Benzylpiperidine-Linked Diarylthiazoles as Potential Anti-Alzheimer's Agents: Synthesis and Biological Evaluation

Article abstract of DOI:10.1021/acs.jmedchem.6b00426

A novel series of hybrid molecules were designed and synthesized by fusing the pharmacophoric features of cholinesterase inhibitor donepezil and diarylthiazole as potential multitarget-directed ligands for the treatment of Alzheimer's disease (AD). The compounds showed significant in vitro anticholinesterase (anti-ChE) activity, the most potent compound (44) among them showing the highest activity (IC₅₀ value of 0.30 ± 0.01 μM) for AChE and (1.84 ± 0.03 μM) for BuChE. Compound 44 showed mixed inhibition of AChE in the enzyme kinetic studies. Some compounds exhibited moderate to high inhibition of AChE-induced Aβ_1-42 aggregation and noticeable in vitro antioxidant and antiapoptotic properties. Compound 44 showed significant in vivo anti-ChE and antioxidant activities. Furthermore, compound 44 demonstrated in vivo neuroprotection by decreasing Aβ_1-42-induced toxicity by attenuating abnormal levels of Aβ_1-42, p-Tau, cleaved caspase-3, and cleaved PARP proteins. Compound 44 exhibited good oral absorption and was well tolerated up to 2000 mg/kg, po, dose without showing toxic effects.

Full text of DOI:10.1021/acs.jmedchem.6b00426

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Article
Benzylpiperidine-linked diarylthiazoles as potential anti-
Alzheimer’s agents-synthesis and biological evaluation
Mahesh Shidore, Jatin Machhi, Kaushik Shingala, Prashant Murumkar, Mayank Kumar Sharma,
Neetesh Agrawal, Ashutosh Tripathi, Zalak Parikh, Prakash Pillai, and Mange Ram Yadav
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00426 • Publication Date (Web): 02 Jun 2016
Downloaded from http://pubs.acs.org on June 3, 2016
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Benzylpiperidineꢀlinked diarylthiazoles as potential antiꢀAlzheimer’s agentsꢀ
synthesis and biological evaluation
a
a
a
a
a
Mahesh Shidore , Jatin Machhi , Kaushik Shingala , Prashant Murumkar , Mayank Kumar Sharma ,
a
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Neetesh Agrawal , Ashutosh Tripathi , Zalak Parikh , Prakash Pillai , and
a
Mange Ram Yadav *
aFaculty of Pharmacy,
Kalabhavan Campus,
The Maharaja Sayajirao University of Baroda,
Vadodara – 390 001, India
bZoology Department,
Faculty of Science,
The Maharaja Sayajirao University of Baroda
Vadodara – 390 001, India
KEYWORDS: Alzheimer’s disease, Aβ1ꢀ42, pꢀTau, 2′,7′ꢀDichlorofluorescine diacetate assay.
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ABSTRACT
A novel series of hybrid molecules were designed and synthesized by fusing the pharmacophoric
features of cholinesterase inhibitor donepezil and diarylthiazole as potential multiꢀtargetꢀdirected
ligands for the treatment of Alzheimer’s disease (AD). The compounds showed significant in
vitro antiꢀcholinesterase (antiꢀChE) activity; the most potential compound (44) among them
showing the highest activity (IC50 value: 0.30±0.01 µM) for AChE and (1.84±0.03 µM) for
BuChE. The compound (44) showed mixed inhibition of AChE in the enzyme kinetic studies.
Some compounds exhibited moderate to high inhibition of AChEꢀinduced Aβ1ꢀ42 aggregation,
and noticeable in vitro antiꢀoxidant and antiꢀapoptotic properties. Compound (44) showed
significant in vivo antiꢀChE and antiꢀoxidant activities. Furthermore, compound (44)
demonstrated in vivo neuroprotection by decreasing Aβ1ꢀ42ꢀinduced toxicity by attenuating
abnormal levels of Aβ1ꢀ42, pꢀTau, cleavedꢀcaspaseꢀ3 and cleavedꢀPARP proteins. Compound (44)
exhibited good oral absorption and was well tolerated up to 2000 mg/kg, p.o. dose without
showing toxic effects.
