Multifunctional Mono-Triazole Derivatives Inhibit AÎ²42 Aggregation

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Multifunctional mono-triazole derivatives inhibit A#42 aggregation, Cu2+ mediated A#42 aggregation and protect against A#42 induced cytotoxicity Amandeep Kaur, Simranjeet Singh Narang, Anupamjeet Kaur, Sukhmani Mann, Nitesh Priyadarshi, Bhupesh Goyal, Nitin Kumar Singhal, and Deepti Goyal Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.9b00168 • Publication Date (Web): 12 Aug 2019 Downloaded from pubs.acs.org on August 13, 2019

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Chemical Research in Toxicology

Multifunctional mono−triazole derivatives inhibit Aβ42 aggregation, Cu2+ mediated Aβ42 aggregation and protect against Aβ42 induced cytotoxicity Amandeep Kaur,a Simranjeet Singh Narang,a Anupamjeet Kaur,a Sukhmani Mann,a Nitesh Priyadarshi,c Bhupesh Goyal,*,b Nitin Kumar Singhal,*,c and Deepti Goyal*,a aDepartment

of Chemistry, Faculty of Basic and Applied Sciences, Sri Guru Granth Sahib World University, Fatehgarh Sahib–140406, Punjab, India bSchool

of Chemistry and Biochemistry, Thapar Institute of Engineering & Technology, Patiala–147004, Punjab, India cNational

Agri−Food Biotechnology Institute, S.A.S. Nagar, Punjab, India

*Corresponding authors E-mail address: [email protected], [email protected], [email protected]

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TABLE OF CONTENTS (TOC)


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ABSTRACT Amyloid beta (Aβ) peptide aggregation is considered as one of the key hallmarks of Alzheimer’s disease (AD). Moreover, Aβ peptide aggregation increases considerably in the presence of metal ions and triggers the generation of reactive oxygen species (ROS), which ultimately leads to oxidative stress and neuronal damage. Based on the ‘multi–target–directed ligands’ (MTDLs) strategy, we designed, synthesized and evaluated a novel series of triazole based compounds for AD treatment via experimental and computational methods. Among the designed MTDLs [4(ax)], the triazole derivative 4v exhibited most potent inhibition of self induced Aβ42 aggregation (78.02%) with an IC50 value of 4.578 ± 0.109 μM and also disassembled the preformed Aβ42 aggregates significantly. In addition, compound 4v showed excellent metal chelating ability and maintained copper in the redox–dormant state to prevent the generation of ROS in copper−ascorbate redox cycling. Further, 4v significantly inhibited Cu2+–induced Aβ42 aggregation and disassembled the Cu2+–induced Aβ42 protofibrils as compared to the reference compound clioquinol (CQ). Importantly, 4v did not show cytotoxicity and was able to inhibit the toxicity induced by Aβ42 aggregates in SH–SY5Y cells. Molecular docking results confirmed the strong binding of 4v with Aβ42 monomer and Aβ42 protofibril structure. The experimental and molecular docking results highlighted that 4v is a promising multifunctional lead compound for AD. INTRODUCTION Various amyloidal diseases, such as Parkinson’s disease, Alzheimer’s disease (AD), type 2 diabetes, Creutzfeldt–Jakob disease, and Huntington’s disease etc. having different etiology are caused due to the self assembly of specific proteins or peptides to form aggregates.1 Among the various neurodegenerative diseases, AD2 accounts for about 60–70% of cases and thus is the most common form of dementia. AD is a progressive neurodegenerative disease, and results into structural and functional demolition of neurons. The patients lose their ability to perform everyday activities and finally become bed ridden at the later stages of the disease. The World Health Organization (WHO) recently reported that about 50 million people are affected with dementia worldwide and this number will likely to reach 152 million by 2050.3 AD is the fifth leading cause of death among the people aged above 65 in the developed countries.4 3 ACS Paragon Plus Environment


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AD has multifactorial and heterogeneous pathology and several hypotheses like abnormally phosphorylated and β–folded tau protein,5 amyloid plaques,6 cholinergic impairment,7 metal dyshomeostasis8 and oxidative stress9 have been proposed to understand the pathomechanism of the disease. The currently administered drugs (e.g. rivastigmine,10 donepezil,11 galantamine12 and memantine13) for AD, treat symptoms rather than dealing with the core pathology of the disease.14 In addition, almost 99.6% of AD drugs have failed in different levels of clinical trials from 2002 to 2012.15 The recent failure of various molecular entities [NAP (NAPVSIPQ),16 LY2886721,17 tarenflurbil,18 semagacestat,19 avagacestat,20 PBT1,21 and bapineuzumab22 etc.] at different level of clinical trials, clearly demonstrates the dire need to identify new molecules for the treatment of AD. Among the various factors responsible for AD progression, deposition of amyloid plaques as well as soluble oligomers is the cardinal feature of AD.6 The amyloid plaques predominantly consists of Aβ peptide (39 to 43 amino acid residues). Aβ is generated via the amyloidogenic pathway by the proteolytic cleavage of the amyloid precursor protein (APP) by β– and γ– secretases.23 The deposition of amyloid species gradually results in neuronal loss and finally neuron death. Hence, various molecules showing ability to inhibit amyloid aggregation has been reported in literature as possible therapeutics against AD.24 The Aβ aggregation increases drastically on complexation of metal ions with Aβ peptide25 and triggers the generation of reactive oxygen species (ROS).26 This eventually leads to oxidative stress and neuron death.27 The dyshomeostasis of brain metal ions [especially copper and zinc] greatly influence the pathogenesis of AD. It has been found that amyloid plaques were enriched with Cu2+ and Zn2+. These metal ions coordinate with histidine residues in senile plaque core.28 As a result, metal ions start accumulating in the extracellular plaques and intracellular copper stores are scarce in AD patients.29 In this regard, various metal chelators have been proposed in the literature, which can modulate the concentration of these biometals.30 However, the complex, multifactorial nature of AD encourages the design of multi−target−directed ligands (MTDLs)31 as the future drug candidates for the treatment of AD. To this purpose, we have designed a library of mono−triazole based compounds as MTDLs for the treatment of AD. The nitrogen heterocycles have been explored against a plethora of biological targets32 and may be a possible starting point for the design of new molecular entities 4 ACS Paragon Plus Environment

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for AD. These ring systems have been extensively explored in the design of scanning probes and drug candidates against various neurodegenerative diseases.33 Jones et al. have evaluated a series of dual functional triazole–pyridine ligands as inhibitor of Aβ aggregation and metal−triggered Aβ aggregation.34 In 2015, Vajragupta and co–workers have reported series of substituted anti– 1,2,3–triazoles (IND, PPRD, and QND) as α7 nicotinic acetylcholine receptor agonists.35 Storr and co-workers have synthesized three bidentate quinoline−triazole derivatives and studied their ability to inhibit amyloid aggregation.36 Recently, Jones et. al. have synthesized three 1,2,3– triazole derivatives (POH, PMorph, PTMorph) and demonstrated that triazole derivatives inhibited Aβ aggregation, metal induced Aβ aggregation, and displayed metal chelation and antioxidant

activity.37

Dyrager

et.

al.

have

synthesized

benzothiazole−triazole

and

benzothiadiazole−triazoles, and evaluated their binding specificity to amyloid aggregates in the brain sections from transgenic mice model of AD.38 In another report, Das and Smid have carried out virtual screening of ZINC chemical database and in vitro studies, and identified dibenzyl imidazolidine and triazole acetamide derivatives as potent Aβ aggregation inhibitors.39 In the present study, we have designed, synthesized and evaluated a library of mono−triazole based compounds 4(a–x) as multifunctional ligands, which will target four major AD hallmarks: Aβ aggregation, metal induced Aβ aggregation, metal dyshomeostasis and oxidative stress. RESULTS AND DISCUSSION Rational design of multifunctional ligands We undertook a rational pharmacophore directed design to develop a drug candidate to target multiple pathological factors of AD.40 The proposed pharmacophore model consists of two parts: hydrophobic part and the metal chelator part (Figure 1). The phenyl groups comprises the hydrophobic part, was introduced to target the hydrophobic pocket of Aβ aggregates. The designed MTDLs will form hydrophobic contacts with Aβ peptide and disrupt the peptide– peptide interaction and produce an anti–aggregation effect. On the other hand, triazole moiety represents the metal chelating part, which chelate metal ions via the donation of lone pair of electron on one of the π–bonded nitrogen atoms present in the triazole ring to the metal ion.41 The metal chelating ability of designed molecules enables them to modulate the metal mediated Aβ42 aggregation. The metal chelating property of the designed triazole based compounds helps to reduce the oxidative stress induced by the generation of ROS, by disrupting the copper– 5 ACS Paragon Plus Environment


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ascorbate redox cycle. The designed triazole based compounds are synthesized by using the highly regioselective Cu(I) catalysed Huisgen 1,3–dipolar cycloaddition reaction.42 Further, a library of triazole based compounds having R group with variable electronic properties are easily assembled. The designed library is evaluated for the amyloid aggregation inhibition, which highlights the effect of R–group variation on the ligand–Aβ42 interactions.

