The Structural Hybrids of Acetylcholinesterase Inhibitors in the Treatment of Alzheimer ’ s Disease : A Review

Alzheimer’s Disease (AD) is characterized by the loss of memory and learning ability in elderly patients affecting large population worldwide. The enzyme Acetylcholinesterase (AChE), (E.C.3.1.1.7) plays a major role in the hydrolysis of the released neurotransmitter acetylcholine. Most of the clinically used drugs to treat AD are Acetylcholinesterase Inhibitors (AChEIs). These drugs can provide symptomatic benefits only and suffer with loss of therapeutic potential with time. Therefore, there is an urgent need of novel cholinesterase inhibitors with wider therapeutic window for the treatment of AD. The strategies targeting the AChE enzyme along with other target(s) like Butyrylcholinesterase (BChE), amyloid-β (Aβ), β-secretase-1 (BACE), metals (Cu2+, Zn2+, or Fe2+), antioxidant properties and free radical scavenging capacity have been mainly focused in the last five years. A number of hybrid molecules incorporating sub-structures with the desired well-established pharmacological profile into a single scaffold have been investigated. The main sub structures used in developing these molecules are derived from diverse chemical classes such as acridine, quinoline, carbamates, huperzine and other heterocyclic analogs. It has been followed by optimization of activity through structural modifications of the prototype molecules for developing the Structure Activity Relationship (SAR). This has led to the development of novel molecules with desired AChE inhibitory activity along with other desirable pharmacological properties. This review summarizes the current therapeutic strategies for the development of these AChEI in the last seven years.


Introduction
Alzheimer's Disease (AD) is the most common form of irreversible neurological disorder of elderly patients that has affected large population worldwide [1].It is clinically characterized by progressive cognitive impairments or defined by a loss of memory and learning ability, together with a reduced ability to perform daily routine activities and adverse array of neuropsychiatric symptoms such as apathy, verbal and physical agitation, irritability, anxiety, depression, delusions and hallucinations [2].Characteristic neuropathologic findings include selective neuronal and synaptic losses, extracellular neuritic plaques containing the β-amyloid peptide and Neurofibrillary Tangles (NFTs) composed of hyperphosphorylated forms of the tau (τ) protein [3].Still existing treatments for mild to moderate AD include the use of Acetylcholinesterase (AChE) inhibitors such as tacrine, donepezil ivastigmine, galanthamine, (Figure1) and the use of N-methyl-D-aspartate (NMDA) antagonist memantine [4].However, these drugs are unable to slow or prevent AD progression, rather can provide symptomatic benefits only and suffer from major drawback of loss of therapeutic potential with time.Thus, increasing daily doses in such circumstances increases the side effects until the pause of the treatment.The major side effects are specifically caused by the peripheral activity of these drugs on cholinesterase enzyme.As the average age is increasing all over the world, and so the AD (66% in the developing countries), there is an urgent need for novel therapeutics, which could act as anti-Alzheimer agents with less side effects than the known commercial drugs for the treatment of the AD.The clinical trial results from current developmental pipeline [5] involving molecules as anti-amyloid, anti-tau, neuroprotective (calpain inhibitor, estrogen receptor beta agonist, Peroxisome Proliferator-Activated Receptor (PPAR)-gamma agonist, Gamma-Aminobutyric Acid (GABA)-receptor modulator), Phosphodiesterase (PDE)-9A inhibitor, Butyrylcholinesterase (BChE) inhibitor, 5-Hydroxytryptamine (5-HT)-6 receptor antagonist etc., but the success rate is very poor (0.4%) due to high attrition rate, 99.6% indicate that the most of the candidate drugs are failing to meet the primary cognitive and functional end points [5][6][7].Hence extensive efforts are made for safe and effective anti-Alzheimer's agents [8].Among several targets explored, the AChE is the major target in providing clinically effective anti-Alzheimer's agents.

Cholinergic Hypothesis
In search for novel therapeutic approaches towards AD, the mechanism-based therapeutic approaches targeting β-amyloid, tau pathologies and cholinergic hypothesis have been mainly addressed in last several years [3].The cholinergic hypothesis [9] has been the most widely used in search of drug(s) for treatment of AD.According to this hypothesis, enzyme AChE hydrolyzes the neurotransmitter ACh and breakdown it into acetic acid and choline (Figure 2) and creates deficiency of the ACh at the synaptic gap which leads to the termination of synaptic transmission in brain.The AD is caused by reduced level of the neurotransmitter Acetylcholine (ACh) at the synaptic gap in central nerve system in mammals.There are two cholinesterase enzymes that metabolize acetylcholine, namely, a) AChE and b) BChE.The AChE is mainly found in neuromuscular junctions and chemical synapses of the cholinergic type and plays a major role in the hydrolysis of the released neurotransmitter ACh in central nervous system.

