa) completely abolished antibacterial efficacy, highlighting a stringent structure–activity relationship (SAR) governed by lipophilic chain length. Conclusions: We have successfully established efficient synthetic routes to access novel lipidated pyrimidine nucleosides. The identification of highly potent aminotridecyl-functionalized thymidine derivatives provides a promising molecular platform for the further development of specialized small-molecule counter-measures against multidrug-resistant Gram-positive infections.