Novel Molecular Hybrids of N-Benzylpiperidine and 1,3,4-Oxadiazole as Multitargeted Therapeutics to Treat Alzheimer's Disease

Article abstract of DOI:10.1021/acschemneuro.9b00430

Multitargeted hybrids of N-benzylpiperidine and substituted 5-phenyl-1,3,4-oxadiazoles were designed, synthesized, and evaluated against Alzheimer's disease (AD). Tested compounds exhibited moderate to excellent inhibition against human acetylcholinesterase (hAChE), butyrylcholinesterase (hBChE), and beta-secretase-1 (hBACE-1). The potential leads 6g and 10f exhibited balanced inhibitory profiles against all the targets, with a substantial displacement of propidium iodide from the peripheral anionic site of hAChE. Hybrids 6g and 10f also elicited favorable permeation across the blood-brain barrier and were devoid of neurotoxic liability toward SH-SY5Y neuroblastoma cells. Both leads remarkably disassembled Aβ aggregation in thioflavin T-based self- and AChE-induced experiments. Compounds 6g and 10f ameliorated scopolamine-induced cognitive dysfunctions in the Y-maze test. The ex vivo studies of rat brain homogenates established the reduced AChE levels and antioxidant activity of both compounds. Compound 6g also elicited noteworthy improvement in Aβ-induced cognitive dysfunctions in the Morris water maze test with downregulation in the expression of Aβ and BACE-1 proteins corroborated by Western blot and immunohistochemical analysis. The pharmacokinetic study showed excellent oral absorption characteristics of compound 6g. The in silico molecular docking and dynamics simulation studies of lead compounds affirmed their consensual binding interactions with PAS-AChE and aspartate dyad of BACE-1.

Full text of DOI:10.1021/acschemneuro.9b00430

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Article
Novel Molecular Hybrids of N-Benzylpiperidine and 1,3,4-Oxadiazole
as Multitargeted Therapeutics to Treat Alzheimer’s Disease
Piyoosh Sharma, Avanish Tripathi, Prabhash Nath Tripathi, SAUMITRA
SEN SINGH, Surya Pratap Singh, and Sushant K. Shrivastava
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00430 • Publication Date (Web): 06 Sep 2019
Downloaded from pubs.acs.org on September 7, 2019
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Novel Molecular Hybrids of N-Benzylpiperidine and
1,3,4-Oxadiazole as Multitargeted Therapeutics to
Treat Alzheimer’s Disease
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‡
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Piyoosh Sharma, Avanish Tripathi, Prabhash Nath Tripathi, Saumitra Sen Singh, Surya Pratap
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‡*
Singh, Sushant Kumar Shrivastava
‡
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Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology
(Banaras Hindu University), Varanasi 221 005, India
†
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Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi 221 005,
India
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ABSTRACT: Multitargeted hybrids of N-benzylpiperidine and substituted 5-phenyl-1,3,4-
oxadiazoles were designed, synthesized, and evaluated against Alzheimer’s disease (AD). Tested
compounds exhibited moderate to excellent inhibition against human acetylcholinesterase
(hAChE), butyrylcholinesterase (hBChE), and beta-secretase-1 (hBACE-1). The potential leads
6g and 10f exhibited balanced inhibitory profiles against all the targets, with a substantial
displacement of propidium iodide from the peripheral anionic site of hAChE. Hybrids 6g and 10f
also elicited favorable permeation across the blood-brain barrier and were devoid of neurotoxic
liability towards SH-SY5Y neuroblastoma cells. Both leads remarkably disassembled Aβ
aggregation in thioflavin T based self- and AChE-induced experiments. Compounds 6g and 10f
ameliorated scopolamine-induced cognitive dysfunctions in the Y-maze test. The ex vivo studies
of rat brain homogenates established the reduced AChE levels and antioxidant activity of both the
compounds. Compound 6g also elicited noteworthy improvement in Aβ-induced cognitive
dysfunctions in Morris water maze test with downregulation in the expression of Aβ and BACE-
1 proteins corroborated by Western blot and immunohistochemical analysis. The pharmacokinetic
study showed excellent oral absorption characteristics of compound 6g. The in silico molecular
docking and dynamics simulation studies of lead compounds affirmed their consensual binding
interactions with PAS-AChE and aspartate dyad of BACE-1.
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KEYWORDS: Multitargeted hybrids; Acetylcholinesterase (AChE), Butyrylcholinesterase
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(BChE); β-secretase-1 (BACE-1); Alzheimer’s disease.
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INTRODUCTION
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Alzheimer’s disease (AD) is the most prevalent neurocognitive disorder characterized by
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memory loss, defective speech, confusion, mood, and behavioral alterations. The AD is
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responsible for functionally and structurally damaging neuronal cells, ultimately leading to cell
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death. Recently published report of WHO predicted an increase the number of patients suffering
from this devastating disorder would be tripled, from 50–150 million, which would create
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significant healthcare, social, and economic burden on the patients and their families. AD is
multifactorial progressive disorder, interconnected with several pathophysiological hallmarks such
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as decline in acetylcholine (ACh) levels, deposition and aggregation of amyloid-beta (Aβ), N-
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methyl-D-aspartate receptor (NMDAR) activation, tau hyperphosphorylation and production of
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neurofibrillary tangles, inflammation of CNS and neuronal cells, gene transcription by CREB
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(cAMP response element-binding protein) signaling mechanisms, and oxidative stress.
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The currently available therapeutic regimens include AChE inhibitors (donepezil, rivastigmine,
and galantamine), and NMDAR blocker (memantine).11,12 However, the treatment approaches
available for AD are still at its infancy and not affecting the actual course of AD progression. The
recently adopted multitarget-directed ligands (MTDLs) strategy involves simultaneous targeting
at multiple pathogenic mechanisms of AD by a single molecule. This approach was invoked owing
to the complex and multifaceted nature of AD, and is based on simultaneous interaction with
multiple targets; to halt the progression of the disease. The available medications could be taken
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in combination to affect several targets, but combination therapy is also marred with several pitfalls
such as drug-drug interactions, bioavailability issues, metabolism, and patient compliance.13
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The cholinesterase (ChE) enzymes are of two types: acetyl- and butyrylcholinesterase (AChE
and BChE). The ChE inhibitors are the most important therapeutics responsible for halting the
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hydrolytic breakdown of ACh into acetic acid and choline to increase the levels of ACh in neuronal
cells. Several clinical evidences suggested the significant role of BChE in the regulation of ACh
and maintaining normal cholinergic functions, which makes BChE an additional promising target
against AD.14-16 Therefore, dual targeting of both ChEs (AChE and BChE) could render
therapeutic advantage in advanced and late phase AD. Moreover, both ChEs are well-
acknowledged for their substantial role in Aβ aggregation.17 The Aβ is an insoluble protein
fragment formed from catalytic proteolysis of amyloid precursor protein by beta-secretase-1
(BACE-1). The accumulation of Aβ aggregates leads to a sequence of reactions such as the
formation of oligomers, fibrillary rods, or β-sheets, which are responsible for progressive neuritic
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injury, and cognitive impairment. Furthermore, the accumulation of Aβ aggregates could also
lead to the generation of free radicals in mitochondria, and thereby causing oxidative stress.19
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Therefore, multitargeted therapeutics with inhibition of ChEs (dual AChE and BChE), BACE-1,
and Aβ aggregation along with antioxidant potential might prove to be fruitful in modifying the
progression of AD, rather than providing only symptomatic relief.
