Viegas Jr.-MS

Send Orders for Reprints to [email protected] Current Medicinal Chemistry, 2018, 25, 1-35

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REVIEW ARTICLE

Multi-Target Directed Drugs as a Modern Approach for Drug Design Towards Alzheimer’s Disease: An Update Matheus de Freitas Silva, Kris Simone Tranches Dias, Vanessa Silva Gontijo, Cindy Juliet Cristancho Ortiz and Claudio Viegas Jr.* PeQuiM - Laboratory of Research in Medicinal Chemistry, Institute of Chemistry, Federal University of Alfenas, 37133-840, Brazil

ARTICLE HISTORY Received: July 03, 2017 Revised: December 07, 2017 Accepted: January 05, 2018 DOI: 10.2174/0929867325666180111101843

Abstract: Alzheimer’s disease (AD) is a progressive multifactorial neurodegenerative disorder. Currently, no effective treatment is available and this is due to multiple factors involved in pathophysiology and severity of AD. A recent approach for the rational design of new drug candidates, also called multitarget-directed ligands (MTDL) strategy, has been used to develop a variety of hybrid compounds capable to act simultaneously in diverse biological targets. The discovery of drug molecules capable of targeting multiple factors involved in AD pathogenesis would greatly facilitate in improving therapeutic strategies. This review is a complement to another review article, recently published by our group, which covered the previous period of 2005-2012, and highlights recent advances and examples of the exploitation of MTDL approach in the rational design of novel drug candidate prototypes for the treatment of AD

Keywords: Alzheimer’s disease; multi-target directed drugs; rational drug design; multifunctional drugs, multitarget drugs, neurodegenerative diseases, MTDLs. 1. INTRODUCTION Alzheimer’s disease (AD), known as a progressive multifatorial neurodegenerative disorder, is the current most common cause of dementia and is characterized by a progressive cognitive impairment in the elderly people. Currently, more than 35 million people are affected worldwide and this number is estimated to get doubled every 20 years, leading to more than 115 million AD patients in 2050 [1]. Although its etiology is not completely understood, several factors including deficits in acetylcholine (ACh) level, β-amyloid (Aβ) deposits, tau-protein hyperphosphorylation, oxidative stress, mitochondrial dysfunction, imbalance in biometals and cholesterol and neuroinflammation are considered to play significant roles in the pathophysiology and severity of this neurodegeneration. These factors together, accentuate changes in the central nervous sys *Address correspondence to this author at the Institute of Chemistry, Federal University of Alfenas, P.O. Box: 37133-840, AlfenasMG, Brazil; Tel/Fax: + 55 35 37011880, + 55 35 37011881; E-mail: [email protected] 0929-8673/18 $58.00+.00

tem (CNS) together, accentuate changes in the central nervous system (CNS) homeostasis, starting a complex process of multiple interconnected physiological damage, leading to cognitive and memory impairment and neuronal death [2-4]. AD patients present lower levels of acetylcholine (ACh) and an impaired of cholinergic transmission, resulting in learning and memory dysfunction. The possibility of modulation of these related events gave rise to the “Cholinergic Hypothesis” for AD, which calls for enhance cholinergic neurotransmission by the inhibition of the enzyme responsible for the metabolic breakdown of acetylcholine (ACh) [5]. Currently, five drugs (Fig. 1) were approved by the Food and Drug Administration (FDA) for the treatment of DA symptoms. Among them, four drugs are based on strategies of AChEIs, such as donepezil (1), tacrine (2), rivastigmine (3) and galantamine (4), with beneficial effects on cognitive, functional and behavioral symptoms of the disease. Donepezil (1) is the most effective pharmacological agent for AD treatment. How© 2018 Bentham Science Publishers

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de Freitas Silva et al.

ever, it is effective in reversing the symptoms for only a short period of time. Tacrine (2), the first approved anti-Alzheimer drug, was discontinued because it turned out to be hepatotoxic. By contrast, memantine (5) was the last drug approved by the FDA in 2003, and is also the only one that does not inhibit AChE. Memantine acts as an antagonist of glutamate receptors of type N-methyl-D-aspartate (NMDA), avoiding an influx of Ca2+ and protecting cells against excessive glutamate [6-9]. N

N NH2 O

O

O

N

N

O donepezil (1)

rivastigmine (3)

tacrine (2) H3CO

NH2

O H

HO

N

galantamine (4)

memantine (5)

Fig. (1). Chemical structures of the drugs approved by FDA to treat AD: donepezil (1), tacrine (2), rivastigmine (3), galantamine (4) and memantine (5)

Another therapeutic premise, known as “Amyloid Hypothesis” is based on the physiological deregulation in some brain regions, which the origin remains not entirely understood, leading to overproduction of amyloid peptide (Aβ). In AD patients, β- and γ-secretase enzymes abnormally cleave Aβ, producing insoluble fragments of 1-42 amino acid residues that is prone to oligomerization and formation of insoluble neurotoxic aggregates, the so-called amyloid plaques. These extracellular deposits play a critical role in the installation and disease progression, besides a complex neuroinflammatory process and cell death. The neurotoxicity of amyloid plaques is responsible for secondary events such as hyperphosphorylation of the neuronal microtubule constitutive tau protein. This abnormal phosphorylation of tau leads to structural collapse of microtubules and the consequent release of tau protein fragments, which take the form of insoluble fibrillar coiled, depositing intracellular as neurofibrillary tangles [10,11]. It is well known that both Aß deposition and formation of neurofibrillary tangles are related to increased free radical production and oxidative stress diffused by the brain. In recent years, significant research has been devoted to characterize and understand the role of free radical formation, oxidative cell damage, disruption of

homeostasis by metal ions and inflammation in the pathogenesis of AD [12-14]. Regarding the identification of a number of new possible molecular targets for AD therapy reported in literature, peroxisome proliferator-activated receptors (PPARs) are one of the most prominent targets, being considered and evaluated as a suitable therapeutic aim. These proteins belong to the superfamily of phylogenetically related protein termed nuclear hormone factor, which act as lipid sensors [15]. Synthetic ligands for PPARγ include antidiabetic drugs that might be able to counteract the increased AD risk in patients with type 2 diabetes, with useful effects in AD both on core pathological processes in brain and on peripheral factors [16]. The activation of these nuclear receptors has been associated with the reduction of chronic inflammation, ability to reduce the neurotoxicity, improvements in hippocampal neurons and as blood-brain barrier (BBB) traffic modulators. So, PPARs agonist emerge as one of the last new strategies explored in drug design for the treatment of neurodegenerative diseases such as AD [17]. Given the variety of factors associated with the onset, progress and severity of complex diseases such as cancer, diabetes, chronic inflammation, hypertension and neurodegenerative disorders, it has become increasingly apparent that the treatment of these diseases based on the “one-drug-one-target” paradigm has failed. Thus, it becomes unavoidable to adopt a new concept for the rational design of new drugs against AD [18,19]. New therapeutic strategies are being developed to reverse the progress of AD. Considering the multifactorial aspects related to its pathophysiology, one suitable approach could focus on the development of new drugs capable to modulate concomitantly various molecular targets involved in the disease. In this context, the strategy of designing multi-target directed ligands (MTDL) is gaining special attention in the scientific community. This approach is based on molecular hybridization, which is a tool for the design of innovative molecular patterns, retaining structural characteristics of the original bioactive molecular prototypes. This strategy can give rise to molecular hybrids with selective affinity for multiple targets, preferably in different biochemical cascades [20,21]. This review aims to show some examples of the exploitation of MTDLs approach in the rational design of novel drug candidate prototypes for the treatment of AD.

Multi-Target Directed Drugs as a Modern Approach

2. MULTI-TARGET DIRECTED LIGANDS (MTDLS) APPROACH FOR THE TREATMENT OF AD Considering the multifactorial hallmarks of AD, one can establish three reasonable therapeutic approaches: a) the use of two or more associated drugs; b) the use of two or more active principles in the same formulation or c) a single drug containing structural attributes that guarantee a selective multiple target action. The latter workaround is the most recent approach, based on the polypharmacology concept, and has aroused the scientific community interest as a new paradigm in rational molecular design [18]. This strategy is also based on molecular hybridization, which is an important and widely used tool for the design of new molecular scaffolds, that retains partial structural characteristics of the original molecules and preferentially replicate their biological activity [20,21]. Since 2005, the literature shows promising results with the application of MTDLs approach [22]. The design of multifunctional drug candidates based on wellknown drugs such as donepezil, tacrine or rivastigmine [23,24], as well as natural bioactive products like curcumin [25] or resveratrol [26] has grown significantly in recent years. This review is an updated version of our earlier work [18] and aims to report the most recent contributions of the exploitation of MTDLs approach in the rational design of novel drug candidate prototypes for the treatment of AD. 3. MTDLs INSPIRED BY DONEPEZIL Donepezil (Aricept®, 1) arose in the 80’s as a reversible and non-competitive AChE inhibitor (AChEI), and is currently the most used drug for the AD therapy [27]. Computational studies indicate that its mechanism of action is derived from both the N-benzylpiperidine and indanone subunits, which guarantee high affinity and selectivity for AChE28. Clinical studies revealed that the use of donepezil results in significant improvement in memory, concentration, language and reasoning, without signs toxicity, but without curative effect [28]. The various examples in literature highlight how donepezil has been widely used as a prototype in the design of new MTDLs drug candidates planned by molecular hybridization. Wu and co-workers developed new hybrid compounds capable of acting concomitantly as AChEIs, monoamine oxidase (MAO) inhibitors and antioxidants, interacting with different targets involved in AD pathogenesis. The novel hybrid series were planned by structural combination of the N-benzyl-piperidine moi-

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ety from donepezil (1) as an AChEI pharmacophore, a metal chelating portion represented by the 8hydroxyquinoline system (6) and a propargylamine moiety (7) for MAO inhibitory activity (Fig. 2). As a result, six new substances were synthesized with compound 8 stood out from the others, showing a selective inhibition of MAO (IC50 MAO-A= 10.1 ± 1.1 µM and IC50 MAO-B >100 µM) and interesting IC50 values for AChE inhibition (IC50= 0.029 ± 0.003 µM) and BuChE (IC50= 0.039 ± 0.003 µM). Then, the evaluation of metalchelating properties, revealed compound 8 to be capable of chelating Zn +2 and Cu +2 ions. These results explain the observed dose-dependent pharmacological profile, when used in concentrations higher than 0.4 µM, for inhibition of Cu-mediated H2 O2 production. In addition, compound 8 also showed antioxidant activity (1.12 ± 0.43 trolox equivalent by ORAC assay). Finally, we can state that compound 8 represent a genuine multi-target hybrid ligand, being a strong inhibitor of ChEs and a selective inhibitor of MAO-A, with antioxidant and Cu+2 chelating activities [29]. O H3CO H3CO

N

N Donepezil (1)

N

OH 8-hydroxyquinoline (6)

Propargylamine (7)

Molecular hybridization

N N OH

N CN

8 IC50 MAO-A = 10.1 ± 1.1 µM IC50 MAO-B = >100 µM IC50 AChE = 0.029 ± 0.003 µM IC50 BuChE = 0.039 ± 0.003 µM ORAC = 1.12 ± 0.43 trolox equivalent

Fig. (2). Design of a new donepezil-8-hydroxyquinolinepropargylamine hybrid compound (8) with antioxidant, metal-chelating, and selective inhibition of AChE and MAOs properties.

The N-benzylpiperidine moiety, derived from donepezil (1), was also used as scaffold by Pudlo and coworkers for the design of a series of donepezilquinolone hybrid compounds (10) with antioxidant and AChE inhibitory properties (Fig. 3). In general, quinolone derivatives (9) include motifs exhibiting a wide variety of biological activities, such as ROS scavenging ability. All phenolic compounds of the target-series 10 have shown moderate to high radical scavenging activities. Compounds 11 and 12 were the most potent, with compound 11 showing an effective inhibition of AChE and high radical scavenging activity, without altering



the inhibition of AChE. The catechol moiety of compound 12 provided antioxidant activity and was also involved in an additional interaction with AChE. However, despite the multifunctional properties exhibited by both compounds 11 and 12, their AChE inhibitory activities were considered weak, with IC50 values of 0.23 ± 0.03 µM and 0.98 ± 0.35 µM, respectively. By contrast, these results suggested that, due to its ability in radical scavenging, quinolone moiety could be considered an interesting biophore in the design of novel multipotent molecules for the treatment of AD [30]. In 2015, Guzior and co-workers reported the synthesis and pharmacologic evaluation of a new series of donepezil-based compounds endowed with inhibitory properties against cholinesterases and β-amyloid aggregation. The donepezil-based series, illustrated by compound 14 (Fig. 4), consisted of an isoindoline-1,3dione fragment connected to N-methyl-benzylamine subunit by alkyl linkers of different lengths. Crystallographic studies revealed that AChE has two binding sites: catalytic anionic site (CAS) and peripheral anionic site (PAS). AChE is associate with the formation of senile plaques, once it can interacts with the amyloid β-peptide (Aβ) and promotes amyloid fibril formation close to PAS, besides Aβ aggregation process [31]. Thus, dual AChEIs could have pro-cognitive effects as

well as disease-modifying properties by inhibiting Aβ aggregation in AD. These findings showed an additional role of PAS, resulting in the development of novel classes of active bivalent compounds, operating as AChE inhibitors by concomitant interaction with both CAS and PAS. Based on this premise, a series of phthalimide-benzylamine derivatives were designed as dual binding site AChE inhibitors, in which the N-benzylamine moiety could interact with the CAS of the AChE and the isoindoline-1,3dione fragment could bind to the PAS. Biological results disclosed compound 14 (Fig. 4) as the most potent human AChE inhibitor (IC50= 0.361 ± 0.010 µM) with additional properties such as Aβ aggregation inhibition and neuroprotective effect against Aβ toxicity. Kinetic studies revealed that 14 inhibited AChE in a noncompetitive mode and the results from blood-brain barrier (BBB) permeability assay showed their ability to penetrate in the CNS [32]. In another approach, Więckowska and cols. designed and synthesized new compounds as donepezil derivatives containing the N-benzylpiperidine moiety combined with phthalimide (15) or indole moieties (16) (e.g. compounds 17 and 18, Fig. 5). Most of these compounds showed micromolar-range activities towards cholinesterases and Aβ aggregation, along with

H3CO O

H3CO

N OH

O

R3

N H

Donezepil (1) R2

OH

N R1

R3

R2

N

N

O (10)

11 R1 = H, R2= OMe, R3 = OMe IC50 AChE = 0.23 ± 0.03 µM

O

12 R1 = H, R2= OH, R3 = OH IC50 AChE = 0.98 ± 0.35 µM ORAC = 12.2 ± 0.4 trolox equivalent

R1 Quinolones (9)

Fig. (3). Chemical structure of the multifunctional quinolone-benzylpiperidine derivatives (10) and the most active compounds 11 and 12, designed as novel antioxidant and AChE inhibitors for AD. H3CO O

H3CO

N O

Donepezil (1)

N

n

O N

N H O

interactions with the PAS

13

interactions with the CAS

N

O

R

14 n=6 , R=H IC50 AChE = 0.361 ± 0.010 µM

Fig. (4). Design of isoindolino-1,3-dione derivatives and structure of the most active compound 14.



