Violina T. Angelova

Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder that requires therapeutic agents capable of targeting multiple pathological pathways. In this study, a series of cannabidiol (CBD)-like hydrazone derivatives (3a–i) was synthesized and characterized by NMR, HRMS, and single-crystal X-ray diffraction for compound 3i. In silico ADME analysis predicted favorable drug-like properties, including compliance with Lipinski’s Rule of Five, oral bioavailability, and blood–brain barrier permeability. The compounds were evaluated for cholinesterase inhibition, antioxidant activity, cytotoxicity in neuronal cell lines, and binding interactions with human butyrylcholinesterase (hBChE) by molecular docking. Biological evaluation revealed a marked preference for BChE over acetylcholinesterase (AChE). Compound 3f was the most potent BChE inhibitor (IC₅₀ = 1.67 ± 0.11 μM), while compounds 3b and 3f demonstrated high selectivity toward BChE. Antioxidant assays (DPPH, ABTS, FRAP, and FTC) indicated moderate, mechanism-dependent activity. Compounds 3b and 3e showed the strongest ABTS radical-scavenging effects, whereas compounds 3b and 3f provided the greatest protection against lipid peroxidation, surpassing CBD under the tested conditions. Several derivatives, particularly 3a, 3b, 3f, 3h, and 3i, exhibited favorable safety profiles in SH-SY5Y and Neuro-2a cells. Molecular docking supported the experimental findings. Overall, compounds 3b and 3f emerged as promising multifunctional leads for the development of multitarget-directed anti-Alzheimer agents.

Dopamine dysregulation is connected to several neurological disorders, including Parkinson’s disease, Huntington’s disease, and addiction. A new, precise, accurate, and specific reversed-phase high-performance liquid chromatographic method was developed for dopamine determination in different biological media (human/mouse plasma and mouse brain homogenate). The chromatographic assay was performed using Avantor ACE^® RP-18 (250 × 4.6 mm, 5 µm) column equipped with a suitable ODS pre-column. The temperature was ambient, and the mobile phase was composed of 10 mM potassium dihydrogen phosphate buffer (pH = 3) with 0.25 g/L sodium octanesulfonate, methanol, and acetonitrile at a volume-to-volume ratio of 75:20:5. Isocratic elution mode, flow rate 1.0 mL/min, and ultraviolet detection (280 nm) were applied. The procedure was validated for linearity, and all calibration curves were linear over the selected range with determination coefficients greater than 0.998. Intraday repeatability, expressed as the coefficient of variation, did not exceed 4.88% for the plasma and 3.32% for the mouse brain homogenate samples across all tested concentration levels. The proposed chromatographic method was evaluated in terms of greenness, whiteness, and blueness using three ecological metrics (the Analytical Greenness software, White Analytical Chemistry model, and Blue Applicability Grade Index). The optimized procedure was proven to be suitable for implementation in the routine analytical practice.

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.

A newly synthesized indole-based N-benzylpiperidine derivative (3a), acting as a multitarget-directed ligand with acetylcholinesterase inhibitory activity, was encapsulated into solid lipid nanoparticles (SLNs) to improve neuroprotective potential and enable intranasal brain delivery in future in vivo studies. Lipid prescreening was performed, and a spectrophotometric method for the quantification of 3a was developed and validated. SLNs were prepared using the nano-template engineering technique and evaluated for particle size, zeta potential, drug entrapment efficiency, in vitro release, and stability. The optimized formulation (3a-SLNs) was further characterized by transmission electron microscopy (TEM), X-ray powder diffraction (XRD), and differential scanning calorimetry (DSC). The nanoparticles exhibited favorable physicochemical properties, releasing 52% of the drug over 24 h. Permeability studies indicated improved cellular uptake of 3a upon SLN encapsulation. The formulation remained stable for 6 months under refrigerated (4 ± 2 °C) and room temperature (25 ± 2 °C; 60 ± 5% RH) conditions. The neuroprotective potential of 3a-SLNs was evaluated in an H₂O₂-induced oxidative stress model in SH-SY5Y neuronal cells using the MTT assay. Cells were pretreated with free 3a (0.1–50 µM) for 90 min before H₂O₂ exposure (1 mM). Hydrogen peroxide induces oxidative damage via reactive hydroxyl radicals, affecting cellular lipids, proteins, and DNA. 3a-SLNs exhibited superior protective effects compared with free 3a, indicating that nanoparticle encapsulation enhances intracellular availability and antioxidant defense. These findings highlight SLN-encapsulated 3a as a promising intranasal nanocarrier system for Alzheimer’s disease therapy, which will be further investigated in in vivo models. By improving brain delivery potential and reducing oxidative stress–induced neuronal injury, 3a-SLNs offer a strategy to overcome pharmacokinetic limitations of conventional compounds and enhance neuroprotective efficacy.

