Effect of Novel Pyrrolo[3,4- d]pyridazinone Derivatives on Lipopolysaccharide-Induced Neuroinflammation

Abstract

Neuroinflammation is considered to be one of the potential causes for the development of neurodegenerative diseases, including Alzheimer's disease. In this study, we evaluated the effect of four newly synthesized pyrrolo[3,4-d]pyridazinone derivatives on the neuron-like PC12 cells under simulated inflammation conditions by preincubation with lipopolysaccharide (LPS). Our novel derivatives are selective cyclooxygenase-2 (COX-2) inhibitors and have similar effects to nonsteroidal anti-inflammatory drugs (NSAIDs). We assessed viability (LDH assay), metabolic activity (MTT assay), DNA damage (number of double-strand breaks measured by fast halo assay), and the neuronal features of cells (average neurite length and neurite outgrowth measured spectrofluorimetrically). DCF-DA and Griess assays were also performed, which allowed determining the impact of the tested compounds on the level of oxygen free radicals and nitrites. LPS administration significantly negatively affected the results in all tests performed, and treatment with the tested derivatives in most cases significantly reduced this negative impact. Multiple-criteria decision analysis indicated that overall, the best results were observed for compounds 2a and 2b at a concentration of 10 µM. The new derivatives showed intense activity against free oxygen radicals and nitrites. Reduced reactive oxygen species level also correlated with a decrease in the number of DNA damage. The compounds improved neuronal features, such as neurite length and outgrowth, and they also increased cell viability and mitochondrial activity. Our results suggest that derivatives 2a and 2b may also act additionally on mechanisms other than 3a and 3b.

Keywords: Alzheimer’s disease; LPS; NSAID; cyclooxygenase inhibitor; lipopolysaccharide; neuroinflammation; pyridazinone.

Figure 1

Structures of tested pyrrolo[3,4-d]pyridazinone derivatives: (2a) 3,5,7-trimethyl-6-phenyl-1-[[3-[(4-phenylpiperazin-1-yl)methyl]-2-thioxo-1,3,4-oxadiazol-5-yl] methoxy]pyrrolo[3,4-d]pyridazin-4-one; (2b) 6-butyl-3,5,7-trimethyl-1-[[3-[(4-phenylpiperazin-1-yl)methyl]-2-thioxo-1,3,4-oxadiazol-5-yl] methoxy]pyrrolo[3,4-d]pyridazin-4-one; (3a) 1-[[3-[[4-(4-chlorophenyl)-4-hydroxy-1-piperidyl]methyl]-2-thioxo-1,3,4-oxadiazol-5-yl] methoxy]-3,5,7-trimethyl-6-phenyl-pyrrolo[3,4-d]pyridazin-4-one; (3b) 6-butyl-1-[[3-[[4-(4-chlorophenyl)-4-hydroxy-1-piperidyl]methyl]-2-thioxo-1,3,4-oxadiazol-5-yl] methoxy]-3,5,7-trimethyl-pyrrolo[3,4-d]pyridazin-4-one.

Figure 2

Synthesis and structures of examined derivatives of pyrrolo[3,4-d]pyridazinone.

Figure 3

Effect of tested compounds on PC12 cells preincubated with lipopolysaccharide (LPS): (A) cell viability measured in LDH assay, (B) metabolic activity measured in MTT assay; Control—cell culture incubated without LPS and tested substances; * p < 0.05—significant difference compared to control preincubated with LPS; # p < 0.05—significant difference compared to the negative control without LPS.

Figure 4

Effect of tested compounds on neurites in PC12 cells preincubated with LPS: (A) average length of neurites, (B) neurite outgrowth; Control—cell culture incubated without LPS and tested substances; * p < 0.05—significant difference compared to control preincubated with LPS; # p < 0.05—significant difference compared to the negative control without LPS.

Figure 5

Sample microphotographs of cell cultures stained with “Neurite Outgrowth Staining Kit”: (A) negative control without LPS; (B) control with LPS; (C) LPS and compound 2a (50 µM); (D) LPS and compound 2b (50 µM); (E) LPS and compound 3a (50 µM); (F) LPS and compound 3b (50 µM).

Figure 6

The effect of tested compounds on PC12 cell culture incubated with LPS: (A) DCF-DA assay, (B) Griess assay, (C) fast halo assay; Control—cell culture incubated without LPS and tested substances; * p < 0.05—significant difference compared to control preincubated with LPS; # p < 0.05—significant difference compared to the negative control without LPS.

Figure 7

Microphotographs of a nuclear halo: (A) sample cell incubated without LPS and without compounds (negative control); (B) sample cell incubated only with LPS; (C) sample cell incubated with LPS and compound 2a (10 µM); (D) LPS and compound 2b (10 µM); (E) LPS and compound 3a (10 µM); (F) LPS and compound 3b (10 µM).

Figure 8

Multiple-criteria decision analysis (MCDA) of the effect of the tested pyridazinone derivatives; MCDA was calculated based on E/E_LPS ratios determined in individual assays, where E is the sample result, and E_LPS is the result in control preincubated with LPS.