Discovery of imidazopyridines containing isoindoline-1,3-dione framework as a new class of BACE1 inhibitors: Design, synthesis and SAR analysis

Accepted Manuscript Discovery of imidazopyridines containing isoindoline-1,3-dione framework as a new class of BACE1 inhibitors: Design, synthesis and SAR analysis Sara Azimi, Afsaneh Zonouzi, Omidreza Firuzi, Aida Iraji, Mina Saeedi, Mohammad Mahdavi, Najmeh Edraki PII:

S0223-5234(17)30486-5

DOI:

10.1016/j.ejmech.2017.06.040

Reference:

EJMECH 9535

To appear in:

European Journal of Medicinal Chemistry

Received Date: 6 March 2017 Revised Date:

31 May 2017

Accepted Date: 22 June 2017

Please cite this article as: S. Azimi, A. Zonouzi, O. Firuzi, A. Iraji, M. Saeedi, M. Mahdavi, N. Edraki, Discovery of imidazopyridines containing isoindoline-1,3-dione framework as a new class of BACE1 inhibitors: Design, synthesis and SAR analysis, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.040. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Discovery of Imidazopyridines Containing Isoindoline-1,3-dione Framework as a New Class of BACE1 Inhibitors: Design, Synthesis and SAR Analysis Sara Azimia, Afsaneh Zonouzia, Omidreza Firuzib, Aida Irajib, Mina Saeedic,d, Mohammad Mahdavie*, Najmeh Edrakib*

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Dedicated to Professor Abbas Shafiee (1937-2016) for his lifetime achievement in pharmaceutical sciences research and education a

School of Chemistry, College of Science, University of Tehran, PO Box 14155-6455,

b

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Tehran, Iran

Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical

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Sciences, Shiraz, Iran.

Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical

Sciences, Tehran, Iran d

Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences,

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Tehran, Iran

Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical

Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran.

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Corresponding authors:

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Najmeh Edraki, PhD Medicinal and Natural Products Chemistry Research Center, Shiraz university of Medical Sciences, Shiraz, Iran Tel: +98 713 230 3872; Fax: +98 713 230 2225 E-mail: [email protected], [email protected]

Mohammad Mahammad Mahdavi, PhD Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran. E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract Alzheimer’s disease is characterized by chronic neurodegeneration leading to dementia. The main cause of neurodegeneration is considered to be the accumulation of amyloid-β. Inhibiting BACE1 is a well-studied approach to lower the burden of amyloid-β aggregates.

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We designed a series of imidazopyridines-based compounds bearing phthalimide moieties as inhibitors of BACE1. The compounds 8a-o were synthesized by the Groebke–Blackburn– Bienaymé three-component reaction of heteroaromatic amidines, aldehydes and isocyanides.

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Evaluating the BACE1 inhibitory effects of the synthesized compounds revealed that introducing an aminocyclohexyl moiety in the imidazopyridine core resulted in a significant

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improvement in its BACE1 inhibitory potential. In this regard, compound 8e was the most potent against BACE1 with an IC50 value of 2.84 (±0.95) µM. Molecular docking revealed that the nitrogen atom of imidazopyridines and the oxygen atom of the phenoxypropyl linker were involved in hydrogen bound interactions with Asp228 and Asp32 of BACE1 active site,

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respectively. The phthalimide moiety oriented toward the flap pocket and interacted with phe108, lle110, Trp115, Ile118 through van der Waal’s and hydrophobic interactions. These findings demonstrate that imidazopyridines-based compounds bearing phthalimide moiety

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disease.

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have the potential to decrease amyloid-β levels and ameliorate the symptoms of Alzheimer’s

Keywords: Alzheimer’s disease, β-secretase inhibitor, Groebke–Blackburn–Bienayme reaction, imidazopyridines, phthalimide, molecular docking

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ACCEPTED MANUSCRIPT 1. Introduction Alzheimer’s disease is a neurodegenerative disorder and the most common cause of dementia in the elderly [1]. The pathogenesis of the disease is often described by the “Amyloid Cascade Hypothesis”(ACH) [2], which suggests that the deposition of amyloid beta (Aβ) is

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the first pathological event that causes neuronal death and eventually leads to dementia. Two proteases known as β- and γ-secretase endoproteolyze the amyloid precursor protein (APP) to produce the Aβ peptide. β-Secretase is active in most tissues of the body [3, 4]; however, β-

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site amyloid precursor protein cleaving enzyme-1 (BACE1) is the major β-secretase in the CNS compared to BACE2 (close homolog of BACE1), which has a more widespread

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expression pattern? [5]. BACE1 activity is increased in the brains of patients with sporadic Alzheimer’s disease [6]. Consequently, BACE1 inhibitors have emerged as ideal candidates for the treatment of Alzheimer’s disease by preventing Aβ accumulation and aggregation [79]. The first generation of BACE1 inhibitors were designed based on peptide analogs of APP

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[10]. Although they have shown high in vitro inhibitory activity, unfavorable in vivo pharmacological properties were observed due to low blood-brain barrier (BBB) permeability

interest.

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or oral bioavailability. Hence, developing non-peptidic BACE1 inhibitors is of particular

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Among various non-peptidic scaffolds, amidine- or guanidine-containing heterocycles were found to be suitable inhibitors due to the formation of a hydrogen-bond network with the catalytic aspartyl dyad of BACE1 [11-13]. Recently, Merck has introduced a guanidine-based drug for Alzheimer's disease known as Verubecestat (MK-8931) curently in phase II/III trials [14]. Verubecestat is a potent inhibitor of BACE1 with an IC50 value of 0.4 nM [15]. The Xray cocrystal structure confirmed hydrogen-binding interactions between the amidine moiety and the BACE1 catalytic dyad. High-affinity binding toward the relatively hydrophobic S1 and S3 subsites results from a diaryl amide substituent that occupies the above-mentioned

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ACCEPTED MANUSCRIPT subsites in BACE1. MK-8931 has favorable physicochemical properties including stability at physiological pH, oral absorption, high cellular permeability and high BBB penetration [15]. The results suggest that the presence of pyridine and guanidine moieties results in satisfactory curative effects. More recently, Al-Tel et al. introduced imidazopyridines with structure A

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(Figure 1) as novel β-secretase inhibitors [16]. Three derivatives of compound A showed IC50 values of 5.51, 2.48, and 2.24 µM where X was replaced with hydrogen, fluorine, and methoxy, respectively.

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In addition, other studies reported isoindoline-1,3-dione (phthalimide) derivatives (compound C, Figure 1) as cholinesterase and Aβ aggregation inhibitors with neuroprotective effects.

