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9 March 2026

17 Pages

N-Benzylpyrrolidine Compounds with MAO-B Inhibitory Activity in an Experimental Model of Parkinson’s Disease

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1
Institute of Neurobiology, Bulgarian Academy of Sciences, 1000 Sofia, Bulgaria
2
Department of Chemistry, Faculty of Pharmacy, Medical University-Sofia, 1431 Sofia, Bulgaria
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Author to whom correspondence should be addressed.
Int. J. Mol. Sci.2026, 27(5), 2496;https://doi.org/10.3390/ijms27052496
This article belongs to the Special Issue New Therapies and Strategies in the Treatment of Neurodegenerative Diseases
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Abstract

The pathogenesis of Parkinson’s disease (PD) is characterized by progressive degeneration of nigrostriatal dopaminergic signaling, resulting in motor dysfunction. Although monoamine oxidase (MAO) inhibitors are clinically used in PD, their long-term efficacy and safety remain limited. In the present study, three novel N-benzylpyrrolidine derivatives (3e, 3f, and 3i), previously identified as dual MAO-A/B inhibitors in silico and in vitro, were pharmacologically evaluated in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. The compounds were administered intraperitoneally starting 2 days prior to MPTP exposure and continuing for 6 days thereafter. Repeated administration of the compounds did not alter striatal dopamine (DA) levels under basal conditions, indicating no detectable modulation of dopaminergic tone in vivo. All three derivatives ameliorated MPTP-induced motor deficits. Compounds 3f and 3i improved motor function without detectable changes in striatal DA levels, whereas compound 3e partially restored striatal DA levels, similar to the positive control. In addition, compound-specific alterations in hippocampal pro-inflammatory cytokines were observed, including increased levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) following 3e administration. Together, these findings provide in vivo pharmacological characterization of novel MAO-targeting derivatives and reveal differential behavioral, neurochemical, and cytokine profiles among the tested compounds, supporting further mechanistic investigation.

1. Introduction

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in the substantia nigra pars compacta, resulting in a profound depletion of striatal dopamine (DA) and the emergence of hallmark motor symptoms, including bradykinesia, rigidity, and resting tremor [1]. In addition to these motor manifestations, PD is associated with a wide spectrum of non-motor complications, such as cognitive impairment, mood disorders, and autonomic dysfunction, which significantly compromise patients’ quality of life [2]. Despite extensive research efforts, current therapeutic strategies remain largely symptomatic—primarily relying on DA replacement therapy or monoamine oxidase B (MAO-B) inhibitors—without effectively halting or reversing disease progression [3].
Accumulating evidence implicates oxidative stress, neuroinflammation, and dysregulated neurotransmitter metabolism as central contributors to PD pathogenesis. Among the enzymes involved, MAO-B plays a critical role in DA catabolism and the generation of reactive oxygen species (ROS), thereby exacerbating oxidative stress and promoting dopaminergic neuronal loss [4]. Consequently, the development of multifunctional compounds capable of modulating MAO-B activity while simultaneously attenuating oxidative stress and neuroinflammation represents a promising strategy for disease modification in PD [5].
N-Benzylpyrrolidine- and pyrrolidine-based derivatives have been widely reported to exhibit significant inhibitory activity against cholinesterases and MAO, highlighting their value as privileged scaffolds in the design of multitarget-directed ligands [6,7,8,9,10,11,12]. In recent years, hybrid molecules combining two or more bioactive pharmacophores have gained considerable attention as potential therapeutic agents for complex disorders such as PD [13,14]. By integrating the neuroprotective properties of selected pharmacophores with the MAO-inhibitory potential of N-benzylpyrrolidine or N-benzylpiperidine scaffolds, such multifunctional compounds may simultaneously target multiple pathological mechanisms underlying PD, including oxidative stress, enzymatic dysregulation, and neuroinflammation [14,15].
In our previous studies, we reported the design, synthesis, in vitro and in vivo biological evaluation of a series of novel melatonin-based hybrid compounds incorporating N-benzylpyrrolidine and N-benzylpiperidine scaffolds [16,17,18,19]. Compounds 3e, 3f, and 3i were selected for further evaluation based on a multi-criteria prioritization strategy integrating MAO-B inhibition potency, neurotoxicity profile, cellular biocompatibility, antioxidant activity, and in silico ADMET predictions [16,19]. Specifically, the selected compounds were prioritized based on enzymatic inhibitory potency toward MAO-B (low IC50 values) comparable to or exceeding that of the reference inhibitor selegiline (sel) [19] and neurobiological safety profile [16,17], including (a) low cytotoxicity in SH-SY5Y neuroblastoma cells; (b) minimal neurotoxic effects in isolated rat brain synaptosomes, mitochondria, and microsomes; (c) biocompatibility assessment, showing negligible cytotoxicity in CCL-1 and Neuro-2a cells (IC50 > 300–800 μM); as well as (a) favorable in silico ADMET predictions, including acceptable LD50 values, low predicted toxicity class, and reasonable lipophilicity (logP), (b) molecular docking analysis [19], demonstrating effective occupation of both entrance and substrate cavities of MAO-B, and (c) antioxidant potential [16], particularly for compound 3e, which exhibited significant ROS-scavenging activity across multiple systems.
In the present study, the compounds 3e, 3f, and 3i were selected as lead structures for further in vivo evaluation due to their structural versatility and favorable interactions with the target enzymes, possession of a balanced optimization of potency, safety, predicted pharmacokinetics, and multifunctional neuroprotective properties for in vivo evaluation using an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD, with sel employed as a positive control. The selected compounds were assessed using a comprehensive battery of behavioral tests to evaluate their efficacy in ameliorating MPTP-induced impairments in motor activity, motor coordination, dyskinesia, and muscle strength. Additionally, their effects on the pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the hippocampus were examined, along with their impact on MPTP-induced alterations in striatal DA levels. Potential side effects were also evaluated in separate control groups treated with a vehicle (veh) in the absence of the neurotoxin.