5

H3CO O

H3CO

N

O

Donepezil (1)

N

N

O phthalimide moiety (15)

O N O

N

6

indole moiety (16) N

Cl N

6

N H

N H 18

17

AB aggreagation of 56% IC50 AChE = 0.34 ± 0.0097 !M

AB aggreagation of 72.5% IC50 AChE = 0.72 ± 0.038 !M

Fig. (5). Design and structure of the bioactive hybrid donepezil-phthalimide 17 and donepezil-indole derivative 18.

positive results in BBB permeability assays. Derivative 17 is an example of such compounds, as it combines inhibitory activity against BuChE (IC50= 0.72 ± 0.038 µM) with Aβ anti-aggregation activity (72.5% inhibition at 10 µM). Moreover, these compounds exhibited a donepezil similar anti-cholinergic effect in animal model of memory impairment induced by scopolamine [33]. Considering that compound M30 (19), had been already described as a potent selective inhibitor of MAOA and metal chelator, its structural framework was selected as a model for molecular hybridization with the N-benzylpiperidine moiety present in donepezil (1) in the drawing of a novel family of M30-donepezil hybrid ligands (Fig. 6). These compounds were designed to act as multifunctional inhibitors of AChE, MAO-A, metal chelators and inhibitors of Aβ-protein formation. Among seven synthesized compounds, derivative 20 showed the best activity, being able to inhibit AChE (IC50= 1.8 ± 0.1 µM) and BuChE (IC50= 1.6 ± 0.2 µM), without significant selectivity, and MAO-A (IC50= 6.2 ± 0.7 µM) and MAO-B (IC50= 10.2 ± 0.9 µM) with a 2fold selectivity for MAO-A. This compound also showed chelating ability for Cu2+ and Fe3+ ions, when subjected to the toxicity test with HepG2 cells, with lower toxicity than donepezil, but similar oral absorption. Thus, compound 20 exhibited balanced properties as AChE and MAO-A inhibitor, with biometals chelating ability, being an interesting multifunctional ligand for future studies aiming at an improvement of its action profile for AD therapeutics [34]. Other two new hybrid compounds 23 and 24 were designed as multipotent inhibitors of ChEs and MAO

H3C N

N

OCH3

O

OCH3

N OH

Donepezil (1) IC50 AChE = 0.0067 ± 0.0004 !M IC50 BuChE = 7.4 ± 0.1 µM IC50 MAO A = 850 ± 13 µM IC50 MAO B = 15 µM Molecular hybridization

M30 (19) IC50 AChE = >100 µM IC50 BuChE = -IC50 MAO-A = 0.057 ± 0.02 µM IC50 MAO-B = 1.5 µM

N OH

N N CN 20 IC50 AChE = 1.8 ± 0.1 µM IC50 BuChE = 1.6 ± 0.2 µM IC50 MAO-A = 6.2 ± 0.7 µM IC50 MAO-B = 10.2 ± 0.9 µM

Fig. (6). Design and structure of donepezil-M30 hybrid 20 and its inhibitory data for ChEs and MAO enzymes.

based on the structure of ASS234 (22), a MAO-A/B, AChE and BuChE inhibitor, and donepezil (1), an AChE inhibitor, and PF9601N (21), a potent and selective MAO-B inhibitor (Fig. 7). The hybrid 23 was identified as a potent nanomolar range inhibitor of MAO-A (IC50= 5.5 ± 1.4 nM) and moderate inhibitor of MAO-B (IC50= 150 ± 31 nM), AChE (IC50= 190 ± 10 nM), and BuChE (IC50= 830 ± 160 nM). Molecular modeling analysis suggested that compound 23 is a mixed-type AChE inhibitor, and that its linear conformation allows to span both the CAS and PAS, that could explain its superior binding toward AChE in comparison to the



O OCH3

HN O

OCH3

Donepezil (1) IC50 EeAChE = 13 ± 0.01 nM IC50 eqBuChE = 840 ± 0.005 nM

BnN

NH PF9601N (21)

Molecular Hybridization BnN O

N N

ASS234 (22) IC50 EeAChE = 350 ± 0.1 nM IC50 eqBuChE = 460 ± 0.06 nM IC50 hMAO-A = 4.2 ± 0.5 nM IC50 hMAO-B = 39 ± 4 nM BnN

HN

O

N

N

23 IC50 EeAChE = 190 ± 0.01 nM IC50 eqBuChE = 830 ± 0.16 nM IC50 hMAO-A = 5.5 ± 1.4 nM IC50 hMAO-B = 150 ± 0.03 nM

BnN

O

N

N

HN O N

25 IC50 hAChE = 2.8 µM IC50 hBuChE = 4.9 µM IC50 hMAO-A = 6.3 nM IC50 hMAO-B = 183.6 nM

O

24 IC50 EeAChE = 130 ± 0.02 nM IC50 eqBuChE = > 1000 nM IC50 hMAO-A = > 1000 nM IC50 hMAO-B = > 1000 nM

Fig. (7). Design and biological data of the donepezil-PF9601N hybrid compounds 22-25.

less active compound 24. Furthermore, compound 23 was able to establish more interactions with the MAOA active site than to MAO-B, which may indicate more tightly interaction and selectivity with the former enzyme [35]. In another work, Bautista-Aguilera and cols. described the synthesis and evaluation of new donepezil−indolyl hybrids, designed in the basis of the structure of ASS234 (22, Fig. 7) as multifunctional drugs able to bind human MAO-A and ChE enzymes. In that approach, the authors varied the piperidine ring substituents, showing a sound quantitative structure−activity relationship (QSAR) study, leading to identification of compound 25 (Fig. 7) as the leadmolecule. According of QSAR studies the o-Methyl group in 25 improves the ligand recognition, increasing the hydrophobic interaction with hBuChE and π−π stacking interaction in hMAO-A, hMAO-B, and hAChE. The QSAR results were confirmed by biological evaluation, underscoring compound 25 as inhibitor of hMAO-A (6.3 ± 0.4 nM), hMAO-B (183.6 ± 7.4 nM), hAChE (2.8 ± 0.1 µM) and hBuChE (4.9 ± 0.2 µM). Similarly to compound 21, ADMET virtual analysis suggested that compound 25 holds good druggable properties, which was confirmed by in vitro BBB permeation assays36.

A series of 5,6-dimethoxy-indanone-benzamides, planned as analogs of donepezil were also drawn as multifunctional agent candidates for the treatment of AD. In this study, the aim was to evaluate a family of donepezil-like secondary amide compounds that display a potent inhibition of cholinesterases and Aβ, with antioxidant and metal chelation abilities. The authors synthesized some 5,6-dimethoxy-indanone-2carboxamide derivatives containing ortho-, meta-, and para-substituted secondary aromatic amines which could act as potent AChE inhibitors. The results showed that the designed compounds are likely to interact with amino acid residues in the CAS of AChE. Compounds 26 (IC50 BuChE= 2.10 ± 0.015 µM) and 27 (IC50 AChE= 0.08 ± 1.813 µM) (Fig. 8), were found to be the most potent inhibitors in the series, with compound 26 as the most potent antioxidant as well. Compound 27 was selective for AChE, with additional good anti-aggregation activity (55.3% at 25 µM), along with a moderate radical scavenging activity. Additionally, docking studies revealed that compound 27 was able to multiple bonding interactions with both catalytic and peripheral AChE binding sites, which supports its high inhibitory potency and mixed-type inhibition mechanism [37].



Donepezil moiety H3CO

and IC50 BuChE= 2384 ± 52.10 nM) were identified as the most active compounds in the target-series. However, only compound 30 showed a promising multitarget profile, being able to act on 5HT6Rs and AChE. Furthermore, compound 30 was able to permeate BBB in in vitro and in vivo models. In a first result, compound 30 seemed to act with a dose-dependent mode, but additional studies with a fixed dose, revealed that its activity increases with time. Finally, the authors concluded that compound 30 should be considered a promise multi-target ligand, with potent AChE, BuChE and 5HT6Rs inhibitory activities and so, an interesting prototype for the development of new drug candidates for AD [38].

O HN

H3CO

R1

O R3

R2

ortho-, meta-, and para-substituted N-benzylaniline rings 27 R1=R2= H, R3= F IC50 AChE = 0.08 ± 1.813 !M IC50 BuChE = 3.21± 0.104 !M

26 R1=R3= H, R2= OCH3 IC50 AChE = 0.54 ± 0.141 !M IC50 BuChE = 2.10 ± 0.015 !M

Fig. (8). Chemical structures of the most active 5,6dimethoxy-indanone-2-carboxamide derivatives 26 and 27.

Lipoic acid (LA, 31, Fig. 10) is a natural occurring substance known for its therapeutic potential as a direct scavenger of ROS, with fast absorption and fast tissue distribution. It is able to quench free radicals in aqueous and lipid phases, chelate bimetals and regenerate other biogenic antioxidants [39]. Data from literature suggest that LA could also plays remarkable role in neuroprotection and cognitive enhancing effects related to AD pathology [40-42]. In spite of donepezil and AP2238 (32) be AChE inhibitors, they are also potent σ-1 receptor agonist and BACE1 inhibitor, respectively [43]. Therefore, these compounds were taken as molecular prototypes for the designing of two new families of LA-donepezil and LA-AP2238 hybrid compounds (Fig. 10), leading to six novel hybrid molecules, four LA-donepezil and two LA-AP2238 hybrids. Some of them were separated in their enantiomers and tested separately in order to compare their results with those of the racemate mixtures. Among the targetsubstances, compounds 33 and 34 were highlighted due to their best results in AChE inhibition. The racemic

Recently, intense research efforts in neuroscience disclosed serotonin receptors (5HT6Rs) play an important role in the progress of neurodegenerative disorders and as a new suitable molecular target for drug development. These receptors are not only related to the cognitive aspects of AD, but also to its behavioral and pathophysiological aspects. These receptors are distributed almost exclusively in the brain areas, being involved in the learning and memory processes33- [36]. According to this new perspective, the inhibition of 5HT6Rs was aimed at the design of the novel multifunctional AChE inhibitors 29 and 30 (Fig. 9). These hybrids were drawn based on the structure of compound 28, a known 5HT6Rs antagonist, and donepezil (1) or tacrine (2) as AChE inhibitors prototypes. In this way, six tacrine-28 and thirteen donepezil-30 hybrids were synthesized and evaluated for the ability to inhibit concomitantly 5HT6Rs, AChE and BuChE. Compounds 29 (IC50 5HT6= 2.0 ± 0.2 nM, IC50 AChE= 12.9 ± 0.17 nM and IC50 BuChE= 8.2 ± 0.23 nM) and 30 (IC50 5HT6= 2.0 ± 0.3 nM, 37.2% AChE inhibition at 10 µM

NH2 HN

N

N O

N

H3CO

Donepezil (1)

N

S

N

O

O Compound I (28)

H3CO

H N

Tacrine (2)

N

N

H N

N N O

N S O

O

29 IC50 5HT6Rs = 2.0 ± 0.2 nM IC50 AChE = 12.9 ± 0.17 nM IC50 BuChE = 8.2 ± 0.23 nM

7

S

N O

30 IC50 5HT6Rs = 2.0 ± 0.3 nM 37.2% AChE inhibition at 10 µM IC50 BuChE = 2384 ± 52.10 nM

Fig. (9). Molecular design and chemical structure of new multifunctional donepezil-28 (29) and tacrine-28 (30) hybrids.



