Design and synthesis of novel non peptidomimtic beta-secretase inhibitors in the treatment of Alzheimer's disease

The aspartic protease BACE1 (β-amyloid precursor protein cleaving enzyme, β-secretase) is recognized as one of the most promising targets in the treatment of Alzheimer's disease (AD). The accumulation of β-amyloid peptide (Aβ) in the brain is a major factor in the pathogenesis of AD. Aβ is formed by initial cleavage of β-amyloid precursor protein (APP) by β-secretase, therefore BACE1 inhibition represents one of the therapeutic approaches to control progression of AD, by preventing the abnormal generation of Aβ. For this reason, in the last decade, many research efforts have focused at the identification of new BACE1 inhibitors as drug candidates. Generally, BACE1 inhibitors are grouped into two families: substrate-based inhibitors, designed as peptidomimetic inhibitors, and non-peptidomimetic ones. The research on non-peptidomimetic small molecules BACE1 inhibitors remains the most interesting approach, since these compounds hold an improved bioavailability after systemic administration, due to a good blood-brain barrier permeability in comparison to peptidomimetic inhibitors. Very recently, our research group discovered a new promising lead compound for the treatment of AD, named lipocrine, a hybrid derivative between lipoic acid and the AChE inhibitor (AChEI) tacrine, characterized by a tetrahydroacridinic moiety. Lipocrine is one of the first compounds able to inhibit the catalytic activity of AChE and AChE-induced amyloid-β aggregation and to protect against reactive oxygen species. Due to this interesting profile, lipocrine was also evaluated for BACE1 inhibitory activity, resulting in a potent lead compound for BACE1 inhibition. Starting from this interesting profile, a series of tetrahydroacridine analogues were synthesised varying the chain length between the two fragments. Moreover, following the approach of combining in a single molecule two different pharmacophores, we designed and synthesised different compounds bearing the moieties of known AChEIs (rivastigmine and caproctamine) coupled with lipoic acid, since it was shown that dithiolane group is an important structural feature of lipocrine for the optimal inhibition of BACE1. All the tetrahydroacridines, rivastigmine and caproctamine-based compounds, were evaluated for BACE1 inhibitory activity in a FRET (fluorescence resonance energy transfer) enzymatic assay (test A). With the aim to enhancing the biological activity of the lead compound, we applied the molecular simplification approach to design and synthesize novel heterocyclic compounds related to lipocrine, in which the tetrahydroacridine moiety was replaced by 4-amino-quinoline or 4-amino-quinazoline rings. All the synthesized compounds were also evaluated in a modified FRET enzymatic assay (test B), changing the fluorescent substrate for enzymatic BACE1 cleavage. This test method guided deep structure-activity relationships for BACE1 inhibition on the most promising quinazoline-based derivatives. By varying the substituent on the 2-position of the quinazoline ring and by replacing the lipoic acid residue in lateral chain with different moieties (i.e. trans-ferulic acid, a known antioxidant molecule), a series of quinazoline derivatives were obtained. In order to confirm inhibitory activity of the most active compounds, they were evaluated with a third FRET assay (test C) which, surprisingly, did not confirm the previous good activity profiles. An evaluation study of kinetic parameters of the three assays revealed that method C is endowed with the best specificity and enzymatic efficiency. Biological evaluation of the modified 2,4-diamino-quinazoline derivatives measured through the method C, allow to obtain a new lead compound bearing the trans-ferulic acid residue coupled to 2,4-diamino-quinazoline core endowed with a good BACE1 inhibitory activity (IC50 = 0.8 mM). We reported on the variability of the results in the three different FRET assays that are known to have some disadvantages in term of interference rates that are strongly dependent on compound properties. The observed results variability could be also ascribed to different enzyme origin, varied substrate and different fluorescent groups. The inhibitors should be tested on a parallel screening in order to have a more reliable data prior to be tested into cellular assay. With this aim, preliminary cellular BACE1 inhibition assay carried out on lipocrine confirmed a good cellular activity profile (EC50 = 3.7 mM) strengthening the idea to find a small molecule non-peptidomimetic compound as BACE1 inhibitor. In conclusion, the present study allowed to identify a new lead compound endowed with BACE1 inhibitory activity in submicromolar range. Further lead optimization to the obtained derivative is needed in order to obtain a more potent and a selective BACE1 inhibitor based on 2,4-diamino-quinazoline scaffold. A side project related to the synthesis of novel enzymatic inhibitors of BACE1 in order to explore the pseudopeptidic transition-state isosteres chemistry was carried out during research stage at Università de Montrèal (Canada) in Hanessian's group. The aim of this work has been the synthesis of the δ-aminocyclohexane carboxylic acid motif with stereochemically defined substitution to incorporating such a constrained core in potential BACE1 inhibitors. This fragment, endowed with reduced peptidic character, is not known in the context of peptidomimetic design. In particular, we envisioned an alternative route based on an organocatalytic asymmetric conjugate addition of nitroalkanes to cyclohexenone in presence of D-proline and trans-2,5-dimethylpiperazine. The enantioenriched obtained 3-(α-nitroalkyl)-cyclohexanones were further functionalized to give the corresponding δ-nitroalkyl cyclohexane carboxylic acids. These intermediates were elaborated to the target structures 3-(α-aminoalkyl)-1-cyclohexane carboxylic acids in a new readily accessible way.

