Rita Gonçalves-Pereira

Alzheimer’s disease (AD) is the most common age-related cause of dementia among elderly people. AD is a severe neurodegenerative disorder characterized by progressive memory and cognition loss that leads to disability and inevitably to death and is considered as an urgent public health problem. It is the third leading cause of death after cancer and heart disease. According to an update in 2020 of the estimates given in the World Alzheimer Report 2015, there are over 50 million people worldwide living with dementia in 2020. This number will almost double every 20 years, reaching 152 million in 2050. AChE inhibitors are the mainstay drugs for early disease stages. In this work we report on the development of a synthetic route to yield open chain sugar amides from commercially available carbohydrates. The synthetic pathway starts with diacetone glucose (DAG), which is converted into perbenzyl d-glucono-1,4-lactone in six steps. Reaction with aromatic or aliphatic amines in dichloromethane under reflux (0.5 h to 2 h) afforded the corresponding amides in high yield (80–95%). Bis(amidation) of a diamine was also accessed by this procedure in 3 h but the reaction product was isolated in a very low yield (13%). Docking studies and evaluation of acetylcholinesterase inhibition were carried out and the results will be disclosed.

The synthesis and biological evaluation of novel guanidino sugars as isonucleoside analogs is described. 5‐Guanidino xylofuranoses containing 3‐O‐saturated/unsaturated hydrocarbon or aromatic‐containing moieties were accessed from 5‐azido xylofuranoses via reduction followed by guanidinylation with N,N′‐bis(tert‐butoxycarbonyl)‐N′′‐triflylguanidine. Molecules comprising novel types of isonucleosidic structures including 5‐guanidino 3‐O‐methyl‐branched N‐benzyltriazole isonucleosides and a guanidinomethyltriazole 3′‐O‐dodecyl xylofuranos‐5′‐yl isonucleoside were accessed. The guanidinomethyltriazole derivative and a 3‐O‐dodecyl (N‐Boc)guanidino xylofuranose were revealed as selective inhibitors of acetylcholinesterase (K_i=22.87 and 7.49 μM, respectively). The latter also showed moderate antiproliferative effects in chronic myeloid leukemia (K562) and breast cancer (MCF‐7) cells. An aminomethyltriazole 5’‐isonucleoside was the most potent molecule with low micromolar GI₅₀ values in both cells (GI₅₀=6.33 μM, 8.45 μM), similar to that of the drug 5‐fluorouracil in MCF‐7 cells. Moreover, the most bioactive compounds showed low toxicity in human fibroblasts, further indicating their interest as promising lead molecules.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, mainly characterized by motor deficits correlated with progressive dopaminergic neuronal loss in the substantia nigra pars compacta (SN). Necroptosis is a caspase-independent form of regulated cell death mediated by the concerted action of receptor-interacting protein 3 (RIP3) and the pseudokinase mixed lineage domain-like protein (MLKL). It is also usually dependent on RIP1 kinase activity, influenced by further cellular clues. Importantly, necroptosis appears to be strongly linked to several neurodegenerative diseases, including PD. Here, we aimed at identifying novel chemical inhibitors of necroptosis in a PD-mimicking model, by conducting a two-step screening. Firstly, we phenotypically screened a library of 31 small molecules using a cellular model of necroptosis and, thereafter, the hit compound effect was validated in vivo in a sub-acute 1-methyl-1-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) PD-related mouse model. From the initial compounds, we identified one hit—Oxa12—that strongly inhibited necroptosis induced by the pan-caspase inhibitor zVAD-fmk in the BV2 murine microglia cell line. More importantly, mice exposed to MPTP and further treated with Oxa12 showed protection against MPTP-induced dopaminergic neuronal loss in the SN and striatum. In conclusion, we identified Oxa12 as a hit compound that represents a new chemotype to tackle necroptosis. Oxa12 displays in vivo effects, making this compound a drug candidate for further optimization to attenuate PD pathogenesis.