Background: Breast cancer continues to pose a significant global health challenge despite advances in early detection and targeted therapies. The development of novel chemotherapeutic agents remains crucial, particularly those with selective cytotoxicity toward specific breast cancer subtypes. Methods: A series of ten hybrid indolyl-methylidene phenylsulfonylhydrazones and one bis-indole derivative were designed, synthesized, and structurally characterized using NMR and high-resolution mass spectrometry (HRMS). Prior to synthesis, in silico screening was performed to assess drug likeness and ADME-related properties. Single-crystal X-ray diffraction was conducted for compound 3e. The cytotoxic potential of the synthesized compounds was evaluated using the MTT assay against MCF-7 (ER-α⁺) and MDA-MB-231 (triple-negative) breast cancer cell lines. Additionally, quantitative structure–activity relationship (QSAR) analysis was conducted to identify key structural features contributing to activity. Results: Most compounds exhibited selective cytotoxicity against MCF-7 cells. Notably, compound 3b demonstrated the highest potency with an IC₅₀ of 4.0 μM and a selectivity index (SI) of 20.975. Compound 3f showed strong activity against MDA-MB-231 cells (IC₅₀ = 4.7 μM). QSAR analysis revealed that the presence of a non-substituted phenyl ring and specific indolyl substituents (5-methoxy, 1-acetyl, 5-chloro) significantly contributed to enhanced cytotoxic activity and ligand efficiency. Conclusion: The synthesized phenylsulfonylhydrazone hybrids exhibit promising and selective cytotoxicity, particularly against ER-α⁺ breast cancer cells. Structural insights from QSAR analysis provide a valuable foundation for the further optimization of this scaffold as a potential source of selective anticancer agents.

Alzheimer’s disease (AD) is the most common cause of dementia in older adults and is becoming a major public health crisis as life expectancy increases worldwide. A major contributor to this disease is a deficiency in melatonin signaling. We have recently synthesised a series of melatonin analogs containing donepezil fragments. These compounds have been tested both in silico and in vitro. In this study, a particularly potent compound, 3a, was evaluated in a hybrid rat model of melatonin deficiency and AD. Rats underwent pinealectomy followed one week later by bilateral intracerebroventricular infusion of Aβ_1-42 (1 µg/µL). A 14-day subchronic treatment with compound 3a was started simultaneously with the neurotoxin infusion. Melatonin was used as a reference drug, while a matched sham group received vehicle treatment. One week after the Aβ_1-42 infusion, the rats’ cognitive functions were assessed using two Y-maze protocols, object recognition and object location tests. Markers of oxidative stress, including hippocampal glutathione, superoxide dismutase, and malondialdehyde, were assessed by ELISA. Compound 3a effectively prevented cognitive impairment induced by the AD model, and its effects were comparable to those of melatonin. In addition, this melatonin analogue with a donepezil fragment reduced AD-associated oxidative stress and suppressed model-associated increased Aβ_1-42 levels in the hippocampus. Our findings suggest that melatonin analogs containing donepezil fragments are promising therapeutic options for targeting oxidative stress associated with AD.

Given the complex, multifactorial nature of Parkinson’s and Alzheimer’s diseases (PD and AD), along with the limited effectiveness of single-target treatments, multi-target-directed ligands (MTDLs) present a promising alternative approach. Monoamine oxidases A and B (MAO-A, MAO-B), as well as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are key targets in the development of MTDLs. In our previous study, we developed melatonin–donepezil hybrids with hydrazone (3a–r) and sulfonylhydrazone (5a–l) scaffolds, which showed AChE/BChE inhibition, antioxidant activity, low neurotoxicity, blood–brain barrier permeability, and Aβ42 reduction. This study focused on their MAO-B inhibition. Compounds 3e, 3f, 3i, and 3r showed dual MAO-B/MAO-A inhibition, while 5a and 5c were selective for MAO-B. Docking confirmed strong binding, especially for 3e and 3f. Despite moderate CYP3A4 inhibition (~30–35%) by some compounds, 3e, 3f, 3i, 3r, 5a, and 5c exhibited promising multitarget activity and are strong candidates for further optimization to enhance MAO-B selectivity while minimizing drug–drug interaction risks.