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These compounds did not show cytotoxicity and had therapeutic potential for treating Alzheimer’s disease [17]. Our previous study on phenyliminochromene carboxamide derivatives bearing bromophenyl piperazine moieties (compound B, Figure 1) suggested that the introduction of an isoindoline-1,3-dione moiety into the piperazine pendant results in a

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significant improvement in BACE1 inhibitory activity (IC50= 0.098 µM) and suppression of Aβ production in N2a-APPswe cells (% Inhibition of Aβ1–40 production= 39.4 at 10µM). Phthalimide moiety could be considered as a non-peptidyl framework with low-molecular

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weight involved in hydrophobic interaction with hydrophobic residue of the S2 sub-pocket of

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active site and the network of hydrogen bonding interactions with Arg235, Thr23 and Gly230. This observation might partially explain the significant inhibitory potential resulted from the incorporation of phthalimide moiety into the BACE1 inhibitor scaffold [18]. As part of our ongoing research to design and synthesize novel anti-Alzheimer’s agents, [1921] we focused on an imidazopyridine core as a promising scaffold for inhibiting BACE1. To this end, we employed molecular hybridization and bioisosterism replacement approaches to identify novel non-peptidic inhibitors with aspartyl binding motifs as new entities for the inhibition of this enzyme (Figure 1). Our design is based on bioisosteric replacement of the

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ACCEPTED MANUSCRIPT phenyl linker with a phenoxypropyl one (compound A, Figure 1) to increase the flexibility of the linker and to allow the inhibitor to properly access and orient itself within the active site of BACE1. Different secondary amines were incorporated into the structure to investigate the importance of substituted groups at the 3-position of the imidazopyridines. Our pervious

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results [18] prompted us to replace the benzimidazole group of compound A with a phthalimide pendant to improve the anti-BACE1 effects and enhancing the accessibility to

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the S2-sub pocket of the active site [18].

Figure 1. Chemical structure of previously reported BACE1 inhibitors; imidazopyridines A, isoindoline-1,3-dione (phthalimide) containing derivative B, cholinesterase inhibitor with neuroprotective effect C and our newly hybridized imidazopyridine-based derivatives as potential novel anti-Alzheimer agents. 5

ACCEPTED MANUSCRIPT 2. Results and discussion 2.1. Chemistry The synthetic procedure for the preparation of imidazopyridines bearing phthalimide moiety 8 is depicted in Scheme 1. Reaction of phthalimide 1 and 1,2-dibromopropane 2 in the

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presence of K2CO3 in refluxing acetone gave 2-(2-bromopropyl)isoindoline-1,3-dione 3. Next, the reaction of compound 3 and 4-hydroxyaldehyde derivative 4 in the presence of K2CO3 in DMF at 80 °C gave the desired aldehyde 5.

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The target compounds were prepared through the reaction of aldehyde 5, 2-aminopyridins 6,

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and isocyanides 7 in refluxing toluene in the presence of ammonium chloride (NH4Cl

Scheme 1. Synthesis of imidazopyridines bearing phthalimide moiety 8a-o.

2.2. Determination of BACE1 inhibition BACE1 inhibitory effects of the synthesized imidazopyridines bearing a phthalimide moiety via the phenoxypropyl linker were determined using a FRET-based assay kit. The concentration of compounds that produced 50% maximum inhibition of BACE1 activity

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ACCEPTED MANUSCRIPT (IC50) and enzyme inhibition percentages at 50 and 10 µM concentrations of the test compounds were assessed and are summarized in Table 1. Experiments were repeated three to four times for each derivative and mean percent of enzymatic inhibition at 50 and 10 µM were used for comparing the potencies of the test compounds. Compounds 8e and 8c bearing

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amino cyclohexyel pendents and a methyl substitute on imidazo pyridine core were the most potent derivatives against BACE1 (IC50 = 2.84 µM and 5.93 µM; respectively). Investigation of the structure activity relationship of synthesized derivatives resulted in the following

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observations:

Assesment of aminoalkyl substitute (NHR2) at imidazopyridine core:

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Aminocyclohexyl containing derivatives (R2=cyclohexyl) were more potent than their amino t-butyl counterparts in most cases. Compound 8i bearing methoxy, amino cyclohexyl and 6chloro moieties at R1, R2 and R3 respectively, demonstrated a higher BACE1 inhibitory potential (inhibition at 50 µM = 88.10%) over its amino t-butyl containing counterpart 8k

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(inhibition at 50 µM = 37.90%). Similar results were observed in the case of the aminocyclohexyl derivative 8e (R1=H and R3 =7-CH3, 100% BACE1 inhibition at 50 µM; IC50= 2.84 µM) and its amino t-butyl counterpart (inhibition at 50 µM = 18.32%). These

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findings indicate the important influence of the amino cyclohexyl moiety on BACE1

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inhibition. Furthermore, the inappropriate orientation of t-butyl derivatives may hinder access and prevent interaction between important functional groups of the ligand and the catalytic residues of the enzyme’s active site. -

Investigation of substituted moiety (R3) on amino cyclohexyl imidazopyridine core.

The effect of type of substituted group R3 on the aminocyclohexyl imidazopyridine core suggested that the introduction of methyl substitutes (R3=CH3) at different positions of imidazopyridine ring increases the inhibitory potency against BACE1 and the preference

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ACCEPTED MANUSCRIPT order for the position of methyl substitute is 7 > 5 > 6,8. The most potent compound of this series, 7-methylimidazopyridine derivative 8e (100% inhibition at 50 µM) demonstrated superior potency over its 5-methylimidazopyridine (8c), 6-methylimidazopyridine (8d) and 8methylimidazopyridine (8g) counterparts with 87.94, 63.70 and 69.43 % BACE1 inhibition at

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50 µM, respectively. Furthermore, the introduction of a small lipophilic electron-donating moiety such as a methyl group into the 3-aminocyclohexyl imidazopyridine derivatives increased the potency of compounds against BACE1. Finally, no significant difference in

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potency was observed between bromine and chlorine derivatives.

Effect of substituted group R1 into the phenoxy propyl linker

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Introduction of a methoxy substitute into the phenoxypropyl linker of the designed scaffold did not significantly alter the potency of compounds against BACE1. The exception was in the case of the amino cyclohexyl imidazopyridine bearing chlorine group at C6 position of imidazopyridine ring and methoxy group in linker part (compound 8i); 8i was four times

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more potent than its non-methoxylated counterpart in inhibiting BACE1 at 50µM (inhibition was 88.10 and 19.6%, respectively). We attribute this to the structural and conformational changes imposed by the spatial hindrance between the methoxy and cyclohexyl substitutes

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resulting in proper orientation of functional groups into the key residues and pockets of

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BACE1 active site.