The effect of cross-linker on endogenous enzymatic activity within radicular dentin and bond strength between intraradicular post and radicular dentin

The primary aim was to evaluate the effect of 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC) on endogenous enzymatic activity within radicular dentin and push-out bond strength of adhesively luted fiber posts, at baseline and after artificial aging. Additionally, the effect of different cementation strategies on endogenous enzymatic activity and fiber post retention was evaluated. The experiment was carried out on extracted human teeth, following endodontic treatment and fiber post cementation. Three cementation strategies were performed: resin cement in combination with etch-and-rinse (EAR) adhesive system, with self-etch (SE) system and self-adhesive (SE) cement. Each of the mentioned strategies had a control and experimental (EDC) group in which root canal was irrigated with 0.3M EDC for 1 minute. The push-out bond strength test was performed 24h after cementation and after 40.000 thermocycles. In order to investigate the effect of EDC and different cementation strategies, in situ zymography analyses of the resin-dentin interfaces were conducted. Statistical analyses were conducted with the software Stata 12.0 (Stata Corp, College Station, Texas, USA) and the significance was set for p<0.05. The results of statistical analysis (ANOVA) showed that the variables “EDC”, “root region” and “artificial aging” significantly influenced fiber posts’ retention to root canal (p<0.05). The highest values were observed in coronal third. The mean values observed after artificial aging were lower when compared to baseline, however EDC was effective in preserving bond strength. The level of enzymatic activity varied between the groups and EDC had a beneficial effect on silencing the enzymatic activity. Within the limitations of the study, it was concluded that the choice of cementation strategy did not influence posts’ retention, while EDC contributed to the preservation of bond strength after artificial aging and reduced enzymatic activity within radicular dentin. In vivo trials are necessary to confirm the results of this in vitro study.

Cleavage Politics in Changing Times. Political Realignment Processes in Western European Countries

The dissertation fits the political realignment literature and aims to pro-vide further insights into cleavage politics by investigating voting behaviour in the Western European countries’ national elections. In particular, the dis-sertation focuses on the class and value voting patterns and on the change of these patterns in different countries and over the course of time. Peculiar pro-cesses affected all Western European party systems: whilst the «traditional» cleavage theory accounts for National and Industrial revolutions, those pro-cesses assumed to constitute the «societal modernization» determined chang-es in electoral competitions that questioned the relevance of individuals’ so-cial positions to study electoral preferences. Since the associations between social positions and voting behaviour underpin the so-called political cleav-age, the dealignment perspective assumes them to have been eroding since the second half of the XX century. On the other hand, the realignment perspective argues that the cleavage theory still accounts for individuals’ vote choices: of the four «traditional» cleavages, this perspective hypothesizes new class vot-ing patterns and alignments between electoral preferences and a new line of conflict, that is based on values. The dissertation provides a theoretical ac-count of the realignment of the class cleavage and a new conceptualization of value voting. Then, class and value voting patterns are explored. The analyses employ European Social Survey data and detect general and country-specific patterns. The dissertation adopts a mediation perspective and aims to observe how class voting patterns change when controlling for value orientations. The results are provided with a sensitivity analysis, indeed two versions of the measures computed for value orientations are compared. The findings show that social class continues to affect voting behaviour and that value orienta-tions both mediate this effect and affect electoral preferences.

Influence of application mode of universal adhesives on bond strength performances and enzymatic activity of coronal and radicular dentin

Objective: The aims of this thesis were to analyze the application mode of the universal adhesives (UA) and to give instructions for clinical procedures. The etching mode of UA on the bond strength to dentin and on the risk of retention, marginal discoloration, marginal adaptation and post-operative sensitivity (POS) was analyzed by two systematic reviews. Three in vitro studies were conducted: 1) evaporation mode of a UA on coronal dentin; 2) cementation approach on radicular dentin; 3) adhesion of metal brackets to enamel. Materials and methods: Two systematic review were conducted firstly, then in vitro study to investigate the evaporation mode in presence or not of pulpal pressure by means of μTBS, and the enzymatic activity using in situ zymography, at T0 and T6. The cementation of a fiber into radicular dentin with different resin-cements was studied, by push-out bond strength evaluation. Orthodontic brackets were cemented according to 4 adhesive protocols and shear bond strength test was conducted. Two adhesive removal techniques were evaluated, and spectrophotometry was used. Results: The probability of POS occurrence was less in SE. SEE approach seems to perform better than SE. Air-drying resulted in higher μTBS. Suction-evaporation, aging and ER mode increased MMPs activity. Differences in NL expression were present at T0 for fiber post study, and the aging produced an increase in marginal infiltration. Brackets cemented with new universal cement with previous etchant application showed good μTBS values. Conclusion: SEE performed better than SE and TE with UA in terms of uTBS. Evaporating with air-drying is better for UA in terms of uTBS and enzymatic activity. Aging and choice of resin cement for cementation of fiber posts influenced the PBS. Brackets cementation with a new resin- cement seems to offer the highest bond strength and leaves more cement remnants after the bracket removal.