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A series of MTDLs are designed based on the structural framework of donepezil, which is the
most potent and highly prescribed drug for the treatment of advanced stage of AD. An investigation
into the SAR of donepezil suggested a drastic decline in AChE inhibitory activity upon
modification or substitution at the benzyl part of N-benzylpiperidine moiety.20 Also, N-
benzylpiperidine ring of donepezil was observed to be extended into the catalytic anionic site
(CAS), and terminal indanone nucleus was oriented in PAS of AChE.21,22 Moreover, N-
benzylpiperidine ring possess basic nitrogen moiety, which gets protonated at physiological pH
and leads to its increased affinity towards AChE and aspartate dyad of BACE-1 along with
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significant BBB permeability due to acid-base equilibrium. These favorable findings led us to
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select N-benzylpiperidine nucleus as a core group for the design of novel hybrids.
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In our previous study, we have reported MTDLs by tethering N-benzylpiperidine with terminally
substituted phenyl groups attached to variable electron-withdrawing (EWGs) and donating (EDGs)
groups. The investigation revealed the remarkable inhibitory activity of AChE, BACE-1, and Aβ
aggregation by the compounds in which the terminal phenyl group was substituted with EWGs
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(particularly 4-CF , compound A) rather than EDGs. However, the substitution at the terminal
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phenyl group with EWGs translated into limited BACE-1 affinity. Corroborating the structures of
the designed molecules with its pharmacological outcome, leaves room for further optimization
by increasing length of the linker chain, thereby assisting the extension of N-benzylpiperidine
moiety further deep into the CAS. Linker with NH functionality was given prominence based on
its ability to increase the affinity of molecules toward BACE-1.25,26 Also, the presence of a lone
pair of electrons at linker site would provide an edge to the designed molecules by imparting
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antioxidant potential. Based on these postulations, we have initially tethered the pharmacophores
by an open-chain spacer group, methylenehydrazine-1-carboxamide (series I, 5a–h). However,
owing to the presence of an imine functionality, compounds 5a–h were prone to the acidic
hydrolysis and degradation.27,28 Our recently designed 1,3,4-oxadiazole hybrids, compound B and
C, signified remarkable PI displacement from PAS-AChE.29,30 The planer conformation and H-
bond acceptor ability of 1,3,4-oxadiazole makes it a suitable candidate for achieving the requisite
orientation within the active pocket of target enzymes. These findings prompted us to replace the
open-chain linker with 1,3,4-oxadiazole hybrids. Thereby, we have designed series of compounds
bearing N-benzylpiperidine and substituted 5-phenyl-1,3,4-oxadiazoles tethered with —NH linker
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(series II, 6a–h), —NHCH linker (series III, 10a–h), and without linker (series IV, 13a–h). The
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pharmacological outcome owing to the variation in linkers among series I–IV were compared. The
adopted design strategy is depicted in Figure 1.
Asp32
Asp228
O
PAS
BACE-1
O
O
F
F
F
EWGs
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spacer
H
N
EWGs
e: 4-Cl
N
N
N
a: H
b: 4-CN
ABP
Donepezil
f: 2,4-diCl
g: 4-CF3
Compound A24
CAS
c: 4-NO2
d: 2,4-diF
IC50 (hAChE) = 0.11 µM
IC50 (hBACE-1) = 0.22 µM
h: 4-OCF3
anti-A and antioxidant activity
spacer
Series III
H
N
H
N
H
N
O
H
N
Series I
N
H
O
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N
5a-h:
N
10a-h:
O
N
N
N
O
N
N
O
N
H
N
Compound B29
IC50 (hAChE) = 1.098 µM
anti-A and antioxidant activity
Series
a-h:
II
O
Series IV
13a-h:
30
Compound C
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N
N
IC50 (hAChE) = 0.30 µM
anti-A and antioxidant activity
N
N
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00
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Figure 1. Design strategy for N-benzylpiperidine analogs.
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The designed series (I–IV) of ligands were synthesized, characterized, and biologically
evaluated against multiple targets of AD such as hAChE, hBChE, and hBACE-1. The blood-brain
barrier (BBB) permeability was assessed by parallel artificial membrane permeation assay
(PAMPA), and peripheral anionic site (PAS)-AChE binding of active leads was evaluated by
propidium iodide (PI) displacement assay. The active leads were also assessed for their anti-Aβ
activity in self- and AChE-induced experiments by thioflavin T assay. The neurotoxic liability
towards SH-SY5Y neuroblastoma cells was tested at 10–80 µM concentrations by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore, promising
leads were assessed for their ability to ameliorate cognitive dysfunctions in scopolamine- and Aβ-
induced AD rat models. The ex vivo study was performed on hippocampal brain homogenates to
determine the antioxidant activity of leads. The expression levels of Aβ and BACE-1 proteins were
evaluated using Western blot and immunohistochemical analysis. The pharmacokinetic study was
performed to determine the oral bioavailability of lead candidate.
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RESULTS AND DISCUSSION
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Chemistry. The target compounds 5a–h and 6a–h were synthesized following the sequential
reactions mentioned in Scheme 1. The carbamate intermediate 3 was obtained in appreciable yield
(85%) by the reaction of 4-amino-1-benzylpiperidine (1) with phenyl chloroformate (2) and
pyridine in dry DCM. The compound 3 was heated at reflux with hydrazine hydrate (80% v/v) in
absolute EtOH to yield (77%) hydrazinecarboxamide intermediate (4), which was subsequently
heated at reflux in EtOH with numerous aromatic aldehydes (variable EWG) under acidic medium
to yield corresponding semicarbazones (5a–h) in appreciable yield (71–80%). Further, compounds
6a–c (62–68%) were obtained by heating compound 4 at reflux with several benzoic acids in
POCl3. Finally, 6d–h were prepared by oxidative cyclization of 5a–h (65–75%) using
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diacetoxyiodobenzene in dry DCM.
Scheme 1. Synthesis of target compounds 5a–h (series I) and 6a–h (series II)a
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Cl
O
H
H
H
NH2
+
N
O
N
N
O
(I)
(II)
NH2
N
N
O
N
O
(1)
(2)
(4)
(3)
(IV)
EWGs
(III)
5a, 6a: H
5b, 6b: 4-CN
5c, 6c: 4-NO2
H
H
5d, 6d: 2,4-diF
e, 6e: 4-Cl
H
EWGs
O
EWGs
N
N
N
5
(V)
N
5f, 6f: 2,4-diCl
N
N
N
O
N
5
g, 6g: 4-CF3
(5a-h)
Series I
5h, 6h: 4-OCF3
(6a-h)
Series II
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aReagents and conditions: (I) pyridine, DCM, 0–5 °C to room temperature, stirring, 4 h (85%); (II)
hydrazine hydrate (80% v/v), absolute EtOH, heated at reflux, 6 h (77%); (III) substituted
benzaldehydes, glacial acetic acid, absolute EtOH, heated at reflux, 2–4 h (71–80%); (IV)
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substituted benzoic acids, POCl , heated at reflux, 8–10 h (62–68%); (V) diacetoxyiodobenzene,
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DCM, stirring, room temperature, 4–6 h (65–75%).
