August 14, 2025

Comparative Study of Pyridine and Pyrimidine Derivatives as Promising Anti‐Inflammatory Agents: Design, Synthesis, and LPS‐Induced RAW 264.7 Macrophages

FSFarid M. SroorNational Research Centre ASAhmed A. F. SolimanNational Research Centre WKWagdy K. B. KhalilNational Research Centre

Key Points

Pyridine derivative 7a showed significant anti-inflammatory activity, reducing pro-inflammatory gene expression levels.
The most effective compounds, 7a and 9d, decreased IL-6 and TNF-ɑ cytokine levels by 32% and 61%, respectively, in LPS-stimulated cells.
Testing involved nitric oxide assays and real-time PCR for gene expression analysis of key inflammatory markers.
Results suggest that pyridine derivatives may be more effective anti-inflammatory agents than their pyrimidine counterparts.

Abstract

In this study, we aimed to design and synthesize a novel series of pyridine and pyrimidine derivatives and evaluate their anti-inflammatory activity against RAW 264.7 macrophages. Using chalcones (5a-f) as suitable precursors, we disclosed a novel series of pyridine (7a-f) and pyrimidine (9a-e) derivatives via the reaction of 5a-f with 2-cyanothioacetamide or guanidine hydrochloride, respectively. Both pyridines and pyrimidines were tested as anti-inflammatory agents to compare the difference in activity of the pyridine and pyrimidine scaffolds as part of a comparative study. With a percentage of live cells greater than 80%, the pyridines (7a-f) and pyrimidines (9a-e) were found to be safe for RAW cells. Moreover, the anti-inflammatory activity of these compounds was evaluated in lipopolysaccharide (LPS)-stimulated RAW macrophages by performing nitric oxide (NO) assays. Among pyridines, 7a and 7f showed significant inhibition with 65.48% and 51.19%, with IC50 values (IC50 = 76.6 and 96.8 µM), respectively. The pyrimidine derivatives showed promising results as well, 9a and 9d ranking the best activity with 55.95% and 61.90%, respectively, and IC50 values (IC50 = 83.1 and 88.7 µM, respectively). The gene expression levels were assessed for the most promising compounds 7a and 9d using real-time reverse transcription-polymerase chain reaction analysis to measure the mRNA and protein expression levels of inflammatory cytokines, including interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-ɑ), nuclear factor kappa β (NF-kβ), and inducible nitric oxide synthase (INOS). The expression levels of IL-1, IL-6, TNF-ɑ, NF-kβ, and INOS genes were decreased significantly in RAW-treated cells with 7a by 43%, 32%, 61%, 26%, and 53% respectively, compared with negative RAW cells. The expression levels of IL-1, IL-6, NF-kβ, and INOS genes were decreased significantly in RAW-treated cells with 9d by 71%, 48%, 61%, and 65%, respectively, compared with negative RAW cells. However, the expression levels of the TNF-ɑ gene were decreased without significant differences in RAW treated with 9d by 83% (p > 0.05) compared with negative RAW cells. These findings exhibited that 7a was more effective compared with 9d as an anti-inflammatory agent.

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