Structure of Acetylcholinesterase
X-ray crystallography of AChE enzyme in Torpedo californica (eeAChE) co-crystalized with tarcrine has shown that it is a long and narrow gorge lined by 14 amino acid residues.The gorge possesses two separate ligand binding sites, one is Catalytic Active Site (CAS) located at the bottom of the gorge and the second is Peripheral Anionic Site (PAS) which is about 15 Å above the catalytic active site and located at the entrance of the gorge [10].
According to the recently reported the crystal structures of human AChE (hAChE) in complex with Fasciculin 2 (FAS-2) and huprine W (PDB code 4BDT) the catalytic active site is lined by aromatic residues and itself can be divided into two sub-unites namely, catalytic triad (also known as esteratic sub-site) and anionic sub-site.The catalytic triad sub-site consists glutamic acid (E202), serine (S203) and histidine (H447), while anionic sub-site consists tryptophan (W86).The catalytic triad is involved in the hydrolysis of the neurotransmitter acetylcholine, while the quaternary group of the choline moiety interacts with of tryptophan (W86) of the anionic site of the CAS.The adjacent area of catalytic triad is known as "oxyanion hole" which is formed by glycine and alanine residues and interacts with carbonyl oxygen of ACh.Whereas the PAS consisting of tryptophan (W286), tyrosine (Y337) and phenylalanine (F338) is believed to guide the entrance of suitable choline esters into the catalytic domain of AChE [11] (Figure 2).The binding of any ligand at PAS blocks the entry of substrates and the exit of the products from the base of the active site.Some ligands such as bulky bis quaternary compounds bind with PAS and hence block the entrance of acetylcholine in the catalytic gorge therefore partially inhibit activity of AChE.

Selectivity for AChE over BChE
It is evident that AChE promotes amyloid fibril formation, more specifically; the peripheral site of enzyme AChE actively involved in aggregation of amyloid-beta (Aβ) peptides [12].The AChE interacts with growing amyloid fibrils and form AChE-Amyloid complex that accelerates assembly of Aβ peptides which leads to Aβ deposits such as mature senile plaque present in the AD brain [13,14].The AChEIs bind only with CAS prevents the hydrolysis of ACh thus rectify the deficit of ACh in brain.Whereas the AChEI which blocks the entire active gorge including catalytic active site and peripheral site of enzyme of AChE, known as dual binding site inhibitors not only prevent the hydrolysis of ACh but also inhibit Aβ aggregation.So these dual AChEIs are considered more effective anti-Alzheimer's agents than the single biding site inhibitors.
The BChE is produced in liver and is mainly found in blood plasma and plays secondary role in hydrolysis of ACh while AChE found in central nerve system plays a lead role in ACh hydrolysis.Recent research indicated that BChE prevent Aβ-fibril formation and hence delay the formation of Aβ-aggregation in brain which is contrary to AChE [15,16].Therefore, high selectivity of the ligand toward AChE over BChE is highly desired property to treat AD.The BChE possesses some structural similarities at CAS but lacks PAS and it lacks three of four aromatic residues of the AChE PAS.This distinct feature may be used in designing selective AChEIs.The hybrid AChEIs are derived by molecular hybridization of two structural fragments that are well known for their pharmacological profile.These novel hybrid AChEIs incorporating additional pharmacophores for binding at PAS may also offer selectivity over BChE.This review compiling these hybrid AChEIs may provide detailed insight into the design and optimization through Structure-Activity-Relationship (SAR) studies in the search of novel candidate molecules.