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ACCEPTED MANUSCRIPT Table 1. BACE1 inhibitory activity of synthesized compounds 8a-o. N

N

R2 NH

O

R1

N

O

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O

% Inhibition at

% Inhibition at

R1

R2

R3

50 µM

10 µM

IC50 (µM)

8a

H

cyclohexyl

-

<10

<10

-

8b

OCH3

cyclohexyl

-

69.49(±7.52)

29.30(±6.12)

-

8c

H

cyclohexyl

5-CH3

87.94(±6.66)

61.54 (±7.16)

5.93 (±1.93)

8d

H

cyclohexyl

6-CH3

63.70(±5.26)

37.61(±6.62)

29.42 (±8.98)

8e

H

cyclohexyl

7-CH3

100

61.32(±6.14)

2.84 (±0.95)

8f

H

t-Butyl

7-CH3

18.32(±10.89)

24.24(±8.33)

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8g

H

cyclohexyl

8-CH3

69.43(±8.71)

44.42(±5.04)

8h

H

cyclohexyl

6-Cl

19.60(±6.86)

11.50(±13.19)

-

8i

OCH3

cyclohexyl

6-Cl

88.097(±7.53)

26.86(±8.82)

14.90(±8.98)

8j

H

t-Butyl

6-Cl

32.95(±11.30)

17.67 (±11.01)

8k

OCH3

t-Butyl

6-Cl

37.90(±1.38)

19.41(±5.85)

-

8l

H

cyclohexyl

6-Br

45.30(±5.83)

32.16(±10.05)

-

8m

OCH3

cyclohexyl

6-Br

43.17(±1.18)

36.71(±2.71)

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8n

H

t-Butyl

6-Br

34.28(±9.20)

7.87(±0.02)

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Compounds

8o

OCH3

t-Butyl

6-Br

48.72(±12.23)

33.19(±11.12)

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OM99-2

-

-

-

-

-

0.014 (±0.0028)

Values represent mean (± standard error of mean (S.E.M.) of three or four independent experiments.

2.3. Molecular docking study We performed molecular docking to better understand the Structure-activity relationship (SAR) of all of the synthesized compounds. To validate and optimize our docking protocol, 9

ACCEPTED MANUSCRIPT redocking of the co-crystallized conformation of a native ligand into the 4ACU active site was performed [22]. The binding pose of the top rank cognate ligand was superimposed over the X-ray crystallographic structure. The best-docked and the experimental conformations of the inhibitor correlate well with an RMSD of 0.601 Å and binding free energies of −12.934

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kcal/mol. This validated docking procedure was performed on all the compounds under investigation. Estimated free binding energies (∆Gb) and Ki for synthesized molecules are summarized in Table 2. All the ligand/receptor interactions were analyzed using Chimera

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1.11 and Viewer lite 4.2 software and following results were obtained:

- In almost all docked structures containing an amino cyclohexyl pendent at R2 and a

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different motif at R3 (compounds 8a, 8b, 8c, 8d, 8e and 8i) a key hydrogen bond interaction between the nitrogen atom of imidazopyridine and Asp228 of the catalytic dyad residues was observed. The Cyclohexyl moiety in these structures occupies the P'2 pocket

and phthalimide oriented toward flap pocket. The substituted

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imidazopyridines was surrounded by Thr231, Thr232, and Asn233. - As mentioned previously, replacing the cyclohexyl pendant with a t-butyl group reduced the inhibitory activity of the compounds. To investigate this observation, we

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performed computational analysis on of one of the most potent compounds, compound

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8e, (aminocyclohexyl derivative) and its amino t-butyl counterpart 8f. As it is depicted in Figure 2, 8e and 8f demonstrated different binding orientations in the active site of BACE1. As it is shown, the phthalimide moiety of 8e was directed toward the flap pocket of the active site surrounded by Phe108, Phe109 and Ileu118. Furthermore, the methyl imidazopyridine core was oriented toward the P2 pocket and amino cyclohexyl pendant occupied the P'2 pocket surrounded by Thr231, Thr232, Asn233 and Arg235. Moreover, Asp32 and Asp228 are involved in H-bond interaction with the oxygen atom of phenoxypropyl and the nitrogen atom of the imidazopyridine, respectively (Table 2).

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ACCEPTED MANUSCRIPT Surprisingly, the amino t-butyl derivative 8f, is inversely directed in the active site; the phthalimide is oriented toward P'2 pocket, amino t-butyl pendant and imidazopyridine are oriented toward P2 and flap pockets, respectively. Such an orientation partly hindered H-bond interaction of 8c with key catalytic Asp32 and partly justifies its poor

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BACE1 inhibitory potential. We conclude that the introduction of an amino t-butyl pendant into the imidazopyridine core imposes an unfavorable binding mode that hinders proper ligand-receptor interaction.

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– The results indicate that the introduction of a methyl substitute (representing a small electron donating group) into the imidazopyridine ring resulted in additional

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hydrophobic interactions with the hydrophobic residues of P2 sub-pocket, therefore increasing the BACE1 inhibitory activity as observed in the case of compounds 8c, 8d, 8e and 8g. Based on the above findings amino cyclohexyl derivatives 8e and 8c containing 7-CH3 and 5-CH3 on imidazopyridine core, respectively, were the most

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potent inhibitors against BACE1 and demonstrated a favorable orientation and H-bond interactions with key residues of catalytic site. As depicted in Figure 3, 8e was involved in hydrogen bond network with Asp32, Asp228 and Gln73 of catalytic site (∆Gb= -

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11.90 Kcal/mol).

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Table 2. Summary of molecular interactions, binding energy (∆Gb) and inhibition constant (Ki) of synthesize compounds with BACE1 active site

Compounds

8a

8b

Mean binding free energy (∆Gb) (kcal/mol)

-9.52

-11.67

Ki (nM)

Hydrogen bond interactions Atom of the ligand

104.73

2.77

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Amino acid

NH (amino cyclohexyl)

N of Thr 72

N (imidazopyridin)

O of Asp 228

N (imidazopyridin)

O of Asp 228

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-12.09

1.37

NH (amino cyclohexyl)

O of Thr 72

N (imidazopyridin)

O of Asp228

-11.68

2.74

N (imidazopyridin)

O of Asp 228

8e

-11.90

4.38

O (phenoxyethyl)

O of Asp 32

8i

8j

-11.37

-11.32

-10.24

-11.83

-11.02

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8k

8l

8m

8n

-10.89

-9.11

O of Asp 228

CO (phthalimide)

N of Thr 71

NH (amino t-Butyl)

O of Thr 231

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-11.70

N (imidazopyridin)

2.62

N (imidazopyridin)

O of Thr 72

NH (amino cyclohexyl)

O of Gly 34

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8h

20.13

4.59

5.03

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8g

-9.61

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8f

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8d

31.17

2.14

4.46 nM

10.33

209.27

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CO (phthalimide)

O of Asp 32

CO (phthalimide)

O of Gly 230

CO (phthalimide)

N of Thr 72

N (imidazopyridin)

O of Asp 228

CO ( phthalimide)

OH of Tyr 198

NH (amino t-Butyl)

O of Gly 230

CO (phthalimide)

N and O of Thr 232

N (imidazopyridin)

O of Thr 232

CO (phthalimide)

O of Phe 108

CO of phthalimide

O of Gly 230

NH (amino cyclohexyl)

N of Thr 72

CO (phthalimide)

O of Phe 108

CO (phthalimide)

O of Gly 230

N (imidazopyridin)

O of Thr 231

CO (phthalimide)

O of Gly 34

ACCEPTED MANUSCRIPT -11.18

14.67

CO (phthalimide)

O of Gln 34

NH (amino t-Butyl)

O of Gln 73

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8o

Figure 2. Comparative binding orientation of compound 8e (displayed in blue) and 8f

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(displayed in yellow) in the BACE1 active site.