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The molecular hybrids of N-benzylpiperidine and substituted 5-phenyl-1,3,4-oxadiazole
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connected with —NHCH linker (10a–h) were synthesized by reaction sequence mentioned in
2
Scheme 2. In the first step, substituted benzoic acid derivatives (7a–h) were reacted with N-
hydroxybenzotriazole (HOBT) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in
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acetonitrile to yield benzoic acid esters, which were subsequently reacted with hydrazine hydrate
(80% v/v) to obtain corresponding benzoic acid hydrazides (8a–h, yield: 79–87%). Subsequently,
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cyclization of compounds 8a–h by heating chloroacetic acid at reflux in POCl provided
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1
1
1
1
1
1
40
41
42
43
44
45
intermediates (9a–h, yield: 79–92%). Finally, the reaction of 9a–h and 4-amino-1-
benzylpiperidine (1) with KI in DMF afforded the target compounds 10a–h (yield: 66–81%).
The synthetic route for compounds 13a–h (directly linked molecular hybrids of N-
benzylpiperidine and substituted 5-phenyl-1,3,4-oxadiazoles) is mentioned in Scheme 3. As
shown, compounds 8a–h (from Scheme 2) and N-benzylpiperidine-4-carboxaldehyde were heated
at reflux in EtOH under the acidic condition to yield corresponding imines (12a–h), which were
subsequently reacted with chloramine T to obtain target compounds 13a–h in appreciable yields
(65–75%).
146
1
1
47
48
Scheme 2. Synthesis of target compounds 10a–h (series III)a
H
N
EWGs
EWGs
EWGs
NH2
NH
N
O
(II)
Cl
(I)
(III)
N
O
O
N
O
N
N
(9a-h)
OH
7a-h)
(
10a-h)
(
(8a-h)
EWGs
Series III
EWGs
7
7
a, 8a, 9a, 10a: H
b, 8b, 9b, 10b: 4-CN
7e, 8e, 9e, 10e: 4-Cl
7f, 8f, 9f, 10f: 2,4-diCl
7g, 8g, 9g, 10g:4-CF3
7h, 8h, 9h, 10h: 4-OCF3
7c, 8c, 9c, 10c: 4-NO2
d, 8d, 9d, 10d:2,4-diF
7
1
150
49
aReagents and conditions: (I) HOBT, EDC, ACN, hydrazine hydrate (80% v/v), 0–5 °C to room
1
51
52
temperature, stirring, 2–4 h (79–87%); (II) chloroacetic acid, POCl , heated at reflux, 4–6 h (79–
3
1
92%); (III) 4-amino-1-benzylpiperidine, KI, DMF, room temperature, stirring, 6–8 h (66–81%).
1
1
53
54
Scheme 3. Synthesis of target compounds 13a–h (series IV)a
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EWGs
EWGs
NH2
NH
H
N
N
(I)
(II)
O
N
O
+
N
O
N
O
EWGs
N
N
(
8a-h)
(11)
(12a-h)
(13a-h)
Series IV
EWGs
7
7
a, 8a, 9a, 10a: H
b, 8b, 9b, 10b: 4-CN
7c, 8c, 9c, 10c: 4-NO2
d, 8d, 9d, 10d:2,4-diF
7e, 8e, 9e, 10e: 4-Cl
7f, 8f, 9f, 10f: 2,4-diCl
7g, 8g, 9g, 10g:4-CF3
7h, 8h, 9h, 10h: 4-OCF3
7
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
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1
aReagents and conditions: (I) glacial acetic acid, absolute EtOH, heated at reflux, 4–6 h; (II)
chloramine T, absolute EtOH, room temperature, stirring, 1–2 h (65–75%).
157
158
In Vitro Biological Evaluations: ChE Inhibition Assay. Compounds from series I (5a–h), II
(6a–h), III (10a–h), and IV (13a–h) were designed to investigate the inhibitory potential,
selectivity profile, and preliminary SAR studies against hAChE (from human erythrocytes) and
hBChE (from human serum). Donepezil and rivastigmine were used as positive reference
standards. The results of enzyme assays are summarized in Table 1.
1
1
59
60
161
1
1
1
62
63
64
The N-benzylpiperidine was retained as a core nucleus, as studies suggested that modification
of the benzyl ring leads to a significant reduction in ChE inhibition activity.20,21 The SAR studies
were envisaged by modifications of X-spacer and substitution of variable EWGs at terminal phenyl
group. The majority of compounds from series I (5a–h) exhibited lower inhibitory activity against
hAChE in the micromolar range. Among the compounds from series I, 5h with strong EWG, 4–
OCF showed maximum hAChE inhibitory potential (IC = 1.05 µM). The concomitant
165
1
1
66
67
168
3
50
1
1
69
70
modification of open chain methylenehydrazide with the cyclic oxadiazole nucleus increased ChE
inhibitory potential to several folds in compounds from series II, III, and IV. Among the series II
(6a–h) compounds with —NH linker presented optimum inhibitory potency compared to
171
172
173
174
compounds with —NHCH linker (series III, 10a–h) and directly linked congeners (series IV,
2
13a–h). So the order of hAChE inhibitory potency was observed as —NH (series II) > —NHCH2
(series III) > directly linked (series IV), with the exception being 13e (IC : 0.098 µM), which
50
175
76
showed better hAChE inhibitory activity than their respective counterparts (IC , 6e: 0.828 µM
5
0
1
and 10e: 1.64 µM). The spacer group (—NH) and oxadiazole hybrids might have formed
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additional π-cation interactions with PAS-hAChE to increase the anti-hAChE activity. Also, the
—NH spacer may be responsible for providing optimum conformational flexibility to the terminal
phenyl group and N-benzylpiperidine nucleus for their effective interactions with PAS and CAS
residues of AChE, respectively.
179
0
1
2
3
4
5
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9
0
1
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0
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To optimize the interaction of compounds with PAS-hAChE, variable EWGs were substituted
at the terminal phenyl region. Among all the tested compounds, the unsubstituted derivatives
showed moderate to poor hAChE inhibitory potential in micromolar range (IC , 5a: > 5 µM; 6a:
5
0
1.32 µM; 10a: 1.70 µM; 13a: > 5 µM). The introduction of EWGs drastically increased the hAChE
inhibitory potency several folds. Among the series II (6a–h) and III (10a–h), substitution with 4–
CN (IC , 6b: 0.783 µM; 10b: 1.050 µM) and 4–Cl (IC , 6e: 0.828 µM; 10e: 1.640 µM) groups
186
5
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50
1
1
87
88
resulted in moderately active compounds with the inhibitory potential in micromolar to sub-
micromolar range. The substitution with 4-NO , a strong EWG has proven to be slightly more
2
189
potent hAChE inhibitor (IC , 6c: 0.149 µM; 10c: 0.222 µM). Interestingly, di-substitution at
5
0
1
1
1
1
1
1
90
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92
93
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95
positions 2 and 4 with —Cl functionality showed remarkable hAChE inhibitory activity in two-
digit nanomolar concentration range (IC , 6f: 0.075 µM; 10f: 0.086 µM) and exhibited 10–20
5
0
folds higher activity compared to their mono-substituted counterparts 6e and 10e, respectively.