Irreversible, quasi-irreversible and reversible AChE inhibitors
The AChEIs can be divided into three categories, irreversible, quasi-irreversible and reversible based on the mechanism of their interaction with AChE.The irreversible inhibitors covalently bind with serine (S202) of catalytic triad in CAS and hence block the active site permanently, e.g., organ phosphorous.The irreversible AChEIs are used as insecticides pesticides and warfare which is out of the scope of the present review.The reversible and quasi-irreversible inhibitors have two key interactions such as π-cation interaction with tryptophan (W86) and hydrogen bonds with serine (S202) of active gorge of AChE enzyme in huprine W-AChE complex [11].These types of inhibitors have more therapeutic applications as anti-alzheimer's agents due to their ability to modulate ACh to appropriate levels.Thus these inhibitors are discussed in this review.They are mainly of three types based on their pharmacokinetic mechanism, i) competitive, ii) non-competitive and iii) mixed type inhibitors.The detailed studies of pharmacokinetic mechanism are out of the scope of this review article.However, the necessary and relevant information of binding mechanism of AChEIs is discussed at appropriate places in this review.
In search of anti-Alzheimer's drugs, intensive efforts have been focused on the AChE target, because of the importance of cholinergic hypothesis in the development of clinically effective anti-Alzheimer's agents.It resulted in the successful launch of 4 drugs specifically AChE inhibitors such as tacrine (tetra hydro amino acridine or THA) donepezil, rivastigmine, and galanthamine (Figure 1) [4].The tacrine, (Figure 1) was withdrawn from the market due to serious hepatotoxicity [17].The less toxic drugs, donepezil, inhibit only AChE while rivastigmine acts against both enzymes AChE as well as BChE, exhibiting thus a higher efficiency.Overall, these drugs show modest improvement in the cognitive function of alzheimer's disease, and suffer from major drawback of loss of therapeutic potential with time.

Hybrid strategy
In search of safer and more potent anti-Alzheimer's agents than the above existing drugs particularly acting as AChEIs different chemical classes have been investigated and reviewed in last five year (2011-2015) [18][19][20][21][22]. Researchers developed a series of innovative hybrid AChEIs analogs by conjoining various active scaffolds with existing drug molecules.These analogs provided promising leads for the development of anti-Alzheimer's agents.The active scaffold in these analogs included coumarin, flavonoid, phenothiazine, benzothiazole, aryl, pyridine, indole, isoindoline-1,3-dione, melatonin, thiadiazolidinone, β-carboline, ferulic or caffeic acid, thioacetyl, acridine-selegiline, acridine-aminothiazol and piperazine.These hybrid structures exhibited much higher AChE inhibitory activities as compared to their non-hybrid precursors [23,24].In addition, these hybrids acted as multifunctional agents, such as anti-oxidant, metal ion chelator, neuroprotective, anti-Aβ aggregation and anti-inflammatory agents in the treatment of AD and are discussed below.
In this review we have summarized the systematic development of key AChEIs claimed in research papers and major reviews published in last five years (2011)(2012)(2013)(2014)(2015).The potent AChEIs with diverse chemical structures including, hybrid multi target, dual binding site, homo/heterodimers, heterocyclic, small molecules and natural products have been analyzed under broad chemical classes from medicinal chemistry point of view.

