Synthesis of anthraquinone derivatives for Catechol-O-methyltransferase inhibition

• Autor(a) principal: Figueira, Raphael Pinto
• Data de Publicação: 2023
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/14192
• topic: Catecol-o-Metiltransferase; Derivados de Antraquinonas; Doença de Parkinson; Inibidores da Comt; Domínio/Área Científica::Engenharia e Tecnologia::Biotecnologia

Catechol-O-methyltransferase (COMT, EC 2.2.1.6) is the enzyme that O-methylates catecholamine neurotransmitters, such as dopamine, epinephrine and norepinephrine, catechol estrogens and other drugs and substances that have a catecholic structure. Due to its correlation with Parkinson’s disease, cardiovascular diseases, and hormone-dependent cancers, COMT is of great therapeutic interest. Parkinson's disease is a neurological disorder characterized by the degradation of dopaminergic neurons, with a consequent reduction in dopamine levels in the striatum. The most effective treatment for Parkinson’s disease to date involves the administration of levodopa along with COMT and aromatic L-amino acid decarboxylase (AADC) inhibitors to increase the amount of levodopa in the brain, since only 1% of the amount of levodopa administered would reach the brain as it is degraded both by AADC, forming dopamine, and by COMT, whose degradation would lead to the formation of 3-O-methyldopa. However, commercially available COMT inhibitors lack effectiveness, have harmful effects and/or are unable to cross the blood-brain barrier. Thus, it is crucial to find and develop new compounds that are more powerful, less harmful and have better pharmacokinetics properties than the inhibitors currently in clinical use. With this goal in mind, previously, our research group synthesized a class of compounds known as triazolopyrimidines, which can be considered catechol bioisosters and can thus interact with the active site of COMT. In the present work, alizarin, a proven molecule capable of inhibiting COMT, was used as the basis for the synthesis of a novel anthraquinone family compound. The synthesized compound, 3-nitroalizarin, shares many structural similarities with tolcapone. There was also an attempt to synthesize histazarin, an alizarin analogue, as well as the possibility of synthesizing 1-nitrohistazarin. Because of the positions of the hydroxyl and nitro groups, these molecules are more similar to tolcapone than alizarin and histazarin. Unfortunately, histazarin could not be synthesized, which prevented the synthesis of 1-nitrohistazarine. The triazolopyrimidine derivatives and the anthraquinone derivatives were subjected to molecular docking to determine which molecular interactions might occur between the compounds and the active site of the protein and to access their binding affinities. All compounds can be possible inhibitors since it was predicted that they would form molecular interactions with the protein’s active site similar to those that tolcapone established, exhibiting even higher binding energies than tolcapone. Recombinant human MBCOMT lysates were used to test the potency of these compounds, being incubated with the compounds at several concentrations to determine the IC50 values. All compounds had the capability to reduce the MBCOMT activity, however triazolopyrimidine derivatives exhibited lower and constant inhibition across the tested concentrations. Anthraquinone derivatives exhibited the highest potency, with 3-nitroalizarin being the most potent. Subsequently, two different cell lines were used, Normal Human Dermal Fibroblasts (NHDF) and Rat Dopaminergic Neural Cell Line (N27), in cytotoxicity assays for the most promising compounds. These cells were treated with the compounds first in a screening trial and then in a concentration-response assay. While compounds at 10 µM exhibit low cytotoxicity, cell viability dramatically decreases at 100 µM. The results of the concentration-response assays show that the IC50 values are significantly higher than those from the enzymatic assays, proving that these compounds are not hazardous at the concentrations at which they inhibit 50% of COMT activity. Thus, it is concluded that alizarin and 3-nitroalizarin are both very promising molecules and should be used in further research on hepatocyte toxicity testing, co-crystallization, and differential scanning fluorimetry, among others.