2. Results

Compounds 3e, 3f, and 3i, which demonstrated MAO inhibitory activity in our prior in silico and in vitro studies, were selected for in vivo evaluation in an acute MPTP mouse model of PD. The chemical structures of these compounds are presented in Figure 1.
Figure 1. The chemical structures of compounds 3e, 3f and 3i synthesized and reported for the first time by Angelova et al. [16].

2.1. Motor Activity

The experimental design is shown in Figure 2. The test compounds and the positive control sel were administered for two days before MPTP intoxication and continued for six days following neurotoxin exposure. Motor activity was assessed on day 5 post-MPTP administration using an actimeter to measure horizontal locomotor activity, mean velocity (V mean), total distance traveled, and resting time. Motor coordination and potential myorelaxant effects were evaluated using the rotarod test, while MPTP-induced motor impairment was assessed using the pole test. Muscle strength and potential muscle-relaxation effects were evaluated using the grip-strength test. The rotarod, pole, and grip strength tests were performed on day 6 after MPTP exposure.
Figure 2. Experimental timeline.
The effects of the novel MAO-A/B-related compounds on locomotor activity under control conditions and in the acute MPTP-induced model are shown in Figure 3A–D. Mice in the MPTP-vehicle group exhibited a significant reduction in motor activity compared to the control group (MPTP-veh group vs. C-veh group: locomotion: p = 0.029; V mean: p = 0.044; resting time: p = 0.05).
Figure 3. Effect of vehicle (veh), selegiline (sel), 3e, 3f and 3i (n = 8) on locomotion (counts) (A), mean velocity (V mean) (cm/s) (B), distance traveled (cm) (C), and resting time (s) (D) under control (C) and MPTP conditions in mice. o p = 0.029, MPTP-veh vs. C-veh (A); One-way ANOVA on ranks under MPTP conditions: H = 9.957, p = 0.041; o p = 0.044, MPTP-veh vs. C-veh; ** p = 0.008, MPTP-sel vs. MPTP-veh; ** p = 0.01, MPTP-3e vs. MPTP-veh; * p = 0.020, MPTP-3f vs. MPTP-veh (B); ** p = 0.008, MPTP-veh vs. MPTP-sel; * p = 0.049, MPTP-3f vs. MPTP-veh (C); One-way ANOVA on ranks under MPTP conditions: H = 9.324, p = 0.05, o p = 0.041 MPTP-veh vs. C-veh, ** p = 0.007, MPTP-sel vs. MPTP-veh, * p = 0.016, MPTP-3e vs. MPTP-veh, * p = 0.040, MPTP-3f vs. MPTP-veh (D).
Treatment with the tested compounds did not affect locomotor activity under control conditions, indicating the absence of sedative effects. Notably, locomotor activity was restored to control level in the 3e (V mean, p = 0.013, MPTP-3e vs. MPTP-veh; resting time: p = 0.016, MPTP-3e vs. MPTP-veh) and 3f-treated (V mean: p = 0.020, MPTP-3f vs. MPTP-veh; distance: p = 0.049, MPTP-3f vs. MPTP-veh; resting time: p = 0.040, MPTP-3f vs. MPTP-veh) MPTP groups. These effects were comparable to those observed with the positive control sel (V mean and distance: p = 0.008, MPTP-sel vs. MPTP-veh; resting time: p = 0.007, MPTP-sel vs. MPTP-veh).