Tacrine Analogs
This chemical class gave the first clinically useful drug tacrine (Figure 1) for treating Alzheimer's disease.However, Watkins, reported it's some serious toxicity [17], its 6-chloro analogs (1 of Figure 1) showed stronger AChE binding than tacrine [25].The 7-methoxytetrahydroacridine (7-MEOTA, 2, figure 1) was weaker cholinesterase inhibitor in human (hAChE IC 50 = 15000nM, BChE IC 50 = 21000 nM) than tacrine (AChEIC 50 = 500 nM) in vitro and in vivo studies [26,27].Most importantly, its lower toxicity and better antioxidant properties, gave advantages over tacrine therefore should be consider for further development.Later on researchers found that the catalytic site of AChE is involved in the hydrolysis of the neurotransmitter acetylcholine, whereas the peripheral site is involved in a binding process with Amyloid-β (Aβ) that accelerates the aggregation of this peptide [12].Thus, simultaneous blockage of the catalytic and peripheral anionic sites of AChE by dual binding site AChEIs resulted in the simultaneous modulation of two important targets of AD pathology, namely Aβ aggregation and the cholinergic deficit [18,19,22].This strategy led to the development of homo/heterodimer or hybrids of two moieties known for their anti-alzheimer properties and such tacrine analogs are discussed below [28,29].
A series of bis-tacrines homodimers linked with 6-8 methylene units showed potent inhibition towards AChE [30].Among these heptylene-linked bis-tacrineor bis(7)-tacrine (B7T, 3a in Figure 3 and Table 1) was 1,000-fold more potent than tacrine in inhibiting rat brain rAChE (IC 50 = 0.2 nM) and rat serum BChE (IC 50 = 315 nM) with high selectivity index (SI = 1369.6)towards AChE over BChE, indicating that it is more selective and effective AChE inhibitor than tacrine (rAChE, IC 50 = 250 nM, rBChE IC 50 = 40 nM, SI = 0.2).This may be due to its interaction with both catalytic and peripheral anionic sites.These dual site binding AChE inhibitors led to a new therapeutic option which later culminated in designing of a large number of dual-site binding AChEIs.Using this strategy, a huge number AChEIs have been extensively coupled with known pharmacophore entities with well-established biological activities, to develop homo-and heterodimers with improved pharmacological profile.In the recent past, researchers developed series of innovative acridine-homo/heterodimer analogs by conjoining various heterocyclic moieties with tacrine [28][29][30][31][32][33].These analogs have been helpful in providing promising leads for the development of anti-alzheimer's agents [28,29,[31][32][33][34].In 2012, Hamulakova et al., reported a series of tacrine homodimers (4a-b in Figure 3 and Table 1) linked through a piperazine/thiourea linker, among them the bis tacrine analog 4a having piperazine linker, displayed 111 fold more hAChE (IC 50 = 4.49 nM) inhibitory activity than its parent drug tacrine (IC 50 = 500 nM) and showed 338.5 times more selectivity towards AChE over BChE (IC 50 = 1520 nM) [35].While the other bis tacrine analog 4b having ethylene-thiourea linker showed 250 fold more hAChE (IC 50 = 2 nM) inhibitory activity than tacrine (IC 50 = 500 nM) with poor selectivity (SI = 0.25) over BChE (IC 50 = 8 nM) [36].These results indicated that piperazine linker was better choice to achieve high selectivity.Recently, a series of bistacrine homodimers (5a-f of Figure 3) with various linkers (alkyl, ether, amine or amide) between two tacrine moieties, were investigated for their pharmacological, pharmacokinetic and physicochemical properties [37].Among them the 5c showed highest AChE inhibitory activity (IC 50 = 1.1 nM) and 213 fold selectivity over BChE (IC 50 = 234 nM).All the tested dimers showed significantly lower cytotoxicity, intestinal permeability than that of tacrine.It was observed that replacement of tetra hydro ring of tacrine moiety dramatically decreased the AChE inhibitory activity.
Additionally, it has good BBB penetrability and may be considered as a promising multifunctional lead compound.A series of tacrine-flavonoid hybrids (18 of Figure 6, Table 1) was reported for its effective AChE, BChE, BACE-1 inhibition, Oxygen-Radical Absorbance Capacity (ORAC) and in vitro Central Nervous System (CNS) penetration [51].Among them the most effective analog 18a with hAChE inhibition (IC 50 = 0.035 nM) was 10,000 fold better than tacrine and was 143 times more selective for hAChE over hBChE (IC 50 = 5.0 nM).The strong AChE inhibitory activity and selectivity of these analogs may be because of better fitting of flavonoid moiety in the PAS of AChE enzyme.In addition, this analog 18a also showed effective antioxidant activity (0.  6) where tacrine and flavonoid moiety were connected through a piperazin linker, moderately inhibited ChE (eeAChE IC 50 = 133, esBChE IC 50 = 558 nM respectively) [53].Moreover, it also showed significant metal chelating (Cu 2+ and Fe 2+ ), inhibition of ChE and self-induced Aβ aggregation and low neuroblastoma cell toxicity in MTT assay.A series of novel tacrine-coumarin heterodimers were designed, synthesized and biologically evaluated for their potential cholinesterase inhibitory activity.Among them the tacrine-coumarin heterodimer 20a (Figure 6) where tacrine was connected with 7-position of coumarin moiety through piperazine spacer, displayed the significant ability to inhibit ChE (AChE, BChE, IC 50 = 92, 234 nM respectively).Additionally it also showed a good Aβ  aggregation inhibition (67.8%, at 20 µM), metal (Cu +2 , Fe +2 ) chelation and non-toxicity towards SH-SY5Y cells [54].The other tacrine-coumarin analog of the series 20 b (Figure 6) having 3,4-dimethyl substituted coumarin moiety, exhibited better eeACh inhibition (IC 50 = 33.63nM, esBChE IC 50 = 80.72 nM) than the analog 20a but with low selectivity (SI = 7.6) [55].Moreover, this analog 20b exhibited selective Monoamine Oxidase-B (MAO-B) inhibition (IC 50 = 240 nM), which was approximately 32 times higher than the reference iproniazid (IC 50 = 7590 nM) with low toxicity and good in vitro BBB permeability (P e = 4.75µM/S).It was a competitive inhibitor and acted through mixed-type mechanism with binding affinity (k i = 34.4nM) towards eeAChE and molecular modeling studies indicated that it was potential multi-target lead compound.The reported tacrine-coumarin heterodimer 21(Figure 6) where tacrine was connected with 4-position of coumarin, showed excellent hChE inhibition (AChE IC 50 = 15.4 nM, BChE IC 50 = 328 nM) which was approximately 32 times higher than tacrine with high selectivity index for AChE (SI = 21.3)[56].The high dissociation constant (K i = 4.1 nM) for AChE-inhibitor complex indicated its strong binding ability with hAChE which was 55 times higher than tacrine.The next hybrid 22 (Figure 6) where tacrine was connected with coumarin moiety at 3-position through an amide linker, displayed higher binding free energy (ΔG cal = -12.7 kcal/ mol) than tacrine (ΔG cal = -11.9kcal/mol) in AChE.Its high dissociation constant (K i =16.7 nM) of AChE-inhibitor complex, explained its higher potency over tacrine [57].The molecular modeling, simulation and kinetic studies of the above mentioned tacrine-coumarin analogs indicated that the potency of the compound largely depends on binding mode of the hybrid with the enzyme AChE.The proper orientation of tacrine moiety in CAS, coumarin moiety in the PAS of the gorge and linker of appropriate length having nitrogen atom are key features of the active compounds.

