Synthesis and evaluation of novel 1,2,3-triazole-based acetylcholinesterase inhibitors with neuroprotective activity

Synthesis and evaluation of novel 1,2,3-triazole-based acetylcholinesterase inhibitors with neuroprotective activity

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journ...

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Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl

Synthesis and evaluation of novel 1,2,3-triazole-based acetylcholinesterase inhibitors with neuroprotective activity Jia-Cheng Li a,y, Juan Zhang b,y, Mosar Corrêa Rodrigues b, De-Jun Ding c, João Paulo Figueiró Longo b, Ricardo Bentes Azevedo b, Luis Alexandre Muehlmann b,d, Cheng-Shi Jiang a,⇑ a

Department of Pharmaceutical Engineering, School of Biological Science and Technology, University of Jinan, Jinan 250022, China Institute of Biological Sciences, University of Brasília, Brasilia 70910900, Brazil College of Pharmacy, Weifang Medical University, Weifang 261042, China d Faculty of Ceilandia, University of Brasilia, Brasilia 72220-900, Brazil b c

a r t i c l e

i n f o

Article history: Received 9 June 2016 Revised 4 July 2016 Accepted 6 July 2016 Available online xxxx Keywords: 1,2,3-Triazole Acetylcholinesterase Amyloid-b-peptide Neuroprotective Alzheimer’s disease

a b s t r a c t A series of new 1,2,3-triazole derivatives were synthesized and evaluated for anticholinesterase and neuroprotective activities. Some synthetic derivatives, especially compound 32, exhibited improved acetylcholinesterase (AChE) inhibitory activity by comparison with the hit 1, high selectivity toward AChE over butyrylcholinesterase (BuChE), and suitable in vitro neuroprotective effect against amyloid-b25–35 (Ab25–35)-induced neurotoxicity in SH-SY5Y cells. Furthermore, these molecules have desired physicochemical properties in the range of CNS drugs and showed no cytotoxicity against two normal cells, including human keratinocytes HaCaT and murine fibroblasts NIH-3T3. The preliminary bioassay results and docking study indicated that compound 32 might be a promising lead compound with dual action for the treatment of Alzheimer’s disease. Ó 2016 Elsevier Ltd. All rights reserved.

Alzheimer’s disease (AD), the most common form of dementia, is a chronic neurodegenerative disorder, which is clinically characterized by impairment in memory, complex cognition, language, emotion, and behavior.1 In 2015, approximately 46.8 million people worldwide were believed to suffer from AD, and this number was expected to triple by 2050 with the aging of the population.2 Several factors, such as levels of acetylcholine (ACh)3 and amyloid-b-peptide (Ab) deposits,4 play a significant role in the occurrence of AD. Although the pathogenesis of AD is not fully understood, currently the most efficacious treatment approach for AD is considered to increase cholinergic neurotransmission in the brain by lowering Ach hydrolysis.5 ACh can be degraded by acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Compared with BuChE, AChE draws more attention from pharmaceutical chemists since it accounts for nearly 80% ACh hydrolysis in the brain.5b In this context, four AChE inhibitors including tacrine, donepezil, rivastigmine and galantamine had been clinically approved by the FDA.5c However, tacrine is rarely used due to its potential liver toxicity, and the others could only modestly improve memory and cognitive function in AD ⇑ Corresponding author. y

E-mail address: [email protected] (C.-S. Jiang). These authors contributed equally to this work.

treatment but do not appear to stop or slow down the progress of AD. Because the pathogenesis of AD is very complicated and related to multiple systems’ dysfunctions, there is still no ideal drug that can completely prevent and treat AD. Therefore, there is an urgent need for developing new effective anti-AD drugs. Many studies indicate another hypothesis for AD pathogenesis, the so-called amyloid hypothesis.6 Accumulated amyloid plaques made up of Ab is a major hallmark of AD.6a Ab is derived from amyloid precursor protein (APP) via sequential proteolytic cleavage by b- and c-secretases.6b It is hypothesized that the accumulated Ab is one of the primary causes of AD because its accumulation in the brain could trigger critical intracellular signaling pathways, resulting in neural cell stress and apoptosis.6c Thus, drugs which specifically protect neurons from injury and apoptosis induced by Ab could be useful for both the prevention and treatment of AD. Recently, a new synthetic 1,2,3-triazole derivative 1 with a morpholinoethanamine side chain (shown in Fig. 1) was screened out from our in-house compounds library. This compound showed AChE inhibitory activity (IC50 = 27.35 lM) and neuroprotective effect against Ab25–35-induced injuries in SH-SY5Y neuronal cells (85.2% of cell viability at 10 lM), indicating it was a potential dual-action anti-AD lead compound. As well as known, the 1,2,3triazole scaffold is commonly found in synthetic products and

http://dx.doi.org/10.1016/j.bmcl.2016.07.017 0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

