Tacrine was first described pharmacologically by Shaw and Bently in Australia [1] in 1949 as an analeptic capable of causing rapid arousal in morphinized dogs and cats. Early clinical applications of tacrine were in the treatment of anesthetic induced delirium [2] and the potentiation of the muscle relaxing effects of succinylcholine [3].

Heilbronn in 1961 [4] was the first to describe the effect of tacrine upon acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). She demonstrated that the compounds act as a reversible inhibitor being partly competitive with the substrate acetylcholine (ACh) and a more potent inhibitor of BuChE than of AChE [5].

Other than being an enzyme inhibitor, tacrine was found to have other mechanisms which could explain its cholinergic effect. In electrophysiological studies with neurons of Lymnaea stagnalis, tacrine inhibits the slow outward K⁺ current and consequently increases the duration of the action potentials. Moreover, tacrine inhibits the uptake of noradrenaline, dopamine and serotonine. Such uptake inhibition does not seem to occur at the level of the axonal membrane, but instead at the level of the monoaminergic storage granules [6], and thus tacrine might well have some monoaminergic effect [7]. Tacrine was one of the first ChEI to be tested clinically for the treatment of Alzheimer's disease (AD). Numerous studies [8] had been performed on a small group of patients, with physostigmin alone or in combination with lecithin. The initial report by Summers et al. [9] which suggests that a combined oral treatment of tacrine and lecithin improved the condition of patients with AD has attracted considerable interest, although later studies have failed to demonstrate such marked improvements. Several reasons have been proposed to explain the negative results of these studies; the small sample size used in the research being among the most significant, and the fact that many of the subjects chosen for the study were already in advanced stages of the disease. Also, multiple transmitter deficits are probably present in AD in addition to a loss of neurons in critical areas of the brain, and counteracting the former with tacrine alone does not bring about clinically demonstrable benefits [7]. There is also a high incidence of side effects in the use of tacrine–autonomic symptoms such as gastrointestinal disturbance and hypotension. Additionally, the therapeutic index of tacrine is so small that it is both critical and difficult to find the optimum dose, as individual patients vary greatly in their responsiveness to the treatment [10-12].

In this review, we will summarize the research into the development of tacrine derivatives with cholinesterase and amyloid aggregation inhibiting activity in addition to discussing their neuroprotective properties. Although many reviews have been published on this topic with medicinal or pharmaceutical focuses, we will try to contribute from a different view of point, namely from a bioorganic perspective.

Acetylcholinesterase (AChE, E.C.3.1.1.7) and butyrylcholinesterase (BuChE, E.C.3.1.1.8) are enzymes which constitute the group of the cholinesterases. Acetylcholinesterase hydrolyses acetylcholine (AChE) and is mainly associated with nerves and muscles, being typically found on the synapsies, while butyrylcholinesterase, also known as plasma cholinesterase or pseudocholinesterase, hydrolyses butyrylcholine and is synthesized by the liver, being found in large concentrations in serum [13].

All current kinetic models for AChE propose the existence of at least two substrate-binding sites; the active site, near the bottom of the active-site gorge, and the peripheral anionic site (PAS), near its entrance. Binding the ligands at the PAS affects catalytic activity [14-15]. The active site is composed of two subsites. In the catalytic anionic subsite (CAS), it has been proposed that the choline moiety of AChE is stabilized principally via a cation-π interaction with Trp84, and also interacts with Glu199 and Phe330 [16]. A similar cation-π interaction occurs in human BuChE, where Trp82 interacts with the product choline and the substrate butyrylthiocholine [17]. The PAS contain three principal amino acids; Trp279, Tyr70 and Asp72. This was established by site-directed mutagenesis and by binding of inhibitors. AChE, one of the most crucial enzymes for nerve response and function, catalyzes the hydrolysis of acylcholinesters with a relative specificity for acetylcholine.

Szegletes et al. [18] demonstrated that the primary physiologic role of the PAS is to accelerate the hydrolysis of acetylcholine at low concentrations of acetylcholine. The gorge of the active site is so deep and broad that it can bind many different substrates and inhibitors. The initial step in the catalytic pathway is substrate binding to the PAS, therefore the designing of new type inhibitors of AChE should be based on the hypothesis of dual binding of AChE inhibitors. The PAS can act as an uncompetitive inhibitor binding site. Strong binding of inhibitors at the peripheral site may hinder the entry of acetylcholine to the enzyme gorge. Such types of inhibitors which are able to bind to PAS can act as a steric blocker.