Further, substitutions with highly lipophilic and strong EWGs, 4–CF (IC , 6g: 0.055 µM; 10g:
3
50
0.144 µM) and 4–OCF (IC , 6h: 0.096 µM; 10h: 0.119 µM) turned out to be excellent inhibitor
3
50
of hAChE. The presence of halogen functional groups (F, Cl, CF , and OCF ) was demonstrated
3
3
196
to be indispensable for hAChE inhibitory activity.
1
97
98
The compounds from series IV (13a–h) with directly linked 5-phenyl-1,3,4-oxadiazoles and N-
benzylpiperidines hybrids also showed moderate to excellent hAChE inhibition. The di-
substitution was found to be unfavorable in series IV compared to series II and III with 2,4–diF
1
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(IC , 13d: 3.49 µM) and 2,4–diCl (IC , 13f: 3.26 µM) functionalities exhibited poor anti-hAChE
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5
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7
8
9
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1
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1
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1
1
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
activity. The substitution at para position with 4–NO (IC , 13c: 0.235 µM), 4–CF (IC , 13g:
2
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50
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0.207 µM), and 4–OCF (IC , 13h: 0.162 µM) groups enhanced the hAChE inhibitory activity up
3 50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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to three-digit nanomolar concentration. The compound 13e bearing a 4–Cl substitution was a most
pronounced inhibitor of series IV with IC = 0.098 µM. Overall, 6f, 6g, 6h, 10f, and 13e were
5
0
found to be the potent hAChE inhibitors with two-digit nanomolar IC values comparable to that
50
of donepezil (IC = 0.046 µM). These compounds also complement our designing concept with
5
0
the observation of IC values in two-digit nanomolar concentrations than our previously reported
5
0
2
4
compound A (IC = 0.107 μM).
50
209
In addition to the hAChE, several clinical evidences suggested that BChE also plays a significant
role in the regulation of ACh and maintaining normal cholinergic functions in AD.14-16 The activity
2
10
11
3
1
2
of BChE was observed to be elevated during AD. Therefore, dual inhibition of AChE and BChE
can be considered as a potential therapeutic advantage in advanced and late phase AD.32-34 All the
tested compounds exhibited comparatively lower hBChE inhibition than hAChE except being
compound 10e, which has more selectivity toward hBChE. Compounds from series I (5a–h) were
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213
214
215
216
217
218
not found active against hBChE (IC > 10 µM). The order of activity against hBChE was increased
50
with lengthening the chain of the linker, i.e. series III > series II > series IV, which can be ascribed
to the larger catalytic gorge of hBChE to occupy the lengthy molecule. The compound 13e from
series IV being an exception, which demonstrated higher hBChE inhibition (IC = 0.418 µM) as
50
219
compared to its counterparts from series II (6e: IC = 4.28 µM) and III (10e: IC = 1.49 µM).
5
0
50
2
20
21
Apart from 13e, compounds with 2,4–diCl, 4–CF , and 4–OCF substitutions in series II (6f: IC
3 3 50
2
= 0.336 µM; 6g: IC = 0.186 µM; 6h: IC = 0.796 µM) and series III (10f: IC = 0.143 µM;
5
0
50
50
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10g: IC = 0.220 µM; 10h: IC = 0.751 µM) were observed to be potent hBChE inhibitors with
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0
50
2
IC in three-digit nanomolar range.
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224
Enzyme Kinetics Study on hAChE and hBChE. To gain insight into the mechanism of
inhibition for the most potent inhibitor 6g, enzyme kinetics study was performed against hAChE
and hBChE. The type of inhibition was elucidated by Lineweaver-Burk double reciprocal plot
between the initial velocity of the substrate (y-axis) at increasing concentrations (x-axis;
acetylthiocholine iodide, ATCI for hAChE; butyrylthiocholine iodide, BTCI for hBChE). The
0
1
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5
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1
2
3
4
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8
9
0
1
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2
2
2
2
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plots revealed decreased Vmax, while K increased with increasing concentration of 6g, the trend
m
being attributed to the mixed-type of inhibition for hAChE and hBChE (Figures 2A and 2B). The
Dixon plots were constructed between Lineweaver-Burk slope and inhibitor concentration
(Figures S51A and S51B, Supporting Information). The intersection point at x-axis of the plot
231
2
2
32
33
considered as dissociation constant K for the inhibitor, which was estimated to be 0.026 µM and
i
234
0.115 µM for hAChE and hBChE, respectively.
2
35
Table 1. The results of hAChE, hBChE, and hBACE-1 inhibition assays by target compounds.
H
EWGs
X
N
X
O
N
EWGs
N
O
N
N
N
5a-h
6a-h, 10a-h and 13a-h
2
36
a
IC ± SEM (µM)
selectivity
for
hAChE
5
0
compd EWGs
X-group
b
hAChE
hBChE
hBACE-1
5
5
5
5
5
a
b
c
-H
NH
NH
NH
NH
NH
>5
>10
>10
>10
>10
>10
nd
--
--
--
--
--
4-CN
4-NO2
2,4-diF
4-Cl
>5
nd
1.56 ± 0.068
2.03 ± 0.034
4.20 ± 0.044
5.07 ± 0.169
8.10 ± 0.143
>10
d
e
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6
1
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1
f
2,4-diCl
4-CF3
4-OCF3
-H
NH
2.10 ± 0.085
1.78 ± 0.062
1.05 ± 0.040
1.32 ± 0.029
>10
>10
>10
>10
4.97 ± 0.253
1.36 ± 0.108
1.37 ± 0.080
7.33 ± 0.091
6.17 ± 0.105
2.35 ± 0.054
3.93 ± 0.046
4.06 ± 0.213
--
g
NH
--
h
NH
--
a
NH
--
1
1
1
1
1
1
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2
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2
2
2
2
2
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2
2
3
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3
3
3
3
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4
5
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b
4-CN
NH
0.783 ± 0.044 >10
--
c
4-NO2
2,4-diF
4-Cl
NH
0.149 ± 0.009 1.69 ± 0.078
0.264 ± 0.017 2.68 ± 0.117
0.828 ± 0.054 4.28 ± 0.081
11.3
10.2
5.2
d
NH
e
NH
f
2,4-diCl
4-CF3
4-OCF3
-H
NH
0.075 ± 0.003 0.336 ± 0.018 0.236 ± 0.007 4.5
0.055 ± 0.003 0.186 ± 0.005 0.146 ± 0.012 3.4
g
NH
h
NH
0.096 ± 0.005 0.796 ± 0.036 1.34 ± 0.051
8.3
--
0a
0b
0c
0d
0e
0f
0g
NHCH2
NHCH2
NHCH2
NHCH2
NHCH2
NHCH2
NHCH2
NHCH2
--*
1.70 ± 0.092
1.05 ± 0.029
>10
>10
4.92 ± 0.116
1.94 ± 0.111
1.39 ± 0.066
1.36 ± 0.084
4-CN
--
4-NO2
2,4-diF
4-Cl
0.222 ± 0.014 1.00 ± 0.028
0.356 ± 0.035 1.25 ± 0.059
4.5
3.5
1.64 ± 0.104
1.49 ± 0.065
0.296 ± 0.006 0.90
2,4-diCl
4-CF3
4-OCF3
-H
0.086 ± 0.007 0.143 ± 0.004 0.114 ± 0.004 1.7
0.144 ± 0.007 0.220 ± 0.003 0.186 ± 0.006 1.5
0.119 ± 0.008 0.751 ± 0.012 0.931 ± 0.037 6.3
10h
13a
13b
13c
13d
13e
13f
>5
>10
nd
--
4-CN
--*
1.37 ± 0.125
9.09 ± 0.272
7.51 ± 0.178
7.44 ± 0.197
>10
6.6
24.3
2.0
4-NO2
2,4-diF
4-Cl
--*
0.235 ± 0.015 5.71 ± 0.075
3.49 ± 0.134 7.13 ± 0.099
--*
--*
0.098 ± 0.005 0.418 ± 0.006 0.690 ± 0.056 4.3
2,4-diCl
4-CF3
4-OCF3
--*
3.26 ± 0.059
6.83 ± 0.081
8.87 ± 0.369
1.83 ± 0.100
5.94 ± 0.148
2.1
13g
13h
--*
0.207 ± 0.006 1.20 ± 0.059
0.162 ± 0.004 4.23 ± 0.126
5.8
--*
26.1
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donepezil
0.046 ± 0.003 1.94 ± 0.093
0.220 ± 0.006 42.2
nd 0.41
rivastigmine
2.58 ± 0.032
1.07 ± 0.054
a
2
2
2
2
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40
The assays were performed on hAChE, hBChE, and hBACE-1. Data are expressed as the mean
b
IC ± SEM of three separate experiments (n = 3); selectivity ratio = IC of hBChE/hAChE;
5
0
50
*
compounds 13a–h having directly linked N-benzylpiperidine and substituted 5-phenyl-1,3,4-
oxadiazoles without X-spacer group; nd = not determined.