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J.-C. Li et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx

Figure 1. The structure of 1 and its structural derivatization.

represents an important pharmacophore in drug discovery.7 Until now, lots of AChE inhibitors and neuroprotectants with 1,2,3triazole unit have been reported.8,9 Recently, Yadav et al. reported that a diaryltriazine possessing a morpholinoethanamine side chain showed potential multitarget-directed therapeutics, such as AChE inhibitory activity and neuroprotective effect against Ab1–42-induced apoptosis neurotoxicity, for the treatment of AD.10 Basic side chain containing an amino group is an integral part of the structure of several reported AChE inhibitors such as donepezil, tacrine, and galantamine. The nitrogen atom was considered to play an important role in enzyme–inhibitor interaction.11 In addition, the substituents attached to the 1,2,3-triazole unit usually affect the bioactivity.12 Based on above observations and our experiences in the synthesis of neuroprotective agents,13 a further improvement on the bioactivity profile of 1 through preliminary structural derivatization was carried out, by introducing different commercially available benzyl or naphthyl group into the 1,2,3triazole unit. Herein, we present the synthesis and biological evaluation of a series of new 1,2,3-triazoles as selective AChE inhibitors with neuroprotective property, and molecular docking as well. The synthetic strategy for target 1,2,3-triazoles 6–32 is depicted in Scheme 1. Briefly, it was started from the Sonogashira coupling reaction14 of 5-bromo-2-methylbenzoic acid 2 and trimethylsilylacetylene to give 3, which was subsequently deprotected and hydrolyzed in one step under basic condition15 to yield 4. Then compound 4 was converted into amide 5 by reacting with 2-morpholinoethanamine in the presence of EDCI and HOBT.16 Finally, the addition 5 and CuI into the azide intermediates, which were freshly prepared from different bromides (ArCH2Br) and NaN3 in the mixture of H2O/DMSO, yielded the corresponding target compounds 6–32 in the CuI-catalyzed Azide–Alkyne Cycloaddition.17 The inhibitory activities of 5–32 against AChE (from electric eel) and BuChE (from equine serum) were evaluated according to the Ellman’s method18 with galanthamine as the reference compound. The results were summarized in Table 1. All target compounds 6–32 showed moderately potent inhibition of AChE with IC50 values ranging from 64.17 to 7.23 lM, but stronger than that of their precursor compound 5, clearly indicating that the introduction of substituted-1,2,3-triazole moiety could significantly increase their inhibitory activity.

Among the derivatives 1, 6, 8–13, the compounds 8 with para-F and 11 with para-Br were the most potent inhibitor for AChE, revealing that the para-substituted group seems to be beneficial for the AChE inhibitory activity. However, the compounds 16–18 showed decreased activity by comparison with the above compounds bearing electron-withdrawing substituent, indicating that the electron-withdrawing substituent in benzene plays an important role in their bioactivities. This deduction was also supported by the observation that the activities of 11 and 13 were decreased when additional electron-donating groups (e.g., methoxyl or a methyl group) were introduced in their benzene ring to form 19 and 20, respectively. Also, the selectivity of 6–20 toward AChE was not so satisfactory. Based on the observation that fluorinated functionalities are usually the key structural units, found in about 20% pharmaceuticals on the market,19 the para-fluoro was conversed and a second small electron-withdrawing atom (such as F, Cl, and Br) was introduced to form 21–25. To our light, the compounds 23 and 24 were found to show increased inhibitory activity against AChE, and higher selectivity toward AChE over BuChE by 6.2- and 6.6-fold compared with the positive control galanthamine, respectively. The para-fluor was further replaced by a more strong electron-withdrawing groups to yield derivatives 26–30. Among them, compound 28 with para-CN also exhibited high inhibitory activity and selectivity toward AChE. Furthermore, the effect of aromatic property of the functional group on bioactivity was investigated by the introduction of naphthyl group, consequently resulting in the production of 31 and 32. Surprisingly, compound 32 with 2-position substitution in naphthalene ring showed the strongest inhibition of AChE (IC50 = 7.23 lM) and highest selectivity toward AChE over BuChE by 12.6-fold among all the tested compounds. However, compound 31 showed significantly decreased activity, probably due to the steric hindrance resulting from 1-position substitution in naphthalene ring, compared with 32. The molecular docking was conducted to investigate the potential binding mode of compound 32 with the catalytic domain of AChE (PDB: 4EY7),20 which was performed by using Autodock 4.221 with structure images created by Pymol 1.5. As shown in Figure 2, compound 32 was located in the active-site gorge of AChE, with 1,2,3-triazole pharmacophore oriented in the peripheral anionic site (PAS) and morpholine group oriented in catalytic active sites (CAS). This compound could form two kinds of primary