The synthesis and anticancer evaluation of new series of nucleosides constructed on 5/6-azidoglycosyl or glucuronamide moieties and containing an O- or an N-dodecyl chain, respectively, are disclosed. Based on our previous results, their structures were planned to preclude them to act via a similar metabolic pathway than that of clinically used nucleoside antimetabolites, against which cancer cells frequently acquire resistance. Xylo and gluco-configured 5/6-azido-1,2-di-O-acetyl furanosyl and pyranosyl donors containing a 3-O-dodecyl group were synthesized from diacetone-d-glucose and were subsequently coupled with silylated uracil or 2-acetamido-6-chloropurine. N-Dodecyl glucuronamide-based nucleosides were accessed from acetonide-protected glucofuranurono-6,3-lactone, which was converted in few steps into O-benzylated 1,2-di-O-acetyl furanuronamide or pyranuronamide derivatives to undergo further N-glycosylation. Both types of nucleosides demonstrated notorious antiproliferative effects in chronic myeloid leukemia (K562) and in breast cancer (MCF-7) cells. The most potent molecules were a 6ʹ-azidoglucopyranosyl N⁷-linked purine nucleoside and glucofuranuronamide derivatives comprising N¹-linked uracil and N⁷-linked purine units with activities in the single-digit micromolar order of concentration against both cell lines. Their GI₅₀ values in MCF-7 cells were similar or ca. 3-fold lower than that of the standard drug 5-fluorouracil. Cell cycle studies and immunoblotting analysis of apoptosis-associated proteins in treated K562 cells indicated that the antiproliferative effect of the most effective nucleosides is based on apoptosis induction.

The synthesis of novel types of furanosyl nucleoside analogues, namely N‐(benzyltriazolyl)methyl glucuronamide derivatives, N‐dodecyl glucuronamide‐based phenyltriazole nucleosides, and theobromine xylosyl 5′‐isonucleosides, as potential cholinesterase inhibitors is described herein. O‐Substituted and partially O‐substituted N‐propargyl glucuronamides, accessed from glucofuranurono‐6,3‐lactone, were engaged in Cu^I‐catalyzed cycloaddition with benzyl azide, whereas their N‐dodecyl uronamide counterparts were converted in three steps into glycosyl azides, which were subjected to cycloaddition with phenylacetylene. A xylofuranose derivative having a free 5‐OH group was coupled with theobromine by Mitsunobu reaction and the obtained isonucleoside was functionalized at C‐1′ with a sulfonamide moiety, leading to a prospective nucleotide mimetic. Five compounds displayed selective inhibition of acetylcholinesterase in the micromolar concentration range, with an α‐glycosyl triazole (K_i = 3.53 µm) and its 1‐azido‐uronamide precursor (K_i = 1.73 µm) being the most active. Docking studies were performed to give insights into the different inhibitory behavior within glycosyl azide anomers. Two of the best inhibitors showed low toxicity in both a neural cell line and human fibroblasts, rendering them promising lead compounds and supporting further investigations.

New xylofuranosyl and glucopyranosyl nucleoside phosphoramidates were synthesized as potential mimetics of nucleoside 5′-monophosphates. Their access involved N-glycosylation of uracil and 2-acetamido-6-chloropurine with 5′/6′-azido-1,2-di-O-acetyl glycosyl donors and subsequent Staudinger-phosphite reaction of the resulting azido nucleosides. The coupling of the purine derivative with the pyranosyl donor furnished N⁹- and N⁷-linked nucleosides in 1:1 ratio, whereas with the furanosyl donor, the N⁹-nucleoside was the major regioisomer formed. When using uracil, only 5′/6′-azido N¹-linked nucleosides were obtained. The purine 5′/6′-azido nucleosides were converted into corresponding phosphoramidates in good yields. The antiproliferative effects of the nucleoside phosphoramidates and those of the azido counterparts on cancer cells were evaluated. While the nucleoside phosphoramidates did not show significant activities, the purine 5′/6′-azido nucleosides displayed potent effects against K562, MCF-7 and BT474 cell lines. The 5′-azidofuranosyl N⁹ and N⁷-linked purine nucleosides exhibited highest activity towards the chronic myeloid leukemia cell line (K562) with GI₅₀ values of 13.6 and 9.7 μM, respectively. Among pyranosyl nucleosides, the N⁷-linked nucleoside was the most active compound with efficacy towards all cell lines assayed and a highest effect on K562 cells (GI₅₀=6.8 μM). Cell cycle analysis of K562 and MCF-7 cells showed that the most active compounds cause G2/M arrest.

The first examples of amino acid (Ser/Thr)–sp²-iminosugar glycomimetic conjugates featuring an α-O-linked pseudoanomeric linkage are reported. The key synthetic step involves the completely diastereoselective α-glycosylation of Ser/Thr due to strong stereoelectronic and conformational bias imposed by the bicyclic sp²-iminosugar scaffold. Mucin-related glycopeptides incorporating these motifs were recognized by the monoclonal antibody (mAb) scFv-SM3, with activities depending on both the hydroxylation pattern (Glc/Gal/GlcNAc/GalNAc) of the sp²-iminosugar and the peptide aglycone structure (Ser/Thr).