For this endeavor, we were inspired by the structures of rivastigmine (1) and the alkaloid (−)-3-O-acetylspectaline (21), obtained in large quantities from flowers and green fruits of Senna spectabilis. Given the structural similarities between 21 and saturated cardanol (15a), this latter has been selected as a suitable starting point to develop CNSL-derived rivastigmine analogues [79,80]. Paula et al. [79,80] designed new potential AChE inhibitors by means of theoretical calculations. Particularly, the target compounds were modeled following a molecular hybridization strategy between 1 and cardanols (15a). To explore structure-activity relationships, the phenolic hydroxyl of 15a was replaced by methoxy (22), acetyl (23), and N,N-dimethylcarbamoyl groups (24). As for secondary amines, alicyclic and heterocyclic amines (N,N-dimethylamine (a), N,N-diethylamine (b), pyrrolidine (c), and piperidine (d)) were selected to study the conformational flexibility of this subunit. The aromatic N-methylbenzylamine (e) was also considered for evaluating whether an additional Π-Π interaction with the Trp84 or Phe330 residues could be established (Figure 6). The calculations of the electronic structures of the fifteen designed derivatives were initially performed at RHF level using 6-31G, 6-31G(d), 6-31+G(d) and 6-311G(d,p) base functions, then B3LYP level with the 6-31G, 6-31G (d) and 6-311+G(2d, p). Among the proposed compounds, the structures featuring the N,N-dimethycarbamoyl, N,N-dimethylamine and pyrrolidine groups showed a better correlation with 1. Based on the descriptors E (HOMO-1), E (LUMO+1), C–O₅₆, C–NR₂, E(LUMO), and ΔL+1, obtained in the principal component analysis (PCA), the N,N-dimethylcarbamates 24a–e emerged to be the most promising anticholinesterase candidates. To validate the theoretical study, the compounds were synthesized as racemic mixtures and evaluated against EeAChE. Compounds 24a–c inhibited AChE in a concentration-dependent manner, whereas 24d and 24e showed negligible activity, by inhibiting the enzyme by less than 25% at 100 μM. As predicted by theoretical studies, the N,N-dimethylamino derivative 24a turned out to be the most potent inhibitor (IC₅₀ = 50 μM) of the series, followed by the pyrrolidinyl derivative 24c (IC₅₀ = 84 μM), and the diethylamino 24b (IC₅₀ = 251 μM) [73].

Motivated by the above results, we considered cardanols 15b–d as suitable frameworks for the design of AChE inhibitors with higher potency. To this end, we envisaged that combining two different pharmacophoric units could be a valuable strategy to increase activity. Particularly, we sought to link the cardanol skeleton, which might interact with the PAS through its aromatic end, with a fragment able to fish the CAS, to obtain dual binding AChE inhibitors (Figure 7) [81]. As a CAS key molecular feature, we selected fragments bearing a cationic head, which included a protonatable amino moiety belonging to different systems: heterocyclic amines such as pyrrolidine (25), piperidine (26), morpholine (27), thiomorpholine (28), N-substituted piperazines (29–33), hydroxylated pyrrolidine (34) and piperidines (35–37) and their corresponding O-acetyl (38–41) and O-dimethylcarbamates (42–45). Since the N-ethyl-N-(2-methoxybenzyl)amino moiety has been successfully exploited by us and others to obtain powerful dual binding AChEIs [82,83,84,85,86,87], we also synthesized hybrids 46–48 (Figure 7).

Collectively, twenty-four compounds were synthesized from the mixture of cardanols 15b–d and evaluated in terms of EeAChE inhibition profile at 100 µM. Of these, nineteen compounds inhibited the enzyme by more than 50%.

We report in Table 3 the best performing derivatives, with IC₅₀ values below 20 µM. Additionally, most of the derivatives did not show appreciable toxicity against HT-29 cells, up to a concentration of 100 μM, which indicates a potential drug-like behaviour [81].

Notably, compound 47, bearing a N-ethyl-N-(2-methoxybenzyl)amine moiety, showed the highest inhibitory activity against EeAChE, with a promising IC₅₀ of 6.6 μM, and a similar inhibition profile against the human isoform (IC₅₀ = 5.7 μM). Furthermore, 47 was predicted to cross the blood-brain barrier (BBB) by a PAMPA-BBB assay [81]. All in all, these data suggested that the approach of obtaining potential anti-AD compounds from CNSL was worth of further pursuit and development.