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INTRODUCTION
In the current era of innovative therapeutics, Alzheimer’s disease (AD) has emerged as a
challenging disease to treat among the neurodegenerative disorders. As on today almost 46
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million people worldwide live with dementia. It is expected that by 2050, AD affected
population would multiply three fold, especially in the developed countries like U.S. and
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, 3
Europe.
Although the exact cause of AD is not yet fully known, reports suggest the
involvement of several factors for the development of the disease, including low levels of
acetylcholine (ACh) in the hippocampus and cortex area of the brain. The deposition of amyloid
β (Aβ) peptide, neurofibrillary tangles (pꢀTau) and oxidative stress are also known to play vital
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roles in the pathogenesis of AD. As per the cholinergic hypothesis, the disease is attributed to
the reduced levels of ACh, an important neurotransmitter involved in memory and learning in the
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brain. Acetylcholinesterase (AChE) is a hydrolase enzyme that hydrolyzes ACh and quickly
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terminates the cholinergic synaptic transmission. Studies also suggest that butyrylcholinesterase
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BuChE), another cholinesterase present in the brain is also responsible for hydrolysis of ACh,
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ꢀ10
and its inhibition therefore may further enhance cholinergic transmission in AD.
Thus, the use
of acetylcholinesterase inhibitors (AChEIs) and butyrylcholinesterase inhibitors (BuChEIs) has
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become currently the foundation for the management of AD.
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Researchers have also widely explored the amyloid hypothesis in AD.
As per this
hypothesis, accumulation of Aβ peptide aggregates leads to numerous pathophysiological
changes causing cognitive dysfunction. Secretase proteases are responsible for the formation of
Aβ in the brain, leading to increased levels of Aβ in senile plaques which ultimately lead to
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development of AD. Studies have also indicated that AChE increased the formation of Aβ
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fibrils and Aβ plaques in the cerebral cortex of transgenic mouse models of AD.
Depleting
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the Aβ peptide from the human brain is considered as a rational therapeutic approach for the
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treatment of AD.
Pathogenic role of oxidative damage in the progression of neurodegeneration has also
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been well reported.
Oxidative stress leads to deposition of senile plaques, neurofibrillary
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tangles,
and the deposition of Aβ in the brain. Some studies have suggested that the
neurotoxicity of aggregated Aβ is mediated through its ability to induce oxidative stress via
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spontaneous generation of free radicals and reactive oxygen species (ROS).
Oxidative and
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nitrative stress resulting in the formation of ROS and reactive nitrogen species (RNS) cause
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oxidative damage to the proteins leading to cellular dysfunction and cell death.
Thus,
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successful protection of the neuronal cells from oxidative damage could probably prevent AD.
Oxidative stress and apoptosis are closely linked physiological phenomena which are implicated
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in various pathological conditions including AD. Apoptotic cell death comprises of a sequence
of events leading to the activation of caspase cascade which initiates the fragmentation of
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cellular proteins and DNA, leading to the disintegration of the cell ultimately. It is now well
accepted that massive neuronal and glial cell death in the brain due to apoptosis is a common
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characteristic feature in patients suffering from AD.
Thus, a significant level of
neuroprotection could be achieved by employing chemical entities exhibiting antiꢀoxidant and
antiꢀapoptotic potentials in the brain.
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Various drugs have been used over the past several years for the treatment of AD. But,
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urrently, there is no drug which can completely cure AD. Primary therapeutic option available
for the treatment of AD is the use of USꢀFDA approved cholinesterase inhibitors (Fig. 1) like
tacrine (1), donepezil (2), rivastigmine (3) and galantamine (4). Tacrine (1) exhibited
hepatotoxicity via elevation of serum alanine aminotransferase levels, forcing its limited clinical
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use and consequent withdrawal from the market shortly after its approval. Donepezil (2) is the
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most effective pharmacological agent among the list of approved drugs to treat AD efficiently.