Figure 3. Binding orientation of compound 8e (displayed in violet) in BACE1 active site. Hbonds are shown as green dash lines.. 13

ACCEPTED MANUSCRIPT Conclusion In this study, we synthesized and evaluated the anti-BACE1 activity of fifteen hybrid imidazopyridines containing phthalimide moieties. The goal was to discover novel small compounds with potentially improved BACE1 inhibitory properties. Our preliminary BACE1

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inhibitory results suggested that a cyclohexyl substitution at R2 was a key structure for improving the inhibitory activity. In the case of aminocyclohexyl containing derivatives, introducing a methyl substitute at 6 or 7 positions of the imidazopyridine core (as in 8d and

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8e) resulted in considerable improvement of BACE1 inhibitory potential. Besides, molecular docking simulation indicated that the amino t-butyl derivatives have different binding

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orientations in the active site of BACE1 in comparison with aminocyclohexyl derivatives. Moreover, all aminocyclohexyl derivatives showed favorable binding interactions with P2, P'2 and stacking pockets; imidazopyridines and phenoxypropyl linkers were involved in hydrogen bond interaction with Asp228 and Asp32 of BACE1 active site, respectively. The

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Phthalimide portion was oriented toward the flap pocket and demonstrated van der Waal’s and hydrophobic interactions with phe108, lle110, Trp115, Ile118. The results of the present study could guide the rational design of more potent BACE1 blocking agents.

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3.1. Chemistry

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3. Experimental Section

All chemicals were purchased from commercial sources (Merck and Aldrich) and used without further purification. Melting points were taken on a Kofler hot stage apparatus and were uncorrected. 1H- and 13CNMR spectra were recorded on Bruker FT-500 (Germany), using TMS as an internal standard. The IR spectra were obtained on a Nicolet MagnaFTIR 550 spectrometer (KBr disks). The elemental analysis was performed with an Elementar Analysensystem GmbH VarioEL CHNS mode (Germany).

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ACCEPTED MANUSCRIPT Synthesis of aldehyde 5: A mixture of phthalimide 1 (1 mmol), 1,2-dibromopropane 2 (1 mmol), and K2CO3 (1.3 mmol) in acetone (15 mL) was heated at reflux for 8 h. After completion of the reaction (checked by TLC), the mixture was poured into crushed ice and the precipitate was filtered of and dried to give 2-(3-bromopropyl)isoindoline-1,3-dione 3.

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Next, mixture of compound 3 (1 mmol), 4-hydroxyaldehyde derivative 4 (1 mmol), and K2CO3 (1.3 mmol) in DMF (10 mL) was heated at 80 °C for 8 h. Upon completion of the following reaction, the mixture was poured into crushed ice and the precipitate was filtered of

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and dried affording aldehyde 5.

Synthesis of imidazopyridine derivatives 8: A mixture of aldehyde 5 (1 mmol), 2-

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aminopyridins 6 (1 mmol), isocyanides 7 (1.2 mmol), NH4Cl (1 mmol) and toluene (10 mL) was heated under reflux conditions for 8-12 h. After completion of the reaction (checked by TLC), the solvent was evaporated under vacuum and the residue was recrystallized from ethanol to afford pure imidazopyridines 8.

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2-(3-(4-(3-(Cyclohexylamino)imidazo[1,2-a]pyridin-2-yl)phenoxy)propyl)isoindoline1,3-dione (8a). Yield: 79%, Mp: 182-184 °C, IR (KBr): 3205, 2917, 2850, 1771, 1705, 1610, 1523 cm-1; MS: m/z (%) 495 (M+, 100), 412(20), 385(24), 264(10), 225(7), 188(100), 170(6),

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160(55), 142(23), 130(12), 104 (4), 90(11), 79(26), 55(13); 1H NMR (CDCl3, 500 MHz):

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δH(ppm)= 8.50-8.46 (m, 3H, H5, H7, H8), 8.02 (d, J= 8.5 Hz, 2H, H3´, H5´), 7.86-7.85 (m, 2H, H1″, H4″), 7.74-7.72 (m, 2H, H2″, H3″), 6.87-6.85 (m, 3H, H2´, H6´, H6), 4.08 (t, J = 6.5 Hz, 2H, CH2), 3.94 (t, J = 6.5 Hz, 2H, CH2), 3.51 (bs, 1H, NH), 2.96-2.94 (m, 1H, CH), 2.22 (q, J= 6.5 Hz, 2H, CH2), 1.79-1.14 (m, 10H, H(cyclohexyl));

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C NMR (CDCl3, 125

MHz): δc(ppm) = 168.5, 159.1, 134.0, 132.1, 130.8, 129.1, 128.8, 127.3, 127.2, 123.3, 123.2, 122.1, 119.1, 117.9, 114.5, 65.6, 56.8, 35.4, 34.0, 28.3, 25.6, 24.7. Anal. Calcd for C29H28N4O3: C, 72.48; H, 5.87; N, 11.66. Found: C, 72.69; H, 5.64; N, 11.47.

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ACCEPTED MANUSCRIPT 2-(3-(4-(3-(Cyclohexylamino)imidazo[1,2-a]pyridin-2-yl)-2methoxyphenoxy)propyl)isoindoline-1,3-dione (8b). Yield: 71%, Mp: 181-183 °C, IR (KBr): 3228, 2923, 2849, 1773, 1708, 1611, 1503 cm-1; MS: m/z (%) 525 (M+, 30), 415 (4), 248(5), 228(7), 188(100), 160(45), 130(15), 104(8), 79(15), 57(10); 1H NMR (CDCl3, 500

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MHz): δH(ppm)= 8.47-8.44 (m, 3H, H5, H7, H8), 7.86-7.84 (m 2H, H1″, H4″), 7.76 (s, 1H, H3´), 7.73-7.72 (m, 2H, H2″, H3″), 7.59 (d, J= 8.0 Hz, 1H, H5′), 6.90 (m, 2H, H6 , H6´), 4.14 (t, J = 5.0 Hz, 2H, CH2), 3.95 (t, J = 5.0 Hz, 2H, CH2), 3.79 (s, 3H, OCH3), 3.48 (bs, 1H,

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NH), 3.00-2.99 (m, 1H, CH), 2.28 (q, J= 6.5 Hz, 2H, CH2), 1.81-1.16 (m, 10H,

H(cyclohexyl)); 13C NMR (CDCl3, 125 MHz): δc(ppm) = 168.4, 165.7, 160.1, 150.0, 149.5,

M AN U

133.8, 132.2, 131.6, 130.4, 127.5, 123.1, 122.8, 119.5, 117.8, 115.9, 112.7, 111.1, 66.9, 56.8, 56.0, 35.6, 34.1, 28.4, 25.6, 24.7. Anal. Calcd for C30H30N4O4: C, 70.57; H, 5.92; N, 10.97. Found: C, 70.34; H, 6.18; N, 11.23.