O N

O H3CO

OH

N

H3CO

S

CH3

S

Donepezil (1)

Lipolic Acid (31)

H3CO


O

H3CO

O

N

S S

N H

33 (R,S)-4 IC50 AChE = 0.39 ± 0.03 µM IC50 BuChE = 1.23 ± 0.14 µM IC50 BACE1 = 5.65 ± 0.26 µM (R)-4 IC50 AChE = 0.43 ± 0.11 µM IC50 BuChE = 0.79 ± 0.20 µM IC50 BACE1 = 8.11 ± 0.26 µM (S)-4 IC50 AChE = 0.21 ± 0.09 µM IC50 BuChE = 0.63 ± 0.09 µM IC50 BACE1 = 9.92 ± 0.39 µM

O AP2238 (32)


N

S

O

S

N H

CH3

34 IC50 AChE = 2.43 ± 0.12 µM IC50 BuChE = 4.87 ± 0.23 µM IC50 BACE1 = >10 µM

Fig. (10). Chemical structures and biological data for the most potent LA-donepezil-AP2238 hybrids 33 and 34

form of 33 showed a selective enzyme inhibitory activity with IC50 AChE= 0.39 ± 0.03 µM, IC50 BuChE= 1.23 ± 0.14 µM, IC50 BACE1 = 5.65 ± 0.26 µM, with the Sisomer showing to be the eutomer (IC50 AChE= 0.21 ± 0.09 µM; IC50 BuChE= 0.63 ± 0.09 µM and IC50 BACE1= 9.92 ± 0.39 µM), whereas the distomer R-33 showed 2fold and 1.4-fold weaker potency and selectivity towards AChE (IC50= 0.43 ± 0.11 µM) and BuChE (IC50= 0.79 ± 0.20 µM), respectively, as well as on BACE1 (IC50= 8.11 ± 0.26 µM). In the same assay, compound 34 showed a weaker enzymatic inhibitory potency with IC50 AChE= 2.43 ± 0.12 µM, IC50 BuChE= 4.87 ± 0.23 µM, IC50 BACE1 >10 µM, in comparison to racemic 33 and its single isomers. Furthermore, compounds (R)-33, (S)-33, (R,S)-33 and 34 were subjected to the inhibition test of σ1R and σ2R, showing affinities of Ki σ1 = 8.90 ± 0.45 nM, Ki σ2= 232 ± 27 nM for (R,S)-33, Ki σ1= 7.56 ± 0.98 nM, Ki σ2= 205 ± 42 nM for (R)-33, Ki σ1 = 15.1 ± 1.4 nM, Ki σ2 = 289 ± 51 nM for (S)-33 and Ki σ1 = 21.0 ± 2.6 nM, Ki σ2 = 1400 ± 230 nM for compound 34. All tested substances showed selectivity for AChE and for σ-1 receptor agonist, with weak antioxidant activities and good CNS permeability [44]. In a recent work, we reported the synthesis and pharmacological evaluation of a new series of feruloyldonepezil hybrids based on the combination of the pharmacophoric N-benzylpiperidine subunit from donepezil (1) and the feruloyl subunit present in ferulic acid (36) and curcumin (35) [45]. Curcumin (35), an abundant natural polyphenol found in Curcuma longa rhizomes, is also widely used as scaffold for the plan-

ning of new MTDLs for AD due its potent antioxidant and anti-inflammatory properties, playing an important role in the decrease of oxidative damage, inflammation and amyloid accumulation with an additional biometals chelating ability. The feruloyl moiety present in curcumin (35) is responsible for its antioxidant activity and is also present in the ferulic acid (36) structure (Fig. 11), a natural compound with potent antioxidant activity. Based on these findings, a novel series of feruloyl-donepezil hybrid compounds were designed as multitarget drug candidates for the treatment of AD. In vitro results revealed potent AChE inhibitory activity for some of these compounds and all of them showed moderate antioxidant properties. Compounds 37, 38 and 39 (Fig. 11) were the most potent AChE inhibitors, highlighting 37 with IC50 = 0.46 µM. Kinetic and molecular docking studies revealed that compounds 37 and 39 are non-competitive inhibitors, capable to interact with PAS of AChE. In addition, these three most promising compounds exhibited significant in vivo antiinflammatory activity in the mice paw edema, pleurisy and formalin-induced hyperalgesy models, in vitro metal chelation activity for Cu2+ and Fe2+, and neuroprotection of human neuronal cells against oxidative damage. Based on these data, 37 was elected as a leadcompound in the series due to its best inhibitory activity of AChE, also displaying high antioxidant activities in neuronal SH-SY5Ycells, in both direct and indirect mode (activating the Keap1/Nrf2/ARE pathway), besides being a good biometal chelator and with significant in vivo anti-inflammatory activity in different animal models [45].

Multi-Target Directed Drugs as a Modern Approach O


9

O

O H3CO

HO

H3CO

OH curcumin (35)

OCH3

N

donepezil (1)

OCH3 molecular hybridization

O OH

O

HO

R1

O OCH3

Ferulic acid (36)

N HO OCH3 37 R1= H IC50 AChE= 0.46 µM IC50 BuChE= 24.97 µM EC50 DPPH = 49.41 µM

38 R1= NO2 IC50 AChE= > 30 IC50 BuChE= -EC50 DPPH = > 100

39 R1= Br IC50 AChE= > 30 IC50 BuChE= -EC50 DPPH = > 100

Fig. (11). Design and chemical structures of compounds 37-39 as new donepezil-feruloyl hybrids with neuroprotective, metal chelating, antioxidant, anti-inflammatory and AChE inhibitory properties

In another recent approach, Xu and cols. synthesized and evaluated a novel family of donepezil-ferulic acid hybrids as MTDLs against Alzheimer's disease (Fig. 12). In vitro results indicated that some of these molecules exhibited potent cholinesterase inhibitory activities, outstanding radical scavenging activities and good neuroprotective effects on PC12 cells, and could penetrate the CNS. Interestingly, the compounds of series 41 without hydroxyl substituents exhibited better ChEs inhibition than the parent compounds with hydroxyl substituents. Compound 41b, bearing two methoxy groups on the R2 and R4 positions and one hydroxyl group on the R3 position, has better ChE inhibitory activity (IC50= 0.398 ± 0.028 µM for AChE; IC50= 0.976 ± 0.102 µM for BuChE). On the other hand, compounds of series 42 exhibited only moderate inhibitory activities towards both ChEs (42a, IC50= 2.60 ± 0.37 µM for AChE; IC50= 1.08 ± 0.16 µM for BuChE), suggesting that the β-diketone bonding functionality is not needed to induce inhibition for these analogues. Additionally, compounds 40a-c, 41a-c, 42a and 42b showed ability to scavenge the ABTS radical with 1.41, 1.81, 1.65, 1.39, 1.78, 2.10, 0.76 and 7 values, respectively. Results from compounds 40a,b and 41a,b, which bear a phenolic hydroxyl group or methoxy group as R2 substituent, clarified that the nature of the substituent at the ortho position of the phenolic subunit is crucial. Moreover, the potency of compound 41b (IC50= 24.9 ± 0.4 µM) indicated that the locations of the phenolic hydroxyl and methoxy groups are non-adjustable. It can be seen that compounds 41a (5.16 × 10−6 cm.s −1), 41b (7.68 × 10 −6 cm.s−1) and 41c (5.38 × 10−6 cm.s −1) might be able to cross the BBB. Comparatively, the results obtained for compounds 41b

suggested that this ligand could represent a potential multifunctional neuroprotective agent as a new lead candidate for the treatment of AD [46]. Similar strategy was used by Wang and co-workers on taking advantage of the neurogenic potential profile of melatonin-based hybrids, which are endowed with additional anticholinergic properties. Thus, they designed a novel series of compounds (44-47, Fig. 13) obtained by fusing the N-benzylpiperidine moiety of the AChEI donepezil (1) and the indole subunit of the antioxidant melatonin 43. The design was based on the anticipation that melatonin-indole subunit could ensure neuroprotective features and could also interact with the AChE-PAS via π-π aromatic stacking for its aromatic character. On the side, the protonable N-benzyl piperidine moiety from donepezil, could be responsible for the interaction with the AChE-CAS through cationπ interaction. By this way, the new molecular scaffold could be able to a dual interaction with PAS and CAS of AChE. Biological evaluation of this series led to compound 47 with the most promising multifunctional profile, showing inhibitory activity of AChE (IC50= 193 ± 0.2 nM (EeAChE) and 273 ± 0.5 nM (hAChE)), with higher selectivity for BuChE (IC50= 73 ± 0.1 nM (eqBuChE) and 56 ± 0.1 nM (hBuChE)), along with moderate inhibition of Aβ1-42 selfaggregation (56.3% at 20 µM), good antioxidant activity (3.28 trolox equivalent by ORAC assay) and good biometal-chelating ability, also reducing PC12 cells death induced by oxidative stress and adequate BBB permeability [47]. Genistein (48) is a flavonoid found in soybeans and other plants that contain red clover. This substance pre-


de Freitas Silva et al. O

HO OH

H3CO

N

H3CO Ferulic acid (36)

O

Donepezil (1) H3CO Molecular hybridization

R2 R3

R1

R2

N H N

R4

n O

N H N

R1

n O

40a-c n=0 41a-d n=2

O

42a R1= OMe, R2= OH, n= 2 (, IC50= 2.60 ± 0.37 µM for AChE; IC50= 1.08 ± 0.16 µM for BuChE) 42b R1= OMe, R2= OH, n= 0 42c R1= H, R2= OH, n= 0

40a R1=OMe, R2=OH, R3=R4=H 40b R1=R2=OH, R3=R4=H 41a R1=R3=OMe, R2=OH, R4=H 41b R1= R4=H, OH, R2= OMe, OH, R3= OH (IC50= 0.398 ± 0.028 µM for AChE; IC50= 0.976 ± 0.102 µM for BuChE) 41c R1=H, R2= R4= OMe, R3= OH 41d R1= R4=H, R2=R3= OH

Fig. (12). Design strategy for a new series of donepezil-ferulic acid hybrid compounds 40-42. O HN

O

CH3 H3CO

H3CO

H3CO

N H

Donepezil (1)


Melatonin (43)

Antioxidant Anti-A! aggregation Neuroprotectant AChE-PAS binder

N

AChE inhibitor AChE-CAS binder

R3 H N HN

(44)

O

R2

R3

(45)

O

N

H N HN

R1

N

N H HN O

n

(46) n= 0

N

(47) n= 2 IC50 EeAChE = 193 ± 0.2 nM IC50 hAChE = 273 ± 0.5 nM IC50 eqBuChE = 73 ± 0.1 nM IC50 hBuChE = 56 ± 0.1 nM

Fig. (13). Design and chemical structures of melatonin-donepezil hybrid compounds 44-47.

sents several pharmacological activities, including antioxidant, anti-inflammatory activity, metal chelator and neuroprotective against Aβ-protein. These characteristics make 49 a good prototype for planning antiAlzheimer drugs. However, its lack in the inhibition of AChE prevents their direct use in the treatment of AD. For this reason, the N-benzylpiperidine fragment, that is one of the well-known pharmacophore group of donepezil (1), which tertiary amino group is related to adequate AChE inhibition, was used for a rational molecular hybridization with 48 aiming new genisteindonepezil hybrids designed as MTDLs with innovative structural pattern (Fig. 14). Thus, three families of hybrid compounds were prepared, with compounds 49 (IC50 hAChE= 5.80 ± 0.65 µM), 50 (IC50 hAChE= 3.88 ±

0.64 µM) and 51 (IC50 hAChE= 0.35 ± 0.03 µM) being the most active in each family for AChE inhibition. None of the synthesized compounds showed good antioxidant activity. Meanwhile, compound 51, in addition to a significant AChE inhibitory activity, also showed ability in Cu2+ chelation, with neuroprotective activity against Aβ-protein greater than curcumin. Moreover, despite the low antioxidant activity, compound 51 proved to be promising for further studies, as it has high ability to inhibit AChE and moderate ability to inhibit aggregation of Aβ-protein, both self-induced aggregation and Cu2+ induced aggregation [48]. Docking-assisted hybridization strategy of 52 and donepezil (1) (Fig. 15), was used by Farina and co-



11

O OH

OH

O

N

H3CO HO

O

H3CO

Donepezil (1) IC50 hAChE = 0.01 µM

Genistein (48)

N

H3C

OH

C6H12

N

O

OH

C4H8

O

H3CO

O

O

OH

O

OCH3

O

49 IC50 hAChE = 5.80 ± 0.65 µM

O

51 IC50 hAChE = 0.35 ± 0.03 µM

OH

C4H8

N

H3CO

O HO O O

C4H8

N

50 IC50 hAChE = 3.88 ± 0.64 µM

Fig. (14). Design and chemical structure of the multifunctional hybrids genistein-donepezil 50-51. O

Cl

N

O O

OCH3

Donepezil (1) (AChEI) AChE IC50 = 5.3 µM BChE IC50 = 2250 µM

O

MC 1095 (MAO-BI) (52) MAO-A IC50 = 8900 µM MAO-B IC50 = 5.1 µM

OCH3 Molecular hybridization

Catalytic site MAO-B R2

Entrance cavity MAO-B R3

N CAS AChE

SPACER

53

O

R1 O O PAS AChE

R2 R2

N R1

X

O

O

R1

O R4

54a R1= Me, R2= H; rMAO-A IC50 = 0.51 µM, AChE IC50 = 0.095 µM, BuChE IC50 = 0.67 µM 54b R1= Et, R2= H; rMAO-A IC50 = 2.1 µM, AChE IC50 = 0.32 µM, BuChE IC50 = 0.49 µM

N

X

O

O

R N

O

Y

O

X

O

56 R1=H, R2= CH2OH, R3= Me, R4= Bn R3 55a R1= Me, R2= Me, R3= Me, R4= Bn rMAO-B IC50 = 0.41 µM, AChE AChE IC50 = 0.10 µM IC50 = 0.42 µM, BuChE IC50 = 1.1 µM 55b R1= H, R2= H, R3= Me, R4= Me , BuChE IC50 = 0.24 µM

Fig. (15). Chemical structures of new aminocoumarins 54-56, as representative hybrid compounds derived from molecular hybridization of donepezil (1) and aryloxy-coumarin 52.

workers to draw the novel molecular pattern 53 (Fig. 15) to access inhibition of MAO-A and B, AChE and BuChE. Compounds 54-56 displayed from low to submicromolar potencies against rat MAO-B (rMAO-B) and EeAChE, whereas potencies against rat MAO-A (rMAO-A) and EeBuChE were slightly lower. Compounds with the longest linker, such as 54a and 54b, displayed the highest inhibitory potency on rMAO-A (IC50= 0.51 and 2.1 µΜ, respectively) and ChEs (IC50 AChE= 0.095 and 0.32 µΜ, respectively, and IC50 BuChE= 0.67 and 0.49 µΜ, respectively). Compounds with substituents in meta and para positions of the 7-benzyloxy subunit of the coumarin ring also were evaluated, with

eleven substances showing significant inhibition of AChE, and compound 55a as the most potent AChE inhibitor (IC50= 0.10 µM). In contrast, only six compounds exhibited a sub-micromolar activity over BuChE, with compound 55b showing the highest inhibitory potency (IC50= 0.24 µM). Moreover, compound 56 showed a good inhibitory activity over the three target enzymes with IC50=0.41, 0.42 and 1.1 µM for rMAOB, EeAChE and EeBuChE, respectively. Thus, some of these multipotent inhibitors, especially compound 55a, were identified as promising novel drug candidate prototypes, suitable for further pre-clinical studies in cognitive and neurodegenerative disease models [49].