Figure 1: Design strategy for mono−triazole based compounds as MTDLs in AD. The designed MTDLs 4(a−x) were synthesized according to the synthetic sequence depicted in Scheme 1. The library of mono−triazole based compounds 4(a−x) were synthesized using copper catalysed 1,3−dipolar cycloaddition reaction between alkyne based building block 2 and various azides 3(a−x). In order to realize the designed strategy, the alkyne building block 2 was assembled from diethyl acetamidomalonate 1.43 Propargylation of 1 in the presence of Cs2CO3 furnished the mono−alkyne building block 2. Simultaneously, various azides 3(a−x) were prepared from the corresponding amines by following the literature procedure.44 Finally, the mono−alkyne building block 2 was reacted with differently substituted phenyl azides 3(a−x) to deliver the library of designed mono−triazole based compounds 4(a−x), listed in Table 1. The formation of compounds 4(a−x) were confirmed by complementary spectral data (1H NMR, 13C NMR and HRMS). Having the library of designed compounds in our hands we next carried out the biophysical studies to investigate the efficacies of synthesized compounds to inhibit Aβ42 aggregation, disaggregation of preformed Aβ42 fibrils, and inhibition of metal induced Aβ42 6 ACS Paragon Plus Environment

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aggregation, disaggregation of preformed Aβ42 fibrils in the presence of metal, metal chelating properties and prevention of ROS generation. N3 R

R EtO2C

EtO2C

a

EtO2C NHAc 1

3(ax) b

EtO2C NHAc 2

N N N EtO2C NHAc 4(ax) EtO2C

Scheme 1: Synthetic route to generate compounds 4(a−x): Reagents and conditions: (a) Propargyl bromide, CS2CO3, CH3CN, 18 h, rt, 100%; (b) Cu(OAc)2, sodium−ascorbate, t−BuOH/H2O (1:1), rt. Table 1: List of mono−triazole derivatives 4(a−x) Cpd

R

Mono−triazole derivatives

Yield (%)a

Cpd

R

Mono−triazole derivatives

Yield (%)a

F

4a

H

N N N EtO2C NHAc

78

EtO2C

4m

o−F

F

H3C

4b

o−CH3

66

N N N EtO2C NHAc EtO2C

40


4n

m−F

62


F

H3C

4c

m−CH3

76

N EtO2C N N EtO2C NHAc

4o

p−F

N N N EtO2C NHAc

CH3

4d

p−CH3


Cl

53

4p

o−Cl

o−OCH3


60


Cl

H3CO

4e

75

EtO2C

51

4q

m−Cl


61



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Cl

H3CO

4f

m−OCH3

51


4r

p−Cl

N N N EtO2C NHAc

OCH3

4g

p−OCH3

N N N EtO2C NHAc

Br

61

EtO2C

4s

o−Br

o−CF3

45


Br

F3C

4h

66

EtO2C

56


4t

m−Br

46


Br

F3C

4i

m−CF3

88

N EtO2C N N EtO2C NHAc

4u

p−Br

N N N EtO2C NHAc

CF3

4j

p−CF3

I

53


4v

o−I

m−NO2

I

52


4w

m−I

aYields

p−NO2


45


I

NO2

4l

71


O 2N

4k

83

EtO2C

80

4x

p−I


95

were of isolated and purified products.

Inhibition of self mediated Aβ42 aggregation by designed mono−triazole derivatives Thioflavin T (ThT) fluorescence assay was used to study the effect of compounds 4(a−x) on the aggregation propensity of Aβ42. ThT, a benzothiazole dye, has been used for the in vitro identification of amyloid fibrils and was used to monitor the kinetics of fibril formation.45 ThT binds selectively to amyloid fibrils and undergoes a huge increase of its fluorescence emission. A known amyloid aggregation inhibitor, curcumin, was used as reference compound in the present 8 ACS Paragon Plus Environment

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assay.46 The experimental conditions such as concentration of Aβ42 and ThT to be used, and time of incubation were optimized and were later used in the studies. The results shown in Figure 2A indicated that 4f, 4j, 4p and 4v in the series exhibited more than 50% inhibition of Aβ42 aggregation. The results highlighted that 4v was the most potent inhibitor of Aβ42 aggregation (78.02% inhibition) among the series of compounds featuring o−iodo group on the phenyl ring. The complete dose–response curve of compound 4v was evaluated. The curve indicated that 4v inhibited Aβ42 aggregation with an IC50 value of 4.578 ± 0.109 μM as compare with the control, curcumin (95% inhibition with IC50 value= 1.482 ± 0.01 μM). In addition, transmission electron microscopy (TEM) assay was performed to observe the morphologies of Aβ42 aggregates. TEM images obtained after incubating fresh Aβ42 monomer showed complex network of long rope−like amyloid fibrils (Figure 2Bb). However, a considerable reduction in the formation of amyloid fibrils was observed in the presence of compound 4v (Figure 2Bc). The results of the ThT assay and TEM clearly proved the potential of 4v to inhibit Aβ42 aggregation. The ability of 4v to inhibit Aβ42 aggregation may be attributed to the presence of halogen on the phenyl ring. The substitution of halogen on the phenyl ring is known to influence the aromatic interaction mediated self assembly processes in amyloid fibril formation.47



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Figure 2: Inhibition of self mediated Aβ42 aggregation: (A) The results of ThT fluorescence assay. Statistical significance was analyzed by one−way analysis of variance (ANOVA) followed by Dunnett’s test: (**) p < 0.01 and (*) p < 0.5, versus Aβ42 alone. [Aβ42]= 10 µM, [4(a−x)]= [Curcumin]= 50 µM. (B) TEM images: [Aβ42]= 10 µM, [4v]= 20 µM, 37 °C, 24 h, constant agitation (a) Aβ42, 0 h; (b) Aβ42, 24 h; (c) Aβ42 + 4v, 24 h. Scale bar: 0.2 µm.


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Disaggregation of self mediated Aβ42 aggregation fibrils by compound 4v The ability of compound 4v to disaggregate self mediated Aβ42 aggregation fibrils was investigated using ThT fluorescence assay. In this regard, Aβ42 monomer was incubated at 37 °C for 24 h to generate Aβ42 fibrils. The test compound 4v and the reference compound curcumin were then added separately to the samples before incubating for another 24 h at 37 ˚C. The ThT binding assay highlighted that 4v could disaggregate Aβ42 fibrils by 71.23% at 20 µM, as compared to curcumin (82.75% at 20 µM, Figure 3A). Further, TEM assay was performed to support the results obtained in ThT assay. A bundle of amyloid fibrils can be seen in a TEM image (Figure 3Bb) obtained after incubation fresh Aβ42 monomer for 24 h. However, addition of 4v to a sample of the preformed fibrils resulted in significant reduction in the number of Aβ42 fibrils (Figure 3Bc). The results of ThT assay and TEM analysis clearly indicated that compound 4v can effectively disassemble the self mediated Aβ42 fibrils.