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9
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9
0
1
2
3
4
5
6
7
8
9
0
[A] 6g-hAChE
[B] 6g-hBChE
6
µM)
g (0.300
2
2
1
50
00
50
6g (0.090
µM)
3
50
6g (0.200
µM)
6g (0.060
µM)
6g (0.030
µM)
250
150
6g (0.100
µM)
100
Control
Control
5
0
5
0
-10
-5
5
10
15
-1
[
ATCI] mM
-10
-5
5
10 -1
15
-50
-50
[BTCI] mM
2
41
42
2
Figure 2. Lineweaver-Burk double reciprocal plot showing the mechanism of enzyme inhibition
by compound 6g; [A] hAChE inhibition with acetylthiocholine iodide (ATCI) as substrate; [B]
hBChE inhibition with butyrylthiocholine iodide (BTCI) as substrate.
2
43
244
245
hBACE-1 Inhibition Assay. In our quest to search for MTDLs, the FRET-based fluorometric
assay was performed to determine the hBACE-1 inhibitory potential of synthesized compounds
using donepezil as a reference standard. The results of hBACE-1 inhibition assay are summarized
in Table 1. The length of linkers in the designed series was varied with an aim to improve the
interaction with aspartate dyad (Asp32 and Asp228) of hBACE-1.
2
2
46
47
248
2
2
49
50
The modification of linker chain length from —NH (series II) to —NHCH (series III) has quite
2
251
significant influence on hBACE-1 inhibitory potential as compounds from series III showed
remarkably higher activity compared to series I, II, and IV, exception being the compound 6g
(series II, IC = 0.146 μM), which showed slightly higher inhibitory potential than its counterpart
2
2
2
52
53
54
5
0
10g (series III, IC = 0.186 μM). Most of the compounds, almost ten of them (6b,6c, 6d, 6e, 6h,
50
255
10c, 10d, 13c, 13h, and 13e) were found to be a good inhibitor of hAChE, but unfortunately,
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devoid of activity against hBACE-1 (IC50 > 1 μM). Conversely, one of the compound 10e
displayed excellent hBACE-1 inhibition (IC = 0.296 μM), but remarkably decreased anti-hAChE
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
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2
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activity (IC > 1 μM). Apart from 6g, 10e, and 10g, four more compounds 6f (IC = 0.236 μM),
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10f (IC = 0.114 μM), 10h (IC = 0.931 μM), and 13e (IC = 0.690 μM) displayed significant
50 50 50
hBACE-1 inhibition with IC50 in three-digit nanomolar range. It is also worth noticing that
compounds with dual (hAChE and hBChE) inhibitory potential were also a good candidate to
provide activity against hBACE-1 and common compounds (6f, 6g, 10f, 10g, 10h, and 13e) were
selected as MTDLs by the same virtue to be investigated further.
PI Displacement Assay. To affirm our hypothesis that designed ligands would bind effectively
with PAS-AChE, PI displacement assay was conducted for the selected MTDLs using method
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described by Taylor et al. PI is the known ligand, selectively binds to PAS-AChE and displayed
increased fluorescence intensity up to eight to ten folds.36,37 The decreased fluorescence intensity
can be estimated as percentage PI displacement from PAS-hAChE due to presence of inhibitor at
10 µM and 50 µM concentrations. Donepezil was used as the reference standard. Among the tested
compounds, 6f, 10h, and 13e have considerably lesser (< 25%), while 10g (10 µM: 17.79%, 50
µM: 27.37%) exhibited slightly higher PI displacement from PAS-hAChE. The results
demonstrated remarkable PI displacement capability of 6g (10 µM: 32.41%, 50 µM: 55.81%) and
10f (10 µM: 27.70%, 50 µM: 51.42%) compared to donepezil (10 µM: 23.23%, 50 µM: 32.58%).
The results of PI displacement assay are summarized in Table 2.
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Table 2. The percentage PI displacement from PAS-hAChE and prediction of BBB permeation of
selected MTDLs.
compd.
PI displacement from PAS-hAChE (%)a
PAMPA-BBB permeability
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19.01 ± 1.14
55.81 ± 2.15
51.42 ± 1.69
27.37 ± 1.83
18.28 ± 1.42
22.50 ± 2.00
32.58 ± 1.94
Pe(exp) (10 cm s ) prediction
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6.9 ± 0.22
5.9 ± 0.18
5.3 ± 0.24
6.2 ± 0.29
3.9 ± 0.32
6.7 ± 0.18
CNS+b
CNS+b
g
CNS+b
CNS+b
CNS+b
CNS±c
CNS+b
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donepezil 23.23 ± 1.31
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The PI assay was conducted to investigate the ability of compounds to displace PI from PAS-
AChE at 10 µM and 50 µM; ‘CNS+’ (prediction of excellent brain permeability with Pe > 4.4 ×
b
-
6
-1
c
-
10 cm s ); ‘CNS±’ (prediction of uncertain brain permeability with P between 4.4 to 1.8 × 10
e
6
-1
cm s ). Data are expressed as the mean ± SEM of three separate experiments (n = 3).