O

OCH3 O

H3CO

H3CO

N

H2N Donepezil (1)

N

57

Cl

57 and 59 Ki(5 HT4R) ! 20 nM IC50(AChE) ! 400 nM

OCH3 O OCH3 O N

H2N N

H2N Cl

Cl

59

Donecopride (58)

AChE (45% at 10-5 M) 5-HT4R (Ki = 2.25 nM)

Fig. (16). Design of compounds 57 and 59 by molecular hybridization of donepezil (1) and donecopride (58).

Considering a number of recent findings that points a potential role of serotonin receptors in neurophysiology of AD [50,51], Rochais and co-workers reported the synthesis of a novel series of MTDLs displaying dual-binding site (DBS) inhibition of AChE and a partial 5-HT4R agonist activity (Fig. 16). Rational molecular design was based on the structure of the AChE inhibitor donepezil (1) and donecopride (58), a novel drug candidate exhibiting, for the first time, both in vitro DBS AChE inhibitory activity and a serotonergic subtype-4 receptor (5-HT4R) partial agonist effect [52], to produce the new hybrid compounds 57 and 59. Compound 57 could be considered as MTDL since they exhibit both 5-‐HT4R (Ki ≤ 20 nM) and AChE (IC50 ≤ 400 nM). Conversely, compound 59 was more potent in AChE inhibition (45% of enzyme activity at 10 µM), but showed a very weak affinity for 5-HT4R. Face to this results, compound 57 (Fig. 16) seems to have a symptomatic and disease-modifying effect, sustaining the AChE inhibitory activity and 5-HT4R antagonist activity, characteristic from the molecular prototypes donepezil (1) and donecopride (58), respectively, and thus representing a potentially promising drug candidate for AD [53].

of natural β-carboline-(pyrido [3,4-b]-indoles (62) and tacrine (2) (Fig. 17). Tacrine was used for the inhibition of ChE through its binding ability to the CAS of AChE, while β-carboline (60) was used for its potential interaction with the PAS due to its aromatic character. In vitro evaluation showed that most of the targetcompounds exhibited significant inhibition of AChE (EeAChE and hAChE), BuChE, self-induced Aβ aggregation, along with Cu2+ -induced Aβ1-42 aggregation and metal chelating ability. Compound 62 showed the highest ChE inhibitory activity (IC50 = 21.6 ± 0.8 nM (EeAChE), 63.2 ± 2.5 nM (hAChE) and 39.8 ± 1.6 nM (BuChE), with significant inhibition of Aβ aggregation (65.8% at 20 µM) and antioxidant activity (1.57 trolox equivalents). Kinetic and molecular modeling studies indicated that compound 62 was a mixed-type inhibitor, binding simultaneously to CAS and PAS of AChE. Besides, compound 62 showed to be capable to reduce PC12 cells death induced by oxidative stress and penetrate the BBB in vitro [57]. NH2 N N H

N

!"carboline (60)

Tacrine (2)

4. MTDLs INSPIRED BY TACRINE Tacrine (2, THA, Cognex®) was the first cholinesterase inhibitor approved by the FDA for the treatment of AD. However, in a few years it was banned in some countries due to its high hepatotoxicity and low bioavailability. In recent years, tacrine has been widely used as a scaffold for the development of new multifunctional agents [24,54-56]. One recent example is the work of Lan and coworkers that outlined novel tacrine-β-carboline hybrid compounds (61), designed from the structural feature

O HN NR2 R1

N N H

61

4

N H

62 R1= H R2= H

eeAChE IC50 = 21.6 ± 0.8 nM hAChE IC50 = 63.2 ± 2.5 nM BuChE IC50 = 39.8 ± 1.6 nM A! aggregation (65.8% at 20 !M) Antioxidant activity (1.57 trolox equivalents)

Fig. (17). Structural design of a novel series of tacrine-βcarboline hybrids (62) from tacrine (2) and natural βcarbolines (60).


Chioua and co-workers also used tacrine (2) as a scaffold for planning a series of eight tacrinepyranopyrazole derivatives (Fig. 18). All compounds were initially submitted to toxicity tests by using cell viability assay with Hep G2 cells (300 µM). The results showed that all tacrine derivatives showed to be safe, with cell viability ranging from 76 to 95%, in comparison to tacrine (40%). Then, AChE inhibition assay was carried out, revealing compounds 63 (IC50= 0.17 ± 0.04 µM) and 64 (IC50= 1.52 ± 0.49 µM) as best inhibitors, with compound 63 being almost 9-fold more potent than 64, with 82% of cell viability. Both compounds were additionally tested for inhibition of Aβ aggregation, reducing by half the amount of Aβ aggregates induced by AChE. Compound 63 was additionally evaluated for its neuroprotective activity, showing 164% of cellular viability in cortical neurons after 24h of treatment (10 µM). This activity showed to be dose dependent, reaching its maximum effect at 10 µM with an EC50= 0.25 ± 0.06 µM. Taking all these results, compound 63 was selected as the most promising tacrinepyranopyrazole derivative, with a genuine structural framework and a singular and safe multifunctional mode of action, inhibiting AChE and AChE-induced Aβ aggregation, with neuroprotective activity [58]. NH2

N Tacrine (2)

NO2

NH2

H3CO

NH2

HN

HN N

O

N

N

O

N

63 IC50 AChE = 0.17 ± 0.04 µM 64 IC50 AChE = 1.52 ± 0.49 µM

Fig. (18). Chemical structures of novel tacrinepyranopyrazole multifunctional hybrid compounds 63 and 64.

Ferulic acid (36) acid was also used as a molecular scaffold by Fu and co-workers in the design of a new series of tacrine-ferulic acid hybrids (Fig. 19) with expected multi-target effects in the inhibition of cholinesterases, reduction of self-induced β-amyloid (Aβ) aggregation, chelation of Cu2+ and neuroprotection. Among all the synthetized compounds, 65 and 66 displayed the highest selectivity in inhibiting AChE over BuChE (SI = 4.087 and 1.733, respectively). Moreover, compound 66 also showed significant inhibition of self-Aβ aggregation (37.2 ± 0.9 % at 20 µM), Cu2+


13

chelating activity and the best activity against Aβinduced neurotoxicity in neuro-2A cells [59]. NH2

H3CO

COOCH3

HO N Ferulic acid (36)

Tacrine (2)

H3CO

O HN

N

COOCH3

n O

N 65, n=4 66, n=5 A! aggregation (37.2 ± 0.9 % 20 !M)

at

N

Fig. (19). Design and chemical structures of new multifunctional tacrine-ferulic acid hybrid compounds 65 and 66.

In another approach, Benchekroun and co-workers selected ferulic acid (36) and melatonin (43) for drawing a new multifunctional tacrine-derived hybrid compounds for AD. Melatonin (43) is a molecule produced by various human organs and tissues, and is involved in many physiological processes, such as the modulation of endogenous antioxidants and immune system regulation. With aging, human beings experience a natural decline in the melatonin levels, which has been associated with the development of neurodegenerative diseases such as AD. Melatonin has been shown to be capable of quenching free radicals, stimulating the biosynthesis of antioxidant enzymes, reducing the hyperphosphorylation of neurofilaments and protective activity against Aβ protein. Moreover, melatonin could induce the proliferation and differentiation of neural cells in the hippocampus of adult mice. Given these data, a new ferulic acid−tacrine−melatonin hybrids (FATMHs) (67, Fig. 20) were synthetized and evaluated for their abilities in the inhibition of AChE and BuChE, toxicity towards HepG2 cells, neuroprotection in SHSY5Ycells, Nrf2 pathway induction in AREc32 cells, BBB permeability as well as their antioxidant capacity through oxygen radical absorbance capacity (ORAC). Among all tested compounds, derivative 68 was identified as the most potent and selective human ChE inhibitor (IC50 hAChE= 1.29 ± 0.070 µM; IC50 hBuChE= 0.234 ± 0.008 µM), strong antioxidant (9.11 ± 0.21 µM), with adequate BBB permeability. This compound also showed the best in vitro neuroprotective profile against toxic insults mediated by H2O2 (300 µM), Aβ1−40 and Aβ1−42 (30 µM) at 1 µM, with significant induction of Nrf2 transcriptional pathway (at 3 µM) in AREc32 cells [60].



H3CO

OCH3

NH2

O

H N

OH N

HO Ferulic acid (36)

O Melatonin (43)

Tacrine (2)


R1 O

NH OCH3

O H N

N 7 NH

O

67 NH

H3CO

HO

N 68 R1= OCH3 IC50 hAChE= 1.29 ± 0.070 !M; IC50 hBuChE= 0.234 ± 0.008 !M Antioxidant activity = 9.11 ± 0.21 !M

Fig. (20). Design of the new MTDL 68 based on the structure of tacrine (2), ferulic acid (36) and melatonin (43).

Quinolines are also considered privileged heterocyclic structures present in different natural or synthetic bioactive substances used in the treatment of different pathologies, including neurodegenerative diseases. Thus, chloro-quinoline 69 was used for molecular hybridization with the structure of tacrine (2) to generate a new family of tacrine-quinoline hybrids, designed as AChE inhibitors and neuroprotector MTDLs candidates (Fig. 21). Thirteen new substances were synthetized and initially submitted to cell viability assays with Hep G2 cells, in order to verify their safety and their hepatotoxicity. Almost all substances showed cytotoxity lower than tacrine (EC50= 179 µM), and were then evaluated for their AChE inhibitory properties. The lowest toxic compound 70 exhibited a very significant AChE inhibition (IC50 AChE= 0.48 ± 0.05 µM), similarly to its methoxy-derivative 71 (IC50 AChE= 0.47 ± 0.13 µM), were identified as the most AChEI inhibitors. All molecules were also subjected to neuroprotection assays for cell death mediated by oxidative stress and, once again, compounds 70 (EC50= 1.59 ± 0.39 µM) and 71 (EC50= 0.63 ± 0.13 µM) have stood out and were considered as innovative candidates for the development of novel MTDLs for AD therapeutics [61]. Resveratrol (72), a potent phenolic natural plant metabolite, with remarkable antioxidant and antiinflammatory properties, has been recently described as an inhibitor of Aβ aggregation. Thus, tacrine (2) and resveratrol were used as molecular prototypes for molecular hybridization in the design of novel tacrineresveratrol hybrids with improved biological properties against AD (Fig. 22). In this context, thirty-six hybrids

were synthesized and tested for their antioxidant activity and ability to inhibit Aβ aggregation. Compounds 73-75 were the most active in the inhibition of Aβ aggregation with IC50 values of 10.8 ± 1.5 µM, 9.7±1.2 µM and 10.3±0.9 µM, respectively. However, the antioxidant activity of all hybrids was lower than that of resveratrol. Compounds 74 and 75 were evaluated toward ChE activity, both exhibited a moderate activity and slightly selective by BuChE with IC50 values of 64.0 ± 0.1 µM (AChE), 0.2 ± 0.1 µM (BuChE) and 68.3 ± 0.1 µM (AChE), 1.0 ± 0.1 µM (BuChE), respectively [62]. N

NH2

Cl

O

N Tacrine (2)

Chloro-Quinoline (69)

N H2N

N

N O

Cl

CO2C2H5

70 IC50 AChE = 0.48 ± 0.05 µM EC50 Nneurop.= 1.59 ± 0.39 µM

H2N

H3CO

N

O

Cl

COCH3

71 IC50 AChE = 0.47 ± 0.13 µM EC50 Nneurop.= 0.63 ± 0.13 µM

Fig. (21). Tacrine-Quinoline hybrids and more potent derivatives (70 and 71).

Coumarins are an important class of natural metabolites with a wide spectrum of biological properties and,



15

OH

NH2 HO

N Tacrine (2)

OH

Resveratrol (72)

CH3 N

N

N

N

N

N

N

N 73 IC50 A!-aggregation =10.8 ± 1.5 µM IC50 AChE = -IC50 BuChE = --

N

N 74 IC50 A!-aggregation =9.7 ± 1.2 µM IC50 AChE = 64.0 ± 0.1 µM IC50 BuChE = 0.2 ± 0.1 µM

75 IC50 A!-aggregation = 10.3 ± 0.9 µM IC50 AChE = 68.3 ± 0.1 µM IC50 BuChE = 1.0 ± 0.1 µM

Fig. (22). Design, chemical and biological data of the tacrine-resveratrol hybrids 73-75. NH2

N

O

Tacrine (2)

Coumarin (76) Molecular hybridization CH3

CH3 HN

CH3

N N

N

HN

Cl

N N

O

O

O

77 IC50 AChE = 16.11 ± 0.09 nM IC50 BuChE = 112.72 ± 0.93 nM IC50 MAO A = 15.07 ± 0.88 µM IC50 MAO B = 0.24 ± 0.01 µM

N

O O 78 IC50 AChE = 24.37 ± 0.23 nM IC50 BuChE = 124.32 ± 1.19 nM IC50 MAO A = 53.07 ± 1.24 µM IC50 MAO B = 0.70 ± 0.02 µM

O

Fig. (23). Design of the dual AChE and MAO-B inhibitors 77 and 78 by molecular hybridization of tacrine (2) and coumarin (76).

recently, they have been drawing special attention due to their biological activity related to neuronal disorders [63]. Coumarin derivatives have been reported as excellent MAO inhibitors, especially for 7-substitutedcoumarins [64]. Structure-activity studies suggested that substituents at 3 and/or 4 positions may increase the selectivity to MAO-B, along with interaction with the AChE-PAS [65,66]. Therefore, a series of tacrinecoumarin hybrids were designed aiming novel dual inhibitors of AChE and MAO-B (Fig. 23). Among twenty new tacrine-coumarin hybrids, in vitro evaluation disclosed compounds 77 (IC50 AChE= 16.11 ± 0.09 nM, IC50 BuChE= 112.72 ± 0.93 nM, IC50 MAOA= 15.07 ± 0.88 µM and IC50 MAO-B= 0.24 ± 0.01 µM) and 78 (IC50 AChE= 24.37 ± 0.23 nM, IC50 BuChE= 124.32 ± 1.19 nM, IC50 MAO-A= 53.07 ± 1.24 µM and IC50 MAO-B= 0.70 ± 0.02 µM) as the most promising derivatives, showing the desired dual inhibitory profile, with good BBB permeability [67].