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Figure 3: Disaggregation of self mediated Aβ42 aggregation: (A) The results of ThT binding assay. Statistical significance was analyzed by one−way analysis of variance (ANOVA) followed by Dunnett’s test: (**) p < 0.01, versus Aβ42 alone. Aβ42= 10 μM, [4v]= [Curcumin]= 20 μM. (B) TEM images: [Aβ42]= 10 µM, [4v]= 20 µM, 37 °C, 24 h, constant agitation (a) Aβ42, 0 h; (b) Aβ42, 24 h; (c) Aβ42 fibrils + 4v, 24 h. Scale bar: 0.2 µm. Metal chelating characteristics of compound 4v The UV−Vis spectroscopy was used to investigate the metal chelating ability of 4v.48 The observed spectral peaks of 4v alone and in the presence of metal ions were shown in Figure 4a. The specific absorbance peaks of 4v were observed at 226 nm and 270 nm as shown in Figure 4a. After addition of CuSO4 to a solution of 4v, there is a considerable decrease in the absorbance at 226 nm indicating an interaction between 4v and Cu2+. On the contrary, the absorbance at 226 nm increases on addition of FeSO4 and ZnCl2 to a solution of 4v, suggesting that 4v binds to Fe2+ and Zn2+. The metal chelating ability of 4v could be due to the donation of lone pair of electron on one of the π–bonded nitrogen atoms present in the triazole ring. Further, the stoichiometry of the 4v–Cu2+ complex was determined using Job’s method by preparing a series of solutions that maintained the total concentration of 4v and CuSO4 constant but vary their proportion.49 The absorbance of the 4v–Cu2+ complex at varying concentrations was recorded on UV−Vis spectrophotometer (Figure S1, Supporting Information). The absorbance readings at 226 nm vs mole fraction of Cu2+ was plotted in Figure 4b. The break at 0.3 in the Job plot for 4v and CuSO4 indicated a 2:1 stoichiometry for 4v–Cu2+ complex.


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Figure 4: Metal chelating properties: (a) The UV−Vis spectrum of compound 4v alone or in presence of 20 µM CuSO4, ZnCl2, or FeSO4 in buffer (20 mM HEPES, 150 mM NaCl, pH 7.4); (b) Determination of stoichiometry of 4v−Cu2+ complex using Job’s method. Suppression of copper−based redox activity in the presence of compound 4v A number of studies have highlighted that the generation of excess free radicals play a major role in causing neuronal cell death in AD patients.26 Among the several biometals, Cu2+ play an important role in catalyzing the generation of ROS, leading to an aggravated neuron toxicity and oxidative stress.8 Therefore, investigating the redox silencing property of 4v is important in assessing its antioxidant property. To estimate the redox silencing property of 4v copper– ascorbate redox system described in Scheme 2 has been used as a model system.50 In the presence of ascorbate (reducing agent), Cu2+ is reduce to Cu+, which being unstable oxidize back to Cu2+ and thus generates •OH (major form of ROS accounting to the toxicity in AD) in the process. Coumarin−3−carboxylic acid (CCA) was used to detect the •OH in vitro, as •OH convert CCA to a fluorescent 7−hydroxyl−CCA (emission at 450 nm). As seen in Figure 5, the fluorescence intensity increases steadily with time and reached a plateau at nearly 12 min. This indicate that the •OH produced by copper and ascorbate increases with time and then achieved a plateau. However, solutions treated with 4v showed negligible fluorescence, highlighting that 4v−bound Cu2+ (redox dormant state) was not involved in the copper–ascorbate redox cycle to generate •OH even under highly reducing conditions.



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Scheme 2: The generation of hydroxyl radical (•OH) in the presence of ascorbate via copper−ascorbate redox cycling.

Figure

5:

Fluorescence

intensity

of

7−hydroxy−coumarin−3−carboxylic

acid

in

copper−ascorbate and copper−ascorbate−4v system. CCA [50 μM] and ascorbate [150 μM] were incubated in each system. [Cu2+]= [5 μM], [4v]= 15 μM. PBS buffer, pH= 7.4. Excitation wavelength= 395 nm and emission wavelength= 450 nm. The effects of compound 4v on Cu2+−mediated Aβ42 aggregation Next, we investigated the effect of compound 4v on Cu2+−mediated Aβ42 aggregation by using ThT fluorescence assay. A know metal chelator, clioquinol48a (CQ), was used as a reference compound in the present study. Aβ42 was first treated with one equivalent of Cu2+ for 2 min at rt and later incubated with or without the test compounds (4v and CQ) for 24 h at 37 °C. According to the ThT assay, the percentage of formation of Aβ42 fibrils is 32% higher in the presence of Cu2+ than Aβ42 alone (Figure 6). On the contrary, the fluorescence of Aβ42 treated with Cu2+ and test compounds decreased dramatically (fluorescence after treatment with 4v, and CQ was 14 ACS Paragon Plus Environment

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44.24% and 45.06%, respectively). These results clearly indicated the capability of 4v to inhibit Cu2+−induced Aβ42 aggregation by effectively chelating the Cu2+.

Figure 6: Inhibition of Cu2+−mediated Aβ42 aggregation: The results of ThT fluorescence assay. Statistical significance was analyzed by one–way analysis of variance (ANOVA) followed by Dunnett’s test: (**) p < 0.01 and (***) p < 0.001, versus Aβ42 + Cu2+ alone. Aβ42= 10 μM, Cu2+= 10μM, [4v]= [CQ]= 20 μM. The effect of compound 4v on Cu2+−mediated Aβ42 aggregation fibrils ThT fluorescence assay was used to investigate the ability of compound 4v to disaggregate Cu2+−mediated Aβ42 aggregation fibrils. In this assay, Aβ42 monomer was incubated with one equivalent of Cu2+ for 24 h at 37 °C to generate the Aβ42 fibrils. Later, 4v and CQ (20 µM) were added separately to Aβ42 fibrils and incubated for another 24 h at 37 °C. The results (Figure 7) indicated that compound 4v manifested excellent disaggregation potency (64.02%) as compared 15 ACS Paragon Plus Environment


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to the reference CQ (58.95%). Therefore, it is concluded that compound 4v has the ability to inhibit Cu2+−mediated Aβ42 aggregation and disrupt well structured Aβ42 fibrils.

Figure 7: Disaggregation of Cu2+−mediated Aβ42 aggregation fibrils by 4v: The results of ThT binding assay. Statistical significance was analyzed by one–way analysis of variance (ANOVA) followed by Dunnett’s test: (**) p < 0.01, Aβ42= 10 μM, Cu2+= 10 μM, [4v]= [CQ]= 20 μM. The effect of compound 4v on neuronal cell viability and on Aβ42 mediated neurotoxicity After establishing the potential of 4v as an effective MTDL in AD, a study was conducted to check the cytotoxicity of compound 4v and its efficiency to ameliorate the neurotoxicity induced by Aβ42 aggregates in cellular model. In this regard, 3–(4,5–Dimethylthiazol–2–yl)–2,5– diphenyltetrazolium bromide (MTT) reduction assay51 using SH–SY5Y cells were performed. The results shown in Figure 8a, highlighted that the incubation of the SH–SY5Y cells with upto 25 µM concentration of the compound 4v did not influence the cell viability of the stain significantly (cell viability in the presence of 4v: 91% at 5 µM and 90% at 25 µM). 16 ACS Paragon Plus Environment