Development of multiꢀtargetꢀdirected ligands is considered one of the most promising
drug discovery approaches for diseases with complex etiology like AD. Singleꢀtargeted drugs
might not always alter the complex diseased system adequately, even if they modulate their
targets with high affinity and selectivity. In contrast, in case of targets connected in a network, a
multiꢀtargetꢀdirected ligand interacting with lower but balanced affinities can still exert superior
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effects compared to a singleꢀtargeted molecule.
Multiꢀtargetꢀdirected drugs have a better
ability to affect the complex equilibrium of whole cellular network than singleꢀtargeted drug
because of their simultaneous effects on several therapeutic targets. Lowꢀ, but balancedꢀaffinity
multiꢀtargetꢀdirected drugs demonstrate weak links with several targets to stabilize the complex
networks of a biological system. A multiꢀtargetꢀdirected drug can actually have a better
efficacy/safety ration than a monoꢀtargeted drug. A balanced, mild activity for multiple
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therapeutic targets might have better safety and reduces the risk of therapeutic resistance.
Hence, there is a need to design such compounds that can act on different causative targets of
AD simultaneously with balanced affinity. This can probably be achieved by linking together
various active moieties having affinity for different targets. The resulting hybrids with balanced
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affinity for different targets could be beneficial to take care of a complex disease like AD.
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NH2
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MeO
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Tacrine (1)
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Figure 1. AChE inhibitors (1ꢀ4) used for the management of AD.
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Recently various bispharmacophore/bivalent ligands
have been reported in the
literature wherein two of the same or different pharmacophoric moieties like donepezilꢀ
propargylamine, donepezilꢀpyridyl, donepezilꢀebselen, tacrineꢀferulic acid, huprineꢀtacrine etc
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are linked together via a spacer for the treatment of AD.
Taking a cue from these
studies, it was planned to synthesize some novel diarylthiazolꢀbenzylpiperidine hybrids and to
assess their potential use in the treatment of AD by performing various in vitro and in vivo
experiments.
RESULTS AND DISCUSSION
Designing considerations. Thiazole ring is present in different chemical entities having a broad
spectrum of biological properties such as antimicrobial, anticonvulsant, antituberculor,
antiinflammatory, antiviral, antimalarial, anticancer, antihypertensive, antiꢀHIV, antiꢀ
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schizophrenia, hypnotic, antinociceptive, and bacterial DNA gyrase B inhibitory activity.
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Acotiamide is a thiazole ring containing drug having potent AChE inhibitory activity.
There
are several other reports which suggest that the thiazole containing compounds can be used for
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the ChE inhibiting activity.
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Some vicinal diaryl triazine derivatives have been reported as potent neuroprotective
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agents. Our laboratory has also reported recently the design and development of vicinally
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substituted diaryltriazines (5) as potential multiꢀtargetꢀdirected therapeutics for the treatment of
AD. It was inferred from the study that the multipotent activity of these diaryltriazines (5)
resided in the diaryltriazine scaffold, and not in the morpholino/piperazinoethyl side chain. A
preliminary molecular modeling study demonstrated that replacement of the
morpholino/piperazinoethyl side chain of compound (5) by benzylpiperidine tail of donepezil
offered a hybrid molecule in which the diaryltriazine and benzylpiperidine portions were bound
to the peripheral anionic site (PAS) and the catalytic active site (CAS) respectively with a Gꢀ
score of ꢀ9.21. In a parallel study, replacement of the diaryltriazine moiety with the
diarylthiazole scaffold exhibited a slightly better Gꢀscore of ꢀ9.38 in the active site of AChE.
Considering these preliminary results and the earlier reports on AChE inhibitory potential of
thiazoleꢀcontaining derivatives, it was persumed that diarylthiazole scaffold could probably be a
better CNSꢀactive moiety for the development of novel multiꢀtargetꢀdirected ligands against AD.