2.2. Rotarod Test

The effects of the three MAO-A/B inhibitors on motor coordination were evaluated by measuring latency to fall from a rotating rod at constant speed under both control and MPTP-induced conditions. Assessments were performed two days before veh/MPTP administration (Day-2), on the day of repeated veh/MPTP injection (Day 0), and six days after injection (Day 6), as shown in Figure 4. No significant differences were observed among treatment groups under either control or MPTP conditions at Day-2 (Figure 4A), indicating that the tested compounds did not induce neurotoxicity. On the day of MPTP administration (Day 0), MPTP-injected mice receiving vehicle exhibited a significant reduction in latency to fall compared with the control group (p < 0.001) (Figure 4B), indicating impaired motor coordination. This MPTP-induced deficit was reversed only by the positive control sel (p = 0.021 vs. MPTP-veh group) (Figure 4B).
Figure 4. Effect of veh, sel, 3e, 3f and 3i (n = 8) on average time before falling off (s) measured in rot-arod test under control (C) and MPTP conditions in mice: no significant difference between groups p > 0.05 (A); ooo p < 001, MPTP-veh vs. C-veh; * p = 0.021, MPTP-sel vs. MPTP-veh (B); One-way ANOVA on ranks under MPTP conditions, H =13.649, p = 0.009; ooo p < 001, MPTP-veh vs. C-veh; * p = 0.017, MPTP-sel vs. MPTP-veh; * p = 0.013, MPTP-3e vs. MPTP-veh; * p = 0.015, MPTP-3i vs. MPTP-veh (C).
By day 6 post-MPTP injection, treatment with compounds 3e and 3i significantly improved motor coordination, as evidenced by increased latency to fall compared with the MPTP-veh group (p = 0.013 and p = 0.015, respectively) (Figure 4C). The effects of the compounds were comparable to those observed with sel (p = 0.017 vs. MPTP-veh group).

2.3. Pole Test

Under control conditions, treatment with the three MAO-B inhibitors or the positive control sel did not significantly affect pole test performance compared with the veh-treated control group (p > 0.05 vs. C-veh) (Figure 5A–C). Consistently, no significant differences among groups were observed on day 2 (Figure 4A). Repeated MPTP injections significantly reduced the latency to fall on day 0 compared with the control group (p = 0.0177, MPTP-veh vs. C-veh) (Figure 5B). All MAO-B inhibitors showed a tendency to reverse the MPTP-induced impairment, with effects comparable to those of the positive control sel.
Figure 5. Effect of veh, sel, 3e, 3f and 3i (n = 8) on average time measured in Pole test under control and MPTP conditions in mice. no significant difference between groups p > 0.05 (A); oo p = 0.005, MPTP-veh vs. C-veh (B); One-way ANOVA on ranks under MPTP conditions: H = 15.548, p = 0.004, oo p = 0.005, MPTP-veh vs. C-veh, *** p < 0.001, MPTP-3i vs. MPTP-veh (C).
To evaluate dyskinesia, the pole test was also performed 6 days after MPTP administration. On Day 6, the acute MPTP model treated with veh exhibited significantly higher scores than the veh-treated control group (p = 0.018, MPTP-veh vs. C-veh) (Figure 5C). Treatment with compound 3i significantly ameliorated MPTP-induced dyskinesia (p < 0.001 vs. MPTP-veh). In contrast, neither sel nor compounds 3e and 3f produced significant improvements compared with the MPTP-veh group (p > 0.05), although a trend toward reduced latency to fall was observed on day 6.

2.4. Grip Strength

Except for C-3i, the administration of the three novel MAO-A inhibitors or the positive control sel did not induce muscle relaxation under control conditions (p > 0.05 vs. C-veh; Figure 6A–C). Thus, 3 days after starting exposure to 3i, rats in this group demonstrated diminished muscle strength compared with the controls (Day 0: p < 0.001; Day 6: p < 0.001). The MPTP-induced reduction in grip strength in the veh-treated group was examined on Days 0 (p = 0.036, MPTP-veh vs. C-veh) and Day 6 (p < 0.001, MPTP-veh vs. C-veh) (Figure 6B,C). Moreover, grip strength was significantly restored on Day 6 in groups treated with the three compounds (p < 0.001, MPTP-3e and MPTP-3f vs. MPTP-veh; p = 0.004 for MPTP-3i vs. MPTP-veh; Figure 6C). A comparable protective effect against MPTP-induced toxicity was observed following treatment with sel (p = 0.006 vs. the MPTP-veh group).
Figure 6. Effect of veh, sel, 3e, 3f and 3i (n = 8) on average time measured in Grip strength test under control (C) and MPTP conditions in mice. no significant difference between groups p > 0.05 (A); One-way ANOVA under C conditions: F4,39 = 3.209, p = 0.025, * p = 0.025, C-3i vs. C-veh, o p = 0.036, MPTP-veh vs. C-veh (B); One-way ANOVA under C conditions: F4,39 = F4,39 = 9.475 p < 0.001, *** p < 0.001 C-3i vs. C-veh, ooo p < 0.001, MTPT-veh vs. C-veh; One-way ANOVA on ranks under MPTP conditions: H = 20.350, p < 0.001 ** p = 0.006, MPTP-sel vs. MPTP-veh, *** p < 0.001, MPTP-3e and MPTP-3f, resp. vs. MPTP-veh, ** p = 0.004, MPTP-3i vs. MPTP-veh (C).