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Figure 8: Cell viability assay: (a) The cell viability in the presence of 4v alone at 5 µM, 25 µM, 50 µM and 100 µM after 48 h of incubation; (b) Cell viability in the presence of Aβ42 [5µM] alone and mixture of Aβ42–4v at molar ratios 1:1, 1:5, 1:10 and 1:100. Data is expressed as mean values ± SEM from at least three independent experiments. Statistical significance was analyzed by one–way analysis of variance (ANOVA) followed by Dunnett’s test: (**) p < 0.01, In both panel (a) and (b) error bars represent the average of three replicate experiments. Further, the ability of 4v to rescue SH−SY5Y cells from Aβ42 mediated toxicity was studied by MTT assay. As shown in Figure 8b, the cell viability reduces to 58% as compared to the control (100%) in the presence of Aβ42 aggregates. The reduction in cell viability indicates that Aβ42 aggregates were toxic to the SH–SY5Y cells. However, the cell viability significantly improved (72%) when cells were treated with compound 4v (5 µM). The cell viability increases with increasing the molar ratio of 4v to Aβ42. The cell viability increases to 78% at Aβ42/4v = 1:10; however, a further increase in the concentration of 4v (> 50 µM) did not affect the cell viability significantly. These results highlighted the potential of compound 4v to rescue SH–SY5Y cells from Aβ42 mediated toxicity. Molecular docking analysis of compound 4v with Aβ42 monomer and Aβ42 protofibril Molecular docking is an indispensable tool to understand the binding regions and key interactions of ligand with protein.52 After getting the satisfactory results in various biophysical studies, we further explored the possible modes of interaction of 4v with Aβ42 monomer and Aβ42 protofibril structure using molecular docking analysis, via AutoDock 4.253 In this respect, 17 ACS Paragon Plus Environment


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4v was docked to Aβ42 monomer (PDB ID: 1IYT) and the observed binding energy was −5.12 kcal/mol (Table 2). The negative binding energy highlights the favorable binding of 4v with Aβ42 monomer. Compound 4v formed two hydrogen bonds with Aβ42 monomer. The first one was formed between the nitrogen atom of triazole ring of 4v with NH of the backbone of Gln15 of Aβ42 monomer. The second hydrogen bond is formed between the carbonyl oxygen atom of ethyl acetate moiety of 4v with NH of the backbone of Lys16 residue of Aβ42 monomer (Figure 9a and Table 2). Hydrophobic contacts also play an important role in the effective binding of 4v with Aβ42. The hydrophobic contacts were analyzed using LigPlot+ software. The 2D interaction map displayed the hydrophobic contacts of 4v with Ser8, Glu11, Val12, Gln15, Lys16, Phe19 and Phe20 residues of Aβ42 monomer (Figure 9b). Table 2: Molecular docking analysis of 4v with Aβ42 monomer and Aβ42 protofibril structure. Protein structurea

AutoDock binding energy (kcal/mol)

Aβ42 residues involved in intermolecular hydrogen bonding with 4v Residue

Atomsb

Distance (nm)

Aβ42 monomer

−5.12

Gln 15 Lys16

N : HN O : HN

0.28 0.17

Aβ42 protofibril

−6.54

aThe bThe

Aβ42 residues involved in intermolecular hydrophobic contacts with 4v

Ser8, Glu11, Val12, Gln15, Lys16, Phe19, Phe20 Leu17 (C, D, E), Val18 (C, E), Phe19 (C, D, E), Gly37 (E), Gly38 (E), Val39 (D), Val40 (E)

PDB ID for Aβ42 monomer and Aβ42 protofibril used in the present study are 1IYT and 2BEG, respectively. atoms on left represent ligand atoms, and on the right represent Aβ42 residue atoms.


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Figure 9: (a) The docked complex of 4v with Aβ42 monomer (PDB ID: 1IYT) highlights the best docked conformations. The Aβ42 monomer is shown in cartoon representation and compound 4v is shown in stick representation; (b) The 2D interaction maps of 4v displays the formation of hydrogen bond (green line) and hydrophobic contacts with 1IYT. The maps are generated using LigPlot+ software. To elucidate the binding regions of 4v with Aβ42 protofibril, 4v was docked to Aβ42 protofibril (PDB ID: 2BEG). The Aβ42 protofibril (PDB ID: 2BEG) model consist of two β−strand regions (Leu17−Ser26 and Ile31−Ala42) linked with the bend region (Asn27−Ala30). The binding energy of −6.54 kcal/mol was observed in best docked pose of 4v with Aβ42 protofibril structure (Figure 10a, Table 2). Compound 4v shows hydrophobic contacts with Leu17 (C, D, E), Val18 (C, E), Phe19 (C, D, E), Gly37 (E), Gly38 (E), Val39 (E) and Val40 (E) residues of Aβ42 protofibril (Figure 10b). The molecular docking studies highlighted that 4v binds preferably to the central hydrophobic core (CHC) of Aβ42 monomer via the hydrogen bond and through hydrophobic contacts. The CHC of Aβ42 is known to play a pivotal role in Aβ42 aggregation.54 Further, the docking results indicated that 4v binds to the even edge (chain E) of the Aβ42 protofibril. The fibril structure grows faster from the even edge of Aβ42 protofibril as compared to the odd edge.55 19 ACS Paragon Plus Environment


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Figure 10: (a) The docked complex of 4v with protofibril (PDB ID: 2BEG) Aβ42 protofibril (PDB ID: 2BEG); (b) The 2D interaction maps displaying the hydrophobic contacts of 4v with Aβ42 protofibril (PDB ID: 2BEG). The maps are generated using LigPlot+ software. CONCLUSION On the basis of knowledge that triazole moiety possesses a metal chelating properties, we have developed a novel series (24 variants) of triazole based compounds as multitarget agents for the treatment of Alzheimer’s disease (AD). The compounds were designed by combining the metal chelating triazole moiety and an anti–amyloid aggregation pharmacophore into one molecule. Among the synthesized library, compound 4v exhibited significant inhibition of Aβ42 aggregation and disaggregation of Aβ42 fibrils. Compound 4v inhibited Aβ42 aggregation with an IC50 values of 4.578 μM. Compound 4v showed efficient metal chelating ability, due to the donation of lone pair of electron on one of the π–bonded nitrogen atoms present in the triazole ring to the metal ion. Compound 4v binds Cu2+ and maintained it in a redox–dormant state even under highly reducing conditions, and thus effectively prevented the production of reactive oxygen species (ROS). Similar to clioquinol (reference compound), 4v inhibited Cu2+–induced Aβ42 aggregation and disassembled preformed Cu2+–induced Aβ42 aggregates. The cytotoxicity of compound 4v and its ability to inhibit Aβ42 induced toxicity in vitro was further evaluated in 20 ACS Paragon Plus Environment

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SH–SY5Y cells. Compound 4v did not influence the cell viability of the SH–SY5Y cells significantly even at 25 µM concentration. In addition, 4v was able to rescue the cells from the toxicity induced by Aβ42 aggregates and the cell viability improves to 78%. Further, the molecular docking analysis highlighted that 4v binds with the central hydrophobic core (CHC) region of Aβ42 monomer and showed interactions with the even edge in the Aβ42 protofibril structure. In conclusion, the ability to inhibit Aβ42 aggregation, metal−induced Aβ42 aggregation, disaggregation of Aβ42 fibrils, chelation of Cu2+, transforming it into a redox−dormant state, and controlling the generation of ROS proved the multifunctional nature of 4v. Furthermore, ability of 4v to rescue SH–SY5Y cells from Aβ42 induced toxicity makes it a promising candidate for further evaluation. Overall, the designed scaffold showed satisfactory profile in the various assays and hence, could be important multi−target−directed ligands (MTDLs) in AD. EXPERIMENTAL SECTION General Methods Human Aβ42 was purchased from Anaspec. All the reagents were purchased from Sigma Aldrich and were used without further purification. The progress of the chemical reactions were monitored by thin layer chromatography (TLC) using an appropriate solvent system for development. The reported yields of the synthesized compounds were the isolated yields. In the 1H

NMR the coupling constants (J) were given in hertz (Hz) and chemical shifts were stated in

parts per million (ppm). The abbreviations s, d, t, q and m stand for singlet, doublet, triplet, quartet and multiplet, respectively. 1H NMR (400 MHz or 500 MHz, CDCl3 or DMSO−d6) and 13C

NMR (100 MHz or 125 MHz, CDCl3 or DMSO−d6) spectra were recorded on a Bruker

NMR spectrometer. I.R. spectra were recorded using Perkin Elmer. HRMS data were recorded using electrospray ionization (ESI) on Bruker MaXis Impact spectrometer and Agilent 6520 Q−TOF instrument. Melting points were recorded with a Perfit apparatus. HPLC analysis was recorded on Agilent Technologies 1260 Infinity series system, reverse phase C18 column eluted with 100% acetonitrile at a flow rate of 1mL/ min. General procedure for synthesis of aryl azides 3(a−x) The solution of aryl amine (13.6 mmol) in 50 mL HCl:H2O (1:1) was cooled at −5 °C using ice−salt mixture. A solution of sodium nitrite (27.2 mmol) in water (15 mL) was added slowly at 21 ACS Paragon Plus Environment


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−5 °C and the reaction mixture was stirred for a period of 1 h. After this, the reaction mixture was neutralized using sodium acetate (272 mmol). Later, a solution of NaN3 (27.2 mmol) in water (15 mL) was added slowly over a period of 30 min by maintaining the temperature at −5 °C.

