Design, synthesis, cholinesterase inhibition and molecular modelling study of novel tacrine hybrids with carbohydrate derivatives

Bioorganic & Medicinal Chemistry 26 (2018) 5566–5577

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Design, synthesis, cholinesterase inhibition and molecular modelling study of novel tacrine hybrids with carbohydrate derivatives

T

João Paulo Bizarro Lopesa, Luana Silvaa, Gabriela da Costa Franarina, Marco Antonio Ceschia, , Diogo Seibert Lüdtkea, Rafael Ferreira Dantasb, Cristiane Martins Cardoso de Sallesc, ⁎ Floriano Paes Silva-Jrb, Mario Roberto Sengerb, , Isabella Alvim Guedesd, ⁎ Laurent Emmanuel Dardenned, ⁎

a

Instituto de Química, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9500, Campus do Vale, 91501-970, Porto Alegre, RS, Brazil Laboratório de Bioquímica Experimental e Computacional de Fármacos, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Av. Brasil, 4365, 21040-360, Rio de Janeiro, RJ, Brazil c Instituto de Ciências Exatas, Universidade Federal Rural do Rio de Janeiro, BR 465, Km 7, Campus Universitário, 23890-000 Seropédica, RJ, Brazil d Laboratório Nacional De Computação Científica-LNCC, Av. Getúlio Vargas, 333, Petrópolis 25651-075, RJ, Brazil b

ARTICLE INFO

ABSTRACT

Keywords: Tacrine Carbohydrate Xylose Ribose Galactose Cholinesterases Molecular modeling Alzheimer

A series of hybrids containing tacrine linked to carbohydrate-based moieties, such as D-xylose, D-ribose, and Dgalactose derivatives, were synthesized by the nucleophilic substitution between 9-aminoalkylamino-1,2,3,4tetrahydroacridines and the corresponding sugar-based tosylates. All compounds were found to be potent inhibitors of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the nanomolar IC50 scale. Most of the D-xylose derivatives (6a-e) were selective for AChE and the compound 6e (IC50 = 2.2 nM for AChE and 4.93 nM for BuChE) was the most active compound for both enzymes. The D-galactose derivative 8a was the most selective for AChE exhibiting an IC50 ratio of 7.6 for AChE over BuChE. Only two compounds showed a preference for BuChE, namely 7a (D-ribose derivative) and 6b (D-xylose derivative). Molecular docking studies indicated that the inhibitors are capable of interacting with the entire binding cavity and the main contribution of the linker is to enable the most favorable positioning of the two moieties with CAS, PAS, and hydrophobic pocket to provide optimal interactions with the binding cavity. This finding is reinforced by the fact that there is no linear correlation between the linker size and the observed binding affinities. The majority of the new hybrids synthesized in this work do not violate the Lipinski's rule-of-five according to FAF-Drugs4, and do not demonstrated predicted hepatotoxicity according ProTox-II.

1. Introduction Neurodegenerative disorders have complex pathological mechanisms which result in progressive and irreversible loss of physiologically related neuronal systems, consisting in one of the greatest challenges of medical sciences. Known as the most common and devastating disorder of this type, Alzheimer's Disease (AD) affects about 46.8 million people in the world, and it is expected to increase at least 3-fold by 2050, according to recent reports.1,2 Currently, there is no cure for AD and several hypotheses have been directing research efforts, such as amyloid cascade, tau hyperphosphorylation, metal imbalance, oxidative stress and cholinergic hypotheses, among others.3 The cholinergic hypothesis is the most related to the symptoms, which suggests that the progressive cognitive impairment of AD results from a deficit in the ⁎

neurotransmitter acetylcholine (ACh), and consequent synaptic failure.4,5 The cholinesterase inhibitors (ChEI) promote the stimulation of the cholinergic receptors, prolonging the availability of ACh in synaptic cleft, hence decreasing the AD associated symptoms.6 The cholinergic theory remains the most effective therapeutic approach for the symptomatic treatment of AD. Among the four drugs that have been used so far, three are ChEI: donepezil (Aricept®), rivastigmine (Exelon®) and galantamine (Razadyne®). Lately, memantine has been introduced into the market as an alternative therapy acting as an antagonist of the NMDA receptor, thereby protecting the nervous tissue against glutamate mediated excitotoxicity with limit therapeutic effects.7 Tacrine (chemically, 1,2,3,4-tetrahydroacridine, THA, Fig. 1) was, in 1993, the first drug approved for the treatment of AD, and is one of the most efficient cholinesterase inhibitors, with high efficacy in

Corresponding authors. E-mail addresses: [email protected] (M. Antonio Ceschi), [email protected] (M. Roberto Senger), [email protected] (L. Emmanuel Dardenne).

https://doi.org/10.1016/j.bmc.2018.10.003 Received 13 July 2018; Received in revised form 5 October 2018; Accepted 7 October 2018 Available online 09 October 2018 0968-0896/ © 2018 Elsevier Ltd. All rights reserved.

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synthesized bis(n)-tacrine analogues linked by an alkylene chain which were up to 10,000-fold more selective and 1,000-fold more potent than THA, inasmuch as it has a double interaction in the enzyme, with both CAS and PAS.24 Recently, chiral bis(7)-tacrine analogues have been found to be potent and selective AChE inhibitors in both enantiomeric series.25 Moreover, the bis(7)-tacrine (Fig. 1) possesses remarkable neuroprotective activities, such as inhibition of NO synthase, binding as antagonist of NMDA receptor and inhibition of Aβ synthesis and fibrils formation.26–28 Furthermore, in vivo studies showed that bis(7)-tacrine was able to reach the AChE only 15 min after the single-dose and intraperitoneal administration, indicating that it may easily cross the blood–brain barrier.29 Since then, bis(7)-tacrine has been the source of inspiration for the design of new potential drugs for the treatment of AD and cognitive impairment. The major strategy relies in the replacement of one tacrine moiety by a pharmacophore or a biologically active nucleus that might be able to bind to PAS.14 In this context, several tacrine-based hybrids have been synthesized in the latest years.22,30–37 A series of tacrinelophine and tacrine-tianeptine hybrids, synthesized recently by our group, has demonstrated to be potent and selective inhibitors of AChE and BuChE with IC50 in the nanomolar concentration scale.38,39 Regarding the great efforts to delineate structural requirements for optimal binding to the active site of AChE and BuChE, a good understanding of their function and structure is essential to interweave salient details together into a coherent predictive tool.12 Since diverse experimentally determined structures of AChE have been known and with the advancement of computational chemistry, molecular modelling has been widely used to predict the binding modes and affinity between the enzyme and inhibitors with different scaffolds, guiding the optimization and development of new compounds. The interactions of the THA moiety within the AChE binding site is highly consistent in most of the compounds, being lodged in the CAS.22 The THA binding mode is mainly characterized by a π-π stacking with the Trp84 and Phe330 side chain, and a hydrogen bond between the protonated aromatic nitrogen of the quinoline ring and the carbonyl oxygen of the H440 main chain.40–42 In addition, it has been found that the secondary amino group present in many potent inhibitors also makes important interactions such as hydrogen bonds with different amino acid residues and/or water molecules. Taking into consideration the potential of reported tacrine-based molecules in the search of novel cholinesterase inhibitors, we designed new hybrids connecting tacrine nucleus with sugar derivatives as showed in Fig. 2. We believe that the biocompatibility of the carbohydrates might increase the inhibitor's bioavailability as well as their permeability in biological barriers. In addition, we also explore the intermolecular interactions between sugar moiety and AChE, predicting the possible binding modes by molecular docking. Besides the wellknown hydrogen bonds between hydroxyl groups of the carbohydrates and polar atoms in proteins, we also studied the less explored CH/π interactions with aromatic residues, mainly tryptophan.43–46 Moreover, carbohydrates are multifunctionalizable structures, which allow the substitution at various positions of the sugar ring and further structural modifications. Recently, compounds containing carbohydrate scaffolds have been studied as promising candidates for Alzheimer's therapy. O-flavonol glycosides were synthesized by Mughal and coworkers and most of the derivatives were potent inhibitors of AChE and BuChE with varying degree of IC50 values.47 Several purine nucleosides were synthesized by N-glycosylation and showed to be potent inhibitors of both AChE and BuChE.48,49 Iminossugar-based compounds were synthesized and evaluated as cholinesterase inhibitors and seven of the twenty-three compounds studied had IC50 values in the micromolar range with significant selectivity for BuChE over AChE. In general, the deprotection of the hydroxyl groups led to the reduction or absence of enzyme inhibition.50 Several studies with selenofuranosides have demonstrated diverse activities associated with possible therapies for AD, such as