2.5. Interleukin Beta (IL-1β) and Tumor Necrotic Factor Alpha (TNFα)

Following the behavioral assessments, the effects of compounds 3e, 3f, and 3i on hippocampal pro-inflammatory cytokines, IL-1β and TNF-α, were evaluated seven days after MPTP administration under both control and acute MPTP-induced conditions. The positive control sel did not alter the levels of these pro-inflammatory cytokines under control conditions. However, treatment with 3f significantly elevated IL-1β levels compared with the C-veh group (Figure 7A). In addition, the novel compounds significantly increased TNF-α levels (p = 0.009, C-3e vs. C-veh; p < 0.001, C-3f vs. C-veh; p = 0.005, C-3i vs. C-veh) (Figure 7A,B). In the acute MPTP model, MPTP administration did not change the levels of the two cytokines in the hippocampus (p > 0.05 vs. C-veh group) (Figure 7A,B). Moreover, treatment with 3e exacerbated the inflammatory response in MPTP-treated animals, as evidenced by further increases in IL-1β (p = 0.032) and TNF-α (p = 0.004) levels compared with the MPTP-veh group (Figure 7A,B).
Figure 7. IL-beta and TNF-α measured in veh, sel, 3e, 3f and 3i (n = 8) under C and MPTP conditions in mice; ** p = 0.005, C-3f vs. C-veh, *** p < 0. 001, MPTP-3e vs. MPTP-veh (A); ** p = 0.004, C-3e vs. C-veh, *** p < 0.001, C-3f vs. C-veh, *** p < 0.001 C-3i vs. C-veh, ** p = 0.004, MPTP-3e vs. MPTP-veh (B).

2.6. Dopamine (DA) Levels

Striatal DA content was quantified by high-performance liquid chromatography (HPLC) under control conditions and in the acute MPTP model. Representative chromatograms obtained during the analysis are shown in Figure 8A,B.
Figure 8. Typical chromatograms obtained from the analysis of C-veh (A) and MPTP-3e (B) samples. The retention times of the internal standard and DA were 6.8 and 7.8 min, respectively.
Treatment with the positive control sel increased DA levels in both control (C-sel) and MPTP-sel groups. In contrast, among the novel compounds, a significant elevation in striatal DA content was observed only after administration of compound 3e in MPTP-treated animals (Figure 9). Compounds 3f and 3i did not significantly affect striatal DA levels under either condition.
Figure 9. Dopamine levels measured in µg/mL by HPLC in veh, sel, 3e, 3f and 3i (n = 8) under control (C) and MPTP conditions in mice. One-way ANOVA in C conditions: F4,39 = 3.263, p = 0.030, * p = 0.041, C-sel vs. C-veh; MPTP conditions: ANOVA on ranks: H = 19.274, p < 0.001, *** p = 0.001, MPTP-sel vs. MPTP-veh; p = 0.001, MPTP-3e vs. MPTP-veh.

2.7. BBB Permeability Assessment (PAMPA-BBB Assay)

Following favorable in silico predictions [16], the BBB permeability of compound 3i was experimentally evaluated using the PAMPA-BBB assay. Compound 3i exhibited high permeability, with a −logPe value of 4.942 (Table 1), comparable to the high-permeability reference propranolol (−logPe = 4.949) and markedly lower than the medium- and low-permeability controls corticosterone (−logPe = 5.282) and theophylline (−logPe = 6.960), respectively. These results indicate efficient passive diffusion of compound 3i across membranes mimicking BBB properties.
Table 1. Experimentally measured BBB permeability.