Boulebd and co-workers described the synthesis and in vitro biological evaluation of thirteen new racemic and diversely functionalized imidazolyl-pyrano-tacrine derivatives 79-91 as non-hepatotoxic multipotent compounds (Fig. 24). Compound 82 was highlighted due to its selective, but moderate AChE inhibitory activity (IC50= 38.7 ± 1.7 µM), with a very potent antioxidant activity on the basis of the ORAC test (2.31 ± 0.29 µmol Trolox/µmol compound). In the search for enhanced inhibitory and antioxidant properties, other tacrine analogues, such as compounds 86 and 99, were obtained and showed to be significantly more potent AChEIs than 82, with high antioxidant activity. Unfortunately, these compounds exhibited a high hepatotoxicity, comparable to tacrine, at high concentrations (≥ 300 µM) [68]. Spilovska and co-workers reported the synthesis and pharmacologic evaluation of a new series of cholinesterase inhibitors acting as dual binding site heterodi-



n

n

N

N H2N

H2N

O

N

O

H2N

H2N

O

79 R= CO2Et (n=0) 80 R= CO2Et (n=1) 81 R= CO2Et (n=2) 82 R= CO2Me (n=1) AChE IC50 = 38.7 ± 1.7 !M ORAC IC50 = 2.31 ± 0.29 !mol Trolox/!mol

X N

N

O

O

N

N N

R

N

N

N

N

n

n

83 (n=0) 84 (n=1)

88 (X=CH, n=0) 89 (X=CH, n=2) 90 (X=N, n=0) 91 (X=N, n=2)

85 (n=0) 86 (n=1) 87 (n=2)

Fig. (24). Chemical structures of imidazo-tacrine analogues 79-91 designed as multifunctional AChE inibitors with antioxidant properties. NH2 H3CO

NH2

NH2 N 7-methoxytacrine (92)

Amantadine (93)

Memantine (5)

O

S HN

n

N H

H3CO

N n= 2-8 7-MEOTA-amantadine thioureas 94-100 7-MEOTA IC50 5.02 to 0.47 !M

HN

n

N H

H3CO

N n= 2-8 101-107 7-MEOTA IC50 4.98 to 0.69 !M

Fig. (25). Design of 7-methoxytacrine (7-MEOTA)-amantadine urea (94-100) and 7-MEOTA-amantadine thiourea (101-107) derivatives.

mers for the management of AD. Based on the MTDLs strategy, a series of 7-MEOTA-amantadine urea-linked derivatives were designed, synthesized and evaluated for AChE/BuChE inhibition (Fig. 25). All fourteen 7MEOTA-amantadine derivatives were compared with 7-MEOTA-amantadine thioureas, 7-MEOTA (92), tacrine and galantamine-memantine dimers. The new hybrids showed to be more potent hAChE and hBuChE inhibitors than 7-MEOTA, with IC50 values ranging from 5.02 to 0.47 µM for thioureas (94-100) and from 4.98 to 0.69 µM for urea-derivatives (101-107). In the 7-MEOTA-amantadine thioureas series, five compounds showed IC50 values in sub-micromolar range for hAChE. Only two derivatives (94 and 96) exhibited inhibitory potency in sub-micromolar range for hAChE. However, compounds 94, 96-100 showed poor selectivity, with sub-micromolar inhibitory potency also for hBuChE. The best inhibitory activity was identified for compound 96 (IC50= 0.11 ± 0.02 µM), bear-

ing five methylene groups in the linker, that displayed inhibitory potency in the same magnitude order of THA for hAChE [69,70]. Memoquim (108) was described in 2007 as one of the first molecules rationally designed following the MDTL hypothesis. In vitro and in vivo studies demonstrated that has good ability to inhibited Aβ aggregation and AChE and a potent free-radical scavenger activity. Thanks for these characteristics, memoquin inspired the design of other MTDLs, such as compound 109, that disclosed an optimal anti-cholinesterase and anti-aggregating feature, retaining a promising anti-amyloid profile. Nep-

ovimova and co-workers propose a new optimized series as a result of molecular hybridization between the memoquin analog 109 and tacrine (2) (Fig 26). From this series, compound 110 showed the best results for selective inhibition of hAChE (IC50 = 0.72 ± 0.06 nM, IC50 hBuChE= 542 ± 16 nM) and Aβ self-aggregation (37.5 ± 4.9% at 10 µM). However, this compound showed to be toxic at



OCH3

O

H N

N CH3

O

17

CH3

Memoquim (108)

N

N H

O

OCH3

NH2

OCH3

H N

N

109

N Tacrine (2)

H3 C O


O

OH

O

H N

N N H

110 Cl IC50 hAChE = 0.72 nM IC50 BuChE = 542 nM inhibition of Aß self-aggregation (10µM) = 37.5%

Fig. (26). Design of Quinone−Tacrine Hybrid (110) as a potential hAChE inhibitor. 10 µM similarly to tacrine, suggesting that further studies are needed to overcome toxicity [71].

As discussed earlier, resveratrol (72) is a natural product with a comprehensive range of biological proprieties. Based on this, Jeřábek and co-workers proposed a new series of hybrids from resveratrol and tacrine (Fig 27) with the goal of access novel molecules with the neuroprotective and anti-inflammatory effect of resveratrol and the biological properties from tacrine. In this scenario, it was obtained compound 111, with a good inhibitory activity on hAChE (IC50= 8.8 ± 0.4 µM) and on Aβ self-aggregation (31.2 ± 9.0% at 10 µM). Additional studies indicated its good BBB permeability and low neuronal and hepatic toxicity, with anti-inflammatory and immunomodulatory properties against microglial activation [72]. OH NH2 HO

N OH

Resveratrol (72)

Tacrine (2) OCH3

HN Cl

N

111 IC50 hAChE = 8.8 µM OCH3 inhibition of Aß self-aggregation (10 µM) = 31.2%

Fig. (27). Design of Tacrine-resveratrol fused hybrid (111) as multi-target-directed ligands against AD.

5. MTDLs INSPIRED BY NATURAL PRODUCTS (NPs) Nature is rich in secondary metabolites from plants, animals and microorganisms, with a great variability of chemical classes, structural pattern and complexity. Many of these natural products (NPs) have been identified for their AChE inhibitory activity, as well as other relevant properties for neurodegenerative diseases, including anti-inflammatory, neuroprotective, antioxidant and metal chelating ability. These properties are not restricted to few or specific chemical classes, but are intrinsically observed in various naturally occurred compounds of different chemical patterns and classes, specially alkaloids, phenolics and terpenoids, among others. Due to this broad chemical diversity, natural products that have been used as source of bioactive molecules, used directly as medicines or isolated active principles, starting materials for semi-synthesis and, as molecular models for inspiration in drug design [73]. One such example is melatonin (43), a molecule produced by various animal organs and tissues. It is involved in many physiological processes, such as the endogenous antioxidants and the immune system regulation. With aging, we have a natural decline in the levels of melatonin, which has been associated with the development of neurodegenerative diseases such as AD. Melatonin has been shown to be capable of capturing free radicals, stimulating the synthesis of antioxidant enzymes, reducing the neurofilaments hyperphos-



OCH3 N H3CO H N

HN

CH3

CH3

H3CO

O

O

AP2238 (112) IC50 hAChE = 0.044 ± 0.006 µM

O

Melatonin (43)

H3CO

H3CO N

H N HN

N

H N

H3C O

HN

OCH3

H3C O

113 IC50 hAChE = 6.1 µM IC50 hBuChE = 7.8 µM

114 IC50 hAChE = 6.8 µM IC50 hBuChE = > 10 µM OCH3

H N

N H3C

HN

O

115 IC50 hAChE = 4.7 µM IC50 hBuChE = 8.2 µM

Fig. (28). Chemical structures of compounds 113-115, designed as MTDLs melatonin-AP2238 hybrids.

phorylation and having protective activity against amyloid-β (Aβ) protein. Moreover, melatonin was able to induce the proliferation and differentiation of neural cells in the hippocampus of adult mice. N, N-dibenzyl (N-methyl) is a protonable amine present in the structure of the known AChE inhibitor AP2238 (112, IC50 = 0.044 ± 0.006 µM), capable to interact with the CAS of AChE [74-76]. Thus, a series of melatonin-AP2238 (Fig. 28) were planned as novel AChE inhibitors, assembling the endogenous characteristics of both bioactive molecular prototypes. Hence, fourteen new substances were synthesized and evaluated, all showing anticholinesterase activities with IC50 values in µM order, but less potent than AP2238 (112). Permeability assay proves that all molecules were able to cross Blood-Brain barrier (BBB), with additional antioxidant activity close to the reference value of melatonin, but with lower neuroprotective activity than melatonin. Finally, considering that methoxy group is indicated as crucial for neurogenic activity of melatonin, the methoxy-substituted hybrids 113-115 were submitted to neurogenic studies. The results evidenced that these three compounds were able to induce neurogenesis, but compound 115 excelled in ability to induce maturation of these cells. Therefore, one can expect that hybrids 113, 114, and especially compound 115 could represent innovative multifunctional drug candidates capable to repair CNS damage caused by neurodegenerative diseases and protect neuronal cells from oxidative processes [77]. In another work, besides melatonin (43) as an antioxidant prototype molecule, benzylpyridinium salt (116) has been considered a privileged structure in the development of AChE inhibitors. In this context, ben-

zylpyridinium-melatonin hybrids were planned with the aim of obtaining novel antioxidant-AChE inhibitor molecules. Twenty-three new substances were synthesized (Fig. 29), highlighting compounds 117 and 118 that showed high ChE inhibitory activities with IC50 values of 0.11 ± 0.001 µM (AChE), 1.1 ± 0.1 µM (BuChE) and 1.3 ± 0.1 µM (AChE), 0.08 ± 0.001 µM (BuChE), respectively, with pronounced antioxidant activity (117, ORAC= 3.41 ± 0.05; 118, ORAC = 2.04 ± 0.004). Toxicity was also evaluated in neuroblastoma cell cultures, with both compounds 117 and 118, showing similar toxicity to or less than melatonin [78]. In 2014, Pan and co-authors have already reported a series of resveratrol (72) derivatives, (Fig. 30) with potential therapeutic use for AD treatment. Basically, the derivatives 119 were planned by change the 3,5-dihydroxy groups for 3,5-di-methoxy substituents on the ring A of resveratrol and the insertion of an alkylamino side chain with different lengths attached to the oxygen on the ring B. Among them, compound 119 exhibited the best biological results, with significant inhibitory activity of cholinesterases (IC50 AChE= 6.55 ± 0.16 µM; IC50 BuChE= 8.04 ± 0.22 µM), Aβ42 aggregation (57.78% ± 2.36 at 20 µM) and monoamine oxidases (IC50 MAO-A= 17.58 ± 0.76 µM; IC50 MAO-B= 12.19 ± 0.40 µM) [79]. Memoquin (108) is considered an example of success in modern medicinal chemistry, being one of the first AD multitarget-drug discovery efforts. It interacts with three molecular targets involved in AD pathology: acetylcholinesterase (AChE), β-amyloid (Aβ) and βsecretase (BACE-1). Ferulic acid 36 has antioxidant and anti-inflammatory effects, inhibits Aβ fibril aggregation, and prevents Aβ-mediated toxicity both in vitro and in vivo. Based on these data, Pan and co-workers



H3CO

19

O R' H N

R

CH3

HN

O

N

O

Br

O Melatonin (43)

Coumarin-based compounds (116) Molecular hybridization

H3CO N H N

HN

F Br

O H3CO

117 IC50 AChE = 0.11 ± 0.001µM IC50 BuChE = 1.1 ± 0.1 µM ORAC = 3.41 ± 0.05

N H N

OOCN(CH3)2 O

HN

118 IC50 AChE = 1.3 ± 0.1 µM IC50 BuChE = 0.08 ± 0.001 µM ORAC = 2.04 ± 0.004

Fig. (29). Design of novel benzylpyridinium-melatonin hybrids 117 and 118 with anticholinesterase and antioxidant properties. OH

OCH3

A HO Resveratrol (72)

A H3CO

B

115

OH

B O

N

IC50 AChE= 6.55 ± 0.16 !M IC50 BuChE= 8.04 ± 0.22 !M A!42 aggregation (57.78 % ± 2.36 at 20 !M) Monoamine oxidases (IC50 MAO-A= 17.58 ± 0.76 !M; IC50 MAO-B= 12.19 ± 0.40 !M).

Fig. (30). Chemical structures of 3,5-dimethoxy-4’-O-alkylamine-resveratrol derivatives (119) with AChE and Aβ inhibitory properties.

selected ferulic acid (36) to combine with different alkyl-benzylamines fragments to design a series of novel ferulic acid-memoquin hybrids (120, Fig. 31), that are expected to show potentially applicable multifunctional properties towards AD. All the target-compounds exhibited more potent inhibitory activities than ferulic acid (36, IC50 > 100 µM), but lower inhibitory activities than memoquin (108, IC50 = 6.7 ± 0.0001 µM). Compound 121 showed the strongest inhibitory activity with IC50= 3.2 ± 0.002 µM. In addition, 121 was able to disaggregate Aβ fibrils (45.5% ± 0.03 disaggregation at 25 µM), with significant antioxidant potential (ORAC of 1.2 ± 0.001) and adequate BBB permeability in vitro. At a concentration of 10.0 µM, compound 121 exhibited significant neuroprotective effect and cell viability (88.3 ± 6.5%). It is important to note that compound 121 exhibited interesting multi-target ligand profile, without restrictions of solubility, that is a great advantage related to memoquin (108) that failed in preclinical trials due to its very low solubility [80].