2-(3-(4-(3-(Cyclohexylamino)-5-methylimidazo[1,2-a]pyridin-2-

TE D

yl)phenoxy)propyl)isoindoline-1,3-dione (8c). Yield: 86%, Mp:204-206 °C, IR (KBr): 3325, 3050, 2926, 2851, 1770, 1712, 1609, 1562 cm-1; MS: m/z (%) 508 (M+, 88), 425(100),

EP

398(25), 264(7), 238(10), 211(12), 188(62), 160(37.5), 130(14), 92(50), 65(14). 1H NMR (CDCl3, 500 MHz): δH(ppm)= 7.83-7.85 (m, 4H, H3´, H5´, H1″, H4″), 7.73-7.71 (m, 2H,

AC C

H3″, H2″), 7.46 (d, J= 7.0 Hz, 1H, H6), 7.03 (t, J = 7.0 Hz, 1H, H7), 6.84 (d, J= 8.5 Hz, 2H, H2´, H6´), 6.45 (d, J= 7.0 Hz, 1H, H8), 4.07 (t, J = 6.5 Hz, 2H, CH2), 3.94 (t, J = 6.5 Hz, 2H, CH2), 3.25 (s, 1H, NH), 2.94 (s, 3H, CH3), 2.76-2.74 (m, 1H, CH), 2.21 (q, J= 6.5 Hz, 2H, CH2), 1.65-1.04 (m, 10H, H(cyclohexyl)); 13C NMR (CDCl3, 125 MHz): δc(ppm) =168.3, 161.1, 158.2, 149.3, 142.3, 136.6, 133.9, 132.1, 128.8, 125.9, 124.9, 123.2, 114.8, 114.2, 113.9, 65.5, 58.4, 35.5, 32.1, 28.3, 25.7, 24.8, 20.1. Anal. Calcd for C30H30N4O3: C, 72.85; H, 6.11; N, 11.33. Found: C, 73.10; H, 6.31; N, 11.56.

16

ACCEPTED MANUSCRIPT 2-(3-(4-(3-(Cyclohexylamino)-6-methylimidazo[1,2-a]pyridin-2yl)phenoxy)propyl)isoindoline-1,3-dione (8d). Yield:81%, Mp:175-177 °C, IR (KBr): 3462, 2931, 2853, 1771, 1708 1600, 1561 cm-1; MS: m/z (%) 508 (M+, 1), 482(14), 374(10), 212(7), 188(100), 160(40), 130(12), 104(3), 83(4), 55(14). 1H NMR (CDCl3, 500 MHz):

RI PT

δH(ppm)= 8.57 (d, J= 9.0 Hz, 2H, H3´, H5´), 7.97 (s, 1H, H5), 7.86-7.84 (m, 2H, H1″, H4″), 7.74-7.72 (m, 2H, H3″, H2″), 7.27-7.26 (m, 2H, H6, H7), 6.87 (d, J= 9.0 Hz, 2H, H2´, H6´), 4.61 (s, 1H, NH), 4.13 (t, J = 6.5 Hz, 2H, CH2), 3.93 (t, J = 6.5 Hz, 2H, CH2), 2.27 (q, J= 6.5

SC

Hz, 2H, CH2), 2.24 (s, 3H, CH3), 2.26-2.22 (m, 1H, CH), 1.91-1.25 (m, 10H, H(cyclohexyl)); 13

C NMR (CDCl3, 125 MHz): δc(ppm) = 170.7, 169.3, 165.8, 163.4, 157.0, 151.9, 140.1,

M AN U

138.2, 134.0, 133.0, 132.0, 123.4, 133.3, 117.8, 114.7, 65.9, 60.6, 35.2, 33.0, 28.4, 28.1, 25.5, 23.9. Anal. Calcd for C30H30N4O3: C, 72.85; H, 6.11; N, 11.33. Found: C, 73.11; H, 6.21; N, 11.12.

2-(3-(4-(3-(Cyclohexylamino)-7-methylimidazo[1,2-a]pyridin-2-

TE D

yl)phenoxy)propyl)isoindoline-1,3-dione (8e). Yield: 67%, Mp:193-195 °C, IR (KBr): 3224, 2926, 2851, 1769, 1712, 1656, 1607 cm-1; MS: m/z (%) 508 (M+, 85), 425 (100), 1

H NMR

EP

398(20), 258(5), 237(5), 211(9), 188(83), 160(50), 130(10), 92(40), 65(10).

(CDCl3, 500 MHz): δH(ppm)= 8.25 (d, J = 7.0 Hz, 1H, H5), 7.97 (d, J= 8.5 Hz, 2H, H3´,

AC C

H5´), 7.88-7.86 (m, 2H, H1″, H4″), 7.77-7.75 (m, 2H, H2″, H3″), 7.68 (s, 1H, H8), 6.86 (d, J= 7.0 Hz, 1H, H6), 6.72 (d, J= 8.5 Hz, 2H, H2´, H6´), 4.84 (bs, 1H, NH), 4.02 (t, J = 6.5 Hz, 2H, CH2), 3.94 (t, J = 6.5 Hz, 2H, CH2), 2.86-2.82 (m, 1H, CH), 2.37 (s, 3H, CH3), 2.22 (q, J= 6.5 Hz, 2H, CH2), 1.74-1.10 (m, 10H, H(cyclohexyl));

13

C NMR (CDCl3, 125 MHz):

δc(ppm) =168.4, 159.2, 143.7, 136.6, 134.1, 132.0, 128.2, 126.8, 124.0, 123.5, 123.3, 118.5, 118.2, 114.5, 111.2, 65.6, 56.2, 35.3, 33.7, 28.3, 25.5, 24.6, 21.4. Anal. Calcd for C30H30N4O3: C, 72.85; H, 6.11; N, 11.33. Found: C, 72.64; H, 6.31; N, 11.18.

17

ACCEPTED MANUSCRIPT 2-(3-(4-(3-(tert-butylamino)-7-methylimidazo[1,2-a]pyridin-2yl)phenoxy)propyl)isoindoline-1,3-dione (8f). Yield: 79%, Mp:201-203°C, IR (KBr): 3297, 3058, 2927, 2852, 1773, 1712, 1606, 1584 cm-1; MS: m/z (%) 482 (M+, 20), 425 (100), 398 (15), 237(3), 211(3), 188(50), 160(30), 130(7), 92(25), 65 (10). 1H NMR (CDCl3, 500 MHz):

RI PT

δH(ppm)= 8.07 (d, J= 7.0 Hz, 1H, H5), 7.85-7.83 (m, 2H, H1″, H4″), 7.79 (d, J= 8.5 Hz, 2H, H3´, H5´), 7.72-7.70 (m, 2H, H2″, H3″), 7.28 (s, 1H, H8), 6.58 (d, J= 8.5 Hz, 2H, H2´, H6´), 6.58 (d, J = 7.0 Hz, 1H, H6), 4.07 (t, J = 6.5 Hz, 2H, CH2), 3.93 (t, J = 6.5 Hz, 2H, CH2),

SC

3.05 (s, 1H, NH), 2.28 (q, J= 6.5 Hz, 2H, CH2), 2.37 (s, 3H, CH3), 1.02 (s, 9H, H(t-Bu)); 13C NMR (CDCl3, 125 MHz): δc(ppm) = 168.3, 157.9, 142.1, 138.6, 134.7, 133.8, 132.1, 129.1,