After stirring the reaction mixture for 30 min, the solution was allowed to warm at room

temperature. The aqueous layer was extracted with ethyl acetate (100 mL x 2), dried over sodium sulphate and evaporated with utmost care to yield the corresponding azide. General procedure for synthesis of mono−triazole based derivatives 4(a-x) The mono-alkyne precursor (1 mmol) was dissolved in t−BuOH/H2O (1:1 mL) and the azide (1.1 mmol), Cu(OAc)2 (0.1 mmol) and sodium ascorbate (0.2 mmol) were added. The resulting mixture was stirred at rt, until TLC indicated completion of reaction. The mixture was diluted with ethyl acetate and washed with aq NH4OH (0.2%) and brine. The aqueous phases were extracted with ethyl acetate (2 x 10 mL). The combined organic extracts were dried over Na2SO4 and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel. Compound (4a). White solid. 1H NMR (400 MHz, DMSO−d6): δ= 1.19 (t, J= 7.0 Hz, 6H), 1.95 (s, 3H), 3.60 (s, 2H), 4.18 (q, J= 7.0 Hz, 4H), 7.46−7.57 (m, 1H), 7.62−7.87 (m, 2H), 7.87−7.89 (m, 2H), 8.21 (s, 1H), 8.54 (s, 1H) ppm.

13C

NMR (100 MHz, DMSO−d6): δ= 13.79, 22.15,

28.96, 61.85, 65.88, 119.78, 122.13, 128.47, 129.85, 136.58, 141.77, 166.82, 169.48 ppm. I.R: 1187, 1513, 1638, 1736, 3115, 3216 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H23N4O5 [M+H]+ 375.1624, found: 375.1660. Rf. 0.39 (30% ethyl acetate/ petroleum ether). Mp: 101−102 °C. Compound (4b). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.30 (t, J= 7.1 Hz, 6H), 1.63 (s, 3H), 2.00 (s, 3H), 3.87 (s, 2H), 4.31 (q, J= 7.1 Hz, 4H), 6.80 (s, 1H), 7.41−7.44 (m, 1H), 7.49−7.53 (m, 1H), 7.68−7.70 (m, 2H), 7.75 (s, 1H) ppm. 13C NMR (100 MHz, DMSO−d6): δ= 13.79, 17.41, 22.10, 28.89, 61.83, 65.95, 125.42, 125.80, 126.89, 129.59, 131.33, 132.94, 136.28, 140.72, 166.84, 169.39 ppm. I.R: 1050, 1293, 1502, 1666, 1741, 3378 cm-1. HRMS (Q−TOF) m/z: calcd. for C19H25N4O5 [M+H]+ 389.1780, found: 389.1811. Rf. 0.33 (50% ethyl acetate/ petroleum ether). Mp: 89−90 °C. Compound (4c). White solid. 1H NMR (400 MHz, DMSO−d6): δ= 1.19 (t, J= 7.0 Hz, 6H), 1.95 (s, 3H), 2.41 (s, 3H), 3.60 (s, 2H), 4.18 (q, J= 7.0 Hz, 4H), 7.28−7.30 (m, 1H), 7.48 (m, 1H), 22 ACS Paragon Plus Environment

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7.66−7.68 (m, 1H), 7.72 (s, 1H), 8.21 (s, 1H), 8.51 (s, 1H) ppm.

13C

NMR (100 MHz,

DMSO−d6): δ= 13.79, 20.90, 22.16, 28.96, 61.84, 65.88, 116.90, 120.21, 122.08, 129.03, 129.62, 136.57, 139.56, 141.68, 166.82, 169.47 ppm. I.R: 1193, 1514, 1641, 1739, 3142, 3250 cm-1. HRMS (Q−TOF) m/z: calcd. for C19H25N4O5 [M+H]+ 389.1780, found: 389.1821. Rf. 0.33 (30% ethyl acetate/ petroleum ether). Mp: 130−132 °C. Compound (4d). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.30 (t, J= 7.1 Hz, 6H), 2.02 (s, 3H), 2.41 (s, 3H), 3.85 (s, 2H), 4.32 (q, J= 7.1 Hz, 4H), 6.79 (s, 1H), 7.28−7.30 (m, 2H), 7.54−7.57 (m, 2H), 7.69 (s, 1H) ppm.

13C

NMR (100 MHz, CDCl3): δ= 14.02, 20.96, 23.41,

29.39, 38.14, 63.14, 120.27, 120.97, 130.32, 142.77, 167.65, 169.47 ppm. I.R: 1197, 1465, 1668, 1739, 3142, 3375 cm-1.HRMS (Q−TOF) m/z: calcd. for C19H25N4O5 [M+H]+ 389.1780, found: 389.1813. Rf. 0.36 (50% ethyl acetate/ petroleum ether). Mp: 126−128 °C. Compound (4e). White solid. 1H NMR (400 MHz, DMSO−d6): δ= 1.19 (t, J= 7.0 Hz, 6H), 1.94 (s, 3H), 3.61 (s, 2H), 3.85 (s, 3H), 4.18 (q, J= 7.0 Hz, 4H), 7.12−7.16 (m, 1H), 7.31−7.33 (m, 1H), 7.49−7.54 (m, 1H), 7.58−7.61 (m, 1H), 8.12 (s, 1H), 8.20 (s, 1H) ppm.

13C

NMR (100

MHz, DMSO−d6): δ= 13.78, 22.09, 28.81, 56.02, 61.83, 65.96, 113.03, 120.87, 125.25, 125.59, 125.68, 130.42, 140.47, 151.23, 166.87, 169.42 ppm. I:R: 1200, 1265, 1504, 1678, 1741, 3056, 3410 cm-1. HRMS (Q−TOF) m/z: calcd. for C19H25N4O6 [M+H]+ 405.1729, found: 405.1765. Rf. 0.32 (30% ethyl acetate/ petroleum ether). Mp: 78−80 °C. Compound (4f). White solid. 1H NMR (400 MHz, DMSO−d6): δ= 1.19 (t, J= 7.0 Hz, 6H), 1.95 (s, 3H), 3.59 (s, 2H), 3.85 (s, 3H), 4.18 (q, J= 7.0 Hz, 4H), 7.03−7.06 (m, 1H), 7.45−7.52 (m, 3H), 8.21 (s, 1H), 8.58 (s, 1H) ppm. 13C NMR (100 MHz, DMSO−d6): δ= 13.79, 22.15, 28.92, 55.55, 61.84, 65.87, 105.42, 111.73, 114.06, 122.29, 130.80, 137.62, 141.72, 160.13, 166.80, 169.49 ppm. I.R: 1190, 1235, 1497, 1639, 1742, 3241 cm-1. HRMS (Q−TOF) m/z: calcd. for C19H25N4O6 [M+H]+ 405.1729, found: 405.1754. Rf. 0.24 (30% ethyl acetate/ petroleum ether). Mp: 120−122 °C. Compound (4g). White solid. 1H NMR (500 MHz, CDCl3): δ= 1.30 (t, J= 7.1 Hz, 6H), 2.03 (s, 3H), 3.87 (s, 2H), 3.88 (s, 3H), 4.32 (q, J= 7.1 Hz, 4H), 6.82 (s, 1H), 7.01−7.03 (m, 2H), 7.59−7.61 (m, 2H), 7.68 (s, 1H) ppm.