3. Discussion

Based on in silico and in vitro screening identifying newly synthesized compounds as potent MAO-A/B inhibitors, three candidates were selected for in vivo evaluation using an acute MPTP mouse model of PD. The neurotoxin was administered at a dose of 20 mg/kg every 2 h over a single day, following the protocol described by Anderson et al. [20]. In this model, MPTP is metabolized by MAO-B to 1-methyl-4-phenylpyridinium (MPP+), a selectively toxic metabolite for dopaminergic neurons, leading to pronounced oxidative stress and striatal neuronal loss.
The results of the present study demonstrate that the selected compounds 3e, 3f, and 3i, when administered both before and after MPTP exposure, exerted behavioral neuroprotective effects. Unexpectedly, despite their demonstrated MAO-A/B inhibitory activity in vitro, repeated administration of these compounds did not modify striatal DA levels under basal (control) conditions, suggesting an absence of functional monoaminergic modulation in vivo. Furthermore, hippocampal levels of the pro-inflammatory cytokines IL-1β and TNF-α were significantly increased compared to veh-treated mice. Notably, the positive control sel increased striatal DA content without affecting hippocampal cytokine levels. Collectively, these findings indicate that the hippocampal pro-inflammatory response induced by the novel compounds in basal conditions, particularly compound 3f, is independent of classical MAO inhibition and likely reflects compound-specific off-target effects. A recent study reported distinct pharmacokinetic profiles for a series of N-benzylpirrolidine-based hydrazide–hydrazone derivatives, with compounds 3e and 3i predicted in silico to cross the BBB, supporting their potential central nervous system activity [16]. In contrast, compound 3f, despite exhibiting high gastrointestinal absorption, showed no predicted BBB permeability. BBB permeability for the lead compound 3i was experimentally evaluated using the PAMPA-BBB assay. Compound 3i demonstrated high BBB permeability, exhibiting a −logPe value of 4.942, which is comparable to the high-permeability reference propranolol (−logPe = 4.949) and markedly lower than those of the medium- and low-permeability controls corticosterone (−logPe = 5.282) and theophylline (−logPe = 6.960), respectively. These results indicate that compound 3i undergoes efficient passive diffusion across membrane systems that mimic BBB properties. The strong agreement between in silico predictions and experimental PAMPA-BBB data strengthens the classification of 3i as a CNS-relevant candidate. Collectively, these findings provide a rationale for advancing compounds 3e and 3i to in vivo studies to confirm their therapeutic potential.
The model group administered with veh showed decreased motor activity in the actimeter, impaired motor coordination in the rotarod test, and dyskinesia in the pole test, as compared with the controls on the day of neurotoxin administration and 6 days later. In addition, MPTP-treated mice demonstrated reduced muscle strength in the grip strength apparatus after repeated administration of the neurotoxin and on day 6. The neurotoxicity induced by MPTP treatment causes a depletion of DA in the striatum, underlying motor dysfunction including bradykinesia, coordination deficits, and reductions in muscle strength consistent with PD-like motor impairments [21,22,23,24,25,26,27]. However, in our study, the observed clear motor deficit was not accompanied by hippocampal IL-1β/TNF-α elevation or striatal DA depletion in the model group (MPTP-veh), suggesting functional dopaminergic dysfunction without overt neurochemical loss. A reduced DA level following a single day of repeated MPTP injections in mice was reported on the 15th day [28]. The detected behavioral deficits on the day of treatment and 5 and 6 days later might precede measurable biochemical alterations in our study and can be explained by altered firing of nigrostriatal neurons or by turnover. The literature reports that acute MPTP effects cause a rapid onset of striatal DA loss shortly after dosing. However, the depth of depletion and timeline vary with regimen and mouse strain [29,30,31]. The discrepancy between our results and others in the acute MPTP model may be due to methodological differences, particularly strain differences (e.g., C57BL/6 vs. Swiss), which should influence sensitivity to MPTP toxicity and DA depletion. In our model, the detected motor dysfunction in the acute MPTP model might reflect early functional impairment of motor circuits rather than overt dopaminergic neurodegeneration or hippocampal inflammation.
On the day of neurotoxin administration, pretreatment with the three compounds exhibiting MAO A/B inhibitory activity did not affect the reductions in motor coordination or dyskinesia induced by MPTP. In contrast, the reference drug sel only restored the impaired motor coordination that had been observed in the rotarod test. Continued administration of the compounds corrected the motor deficit 5–6 days after MPTP exposure, demonstrating an effect comparable to sel. The three compounds had different beneficial effects on the rotarod and pole tests, but all improved motor weakness induced by MPTP, as shown by the grip strength test.
Research from human studies and animal models indicates that chronic inflammation is a key factor in the onset and progression of PD [32]. The outcome of central and peripheral inflammatory response is loss of dopaminergic neurons in the substantia nigra pars compacta [33,34]. Enhanced secretion of IL-1β and TNF-α was observed in patients with PD [35]. The two cytokines are key signaling molecules secreted by activated microglia, thereby contributing to immune dysfunction and concomitant progressive dopaminergic neuronal degeneration [36]. However, IL-1β release can also be induced in neurons [37]. Furthermore, the transcript of the transmembrane receptor for IL-1β was detected in the rat hippocampus [38]. Given that these two cytokines play a crucial role in severe inflammation resulting from microglial activation, which is involved in the pathogenesis of PD, drugs targeting them are considered potential neuroprotective agents against dopaminergic neurodegeneration [39]. Factors that trigger overproduction of IL-1β, but not its acute expression, have a damaging impact on SN and subsequent motor behavior. In the present study, the acute MPTP protocol in mice did not produce a significant surge in the two pro-inflammatory cytokines, IL-1β and TNF-β, in the hippocampus, nor did it induce DA loss in the striatum. The impact of IL-1β on dopaminergic cells might vary depending on factors that affect its pharmacokinetic or pharmacodynamic profile. Our results are consistent with reported data showing that IL-1β produced in response to acute 6-OHDA or lipopolysaccharide (LPS) treatment is insufficient to damage the SN [40,41]. In addition, 6-OHDA-induced neuroinflammation in the SN altered the transcription. Still, translation of this cytokine [42] and additional stimuli, such as a low, ineffective dose of LPC applied in the SN, might trigger the protein synthesis [41].
The signaling pathways triggered by TNF-α might have either damaging or protective effects depending on the activation of TNFR1 or TNFR2 receptors in tissues [43]. TNFR1 signaling is associated with pro-inflammatory, tissue-injuring responses, whereas TNFR2 signaling can be protective, supporting tissue repair and countering deleterious effects. In addition, data on the role of this cytokine expression induced by MPTP in TNFR knock-out mice are ambiguous [44,45], suggesting complex relationship between TNF-α and MPTP with either protection or neurotoxicity depending on the duration of exposure to MPTP.
Within the context of the acute MPTP model, the observed increase in IL-1β and TNF-α in the hippocampus following MAO-A/B inhibition is biologically plausible. This phenomenon may be attributed to multiple non-mutually exclusive mechanisms. It is important to note that this does not necessarily contradict the concept of neuroprotection. In the present study, while the compound 3e effectively ameliorated motor deficits and increased striatal DA levels, it also induced pro-inflammatory cytokine expression in the hippocampus. This suggests that its motor benefits and inflammatory effects arise from dissociable mechanisms. Despite increased levels of IL-1β and TNF-α in the hippocampus, MAO-A/B inhibition robustly restored MPTP-induced motor deficits and normalized striatal DA content. This apparent dissociation suggests that cytokine induction in this context does not reflect deleterious neuroinflammation but rather a region-specific, adaptive immune response. The inhibition of MAO-A and MAO-B has been shown to elevate monoaminergic tone and reduce MAO-derived oxidative stress. This, in turn, has been demonstrated to preserve nigrostriatal dopaminergic function. It is this latter function that is the primary determinant of motor performance in the acute MPTP model. Concurrently, transient elevations in hippocampal IL-1β and TNF may indicate controlled glial activation, a process known to be particularly prominent in the hippocampus and implicated in synaptic plasticity and stress adaptation. It is noteworthy that TNF family signaling, particularly via TNFR2, and low-to-moderate IL-1β signaling have been implicated in neuroprotective and homeostatic functions rather than neurotoxicity when temporally restricted [44,45]. Consequently, the behavioral recovery observed in the present study is consistent with effective dopaminergic rescue in the striatum with 3e compound treatment. At the same time, the elevation of cytokines in the hippocampus is likely to represent compensatory immune-neuromodulatory signaling rather than pathology.