PAMPA-BBB Assay. The penetrability through BBB is the major concern for the development
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of drugs to treat AD. To explore the permeability characteristics of selected MTDLs (6f, 6g, 10f,
10g, 10h, and 13e), PAMPA-BBB assay was performed following the method reported by Di et
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al. The assay is based on passive diffusion of compounds from donor to acceptor compartment
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through a parallel artificial membrane made up of porcine brain lipid. The experimental (Pe(exp)
)
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and reference (Pe(ref)) permeability values of nine commercial drugs were compared for assay
2
validation (Table S2, Supporting Information). The plot between experimental and reported
-
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-1
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permeability values was constructed and a threshold limit (P > 4.4 × 10 cm s ) was designated
e
-6
-1
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for compounds with excellent brain permeability. While permeability values < 1.8 × 10 cm s
-6
-1
was considered to have poor penetrability and the values between 1.8–4.4 × 10 cm s considered
as uncertain permeability.39,40 The results of PAMPA-BBB assay demonstrated the excellent brain
permeability (CNS+) for all the tested compounds 6f, 6g, 10f, 10g, and 10h, the exception being
13e, which showed uncertain brain permeability (CNS±). The significant brain permeability may
be attributed to the presence of lipophilic functional groups (2,4-diCl, 4–CF , and 4–OCF ) at the
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terminal phenyl group of compounds. The results of PAMPA-BBB assay are summarized in Table
2.
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Considering the balanced multifunctional enzyme inhibitory potential (dual ChE and hBACE-
1), significant displacement of PI from PAS-hAChE, and excellent brain permeability
characteristics, compounds 6g and 10f were selected for further pharmacological investigations.
Inhibition of Aβ Aggregation: The deposition and aggregation of Aβ can be considered as a
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major detrimental factor in AD. The results of PI displacement assay signified the remarkable
binding capability of compounds 6g and 10f with PAS-hAChE, and it is a known fact that PAS
binding of the inhibitor will not only result in AChE inhibition but also have a significant role in
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prevention of Aβ aggregation. Moreover, Compounds 6g and 10f were also observed to be
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potential inhibitors of hBACE-1, an enzyme responsible for cleavage of amyloid precursor protein
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into Aβ. All these outcomes have prompted us to determine the anti-Aβ aggregation activity
these compounds, using thioflavin T assay in self– and hAChE–induced experiments. The tests
were performed at three varied concentration ratios of Aβ and inhibitor (10:5 µM, 10:10 µM, and
10:20 µM, respectively) and the results were reported as normalized fluorescence intensity (NFI)
(Figures 3A and 3B) and % inhibition of Aβ aggregation (Figures S52A and S52B, Supporting
Information). The results demonstrated concentration-dependent inhibition of Aβ aggregation with
the maximum activity being observed at 20 µM inhibitor concentration (Aβ:inhibitor, 10:20 µM).
A slightly higher Aβ inhibition for compound 10f (self-induced: 14.66–49.19; hAChE-induced:
33.73–78.65%) than donepezil (self-induced: 16.96–38.46%; hAChE-induced: 24.17–60.55%)
was observed, while compound 6g displayed remarkable anti-Aβ aggregatory activity in self–
(17.83–56.03%) and hAChE-induced (45.60–89.92%) experiments. The observed results were in
accordance with the assays of hAChE, hBChE, hBACE-1, and PI displacement.
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Congo Red Binding Assay: The aggregation kinetics of Aβ was determined using Congo red
3
binding assay. Congo red is an azo dye that specifically binds to many amyloid proteins due to
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their extensive β-sheet structure. The absorbance spectrum of Congo red changes upon binding
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to Aβ proteins, which is used to determine the extent of amyloid formation at several time points.45
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The intensity of Congo red was measured for incubated samples of Aβ at 0 (freshly prepared), 1,
4, 7, and 10 days (Figure 3C). The results indicated that the intensity of freshly prepared Aβ sample
with Congo red was minimal, which was significantly increased after 4 days, suggesting the
existence of Aβ aggregation due to β-sheet structures. The maximum intensity of Congo red was
observed in the incubated samples on days 7 and 10, suggesting maximum Aβ aggregation and
formation of large oligomers and fibrils during that period.
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AFM Studies. The AFM analysis was performed to validate the Aβ-inhibitory potential of
compound 6g by visualizing the surface topographical maps for the samples of Aβ (10 µM) in the
presence or absence of inhibitor (Figure 3D). The morphological changes of Aβ aggregates in the
presence or absence of inhibitors over a different incubation time points (0, 4, 7 and 10 days)
exhibited a positive correlation with its aggregation phenomenon, i.e., the formation of Aβ
aggregates, oligomers, or fibrils as evidenced in the Congo red binding assay. The estimation of
maximum peak height and roughness index provided the actual size of the deposited aggregates.
The results revealed the formation of larger aggregates and oligomers for the samples of Aβ with
the exponential rise in maximum peak height (22.84–527.46 nm) and roughness index (1.08–27.52
nm) throughout ten days incubation. The presence of inhibitor (6g) displayed remarkable cessation
in oligomer formation and deposition of Aβ aggregates, which was ascertained by several folds
reduction in maximum peak height (28.31–182.96 nm) and roughness index (2.88–5.82 nm) over
ten days incubation period.
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A Incubation Time (days)
A : DONE
M)
A : 6g
(M)
A : 10f
(M)
A : DONE
(M)
A : 10f
(M)
A : 6g
M)
(
(
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Figure 3. The anti-Aβ aggregation activity of compounds 6g and 10f: [A] self-induced and; [B]
hAChE-induced aggregation inhibition experiments. [C] Congo red binding assay of pre-aged Aβ
samples as a function of incubation time. Data are expressed as the mean ± SEM of three
independent experiments (n = 3); DONE = donepezil. [D] AFM studies showing aggregation of
Aβ fibrils (10 µM) samples in the presence or absence of inhibitor after incubation at different
time points, i.e., on days 0, 4, 7, and 10.
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Neuronal Viability on SH-SY5Y Cells. To ascertain the neurotoxic liability of compounds 6g
and 10f, cell viability assay based on MTT was performed on neuroblastoma SH-SY5Y cell lines.
The cell viability was determined at concentrations of 10, 20, 40, and 80 µM of test compounds
with parallel to standard drug donepezil. The outcome of assay demonstrated a non-significant
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reduction in cell viabilities towards neuroblastoma cell lines by both the compounds (6g and 10f)
up to the maximum tested concentration of 80 µM (Figure 4). The results ascertained both the
compounds (6g and 10f) were non-neurotoxic and can be considered for further preclinical
investigations.
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10 20 40 80 10 20 40 80
donepezil, M
10f, M
donepezil, M
6g, M
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Figure 4. The results of % cell viability by compounds 6g and 10f parallel to donepezil measured
by MTT assay. The tested concentrations of compounds are 10, 20, 40, and 80 µM, seeded with
neuroblastoma SH-SY5Y cell lines for the incubation period of 48 h. Data are expressed as the
mean ± SEM of three independent experiments (n = 3).
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In Vivo Biological Evaluations: Acute Oral Toxicity Study. The acute toxicity study was
accomplished to evaluate the safety profile of compounds 6g and 10f in female Wistar rats
following an oral administration. The results demonstrated no toxic or abnormal reactions on
animals after administration of compounds in graded doses of 677, 1333, 2000 mg/kg, p.o.
Animals of treatment groups lost slight weight 10-20% initially in the first few days, which was
entirely regained within the next 14 days of study without any clinical signs of toxicity. Overall
results suggested that both the compounds can be well tolerated up to the maximum dose of 2000
mg/kg.
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Scopolamine-induced Y-Maze Test. The in vivo efficacy of compounds 6g and 10f to improve
learning and memory was evaluated by scopolamine-induced Y-maze test in healthy male Wistar
rats. Scopolamine is the most commonly used drug for inducing the cognitive impairment from
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cholinergic deficit. Donepezil was used as a standard (5 mg/kg, p.o.) and the compounds were
evaluated at a dose range of 2.5, 5, and 10 mg/kg, p.o.