Based upon these preliminary studies, it was planned to link the diarylthiazole ring with the
benzylpiperidine tail of donepezil to obtain novel diarylthiazolꢀbenzylpiperidine hybrids (Fig. 2).
It was considered that the diarylthiazole core of the designed hybrids would mimic the
dimethoxyindanone scaffold of donepezil (2) into the PAS of AChE and the benzylpiperidine
fragment of the designed compounds would interact with the CAS similar to benzylpiperidine
tail of donepezil, retaining the same πꢀstacking interactions. Thus, the designed diarylthiazolꢀ
benzylpiperidine hybrids could show multiꢀtargetꢀdirected potentials for the management of AD.
The designed molecules are novel because such vicinal diarylthiazoles have not yet been
reported as ADꢀresponsive agents.
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The resulting compounds were planned to be evaluated for their antiꢀAlzheimer’s
potentials. Hence, we report herewith an approach wherein a vicinal diarylthiazole scaffold is
linked to a benzylpiperidine moiety to get novel hybird molecules of type (A) (Fig. 2) as
potential multiꢀtargetꢀdirected antiꢀAlzheimer’s agents.
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Figure 2. Designing of a novel series of compounds as multiꢀtargetꢀdirected potential antiꢀAlzheimer’s agents.
Chemistry. The targeted compounds were synthesized as per Scheme 1ꢀ3. In Scheme 1, the
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67
amines
(6ꢀ8) were treated with 1ꢀ(t.butoxycarbonyl)piperidineꢀ4ꢀcarboxylic acid (9) in
presence
of N,Nꢀdiisopropylethylamine and (benzotriazolꢀ1ꢀ
yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) as the coupling reagent to
get the amides (10ꢀ12). Deprotection of the tertꢀbutoxycarbonyl (BOC) group was performed
using trifluoroacetic acid (TFA) in dichloromethane (DCM) to get the amine salts (13ꢀ15) which
were treated further with substituted benzyl bromides using potassium carbonate and
dimethylformamide (DMF) to yield the desired compounds (16ꢀ41).
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Scheme 1. Synthesis of 1ꢀsubstituted benzylꢀNꢀ[4,5ꢀbis(substituted phenyl)thiazolꢀ2ꢀyl]piperidineꢀ4ꢀ
carboxamides (16ꢀ41)
X
HO
O
X
(a)
N
S
N
S
Me
Me
Me
NH2
NH
O
O
O
N
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(
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(10ꢀ12)
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Me
Me
(
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(9)
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(c)
NH
O
N
S
NH . TFA
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(
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R
Br
(13ꢀ15)
X
Cl
X
Cl
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ꢀ
R
R
Me
OMe
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Me
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OMe
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ꢀ
ꢀ
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41
ꢀ
Compd
X
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ꢀ
2ꢀMe
2ꢀCF3
3ꢀF
3,5ꢀDiF
2ꢀCl, 4ꢀF
4ꢀF
4ꢀCN
4ꢀNO2
4ꢀCF3
2ꢀCN
4ꢀF, 2ꢀCF3
4ꢀOCF3
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7
, 10, 13 Me
, 11, 14 Cl
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8, 12, 15
OMe
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ꢀ
ꢀ
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2ꢀCl, 6ꢀF
o
Reagents and Conditions: a) BOP reagent, N,N,ꢀdiisopropylethylamine, dry acetonitrile, C to rt; b) TFA:DCM
o
(
70:30), rt; c) Potassium carbonate, dry DMF, substituted benzyl bromides, rt to heating 60 C.
Nꢀ[(1ꢀSubstituted benzyl)piperidinꢀ4ꢀyl]methylꢀ4,5ꢀdiarylꢀ2ꢀamines (42ꢀ52) were
prepared by reduction of 1ꢀsubstituted benzylꢀNꢀ[4,5ꢀbis(substituted phenyl)thiazolꢀ2ꢀ
yl]piperidineꢀ4ꢀcarboxamides (16, 17, 19, 21, 26, 29, 30, 32, 34, 35 and 39) in presence of
o
boraneꢀdimethyl sulphide complex in THF at 0 C as shown in Scheme 2.