The hydrolysis reaction proceeds with the nucleophilic attack on the carbonyl carbon, acylating the enzyme and liberating choline. This is followed by a rapid hydrolysis of the acylated enzyme yielding acetic acid, and the restoration of the esteratic site. The pathway is similar to a pipeline, where the substrate goes in one end and the products come out at the other end through conformational changes, along with the effect of hydrophobic and electrostatic forces. In mammals, the alternative splicing of a single gene gives rise to three different variants of AChE (types R, H/E, and T/S), which possess the same catalytic activity, but with different tissue specific distributions. AChE is a glycoprotein which is present in cells as either a monomer or an oligomer, depending on the type of cells and on the variant of enzyme, but all of them is membrane-bound.

Recent findings indicate that AChE is involved not only in the correct functioning of the central and peripheral nervous systems, but that it is a ubiquitous molecule which takes part in the regulation of several other processes such as cell growth, locomotion or apoptosis [19].

While AChE has a well established “classical” esterase activity, the physiologic role of BuChE is still controversial. BuChE is an enzyme which is also involved in cholinergic neurotransmission, and which has received increasing attention in recent years [20].

However, BuChE may have a compensatory role in the hydrolysis of ACh in brains with degenerative changes. Although BuChE activity is lower than that of AChE in the normal human brain, BuChE activity is greatly increased in AD [21-22]. It is possible that these divergent changes are related to the loss of cholinergic synapses and neurons and the increases in glial cells in the AD brain, which suggests that inhibition of BuChE (in adition to AChE) may become more important as AD progresses. This has raised the hypothesis that inhibitory action on both enzymes could lead to improved therapeutic benefits [20,23-24].

The current therapeutic options for the treatment of AD are acetylcholinesterase inhibitors, which increase neurotransmission at cholinergic synapses in the brain and reduce temporarily the cognitive deficit [25].

Cholinergic dysfunction can be associated with a decrease in the production of the neurotransmitter acetylcholine. Many approaches leading to enhancing concentration of acetylcholine at the synaptic cleft have been suggested. An understanding of the protein structure and mechanism of catalytic action of AChE is important for the design of a new potent inhibitor of AChE.

Amyloid aggregation of proteins is a complex process associated with amyloid-related neurodegenerative disorders such as Alzheimer's and prion diseases. The conversion of a specific protein or protein fragment from soluble native states into amyloid aggregates results in the formation of amyloid deposits with a single predominant protein compound characteristic of each disease. In AD, two types of aggregates have been identified in the brain: intracellular neurofibrillary tangles created by tau protein and extracellular senile plaques NFTs consisting of Aβ peptide. Tau protein is also involved in various tauopathies. The fibrillar form of huntingtin is associated with Huntington's disease, and transthyretin with familial amyloid polyneuropathy. Amyloid deposits formed by alpha-synuclein are found in patients with Parkinson's disease [80].

The formation of amyloid aggregates is triggered by nucleation-dependent polymerization from subunit protein by axial stacking of beta strands, generating a cross-beta-sheet structure at the core of the filaments [81]. The process of aggregation involves a series of steps during which many intermediate aggregation states are populated. Recent evidence points to these intermediate states as the toxic moieties primarily responsible for cell damage or cell death, which are critical steps in the origin and progression of amyloid-related neurodegenerative disorders [82]. Recently, growing evidence has emerged that soluble oligomers may be the main toxic species [83,84]. The reason why early aggregates are more toxic than mature amyloid fibrils is not yet clear, although it is likely that it arises from the accessibility of oligomers to interact inappropriately with cellular species leading to deregulation of cell homeostasis [85-87].

Currently, the precise mechanisms of toxicity have not been fully elucidated; however, there is evidence that reduction of amyloid deposits lead to alleviation of disease's symptoms [88,89]. As a consequence, considerable effort has been directed toward the identification of the substances able to reduce amyloid aggregation. In the past few years, a range of diverse small molecules have been found to inhibit or reduce the amyloid aggregation of various proteins, particularly in relation to Aβ deposition, the aggregation of transthyretin and the formation of protease-resistant forms of the prion protein [90-94]. Kim [93] investigated the effect of several classes of naturally occurring compounds on Aβ amyloid aggregation. The results of his study showed that curcumins, flavones type flavonoids, and naphthoquinones are potent inhibitors of Aβ fibrillization formation in vitro. Doplhin et al. [94] demonstrated that longer trioxime oligomers showed an effective reduction of Aβ amyloid fibrils formation. One of the first compounds to be identified with blocked tau-tau interaction was the phenothiazine methylene blue [95]. This compound was shown to partially disrupt the structure of isolated paired helical filaments and could affect tau fibrillization. Interestingly, this dye molecule has now progressed into clinical testing in AD patients [96]. In a screen of 42 compounds, Taniguchi et al. [97] identified small molecules categorized as phenothiazines, polyphenols and porphyrins which were able to inhibit tau amyloid polymerization. It was found that a number of anthraquinones were able to inhibit tau aggregate formation and could also induce disassembly of pre-formed tau fibrils. A similar effect was observed for cyanine dyes [98] and rhodamines [99].