Quinoline Analogs
Earlier studies revealed that the FDA approved drug rivastigmine is a reversible inhibitor for both AChE and BChE.It is being used for the treatment of mild to moderate AD.It helps in cognition improvement and activities in daily life in AD patients [89].In order to identify the active substructure of tacrine, the six member saturated ring of tacrine was replaced by more flexible open ended chains which resulted into quinoline.Therefore, quinoline and carbamate substructures were rationally integrated by molecular hybridization to produce a large number of rivastigmine-quinoline analogs, which were investigated for their AChE inhibitory activity and are discussed below in details.

Rivastigmine Analogs
Earlier studies revealed that the FDA approved drug rivastigmine is a reversible inhibitor for both AChE and BuChE, is being used for the treatment of mild to moderate AD.It helps in cognition improvement and activities in daily life in AD patients.The carbamate fragment of rivastigmine binds to the catalytic triad of AChE enzyme for much longer than the acetate fragment of ACh during the hydrolysis of ACh and is responsible for its AChE activity for longer duration.Additionally, rivastigmine shows relatively low protein-binding affinity and does not interfere in other medication for concurrent illness.The enzyme AChE exists in several forms and rivastigmine exhibits selectivity for the G1 form which progressively increases with advances of AD, than G4 form which is found in abundance in normal human.Hence, it plays more important role in hydrolyzing ACh at cholinergic synapses during the progression of AD [89].All these unique qualities of rivastigmine increases its bio-efficacy and attracted researcher's attention to explore its derivatives.Some potent rivastigmine or carbamate analogs appeared in last five years (2011-2015) as anti-Alzheimer's agents, are discussed below.
Various substituted 1,2,3,4-tetrahydroquinolin-6(or-7)-yl carbamates (54-57 of Figure 12, Table 3) have been reported as anti-alzheimer's agents [98][99][100][101].Their design and synthesis was based on a systematic Virtual Screening (VS) experiments, consisting of the development of 3D-pharmacophore models, screening of virtual library, synthesis and pharmacology.The initial lead compound 2-chlorophenylcarbamic acid 1-benzyl-1,2,3,4-tetrahydroquinolin-6-yl (54 of Figure 12) showed IC 50 value 3.31 µM [100].Considering the high aromatic content at the active site gorge and the dominating hydrophobic interactions of AChE with its inhibitors, the modifications were targeted at site-1 and site-2 of the lead compound 54.The comparatively more hydrophobic propargyl and 2,4-dichlorobenzyl groups were the two highly suited groups at site-1 as R 1 .The attempted major modifications at the site-2 of the lead compound 54 where both substituted aromatic and long-chain (C6-C8) alkyl groups were incorporated which led to the invention of a series of novel compounds 55 and 56 (Figure 12).Among these molecules the three promising compounds, 55 (AChE, IC 50 = 0.70µM) showed almost comparable AChE inhibitory activity in Swiss albino mice brain (mAChE) with the drug rivastigmine (mAChE, IC 50 = 1.11 µM) in vitro studies [100].Interestingly, through docking studies in the substituted 1,2,3,4-tetrahydroquinolin-7-yl carbamates analogs 56 (Figure 12) [101] were identified to be more potent than their analogs 55.Among these the molecule 56 (mAChE, IC 50 = 0.10 µM) possessing benzyl group on the carbamate end exhibited 11-fold better AChE inhibitory activity compared to the currently used drug rivastigmine and almost equal or even slightly better AChE inhibitory activity compared to the drug tacrine.The compound 56 has also shown potential towards the improvement in learning in mice in behavioral study which was almost  comparable to the drug rivastigmine and tacrine in vivo studies.The molecules 55-57 