Design, synthesis and biological evaluation of novel selective inhibitors of the hidrolase O-GlcNAcase (OGA)

Author(s)

Michelle Ogava Igual

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2018-04-19

Examining board members

Ivone Carvalho; Daniel Fabio Kawano

• Advisor: Ivone Carvalho

Abstract

O-GlcNAcylation, or O-GlcNAc modification, consists of the glycosylation of proteins involved in fundamental cellular processes, and its deregulation has been linked to important diseases such as type 2 diabetes, neurodegenerative, cardiovascular diseases, and cancer. O-GlcNAcylation is regulated by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Many OGA inhibitors described exhibit lack of selectivity between OGA and the lysosomal isoenzymes Hex A and B, which can result in the accumulation of gangliosides within the lysosomes and neurodegenerative diseases. Therefore, aiming to obtain more selective inhibitors, this study proposed the synthesis and biological evaluation of N-acetylglucosamine derivatives, containing at the C-2 position the 1,4-di-substituted 1,2,3-triazole ring bearing different side chains, most of them containing aromatic ring at their end, and the investigation of the side chain extension influence between the rings into OGA active site pocket. It was obtained the triazole derivatives 1, 2 (in the previous study) and 8a-j (in the current project) in good to moderate yield through the click chemistry strategy, involving the CuAAC reaction between azide glucopyranoside intermediate 6 and ten different alkynes, which five were previously synthetized (9 - 11, 14 and 15), and five commercial available. For the synthesis of the acetylenic precursors 9 - 11, 14 and 15 it was employed different synthetic strategies according to each alkyne, for example nucleophilic substitution reactions and reductive amination. The triazole derivatives were obtained as a mixture of ?:? anomers in the proportion of 10:1, respectively. Due to the OGA selectivity to ? substrates, compounds 8a-h were purified by HPLC-DAD in order to separate the mixtures. Later in this study, it was possible to obtain the crystallization of azide 6 in ethanol, resulting in the separation of the ? anomer from this precursor, which assisted in the synthetic step of the derivatives 8i and 8j as pure anomers. The MTT assays did not show cytotoxicity for the synthesized compounds, evaluated at 1.0 ?M. The western blot and the enzymatic assays, evaluated for compounds 1, 2 and 8a-j, demonstrated that only compounds 1, 8i e 8j were active towards OGA, with IC50 values of 0.49, 0.52 e 0.72 ?M, respectively. From this result, it was possible to suggest that the ideal side chain extension linked between the triazole and aromatic rings is of two-carbon atoms (1), being able to accommodate one heteroatom of nitrogen (8i) or oxygen (8j). Furthermore, the enzymatic assays, evaluated for compounds 1, 8i e 8j against Hex A and B, exihibted IC50 of 550, 569 e 571 ?M, respectively, suggesting a high selectivity of these compounds to OGA rather than the two lysosomal isoenzymes. In the project developed in partnership with Dra. Nuria E. Campillo (CSIC) it was designed new non-carbohydrates compounds capable of interacting with important residues of the OGA catalytic site with potential biological activity. (AU)

• FAPESP's process: 16/04003-2 - Design, synthesis and biological evaluation of novel selective inhibitors of the hydrolase O-GlcNAcase (OGA)
• Grantee: Michelle Ogava Igual
• Support Opportunities: Scholarships in Brazil - Master