Fig. 1. Structures of tacrine and bis(7)-tacrine.

restoring ACh. However, it has been withdrawn from the market due to its hepatotoxicity, mainly by metabolism to toxic quinone-type metabolites species through the action of cytochrome P450 enzyme CYP1A2.8 Acetylcholinesterase (AChE, 3.1.1.7) belongs to the α/β hydrolase protein fold and has been purified and crystallized in the monomeric, dimeric, and tetrameric forms.9 The active site is a narrow gorge with a length of approximately 20 Å, which penetrates more than half-way into the enzyme, containing two main subsites: the catalytic active site (CAS), located at the bottom of the cavity, and the peripheral anionic site (PAS), near the entrance of the gorge.10,11 Besides the catalytic triad, composed by Ser200, His440 and Glu3271, there are amino acids residues in the CAS strictly positioned to confer high selectivity to acetyl esters, in order to preclude the hydrolysis of bulky esters and to stabilize the acetate leaving group. These residues comprise, respectively, the acyl pocket (Phe288, Phe290, Phe331) and the oxyanion hole (Gly118, Gly119, Ala201).12 One of the essential regions to the catalytic process is the anionic subsite, composed mainly by Trp84, placed at the bottom of the gorge, which plays a central role to attract ACh to CAS, interacting with the ACh quaternary nitrogen.10,12 Moreover, crystallographic and molecular dynamics (MD) simulation data of TcAChE have pointed to the distance alternation between Tyr442 and Trp84 as a backdoor, whose opening allows the substrate or product trafficking into or out of the cavity.13 Several studies have shown that AChE is not merely the enzyme which hydrolyzes ACh, but also plays an essential role on the Aβ peptide assembly, acting as a chaperone in Aβ folding.14 It has been suggested that the PAS is the enzymatic region responsible for this role, which has been strengthened by the fact that this chaperone effect is sensitive only to drugs that block the PAS of the AChE.15,16 Butyrylcholinesterase (BuChE, E.C. 3.1.1.8) is also involved in the hydrolysis of the neurotransmitter ACh. It has been suggested that BuChE activity rises in the elderly and in AD patients’ brains, while AChE activity declines, indicating that BuChE plays a critical role for ACh hydrolysis in the late stage of AD. Therefore, compounds that are able to inhibit both AChE and BuChE might have better long-term therapeutic efficacy.17–19 The structure of human BuChE (HsBuChE) is very similar to HsAChE, sharing 65% amino acid sequence homology and having the same kind of residues in the catalytic triad: Ser198, His438, and Glu325 in HsBuChE.19,20 The absence of bulky aromatic amino acids in the PAS, especially in the acyl pocket of CAS, is the main difference of BuChE when compared with AChE, being postulated as a primary source of enzymatic selectivity.12,21 After tacrine has been approved for the treatment of AD, several tacrine-based compounds have been studied boosted by their synthetic ease, highly inhibitory profile and binding affinity with CAS and PAS of AChE. Despite its hepatotoxicity, tacrine remains a reference and a good chemical scaffold to design novel and more potent cholinergic prototypes with suppressed side effects.14,22 Recently, several 9-acridines derivatives were found as selective cholinesterase inhibitors together with free radical scavenging, which makes promising the use of the acridine scaffold to obtain multifunctional drugs for the therapy of neurodegenerative diseases.23 In 1996, Pang and co-workers

1 All residues described in this text belong to AChE from Torpedo californica (TcAChE).

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Fig. 2. Carbohydrates scaffolds used in this work and the general structure of the synthesized tacrine-carbohydrate hybrids.

antioxidant, inflammation involvement, antidepressant-like effect and protection against memory loss in mice.51–55

only a complex mixture, mainly by degradation of the sugar moiety. We also intend to perform further studies to enable the access of the deprotected compounds.

2. Results and discussion

2.2. AChE and BuChE inhibition evaluation

2.1. Chemistry

The inhibitory activities against MmAChE and MmBuChE of new tacrine-(carbohydrate-derived) hybrids, together with the reference compounds bis(7)-tacrine and tacrine, are reported in Table 1. (For the corresponding inhibitor titrating curves for IC50 determination, see supporting information, Fig. S1). All compounds were found to be potent inhibitors of both cholinesterases with IC50 values in the nanomolar concentration scale. Moreover, six of nine compounds were able to inhibit AChE in the same order of magnitude than the bis(7)-tacrine. In general, the D-xylose derivatives (6a-e) exhibited AChE modest selectivity and the compound 6e (n = 6, Entry 5) showed an IC50 of 2.2 nM against AChE and of 4.93 nM against BuChE, being the most potent inhibitor of both AChE and BuChE in the assayed series of tacrine-carbohydrate hybrids. Despite the higher potency, compound 6e showed the lower selectivity. The compound 6b (n = 5, Entry 2) was the most selective for BuChE (IC50 ratio of 0.14) and showed a good inhibitory activity for this enzyme (IC50 = 11.7 nM). The D-galactose derived 8a (n = 6, Entry 8) was found to be the most selective AChE inhibitor, exhibiting an IC50 ratio of 7.6 for AChE over BuChE. Compounds derived from D-ribose (7a and 7b, Entries 6 and 7) showed slight preference for BuChE (Table 2).