Limitation of the Study

Several limitations of this study should be considered when interpreting the present findings. First, under the present experimental conditions, the acute MPTP paradigm resulted in motor deficits without measurable changes in striatal dopamine content or overt hippocampal cytokine elevation in vehicle-treated MPTP animals. Therefore, the findings should be interpreted within the context of toxin-induced functional motor disturbances rather than established dopaminergic neurodegeneration or chronic Parkinsonian pathology. Second, cytokine measurements were conducted in whole hippocampal homogenates, precluding identification cellular sources or region-specific inflammatory mechanisms. Future studies employing histological, cell-type-specific or spatial transcriptomic approaches will be required to define the cellular pathways involved. Third, although compound 3e induced hippocampal IL-1β and TNF-α levels, the functional consequences of this molecular response were not directly assessed. The present study establishes an association between cytokine elevation and compound exposure but does not demonstrate causal linkage to behavioral outcomes or long-term safety. Finally, while sel was included as a reference compound, differences in pharmacokinetic properties and off-target profiles among tested compounds were not systematically evaluated and may contribute to the observed pharmacological profiles. All experiments were conducted with appropriate vehicle-treated, MPTP-only, and positive control groups (selegiline) under standardized conditions. The inclusion of selegiline as a clinically established MAO-B inhibitor confirmed pharmacological responsiveness of the behavioral paradigm. Nevertheless, additional independent replication and expanded mechanistic analyses would further strengthen the robustness and interpretability of these findings. Despite these limitations, the findings provide a comprehensive pharmacological characterization of the compounds’ motor efficacy and identify a compound-specific cytokine alterations that appear dissociated from motor efficacy.

4. Materials and Methods

4.1. Drugs and Treatment

Reagents used in the synthetic, pharmacological experimental work, and bioanalysis of DA were purchased from E. Merck (Darmstadt, Germany), and Sigma Aldrich (St. Louis, MO, USA). The structure and purity of the newly synthesized compounds 3e, 3f, and 3i (Figure 1) were confirmed by 1H NMR, 13C NMR, and high-resolution mass spectrometry (HRMS) [16]. The NMR spectra were recorded in DMSO-d6 on a Bruker Avance III 600 MHz spectrometer (Bruker, Billerica, MA, USA), using tetramethylsilane (TMS) as an internal standard. HRMS analysis was performed using an Agilent Accurate-Mass Q-TOF LC/MS G6520B system equipped with a dual electrospray ionization (DESI) source (Agilent Technologies, Santa Clara, CA, USA).

4.2. Animals

Young adult male Swiss mice (25–30 g) were purchased from the vivarium of the Institute of Neurobiology, BAS, and were left untreated for a week after arrival. They were accommodated in standard conditions before the experiments as follows: transparent cages (8–10 mice per cage), a strict diurnal regime of 12:12 light/dark exposure, temperature (22 ± 1 °C), and access to animal pellets and tap water ad libitum. The procedures were conducted in agreement with the Declaration of Helsinki on the care and use of animals (DHEW publication, NHI 80-23) and the EC Directive 2010/63/EU on animal experimentation. The experimental design was approved by the Bulgarian Food Safety Authority (license number: 354/2023).