Pérez-Areales and co-workers described the design, synthesis and pharmacological evaluation of a short series of multitarget anti-Alzheimer hybrid compounds that combined a fragment of the highly potent AChE inhibitor huprine Y (123) with the 4-hydroxy-3methoxyphenylpentenone moiety of shogaols (122) (Fig. 32). A series of hybrids 124-126, differing in the nature and size of the spacer between the huprine (123) and shogaol (122) fragments were docked in the hAChE models. All three compounds turned out to be potent hAChE and hBuChE inhibitors with IC50 values of 6.7 ± 0.1, 18.3 ± 2.0 and 21.1 ± 1.9 nM, respectively, and potent antioxidant agents, even though this hybridization strategy led to slightly decreased hAChE inhibitory activity relative to the parent huprine Y. The presence of an additional aromatic ring in the linker subunit of hybrids 125 and 126, which led to increased antioxidant activity, seemingly enhances their interaction with Aβ42 and tau protein, leading to potent Aβ42 and tau anti-aggregating activities. The Aβ42 and tau anti-aggregating activities were in the ranges 39-71%

20 Current Medicinal Chemistry, 2018, Vol. 25, No. 00 AChEI

OCH3

Antioxidant and A! inhibition H3CO


COOH

O H N

N

CH3

CH3

HO

Memoquin (108) Ferulic acid (36)

O

OCH3

AChE IC50 = 6.7 ± 0.1 nM

Molecular hybridization O

H3CO

N

N H

R1 N H

n

N R2

HO

Ferulic acid-memoquin hybrids (120) 121 - n= 6, R1 = CH3O, R2 = C2H5 AChE IC50= 3.2 ± 0.02 !M ORAC of 1.2 ± 0.001

Fig. (31). Design strategy for ferulic acid-memoquin hybrids (120) and structure of the most active hybrid 121. OH OCH3 Cl H2N N O

(123) huprine Y

(122) [6]-shogaol


NH

HN

N R

N

Cl O 124

OH

HO

Cl OCH3

H3CO

O

(125) R= H; AChE IC50 = 18.3 ± 2.0 nM (126) R= CH2NMe2; AChE IC50 = 21.1 ± 1.9 nM

AChE IC50 = 6.7 ± 0.1 nM

Fig. (32). Structures of the natural antioxidant [6]-shogaol, the AChE inhibitor huprine Y and shogoal-huprine Y hybrids 124126.

and 35-51%, respectively, using a 10 µM concentration of the hybrids, they being clearly more potent than the parent huprine Y (123) and shogaol (122) [81]. Viayna and collaborators designed a family of rhein−huprine Y hybrids (128, Fig. 33) to hit several key targets for AD. All the racemic hybrids (±)-129132, obtained from racemic huprine Y (122), turned out to be potent inhibitors of hAChE, with IC50 values in the low nanomolar range. Hybrid (±)-129, the most potent hAChEI of the series (IC50= 1.07 nM), was equipotent to the parent racemic huprine Y [(±)-122]. Additionally, the beneficial effects of (+)-129 on synaptic integrity were apparent in the context of LTP induction. Finally, in vivo experiments with transgenic APPPS1 mice have shown that (+)- and (−)-129 are able to lower the levels of hippocampal total soluble Aβ and

increase the levels of APP both initial and advanced stages of this AD model, thus suggesting a reduction of APP processing, as expected from their potent BACE-1 inhibitory activity. Overall, the novel rhein−huprine hybrids (+)- and (−)-129 emerge as very promising multitarget anti Alzheimer drug candidates with potential to positively modify the underlying mechanisms of this disease [82]. To date, it is well known that MAO-B activity can increase up to 3-fold in the AD patients compared with controls. This increase in MAO-B activity produces higher levels of H2O2 and oxidative free radicals, which have been correlated with the development of oxidative stress [83]. In addition, highly concentrated metal ions, (e.g. Cu2+, Zn2+ and Fe3+) in the neuropil and plaques of the brain are closely associated with the formation of



Aβ plaques and neurofibrillary tangle, as well as linked to the production of reactive oxygen species (ROS) and oxidative stress [84,85]. According to the studies of Huangi and co-workers, a series of hybrid derivatives (134) with the pharmacophore moiety of metal chelator clioquinol (CQ) (133) and coumarin (76) (Fig. 34) was rational planned to generate coumarin derivatives with expected biometal chelation ability, along with inhibitory activity of MAO-B and Aβ anti-aggregation. Compound 135 showed the greatest potential to inhibit hMAO-B (IC50 = 0.081 ± 0.0002 µM) with >1234-fold selectivity over MAO-A (IC50 = 3.5 ± 0.1 µM), as well as good inhibition of Aβ1-42 aggregation (52.9% ± 2.2 at 20 µM), low cell toxicity in rat pheochromocytoma (PC12) and SH-SY5Y cells and BBB permeability [86]. OH

O

NH2

OH

Cl

N

HO2C

Huprine Y (123)

O Rhein (127)

HO O HO

O 128

HN HN

X

O

Cl

N

129 X= (CH2)5 130 X= (CH2)9 131 X= (CH2)10 132 X= (CH2)11

Fig. (33). Design of the new series of derivatives of rhein (128) and Huprine Y (123). Metal Chelation Cl

R1 R2

N

I

N

OH R3

Clioquinol (133)

OH

O

O

(134) R1

MAO Inhibitory, A! Interaction

R2

R1 N H

R2

R

O Coumarins (76)

O

O

O

OH (135) R1= R2=(CH2)3, R3= H hMAO-B (IC50 = 0.081 ± 0.0002 !M) MAO-A (IC50 = 3.5 ± 0.1 !M) A!1-42 aggregation (52.9% ± 2.2 at 20 !M)

Fig. (34). Structure of coumarin (76) and the most active compounds 134 and 135 designed by molecular hybridization of clioquinol (133) and coumarins (76).

21

Aurones, 2-benzylidenebenzofuran-3(2H)-ones (136), which are structural isomers of flavones present in vegetables and flowers, have attracted considerable attentions due to their wide range of bioactivities associated with neurological diseases. Li and co-workers synthesized and evaluated a series of 4-hydroxy-aurone derivatives 137-138 (Fig. 35), designed as potential multifunctional agents for the treatment of AD. They observed that all derivatives showed high antioxidant activities, ranging from 1.00 to 3.56-fold of Trolox, especially compound 137e that showed an antioxidant activity 1.90-fold higher than trolox, along with a good inhibitory activity of self- and Cu2+ induced Aβ1-42 aggregation with 99.2% ± 1.1 and 84.0% ± 1.5 at 25 µM, respectively. In addition, 137e also showed remarkable inhibitory activities of both MAO-A and B with IC50 values of 0.271 ± 0.013 µM and 0.393 ± 0.025 µM, respectively. Furthermore, compound 138b exhibited high selectivity to MAO-B over MAO-A, which may serve as potential MAO-B selective inhibitors. These lead compounds 137e and 138b also showed good metal-chelating properties and BBB permeability [87]. Li and co-workers reported the design, synthesis and biological evaluation of a new series of pterostilbene-benzylamines hybrid derivatives (141-144, Fig. 36). Pterostilbene (139) is a dimethoxy-derivative of resveratrol (72) and it was used as molecular model for planning new MTDL candidate prototypes with inhibitory activity of AChE and BuChE, along with antioxidant and Aβ aggregation inhibitory effects. The design approach was based on the connection of pterostilbene 139 with benzylamines 140a-d by using an amide functionality as spacer subunit with different chain lengths. Pharmacological evaluation revealed compound 144d as the best selective AChE inhibitor (IC50= 0.06 ± 0.03 µM), with good selectivity ( (IC50 BuChE= 28.04 ± 1.71 µM). Both, inhibition kinetic analysis and molecular modeling study indicated that these compounds showed mixed-type inhibition mode, binding simultaneously to the CAS and PAS of AChE. In addition to cholinesterase inhibitory activities, these compounds showed different levels of antioxidant activity (ORAC 0.51 ± 0.03 of Trolox equivalent). SAR studies suggested that the introduction of an amide function on the side-chain of the pterostilbene fragment led to significantly increased enzymatic activity, as well as increasing the length of the linker may give rise to a better activity [88]. Chromone 145, a privileged scaffold in Medicinal Chemistry, is the core fragment of several flavonoid derivatives such as flavones and isoflavones. Liu and co-workers selected chromone-2-carboxamide moiety


de Freitas Silva et al. R1=

O CH3

N

N

CH3

O

A

Aurone scaffolds 136

CH3

N

CH3

CH3

CH3

B

C

N

D

N

N

R1

N

O F

E

O

O

CH3

G 137e A!1-42 aggregation with 99.2% ± 1.1 !M (Self-induced) A!1-42 aggregation with 84.0% ± 1.5 at 25 !M (Cu2+-induced) MAO-A IC50 = 0.271 ± 0.013 !M MAO-B IC50 = 0.393 ± 0.025 !M

137 a-g R = OH, R1 =4´-A-G 137 h R = OH, R1 =2´-A 138 a-c R = OCH3, R1 =4´-A, E, F

R

N

Aurone derivates 137-138

Fig. (35). Structure of multifunctional aurone derivatives 137-138 designed as multifunctional drug candidates for AD. X X X

X X

X

X

X

X

X

X

X

X

X

Benzylamine series 140

X

X X

X

X X

X X

X X

a

X

X

X

X

X

X

X

X X

X

X

X X X X X X X X X X X X X X X X X X X X X X X X

X X b

c

d

Pterostilbene (139)

Molecular hybridization X X X

X X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

nX

X

X 141 142 143 144

a-d, n = 2 a-d, n = 3 a-d, n = 4 a-d, n = 6

144d AChE IC50 = 0.06 ± 0.03 !M BuChE IC50 = 28.04 ± 1.71 !M ORAC 0.51 ± 0.03 of Trolox equivalent

Fig. (36). Design of the Pterostilbene-benzylamines hybrid multifunctional derivatives 141-144.

to combine with alkyl-benzylamine fragments of different length (a-h), inspired in the structure of genistein derivatives 146, to design a potential multifunctional series of novel chromone-2-carboxamidoalkylbenzylamine series (147, Fig. 37). In vitro biological evaluation showed that all the targetcompounds significantly inhibited AChE activity in a sub micromolar to micromolar range, with good selectivity. Compound 148 exhibited the best inhibitory potency over rat cortex homogenate AChE (RatAChE) (IC50 = 0.07 ± 0.002 µM), with 736-fold higher selectivity for AChE over BuChE. Kinetics studies revealed a mixed-type inhibition mode for this compound, being capable to access both the CAS and PAS of AChE. In addition, 148 exhibited a moderate anti-oxidative activity, selective biometal chelating ability, along with excellent self-induced and good Cu2+-induced Aβ aggregation inhibitory activity 63.0% ± 1.3 and 55.6% ± 1.3, respectively) [89].

Synthetic and natural coumarins (76) have recently received special attention due to their wide biological employability. Some of their derivatives have been described as good AChE inhibitors, Aβ-aggregation inhibitors and neuroprotectors against oxidative damages. Thus, coumarin was used as the basic scaffold in the drawing of a series of innovative and with singular structural pattern coumarin derivatives. Nineteen new coumarin derivatives were synthesized and showed a very selective and potent AChE inhibition profile (Fig. 38). Among all these, compounds 149 (IC50= 0.016 ± 0.0021 µM), 150 (IC50= 0.003 ± 0.0007 µM), 151 (IC50= 0.012 ± 0.0018 µM) and 152 (IC50= 0.019 ± 0.001 µM), (Fig. 38) were the most prominent AChE selective inhibitors (IC50 BuChE >100 µM). These most active substances were submitted to cell viability assay (MTT) and H2 O2-induced oxidative damage in human neuroblastoma cells to verify possible neurotoxic effects, among the four, one presented a profile similar to



23

OCH3 OH

O

O

O

N

CH3

H3C

N

O

O OCH3

Chromone (145)

7,4'-modified genistein derivatives (146)

-NR1R2 =

n = 3,4, 6 OH

O

O

R

O

N

R1

H N

N n

N

a

N

b

R2 N

O

c

N

d

O

N

O

(147) OH

O

N

N

e

f

N

N

g

Oh

O

O

H N

O

(148) AChE (RatAChE) (IC50 = 0.07 ± 0.002 !M) A! aggregation with 63.0% ± 1.3 at 25 !M (self-induced) A! aggregation with 55.6% ± 1.3 at 25 !M (Cu2+-inducec)

N 6

O

Fig. (37). Structure of chromone (145), and 7,4’-O-modified genistein derivative 146, used as molecular prototypes in the design of chromone-2 carboxamide-alkyl-benzylamine derivatives 147 and the most active compound 148.