4ꢀPiperidinecarboxamide (53) was treated with substituted benzyl bromides (54ꢀ59) using
potassium carbonate in methanol to get 1ꢀ(1ꢀsubstituted phenyl)piperidineꢀ4ꢀcarboxamides (60ꢀ
65), which were treated further with bis(triacetoxyiodobenzene) and benzoyl isothiocyanate to
obtain 1ꢀ(substituted benzyl)piperidinꢀ4ꢀylthioureas (66ꢀ71). The thiourea derivatives (66ꢀ71)
were then reacted with 2ꢀbromoꢀ1,2ꢀdiarylethanone (72aꢀc) in dry ethanol to obtain the desired
compounds (73ꢀ82) as shown in Scheme 3.
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Scheme 2. Synthesis of Nꢀ[(1ꢀsubstitued benzyl)piperidinꢀ4ꢀylmethyl]ꢀ4,5ꢀbis(substituted phenyl)ꢀ2ꢀ
ylamines (42ꢀ52)
X
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(a)
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
X
X
R
R
(16, 17, 19, 21, 26, 29, 30, 32, 34, 35, 39)
(42ꢀ52)
X
Cl
R
Me
OMe
1
6, 42 26, 46 34, 50 2ꢀMe
17, 43 35, 51 2ꢀCF3
19, 44 29, 47
ꢀ
ꢀ
3,5ꢀdiF
4ꢀF
21, 45
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
30, 48
32, 49
ꢀ
ꢀ
ꢀ
2ꢀCl, 4ꢀF
2ꢀCF , 4ꢀF
3
39, 52 2ꢀCl, 6ꢀF
o
Reagents and Conditions: a) i) BH ꢀDMS, dry THF, 0 C to rt; ii) 1N HCl, Reflux 4 hrs; iii) 5 % sodium
3
bicarbonate, rt to reflux, 2 hrs.
Scheme 3. Synthesis of 1ꢀ(substituted benzyl)ꢀNꢀ[4,5ꢀbis(substituted phenyl)thiazolꢀ2yl]ꢀpiperidinꢀ4ꢀ
ylamines (73ꢀ82)
S
H N
2
O
H N
2
O
Br
HN
N
NH2
(a)
(b)
X
X
N
O
N
R
H
(c)
(53)
(54ꢀ59)
Br
R
R
(60ꢀ65)
(66ꢀ71)
72(aꢀc)
Compd
60 66
R
Compd
72a
X
X
X
R
R
Me Cl OMe
75 H
5
5
5
5
5
5
4
5
6
7
8
9
H
Me
Cl
OMe
7
74
ꢀ
ꢀ
ꢀ
3
ꢀ
7
7
2b
2c
N
S
61 67 4ꢀMe
62 68 4ꢀOMe
63 69 2ꢀCl, 4ꢀF
64 70 4ꢀCN
65 71 4ꢀCF3
H
N
76 80 4ꢀMe
77 81 4ꢀOMe
ꢀ
78
79
N
82 4ꢀCF3
ꢀ
ꢀ
4ꢀCN
2ꢀCl, 4ꢀF
X
(73ꢀ82)
ꢀ
Reagents and Conditions: a) Potassium carbonate, methanol, reflux; b) i) Bis(triacetoxyiodobenzene), Potassium
carbonate, ACN/water, rt to reflux; ii) Benzoyl isothiocyanate, dry DCM iii) THF/1N NaOH, reflux, 4 hrs; c) Dry
ethanol, reflux.