The synthesis of tacrine-based hybrids required the previous preparation of precursor 9-chloro-1,2,3,4-tetrahydroacridine (1) through the classical Niementowski reaction between anthranilic acid and cyclohexanone mediated by POCl3.56 Treatment of the chloride with an excess of 1,n-diaminoalkanes under reflux for 18 h provided 9-amino1,2,3,4-tetrahydroacridines (2a-e) in good yields (78%–95%), as depicted in Scheme 1. This step was carried out in the presence of catalytic amount of KI.57 The natural carbohydrates D-xylose, D-ribose and D-galactose were straightforwardly converted into their tosylates, respectively 3, 4 and 5, by a short synthetic sequence, according to reported protocols (Scheme 2).58–60 With the amines 2a-e and tosylates 3, 4 and 5 in hands, we turned our attention to the connection of both nuclei tacrine and sugar derivatives through the nucleophilic substitution of the tosylate leaving group. The general synthesis of the novel tacrine-(carbohydrate-derived) hybrids is depicted in Scheme 3. The reactants were mixed and solved in isopropanol in a sealed flask, and tosylates 3, 4 and 5 were then converted into tacrine-based hybrids derived from D-xylose, D-ribose and D-galactose, respectively. In this work, we opted for the functionalization in the primary hydroxyl, keeping the anomeric position protected. This choice was made to allow the stereochemical control, preclude the oxidation in the physiological medium and enable further structural studies. The acetonide group was chosen as the protecting group for the hydroxyl due to their small size when compared to others, such as benzyl and acetyl. We also studied the deprotection of the hydroxyl groups using the most common procedures to remove the acetonide group of carbohydrates, involving the reaction with trifluoroacetic acid (TFA) in methanol or water, in short reaction times. Unfortunately, the product was not obtained from anyone of the evaluated conditions and we observed

2.3. Molecular modelling 2.3.1. Redocking studies We carried out redocking experiments for the AChE and BuChE structures used in this work to validate the docking protocol adopted (i.e., GOLD docking program with the ChemPLP scoring function). In all four complexes (three structures of AChE and one structure of BuChE), our docking methodology was able to predict the top-ranked pose close to the experimental conformation observed for the reference ligand present on each crystallographic structure (Fig. 3), even for the highly Scheme 1. Preparation of tacrine derivative precursors (2a-e).

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Scheme 2. Preparation of tosylates from natural sugars D-xylose, D-ribose and D-galactose.

flexible AChE inhibitors. In general, docking studies are considered successful when the root-mean-square deviation (RMSD) between the docked pose and a reference conformation is less than 2 Å.

AChE inhibitors as the top-ranked compounds. For BuChE, all compounds obtained significantly worst scores, and no correlation between them and the experimental binding data was found. For instance, 6b exhibit selectivity against BuChE over AChE (IC50 BuChE/AChE ratio of 0.14), but ChemPLP predicted better score for AChE than BuChE. This might indicate that further experiments are needed to explore additional BuChE conformations, since there are no BuChE crystal structures available with such large compounds.

2.3.2. Ensemble docking According to the ensemble docking strategy adopted herein for the AChE, the top-scored binding mode of all compounds predicted by the ChemPLP scoring function was found for the 1ZGC conformation (Table S1). In our docking strategy, we were able to find three highly potent

Scheme 3. Synthesis of tacrine-(carbohydrate-derived) hybrids.

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Table 1 Inhibitory activity on AChE and BuChE, docking results against AChE (PDB ID 1ZGC) and BuChE structures (5K5E), and the IC50 ratio of the studied compounds. Entry

Compound

1 2 3 4 5 6 7 8 9 10 11

n

6a 6b 6c 6d 6e 7a 7b 8a 8b Tacrine Bis(7)tacrine

4 5 6 7 8 6 7 6 7 – –

IC50 (nM) [confidence interval]

Docking Score*

MmAChE

MmBuChE

TcAChE

TcBuChE

8.7 [7.3–10.5] 84.4 [63.6–113.3] 5.1 [3.6–7.6] 10.2 [7.2–14.5] 2.2 [1.8–2.7] 89.9 [62.4–131.0] 65.5 [45.2–93.7] 4.8 [3.2–7.4] 7.6 [5.6–10.3] 34.3 [14.9–107.8] 1.9 [1.5–2.4]

27.6 [19.3–39.7] 11.7 [8.7–15.9] 25.8 [18.5–36.0] 30.8 [20.4–46.6] 4.93 [4.0–6.1] 36.9 [21.8–62.5] 64.3 [50.2–82.5] 36.7 [22.9–58.7] 26.2 [19.1–36.1] 62.5 [44.0–89.0] 9.1 [6.6–12.5]

118.31 121.77 120.60 125.38 131.44 125.11 129.19 131.93 131.17 – –

93.57 97.34 98.34 94.55 95.69 95.29 96.15 93.97 96.56 – –

IC50 ratio BuChE/AChE

3.2 0.14 5.0 3.0 2.2 0.41 0.98 7.6 3.4 1.8 4.8

* Docking results obtained using the GOLD program with the ChemPLP scoring function. Higher score value correspond to better protein–ligand binding strength.

imparted on the neighboring carbon atoms, increasing the strength of the CH/π interactions and helping to stabilize the sugar moiety with PAS. Many theoretical and experimental studies highlighted the importance of CH/π interactions in molecular recognition and binding affinity, with tryptophan being the most frequent aromatic residue found in contact with carbohydrates43,46,61,62. Despite the potent compound 6c obtained a relatively poor score (120.6), it was found as one of the most potent AChE inhibitors of the series (IC50 = 5.1 nM). In addition to the interaction with Trp279, the binding mode predicted for this compound exhibits an intramolecular interaction between the protonated nitrogen atom and the hydroxyl group from xylose, at the same time as this oxygen atom is within hydrogen bond distance from the Tyr70 hydroxyl group (2.6 Å). For the compound 8a, besides the CH/π interaction with Trp279, the acetonide group also interact with a hydrophobic pocket formed by Ile287 and Phe331 (Fig. 4B). These nonpolar contacts are also observed for the less potent inhibitor 7a. The reference ligand bound in the 1ZGC complex has a carbonyl oxygen atom in the same region interacting with the Phe285 backbone NH atom through a hydrogen bond (3.2 Å) (Fig. 4C).63 However, in the compounds 8a and 7a, the nearest oxygen atoms of the acetonide group to the Phe288 NH (4.0 and 5.0 Å, respectively) are more distant than the limiting criteria for hydrogen bond formation. Thus, further structural modifications guided by molecular docking could even improve the potency of 8a and 7a against AChE. Fig. 4. Docking results for the compounds 6c (pink), 7a (yellow) and 8a (green) against AChE (PDB ID 1ZGC). A) Predicted binding mode of the compounds interacting with the entire AChE gorge. B) Interaction between the compounds and a methyl group from acetonide with the hydrophobic pocket formed by Ile287 and Phe331. C) Superposition of

Table 2 Drug likeness of the carbohydrate derivatives, according to the Lipinski’s rules evaluated by the FAFDrugs4 webserver. Entry

Compound

MW

HBD

HBA

ClogP

Violations

1 2 3 4 5 6 7 8 9

6a 6b 6c 6d 6e 7a 7b 8a 8b

447.61 461.64 475.66 489.69 503.72 489.69 503.72 545.75 559.78

4 4 4 4 4 3 3 3 3

7 7 7 7 7 7 7 8 8

2.96 3.32 3.68 4.22 4.76 3.66 4.20 4.07 4.61

0 0 0 0 1 0 1 1 1

2.3.3. Contribution of the carbohydrate moiety (compounds with linker size n = 6) We analyzed the influence of the carbohydrate moiety in the predicted binding mode and affinity against AChE for the 6-carbon linker compounds 8a, 7a and 6c (Table 1). These three compounds exhibit similar overall binding modes (Fig. 4A): (i) the tacrine moiety interact at the bottom of the gorge through the conserved interactions already described in the literature; (ii) the linker is located at the middle of the gorge performing hydrophobic contacts mainly with Tyr334; (iii) the carbohydrate moiety interacts in the PAS region. Although ChemPLP is not able to compute specific contributions from CH/π interactions and correctly rank these derivatives, the binding modes suggested by docking indicated this kind of interactions between the carbohydrates and the Trp279 from PAS (Figs. 4B and 5). Furthermore, it is possible that the positive charge of the protonated nitrogen atom of the linker is