O Coumarin (76)

O

O

HN

O Br

O

N H

149 IC50 AChE = 1.6 ± 0.21µM

O

O

O

O

O

O

HN

HN O

N H

150 IC50 AChE = 0.3 ± 0.07 µM

Br

O Cl

O

N H

151 IC50 AChE = 1.2 ± 0.18 µM

O

O

O

HN O

N H

Cl

152 IC50 AChE = 1.9 ± 0.1 µM

Fig. (38). Chemical structural of more potent derivatives of coumarin (149-152).

that of galantamine and another three had better results, especially 150. Taking all data set, compound 151 exhibited the best MTDL profile, with lower toxicity than galantamine, and could be considered an interesting prototype candidate in the process of drug development for AD therapeutics [90]. 6. MTDLs INSPIRED BY OTHER POLICYCLIC STRUCTURES The aspartyl protease β-secretase (BACE-1) is the enzyme responsible for Aβ formation and it has become an attractive drug target for AD. Besides, the enzyme knows as glycogen synthase kinase-3β (GSK-3β) is involved in the tau hyperphosphorylation process, promotes tau detachment from the microtubules and then the tangles aggregates formation. In 2015, Prati

and co-workers discovered a new series of 6-amino-4phenyl-3,4-dihydro-1,3,5-triazin-2(1H)-ones 155 by hybridization of pharmacophoric features responsible for the BACE-1 and GSK-3β activity, such as the guanidine 65 and the cyclic amide group 66, respectively, into a single scaffold (Fig. 39). Compound 156 was the most potent activity with the ability to inhibit both enzymes simultaneously BACE-1 and GSK-3β (IC50 of (18.03 ± 0.01µM) and (14.67 ± 0.78 µM) respectively), in addition, 156 has shown good profile of neuroprotection, neurogenesis, have no neurotoxicity, and good CNS permeability [91]. The combination of the quinoline (157) and triazolopyrimidine (158) groups has different biological effects described, among them we can highlight neuro-

24 Current Medicinal Chemistry, 2018, Vol. 25, No. 00 Guanidino motif (153)

O HN R2

NH N

R1

N H

(155) 156 R1= H, R2= 4-F BACE-1 IC50 = 18.03 ± 0.01!M GSK-3! IC50 = 14.67 ± 0.78 !M

H N

NH2 N

de Freitas Silva et al. N

N

N

HN

NH

N

N Triazolopyrimidine (158)

Quinoline (157)

Cyclic Amide Group (154)

Piperazine (159)

F

O

N

N

NH

Fig. (39). Design strategy of dual BACE-1 and GSK-3β Inhibitors compounds 155 and structure of the most potent derivate 156.

protection, the AChE inhibitory activity, the free radical scavenging effect and the complexing ability with metals. Quinoline structure was expected to interact with AChE via π-π interaction. Besides this it was supposed to intercalate between Aβ sheets and was expected to enhance its disaggregation because of its planner structure. Triazolopyrimidine scaffold was anticipated to interact with the important residues of AChE. Indeed, triazole scaffold has an excellent previous record in the inhibition of AChE and BuChE. In this context, this work sought to develop quinolinetriazolopyrimidine hybrids connected by a piperazine (159) as multi-target candidates for the treatment of AD (Fig. 40). Eleven new hybrids were synthesized and tested for inhibition of AChE, BuChE, self-induced Aβ-aggregation, AChE-induced Aβ-aggregation and antioxidant properties. Among the hybrids synthesized substance 160 stands out for having IC50 values for ChE inhibition very low (IC50 AChE= 0.042 ± 0.79 µM IC50 BuChE = 0.51 ± 0.91 µM), its AChEI activity is close to donepezil (IC50 AChE= 0.038 ± 0.003 µM IC50 BuChE = 2.58 ± 0.65 µM) and 160 has a selectivity of 12.14 for AChE/BuChE. As for the inhibition of Aβpeptide aggregation assay, compounds were tested at concentrations of 25 µM for self-induced Aβaggregation inhibition and at the concentration of 100 µM for Inhibition of AChE-induced Aβ-aggregation. Once again compound 160 stood out by showing a reduction in the amount of Aβ-aggregation of 72.86% in the first test and of 80.45% in the second test. Antioxidant activity assays were performed by analyzing cell viability in MTT test in human neuroblastoma cells (SH-SY5Y), with substances given at concentrations of 1, 5, 10, 20 and 25 µM. Compound 160 showed a dosedependent activity, about 2.5-fold higher than Trolox, suggesting a very interesting multi-target profile to be considered for further development of new drug candidates against AD [92].

N

N O O

N

N CH3

160 IC50 AChE = 0.042 ± 0.79 µM IC50 BuChE = 0.51 ± 0.91 µM ORAC = 2.43 ± 0.61 Selectivity of 12.14 for AChE/BuChE A! aggregation of 72.86% (self-induced) A! aggregation of of 80.45% (Cu2+-inducec)

Fig. (40). Design of novel quinoline-triazolopyrimidinepiperazine hybrids and chemical structure of the potent multifunctional lead-derivative 160.

In another approach to pull of novel tacrine derivatives with MTDL profile, Liao and collaborators studied new family tacrine-flavonoid hybrids designed to act as selective AChE inhibitors, antioxidants and inhibitors of self-induced β-amyloid peptide (Aβ) aggregation. The structural architecture of this new series was based on the molecular hybridization of the 5,6,7trimethoxyflavone (161) with 6-chlorotacrine (162, Fig. 41). From the target-hybrids 163a-d and 164a-d, compound 162, with five methylene groups into the spacer subunit between the 5,6,7-trimethoxyflavone and 6-chlorotacrine moieties, showed the strongest selective inhibitory potency for AChE with IC50= 12.8 ± 0.05 nM, being 6-fold more potent than 6-chlorotacrine (IC50= 78.5 ± 0.04 nM). Furthermore, this compound also showed significant antioxidant activity, a remarkable inhibition of self-induced Aβ1-42 aggregation (33.8% ± 0.06 at 25 µM), good neuroprotective effect against H2 O2-induced PC12 cell injury and BBB permeability [93]. The structural pattern of natural glycosylated dihydroxy flavone inspired Sang and co-workers in the combination of the structure of scutellarin (165) and rivastigmine (3) to draw a new molecular architecture of MTDL candidates (Fig. 42). Scutellarin is a natural compound with a number of pharmacological properties related to neurological disorders, such as free radical scavenging and metal chelating activities, antiinflammatory effects, neuroprotective action and inhibition of Aβ fibril formation. Thus, a series of scutellarin-rivastigmine (166) based carbamates were synthetized and evaluated for multifunctional biological prop-



erties, including AChE and BuChE inhibition, metalchelating properties, anti-oxidative and neuroprotective effects against H2O2-induced PC12 cell injury and BBB permeability. Compounds 167c and 168c, containing the N,N-diethyl carbamate moiety, showed the most potent and selective inhibition of AChE over BuChE, with 167c exhibiting an IC50 value of 0.34 µM for AChE and a 24.1-fold higher selectivity, followed by compound 168c with a 39.7-fold higher selectivity for AChE (IC50= 0.57 µM). Kinetic studies demonstrated that compound 168c could interact concomitantly with the CAS and PAS of AChE, which is consistent with molecular modeling results. Compound 168c also exhibited a good antioxidative activity with a value 1.3fold of Trolox, and ability for selective biometal chelation. Furthermore, compound 168c showed neuroprotective effect against H2O2-induced PC12 cell injury and adequate in vitro BBB permeability [94]. OCH3 O

NH2

H3CO

H3CO

O

Cl

N

[80,98,99]. On this basis, novel rivastigminehydroxycinnamic acid hybrid compounds were planned as inhibitors of AChE, BuChE, Aβ aggregation and ROS scavengers (Fig. 43). Among eight hybrids, compound 172 stood out, exhibiting moderate AChE inhibition rate (23.42% at 1µM and 23.99% at 5 µM) and a higher and selective BuChE inhibitory activity (76.32% at 1µM and 91.95% at 5 µM), with additional good ability to inhibit Aβ self-aggregation (85.3% of inhibition at 10µM). All compounds showed good safety in the MTT cell viability assay with hippocampus neuronal cells (HT22 cells). Finally, a neuroprotection test was performed with the lead-compound 172, that showed neuroprotective effects on HT22 cells against cell injury induced by glutamate and against H2 O2 induced cell death. In spite of the moderate AChE inhibitory activity, compound 172 was able to inhibit the hydrolytic activity of ChEs, to potentially prevent Aβ self-aggregation, to protect HT22 cells from glutamate and H2 O2 induced cell death. These data support that compound 172 could be an attractive lead compound for further optimization in the drug discovery process [100]. OH

6-chlorotacrine (162)

5,6,7-trimethoxyflavone (161)

NMe2 COOH O

H3CO

Spacer linking

O

O

OCH2CONH(CH2)nNH

OH

Et

OH

O AChE inhibitor

Scutellarin (165)

Rivastigmine (3) Molecular hybridization

OR

163a-d n=3, 4, 5, 6 spacer linking in Para position 164a-d n=3, 4, 5, 6 spacer linking in Meta position 163c AChE with IC50= 12.8 ± 0.05 nM A!1-42 aggregation 33.8% ± 0.06 at 25 !M

Me O

H3C

Antioxidant Metal chelation

OH

OH

O

O

HO

AChE IC50 = 78.5 ± 0.04 nM

OCH3 O

25

O

H3CO O

Cl

N

Fig. (41). Design strategy for the 5,6,7-trimethoxyflavone-6chlorotacrine hybrids 163a-d and 164a-d.

Butyrylcholinesterase (BuChE) has several neural and non-neural functions. Recent observations suggest that rather than selective inhibition of AChE, BuChE inhibition may be more effective for the treatment of neurodegenerative diseases such as AD [95]. Studies indicate that high levels of BuChE in the cortex are related to some DA markers such as the extracellular deposition of the Aβ and the aggregation of hyperphosphorylated tau protein [96]. According to these statements, the use of non-selective ChEs inhibitors would lead to more expressed improvements than the use of selective AChE inhibitors [97]. Previously, some researchers reported that hybrid compounds combining hydroxycinnamic acids such as 171 and ChE inhibitory pharmacophores like in rivastigmine (3) could have potential multi-target profile for the therapy of AD

H3CO

O

N

O

R2

m, p

166 CH3 N(CH3)2

R1R2N=

a

b

N

O e

N(CH2CH3)2

N CH3

N

c

167a-h 168a-h 169a-h 170a-h

N(i-Pr)2 d

N f

R1

NCH3 g

N

NCH2Ph

R=CH3, 4' R=H, 4' R=CH3, 3' R=H, 3' 167c AChE IC50 = 0.34 !M 168c AChE IC50 = 0.57 !M

h

Fig. (42). Molecular hybridization of scutellarin (165) and rivastigmine (3) in the design strategy of a new series of hybrid derivatives 167-170.

A series of hybrid compounds, illustrated by derivatives 174 and 175, was synthesized by Wang and coauthors, based on the structural scaffold of the metal chelator clioquinol (133) and ebselen (173, Fig. 44), a glutathione peroxidase (GPx) mimic. Biological evaluation for their potential inhibitory activity on Aβ142 aggregation, antioxidant and superfast antioxidant catalysts against H2O2 and metalchelating properties were performed. All hybrid compounds exhibited more significant inhibition of Aβ1-42 aggregation than ebselen



(173), clioquinol and ebselen (174-175) + clioquinol (133), specifically, for compounds 174a (IC50= 9.6 ± 0.4 µM) and 174b (IC50= 8.1 ± 0.3 µM). Most of the target compounds demonstrated higher antioxidant activity (0.6-3.5 trolox equivalents), than ebselen, clioquinol and ebselen + clioquinol, with compound 175a showing the best antioxidant activity (3.5 ± 0.1 trolox equivalent). Evaluation of brain penetration ability of the target-hybrids, revealed that compound 174a (permeability > 4.7 x 10-6 cm.s-1) could adequately cross BBB. These properties highlight the potential of compound 174a as an innovative lead-compound for the development of multifunctional drug candidates for AD therapeutics [101]. O H3C

O N

CH3 N

O

CH3

OH

CH3

HO

CH3

Rivastigmine (3) Molecular hybridization

OH Hydroxycinnamic acid (171)

HO H N

HO

O

O

N O

172 Inhibition rate % AChE 1 µM = 23.42% and 5 µM = 23.99% Inhibition rate % BuChE 1 µM = 76.32% and 5 µM = 91.95%

Fig. (43). Design and structure of the most potent rivastigmine-hydroxycinnamic acid hybrid derivative 172. Cl Se N I

N O

OH

Ebselen (173)

Clioquinol (133) Molecular hybridization R1 R2

N

N Se

Se N

OH

N

OH

R3 R4

O

X CQ-ebselen hybrids

174a R1=R2=R3=H, R4=F, X=Cl A!1-42 aggregation = IC50 = 9.6 ± 0.4 !M 174b R1=R3=R4=H, R2=F, X=I A!1-42 aggregation = IC50 = 8.1 ± 0.3 !M

O

X

175a (X=I) ORAC = 3.5 ± 0.1 trolox equivalent

Fig. (44). Chemical structure of compounds 174 and 175, designed from ebselen (173) and clioquinol (133).as novel multi-target active ligands against AD

Coumarins were also exploited as molecular prototypes by Wang and co-authors in the design and synthesis of a series of 3-imine-4-hydroxycoumarin derivatives (176, Fig. 45). The compounds were evaluated as multifunctional agents in MAO, Aβ1-42 and potential biometal chelators and antioxidants and most of

the derivate exhibits activity. In particular, compounds 177 (IC50 MAO-A= 0.673 ± 0.011 µM, IC50 MAO-B = 0.711 ± 0.013 µM), 178 (IC50 MAO-A = 4.97 ± 0.41 µM, IC50 MAO-B = 0.851 ± 0.047 µM) and 179 (IC50 MAO-A = 3.78 ± 0.62 µM, IC50 MAO-B = 1.32 ± 0.18 µM) exhibited high potency in MAO inhibition. All three compounds exhibited moderate to good potencies (20.2% to 82.3% at 20 µM) of inhibit self-induced Aβ1-42, compared to those of resveratrol (67.3 ± 3.4 µM at 20 µM) and curcumin (50.2% ± 5.9 at 20 µM). The chelating effect of compound 177 was studied for Cu2+, indicating that this compound could serve as metal chelator for AD therapy. All of these compounds also demonstrated moderate to good antioxidant activities ranging from 0.12 to 1.57-fold of the trolox values, with the best activity observed for compound 179 (1.57 trolox equivalent). In the DPPH radical scavenging assay, compounds 177 (IC50= 45.8 ± 1.2 µM) and 179 (IC50= 38.6 ± 2.0 µM) exhibited the most potent radical scavenging activities. Taking all these data set, compound 177 seems to be a promising lead compound with balanced properties for AD treatment [102]. The structure of clioquinol (133) was also exploited by Wang and co-authors as a model for molecular hybridization with moracin (180), leading to the novel series of hybrid compounds 181-184 (Fig. 46). Among the four series of compounds (181-184), 181a (PDE4D2 IC50 = 2.31 ± 0.32 µM), 182a (PDE4D2 IC50 = 0.96 ± 0.11 µM) and 183a (PDE4D2 IC50 = 0.32 ± 0.02 µM), which bear a phenolic hydroxyl group on the R3 position, demonstrated better inhibition of PDE4D2 than other analogs in the target-series. The inhibitory activities of the target compounds on Aβ1-42 selfinduced aggregation was first determined by a thioflavin T (ThT) fluorescence assay using curcumin and resveratrol as reference compounds. None of the tested compounds exhibited significant fluorescence signals under the experimental conditions. Moreover, most of the target compounds demonstrated higher antioxidant abilities compared to moracin M (180) and clioquinol (133). Compounds with a phenolic hydroxyl group on the benzofuran moiety, such as 183a, exhibited significantly higher ORAC values (3.6 ± 0.02). The parallel artificial membrane permeability (PAMPA) assay, suggested that most of the target compounds exhibited significant BBB permeability. In addition, compound 183a (19.1 x 10-6 cm.s-1) exhibited a much better BBB permeability and also showed appropriate biometal chelating ability and significative neuroprotective effects against inflammation in microglial cells. All of these results highlighted compound 183a as a potential