M AN U

127.8, 123.1, 122.6, 122.3, 115.3, 114.0, 113.8, 65.6, 56.2, 35.5, 30.2, 28.4, 21.2. Anal. Calcd for C28H28N4O3: C, 71.78; H, 6.02; N, 11.96. Found: C, 71.59; H, 5.87; N, 12.22. 2-(3-(4-(3-(Cyclohexylamino)-8-methylimidazo[1,2-a]pyridin-2yl)phenoxy)propyl)isoindoline-1,3-dione (8g). Yield: 94%, Mp:185-187 °C, IR (KBr):

TE D

3292, 2929, 2851, 1771, 1716, 1610, 1559 cm-1; MS: m/z (%) 508 (M+, 88), 425(100), 398(37), 325(40), 264(5), 238(6), 211(8), 188(47), 160(37), 130(9), 92(45), 65(12); 1H NMR

EP

(CDCl3, 500 MHz): δH(ppm)= 7.96 (d, J= 6.5 Hz, 1H, H5), 7.90 (d, J= 9.0 Hz, 2H, H3´, H5´), 7.86-7.83 (m, 2H, H1″, H4″), 7.74-7.71 (m, 2H, H2″, H3″), 6.91 (d, J= 6.5 Hz, 1H, H7), 6.87

AC C

(d, J= 9.0 Hz, 2H, H2´, H6´), 6.69 (t, J = 6.5 Hz, 1H, H6), 4.08(t, J = 6.5 Hz, 2H, CH2), 3.93 (t, J = 6.5 Hz, 2H, CH2), 3.12 (bs, 1H, NH), 2.92-2.88 (m, 1H, CH), 2.62 (s, 3H, CH3), 2.21 (q, J= 6.5 Hz, 2H, CH2), 1.80-1.13 (m, 10H, H(cyclohexyl));

13

C NMR (CDCl3, 125 MHz):

δc(ppm) =168.3, 158.0, 144.6, 142.0, 133.9, 132.1, 128.4, 126.8, 124.5, 123.2, 122.6, 120.5, 118.8, 114.4, 111.5, 65.6, 56.8, 35.5, 34.1, 28.3, 25.7, 24.7, 16.6. Anal. Calcd for C30H30N4O3: C, 72.85; H, 6.11; N, 11.33. Found: C, 73.10; H, 5.87; N, 11.15. 2-(3-(4-(6-Chloro-3-(cyclohexylamino)imidazo[1,2-a]pyridin-2yl)phenoxy)propyl)isoindoline-1,3-dione (8h). Yield: 69%, Mp:255-257 °C, IR (KBr): 18

ACCEPTED MANUSCRIPT 3250, 3049, 2927, 2852, 1770, 1711, 1608, 1561 cm-1; MS: m/z (%) 528 (M+, 90), 445 (60), 418(20), 258(5), 258(7), 231(14), 188(100), 160(65), 130(15), 112(25), 76(10), 57(15); 1H NMR (CDCl3, 500 MHz): δH(ppm)= 8.15 (s, 1H, H5), 7.90 (d, J= 9.0 Hz, 2H, H5´, H3´), 7.86-7.85 (m, 2H, H1″, H4′′), 7.74-7.72 (m, 2H, H2″, H3′′), 7.52 (d, J= 9.5 Hz, 1H, H8), 7.11

RI PT

(d, J= 9.5 Hz, 1H, H7), 6.87 (d, J= 9.0 Hz, 2H, H2´, H6´), 4.08 (t, J = 6.5 Hz, 2H, CH2), 3.94 (t, J = 6.5 Hz, 2H, CH2), 3.28 (s, 1H, NH), 2.94-2.93 (m, 1H, CH), 2.27 (q, J= 6.5 Hz, 2H, CH2), 1.79-1.15 (m, 10H, H(cyclohexyl));

13

C NMR (CDCl3, 125 MHz): δc(ppm) = 168.4,

SC

158.5, 141.5, 133.9, 132.1, 131.0, 129.0, 128.2, 126.2, 125.9, 124.4, 123.2, 120.7, 116.9, 114.5, 65.6, 56.7, 35.4, 34.0, 28.4, 25.6, 24.7. Anal. Calcd for C29H27ClN4O3: C, 67.63; H,

M AN U

5.28; N, 10.88. Found: C, 67.45; H, 5.41; N, 10.63.

2-(3-(4-(6-Chloro-3-(cyclohexylamino)imidazo[1,2-a]pyridin-2-yl)-2methoxyphenoxy)propyl) isoindoline-1,3-dione (8i). Yield: 82%, Mp: 189-191 °C, IR (KBr): 3295, 3058, 2926, 2853, 1774, 1712, 1605, 1584 cm-1; MS: m/z (%) 558 (M+, 55),

TE D

475(14), 448 (5), 261(7), 188(100), 160(50), 130(10), 112(15), 76(5), 57(8);

1

H NMR

(CDCl3, 500 MHz): δH(ppm)= 8.16 (s, 1H, H5), 7.86-7.84 (m, 2H, H1″, H4″), 7.73-7.71 (m,

EP

2H, H2″, H3″), 7.60 (d, J= 1.5 Hz, 1H, H3´), 7.51 (d, J = 9.0 Hz, 1H, H8), 7.47 (dd, J= 8.0, 1.5 Hz, 1H, H5´), 7.13 (d, J= 9.0 Hz, 1H, H7), 6.88 (d, J= 8.0 Hz, 1H, H6´), 4.13 (t, J = 6.5

AC C

Hz, 2H, CH2), 3.94 (t, J = 6.5 Hz, 2H, CH2), 3.81 (s, 3H, OCH3), 3.46 (bs, 1H, NH), 2.982.97 (m, 1H, CH), 2.22 (q, J= 6.5 Hz, 2H, CH2), 1.80-1.15 (m, 10H, H(cyclohexyl));

13

C

NMR (CDCl3, 125 MHz): δc(ppm) = 168.4, 164.5, 155.8, 150.0, 149.4, 148.1, 137.7, 133.8, 132.2, 131.1, 124.6, 123.2, 120.7, 119.2, 116.6, 112.8, 110.6, 66.8, 56.6, 55.9, 35.5, 34.0, 28.3, 25.6, 24.7. Anal. Calcd for C30H29ClN4O4: C, 66.11; H, 5.36; N, 10.28. Found: C, 65.86; H, 5.58; N, 10.41. 2-(3-(4-(3-(tert-butylamino)-6-chloroimidazo[1,2-a]pyridin-2yl)phenoxy)propyl)isoindoline-1,3-dione (8j). Yield: 74%, Mp: 229-231 °C, IR (KBr): 19

ACCEPTED MANUSCRIPT 3289, 2967, 2877, 1770, 1714, 1612, 1557 cm-1; MS: m/z (%) 502 (M+, 29), 445 (70), 418(15), 258(5), 231(7), 188(100), 160(60), 130(10), 112(20), 76(7), 57(8); 1H NMR (CDCl3, 500 MHz): δH(ppm)= 8.27 (s, 1H, H5), 7.86-7.84 (m, 2H, H1″, H4″), 7.78 (d, J= 8.5 Hz, 2H, H3´, H5´), 7.73-7.72 (m, 2H, H2″, H3″), 7.55 (d, J= 9.5 Hz, 1H, H8), 7.14 (d, J= 9.5 Hz, 1H,