To understand the different AChE inhibitory profiles of benzylamines 46–48, Silva et al. [88] carried out theoretical studies to identify key physicochemical properties underlying the experimental data. Particularly, electronic calculations of molecular descriptors, which included molecular volume, polarizability, polar surface area, dissociation constant (pKa), and distribution coefficient (LogD), and PCA were performed together with molecular dynamics and molecular docking. It was possible to verify that a modified cardanol (obtained from the replacement of the hydroxyl by a methoxyl group) and the protonated benzylamine derivatives assume characteristics of electron donor and electron acceptor, respectively, as revealed by electronic structure calculations. In fact, the energies of the HOMO boundary orbitals, HOMO-1, LUMO and LUMO+1, are important descriptors for measuring molecular similarities and grouping inhibitors according to their inhibitory profiles, as shown in the PCA results. Interestingly, molecular docking calculations confirmed a dual binding mode within the human AChE cavity, a characteristic that seems crucial for the stability of this type of complex [88]. In particular, it was noted that ligands 47 and 48 are more likely to bind in an inward orientation, mimicking the donepezil X-ray pose [88].

As a further step, we were interested in the design of multifunctional CNSL-derived compounds, endowed with additional beneficial properties beyond AChE inhibition. Thus, we rationally manipulated the structure of dual binding AChE inhibitor methoxy–cardanol 47 (LDT161) (Figure 8) [89]. In this novel series, we kept the free phenolic group as in cardanols 15, with the objective of inhibiting Aβ aggregation and preserving the antioxidant properties, commonly found in phenols. Furthermore, we maintained a protonable aminomethyl group, critical for the inhibitory profile against both cholinesterases. Finally, the introduction of a terminal -OH on the C8 aliphatic chain was performed with the aim of enhancing solubility and membrane permeability. This modification might also allow the establishment of an additional H-bond interaction that potentially increases potency towards the target(s). Collectively, the set of cardanol derivatives 49–58 was designed with the goal of obtaining cholinesterase inhibitors with concomitant antioxidant and anti-amyloid properties (Figure 8) [89].

The synthesized compounds 49–58 were tested in vitro for their ability to inhibit human AChE and BChE, using the Ellman assay. Then, their anti-amyloid properties were assesed by transmission electron microscopy and their antioxidant (HORAC) profiles were evaluated in comparison with ferulic acid as reference compound. The antioxidant activity was further confirmed in SHSY-5Y cells. Lastly, the assessment of preliminary drug-like properties—in terms of BBB permeation (PAMPA-BBB assay), toxicity in HepG2 cells, plasma stability and kinetic solubility-, was also performed.

The most interesting results are summarized in Table 4 [89]. As regards to the initial drug-likeness evaluation, they were found potentially BBB permeable, devoid of hepatotoxicity, stable in plasma and soluble. Notably, 52 and 58, which combine cholinesterase activity and anti-amyloid/antioxidant capacity together with good drug-like properties, emerged as the best performing compounds. Thus, we succeeded in identifying phenols 52 and 58 as promising multifunctional cholinesterase inhibitors, modulating amyloid and oxidative cascades.

Based on the promising anti-inflammatory activity of 14a, we applied a framework combination strategy to the design and synthesis of a series of CNSL-derived compounds-tacrine hybrids (63–75) for the treatment of AD [94]. We combined in a single chemical entity tacrine moieties (76–78, Figure 10) with a modified CNSL framework (79–82). Considering that the presence of the long alkyl chain (C15) of 14a might limit drug-likeness due to excessive lipophilicity, we turned our attention to the shorter-chain (C8) CNSL derivatives (76–79). In this way, we aimed to create new CNSL-tacrine hybrids which in principle maintained the tacrines’ anticholinesterase activity, combined with the anti-inflammatory activity of CNSL compounds, and possessed drug-like properties. Notably, screening of anticholinesterase activity highlighted potent and selective AChE/BChE inhibitors (63, 64 and 70), with subnanomolar activities. The excellent BChE activity of 63 (IC₅₀ = 0.0352 nM) was rationalized by the solved X-ray crystal structure. Compounds 63 and 64 exerted neuroprotective/neuroinflammatory effects in microglial BV-2 cells treated with LPS. Particularly, 63 and 64 reduce pro-inflammatory mediators, i.e., TNF-α, IL-1β, iNOS, and COX-2. Furthermore, they acted as anti-neuroinflammatory compounds by inhibiting the transcriptional activation of NF-κB, without causing cytotoxicity in microglial, neuronal, and hepatic cell lines. Lastly, their BBB permeability was also confirmed by BBB-PAMPA assay. All in all, the collected data corroborated our rational design for obtaining effective multitarget anti-AD compounds based on CNSL structure.