Titolo della tesi: New Prospectives on the Benzo[b]thiophene-3-ole Scaffold: Design, Synthesis, and Biological Evaluation of Novel Monoamine Oxidase Inhibitors & Photocatalytic Functionalization of Dehydroalanine-Derived Peptides in Batch and Flow.
Human monoamine oxidases (MAOs) are flavin-containing enzymes located on the outer mitochondrial membrane, which catalyse the oxidative degradation of amines. There are two different isoforms in human organism: hMAO-A and hMAO-B. The two MAO enzymes share similar affinity for dopamine, epinephrine, norepinephrine, and tyramine; serotonin is the preferred substrate for hMAO-A, while hMAO-B has high affinity for benzylamine. The activity of these enzymes significantly impacts the concentration and functional levels of catecholamines in the central nervous system (CNS). As a result, hMAO inhibitors that operate within the CNS are vital for decreasing the degradation of these neurotransmitters, which is essential for treating neurodegenerative conditions such as depression, Alzheimer’s disease, and Parkinson’s disease.
Starting from these evidences, the focus of this project has been the design, synthesis, and biological evaluation of novel monoamine oxidase (MAO) inhibitors based on the open form of benzothiophene-3-ol scaffolds and benzo[b]thiophene-3-ole-2-phenylcarboxamide derivatives, with the aim of developing new effective neuroprotective agents.
Initial studies conducted in 2019 by Professor Daniela Secci’s research group identified the benzo[b]thiophene-3-ol core as a promising scaffold for neuroprotection, demonstrating impressive efficacy in inhibiting monoamine oxidases. [1]
To further enhance the potential of the 2-aroyl-benzo[b]thiophen-3-ol scaffold, we explored chemical modifications inspired by previous structure-activity relationship (SAR) studies, showing that chromone-based compounds featuring an amide spacer at position 3 demonstrated the highest potency and selectivity for MAO inhibition. Building on this finding, we developed a series of carboxamide analogues of the 2-aroyl-benzo[b]thiophen-3-ols by substituting the carbonyl spacer with an amide. [2]
All the derivatives were synthesized through a multi-step process (yields ranging from 33% to 45%) characterized using NMR spectroscopy (1H and 13C) and tested for inhibition of human MAO-A and MAO-B isoforms at a concentration of 10 µM. Most of the compounds demonstrated selectivity for MAO-B, with compound 5 (bearing a 2-methoxy group) achieving complete (100%) inhibition. Structural modifications, such as positional changes of substituents, significantly influenced the activity, with para-methyl (compound 4) showing 65.31% inhibition of MAO-B, while other substitutions were less effective. Additionally, halogenated derivatives showed promising inhibition patterns.
In the second part of the project, we developed novel open-structure analogues of benzo[b]thiophen-3-ols based on the methyl 2-(benzylthio)benzoate scaffold. This flexible structure allowed for greater activity toward hMAO and provided a platform for further chemical modifications. [1]
The new derivatives were obtained through multi-step synthetic procedures, ensuring the reproducibility of the method and reaction yields ranging from 62% to 78%. Each compound was characterized using NMR spectroscopy (1H and 13C) and subsequently evaluated for its inhibitory activity against the A and B isoforms of the monoamine oxidase enzyme through preliminary assays at a fixed concentration of 10 µM. The results from the enzyme inhibition assays revealed that some derivatives, particularly 16B and 20A (methyl 2-((2-(4-bromophenyl)-2-oxoethyl) sulfonyl) benzoate and 2-((2-oxo-2-(p-tolyl) ethyl) sulfinyl) benzoic acid, respectively), exhibited inhibition percentages greater than 50% against the B isoform.
Additionally, for derivatives 16, 16A, and 16B (methyl 2-((2-(4-bromophenyl)-2-oxoethyl)sulfonyl)benzoate, methyl 2-((2-(4-bromophenyl)-2-oxoethyl)sulfinyl)benzoate, methyl 2-((2-(4-bromophenyl)-2-oxoethyl)sulfonyl)benzoate), which showed more than 70% inhibition against the A isoform, further studies were conducted to determine their IC50 values, yielding 3.01 µM, 3.16 µM, and 1.20 µM, respectively. Another promising result was observed for derivative 18A (methyl 2-((2-(4-nitrophenyl)-2-oxoethyl)sulfinyl)benzoate), which demonstrated an inhibition percentage of over 90% against the A isoform, with an IC50 value of 2.86 µM.
This research underscores the potential of these compounds as a monoamine oxidase inhibitor, paving the way for further investigation, including the determination of IC50 values for the most promising derivatives. It also establishes a solid foundation for the continued development of neuroprotective agents aimed at treating neurodegenerative diseases.
The last part of the project has been focused on the Photocatalytic Functionalization of Dehydroalanine-Derived Peptides in Batch and Flow.
Unnatural amino acids, and their synthesis via the late- stage functionalization (LSF) of peptides, play a crucial role in areas such as drug design and discovery. Historically, the LSF of biomolecules has predominantly utilized traditional synthetic methodologies that exploit nucleophilic residues, such as cysteine, lysine or tyrosine. In this study, we present a photocatalytic hydroarylation process targeting the electrophilic residue dehydroalanine (Dha). This residue possesses an α,β-unsaturated moiety and can be combined with various arylthianthrenium salts, both in batch and flow reactors. Notably, the flow setup proved instrumental for efficient scale-up, paving the way for the synthesis of unnatural amino acids and peptides in substantial quantities. Our photocatalytic approach, being inherently mild, permits the diversification of peptides even when they contain sensitive functional groups. The readily available arylthianthrenium salts facilitate the seamless integration of Dha-infused peptides with a wide range of arenes, drug blueprints, and natural products, culminating in the creation of unconventional phenylalanine derivatives. The synergistic effect of the high functional group tolerance and the modular characteristic of the aryl electrophile enables efficient peptide conjugation and ligation in both batch and flow conditions. [3]
1. Guglielmi, P., Secci, D., Petzer, A., Bagetta, D., Chimenti, P., Rotondi, G., ... & Carradori, S. (2019). Benzo [b] tiophen-3-ol derivatives as effective inhibitors of human monoamine oxidase: design, synthesis, and biological activity. Journal of Enzyme Inhibition and Medicinal Chemistry, 34(1), 1511-1525.
2. Reis J, Cagide F, Chavarria D, Silva T, Fernandes C, Gaspar A, Uriarte E, Remião F, Alcaro S, Ortuso F, Borges F. Discovery of New Chemical Entities for Old Targets: Insights on the Lead Optimization of Chromone-Based Monoamine Oxidase B (MAO-B) Inhibitors. J Med Chem. 2016 Jun 23;59(12):5879-93. doi: 10.1021/acs.jmedchem.6b00527.
3. Angew. Chem. Int. Ed. 2024, e202403271