13C

NMR (125 MHz, CDCl3): δ= 14.06, 23.14, 29.12,

55.68, 62.90, 66.13, 114.85, 121.13, 122.00, 130.40, 142.59, 159.76, 167.48, 169.47 ppm. I.R: 23 ACS Paragon Plus Environment


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1198, 1215, 1517, 1643, 1739, 3264 cm-1. HRMS (Q−TOF) m/z: calcd. for C19H25N4O6 [M+H]+ 405.1729, found: 405.1766. Rf. 0.26 (50% ethyl acetate/ petroleum ether). Mp: 132−134 °C. Compound (4h). White solid. H1 NMR (400 MHz, DMSO−d6): δ= 1.16 (t, J= 7.0 Hz, 6H), 1.94 (s, 3H), 3.63 (s, 2H), 4.17 (q, J= 7.0 Hz, 4H), 7.66−7.64 (m, 1H), 7.87−7.84 (m, 1H), 7.96−7.92 (m, 1H), 8.04−8.02 (m, 1H), 8.13 (s, 1H), 8.26 (s, 1H) ppm. 13C NMR (100 MHz, DMSO−d6): δ= 13.70, 22.42, 28.96, 62.28, 66.32, 125.02, 126.45, 127.51, 129.11, 131.02, 133.94, 134.35, 140.86, 166.74, 169.47 ppm. I.R: 1057, 1114, 1497, 1667, 1739, 3407 cm-1. HRMS (Q−TOF) m/z: calcd. for C19H22F3N4O5 [M+H]+ 443.1498, found: 443.1537. Rf. 0.34 (50% ethyl acetate/ petroleum ether). Mp: 102−104 °C. Compound (4i). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.29 (t, J= 7.0 Hz, 6H), 2.01 (s, 3H), 3.87 (s, 2H), 4.30 (q, J= 7.0 Hz, 4H), 6.79 (s, 1H), 7.63−7.70 (m, 2H), 7.83 (s, 1H), 7.89−7.91 (m, 1H), 8.00 (s, 1H) ppm.

13C

NMR (100 MHz, CDCl3): δ= 14.14, 23.26, 29.26,

63.15, 66.15, 117.42, 121.14, 123.43, 125.46, 130.73, 132.82, 137.45, 143.43, 167.48, 169.60 ppm. I.R: 1120, 1159, 1509, 1657, 1740, 3301 cm-1. HRMS (Q−TOF) m/z: calcd. for C19H22F3N4O5 [M+H]+ 443.1498, found: 443.1533. Rf. 0.37 (50% ethyl acetate/ petroleum ether). Mp: 138−140 °C. Compound (4j). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.29 (t, J= 7.0 Hz, 6H), 2.00 (s, 3H), 3.87 (s, 2H), 4.30 (q, J= 7.0 Hz, 4H), 6.79 (s, 1H), 7.76−7.80 (m, 2H), 7.83-7.85 (m, 2H), 7.86 (s, 1H) ppm.

13C

NMR (100 MHz, CDCl3): δ= 14.14, 23.26, 29.26, 63.16, 66.14, 114.96,

120.39, 121.12, 127.28, 127.31, 130.99, 143.46, 144.43, 167.47, 169.61 ppm. I.R: 1123, 1160, 1505, 1660, 1750, 3074, 3403 cm-1. HRMS (Q−TOF) m/z: calcd. for C19H21F3N4O5Na [M+Na]+ 465.1361, found: 465.1357. Rf. 0.33 (40% ethyl acetate/ petroleum ether). Mp: 188−190 °C. Compound (4k). White solid. 1H NMR (400 MHz, DMSO−d6): δ= 1.20 (t, J= 7.0 Hz, 6H), 1.96 (s, 3H), 3.62 (s, 2H), 4.19 (q, J= 7.0 Hz, 4H), 7.89−7.93 (m, 1H), 8.21 (s, 1H), 8.31−8.34 (m, 1H), 8.38−8.40 (m, 1H), 8.71−8.72 (m, 1H), 8.82 (s, 1H) ppm.

13C

NMR (100 MHz,

DMSO−d6): δ= 13.79, 22.20, 28.92, 61.89, 65.82, 114.43, 122.78, 122.97, 125.81, 131.57, 137.13, 142.29, 148.56, 166.77, 169.53 ppm. I.R: 1264, 1540, 1683, 1740 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H22N5O7 [M+H]+ 420.1475, found: 420.1494. Rf. 0.32 (30% ethyl acetate/ petroleum ether). Mp: 150−151 °C. 24 ACS Paragon Plus Environment

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Compound (4l). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.30 (t, J= 7.0, 6H), 2.00 (s, 3H), 3.90 (s, 2H), 4.32 (q, J= 7.0, 4H), 6.81 (s, 1H), 7.89 (s, 1H), 7.92−7.96 (m, 2H), 8.39−8.43 (m, 2H) ppm. 13C NMR (100 MHz, CDCl3): δ= 14.07, 23.26, 29.29, 63.13, 65.86, 120.35, 121.13, 125.80, 141.05, 143.86, 147.28, 167.52, 169.70 ppm. I.R: 1216, 1285, 1445, 1667, 1758, 3389 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H21N5O7Na [M+Na]+ 442.1338, found: 442.1336. Rf. 0.24 (50 % ethyl acetate/ petroleum ether). Mp: 186−188 °C. Compound (4m). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.30 (t, J= 7.2 Hz, 6H), 2.03 (s, 3H), 3.89 (s, 2H), 4.31 (q, J= 7.2 Hz, 4H), 6.77 (s, 1H), 7.28−7.32 (m, 1H), 7.33 (s, 1H), 7.39−7.45 (m, 1H), 7.85 (s, 1H), 7.93−7.98 (m, 1H) ppm.

13C

NMR (100 MHz, CDCl3): δ=

13.97, 22.99, 28.91, 62.94, 66.08, 116.91, 117.11, 124.07, 124.16, 124.59, 125.27, 125.31, 130.01, 130.09, 142.33, 167.32, 169.40 ppm. I.R: 1195, 1275, 1463, 1643, 1740, 3257 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H22FN4O5 [M+H]+ 393.1530, found: 393.1558. Rf. 0.31 (30% ethyl acetate/ petroleum ether). Mp: 64−66 °C. Compound (4n). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.28 (t, J= 7.1 Hz, 6H), 2.00 (s, 3H), 3.85 (s, 2H), 4.29 (q, J= 7.1 Hz, 4H), 6.79 (s, 1H), 7.09−7.14 (m, 1H), 7.45−7.50 (m, 3H), 7.76 (s, 1H) ppm.

13C

NMR (100 MHz, CDCl3): δ= 14.11, 23.21, 29.21, 63.10, 66.14, 108.08,

108.34, 115.60, 115.68, 115.71, 115.81, 121.14, 131.31, 131.40, 143.11, 164.48, 167.46, 169.58 ppm. I.R: 1191, 1234, 1509, 1641, 1740, 3253 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H22FN4O5 [M+H]+ 393.1530, found: 393.1566. Rf. 0.25 (30% ethyl acetate/ petroleum ether). Mp: 128−130 °C. Compound (4o). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.30 (t, J= 7.3 Hz, 6H), 2.01 (s, 3H), 3.86 (s, 2H), 4.31 (q, J= 7.3 Hz, 4H), 6.79 (s, 1H), 7.19−7.23 (m, 2H), 7.65−7.69 (m, 2H), 7.71 (s, 1H) ppm.

13C

NMR (100 MHz, CDCl3): δ= 13.98, 23.09, 29.06, 62.95, 66.01, 116.61,

116.84, 121.14, 122.21, 122.30, 142.84, 167.34, 169.40 ppm. I.R: 1206, 1283, 1510, 1670, 1726, 3394 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H22FN4O5 [M+H]+ 393.1530, found: 393.1563. Rf. 0.22 (50% ethyl acetate/ petroleum ether). Mp: 120−122 °C. Compound (4p). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.30 (t, J= 7.0 Hz, 6H), 2.02 (s, 3H), 3.89 (s, 2H), 4.31 (q, J= 7.0 Hz, 4H), 6.80 (s, 1H), 7.42−7.47 (m, 2H), 7.55−7.58 (m, 1H), 7.60−7.62 (m, 1H), 7.77 (s, 1H) ppm.