4.3. MPTP-Induced Parkinson’s Disease Model

The mice were intraperitoneally (i.p.) injected with either saline or MPTP (FOT Ltd., Sofia, Bulgaria) 4 times at 2 h intervals on day 0 at a dose of 20 mg/kg according to the protocol of Jackson-Lewis and Przedborski [46]. They were euthanized on day 7 after MPTP administration for dissection of the hippocampus and striatum, respectively.

4.4. Experimental Design

The experimental procedures and their sequence are summarized in Figure 1. Selegiline was used as a positive control, and the dose was selected based on previous studies [47]. The compounds were applied in equimolar doses. The mice were randomly distributed in two subsets as follows: five control groups—C-veh (a group treated with vehicle), C-sel, a group treated with veh and sel (10 mg/kg), C-3e, a group treated with veh and 3e (17.27 mg/kg), C-3f, a group treated with veh and 3f (18.1 mg/kg), C-3i, a group treated with veh and 3i (18.9 mg/kg); five MPTP-treated groups—MPTP-veh, a group treated with MPTP and veh, MPTP-sel, a group treated with MPTP and sel, MPTP-3e, a group treated with MPTP and 3e, MPTP-3f, a group treated with MPTP and 3f, MPTP-3i, a group treated with MPTP and 3i. All treatments were administered i.p. at a volume of 10 mL per 10 g body weight.

4.5. Behavioral Tests

4.5.1. Motor Activity

Locomotor activity was assessed 5 days after MPTP treatment. Horizontal activity (locomotion) (counts), as well as mean velocity (mean V) (cm/sec) (distance cm) and resting time (sec), were registered for 10 min by an actimeter (20 cm high, 45 cm wide, and 45 cm long) (Bioseb, Vitrolles, France).

4.5.2. Pole Test

A pole test was conducted three times: 2 days before the MPTP injection, on the day of treatment with MPTP, and 6 days later (Figure 1). The mice were trained for 2 consecutive days before testing on a rod (50 cm high, 0.7 cm wide) for up to 120 s. The time to successfully descend from the top to the bottom of the pole was detected.

4.5.3. Rotarod Test

Before the test on days “−2”, “0” and “6”, mice were trained (3 trials for 1 min each) in a rotarod (3.2 cm in diameter, at a speed of 10 rpm). The tested mouse was considered to have failed if it did not remain on a rotating rod for at least 60 s across three trials. The average time before falling off was measured.

4.5.4. Grip Strength Test

Skeletal muscle strength was assessed using the grip strength apparatus (BioSeb, France) as described earlier [48]. The test was repeated three times per mouse: 2 days before, 1 h after the 4th MPTP injection, and 6 days later. The average of three measurements of muscle strength for each animal was expressed in newtons (N) per 100 g BW.

4.6. Biochemistry

To determine cytokine levels in the hippocampus and DA levels in the striatum, the mice were euthanized by neck dislocation, and the hippocampus and striatum were gently dissected. Then, the tissue was weighed quickly and placed in a refrigerator at −80 °C for use. The mixture was homogenized at 3500 rmp/min for 99 s and centrifuged at 12,000 rmp/min for 20 min in a low-temperature high-speed centrifuge. At last, the supernatant was removed to obtain the biological sample for testing.

4.6.1. Elisa Test

Total protein levels in mouse hippocampal tissue were determined using the Bradford protein assay. All samples were normalized to a final protein concentration of 1.25 mg/mL, relative to the lowest measured protein concentration.
The pro-inflammatory cytokines TNF-α (tumor necrosis factor alpha) and IL-1β (interleukin-1 beta) were quantified using ELISA kits: Mouse TNF-α ELISA Kit (E-EL-M3063) and Mouse IL-1β ELISA Kit (E-EL-M0037) (Elabscience Biotechnology Co., Ltd., Houston, TX, USA). All assays were performed according to the manufacturer’s instructions.

4.6.2. HPLC

The HPLC-UV method was used to determine DA levels using 3,4-dihydroxybenzylamine hydrobromide (DHBA) as the internal standard. The optimal analysis conditions were as follows: reversed-phase chromatographic column Avantor ACE RP-18 (250 mm × 4.6 mm, 5 μm), isocratic elution, flow rate 1.0 mL/min, and ambient temperature. The mobile phase contained a mixture of 10 mM monopotassium phosphate buffer (pH = 3) with 0.25 g/L sodium octanesulfonate: methanol: acetonitrile in a ratio of 75:20:5 (v/v/v). The analysis was performed at 280 nm, and the total running time was less than 10.0 min. The stock solution of DHBA-IS was prepared by dissolving 5 mg in methanol to obtain a concentration of 100 μg/mL. A working solution was prepared by pipetting an aliquot of 0.5 mL, transferring to a 5.0 mL volumetric flask, and diluting with methanol. The obtained concentration was 10 μg/mL. Briefly, the sample preparation procedure included the following steps: mixing 250 μL of a mouse brain homogenate aliquot with 50 μL of the DHBA-IS working solution for 1 min, adding 200 μL of acetonitrile, vortexing for 5 min, and centrifuging for 10 min at 13,000 rpm. The supernatant was pipetted, and 450 μL were filtered through a 0.45 μm syringe filter. RP-HPLC analysis was carried out with an injection volume of 20 μL.

