## 2 Multi-Target Natural and Nature-Inspired Compounds
### Flavonoids and Coumarins  
Flavonoids are known for possessing a wide range of biological activities. Through the years, the compounds belonging to this class have been studied as antioxidants, anti-inflammatory agents and also as AChE inhibitors and, more in general, as promising scaffolds for developing compounds to contrast neurodegeneration through different mechanisms [42; 43]. [PERSON] et al. synthesized a set of drug-like flavonoid derivatives, based on the chromen-4-one scaffold, inhibiting AChE at nM concentrations (IC\({}_{50}\) = 5.87 nM for the best performing compound) and endowed with antioxidant properties. Besides in vitro results, the authors also observed that one of the synthesized compounds was effective in restoring memory in vivo in a mouse model of scopolamine-induced amnesia [3]. On the other hand, the activity of flavonoids as non-specific or specific PDE inhibitors is well-documented. Icarini, icarisid II (Figures 3 and 4) and sophoflavesconj are cGMP-specific inhibitors and isoflav have been studied as PDE4, PDE5 and PDE9 inhibitors. In particular, icarini inhibits PDE5 with an IC\({}_{50}\) value of 5.9 uM [44; 45; 46; 47; 48]. Naringenin (Figure 4), dioclein, epigallocatechin-3-gallate represent some other examples of compounds targeting several PDE isoforms [11; 49; 50].  
Figure 3: 3D structure of PDE5 in complex with icarisid II, a natural inhibitor from _Epimedium brevicorium_ (PDB ID: 2H44). In the artwork, the residues interacting with the ligand (<5 Å) have been labeled.  
Figure 2: Chemical structures of representative alkaloids and derivatives considered in the cited studies: caffeine, theophylline,opentofyline, beta-carboline and vascinone.  
Besides the several available examples of multi-target compounds from this class, it must be pointed out that recent contributions are starting to investigate closely the dual AChE-PDE inhibitory activity of flavonoids. More specifically, [PERSON] et al. reported that rutin and its aglycone quercetin (Figure 4) efficiently reduce AChE activity in rat tissues (25 and 50 mg/kg) and, at the same time, inhibit PDE5. In particular, a more marked inhibitory activity on PDE5 was observed for rutin over quercetin. Moreover, the compounds showed antioxidant activity, as expected. Importantly, even if the focus of the cited article was on the treatment of erectile dysfunction (ED), the results could be translated into the field of central nervous system (CNS) diseases, for which the management of oxidative status is of primary relevance. In neurodegeneration, in particular, antioxidant activity often parallels AChE and PDE inhibition [51, 52, 53, 54].  
Coumarins are a family of naturally occurring small molecules with a wide range of biological activities, including a role in contrasting CNS diseases. This is likely due to their reported effect on enzymes such as AChE and monoamino oxidases (MAOs) [55, 56]. Compounds from this class were also studied both as PDE inhibitors and anti-AD agents [57, 55]. Nevertheless, their dual inhibitory activity on cholinesterase and PDEs, as well as their isoform selectivity, has not been investigated in detail to date. On the other hand, [PERSON] et al. synthesized a set of drug-like, blood-brain barrier (BBB)-permeable coumarin-dithiocarbamate hybrids that act through a combination of mechanisms. In particular, the authors studied the activity of these compounds on AChE (IC\({}_{50}\) = 0.21-36.85 \(\upmu\)M), BuChE (6.21-37.24 % enzyme inhibition) and A\(\otimes\) aggregation [58].