Fig. 3. Top-ranked poses predicted by GOLD::ChemPLP for each AChE (PDB IDs 2CKM, 1Q84 and 1ZGC) and BuChE (5K5E) structure. The predicted and experimentally observed binding mode are highlighted in yellow and green, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. Docking results for the compounds 6c (pink), 7a (yellow) and 8a (green) against AChE (PDB ID 1ZGC). A) Predicted binding mode of the compounds interacting with the entire AChE gorge. B) Interaction between the compounds and a methyl group from acetonide with the hydrophobic pocket formed by Ile287 and Phe331. C) Superposition of the most potent inhibitors 8a and 7a with the co-crystallized ligand in the complex 1ZGC. Hydrogen bonds are represented as yellow dashes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Predicted interactions of the xylose, ribose and galactose moieties of the compounds 6c, 7a and 8a (green) with Trp279 from PAS from AChE (PDB ID 1ZGC). Possible CH/π interactions are represented as pink dashed lines and the carbohydrate moieties are represented as sticks. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

the most potent inhibitors 8a and 7a with the co-crystallized ligand in the complex 1ZGC. Hydrogen bonds are represented as yellow dashes.

the binding data, the linker size of eight carbon atoms led to the most potent xylose derivative 6e (Table 1, IC50 = 2.2 nM, ChemPLP score = 131.4). Strikingly, the compound 6b differs only in one carbon in the linker when compared with 6a and 6c, but exhibits significantly distinct IC50 value against AChE. Unfortunately, we could not find

2.3.4. Influence of the linker size – Xylose derivatives According to the ChemPLP predictions for AChE, in accordance with 5571

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Fig. 6. Docking results of the compounds 6a (purple), 6b (light blue), 6c (pink), 6d (dark green) and 6e (grey) against AChE (PDB ID 1ZGC). Hydrogen bonds are represented as yellow dashes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

correlations between the observed binding affinities and the predicted binding scores for the xylose-derivatives that could explain the influence of the linker size to the potency of these compounds. According to the docking results, the most potent compound 6e is able to interact with both pockets (Fig. 6A): the xylose moiety interacts with the hydrophobic binding site, mainly with Ile287, while the protonated nitrogen atom and neighboring carbon atom interact with PAS, possibly through cation-pi interactions. Additionally, the hydroxyl group from the xylose moiety is exposed to the solvent, thus avoiding any polar desolvation cost. The eight-carbon linker seems to be an adequate long linker, allowing a more efficient blockage of the AChE gorge due to the interaction of the sugar moiety with both pockets in the entrance of the cavity (Fig. 6B). The less potent xylose-derivative, compound 6b, mainly interacts with the entrance of the gorge through two hydrogen bonds with Tyr70 and Tyr121 (Fig. 6C and 6D). The compounds 6a and 6c, which differs from the 6b structure with a single carbon atom in the linker, were predicted to assume significant distinct positions for the xylose moiety. The shortest linker of 6a allowed for a hydrogen bond between the protonated nitrogen atom and the Tyr121 side chain (2.8 Å), while the xylose group is located near the hydrophobic pocket (Fig. 6C). On the other hand, the compound 6c was able to interact with Trp279 and is hydrogen bonded with Tyr70 side chain (2.6 Å) (Fig. 6D), probably stabilized by the intramolecular hydrogen bond discussed elsewhere (Fig. 4B).

In this work, the synthesis was directed to compounds containing the hexa- and heptamethylenic linker, which allow a optimal distance between CAS and PAS of the AChE, as well as bis(7)-tacrine.24 The Dxylose derivatives were chosen to have their scope expanded from n = 4 to n = 8 to evaluate the influence of the linker size. According to our docking results as discussed above, shorter linkers (n = 4 and n = 5) might not furnish the most favorable positioning of sugar moiety in the PAS, with difficulty to establish important interactions. On the other hand, in spite of the larger linker (n = 8) provided a more efficient blockage of the AChE cavity entrance by the sugar moiety, the enzymatic selectivity was lost, possibly by the greater flexibility and ability of the molecule to adjust in both AChE and BuChE active sites. 2.3.5. ADME-Tox prediction We submitted the tacrine-carbohydrate hybrids synthesized in this study through the free FAF-Drugs4 webserver to predict their drug-like properties.64,65 The Lipinski’s “Rule-of-five” concerns a set of molecular descriptors widely used to predict potential oral drugs, such as: molecular weight (MW) ≤ 500 Da, lipophilicity assessed by the ClogP ≤ 5 (the logarithm of the partition coefficient between water and octanol), number of hydrogen bond donors (HBD) and acceptors (HBA), respectively ≤ 5 and ≤ 10.66 According to the predictions, five derivatives do not violate the Lipinski's rules (6a-d and 7a), and four compounds only violate the MW rule (6e, 7a, and 7b). Therefore, all carbohydrate 5572

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derivatives were predicted to have good oral absorption.

liquid chromatography (HPLC) was performed in a Dionex Ultimate 3000 apparatus, equipped with column Shim-pack CLC-ODS (M) (250 mm x 4,6 mm, 5 µm). Stock solutions were prepared by dissolution of 1 mg from each compound in 1 mL of acetonitrile (HPLC grade Vetec), from which the working solutions were prepared by diluting 100 μL of each sample in 1 mL of acetonitrile. The working solutions were filtered in PVDF Flowsupply filter with 0,22 µm of porosity being followed by HPLC-UV analyses at 25 °C, injection's volume of 10 µL, isocratic elution with acetonitrile: water (60: 40), and rate of flow of 1 mL min−1, 10 min of acquisition time and 270 nm. The absence of significant peaks besides the compound peak evidence of sample high purity of compounds. Nitrogen was used as the desolvation gas. Methanol (Tedia HPLC grade) was used as solvent for the analysed samples and filtered prior to injection. Purification by column chromatography was carried out on silica gel 60 Å (70–230 mesh) and analytical thin layer chromatography (TLC) was conducted on aluminium plates with 0.2 mm of silica gel 60F-254 (Macherey-Nagel). Solvents were obtained from Tedia and Nuclear, and reagents were purchased from Sigma-Aldrich, Acros Organics and TCI. The compounds 156, 2a-e57 and tosylates 3, 4 and 558–60, were prepared according to the previous literature procedure.