RI PT

H7), 6.85 (d, J= 8.5 Hz, 2H, H2´, H6´), 4.07 (t, J = 6.5 Hz, 2H, CH2), 3.94 (t, J = 6.5 Hz, 2H, CH2), 3.23 (bs, 1H, NH), 2.28 (q, J= 6.5 Hz, 2H, CH2), 1.04 (s, 9H, H(t-Bu)); 13C NMR

(CDCl3, 125 MHz): δc(ppm) = 168.4, 158.6, 137.9, 133.9, 132.1, 129.3, 128.1, 126.1, 123.3,

SC

123.2, 122.6, 121.4, 120.5, 116.9, 114.3, 65.6, 56.5, 35.4, 30.3, 28.4. Anal. Calcd for

M AN U

C27H25ClN4O3: C, 66.32; H, 5.15; N, 11.46. Found: C, 66.54; H, 5.34; N, 11.67. 2-(3-(4-(3-(tert-butylamino)-6-chloroimidazo[1,2-a]pyridin-2-yl)-2methoxyphenoxy)propyl)isoindoline-1,3-dione (8k). Yield: 67%, Mp:267-269 °C, IR (KBr): 3290, 3079, 2929, 2851, 1772, 1711, 1610, 1559 cm-1; MS: m/z (%) 604 ((M+2)+, 20), 602 (M+, 20), 520 (34), 522(34), 506(7), 504(7), 399(68), 360(56), 325(45), 291(100),

1

TE D

259(10), 261(), 188(100), 160(44), 151(20), 125(75), 104(4), 97(18), 57(12). H NMR (CDCl3, 500 MHz): δH(ppm)= 8.28 (s, 1H, H5), 7.85-7.84 (m, 2H, H1″, H4″), 7.72-

EP

7.71 (m, 2H, H2″, H3″), 7.54 (d, J= 9.0 Hz, 1H, H8), 7.47 (d, J = 1.5 Hz, 1H, H3´), 7.38 (d, J= 8.0 Hz, 1H, H5´), 7.15 (d, J= 9.0 Hz, 1H, H7), 6.87 (d, J= 8.0. Hz, 1H, H6´), 4.12 (t, J =

AC C

6.5 Hz, 2H, CH2), 3.94 (t, J = 6.5 Hz, 2H, CH2), 3.80 (s, 3H, OCH3), 3.34 (bs, 1H, NH), 2.22 (q, J= 6.5 Hz, 2H, CH2), 1.05 (s, 9H, H(t-Bu));

13

C NMR (CDCl3, 125MHz): δc(ppm) =

168.3, 149.5, 148.0, 141.9, 133.8, 132.2, 131.4, 126.8, 125.4, 123.5, 123.2, 122.7, 121.5, 120.5, 116.6, 112.7, 111.7, 66.9, 56.0, 55.9, 35.5, 30.3, 28.2. Anal. Calcd for C28H27ClN4O4: C, 64.80; H, 5.24; N, 10.80. Found: C, 64.59; H, 5.51; N, 10.71. 2-(3-(4-(6-Bromo-3-(cyclohexylamino)imidazo[1,2-a]pyridin-2yl)phenoxy)propyl)isoindoline-1,3-dione (8l).Yield: 85%, Mp: 263-265°C, IR (KBr): 3248, 3045, 2928, 2852, 1770, 1711, 1608, 1561 cm-1; MS: m/z (%) 574 ((M+2)+, 40), 574 (M+, 20

ACCEPTED MANUSCRIPT 40), 493(25), 491(25), 466(10), 464(8), 277(5), 275(5), 188(100), 160(60), 130(20), 104(5), 77(7), 55(10). 1H NMR (CDCl3, 400 MHz): δH (ppm) = 8.28 (s, 1H, H5), 7.91 (d, J = 8.5 Hz, 2H, H3′, H5′), 7.87-8.50 (m, 2H, H1″, H4′′), 7.74-7.72 (m, 2H, H2′′, H3′′), 7.54 (d, J = 8.0 Hz, 1H, H8), 7.17 (d, J = 8.0 Hz, 1H, H7), 6.85 (d, J = 8.5 Hz, 2H, H2´, H6´), 4.08 (t, J = 6.5

RI PT

Hz, 2H, CH2), 3.94 (t, J = 6.5 Hz, 2H, CH2), 3.52 (bs, 1H, NH), 3.00-2.80 (m, 1H, CH), 2.20 (q, J= 6.5 Hz, 2H, CH2), 1.78- 1.15 (m, 10H, H (cyclohexyl)); 13C NMR (CDCl3, 100 MHz): δc (ppm) =168.4, 158.6, 157.2, 144.8, 143.2, 136.3, 135.1, 133.9, 132.1, 128.3, 127.0, 124.3,

SC

123.2, 123.1, 114.6, 65.6, 56.6, 35.4, 34.0, 28.3, 25.5, 24.7.Anal. Calcd for C29H27BrN4O3: C,

M AN U

62.26; H, 4.86; N, 10.01. Found: C, 62.41; H, 4.62; N, 9.86.

2-(3-(4-(6-Bromo-3-(cyclohexylamino)imidazo[1,2-a]pyridin-2-yl)-2methoxyphenoxy)propyl) isoindoline-1,3-dione (8m).Yield: 83%, Mp:196-198°C, IR (KBr): 3294, 3047, 2992, 2926, 2852, 1773, 1710, 1606, 1583 cm-1; MS: m/z (%) 604 ((M+2)+, 20), 602 (M+, 20), 520 (34), 522(34), 506(7), 504(7), 399(68), 360(56), 325(45),

1

TE D

291(100), 259(10), 261(), 188(100), 160(44), 151(20), 125(75), 104(4), 97(18), 57(12). H NMR (CDCl3, 500 MHz): δH (ppm)= 8.24 (s, 1H, H5), 7.86-7.83 (m, 2H, H1″, H4′′),

EP

7.73-7.71 (m, 2H, H2″, H3′′), 7.60 (d, J = 2.0 Hz, 1H, H3′), 7.49 (dd, J = 8.5, 2.0 Hz, 1H, H5´), 7.44 (d, J = 9.0 Hz, 1H, H8), 7.20 (d, J = 9.0 Hz, 1H, H7), 6.89 (d, J = 8.5 Hz, 1H,

AC C

H6´), 4.14 (t, J = 6.5 Hz, 2H, CH2), 3.94 (t, J= 6.5 Hz, 2H, CH2), 3.81 (s, 3H, OCH3), 3.28 (bs, 1H, NH), 3.10-2.98 (m, 1H, CH), 2.24 (q, J= 6.5 Hz, 2H, CH2), 1.80-1.15 (m, 10H, H(cyclohexyl));

