Synthesis and evaluation of lupeol-derived triterpenic azines as potential neuroprotective agents

Abstract

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the selective loss of dopaminergic neurons and the accumulation of α-synuclein aggregates. Current treatments are primarily symptomatic, highlighting the need for new neuroprotective strategies. Natural triterpenes have shown promise in neurodegenerative diseases, and structural modifications can enhance their bioactivity. In this study, we obtained a series of new triterpenic azines (4a-4p) from lupeol, optimizing reaction conditions through microwave-assisted synthesis. The neuroprotective potential of these derivatives was evaluated in human neuroblastoma IMR-32 cells exposed to 6-hydroxydopamine (6-OHDA), a widely used in vitro model of PD. Compounds 4c, 4m, and 4n significantly prevented 6-OHDA-induced cytotoxicity, restoring cell viability at 10 and 50 μM to control levels. Since ferroptosis is a cell death mechanism implicated in PD, we further examined the effects of these compounds in N27 dopaminergic neurons exposed to the ferroptosis inducers RSL3 and erastin. Among the tested derivatives, 4c exhibited a remarkable protective effect against RSL3-induced ferroptosis, which was comparable to ferrostatin-1, displaying an IC₅₀ value of 9.1 μM. These findings support the development of triterpenic azines as neuroprotective agents and warrant further investigation in preclinical PD models.

Scheme 1. Synthesis of compound 2.

Scheme 2. Synthesis of aromatic hydrazones 3a–p.

Scheme 3. Synthesis of triterpenic azines 4a–p.

Fig. 1. Protective effect of compounds 4a–p against 6-OHDA cytotoxicity in IMR-32 cells. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay after cells were pre-treated with compounds 4a–p (10 and 50 μM) for 30 min, and then with 6-OHDA at 25 μM for 24 h. Results are expressed as percentage of the control. The data shown represent the mean ± SD of three independent experiments. ***p < 0.001 compared to 6-OHDA using one-way ANOVA with Tukey's post hoc tests.

Fig. 2. Compound 4c protects neurons from ferroptosis. (A) The MTT reduction assay was used to evaluate cell viability after exposure to compounds 4c, 4m and 4n at 10 μM, followed by the induction of ferroptosis with RSL3 or erastin. Fer-1 (10 μM) was used as the positive control. (B) Cell morphology was analyzed by light field microscopy. (C) Dose–response experiments of 4c in RSL3-induced ferroptosis in N27 cells. The IC₅₀ was determined by non-linear regression (95% CI in parentheses).