2.3.6. Hepatotoxicity prediction To verify a possible hepatotoxic effect of the compounds, we used ProTox-II, a webserver for the prediction of small molecules toxicity67. We utilized the hepatotoxicity predictor and the main results are expressed in Table S2. All of the nine compound synthetized in this work were predicted to have a 5 fold higher LD50 than tacrine. Furthermore, tacrine bellows to a higher predicted toxicity class than the tacrinecarbohydrate compounds. Also, tacrine was predicted to mediate its toxicity by interacting with Amine Oxidase A, fact that was not observed for the new compound class described in this work. 3. Conclusion In this work, we synthesized a series of novel tacrine hybrids with natural-based D-xylose, D-ribose and D-galactose with high AChE and BuChE inhibitory activity in the nanomolar concentration scale. The synthesis of the hybrids is accomplished by the displacement reaction between electrophilic carbohydrate derivatives in the tosylate form with the appropriated 9-alkylamino-1,2,3,4-tetrahydroacridines. All compounds were found to be potent inhibitors of both cholinesterases with IC50 values in the nanomolar concentration scale. Moreover, six of nine compounds were able to inhibit AChE in the same order of magnitude than the bis(7)-tacrine. In general, the D-xylose and D-galactose derivatives exhibited AChE modest selectivity whereas the hybrids derived from D-ribose showed a slight preference for BuChE. Our ensemble docking results indicate that the main contribution of the linker is to enable the correct positioning of the two moieties with CAS, PAS, and hydrophobic pocket to provide optimal interactions with the binding cavity, providing an efficient blockage of the binding site. This finding is reinforced by the fact that there is no linear correlation between the linker size and the observed binding affinities. Also, the sugar moieties are stabilized in the PAS region through cation-π and CH/π interactions with Trp279, hydrophobic interactions from their acetonide protecting groups and hydrogen bonds. Overall, the compounds synthesized in this work, with tacrine connected to a carbohydrate nucleus, represent a novel approach in the literature. The desired hybrids were obtained from simple starting materials in just a few steps and showed high inhibitory potency. Most of them were predicted to obey the Lipinski's rules and do not demonstrated predicted hepatotoxicity, representing new promising prototypes for the treatment of AD.

4.1.1. General procedure for the preparation of tacrine-(carbohydratederivative) hybrids A mixture containing 0.3 mmol of tosylate from D-xylose (3), D-ribose (4) or D-galactose (5) and 0.6 mmol of 9-alkylamino-1,2,3,4-tetrahydroacridines (2a-e) was dissolved in isopropyl alcohol (1.0 mL) and was maintained under stirring at 83 °C during 72 h in a sealed flask. After this time, the reaction was quenched by saturated NaHCO3 (20 mL), washed with brine (20 mL) and water (20 mL) and the product was extracted with dichloromethane. The organic layer was dried by Na2SO4, the solvent was removed under vacuum and the crude product was purified by column chromatography, eluting with hexane: ethyl acetate 1: 1 until remove remaining tosylate, follow by chloroform: methanol 98: 2 to give the desired product. Samples used in biological assays showed high purity as observed by NMR and HPLC analyzes. 4.1.1.1. N1-(5-Deoxy-1,2-O-isopropylidene-α-D-xylofuranose)-5-(N4(1,2,3,4-tetrahydroacridin-9-yl)butane-1,4-diamine) (6a). Intermediate 2a and tosylate 3 were reacted according to general procedure to give the desired product 6a as a yellow oil (54% yield); [α]D20 = +63.6° (c 0.154, CH2Cl2); IR (KBr) νmax/cm−1: 3397, 2920, 2853, 1562, 1499, 1372, 1072, 1009, 752; 1H NMR (CDCl3, 300 MHz) δ 7.97–7.87 (m, 2H), 7.55 (ddd, J = 8.4, 6.9, 1.2 Hz, 1H), 7.39–7.31 (m, 1H), 5.95 (d, J = 3.7 Hz, 1H), 4.49 (d, J = 3.6 Hz, 1H), 4.28 (d, J = 2.8 Hz, 1H), 4.21 (d, J = 2.2 Hz, 1H), 3.98 (br, 2H), 3.56–3.45 (m, 2H), 3.38 (dd, J = 12.9, 3.5 Hz, 1H), 3.11–3.02 (m, 2H), 2.95 (dd, J = 13.0, 1.3 Hz, 1H), 2.75–2.65 (m, 2H), 2.65–2.51 (m, 1H), 1.98–1.86 (m, 4H), 1.77–1.53 (m, 4H), 1.50–1.46 (s, 3H), 1.34–1.24 (m, 4H); 13C NMR (CDCl3, 75 MHz) δ 158.5 (C0), 150.9 (C0), 147.3 (C0), 128.6 (C0), 123.9 (CH), 122.9 (CH), 122.6 (CH), 120.3 (CH), 116.2 (C0), 111.6 (C0), 105.2 (CH), 86.2 (CH), 78.4 (CH), 77.1 (CH), 49.3 (2CH2), 48.7 (CH2), 34.0 (CH2), 29.5 (CH2), 27.2 (CH3), 27.0 (CH3), 26.3 (CH2), 24.9 (CH2), 23.1 (CH2), 22.9 (CH2); HRMS-ESI: calcd for [M−H]+ 442.2700, found 442.2695.

4. Experimental protocols 4.1. Chemistry 1

H NMR and 13C NMR spectra were recorded in CDCl3 solution on a Varian VNMRS 300 MHz spectrometer. The assignment of chemical shifts is based on standard NMR experiments (1H; 13C; 1H, 1H-COSY; 1H, 13 C-HMQC). Chemical shifts (δ) are given in part per million from the peak of tetramethylsilane (δ = 0.00 ppm) as internal standard in 1H NMR or from the solvent peak of CDCl3 (δ = 77.23 ppm) in 13C NMR. NMR multiplicities are given as s (singlet), d (doublet), dd (doublet of doublets), ddd (doublet of doublet of doublets), t (triplet), m (multiplet) or br (broad) and coupling constants (J) are given in Hz. IR spectra were recorded on a Varian 640-IR spectrometer in KBr pellets. Specific rotations were measure on a Jasco P-2000 polarimeter with a 1.0 mL cell at a temperature of 20 °C. High Resolution Mass Spectrometry with Eletrospray Ionization (HRMS-ESI) data on the positive mode was collected on a UHPLC-QTOF/MS Bruker Impact II. Samples were infused from a 100 mL Hamilton syringe at flow rate range from 5 to 10 mL/ min, depending on the sample. The instrument settings were the following: capillary voltage 3000 V, cone voltage 33 V, extraction cone voltage 2.5 V, desolvation gas temperature 100 °C. High performance

4.1.1.2. N1-(5-Deoxy-1,2-O-isopropylidene-α-D-xylofuranose)-5-(N5(1,2,3,4-tetrahydroacridin-9-yl)pentane-1,5-diamine) (6b). Intermediate 2b and tosylate 3 were reacted according to general procedure to give the desired product 6b as a yellow oil (70% yield); [α]D20 = +60.9° (c 0.328, CH2Cl2); IR (KBr) νmax/cm−1: 3381, 2940, 2843, 1572, 1487, 1072, 998, 757; 1H NMR (CDCl3, 300 MHz) δ 7.94 (t, J = 7.8 Hz, 2H), 7.61–7.52 (m, 1H), 7.40–7.30 (m, 1H), 5.95 (d, J = 3.7 Hz, 1H), 4.49 (d, J = 3.6 Hz, 1H), 4.28 (d, J = 2.8 Hz, 1H), 4.20 (d, J = 2.3 Hz, 1H), 4.04 (br, 2H), 3.49 (t, J = 7.2 Hz, 2H), 3.38 (dd, J = 13.0, 3.5 Hz, 1H), 3.07 (s, 2H), 3.00–2.89 (m, 1H), 2.76–2.65 5573

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(m, 2H), 2.61–2.48 (m, 1H), 1.97–1.88 (m, 4H), 1.74–1.61 (m, 2H), 1.58–1.35 (m, 7H), 1.35–1.21 (m, 4H); 13C NMR (CDCl3, 75 MHz) δ 157.9 (C0), 150.9 (C0), 146.7 (C0), 128.4 (CH), 128.0 (CH), 123.6 (CH), 122.9 (CH), 119.9 (C0), 115.6 (C0), 111.3 (C0), 105.0 (CH), 85.9 (CH), 77.9 (CH), 77.0 (CH), 49.2 (CH2), 49.1 (CH2), 48.4 (CH2), 33.6 (CH2), 31.4 (CH2), 29.2 (CH2), 26.8 (CH3), 26.1 (CH3), 24.7 (CH2), 24.4 (CH2), 22.7 (CH2), 22.6 (CH2); HRMS-ESI: calcd for [M−H]+ 456.2857; found 456.2855.