27

Metal A! interaction N

N

N MAO inhibition antioxidant

Metal Chelanting Cl

1,4-diamine (176) R1

OH R

HO

OH

R2 I

N O

R3

O

Coumarins (76)

O

N OH

R4

O

Clioquinol (133)

177 R1=R2=OH, R3=R4=H IC50 MAO-A = 0.673 ± 0.011 !M, IC50 MAO-B = 0.711 ± 0.013 !M IC50 DPPH = IC50= 45.8 ± 1.2 !M 178 R1=R3=Br, R2=R4=H IC50 MAO-A = 4.97 ± 0.41 !M, IC50 MAO-B = 0.851 ± 0.047 !M IC50 DPPH = -179 R1=R2=R4=H, R3=NO2 IC50 MAO-A = 3.78 ± 0.62 !M, IC50 MAO-B = 1.32 ± 0.18 !M IC50 DPPH = IC50= 38.6 ± 2.0 !M

Fig. (45). Design of the new series of derivatives of coumarin-clioquinol hybrids (177-179) with improved activities for MAO inhibition, antioxidant, anti-aggregation of amyloid-β and ion metal chelation.

and promising lead-molecule for the development of orally active therapies for AD [103]. OH HO

N

O Cl Moracin M (180)

R1 R2

OH

OH

Clioquinol (133) I

N O

R

N OH

OH

showing the most promising AChE inhibitory activity (IC50= 0.059 ± 0.003 and 0.080 ± 0.005 µM, respectively), along with high inhibition of Aβ1-42 aggregation (IC50= 10.1 ± 0.09 and 10.9 ± 0.15 µM, respectively). Molecular modelling studies suggested that compounds 187 and 188 have significant binding affinity with both CAS and PAS of the AChE. In addition, results from neuroprotection studies indicated that these derivatives can significantly reduce H2 O2-induced neuronal death mediated by oxidative stress and Aβ1-42 induced cytotoxicity [104].

O

CF3

R3 R4

R5

X

181a R3=OH R1=R2=R4=R5= X=H PDE4D2 IC50 = 2.31 ± 0.32 !M 182a R3=OH,R1=R2=R4=R5= X=Cl PDE4D2 IC50 = 0.96 ± 0.11 !M 183a R3=OH,R1=R2=R4=R5= X=I PDE4D2 IC50 = 0.32 ± 0.02 !M ORAC = 3.6 ± 0.02 !M

OH 184a, R=H PDE4D2 IC50 = 8.86 ± 0.39 !M

Fig. (46). Design of the new series of derivatives of Moracin M (180) and clioquinol (133).

1,3,5-triazine scaffold (185) has been serving medicinal chemists for a long time for the development of antifungal, anticancer, antiviral agents. Regarding neurodegenerative diseases, due to its roughly planar structure, triazine is expected to intercalate Aβ sheets and to enhance the Aβ disaggregation. For this reason, triazine was selected by Maqbool and co-workers as preferred scaffold in the draw of a series of new cyanopyridinetriazine (186) hybrids rationaly designed as persuasive multifunctional agents for the treatment of AD (Fig. 47). All eight derivatives obtained were potent and selective AChE inhibitors, with compounds 187 and 188

N

HN

H N

N

N N

N

N

N

N R1

Triazine moieties (185)

N

R2

Cyanopyridine moieties (186)

N N N

187 = R1=F and R2=Cl AChE IC50 = 0.059 ± 0.003 !M A!1-42 aggregation IC50= 10.1 ± 0.09 !M 188 = R1=H and R2=OCH3 AChE IC50 = 0.080 ± 0.005 !M A!1-42 aggregation IC50=0.9 ± 0.15 !M

Fig. (47). Chemical structures of the multi-target active cyanopyridine-triazine hybrids 187 and 188.

Sheng and co-workers planned a series of novel 1phenyl-3-hydroxy-4-pyridinone (193), as multifunctional agents for AD therapy through incorporation of 3-hydroxy-4-pyridinone moiety from deferiprone (191) into the general scaffold of H3 receptor antagonists (190, Fig. 48). The 3-hydroxy-4-pyridinone moiety


de Freitas Silva et al. O

O

OH

N NR1R2

O

N

O

H3 receptor Antagonism

Metal Chelation Radical Scavenging

O A! Aggregation Inhibition

aminopropoxyphenyl moiety (189)

deferiprone (191)

SKF-64346 (190)

OH

OH R3

O N

N

R1 N Structure modification R2

O 192

O N

n

O

R4

193

194 para, n= 3, NR1R2= pyrrolidinyl, R3= CH3, R4=H. H3 inhibitory potency IC50 = 0.32 ± 0.01 nM. A! 1-42 agregation inhibitory potency IC50 = 2.85 ± 0.22 µM.

Fig. (48). Rational design of 1-phenyl-3-hydroxy-4-pyridinone derivatives (194) as multifunctional agents.

(193) of the commercial metal chelator deferiprone was chosen due to its desirable chelating properties with high affinity for Cu, Zn, and Fe ions, but low affinity for sodium, potassium, magnesium. The generally accepted pharmacophore model of H3 receptor antagonists (191) is composed of a “western part” containing a tertiary basic amine and a spacer, an aromatic central core, and an “eastern part” consisting of a polar group, a second basic amine, or a lipophilic residue. Next, they prioritized the introduction of an appropriate metal chelating pharmacophore 193 according to the “eastern part” attributes to generate novel molecular hybrids with multiple functions. Interestingly, the newly designed 1-phenyl-3-hydroxy-4-pyridinone derivatives share similar structural elements to Aβ aggregation inhibitor SKF-64346 (190). Thus, molecular hybridization of the molecular models 190 and 191 led to compounds 192. Structural modifications on compound 192 were carried out and gave rise to series 193. Among all tested compounds, hybrid 194 displayed excellent selective H3 receptor antagonistic activity (IC50 = 0.32 ± 0.01 nM), efficient ABTS•+ scavenging effect (1.54 ± 0.15), good Cu2+ and Fe3+ chelating properties, and effective inhibitory activity against self- and Cu2+induced Aβ1−42 aggregation (IC50 = 2.85 ± 0.22 µM). More interestingly, an in vivo study revealed that 194 possesses suitable PK profiles in plasma and acceptable BBB penetration behavior, highlighting this compound as a new promising and innovative drug candidate prototype for AD [105]. Wei and co-workers used the structure of oxoisoaporphine (195) for planning two series of 8- and 11substituted-amide derivatives (196 and 197, Fig. 49) as potential inhibitors of AChE and Aβ aggregation, with additional neuroprotective properties. Oxoisoaporphine

is an alkaloid with important properties related to antiAlzheimer drugs [106]. Biological evaluation of the oxoisoaporphine derivatives 196a-e and 197a-e, with the same side-chain, exhibited similar inhibitory potency toward AChE. Compounds 196b and 197b, with two methylene groups in the side chain showed strongest AChE inhibitory potency (IC50 = 28.4 ± 14 nM and 80.8 ± 26 nM, respectively) and a significant inhibition of self-induced Aβ1-42 aggregation (IC50 = 74.6 ± 1.9 and 76.1 ± 1.2, respectively). Moreover, compounds 196b and 197b showed to be able in crossing the BBB to reach their targets in the CNS and a significant reduction in Aβ secretion levels by human neuronal cells (SH-SY5Y), which overexpress the Swedish mutant form of human β-amyloid precursor protein (APPsw) [107]. N

O oxoisoaporphine moieties (195) N N

Structural modification n

O

NH

N

Cl

NH N

O

n O

O (196a-d) n= 1,2,3,4,5

(197a-d) n= 1,2,3,4,5

196b AChE inhibitory potency IC50 = 28.4 ± 14 nM A!1-42 aggregation potency IC50 = 74.6 ± 1.9 nM 197b AChE inhibitory potency IC50 = 80.8 ± 26 nM A!1-42 aggregation potency IC50 = 76.1 ± 1.2 nM

Fig. (49). Structure of 8- and 11-substituted 1azabenzanthrone derivatives 196 and 197, designed as oxoisoaporphine analogues.



In another approach for searching innovative and effective drug candidates for AD treatment, and based in previous research data [108], Hebda and co-workers synthesized two novel series of phthalimide derivatives (198 and 199, A and B, Fig. 50), designed as dual binding site AChE inhibitors. The pyrrolidine substituted derivative 200 (series A) exhibited the best inhibitory activity of AChE (IC50 = 0.276 ± 0.003 µM). Regarding the length of the linker, the best activities were observed for compounds with six to eight carbon atoms. In the series B, compounds with a five carbon atom linker, such as 201a (2-F derivative) and 201d (3-Cl derivative), were the most potent AChE inhibitors with IC50 values of 150 and 70 nM, respectively. Regarding to the inhibition of self-induced Aβ1-42 aggregation, only compound 200n showed a moderate inhibitory activity of 35.8% [109].

prospection of novel drug candidate prototypes, more than 600 compounds were screened by HTS (high throughput screening) for AChE inhibitory activity. The aim was to identify promising structures to be used in a structural optimization study. As a result, the pyrimidine derivative 202 (AChE inhibition: 32.15% at 40 µM) was chosen as initial prototype molecule and fifteen derivatives were synthesized and evaluated for their ability to inhibit AChE (Fig. 51). Aiming to identify the optimal side chain for the desired enzyme inhibition, compound 203 was recognized as the most active of this first series (77.26% of AChE activity at 40 µM and IC50= 8.14 ± 0.40 µM). In a second step, optimization of the lateral 1,2-diazol ring in compound 203, led to additional nine new analogues, with the 1,3diazol derivative 204 disclosing a discreet improving in AChE inhibition (IC50= 1.59 ± 0.02 µM). In sequence, modification in the central ring confirmed that 1,3diazine was the best pharmacophore, and it was conserved in another twenty-four analogues with different substituents at the lateral 1,3-imidazolyl ring. After all

Considering that molecular hybridization plays a highlighted role in the design of novel MTDLs and that the search of new pharmacophores is determinant in the A Series

R

O

N N

NH

O

N

6

N

N

O

O

198

200a-o, n=3-6 IC50 EeAChE = 0.276 ± 0.003 !M

N

B Series

O

S

N

N H

3

R

n

O

N H

O

29

F

O

O

S

2HN

N

199

Cl

2HN

N H

3

O

R

201a-f

O

n=3-5 R= 2-F or 3-Cl 201a EeAChE IC50 = 0.150 ± 0.003 !M 201d EeAChE IC50 = 0.070 ± 0.001 !MnM

Fig. (50). Chemical structure of compounds 200 and 201, designed as dual hybrid inhibitors of AChE and BuChE and amyloid β aggregation. H N

N N

15 compounds

N

H N S

N

202 AChE inhibition: 32.15% at 40 µM

H N

H N

H N

N

N

N

N

H N S

N

H N

H N S

N N

N

204 IC50 AChE = 1.59 ± 0.02 µM 05 compounds

N

205 IC50 AChE = 0.067 ± 0.019 µM

N

H N

09 compounds

N

203 IC50 AChE = 8.14 ± 0.40 M µM

24 compounds S

N

N N

N

N

204 IC50 AChE = 1.59 ± 0.02 µM

Fig. (51). Key transition compounds of the structural optimization of pyrimidine derivatives (202-205).

N


these SAR studies, compound 205 was finally identified with a very improved selective AChE inhibitory activity (IC50= 0.067 ± 0.019 µM), and additional chelating ability for Cu2+ ions, antioxidant activity, inhibitory activity of Aβ-aggregation and low toxicity in human neuroblastoma cells [110]. CONCLUSION In conclusion, the search for more active drugs with longer lasting responses and a possible cure for AD is still a challenge for medicinal chemistry. Considering the most recent findings supporting the multifactorial hallmarks in the pathophysiology of AD, the concomitant modulation of diverse molecular targets and biochemical pathways is still a problem to be overcome in the development of new effective disease modifying drug candidates. This multifactorial aspect supports the rational planning of multi-target substances as the main strategy in obtaining new effective and well tolerated drugs for AD and other neurodegenerative illnesses. The high selectivity of the available drugs used in AD treatment, combined with the multifactorial aspects of the disease, make the current therapeutic approach ineffective, compelling patients to use drug cocktails to control a set of symptoms associated to the progress of the disease. In this way, in consonance with many medicinal chemists worldwide, we can affirm that the AD treatment will be effective only when we find druggable chemical entities capable of interacting and modulate concomitantly different targets, blocking or activating different biochemical interconnected pathways related to the disease progression. In the last years, many efforts have been directed towards to the development of novel bioactive chemical entities, capable of acting on the most distinct, varied and probable therapeutic targets on the AD pathogenesis. Many review papers are available in the literature, giving sufficient arguments for the potential beneficial role of polypharmacology in the control of multifactorial diseases, especially when a single drug, with multiple mode of action, is administered, avoiding adverse effects, toxicity, overloaded metabolism and drug-drug interactions. In this context, several substances with a multi-target profile have been found and several of them have demonstrated interesting and promising pharmacological profiles, making them possible drug candidates as well. Although there are no new drugs for AD on the market, expectations for a multi-target drug are real and this approach is still the most promising tool in Medicinal Chemistry in the challenge of searching new effective and innovative drugs to control and, in our best wishes, to cure AD and other neurodegenerative diseases.


ACKNOLEGDEMENTS The authors are greatul to the Brazilian Agencies CNPq (#454088/2014-0, #400271/2014-1, #310082/2016-1), FAPEMIG (#CEX-APQ-00241-15), FINEP, INCT-INOFAR, PRPPG-UNIFAL and CAPES for financial support and felowships. CONFLICT OF INTEREST The authors declare no conflicts of interest. REFERENCES [1]

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