Development and prevalence of neurodegenerative diseases surge with increasing age. One of the most common age-related diseases is Alzheimer's disease (AD) where it represents the 6th cause of death in US. About 5.8 M Americans; almost 1.6 % of the population, were living with AD in 2020 and about 50 M are diagnosed with AD worldwide. According to National Institute on Aging, these numbers are thought to be doubled every 5 years [1].

AD is characterized with irreversible declination in cognition leading to memory loss in addition to other cognitive disorders that worsen over time; from short-term memory loss to losing the ability to deal with the external environment in late AD stage. AD progression results in neurons death and loss of synapse mainly in hippocampus and cerebral cortex due to its 2 main characteristic hallmarks; extracellular β-amyloid (Aβ) plaques and intracellular neurofibrillary tau tangles (NFTs) [2].

Another characteristic accompanied with AD development is death of cholinergic nerve cells, in addition to increasing the activity of acetylcholinesterase (AChE) in brain resulting in decreasing level of acetylcholine neurotransmitters (ACh). The problem of AChE is not only its high activity but also its ability to form complex with amyloid plaques (AChE–Aβ) increasing the risk of neurotoxicity [3].

Many other factors are suggested as causes for AD development and progression including extensive oxidative stress, chronic neuro-inflammation, mitochondrial dysfunction, calcium and metal dyshomeostasis in addition to other genetic and environmental factors. This makes the pathogensis and trials to find effective curable or preventive measures very difficult. All available medications and trials now are only treating the cognitive symptoms or trying to prevent further progression [4,5].

Various drug discovery approaches are being currently tried like β-amyloid and tau protein-targeted immunotherapies, AChE inhibitors, peptide inhibitors for anti-Aβ amyloidosis, targeting brain metal homeostasis, N-methyl-D-aspartic acid (NMDA) receptor, protein kinase C and epigenetic therapies. However, only four FDA approved drugs are clinically available for treating AD symptoms; three of them are AChE inhibitors which are donepezil, rivastigmine, and galantamine and one is NMDA antagonist which is memantine [6].

Being a complex disease with different druggable targets and no cure until now drives research to find different approaches towards finding AD treatment. One approach is to find a ligand that can have effect on more than one target for the sake of synergistic effects, this ligand is defined as multitarget-directed ligand (MTDL) [7,8]. Based on that, Namzaric; a combination therapy consists of donepezil (a cholinesterase inhibitor) and memantine (NMDA receptor antagonist) has been FDA approved in 2014 as symptomatic management agent for AD [9]. Other clinical trials for combination therapies using mainly cholinesterase inhibitors are running now [10].

Another way for designing a MTDL is creating a small molecule with a merged pharmacophore for multifunctional anti-AD agents. Based on that theory, fragments of anti-AD agents like, tacrine; a strong inhibitor of cholinesterases, donepezil, rivastigmine and 5 membered heteroaryls such as thiazoles and 1,3,4-oxadiazoles are now designed as MTDL to inhibit cholinesterases (acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)) enzymes [11]. Both AChE and BuChE are promising targeting for treating AD symptoms where AD is characterized by high level of AChE and BuChE expression in the plasma while in the brain AChE expression becomes lower and BuChE expression increases causing deregulation in skewing the balance between AChE/BuChE ratio and deficiency in the brain. Rivastigmine was found to have a dual inhibition activity on both AChE and BuChE. In addition to rivastigmine, 1,3,4-oxadiazole derivatives are widely investigated for their dual inhibition activity as MTDL over the last 10 years [12,13].

AChE and BuChE are serine hydrolase enzymes that carry out hydrolysis of ACh in brain and plasma which are reported to show high levels of expression in AD and to be responsible for cognitive decline. Accordingly, drug discovery concentrated on finding hit ligands that dually target AChE and BuChE as MTDLs aiming towards enhancing the cognition and other symptoms associated with AD. Recent researches managed to design 2,5-disubstituted-1,3,4-oxadiazole derivatives as MTDLs anti-AD agents. Mishra et al. designed and synthesized novel 1,3,4-oxadiazole hybrid compounds with 4-aminopyridine (4-AP) and reported their inhibitory activity towards both AChE and BuChE (their scaffold is shown in Fig. 1A) [14].

Another study managed to synthesize and evaluate N-benzylpyrrolidine and 1,3,4-oxadiazole as multi-targeted hybrids for the treatment of AD (their scaffold is shown in Fig. 1B) [15]. A recent study from our lab also managed to synthesize new 2,5-disubstituted-1,3,4-oxadiazoles (their scaffold is shown in Fig. 1C) resulting in discovery of a new hit compound. This study highlighted the new hit's significant AD-biotargeting effects including reducing the elevated levels of lipid peroxidation and glutathione (GSH), normalizing levels of 8-OHdG, and, most importantly, decreasing the levels of the well-known AD hallmark β-amyloid protein [16].

Based on recent finding [17], and in continuation to our previous work to extend on knowledge set forth so far [16], we herein add to the diversity of the oxadiazole-based chemical backbone as a privileged scaffold that can be functionalized and chemically decorated to enhance the ligand-biotarget recognition. This was achieved through newly adding multiple possible interacting functional groups by having a mono- or bi-aryl ring system on the 5-position of the 1,3,4-oxadiazole scaffold. The aim of this structural modification is to add interacting ring systems that can anchor the inhibitor to the binding pocket of the target enzymes competitively inhibiting the binding of the native ligands, acetyl- and butyryl choline in this case. Additionally, the 5-biaryl system was altered between being a biphenyl or a pyridyl-phenyl system to investigate whether introduction of the nitrogen heteroatom will impact the compounds' ability to inhibit the target enzymes. Finally, another front was explored which is the effect of introducing flexibility to the backbone of this chemotype on their biotarget inhibition. This was affected by adding a flexible NH linker at the 2-position of the oxadiazole ring which may also contribute to the molecular recognition and facilitate the binding of these molecules. Thus, we herein describe the design, synthesis and evaluation of new MTDL aniline and 1,3,4-oxadiazoles hybrid molecules as anti-AD agents (Fig. 1D). Various extensive in vitro, in vivo, ex vivo and in silico investigations were performed to assess the potency of these new chemotypes and evaluate the efficiency of their binding, their efficacy on their biological targets and their ability to elicit the biological function they were designed for.