Background/Objectives: New anti-tuberculosis compounds are needed to treat patients infected with multi- or extensively drug-resistant Mycobacterium tuberculosis strains. Studies based on spontaneous in vitro mutagenesis can provide insights into the possible modes of action and resistance mechanisms of such new compounds. We evaluated the primary response of M. tuberculosis in vitro to the action of new aroylhydrazones and nitrofuroylamides. Methods: The reference strain H37Rv was cultured on solid media with compounds at increased concentrations relative to MIC. Resistant clones were investigated using whole-genome sequencing and bioinformatics tools to assess the role and potential impact of identified mutations. Results: Some of the mutations are significant (based on in silico analysis), located in essential genes, and therefore of particular interest. Frameshift mutations were observed in (i) Rv2702/ppgK, which is associated with starvation-induced drug tolerance and persistence in mice, and (ii) Rv3696c/glpK, which has been described as a switch on/off mutation associated with drug tolerance. Nonsynonymous substitutions were found in Rv0506/mmpS2, which belongs to the Mmp protein family involved in transport and drug efflux, and in infB, encoding the translation initiation factor IF-2. Conclusions: The primary adaptation of M. tuberculosis to the selective pressure of the tested compounds is complex and multifaceted. It involves multiple unrelated genes and pathways linked to non-specific drug tolerance, efflux systems, or mechanisms counteracting oxidative stress.

Alzheimer’s disease (AD) involves a complex pathophysiology with multiple interconnected subpathologies, including protein aggregation, impaired neurotransmission, oxidative stress, and microglia-mediated neuroinflammation. Current treatments, which generally target a single subpathology, have failed to modify the disease’s progression, providing only temporary symptom relief. Multi-target drugs (MTDs) address several subpathologies, including impaired aggregation of pathological proteins. In this review, we cover hybrid molecules published between 2014 and 2024. We offer an overview of the strategies employed in drug design and approaches that have led to notable improvements and reduced hepatotoxicity. Our aim is to offer insights into the potential development of new Alzheimer’s disease drugs. This overview highlights the potential of multi-target drugs featuring heterocycles with N-benzylpiperidine fragments and natural compounds in improving Alzheimer’s disease treatment.

Taking into consideration the growing resistance towards currently available antimycobacterials, there is still an unmet need for the development of new chemotherapeutic agents to combat the infectious agents. This study presents X-ray single-crystal analysis to verify the structure of leading sulfonyl hydrazone 3b, which has proven its potent antimycobacterial activity against Mycobacterium tuberculosis H37Rv with an MIC value of 0.0716 μM, respectively, low cytotoxicity, and very high selectivity indexes (SI = 2216), and which has been fully characterized by Nuclear Magnetic Resonance (NMR) and High-Resolution Mass Spectrometry (HRMS) methods. Furthermore, this study assessed the ex vivo antioxidant activity, acute and subacute toxicity, and in vitro inhibition capacity against enoyl-ACP reductase of hydrazones 3a and 3b, as 3a was identified as the second leading compound in our previous research. Compared to isoniazid, compounds 3a and 3b demonstrated lower acute toxicity for intraperitoneal administration, with LD₅₀ values of 866 and 1224.7 mg/kg, respectively. Subacute toxicity tests, involving the repeated administration of a single dose of the test samples per day, revealed no significant deviations in hematological and biochemical parameters or pathomorphological tissues. The compounds exhibited potent antioxidant capabilities, reducing malondialdehyde (MDA) levels and increasing reduced glutathione (GSH). Enzyme inhibition assays of the sulfonyl hydrazones 3a and 3b with IC₅₀ values of 18.2 µM and 10.7 µM, respectively, revealed that enoyl acyl carrier protein reductase (InhA) could be considered as their target enzyme to exhibit their antitubercular activities. In conclusion, the investigated sulfonyl hydrazones display promising drug-like properties and warrant further investigation.