The respective group of rats was administered with test compounds and donepezil for seven
consecutive days. The treatment on the seventh day was followed with an administration of
scopolamine hydrobromide (0.5 mg/kg, i.p.) to induce the cognitive impairment and Y-maze test
was conducted to measure the spontaneous alternations and total arm entries. The increased %
spontaneous alternation was considered as a score of improvement in cognitive deficit.
The cognitive deficit was induced by administration of scopolamine affirmed by a significant
reduction in % spontaneous alternation (Figure 5A, ###p < 0.001) compared to control. The
treatment with donepezil displayed significantly higher (Figure 5A, ***p < 0.001) % spontaneous
alternations compared to scopolamine. Further, the treatment with test compounds 6g and 10f
demonstrated improvement in scopolamine-induced cognitive deficit in a dose-dependent manner
with maximum activity was observed at 10 mg/kg. The treatment with 6g and 10f showed
significantly higher % spontaneous alternations (Figure 5A, ***p < 0.001) compared to
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*
*
scopolamine, exception being the compound 10f at the dose of 2.5 mg/kg, (Figure 5A, p < 0.01).
Interestingly, compound 6g (5 and 10 mg/kg) and 10f (10 mg/kg) demonstrated statistically non-
significant difference in % spontaneous alternation (Figure 5A, ns) than donepezil. Moreover,
similar total arm entries of all the groups (Figure 5B, ns) ascertained that scopolamine did not
impede the locomotive behavior in animals during the experiment.
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***
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
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n
N
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O
D
o
O
o
C
D
C
6g (mg/kg)
10f (mg/kg)
6g (mg/kg)
10f (mg/kg)
Scopolamine (0.5 mg/kg)
Scopolamine (0.5 mg/kg)
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Figure 5. The effect of compounds 6g, 10f on the scopolamine-induced cognitive deficit in Y-
maze test; [A] % spontaneous alternation and; [B] total arm entries. Data are expressed as the mean
± SEM (n = 6); ###p < 0.001 vs control; ***p < 0.001, p < 0.01 vs scopolamine; πππp < 0.001, ππp
< 0.01 vs donepezil (DONE5); ns = non-significant.
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**
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00
Ex Vivo Studies. Immediately after behavioral studies, the rats of the respective groups were
decapitated; the brain hippocampus region was isolated and homogenized. The ex vivo studies
were performed to estimate the AChE levels and oxidative stress biomarkers, in hippocampal
homogenates by the treatment of donepezil (5 mg/kg), 6g (10 mg/kg) and 10f (10 mg/kg).
The ex vivo AChE study was performed following the Ellman assay47 and the results
demonstrated that scopolamine significantly elevated AChE activity (Figure 6A, ###p < 0.001) by
means of extensive substrate hydrolysis compared to healthy control, while treatment with
compounds 6g, 10f, and donepezil showed remarkable attenuation of AChE activity (Figure 6A,
***p < 0.001) compared to scopolamine. Interestingly, compound 6g and donepezil showed a non-
significant difference (Figure 6A, ns) in AChE activity. Overall, the results of ex vivo study were
observed in agreement to in vitro hAChE assay.
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Scopolamine is responsible for the induction of oxidative stress by altering the levels of several
biochemical markers, such as malondialdehyde (MDA), and superoxide dismutase (SOD).4
Therefore, to evaluate the antioxidant potential of compounds 6g and 10f, oxidative stress
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biomarkers such as malonaldehyde (MDA) and superoxide dismutase (SOD) were estimated. The
MDA is an important oxidative stress biomarker formed as a byproduct of lipid peroxidation
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reaction. Whereas SOD specifically catalyzes the dismutation reactions to convert superoxide
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–
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(O ) radicals into free oxygen (O ). The results suggested induction of oxidative stress with
2 2
scopolamine administration due to significant elevation of MDA (Figure 6B, ###p < 0.001), and
substantial attenuation of SOD (Figure 6C, ###p < 0.001) compared to control. The treatment with
compound 6g and 10f showed remarkable attenuation in MDA level (Figure 6B, ***p < 0.001),
while the SOD activity was increased significantly (Figure 6C, ***p < 0.001) compared to
scopolamine. Also, the results signified the non-antioxidant potential of donepezil. The
investigations also demonstrated the slightly higher antioxidant potential of compound 6g than
10f.
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[A]
[C]
[B]

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.008
.006
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#
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Control
DONE5
6g
10f
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10f
Control
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6g
10f
Scopolamine (0.5 mg/kg)
Scopolamine (0.5 mg/kg)
Scopolamine (0.5 mg/kg)
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Figure 6. The effect of 6g and 10f on a hippocampal region of rat brain by ex vivo study; [A]
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AChE activity; [B] thiobarbituric acid reactive substance (TBARS) assay and; [C] SOD assay.
Data are expressed as the mean ± SEM (n = 6). ###p < 0.001, p < 0.01, p < 0.05 vs control; ***p
##
#
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*
π
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< 0.001, p < 0.05 vs scopolamine; p < 0.05 vs donepezil (DONE5); ns = non-significant.
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Aβ-induced Morris Water Maze Test. The Aβ aggregation and deposition is one of the most
crucial pathophysiological mechanism involved in AD, and its production also increased with
BACE-1 activity. Thereby, the effect of most potential lead candidate 6g in ameliorating Aβ-
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induced cognitive deficit was evaluated by Morris water maze test. The AD-like phenotypic
condition was produced by intracerebrovascular (ICV) injection of Aβ (4 μM, 5 μl) in the
hippocampal brain region of rats through stereotaxic surgery. All the groups were administered
Aβ, except for the sham group of animals. The treatment of 6g and donepezil were given to the
corresponding group of animals for nine consecutive days, while rats from model (Aβ) group were
given vehicle only. The Morris water maze test was conducted during the last five days of
treatment and escape latency time (ELT, i.e., time to find the hidden platform), and total platform
crossovers were calculated for 90 seconds. The cognitive impairment induced by Aβ
administration affirmed significant prolongation of ELT (Figure 7A), while total platform
crossovers considerably reduced (Figure 7B) in the model group of animals compared to control
over five days. The treatment with 6g drastically reduced ELT (Figure 7A), while total platform
crossovers were increased significantly (Figure 7B) compared to the model group of animals.
Moreover, the results also demonstrated a slightly better effect of 6g in ameliorating cognitive
deficit than donepezil. Overall results of behavioral studies suggested a remarkable profile of
compound 6g at the tested dosage in improving learning and memory impairment.
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Figure 7. The effect of 6g in amelioration of Aβ-induced cognitive deficit by Morris water maze
test; [A] ELT; [B] total platform crossovers during the last five days of trials. Data are expressed
as the mean ± SEM (n = 10).
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Western Blot Analysis. The elevation of Aβ and BACE-1 are the key pathophysiological
hallmarks of AD. Thereby, at the end of Morris water maze experiment, Aβ and BACE-1
expression levels were estimated in hippocampal tissue samples of all the experimental groups by
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*
**
453
Western blot analysis. A significant elevation Aβ ( p < 0.001, Figures 8A and 8B) and BACE-1
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(***p < 0.001, Figures 8A and 8C) levels was observed in Aβ treated (model) group of animals
*
*
compared to control. The results demonstrated significant attenuation of Aβ ( p < 0.01, Figures
*
*
456
8A and 8B) and BACE-1 ( p < 0.01, Figures 8A and 8C) expression levels and were in accordance
to in vitro assays. Moreover, the results also signified the non-significant difference between 6g
and donepezil in attenuating the expression levels of Aβ and BACE-1.