procedure to give the desired product 7a as a yellow oil (35% yield); [α]D20 = −54.7° (c 1.10, CH2Cl2); IR (KBr) νmax/cm−1: 3386, 2926, 2851, 1566, 1089, 867, 760; 1H NMR (CDCl3, 300 MHz) δ 7.98–7.87 (m, 2H), 7.57–7.51 (m, 1H), 7.37–7.30 (m, 1H), 4.96 (s, 1H), 4.62 (d, J = 6.0 Hz, 1H), 4.57 (d, J = 6.0 Hz, 1H), 4.30 (t, J = 6.7 Hz, 1H), 3.93 (s, 1H), 3.47 (t, J = 7.1 Hz, 2H), 3.33 (s, 3H), 3.09–2.99 (m, 3H), 2.78–2.66 (m, 4H), 2.66–2.51 (m, 2H), 1.98–1.83 (m, 5H), 1.73–1.59 (m, 3H), 1.55–1.28 (m, 10H); 13C NMR (CDCl3, 75 MHz) δ 158.4 (C0), 151.1 (C0), 147.4 (C0), 128.7 (CH), 128.6 (CH), 123.8 (CH), 123.0 (CH), 120.3 (C0), 115.9 (C0), 112.5 (C0), 109.9 (CH), 86.4 (CH), 85.6 (CH), 82.9 (CH), 55.3 (CH2), 53.3 (CH2), 49.9 (CH2), 49.6 (CH2), 34.1 (CH2), 31.9 (CH2), 30.2 (CH2), 27.2 (CH2), 27.1 (CH2), 26.7 (CH3), 25.2 (CH3), 24.9 (CH2), 23.2 (CH2), 22.9 (CH2); HRMS-ESI: calcd for [M−H]+ 484.3170, found 484.3170.

4.1.1.3. N1-(5-Deoxy-1,2-O-isopropylidene-α-D-xylofuranose)-5-(N6(1,2,3,4-tetrahydroacridin-9-yl)hexane-1,6-diamine) (6c). Intermediate 2c and tosylate 3 were reacted according to general procedure to give the desired product 6c as a yellow oil (86% yield); [α]D20 = -65.0° (c 0.303, CH2Cl2); IR (KBr) νmax/cm−1: 3369, 3928, 2847, 2345, 1502, 1081, 1001, 760, 659; 1H NMR (CDCl3, 300 MHz) δ 7.98–7.86 (m, 2H), 7.54 (ddd, J = 8.3, 6.9,1.3 Hz, 1H), 7.35 (ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 5.95 (d, J = 3.6 Hz, 1H), 4.49 (d, J = 3.5 Hz, 1H), 4.28 (d, J = 2.6 Hz, 1H), 4.19 (m, 1H), 3.95 (br, 1H), 3.54–3.42 (m, 2H), 3.36 (dd, J = 13.0, 3.3 Hz, 1H), 3.06 (s, 2H), 2.93 (d, J = 11.7 Hz, 1H), 2.68 (s, 2H), 2.64–2.40 (m, 2H), 1.97–1.84 (m, 4H), 1.72–1.58 (m, 2H), 1.53–1.22 (m, 12H); 13C NMR (CDCl3, 75 MHz) δ 158.4 (C0), 150.8 (C0), 147.4 (C0), 128.6 (CH), 128.3 (CH), 123.6 (CH), 122.9 (CH), 120.2 (C0), 115.8 (C0), 111.4 (C0), 105.1 (CH), 85.8 (CH), 78.1 (CH), 77.0 (CH), 49.4 (CH2), 48.5 (CH2), 34.0 (CH2), 31.7 (CH2), 29.5 (CH2), 26.9 (1CH2 and 1CH3), 26.7 (CH3), 26.2 (CH2), 24.8 (CH2), 23.1 (CH2), 22.8 (CH2); HRMS-ESI: calcd for [M−H]+ 470.3013; found 470.3014.

4.1.1.7. N1-(1-Methyl-5-deoxy-2,3-O-isopropylidene-β-D-ribofuranoside)5-(N7-(1,2,3,4-tetrahydroacridin-9-yl)heptane-1,7-diamine) (7b). Intermediate 2d and tosylate 4 were reacted according to general procedure to give the desired product 7b as a yellow oil (29% yield); [α]D20 = -37.9° (c 0.287, CH2Cl2); IR (KBr) νmax/cm−1: 3370, 2921, 2846, 1554, 1493, 1371, 1098, 863, 750; 1H NMR (CDCl3, 300 MHz) δ 7.98–7.86 (m, 2H), 7.59–7.49 (m, 1H), 7.33 (t, J = 7.2 Hz, 1H), 4.95 (s, 1H), 4.62 (d, J = 6.0 Hz, 1H), 4.57 (d, J = 6.0 Hz, 1H), 4.30 (t, J = 6.9 Hz, 1H), 3.93 (br, 2H), 3.47 (t, J = 7.2 Hz, 2H), 3.31 (s, 3H), 3.10–2.97 (m, 2H), 2.73–2.64 (m, 4H), 2.64–2.52 (m, 2H), 1.98–1.84 (m, 4H), 1.76–1.58 (m, 4H), 1.56–1.19 (m, 12H); 13C NMR (CDCl3, 75 MHz) δ 158.6 (C0), 151.0 (C0), 147.6 (C0), 128.9 (CH), 128.4 (CH), 123.8 (CH), 123.0 (CH), 120.4 (C0), 116.0 (C0), 112.5 (C0), 109.9 (CH), 86.4 (CH), 85.6 (CH), 82.9 (CH), 55.3 (CH2), 53.3 (CH2), 50.0 (CH2), 49.7 (CH2), 34.2 (CH2), 31.9 (CH2), 30.2 (CH2), 29.5 (CH2), 27.4 (CH2), 27.1 (CH2), 26.7 (CH3), 25.2 (CH3), 25.0 (CH2), 23.3 (CH2), 23.0 (CH2); HRMS-ESI: calcd for [M−H]+ 498.3326, found 498.3327.

