# Heterocyclic Nitrogen Compounds as Potential PDE4B Inhibitors in Activated Macrophages
## 3 Results and Discussion
### Compounds **1**-**3** Affect LPS-Induced PDE4B Response  
After finding that none of the molecules significantly altered PBMC-derived macrophage cell viability at 100 nM and 10 uM, we decided to test these concentrations in the following experiments. Given that PDE4B is upregulated in response to LPS stimulation leading to increased secretion of ROS inflammatory mediators [27], we investigated the potential effect of synthesized molecules (**1**-**6**) on ROS production in human PBMC-derived macrophages induced by LPS. In M1 macrophages activated with LPS, there are different sites of ROS production, including NADPH oxidase (NOX), the mitochondrial electron transport chain, lipoxygenase, cyclooxygenasegenases (COX), xanthine oxidase, and monoamine oxidase [28], that regulate the proinflammatory response. The redox balance is essential to restore a physiological condition after an inflammatory response. Since many diseases are characterized by ROS dysregulation, identification of the specific source of ROS as well as its specific inhibitors could be relevant. In this context, NOX and PDE inhibitors are under investigation as treatments for neurological disorders [29,30] and arthritis [1,31], respectively. In our experiments, we observed an approximate 20% increase in ROS levels when macrophages were stimulated with LPS (L, Figure 5A), which is completely abrogated in the presence of hydroxyapatite (HCA), the specific inhibitor of ATP citrate lyase (ACLY), able to decrease ROS levels in M1 macrophages [13]. None of the newly synthesized molecules significantly reduced ROS levels when tested at 100 nM (Figure 5A). However, at 10 uM, **3** reduced ROS to levels comparable to those of unstimulated cells (100.3 \(\pm\) 3.3% of C), while **2** exhibited an even stronger decrease (87.3 \(\pm\) 5.3% of C; Figure 5A). Derivatives **1**, **4**, and **6** had no significant effects on ROS levels, whereas **5** even increased ROS secretion compared to LPS-stimulated cells (Figure 5A). Based on the results obtained, we tested synthetic molecules **1**-**6**, at the concentration of 10 uM, on cell extract from LPS-induced macrophages. Our findings confirmed that PDE4B is activated by LPS treatment. Indeed, an increase in the intensity of the yellow-colored product in LPS-triggered macrophage cell extracts was observed when compared with the control (Figure 5B). Furthermore, a decrease in intensity was observed when  
Figure 4: Effect of derivatives **1**-**6** on cell viability. Cell viability was assessed in untreated PBMC-derived macrophages (no-DMSO), or macrophages treated with the vehicle DMSO (C) or synthetic molecules **1**–**6** at the indicated concentrations (100 nM, 10 uM, and 1 mM) after 72 h exposure. Data are expressed as a percentage of C (mean values \(\pm\) SD) from three independent experiments, each with four replicates. Dots represent the mean values obtained from each experiment. Where indicated, differences were significant according to one-way ANOVA followed by [PERSON]’s multiple comparison test (* \(p\) < 0.05; *** \(p\) < 0.001). compounds **1**-**3** were added to the cell extracts (Figure 5B), suggesting that these molecules could affect PDE4B activity in human PBMC-derived macrophages activated by LPS. In summary, our study of the six molecules (**1**-**6**) suggests that the bicyclic structure is more likely to inhibit the PDE4B activity than a tricyclic one. Molecules containing an indole ring as **1** and **2** or a pyridine ring as **3** showed better inhibitory activity in comparison with those having a carbazole ring system as **5** and **6**. This could be because the larger size of the carbazole system in derivatives **5** and **6** might lead to steric hindrance. This means that the bulky carbazole group could prevent proper interaction with crucial amino acids in the active site, hindering effective binding and potentially causing partial inhibition or even activation (as observed at high concentrations).