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Immunohistochemical Analysis. The expression levels of Aβ and BACE-1 in rat hippocampal
region were further examined by immunofluorescence staining. The results demonstrated elevated
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***
***
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levels of Aβ ( p < 0.001, Figures 8D (a4) and 8E) and BACE-1 ( p < 0.001, Figures 8D (b4)
and 8F) expressions in model group of animals compared to sham group (Figures 8D (a1, b1), 8E
and 8F). The treatment with 6g significantly declined the expression levels of both the target
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proteins, Aβ ( p < 0.01, Figures 8D (a3) and 8E) and BACE-1 ( p < 0.01, Figures 8D (b3) and
8F) compared to the model group of animals. Moreover, the results of compound 6g mediated
expression levels were observed to be non-significant (ns) compared to donepezil (Figures 8D (a2,
b2), 8E, and 8F).
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Figure 8. Effect of 6g on in vivo expression of Aβ and BACE-1 levels in the hippocampal region
of the rat brain. [A] Representative bands in the Western blot analysis; [B] densitometric
quantification showing attenuated levels of Aβ and; [C] BACE-1, which were elevated with icv
administration of Aβ; [D] Immunostaining showing attenuated levels of Aβ and BACE-expression
by 6g at 10× magnification after staining; [E & F] Quantification analysis of immunostains
showing changes in burden (% area) of Aβ and BACE-1 levels, respectively. Data are expressed
as the mean ± SEM (n = 5). ***p < 0.001, p < 0.01 vs model group; ns = non-significant.
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Pharmacokinetic Studies. The pharmacokinetic profiling of lead candidate 6g was performed
in healthy male Wistar rats at the dose of 10 mg/kg, p.o. The blood samples were collected at
several time points (as given in experimental section) from retro-orbital plexus, and several
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pharmacokinetic parameters such as Cmax, Tmax, AUC, t , and MRT were calculated by the
1/2
extravascular non-compartment model using Kinetica 5.0 (Thermo Scientific Kinetica, USA). The
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maximum concentration (Cmax = 245.9 ng/mL) of compound 6g in blood was attained at time Tmax
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= 5.33 h after an oral administration. The elimination half-life t = 45.59 h, mean residence time
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*
483
MRT = 67.39 h, and (AUC)0-24 = 4926 ng/mL h were indicative of significant oral absorption and
excellent bioavailability of compound 6g following an oral administration. The results of the
pharmacokinetic experiment are summarized in Table 3.
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Table 3. Pharmacokinetic evaluation of compound 6g at 10 mg/kg, p.o.
Effect of 6g*
Parameters
Cmax (ng/mL)
Tmax (h)
245.9 ± 8.54
5.33 ± 1.33
1524 ± 10
*
(
AUC)0-8 (ng/mL h)
*
(
AUC)0-24 (ng/mL h) 4926 ± 159
t (h)
45.59 ± 0.995
67.39 ± 2.013
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*Data are expressed as mean ± SEM (n = 3).
Computational Studies. Molecular Docking. To gain insight into the interaction of compounds
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6g and 10f with the active site residues of hAChE and hBACE-1, molecular docking studies were
undertaken using Schrödinger Glide Program. Initially, prepared protein grids were validated by
extracting and re-docking the co-crystallized ligands, i.e., donepezil and F1M for hAChE (4EY7)
and hBACE-1 (2ZJM), respectively. The superposition tool was used to estimate the RMSD values
between the docked and co-crystallized poses of ligands, which were observed within the limit of
491
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2 Å to ascertain the validity of docking protocols and prepared grids (Figures S53A and S53B,
Supporting Information). The ligands 6g and 10f were docked using Glide XP module, and the
results were visualized using XP visualizer tool.
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The results of docking on hAChE revealed that both the compounds 6g and 10f interacted
effectively with residues of PAS and extended into the deep gorge of CAS (Figures S54A an S54B,
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Supporting Information). At PAS, 6g interacted hydrophobically with Tyr72, Tyr124, and Tyr341
residues and its terminal phenyl group formed face-to-face π-π stacking interaction with Trp286
(Figure 9A). In compound 10f, terminal phenyl group interacted with Tyr124 and Trp286
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hydrophobically, while —NHCH linker formed π-cation interaction with Tyr341 (Figure 9B).
2
The 1,3,4-oxadiazole linker extended the ligands deep into the gorge through interactions at
oxyanion hole (Gly120 and Gly121) and acyl binding pocket (ABP, Phe297). At anionic subsite,
N-benzyl nucleus of both 6g and 10f stacked with Trp86 and Glu202 via π-π and electrostatic
interactions, respectively. Moreover, N-benzylpiperidine formed polar interactions with Ser203
and His447 at CAS. Overall, results suggested that —NH linker in 6g provided optimum flexibility
to the terminal phenyl group to interact more effectively with PAS residues than 10f with —
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NHCH linker. These outcomes were consistent with in vitro assay, which suggested the slightly
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higher inhibitory potential of compound 6g (IC = 0.055 μM) compared to 10f (IC = 0.086 μM).
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Figure 9. Docking poses of ligands in the active site of hAChE (4EY7) [A] 6g and; [B] 10f are
shown as pink and blue ligand-bound surface, respectively. The peripheral anionic site (PAS)
residues are indicated as yellow (Tyr72, Tyr124, Trp286, and Tyr341), catalytic active site (CAS)
as red (Ser203, His447, and Glu334), anionic subsite as green (Trp86, Glu202, and Phe338), acyl
binding pocket (ABP) as orange (Phe295 and Phe297), and oxyanion hole as white (Gly120 and
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Gly121). Blue and green dotted lines denoted for π-π stacking and π-cation interactions,
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Additionally, the binding mode interaction of compounds 6g and 10f were observed on hBACE-
1 (2ZJM), and the results are presented in Figures 10A and 10B. The N-atom of the piperidine
nucleus in 6g contacted with Asp32 through the salt bridge, while forming electrostatic interaction
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with Asp332. However, —NHCH linker in 10f responsible for salt bridge formation with catalytic
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dyad (Asp32 and Asp228) and additional H-bonding with Asp32 (Figures S55A and S55B,
Supporting Information). The compound 10f displayed better interaction profile with catalytic
dyad residues than 6g and observed results are in agreement to in vitro hBACE-1 inhibition assay
(IC , 10f: 0.114 μM; 6g: = 0.143 μM).
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Figure 10. Docking poses of ligands in the active site of hBACE-1 (2ZJM) [A] 6g and; [B] 10f
are shown as pink and blue ligand-bound surface, respectively. Aspartate dyad (Asp32 and
Asp228) residues are showing as yellow. The pink and yellow dotted lines are denoting salt bridges
and H-bonding, respectively.
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Molecular Dynamics Simulations Study. The molecular dynamics simulation studies were
performed to affirm the stability of docked complexes of 6g and 10f on both the targets, i.e.,
hAChE and hBACE-1. The RMSD values for each simulation run were calculated to measure the
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