4.1.1.4. N1-(5-Deoxy-1,2-O-isopropylidene-α-D-xylofuranose)-5-(N7(1,2,3,4-tetrahydroacridin-9-yl)heptane-1,7-diamine) (6d). Intermediate 2d and tosylate 3 were reacted according to general procedure to give the desired product 6d as a yellow oil (81% yield); [α]D20 = +87.7° (c 0.106, CH2Cl2); IR (KBr) νmax/cm−1 3423, 2920, 2840, 1621, 1066, 604; 1H NMR (CDCl3, 300 MHz) δ 8.02–7.85 (m, 2H), 7.61–7.48 (m, 1H), 7.41–7.30 (m, 1H), 5.95 (d, J = 3.6 Hz, 1H), 4.49 (d, J = 3.6 Hz, 1H), 4.28 (d, J = 2.7 Hz, 1H), 4.20 (s, 1H), 4.00 (br, 1H), 3.56–3.44 (m, 2H), 3.39 (dd, J = 12.9, 3.3 Hz, 1H), 3.07 (s, 2H), 2.94 (d, J = 13.1 Hz, 1H), 2.79–2.45 (m, 4H), 1.92 (s, 4H), 1.65 (s, 2H), 1.57–1.23 (m, 14H).; 13C NMR (CDCl3, 75 MHz) δ 158.1 (C0), 150.8 (C0), 147.2 (C0), 128.3 (2CH), 123.5 (CH), 122.9 (CH), 120.1 (C0), 115.6 (C0), 111.3 (C0), 105.0 (CH), 86.0 (CH), 77.9 (CH), 77.0 (CH), 49.4 (CH2), 49.3 (CH2), 48.5 (CH2), 33.8 (CH2), 31.6 (CH2), 29.4 (CH2), 29.0 (CH2), 26.9 (CH3), 26.7 (CH3), 26.1 (CH2), 24.7 (CH2), 23.0 (CH2), 22.7 (CH2); HRMS-ESI: calcd for [M−H]+ 484.3170; found 484.3164.

4.1.1.8. N1-(6-Deoxy-1,2:3,4-di-O-isopropylidene-α-D-galactopyranose)5-(N6-(1,2,3,4-tetrahydroacridin-9-yl)hexane-1,6-diamine) (8a). Intermediate 2c and tosylate 5 were reacted according to general procedure to give the desired product 8a as a yellow oil (58% yield); [α]D20 = -26.0° (c 1.09, CH2Cl2); IR (KBr) νmax/cm−1: 3338, 2982, 2928, 2852, 1561, 1383, 1205, 1068, 993, 768, 734, 522; 1H NMR (CDCl3, 300 MHz) δ 7.98–7.85 (m, 2H), 7.57–7.48 (m, 1H), 7.36–7.27 (m, 1H), 5.54 (d, J = 5.1 Hz, 1H), 4.59 (dd, J = 7.9, 2.3 Hz, 1H), 4.31 (dd, J = 5.1, 2.3 Hz, 1H), 4.17 (dd, J = 7.9, 1.8 Hz, 1H), 3.98 (br, 1H), 3.96–3.85 (m, 1H), 3.52–3.37 (m, 2H), 3.05 (s, 2H), 2.95–2.44 (m, 6H), 1.97–1.81 (m, 4H), 1.71–1.56 (m, 2H), 1.55–1.21 (m, 18H); 13C NMR (CDCl3, 75 MHz) δ 158.2 (C0), 150.8 (C0), 147.3 (C0), 128.5 (CH), 128.3 (CH), 123.5 (CH), 122.9 (CH), 120.1 (C0), 115.7 (C0), 109.1 (C0), 108.4 (C0), 96.4 (CH), 72.0 (CH), 70.8 (CH), 70.5 (CH), 66.7 (CH), 49.6 (CH2), 49.4 (CH2), 49.2 (CH2), 33.9 (CH2), 31.7 (CH2), 29.9 (CH2), 27.1 (CH2), 26.8, 26.1, 26.0, 24.9, 24.8, 24.3, 23.0, 22.7 (4CH2 and 4CH3); HRMS-ESI: calcd for [M−H]+ 540.3432; found 540.3435.

4.1.1.5. N1-(5-Deoxy-1,2-O-isopropylidene-α-D-xylofuranose)-5-(N8(1,2,3,4-tetrahydroacridin-9-yl)octane-1,8-diamine) (6e). Intermediate 2e and tosylate 3 were reacted according to general procedure to give the desired product 6e as a yellow oil (29% yield); [α]D20 = -12.5° (c 0.990, CH2Cl2); IR (KBr) νmax/cm−1: 3324, 2914, 2846, 1573, 1561, 1492, 1370, 1068, 1000, 754, 726; 1H NMR (CDCl3, 300 MHz) δ 8.01–7.89 (m, 2H), 7.59–7.51 (m, 1H), 7.40–7.30 (m, 1H), 5.95 (d, J = 3.7 Hz, 1H), 4.48 (d, J = 3.7 Hz, 1H), 4.28 (d, J = 2.9 Hz, 1H), 4.20 (d, J = 2.9 Hz, 1H), 4.04 (br, 1H), 3.53–3.43 (m, 2H), 3.39 (dd, J = 13.0, 3.5 Hz, 1H), 3.11–3.00 (m, 2H), 2.94 (dd, J = 13.0, 1.3 Hz, 1H), 2.78–2.45 (m, 4H), 1.96–1.82 (m, 4H), 1.70–1.55 (m, 2H), 1.52–1.10 (m, 16H); 13C NMR (CDCl3, 75 MHz) δ 158.3 (C0), 151.2 (C0), 147.2 (C0), 128.7 (CH), 128.6 (CH), 123.9 (CH), 123.1 (CH), 120.2 (C0), 115.7 (C0), 111.6 (C0), 105.3 (CH), 86.2 (CH), 78.4 (CH), 77.1 (CH), 49.7 (2CH2), 49.6 (CH2), 48.7 (CH2), 33.9 (CH2), 31.9 (CH2), 29.9 (CH2), 29.7 (CH2), 29.5 (CH2), 29.4 (CH2), 27.2 (CH2), 27.0 (2CH3), 26.3 (CH2), 24.9 (CH2), 23.2 (CH2), 22.9 (CH2); HRMS-ESI: calcd for [M−H]+ 498.3326, found 498.3329.

4.1.1.9. N1-(6-Deoxy-1,2:3,4-di-O-isopropylidene-α-D-galactopyranose)5-(N7-(1,2,3,4-tetrahydroacridin-9-yl)heptane-1,7-diamine) (8b). Intermediate 2d and tosylate 5 were reacted according to general procedure to give the desired product 8b as a yellow oil (34% yield); [α]D20 = -19.8° (c 0.136, CH2Cl2); IR (KBr) νmax/cm−1: 3355, 2928, 2848, 1555, 1499, 1361, 1201, 1064, 991, 758; 1H NMR (CDCl3, 300 MHz) δ 7.96 (t, J = 6.0 Hz, 2H), 7.57 (t, J = 7.7 Hz, 1H), 7.40–7.30 (m, 1H), 5.54 (d, J = 5.0 Hz, 1H), 4.60 (d, J = 7.7 Hz, 1H), 4.31 (d, J = 5.0 Hz, 1H), 4.19 (d, J = 8.0 Hz, 1H), 3.92 (d, J = 4.6 Hz, 1H), 3.51 (dd, J = 12.9, 6.3 Hz, 2H), 3.09 (s, 2H), 2.89 (dd, J = 12.9, 8.7 Hz, 1H), 2.81–2.51 (m, 5H), 2.08–1.83 (m, 5H), 1.75–1.57 (m, 2H), 1.59–1.22 (m, 20H); 13C NMR (CDCl3, 75 MHz) δ 158.0 (C0), 151.4 (C0), 146.9 (C0), 128.8 (CH), 128.2 (CH), 123.9 (CH), 123.1 (CH), 120.0 (C0), 115.5 (C0), 109.4 (C0), 108.7 (C0), 96.6 (CH), 72.2 (CH), 71.0 (CH), 70.7 (CH), 66.9 (CH), 49.9 (2CH2), 49.6 (CH2), 33.7 (CH2), 31.9 (CH2),