Acridine-based compounds were identified as potent inhibitors of protease-resistant forms of prion protein [100]. Anti-scrapie activity, probably through inhibition of the formation of protease-resistant prion protein, has been found also for other acridine derivatives [92,101]. It has been shown that some acridines and bis-acridine derivatives are able to reduce scrapie prion concentration in infected cells [102]. The first acridine derivatives reported to inhibit or interact with the Aβ polymerization process are diaryl heterocyclic compounds and 9-acridone derivatives [103,104]. More recently, a high affinity for Aβ aggregation was detected for fluorinated acridine orange analogue [105,106]. The inhibiting activity was observed for acridine derivatives, namely for quinacrine and quinacrine mustard [107]. They have shown that these compounds are able to inhibit formation of amyloid fibrils of tau and Aβ peptide.

The tacrine dimers 100-102 characterized by AChE inhibitory activities were tested for their ability to inhibit AChE-induced Aβ fibrillogenesis [75]. In particular, 100 μM inhibitors inhibited Aβ aggregation from 53% to 76%, 100 being the most potent and slightly more potent than 17f (76% compared to 68%). These findings suggest that effective inhibitor concentration in the aggregation assay is much higher than the IC₅₀ values in comparison to the enzyme. Muñoz-Ruiz et al. designed dual binding site acetylcholinesterase inhibitors as potent new drugs inhibiting amyloid Aβ peptide aggregation through binding to both catalytic and peripheral sites of the enzyme [72]. The heterodimers showing the best AChE inhibitory potency (49, 50 and 58-60) were selected to assess their ability to inhibit Aβ peptide (Aβ_1–40) aggregation. Results showed that the heterodimers inhibited the Aβ peptide aggregation at 100 μM in a range varying from 49% to 63%, and that they were at least as potent as propidium, which caused 46% inhibitions. The most active compounds were 49 and 50, followed by 58, 59, 60 the latter being around 2-, 3- and 10-fold less active than the former compounds, respectively.

Hybrids 25-28 and 71-81 result in a significant in vitro ability to inhibit the hAChE-induced aggregation of Aβ peptide, with these hybrids exhibiting percentages of inhibition of 23–46%, 38–66%, respectively, at 100 μM. In the first series, the separation of two methylenes between the amido group of the linker and the pyrano[3,2-c]quinoline system leads to a higher Aβ antiaggregating effect. In the second series, the substitution pattern leading to an optimal Aβ antiaggregating effect involves the presence of an indane moiety irrespective of the length of the linker and the substitution at the tacrine unit. These results suggest that the new compounds could bind to the AChE-PAS and could therefore inhibit the Aβ fibril formation promoted by this enzyme.

Recently, we have examined a small group of structurally distinct acridine derivatives which differ in molecule planarity and bulky side groups at the middle ring moiety for their ability to inhibit lysozyme fibril formation in vitro [107,108]. We have found that planar acridine compounds are very effective inhibitors, while spiroacridines have been ineffective in inhibiting fibril formation. Tetrahydroacridines have had no significant effect on the prevention of lysozyme fibrillization; moreover, in the presence of some derivatives, an enhanced degree of aggregation has been detected. Anti-amyloid activity has also been observed for glycosyl acridines [109]. The different activities of the acridine derivatives studied have indicated that the structure of the acridine side chains and planarity of the acridine cyclic core are the crucial elements in determining the extent of amyloid aggregation. The highest inhibiting activity among screened compounds at all was have been detected for dimeric acridine. A similar increase in anti-amyloid activity for bis-acridines was observed by May et al. [102]. They found that covalent acridine dimers could be more potent than monomeric equivalents in decreasing scrapie-prion concentration. Dolphin et al. [110] investigated the potential of a multimeric quinacrine derivative to inhibit Aβ fibril formation in comparison to monomer quinacrine. The multimeric conjugate exhibits a cluster of four quinacrine derivatives on a rigid cyclopeptide core. Their data showed that the multimeric compound substantially enhanced inhibition of Aβ polymerization into amyloid aggregates. It suggests that assembling multiple copies of acridine moieties to a central scaffold allows multiple interactions with protein leading to effective anti-amyloid activity of the compound.