13C

NMR (100 MHz, CDCl3): δ= 13.99, 23.03, 28.98, 25

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62.93, 66.09, 124.98, 127.66, 127.98, 128.29, 130.66, 130.78, 134.86, 141.66, 167.32, 169.33 ppm. I.R: 730, 1210, 1497, 1679, 1742 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H22ClN4O5 [M+H]+ 409.1272, found: 409.1271. Rf. 0.20 (50% ethyl acetate/ petroleum ether). Mp: 84−86 °C.

Compound (4q). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.29 (t, J= 7.1 Hz, 6H), 2.01 (s, 3H), 3.86 (s, 2H), 4.30 (q, J= 7.1 Hz, 4H), 6.78 (s, 1H), 7.37−7.47 (m, 2H), 7.57−7.61 (m, 1H), 7.73−7.74 (m, 1H), 7.75 (s, 1H) ppm.

13C

NMR (100 MHz, CDCl3): δ= 14.01, 23.12, 29.11,

62.99, 66.00, 118.24, 120.62, 120.94, 128.73, 130.91, 135.81, 137.99, 143.16, 167.65, 169.56 ppm. I.R: 730, 1198, 1496, 1595, 1677, 1741 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H22ClN4O5 [M+H]+ 409.1272, found: 409.1276. Rf. 0.34 (50% ethyl acetate/ petroleum ether). Mp: 107−109 °C. Compound (4r). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.30 (t, J = 7.1 Hz, 6H), 2.01 (s, 3H), 3.86 (s, 2H), 4.31 (q, J= 7.1 Hz, 4H), 6.79 (s, 1H), 7.46−7.51 (m, 2H), 7.62−7.67 (m, 2H), 7.73 (s, 1H) ppm.

13C

NMR (125 MHz, CDCl3): δ= 13.98, 22.92, 23.14, 23.85, 29.08, 62.99,

65.27, 66.01, 71.39, 120.92, 121.46, 129.96, 134.45, 135.45, 142.99, 166.66, 167.34, 169.31, 169.45 ppm. I.R: 517, 1091, 1446, 1669, 1744, 3392 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H22ClN4O5 [M+H]+ 409.1272, found: 409.1272. Rf. 0.34 (50% ethyl acetate/ petroleum ether). Mp: 107−109 °C. Compound (4s). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.30 (t, J= 7.3 Hz, 6H), 2.03 (s, 3H), 3.89 (s, 2H), 4.31 (q, J= 7.3 Hz, 4H), 6.80 (s, 1H), 7.36−7.40 (m, 1H), 7.46−7.50 (m, 1H), 7.52−7.55 (m, 1H), 7.36−7.59 (m, 1H), 7.72 (s, 1H), ppm.

13C


14.00, 23.08, 28.99, 62.93, 66.09, 118.38, 125.04, 128.14, 128.54, 131.11, 133.91, 136.51, 141.57, 167.31, 169.31 ppm. I.R: 513, 1195, 1508, 1646, 1741, 3283 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H22BrN4O5 [M+H]+ 453.0767, found: 453.0763. Rf. 0.22 (50% ethyl acetate/ petroleum ether). Mp: 100−102 °C. Compound (4t). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.30 (t, J= 7.0 Hz, 6H), 2.02 (s, 3H), 3.87 (s, 2H), 4.31 (q, J= 7.0 Hz, 4H), 6.79 (s, 1H), 7.36−7.40 (m, 1H), 7.55−7.57 (m, 1H), 7.63−7.65 (m, 1H), 7.75 (s, 1H), 7.89−7.91 (m, 1H), ppm.

13C


13.98, 23.10, 29.07, 62.98, 65.98, 118.72, 120.91, 123.33, 123.42, 131.08, 131.67, 137.86, 26 ACS Paragon Plus Environment

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143.02, 167.31, 169.43 ppm. I.R: 678, 1021, 1486, 1643, 1739, 3255 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H22BrN4O5 [M+H]+ 453.0767, found: 453.0762. Rf. 0.25 (30% ethyl acetate/ petroleum ether). Mp: 116−118 °C. Compound (4u). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.29 (t, J= 7.1 Hz, 6H), 2.01 (s, 3H), 3.86 (s, 2H), 4.31 (q, J= 7.1 Hz, 4H), 6.79 (s, 1H), 7.57−7.60 (m, 2H), 7.62−7.65 (m, 2H), 7.74 (s, 1H) ppm.

13C

NMR (100 MHz, CDCl3): δ= 13.98, 23.08, 29.05, 62.97, 65.99, 120.83,

121.67, 122.28, 132.91, 135.91, 143.01, 167.33, 169.42 ppm. I.R: 664, 1109, 1498, 1667, 1740, 3141, 3386 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H22BrN4O5 [M+H]+ 453.0767, found: 453.0766. Rf. 0.21 (50% ethyl acetate/ petroleum ether). Mp: 148−150 °C. Compound (4v). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.31 (t, J= 7.3 Hz, 6H), 2.04 (s, 3H), 3.90 (s, 2H), 4.31 (q, J= 7.3 Hz, 4H), 6.82 (s, 1H), 7.21−7.24 (m, 1H), 7.41−7.43 (m, 1H), 13C

7.48−7.52 (m, 1H), 7.62 (s, 1H), 7.97−8.00 (m, 1H) ppm.


14.01, 23.18, 29.00, 29.70, 62.95, 66.10, 93.86, 124.92, 127.86, 129.29, 131.48, 140.21, 141.67, 167.29, 169.33 ppm. I.R: 1264, 1516, 1693, 1742, 3380 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H22IN4O5 [M+H]+ 501.0590, found: 501.0606. Rf. 0.21 (30% ethyl acetate/ petroleum ether). Mp: 70−72 °C. HPLC purity: 100%. Compound (4w). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.30 (t, J= 7.2 Hz, 6H), 2.02 (s, 3H), 3.86 (s, 2H), 4.31 (q, J= 7.2 Hz, 4H), 6.78 (s, 1H), 7.21−7.24 (m, 1H), 7.66−7.69 (m, 1H), 7.73 (s, 1H), 7.74−7.77 (m, 1H), 8.08−8.09 (m, 1H) ppm.

13C


13.98, 23.11, 29.06, 62.98, 65.99, 94.44, 119.42, 120.88, 129.13, 131.15, 137.66, 137.71, 142.98, 167.32, 169.43 ppm. I.R: 1047, 1265, 1498, 1678, 1741, 3383 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H22IN4O5 [M+H]+ 501.0590, found: 501.0630. Rf. 0.37 (40% ethyl acetate/ petroleum ether). Mp: 82−84 °C. Compound (4x). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.28 (t, J= 7.1 Hz, 6H), 2.00 (s, 3H), 3.85 (s, 2H), 4.29 (q, J= 7.1 Hz, 4H), 6.79 (s, 1H), 7.44−7.46 (m, 2H), 7.74 (s, 1H), 7.81−7.83 (m, 2H) ppm.

