Classical Paal-Knorr Cyclization for Synthesis of Pyrrole-Based Aryl Hydrazones and In Vitro/In Vivo Evaluation on Pharmacological Models of Parkinson's Disease

Abstract

Some studies performed in our laboratory on pyrrole and its derivatives pointed towards the enrichment of the evaluations of these promising chemical structures for the potential treatment of neurodegenerative conditions in general and Parkinson's disease in particular. A classical Paal-Knorr cyclization approach is applied to synthesize the basic hydrazine used for the formation of the designed series of hydrazones (15a-15g). The potential neurotoxic and neuroprotective effects of the newly synthesized derivatives were investigated in vitro using different models of induced oxidative stress at three subcellular levels (rat brain synaptosomes, mitochondria, and microsomes). The results identified as the least neurotoxic molecules, 15a, 15d, and 15f applied at a concentration of 100 µM to the isolated fractions. In addition, the highest statistically significant neuroprotection was observed for 15a and 15d at a concentration of 100 µM using three different injury models on subcellular fractions, including 6-hydroxydopamine in rat brain synaptosomes, tert-butyl hydroperoxide in brain mitochondria, and non-enzyme-induced lipid peroxidation in brain microsomes. The hMAOA/MAOB inhibitory activity of the new compounds was studied at a concentration of 1 µM. The lack of a statistically significant hMAOA inhibitory effect was observed for all tested compounds, except for 15f, which showed 40% inhibitory activity. The most prominent statistically significant hMAOB inhibitory effect was determined for 15a, 15d, and 15f, comparable to that of selegiline. The corresponding selectivity index defined 15f as a non-selective MAO inhibitor and all other new hydrazones as selective hMAOB inhibitors, with 15d indicating the highest selectivity index of >471. The most active and least toxic representative (15d) was evaluated in vivo on Rotenone based model of Parkinson's disease. The results revealed no microscopically visible alterations in the ganglion and glial cells in the animals treated with rotenone in combination with 15d.

Keywords: Parkinson’s; hydrazone; in vivo; pyrrole; synthesis.

Scheme 1

Paal-Knorr synthesis of the initial hydrazide 15.

Scheme 2

General synthesis of the target hydrazones 15a–15g.

Figure 1

Applied phenyl/substituted phenyl aldehydes a–g.

Figure 2

Visualizations of the intermolecular interactions of 15 within the active site of MAOB (PDB: 2V5Z): (A1) 2D panel; (A2) 3D panel, 11 with the active site of MAOB (PDB: 2V5Z); (B1) 2D panel; (B2) 3D panel and 12 with the active site of MAOB (PDB: 2V5Z); (C1) 2D panel; (C2) 3D panel.

Figure 3

Effect of the test substances applied alone at a concentration of 100 µM on synaptosomal viability. * p < 0.05 vs. control (non-treated synaptosomes).

Figure 4

Effect of the test substances applied alone at a concentration of 100 µM on the level of GSH in isolated rat brain synaptosomes (blue color) and mitochondria (brownish color). * p < 0.05 vs. control (non-treated synaptosomes); * p < 0.05 vs. control (non-treated mitochondria).

Figure 5

Effect of test substances applied alone at a concentration of 100 µM on MDA production in isolated rat brain mitochondria (brownish color) and rat brain microsomes (rufescent color). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control (non-treated mitochondria). ** p < 0.01; *** p < 0.001 vs. control (non-treated microsomes).

Figure 6

Effect of newly synthesized hydrazones (at a concentration of 1 µM) on the activity of human recombinant MAOА enzyme (hMAOA). *** p < 0.001 vs. control (pure hMAOA).

Figure 7

Effect of newly synthesized hydrazones (at a concentration of 1 µM), on the activity of human recombinant MAOB enzyme (hMAOB). *** p < 0.001 vs. control (pure hMAOB).

Figure 8

Effect of the most active and least toxic substances, in combination with 6-OHDA, on synaptosomal viability (A) and level of reduced glutathione (GSH) (B). *** p < 0.001 vs. control (non-treated synaptosomes); ++ p < 0.01 vs. 6-OHDA.

Figure 9

Effect of the most active and least toxic substances, in combination with t-BuOOH, on GSH level (A) and on MDA production (B). *** p < 0.001 vs. control (non-treated mitochondria); + p < 0.01 vs. t-BuOOH; ++ p < 0.01 vs. t-BuOOH.

Figure 10

Effect of the most active and least toxic substances under conditions of non-enzyme-induced lipid peroxidation (Fe²⁺/AA). *** p < 0.001 vs. control (non-treated microsomes); ++ p < 0.05 vs. Fe²⁺/AA.

Figure 11

Histological examination of mouse brains (H&E). (A) Control group of animals: normal histoarchitectonics of the cerebral cortex; (B) Control group of animals: normal histological structure of the cortex of cerebellum; (C) Rotenone-treated animal: loss of intercellular space integrity (arrow) and pyknotic changes in ganglion cells (high magnification); (D) Rotenone-treated animal: degenerative-necrotic area with hemorrhage in brain tissue (arrow); (E) Rotenone-treated animal: glial nodule in brain parenchyma (arrow); (F) Cerebellum of rotenone-treated animal: pyknotic changes in Purkinje cells (arrows); (G) Animal treated with 15d alone: normal brain structure; (L) Animal treated with combination of 15d with rotenone: unaltered brain structure.