were evaluated for in vivo passive avoidance test and aldicarb-sensitivity assay, led to the discovery of orally active novel AChEIs which improved scopolamine-induced cognition impairment in Swiss male mice.The % learning improvement of molecules 56 was comparable to donepezil but weaker than tacrine (Figure 12).The 1,2,3,4-tetrahydroisoquinolin-6-yl carbamate analog 58 (Figure 12) showed potent AChE inhibition (IC 50 = 100 nM) which was approximately 10 times higher active than rivastigmine (IC 50 = 1030 nM) in rat brain and efficiently regulated L-type calcium channel inhibitory activity (53% at 50 µM) [102].The N-substituted carbamoyl derivative 59 (Figure 12) showed very good AChE inhibition (IC 50 = 21800 nM) which was nearly 22 fold higher than rivastigmine (IC 50 = 501000 nM) [103].This compound was highly lipophilic with lipophilicity values (log k = 1.09, log P = 9.13).The 4-methyl-2oxo-2H-chromen-7-yl phenyl carbamate (60 of Figure 13) exhibited strong mAChE inhibition (IC 50 = 13.5 nM) which was 20 times more selective over BChE and also evaluated for memory testing in scopolamine-induced amnesia [104].The incorporation of AChE inhibitory moiety-carbamate with anti-Aβ aggregation/metal chelating pharmacophores thioflavin and deferiprone moieties resulted in compounds 61 and 62 respectively (Figure 13, Table 3).The thioflavin-carbamate 61 was an irreversible AChE inhibitor (IC 50 = 20900 nM) while the deferiprone-carbamate 62 was reversible and mixed non-competitive/ competitive type AChE inhibitor (IC 50 = 48800 nM).These two prodrugs were nontoxic below 200 µM and showed very good biological compatibility data for drug-likeness.A novel phenylcarbamate derivative meserine 62 (Figure 13), showed significant in vitro hAChE inhibitory activity (IC 50 = 274 nM) and significantly improved spatial learning and memory deficits in scopolamine-induced dementia in adult mice at 1 mg/kg, i.p. dose.Furthermore, meserine reduced APP level by 28% at 7.5 mg/kg dose in 3 weeks and Aβ 42 level by 42% in APP/PS1 transgenic mouse [105].Recently Bohn et al., reported selective central AChE inhibitors among them the bio-oxidizable prodrug 63 (Figure 13) was almost inactive against AChE (IC 50 > 1mM) in peripheral system [106].However after crossing the BBB (Blood Brain Barrier), the prodrug 63 oxidised in CNS to produce prodrug 64 which further activated a central cholinergic system by carbamylation of AChE with remarkably high activity (IC 50 = 20 nM) and released a metabolite 5-hydroxyl quinolinium which transported out of the brain easily.Overall, in vivo and ex vivo studies indicated that the "bio-oxidizable" prodrug approach could be a promising strategy for rational designing of selective central AChE inhibitors.

Donepezil Analogs
The FDA approved anti-Alzheimer's drug, donepezil (Aricept) is cis1-benzil-4-[5,6-dimethoxy-(1-indanone)-2-yl]-methylpiperidine hydrochloride (Figure 14, Table 4), which is reversible, non-competitive and very effective AChE inhibitor (IC 50 = 5.7 nM) with less toxicity [107,108].This drug is clinically used for the treatment of AD in patients with mild, moderate and severe stages of cognitive impairment at maximum 23mg/day dose [107].The phenomenon Deuterium Kinetic Isotope Effect (DKIE) has been used on donepezil drug to improve its Pharmacokinetics (PK), Pharmacodynamics (PD), and toxicity profiles [109] viz increased half-life, reduced metabolic rate and thus making it more effective and safer drug.Seeing the high potency and low toxicity of donepezil, later researchers focused on designing new multi-potent AChE inhibitors by combining the structural features of donepezil with bioactive chemical entities.represented by site-1 and site-2 connected through a spacer.The majority of novel donezepil analogs were achieved by structural modifications in these two sub-structures and the spacer.These novel donepezil analogs are discussed below [108].