4.7. Statistical Analysis

All data were represented as means ± SEM. One-way ANOVA was applied separately to the C groups (groups under basal conditions) and the MPTP groups (GraphPad Prism, version 6.0, GraphPad Software, Boston, MA, USA). An independent t-test was also used to compare the C-veh and MPTP-veh groups. The statistically significant difference was accepted at a p-value of <0.05.

4.8. Assessment of BBB Using the In Vitro PAMPA Test

The permeability of the tested compounds across the blood–brain barrier (BBB) was evaluated using a PAMPA Permeability Analyzer (pION Inc., Billerica, MA, USA). Stock solutions of the test compounds and reference controls were prepared at a concentration of 10 mM in DMSO and subsequently diluted with Prisma HT buffer (pH 7.4). The assay was conducted following the standard BBB PAMPA protocol. Briefly, 200 µL of each sample solution was added to the donor wells, and an equal volume of Brain Sink Buffer was introduced into the acceptor wells. The permeation membranes were coated with BBB-1 lipid. The donor and acceptor plates were then assembled in a sandwich configuration. The system was then incubated for 4 h at 25 °C under controlled humidity, without agitation. After incubation, UV absorbance spectra for blanks, standards, donor wells, and acceptor wells were recorded within the range of 250 to 500 nm. Effective permeability values (Pe, expressed in 10−6 cm/s) were calculated and reported as logPe. All measurements were performed in triplicate, and mean values were used forced for analysis. Compounds were classified as highly permeable when −logPe < 5, moderately permeable when 5 ≤ −logPe ≤ 6, and poorly permeable when −logPe > 6 [16]. The reference compounds theophylline, corticosterone, and propranolol HCl served as assay controls to ensure experimental reliability and reproducibility [49].

5. Conclusions

In the present study, acute MPTP administration produced pronounced motor impairments across multiple behavioral paradigms in the absence of detectable striatal DA depletion or overt hippocampal cytokine elevation. These findings indicate that, under the present conditions, the model reflects toxin-induced functional motor disturbances rather than established DAergic neurodegeneration characteristics of PD. Treatment with the tested compounds reversed these motor deficits, demonstrating pharmacological efficacy in this acute paradigm. Compound 3e additionally increased hippocampal IL-1β and TNF-α levels in both control and MPTP-treated mice. This cytokine elevation was observed despite motor improvement and was not reproduced by the positive control sel, indicating that the pro-inflammatory response associated with 3e was not directly correlated with behavioral efficacy or with the DA-elevating effect observed with the reference compound. Importantly, the primary objective of this study was pharmacological characterization rather than definitive mechanistic dissection. Within this defined scope, the findings identify compound-specific behavioral efficacy and differential neurochemical and cytokine profiles that warrant further mechanistic investigation.

Author Contributions

Conceptualization, J.T., V.T.A. and V.M.; methodology, J.T., P.I., V.T.A., S.B., M.S., and V.M.; software, N.B.; validation, P.I., N.B. and J.T.; formal analysis, N.B.; investigation, J.T., P.I., and V.M.; resources, V.M.; writing—original draft preparation, J.T.; writing—review and editing, J.T., V.T.A. and V.M.; project administration, V.M.; funding acquisition, V.M. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Project BG-RRP-2.004-0004-C01: “Strategic Research and Development Innovation Program of Medical University-Sofia”.

Institutional Review Board Statement

The procedures were conducted in agreement with the Declaration of Helsinki on the care and use of animals (DHEW publication, NHI 80-23) and the EC Directive 2010/63/EU on animal experimentation. The experimental design was approved by the Bulgarian Food Safety Authority (license number: 354/2023) on 20 January 2023.

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors thank Iva V. Valkova (Department of Chemistry, Faculty of Pharmacy, Medical University of Sofia, 1000 Sofia, Bulgaria; ivalkova@pharmfac.mu-sofia.bg) for her valuable support in performing the in vitroPAMPA-BBB permeability studies.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
PDParkinson’s disease
MAOmonoamine oxidase
DADopamine
MPTP1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
IL-1βinterleukin-1β
TNF-αtumor necrosis factor-α
ROSreactive oxygen species
selselegiline
HPLChigh-performance liquid chromatography
MPP+1-methyl-4-phenylpyridinium
BBBblood–brain barrier
LPSlipopolysaccharide
SNSubstancia nigra
HRMShigh-resolution mass spectrometry
TMStetramethylsilane
DHBA3,4-dihydroxybenzylamine hydrobromide

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