*3.4. Aβ Aggregation Assay*

The antiaggregant effect of the newly synthesised compounds on the self-induced aggregation process of Aβ was assayed as previously reported [32]. Briefly, Aβ (20 µM), ThT (60 µM) and the compounds of interest were incubated in phosphate buffered saline (pH 7.4) at 37 ◦C in a multiplate reader (Varioskan Flash, Thermo Scientific, Leiden, The Netherlands). The fluorimetric readings (excitation and emission wavelengths were 450 nm and 480 nm, respectively) were collected every 10 min up to 60 h. Data of all the measurements, carried out in triplicate, were fitted to Equation (1).

$$F(t) = F\_0 + \frac{F\_{\max} - F\_0}{1 + e^{\frac{t - t}{k}}} \tag{1}$$

*Fmax* − *F*<sup>0</sup> is the higher fluorescence increment recorded all over the aggregation process; the lag phase (*tlag*) is the time interval preceding the formation of amyloid-type species sensitive to ThT. The *tlag* values were calculated by using Equation (2)

$$t\_{\rm lag} = t - \text{2/k} \tag{2}$$

The parameters of each set of measurements were expressed as the mean ± SD.