Scheme 1. Synthesis of 6–32. Reagents and conditions: (a) trimethylsilylacetylene, Pd(PPh3)4, CuI, DMF, Et3N, 50 °C, 6 h, 73%; (b) KOH, MeOH, 0 °C to reflux 4 h, 98%; (c) 2morpholinoethanamine, EDCI, HOBT, DIEPA, DCM, 12 h, 85%; (d) NaN3,CuI, sodium ascorbate, substituted benzyl bromides for 6–30, 1-(bromomethyl)naphthalene for 31 and 2-(bromomethyl)naphthalene for 32, DMSO, H2O, rt, overnight.

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J.-C. Li et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx Table 1 In vitro inhibition and selectivity of AChE and BuChE

a b

Compound

X,Y

1 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

4-CF3

IC50 (lM)

2-CF3 4-CF3O 4-F 3-F 2-F 4-Br 3-Br 2-Br 3-Cl 2-Cl H 4-CH3 3,5-Me 2-Br, 5-MeO 4-F, 2-Me 4-F, 3-NO2 2,4-F 3,4-F 2-Cl, 4-F 2-Br, 4-F

26

4,

27 28 29 30 31 32 Galanthamine

4-CO2Me 4-CN 4-SO2NH2 4-NO2 — — —

CO2Me

Selectivity for AChEa

AChE

BuChE

27.35 >100 34.15 ± 0.03 30.17 ± 0.21 21.22 ± 0.14 30.12 ± 0.14 39.34 ± 0.05 20.47 ± 0.14 28.43 ± 0.52 35.22 ± 0.02 32.61 ± 0.05 31.33 ± 0.12 45.23 ± 0.04 60.12 ± 0.05 64.17 ± 0.21 40.78 ± 0.04 37.56 ± 0.01 30.12 ± 0.01 24.43 ± 0.34 15.43 ± 0.02 10.43 ± 0.13 31.43 ± 0.01

NTb NT 45.15 ± 0.11 60.23 ± 0.06 43.66 ± 0.08 60.78 ± 0.01 47.23 ± 0.19 45.56 ± 0.09 40.34 ± 0.14 78.56 ± 0.17 36.25 ± 0.89 34.45 ± 0.57 68.12 ± 0.34 83.12 ± 0.08 56.23 ± 0.15 50.76 ± 0.18 60.96 ± 0.06 34.78 ± 0.23 89.56 ± 0.17 95.45 ± 0.03 68.95 ± 0.04 70.76 ± 0.18

— — 1.3 2.0 2.1 1.2 2.0 2.2 2.0 2.2 1.1 1.1 1.5 1.4 0.9 1.2 1.6 1.2 3.7 6.2 6.6 2.3

44.73 ± 0.04

90.86 ± 0.77

2.0

38.37 ± 0.04 12.15 ± 0.14 30.33 ± 0.05 20.45 ± 0.17 46.15 ± 0.01 7.23 ± 0.16 3.56 ± 0.01

67.77 ± 0.15 92.83 ± 0.11 45.52 ± 0.05 60.46 ± 0.01 72.85 ± 0.13 90.76 ± 0.21 16.32 ± 0.23

1.8 7.6 1.5 2.9 1.58 12.6 4.6

IC50 (BuChE)/IC50 (AChE). NT: not tested.

Figure 2. Docking mode of 32 (carbon in green) in the catalytic site of AChE with only the key residues shown. Hydrogen bonds and p–p stacking interactions are displayed as black and brown dashed lines, respectively.

interactions with the key amino acid residues in the active-site of the enzyme, including hydrogen-bond interactions between 1,2,3triazole and Phe295 in PAS, and p–p stacking interactions between 1,2,3-triazole/phenyl and the aromatic residue Tyr341 in the middle of the gorge. Also, this molecule interacts with the key residues Trp286, Phe295, Phe297 in PSA, and Trp86, Tyr337 in CAS through van der Waals interactions, which also contribute to the affinity of 32 to the active site of AChE. In addition, a common feature of numerous AChE inhibitors’ structures is location of a cationic charge center, which can form cation–p interactions with aromatic side chains incorporated into the gorge wall.22 Since the nitrogen

atom in the morpholine moiety should be protonated at the physiological pH,10 in this case the protonated amines might undergo cation–p interactions with the aromatic side chains in CAS, such as Trp86 and Tyr337. Further, the compounds 23, 24, 28 and 32 were selected to be evaluated their neuroprotective activity against Ab25–35-induced neurotoxicity in SH-SY5Y cells.13 The results are shown in Table 2. Except compound 24, the others showed satisfactory neuroprotective activity with the range from 89.4.3% to 96.5% of cell viability at 10 lM, and compounds 23 and 32 could further increase the cell viability (70.3% and 77.9%, respectively) at 1 lM. Among all tested