Huperzine Analogs
(-)-Huperzine A (88; Hup A), a novel alkaloid isolated from a Chinese herb Huperzia Serrata, (Thunb) of Lycopodium alkaloid plant family, is a reversible and selective cholinesterase inhibitor [138].It has also shown "non-cholinergic" effects such as protective effect on neurons against amyloid beta-induced oxidative injury, mitochondrial dysfunction and up-regulation of nerve growth factor [139,140].A comparative in vitro and in vivo studies of Hup A showed that its AChE inhibitory activity is better than tacrine, rivastigmine and galanthamine drugs while poorer than that of donepezil (Figure 18) in rats.Hup A has better penetration through the BBB, higher oral bioavailability and longer duration of AChE inhibitory action compared with tacrine, donepezil and rivastigmine.It has also shown improve cognitive deficits in a broad range of animal models.The clinical trial of Hup A, in China demonstrated the safety, tolerability, and efficacy of huperzine A in mild to moderate AD and in cognitive improvement [141].The 5-Cl-O-vanillin synthetic derivative of huperzine, called prodrug ZT-1(89, IC 50 = 63.6 nM of Figure 18, Table 5 ), also known as DEBIO 9902, was reported to be well-tolerated, rapidly absorbed, and converted into huperzine A, in a small Phase 1 trial [142].However, the clinical development of prodrug ZT-1 has been discontinued.A published meta-analysis of 20 clinical trials conducted with huperzine analogs/derivatives in China, Europe and the United States left open the question of whether huperzines could be therapeutically useful.Trials reporting beneficial effects were small and of short duration [143].In the United States it is commercially permitted as a food supplement, but has not been approved by the FDA as a treatment for AD or other cognition disorders.Huperzine A was modified in a systematic way to improve activity, reduce toxicity and side effects.A series of modified-huperzine has been reported.Among them the recent developments are discussed below.
The molecular hybridization of two known active molecules namely tacrine and huperzine A, resulted into a new lead molecule huprine (90; Figure 18) with increased potency and extended sites for functional modification.
Among more than 40 synthesized analogs, the racemic huprine A (90a; IC 50 = 65 nM), enantiopure, (-)-huprine X (90b IC 50 = 0.32 nM) and Y (90c) (IC 50 = 0.32 nM; Figure 18, Table 5) were promising.The analogs 90b and 90c were 800-and 640-fold more potent than their parental molecules (-)-huperzine A and tacrine, (IC 50 = 260 nM; 205 nM respectively) [144].The high-affinity of huprine X towards inhibition of AChE and low effective dose of rac-huprine Y (90c) in ex vivo studies (ID 50 = 1.09 µmol/kg) indicated that high activity resulted in a much lower dose, may avoid the toxicity problems associated with higher dose of tacrine.The huprine A, X and Y showed cognitive-enhancing properties and their effects on the regulation of several neurochemical processes related to AD in triple transgenic mice (3xTg-AD) [145].Therefore these were selected for further lead optimization.A series of Huprine X with diverse modifications at position-9 and position-12 was evaluated for AChE inhibitory activity [146][147][148].Among them the representative analogs (90d of Figure 18) inhibited AChE at nano molar (hAChE IC 50 = 1.1 nM) with high selectivity towards AChE (SI = 1130) in human and low dissociation constant (K D = 0.87 nM).The molecular studies suggested that the terminal group of alcohol in molecule 90d forms additional favorable hydrogen bonds with the catalytic Ser203 and Gly122 of the oxy anion hole.Its LD 50 in Swiss albino mice was 40 mg/kgip and it was able to cross the BBB at doses above 15 mg/kg [147].
The steric bulky substituent's at position-9 decreased AChE inhibitory potency, as bulk substituent it did not fit in the enzyme's binding pocket.It was proposed that aniline group interacted with residues Thr83, Trp86, Tyr133, Ser125 and Glu202 in catalytic gorge of the enzyme through hydrogen bonding with the help of key water molecules.Therefore the functionality at position-12 decreased the potency

Compound
No.   [148].Hence the substitution at position 9 provides the opportunity to generate a linker that will not disrupt the water network and that can reach the peripheral site without making steric clashes with gorge residues.This led to the development of dual binding huprine-heterodimer inhibitors by coupling huprine with heterocyclic ligands, which are discussed below.

