Design, Synthesis, and Structure–Activity Relationships of Substituted Phenyl Cyclobutylureas as Potential Modulators of Inflammatory Responses

Figures

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  Figure 1. Context of the development of the new ACBUs series.

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  Scheme 1. Synthesis of substituted phenylcyclobutylureas (ACBUs) 2b–24b.

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  Figure 2. Inhibition of IL-6 production by (a) all soluble and non-antiproliferative ACBUs (10 µM), and dexamethasone (Dex, 1 µM) in HaCaT cells stimulated with TNF-α and IL-17A and (b) ACBUs (10 µM) that exhibit similar activity to dexamethasone (Dex, 1 µM) in the same cell model. ns: non-significant. One way ANOVA with Dunnett’s multiple comparisons test against dexamethasone were performed (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

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  Figure 3. Annexin V/propidium iodide (PI) staining of cells to determine their cell death status (% of stained cells) after treatment with ACBUs 4b, 10b and 16b (100 µM) and staurosporine (stau, 500 nM) after a 48 h treatment. Untreated and cells treated with DMSO 0.2% were used as negative controls (ns: non-significant, * p < 0.05, **** p < 0.0001).

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  Figure 4. Representative scatter plots for (a) iNOS+, (b) CD86+, (c) CD163+ and (d) LAP+ cell populations on THP-1 M1-polarized macrophages after 24 h treatment with the selected ACBUs 4b, 10b and 16b (10 µM). THP-1 M1-polarized macrophages treated with dexamethasone (1 µM) were used as positive control while untreated THP-1 M0-polarized macrophages, untreated THP-1 M1-polarized macrophages and THP-1 M1-polarized macrophages treated with DMSO (0.2 %) were used as negative controls. M0: PMA stimulated THP-1 M0-polarized macrophages. M1: PMA, LPS, IFNγ, and TNFα-stimulated THP-1 M1-polarized macrophages. Dex: Dexamethasone.

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  Figure 5. Histograms summarizing the effect of selected ACBUs 4b, 10b and 16b (10 µM) on THP-1 M1-polarized macrophages of the expression of iNOS, CD86, CD163 and LAP after 24 h treatment. THP-1 M1-polarized macrophages treated with dexamethasone (1 µM) were used as positive control while untreated THP-1 M0-polarized macrophages, untreated THP-1 M1-polarized macrophages and THP-1 M1-polarized macrophages treated with DMSO (0.2 %) were used as negative controls. 1 M0: PMA stimulated THP-1 M0-polarized macrophages. 2 M1: PMA, LPS, IFNγ, and TNFα-stimulated THP-1 M1-polarized macrophages. 3 Dex: Dexamethasone. ns: non-significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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  Figure 6. Inhibition of biotransformation of sEH known substrate PHOME (% of inhibition) of ACBUs 4b, 10b and 16b in (a) human and (b) mouse sEH.

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  Figure 7. Predicted binding mode of the reference inhibitor TPPU within the active site of human soluble Epoxide Hydrolase (sEH) (PDB: 3I28). (a) 3D surface representation showing the ligand occupancy in the hydrophobic tunnel. (b) 2D interaction map highlighting the critical hydrogen-bond network (green dashed lines) with the catalytic triad residues Asp335, Tyr383, and Tyr466, as well as key hydrophobic interactions (pink/purple spheres) with Phe267, Val498, and Met339.

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  Figure 8. Molecular docking pose of the reference inhibitor t-AUCB in the sEH catalytic pocket. (a) 3D visualization of the inhibitor fit. (b) 2D diagram detailing the stabilizing interactions; the urea pharmacophore forms strong hydrogen bonds with Asp335, Tyr383, and Tyr466, while the adamantyl group engages in extensive hydrophobic contacts with residues such as Trp336, Val498, and Leu499.

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  Figure 9. Docking pose and interaction profile of compound 4b in the sEH active site. (a) 3D view of the best-ranked conformation (−5.8 kcal/mol). (b) 2D interaction diagram showing a predominance of non-specific hydrophobic interactions (Pi-Alkyl, Alkyl) with Phe267, Phe381, and Met469. Note the absence of the canonical hydrogen-bond triad observed for the reference inhibitors.

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  Figure 10. Predicted binding mode of compound 10b in the sEH hydrolase domain. (a) 3D visualization of the ligand orientation. (b) 2D interaction map for the top-ranked pose (−7.0 kcal/mol), illustrating hydrophobic contacts with Val498 and Trp336. The binding is primarily driven by Van der Waals forces rather than the specific polar anchoring required for high-affinity inhibition.

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  Figure 11. Molecular modeling results for compound 16b within the sEH active site. (a) 3D representation of the docked ligand. (b) 2D diagram summarizing the intermolecular forces; the binding is supported by hydrophobic interactions with residues such as Phe267 and Met339, consistent with the moderate affinity score (−6.3 kcal/mol) and micromolar inhibitory potency.