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Table 2 The neuroprotective effects and structural properties of compounds 1, 23, 24, 28 and 32 Compound

1 23 24 28 32 EGCG Ab25–35 a b c d

Cell viability(%)a/recovery of cell viability (%)b 10 lM

1 lM

85.2/22.9 89.4/27.1 NA 78.9/16.6 96.5/34.2 95.7/33.4

NAc 70.3/8.0 NTd NA 77.9/15.6 NT

MW (Da)

C log P

TPSA (Å2)

441.7 430.50 441.7 457.93 455.55 —

3.47 2.32 2.82 3.34 3.75 —

72.29 96.08 72.29 72.29 72.29 —

62.3 (100 lM)

Cell viability (%): the cell viability in control was taken as 100%. Recovery of cell viability (%): the difference value between the cell viability of compound treated cells and that of Ab25–35 treated cells. NA: not active. NT: not tested.

Figure 3. Viability of human keratinocytes (HaCaT) and murine fibroblasts (NIH-3T3) exposed to different concentrations of 23, 28 and 32 for 24 h. Cell viability was evaluated by the MTT method25 after the end of exposure time to the tested compounds.

derivatives, compound 32 showed the highest activity, similar with that of positive control epigallocatechingallate (EGCG). These results of its selective AChE inhibition and neuroprotective effect indicated that compound 32 could be selected as a potential anti-AD hit. When designing central nervous system (CNS) drugs, it is important to consider the fundamental physiochemical properties of drugs related to their ability to penetrate the blood-brain barrier (BBB).23 Typically, drugs which penetrate the BBB via passive diffusion have molecular weights less than 500 Da, and calculated octanol–water partition coefficient (C log P) values of 3.4.23a Also, molecular topological polar surface area (TPSA) value is another key descriptor that was shown to correlate well with passive molecular transport through membranes and therefore, allows prediction of transport properties of drugs.23b The mean value of TPSA for the marketed CNS drugs is 40.5 Å2 with a range of 4.63– 108 Å2.23c Moreover, poor absorption, and low permeability is predicted for drug with C log P P 5 and TPSA P 140 Å2.23d In our experiments, the C log P and TPSA values of 23, 24, 28 and 32 were calculated by Molinspiration web services.24 From the data presented in Table 2, it is significant that the C log P and TPSA values of all these compounds fall into the range of marketed CNS drugs. In addition, their molecular weights (MW) range from 430.50 to 457.93 Da, which is well below the cutoff for BBBpenetrable molecules. The calculated results indicated that these synthetic molecules should have suitable BBB penetrability. In addition, the toxicity profile of compounds is usually considered during the process of drug research and development. Herein, the cytotoxic activity of compounds 23, 28 and 32 was evaluated in vitro on normal cells, including human keratinocytes HaCaT and murine fibroblasts NIH-3T3. The results showed that at all

the concentrations tested, these compounds did not exhibit significant cytotoxic effects on both HaCaT and NIH-3T3 cell lines, even at 400 lM when compared with control (Fig. 3). In conclusion, a series of new 1,2,3-triazole derivatives were synthesized and evaluated for their anti-AD potential, including selective AChE inhibitory and neuroprotective activity. Meanwhile, their cytotoxicities on normal cells (HaCaT and NIH-3T3) were also tested. C log P and TPSA were calculated to predict their ability to penetrate the BBB. Among all the tested derivatives, compound 32 exhibited the strongest inhibition of AChE with high selectivity, and good in vitro neuroprotective effect against Ab25–35-induced neurotoxicity in SH-SY5Y cells. In addition, this compound showed no cytotoxicity against HaCaT and NIH-3T3 cell lines. Together with the physicochemical properties and docking study, compound 32 can be considered as a potential lead drug with dual action for the treatment of AD. Further investigation on in vitro/in vivo assay of this 1,2,3-triazole series are in progress and will be reported in due course. Acknowledgements This research work was financially supported by the Natural Science Foundation of China (No. 81302692) and by the Brazilian government agencies Fundação de Apoio à Pesquisa do Distrito Federal and CNPq. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.07. 017.

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