Galanthamine Analogs
Galantamine (Figure 2) is an alkaloid isolated from the bulbs and flowers of Galanthus woronowii in plant family Amaryll idaceae [159] and later synthesized by several researchers [160,161].It is a reversible competitive and selective AChE inhibitor.It also inhibited human nicotinic ACh receptors (nAChRs) in AD patients, but has not been explored well due to its toxicity factor [162,163].In 2015, Doytchinova et al., reported the galanthamine-indole heterodimer 101 (Figure 21, Table 6) as a potent AChE inhibitor against enzyme rhAChE experimentally (IC 50 = 11 nM) [164] which is 82 times stronger than galanthamine (IC 50 = 1070 nM) against AD.The galanthamine moiety binds to the CAS and the indole moiety binds to PAS of AChE enzyme.Additionally, it was able to block the Aβ deposition on AChE.

Novel Small Molecules
Apart from the above mentioned homo and hetero dimers, many other small molecules have been explored for the treatment of AD.Some recently reported potent small AChE inhibitors are discussed here.

Novel Heterocyclic Molecules
A number of heterocyclic molecules incorporating various heterocyclic rings, such as imidazole, pyrazole, thiazole, thiophene, pyridine, pyrimidine and triazine have been evaluated against AChE inhibition in search of anti-Alzheimer's agents.Among them some potent heterocyclic molecules reported in last 5 years are described here.

Miscellaneous
Apart from the above mentioned AChE inhibitors, the other potential AChE inhibitors reported in the last years are discussed below.

Conclusion
The published clinical trial statistics indicate that in total 93 molecules have been registered under total 115 different phases of clinical trial for AD therapies till January 4, 2016.Only 24 agents reached in phase III in 36 trials.Most of the diseases modifying treatments (76%, 43% and 57% in phase III, II, I respectively) address amyloid-related targets.These statistics pointed out two things; firstly, that only a small number of molecules being assessed in clinical trial, secondly, that there is very high failure rate in advanced stage drug development for AD.It means that disease-modifying treatments alone could not provide any drug in last one decade which suggests that there was an urgent need for alternative approach.Hence this review underscores the extensive research carried out in search of AChE inhibitors as anti-alzheimer's agents.The major approach for this has been towards the search of dual binding site AChE inhibitors as they not only bind with CAS and PAS of the enzyme but also modulated additional targets such as β-amyloid, MAO-A/B, tau inhibition, reduced the free radicals and oxidative damage.However, a large number of dual binding site AChE inhibitors reported in last decades but none of them have been proved by FDA after 2003 as an effective anti-alzheimer's drug.That indicates that mere inhibitor design strategies based solely on SAR-conclusions are insufficient.A balanced drug profile including AChE inhibitory activity, solubility, cytotoxicity and permeability should be considered in lead optimization and further in vivo investigation.The potent inhibitors which extended beyond the previously established SARs are i) the analog 28 with an excellent AChE inhibition (IC 50 = 0.006 nM) along with effective anti-Aβ (1-42) aggregation activity (52.5% at 10 µM) and anti-tau aggregation activity ( 40.7% at 10 µM) in E. coli., with good BBB permeability (P e =10.5 (10 -6 cm/s) in CNS.ii) the other example is novel templates of carbamates such as the quinoline/tetrahydroquinolin-6(-7)-yl carbamates, 56 (IC 50 = 100 nM) which has shown 8 and 2 times higher therapeutic window than rivastigmine and donepezil respectively.Additionally, the later 56 showed improvement in reversible cognitive impairment induced by scopolamine as compared to existing drugs with less side effects.The other carbamate prodrug 64 acting through central cholinergic system with remarkably high AChE inhibitory activity (IC 50 = 20 nM) suggest that the pro drug approach could also be an alternative for rational designing of selective central AChE inhibitors.

Figure 2 :Volume 4 •
Figure 2: Diagrammatic view of AChE enzyme active site and catalytic hydrolysis of ACh.

Figure 6 :
Figure 6: Structures of tacrine analog with their IC 50 values.

Table 1 :
The screening model and activity IC 50 of Tacrine Analogs.

Table 2 :
The screening model and activity IC 50 of quinoline analogs.

Table 3 :
The screening model and activity IC 50 of rivastigmine analogs.

Table 4 :
The screening model and activity IC 50 of donepezil analogs.

Table 5 :
The screening model and activity IC 50 of huperzine analogs and huprine heterodimers.

Table 6 :
The screening model and activity IC 50 of galanthamine analogs.Saxena AK, Saini R (2018) The Structural Hybrids of Acetylcholinesterase Inhibitors in the Treatment of Alzheimer's Disease: A Review.J Alzheimers Neurodegener 4: 015.

Table 7 :
The screening model and activity IC 50 of novel heterocyclic molecules.

Table 8 :
The screening model and activity IC 50 of some miscellaneous.