4.1.1.6. N1-(1-Methyl-5-deoxy-2,3-O-isopropylidene-β-D-ribofuranoside)5-(N6-(1,2,3,4-tetrahydroacridin-9-yl)hexane-1,6-diamine) (7a). Intermediate 2c and tosylate 4 were reacted according to general 5574

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30.0 (CH2), 29.4 (CH2), 27.4, 27.0, 26.3, 26.2, 25.1, 24.8, 24.5, 23.1, 22.8 (5CH2 and 4CH3); HRMS-ESI: calcd for [M−H]+ 554.3588, found 554.3587.

modes in our redocking studies (results not shown). For the BuChE, we selected the conformation complexed with the largest inhibitor 6QS (PDB ID 5K5E).73 Three-dimensional structures of the compounds were drawn using ChemBio3D Ultra 14 Suite (PerkinElmer, Waltham, MA, USA, 2014) and isomers, protonation states, and tautomer’s of the ligands were manually set using Maestro (Maestro, Schrödinger, LLC, New York, NY, 2014). The AChE and BuChE structures were taken from our previous study (Ceschi et al.)39 which were prepared with Protein Preparation Wizard tool (Schrödinger Suite 2014-1 Protein Preparation Wizard; Impact version 6.2, Schrödinger, LLC, New York, NY, USA, 2014). The protonation states of the amino acid residues were predicted using PROPKA with pH = 7 while respecting the protonation state of the active site residues already described in the literature: Glu202 and Glu327 negatively charged and neutral His440:ND1. Tacrine moiety of all inhibitors was predicted to be protonated in the previous study according to the Epik prediction.74 This protonation state is essential since tacrine moiety donates a hydrogen bond to the carbonyl oxygen of the catalytic His440 main chain.63 Optimization of the hydrogen bond network between protein and inhibitor was performed to adjust the orientation of the hydrogen atoms, followed by energy minimization with heavy atoms fixed. In this work, the docking experiments were performed with the molecular docking program GOLD75 considering 200% of efficiency – indicated for highly flexible ligands, setting early termination disabled and using ChemPLP to score the ligand poses and to rank the different compounds. We also redocked the co-crystallized ligands into their respective AChE and BuChE conformation to validate the docking protocol adopted herein.

4.2. AChE and BuChE inhibition assay Swiss Webster mice provided by the Central Animal Laboratory of the Oswaldo Cruz Foundation/RJ (Fiocruz) were used in this study. Animals were held in light/dark cycle of 12/12 h and fed ad-libitum. All experiments were performed in accordance with the Ethics Committee for Animal Use of Fiocruz (CEUA, license number L044/2015 with the additive license LA-010/2016). After euthanasia, brain and blood were collected and used for the preparation of biological fractions used in enzymatic assays. A cerebral homogenate was prepared in distilled water then centrifuged at 20,000 G for 60 min at 4° C. The supernatant was discarded and the pellet resuspended in 2 mL of 1% Triton X-100. After, this fraction was again centrifuged at 14000 rpm (20800 G) for 90 min. The pellet was discarded and the supernatants were used for enzymatic assays. After allow the whole blood to clot at room temperature, the sample was centrifuged at 2,000×g for 10 min in a refrigerated centrifuge (4 °C). The resulting supernatant, named serum fraction, was maintained in polypropylene tubes until the enzymatic assays. The activities of the AChE enzyme in the brain and BuChE in the serum of mice were determined using a modified Ellman method.68 Both brain extract and blood serum were added to their respective reaction medium containing sodium phosphate buffer 0.1 M pH 7.5 in a 96-well plate, as well as the synthetic compounds to be analyzed. Control groups did not have the addition of compounds. DTNB (5,5′dithio-bis-(2-nitrobenzoic acid)) in the concentration of 0.32 mM was added and the reaction medium preincubated at 25 °C for 10 min. The enzymatic assays were initiated by addition of the substrates, acetylthiocholine iodide or butyrylthiocholine iodide at a final concentration of 1.5 mM, and a further incubation was carried out for 10 min. After incubation, optical density was measured at wavelength 412 nm and blank wells (without enzymatic activity) had their values discounted. Compounds were first solubilized in 100% Dimethyl sulfoxide (DMSO). Afterwards, they were diluted again in Milli-Q® water and tested against inhibition on the enzymatic activities at the final concentrations ranging from 0.01 to 10,000 nM. Data analysis was performed using GraphPad Prism version 6.0 (GraphPad Software, Inc., San Diego, CA).

Acknowledgements We would like to thank the following Brazilian agencies for financial support and fellowships: CNPq, FAPERGS, CAPES, PROPESQ – UFRGS, LNCC, FIOCRUZ. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmc.2018.10.003. References 1. Jameel E, Meena P, Maqbool M, et al. Rational design, synthesis and biological screening of triazine-triazolopyrimidine hybrids as multitarget anti-Alzheimer agents. Eur J Med Chem. 2017;136:36–51. 2. Martin P, Adelina C, Martin K, Maëlenn G, Maria K. World Alzheimer Report. 2016:1–140. 3. Lv ZY, Tan CC, Yu JT, Tan L. Spreading of pathology in Alzheimer's disease. Neurotox Res. 2017;32:707–722. 4. Bartus RT, Dean III RL, Beer B, Lippa AS. The cholinergic hypothesis of geriatric memory dysfunction. Science. 1982;217:408–417. 5. Bartus RT. On neurodegenerative diseases, models, and treatment strategies: lessons learned and lessons forgotten a generation following the cholinergic hypothesis. Exp Neurol. 2000;163:495–529. 6. Shah AA, Dar TA, Dar PA, Ganie SA, Kamal MA. A current perspective on the inhibition of cholinesterase by natural and synthetic inhibitors. Curr Drug Metab. 2017;18:96–111. 7. Anand A, Patience AA, Sharma N, Khurana N. The present and future of pharmacotherapy of Alzheimer's disease: a comprehensive review. Eur J Pharmacol. 2017;815:364–375. 8. McEneny-King A, Osman W, Edginton AN, Rao PPN. Cytochrome P450 binding studies of novel tacrine derivatives: Predicting the risk of hepatotoxicity. Bioorg Med Chem Lett. 2017;27:2443–2449. 9. Gorfe AA, Chang CA, Ivanov I, McCammon JA. Dynamics of the acetylcholinesterase tetramer. Biophys J. 2008;94:1144–1154. 10. Dvir H, Silman I, Harel M, Rosenberry TL, Sussman JL. Acetylcholinesterase: from 3D structure to function. Chem Biol Interact. 2010;187:10–22.

4.3. Molecular modelling We selected three representative conformations of AChE following an ensemble docking strategy adopted on our previous study39 to consider the significant conformational changes observed on the peripheral anionic site (PAS).{Haviv, 2005, Crystal packing mediates enantioselective ligand recognition at the peripheral site of acetylcholinesterase}69 This methodology consists of docking compounds into each representative conformation of the receptor, aiming to include implicitly the protein flexibility.70,71 The selected structures in this work were 1ZGC (Torpedo californica),63 2CKM (Torpedo californica),41 and 1Q84 (Mus musculus).72 Structures from Homo sapiens were not selected since they are complexed with smaller inhibitors when compared with the compounds tested herein. All inhibitors from the three representative conformations of AChE (i) interact with the CAS, (ii) interact with the PAS, and (iii) are similar to the tacrine inhibitor. No water molecules were considered in this work since they demonstrated not to be important to predict experimental binding

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