13C

NMR (100 MHz, CDCl3): δ= 14.13, 23.23, 29.22, 63.10, 66.14,

93.60, 120.94, 121.95, 136.73, 139.01, 143.14, 167.46, 169.55 ppm. I.R: 663, 1051, 1498, 1667, 1740, 3142, 3385 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H21IN4O5Na [M+Na]+ 523.0454, found: 523.0449. Rf. 0.29 (30% ethyl acetate/ petroleum ether). Mp: 152−154 °C. 27 ACS Paragon Plus Environment


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The Thioflavin T assay Aβ42 peptide was purchased from Anaspec and dissolved in 1,1,1,3,3,3−hexafluoro−2−propanol (HFIP). HFIP treated Aβ42 peptide was further evaporated and stored at −20 ˚C. The stock solution of Aβ42 (221.5 μM) was prepared by dissolving 0.1 mg Aβ42 in 100 μL NaOH (10 mM) and stored at −80 ˚C for further use. Then 2 mM stock solutions of all the test compounds and controls were prepared in DMSO. 1 mM stock solution of ThT was prepared in 50 mM PBS buffer (pH 7.4) then diluted to 200 µM stock. For self induced aggregation assay, mixture of Aβ42 (9 µL, 10 µM) and ThT (20 µL, 20 µM) with or without the presence of test compounds and controls (2.5 µL, 50 µM) were diluted to final volume of 200 µL with 50 mM PBS buffer (pH 7.4) in black 96−well plate and incubated for 24 h at 37 ˚C with constant agitation (180 rpm). For disaggregation assay, the monomer Aβ42 was incubated for 24 h at 37 ˚C to form fibrils. Then, the test compound and control were added to the aliquots of Aβ42 fibrils and incubated for another 24 h under same conditions. The fluorescence intensities were recorded in SpectraMax M5e spectrophotometer with excitation and emission wavelengths at 450 nm and 485 nm respectively. Percentage inhibition was calculated by using the formula (1– Fsample/Fcontrol)*100. For the inhibition of Cu2+–mediated Aβ42 aggregation, the Aβ42 stock solution was diluted in 20 μM HEPES (pH 6.6) with 150 μM NaCl. The mixture of the Aβ42 (9 μL, 10 μM) with copper (2.5 μL, 10 μM) with or without the presence of the test compounds (5 μL, 20 μM) were incubated at 37 °C for 24 h. Then 16.5 μL of the sample was diluted to a final volume of 200 μL with 50 mM glycine−NaOH buffer (pH 8.0) containing thioflavin T (20 μM). For disaggregation assay, the monomer Aβ42 was incubated with copper for 24 h at 37 °C to form fibrils. Then, the test compound and control were added to the aliquots of Aβ42 fibrils and incubated for another 24 h under same conditions. Statistical Analysis Statistical analysis was performed with one–way analysis of variance (ANOVA) followed by Dunnett’s test on at least three different measurements (**) p < 0.01 and (***) p < 0.001; Mean±SD for n= 3 experiments). ThT Data analysis and graph production was done by using IBM SPSS Statistics (version 21) for Windows. 28 ACS Paragon Plus Environment

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Transmission electron microscopy assay Transmission electron microscopy was performed to visualise Aβ42 fibrils in the presence of compound 4v. For self induced Aβ42 aggregation assay, Aβ42 monomer (10 μM) was incubated alone or in the presence of 4v (20 μM) in 50 mM PBS buffer (pH 7.4) at 37 °C for 24 h. For Aβ42 disaggregation assay, Aβ42 fibrils (10 μM) was incubated in the presence of 4v (20 μM) in 50 mM PBS buffer (pH 7.4) at 37 °C for 24 h. 5 μL of the samples were placed on a carbon–coated 200 mesh (agar scientific) copper grid for 2 min and allowed to dry for 30 min. Each grid was stained with 2% phosphotungstic acid for 45 seconds. After draining off the excess staining solution, the specimen was loaded to grid holder and transferred for imaging by transmission electron microscopy (JEOL JEM–2100) operating at 200 kV. Metal chelating studies The chelating ability of compound 4v towards biometals such as Cu2+, Zn2+, Fe2+ was examined by UV−Vis spectroscopy (Ultrospec 3000). The stock solution of compound 4v (4 mM), metal ions (8 mM) were prepared in methanol. The solution of 4v (20 µM, final concentration) alone or in the presence of CuSO4, ZnCl2 or FeSO4 (20 µM, final concentration) in (20 mM HEPES, 150 mM NaCl, pH 7.4) were incubated for 30 min at 25 °C. The spectra of each sample were recorded with wavelength ranging from 200 nm to 600 nm using blank containing 20 mM HEPES, 150 mM NaCl, pH 7.4. The stoichiometry of the 4v−Cu2+ complex was determined by Job’s method, by preparing the separate solutions of 4v and CuSO4 in which the total concentration remain constant (40 µM) but their proportion of each component varied from 0% to 100%. The absorbance at 226 nm was plotted against mole fraction of Cu2+. The breakpoint displays the stoichiometry of the complex. Ascorbate studies The ascorbate study was performed by using SpectraMax M5e spectrophotometer. The fluorescence intensities were recorded with excitation and emission wavelengths at 395 nm and 450 nm for the period of 16 min. The stock solution of 4v (in methanol), CuSO4 (in Milli−Q water), CCA and ascorbate were dissolved and diluted in 50 mM PBS buffer (pH 7.4). The final volume of the sample was 200 µL. The production of hydroxyl radical was measured as the 29 ACS Paragon Plus Environment


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change of CCA into 7−hydroxy−CCA. General order of addition follows: CCA (50 µM), ligand (15 µM), or copper (5 µM) and ascorbate (150 µM). All test solutions contained 1 μM desferryl. MTT assay The potential role of 4v in the Aβ42−induced cell toxicity was studied using the MTT assay with SH−SY5Y cell line according to the literature procedure.51 In high glucose DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 U/mL streptomycin, the SH−SY5Y cells were maintained at 37 °C under 5% CO2 in a CO2 cell culture box. In a polystyrene 96− well plate, a total of 5 × 103 cells (90 μL) were seeded for 24 h and then, the cells were treated with Aβ42 and 4v + Aβ42 (2.5 μL, 4v of different concentration was co−incubated with Aβ42 (2.25 μl) monomers at 37 °C for 24 h). The incubation of cells was continued for an additional 24 h, and then 10 μL of MTT solution at the concentration of 5.5 mg/ mL in PBS was added into each well and followed by another 4 h of incubation. Then the medium was discarded, and 100 μL of DMSO was used to dissolve the cells till the complete dissolution of purple crystals. By Plate Reader, the absorbance at 570 nm was measured and using signals at 570 nm, cell viability was evaluated. From each reading, the wells containing medium only were subtracted as the background. The cell viability data were normalized as a percentage of the control group without Aβ42 and inhibitor. Molecular docking The molecular docking was performed using AutoDock 4.2.53 The NMR structure of Aβ42 monomer (PDB ID: 1IYT) and Aβ42 protofibril (PDB ID: 2BEG) structures were used. The 3D structure of 4v was first optimized by Gaussian using density functional theory (DFT) methods with basis set LanL2DZ. The grid spacing was kept default (0.375 Å) and dimension of the box for 1IYT was set to 110 Å × 62 Å × 126 Å with grid center defined at x= −4.994, y= −0.065 and z= 1.577 and for 2BEG, dimension of the box is 126 Å × 74 Å × 86 Å with grid center defined at x= −0.503, y= 0.250 and z= −8.949. The population of 150 individuals was used to generate 100 conformations for 27,000 generations with 2,500,000 energy evaluations. The mutation rate of 0.02, a crossover rate of 0.80, and reference root−mean−square deviation (RMSD) were kept as default. Among stochastic search algorithms available in AutoDock, Lamarckian Genetic Algorithm which utilizes global search (Genetic Algorithm) and local search (Solis and Wets 30 ACS Paragon Plus Environment

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algorithm) was chosen.56 The docked poses of 4v were clustered using a tolerance of 0.2 nm for RMSD and ranked on the basis of binding energy. The molecular structures were visualized by PyMOL. ASSOCIATED CONTENT Supporting Information 1H/ 13C

NMR of synthesized compounds and Figure S1 and S2.

ACKNOWLEDGMENTS DG and BG gratefully acknowledges Science and Engineering Research Board (SERB), Department of Science & Technology (DST), Government of India for the financial support (Grant: YSS/2015/000320 and Grant: SB/FT/CS–013/2014). The authors acknowledge Department of Chemistry and Department of Biotechnology, Sri Guru Granth Sahib World University, Fatehgarh Sahib, Punjab, India and School of Chemistry and Biochemistry, Thapar Institute of Engineering & Technology, Punjab, India for providing the research facilities. The authors express sincere gratitude toward National Agri–Food Biotechnology Institute, S.A.S. Nagar, Punjab, India for giving access to the instrumental facilities. REFERENCES

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