13

C NMR (CDCl3, 125 MHz): δc(ppm) =168.3, 155.8, 149.4, 148.0, 145.1,

143.5, 139.8, 133.8, 132.1, 128.0, 124.4, 123.1, 122.8, 119.2, 117.2, 112.8, 110.62, 66.8, 56.7, 55.9, 35.5, 34.0, 28.1, 25.6, 24.7.Anal. Calcd for C30H29BrN4O4: C, 61.13; H, 4.96; N, 9.50. Found: C, 61.37; H, 5.27; N, 9.74. 2-(2-(4-(6-Bromo-3-(tert-butylamino)imidazo[1,2-a]pyridin-2yl)phenoxy)propyl)isoindoline-1,3-dione (8n).Yield: 78%, Mp: 233-235°C, IR (KBr): 21

ACCEPTED MANUSCRIPT 3231, 3066, 2926, 2852, 1769, 1714, 1657, 1606, 1572 cm-1; MS: m/z (%) 548 ((M+2)+, 25), 546 (M+, 25), 491(30), 489(30), 464(12), 462(12), 411(7), 341(5),188(100), 160(80), 130(15), 85 (20), 77(23), 55(40); 1H NMR (CDCl3, 500 MHz): δH(ppm)= 8.35 (s, 1H, H5), 7.86-7.84 (m, 2H, H1″, H4′′), 7.78 (d, J= 8.0 Hz, 2H, H3´, H5´), 7.73-7.72 (m, 2H, H2″,

RI PT

H3′′), 7.48 (d, J= 9.0 Hz, 1H, H8), 7.21 (d, J=9.0 Hz, 1H, H7), 6.85 (d, J= 8.0 Hz, 2H, H2´, H6´), 4.07 (t, J = 6.5 Hz, 2H, CH2), 3.93 (t, J = 6.5 Hz, 2H, CH2), 3.21 (bs, 1H, NH), 2.22 (q, J= 6.5 Hz, 2H, CH2), 1.04 (s, 9H, H (t-Bu)); 13C NMR (CDCl3, 125 MHz): δc(ppm) = 168.3,

SC

158.5, 150.6, 143.1, 139.2, 136.1, 133.9, 132.1, 129.3, 128.2, 123.7, 123.2, 117.2, 115.9, 114.3, 65.6, 56.5, 35.5, 30.3, 28.3. Anal. Calcd for C27H25BrN4O3: C, 60.79; H, 4.72; N,

M AN U

10.50. Found: C, 60.58; H, 4.58; N, 10.74.

2-(3-(4-(6-Bromo-3-(tert-butylamino)imidazo[1,2-a]pyridin-2-yl)-2methoxyphenoxy)propyl) isoindoline-1,3-dione (8o). Yield: 71%, Mp:165-167°C, IR (KBr): 3295, 3048, 2992, 2926, 2852, 1774, 1710, 1606, 1583 cm-1; MS: m/z (%) 578

TE D

((M+2)+, 10), 576 (M+, 10), 521(20), 519 (18), 441(4), 188(100), 160(40), 130(7), 104(4), 77(7), 55(9); 1H NMR (CDCl3, 500 MHz): δH(ppm)= 8.35 (s, 1H, H5), 7.85-7.83 (m, 2H,

EP

H1″, H4′′), 7.72-7.71 (m, 2H, H2″, H3′′), 7.47-7.45 (m, 2H, H8, H3´), 7.36 (d, J= 8.0 Hz, 1H, H5´), 7.21 (d, J= 9.5Hz, 1H, H7), 6.89 (d, J= 8.0 Hz, 1H, H6´), 4.13 (t, J= 6.5 Hz, 2H, CH2),

AC C

3.94 (t, J= 6.5 Hz, 2H, CH2), 3.80 (s, 3H, OCH3), 3.21 (bs, 1H, NH), 2.21 (q, J= 6.5 Hz, 2H, CH2), 1.05 (s, 9H, H(t-Bu)); 13C NMR (CDCl3, 125 MHz): δc(ppm) = 168.3, 161.2, 149.4, 148.1, 139.5, 135.9, 133.8, 132.2, 127.9, 123.6, 123.3, 123.1, 120.5, 117.3, 114.8, 112.7, 111.7, 66.9, 56.5, 55.9, 35.5, 30.3, 28.3. Anal. Calcd for C28H27BrN4O4: C, 59.69; H, 4.83; N, 9.94. Found: C, 59.27; H, 4.62; N, 10.24. 3.2. BACE1 enzymatic assay Enzyme inhibition assay was carried out using BACE1 (β-secretase) FRET assay kit, from Invitrogen (former Pan Vera, Madison, WI) according to the manufacturer instructions [16, 22

ACCEPTED MANUSCRIPT 23]. The analysis was carried out according to the manufacturer’s instructions with small modifications. Stock solutions of all tested compounds were prepared in DMSO and further diluted with the assay buffer (50 mM sodium acetate; pH 4.5). Ten µl of BACE1 substrate (Rh-EVNLDAEFK-Quencher) was mixed with 10 µL of each tested compound and then 10

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µL of enzyme (1 U/mL) was added to start the reaction. After around 90 min of incubation at room temperature, 10 µL of the stop solution (2.5 M sodium acetate) was applied to stop the reaction. The fluorescence was monitored at 545 nm (Ex) and 585 nm (Em) [19, 27]. The

plates and OM99 was used as a reference inhibitor agent.

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3.3. Molecular docking

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assays were performed in triplicate for each concentration in a 96-well polystyrene black

Flexible-ligand docking studies were carried out using AutoDock version (1.5.4). X-ray crystallographic holo structures of BACE1 were retrieved from the Brookhaven Protein Data Bank (4acu; http://www.rcsb.org/). Water molecules and cognate ligand were removed from

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the 4acu and hydrogen atoms were added to the receptor PDB. Next, non-polar hydrogens were merged into related carbon atoms of the receptor and Kollman charges were also assigned. Structures of the desired ligands were constructed using HyperChem software

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version 8.0.10 and energetically minimized using MM+ force field. The active sites were

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defined based on the center and radius of cognate ligand which are mentioned in X-ray crystal structure. Docking computations were performed using the Lamarkian genetic algorithm with grid sizes 60× 60×60 (grid spacing 0.375 Å), the maximum number of evaluations were set to 2500000, the number of GA runs were 150 and the maximum number of generations were set as 27000 and all other options were set as default. The lowest energy conformation of the highest populated cluster was selected for analysis. Schematic 2D representations of the ligand–receptor interactions were all generated using Chimera 1.11. Acknowledgement 23

ACCEPTED MANUSCRIPT The authors gratefully acknowledge financial support from Tehran University of Medical Sciences. The authors also acknowledge the financial support of Shiraz University of Medical Sciences, Vice Chancellor of Research (Grant: 12-12200). We thank Alireza Edraki and

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Samantha J. Nelson from Umass Medical School, MA, USA for thorough editing of this manuscript

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ACCEPTED MANUSCRIPT Highlights

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A novel class of imidazopyridine- phthalimide hybrids was rationally designed and synthesized as novel anti-Alzheimer agents. All compounds were examined for their BACE1 inhibitory potential. Compound 8e was the most potent against BACE1 with an IC50 value of 2.84 (±0.95) µM. Molecular docking revealed the key binding interactions of 8e in BACE1 active site

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