The typical role of AChE is to terminate cholinergic neurotransmission, but AChE has also been observed in non-neuronal tissues (erythrocytes, megakaryocytes and the others), and there is significant evidence for additional noncatalytic functions of AChE. The level of this enzyme changes during proliferation, neurite growth, apoptosis, differentiation and also the expression of AChE is changed at neurodegenerative diseases [111-116]. Cholinergic neurons in brains affected by AD express more AChE than other neuron types and it has been confirmed that these neuronal cells have higher levels of AChE-S isoform. Apoptosis has been proposed to explain the loss of the cholinergic neurons but the exact mechanisms which regulate the expression of AChE during apoptosis have not been known. Recently, it has been confirmed that up-regulation of AChE-S is associated with activity of GSK-3 which plays a central role in ER-stress induced activation of caspase 3 [117]; as a result, the inhibition of caspases has been suggested as a therapeutic strategy in neurodegenerative diseases, although the possibility of caspase-independent death means that caspase inhibition can offer only transient protection. The search for new inhibitors of AChE has become more intense since AChE was characterized as a multifaceted protein, and also since the discovery of AChE in AD plaque formation and in the neurofibrillary tangles and the amyloid-positive vessels. Even when AChE-peptide was added at nanomolar concentrations, cells died via an apoptotic pathway [118-121]. In light of the non-classical role of AChE, AChEIs could act as multifunctional agents and some of them could possess neuroprotective effects in addition to their AChE-inhibiting action. Therefore the development of new AChEI, including derivatives of tacrine is aimed not only at improving selectivity for AChE and the better side effect profile/low cytotoxicity, but also the potency of their their neuroprotective [49,50,122-126]. Oxidative stress is an early event in AD pathogenesis and therefore new hybrid molecules of tacrine with antioxidant capacities are being synthesized and their neuroprotective effect studied. The neuroprotectivity of new derivatives of tacrine which inhibit Aβ aggregation and the state of intracellular concentration of Ca²⁺ in neuronal tissue are also being researched.

In this review, recent findings concerning new tacrine hybrids which act as cholinesterase (AChE and BChE), and β-amyloid aggregation inhibitors are summarized. Our attention is focused on dividing these derivatives in following groups: monomeric, homodimeric and heterodimeric congenes, which have the ability to simultaneously bind to both the active and peripheral binding sites of AChE. It is of interest to note the impact of the length and nature of the chain connecting the two skeletons (anchor groups) in relation to the increase in their inhibitory effect. The data suggests that the optimal length of the tether is from 6 to 11 units (methylenes, amides and other). The desolvatation of free energy is also important in this context, suggesting the importance of ligand hydrophobicity for effective interaction with the peripheral side. The most effective inhibitors were found to be the hybrids of tacrine moiety with indanone, 8-hydroxychinoline, indole 3-methoxy-substituted benzene and others. Furthermore, numerous tacrine hybrids have been developed with the aim of improving and enlarging the biological profile beyond its ability to act as ChEIs. Amyloid aggregates are toxic for neurons and influence the direct or indirect origins of the pathological conditions associated with the disease. The precise mechanisms of toxicity have not been fully elucidated; however, there is evidence that the reduction of amyloid deposits leads to the alleviation of the disease's symptoms. Anti-amyloid activity has been observed for several tacrine-based compounds. It was found that both the structure of tacrine derivate and the formation of dimeric or multimeric tacrine conjugates affect the extent of the inhibiting properties of these tacrine derivatives. The development of amyloid aggregation inhibitors would also be able to disaggregate filaments and provide an alternative to existing pharmaceutical strategies. In principle, the inhibitory effect of compounds could take place on different levels, for example, interference with the association of dimmers or oligomers and elongation of filaments. This can be achieved by tight binding of the compounds to the protein monomer and oligomer.

One of the hallmarks of AD is the loss of cholinergic neurons and it is well known that Aβ peptides and higher level of AChE (confirmed in cholinergic neurons in brains affected by AD) are able to induce cell death. The elucidation of the mechanism of neuronal death in brains affected by AD and new functions of AChE are of importance for the development of new drug designs. New types of multifunctional AChEI, with antioxidant potential, an ability to reduce of amyloid aggregation and to interrupt the aberrant influx of Ca²⁺ could be a promising treatment for AD, which explains why hybrid molecules of tacrine are under development as promising drugs.