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Biological Evaluation. In vitro AChE and BuChE inhibition assays. The synthesized
compounds (16ꢀ52 and 73ꢀ82) were assessed to determine their antiꢀcholinesterase (antiꢀChE)
activity. AChE and BuChE inhibition activities were evaluated by the method described by
4
5
6
7
8
9
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
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
68
Ellman wherein tacrine and donepezil were used as reference standards (Table 1). Compounds
(43ꢀ45, 48 and 52) having benzylpiperidine and 4,5ꢀdisubstituted thiazolylꢀ2ꢀamine linked with
aminomethylene spacer exhibited higher AChE inhibitory activity [IC values (µM): 0.40±0.01,
5
0
0.30±0.01, 0.37±0.01, 0.52±0.01 and 0.36±0.01 respectively] than the rest of the derivatives.
Compounds (16ꢀ18, 20 and 25) having 4,5ꢀbis(pꢀtolyl)thiazolꢀ2ꢀylamine and substituted
benzyl piperidines linked through the carboxamide group were found to be inactive [IC value
5
0
(µM): >10]. Compounds (80ꢀ82) having 4,5ꢀbis(4ꢀmethoxypheny)thiazolꢀ2ꢀylamine and
substituted benzylpiperidines without a carbon spacer showed good inhibitory activities [IC50
values (µM): 0.83±0.03, 0.77±0.03, 0.75±0.03 respectively].
It has been reported that BuChE level remains constant or increases in advanced AD
8
ꢀ10
while AChE concentration in certain brain regions decreases.
These reports concluded that
balanced inhibition of both AChE and BuChE could be beneficial for the treatment of patients
with cognitive deficit observed in AD. Thus, it was decided to check the test compounds for
BuChE inhibitory activities also. Majority of the synthesized compounds exhibited good BuChE
inhibitory activity. Compounds (16, 26, 28, 36 and 39) showed better BuChE inhibitory activity
[IC value (µM): 1.50±0.02, 1.04±0.02, 0.72±0.01, 1.54±0.03 and 1.57±0.04 respectively] than
5
0
the rest.
The two test compounds (44, 48) which showed the best AChE inhibitory activities [IC50
values (µM): 0.30±0.01 and 0.52±0.01 respectively] also showed reasonably good BuChE
inhibitory activities [IC values (µM): 1.84±0.03 and 0.74±0.01 respectively].
5
0
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1
1
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1
1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
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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
To get an insight into the mechanism of AChE enzyme inhibition, the most potent
compound (44) was subjected to a kinetic study. LineweaverꢀBurk reciprocal plots were
generated by plotting the reciprocal of the reaction rates versus the reciprocal of substrate
concentrations using different concentrations of the test compound (inhibitor, 44). The plots
showed that with increasing concentrations of the inhibitor (44) there occurred increase in slope
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
(
decreased Vmax) and the intercept (higher K ) (Fig. 3). This characteristic pattern indicated a
m
mixed type of an inhibition which supported binding of the compound (44) to AChE at both the
39, 69, 70
sites (CAS and PAS).
Figure 3. Kinetic study of the mechanism of AChE inhibition by compound (44). LineweaverꢀBurk reciprocal plots of the AChE
initial velocity at increasing substrate concentrations (0.1ꢀ1 mM) in the absence and presence of 44 (0.05ꢀ0.4 µM) are shown.
Table 1. AChE and BuChE inhibitory activities (IC values), inhibition of AChEꢀinduced Aβ1ꢀ42
5
0
aggregation and selectivity ratio of the compounds (16ꢀ52 and 73ꢀ82)
b
c
Compound
AChE
BuChE
Selectivity ratio
Aβ1ꢀ42 aggregation
% Inhibition
a
a
(
IC µM) ± SEM
(IC µM) ± SEM
50
50
d
16
17
18
>10
1.50±0.02
1.88±0.03
1.97±0.02
7.0
4.0
5.1
nd
d
7.57±0.03
>10
nd
d
nd
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7
8
9
d
d
d
d
d
d
d
d
1
2
2
2
2
2
2
2
2
9
0
2
3
4
5
6
7
8
2.50±0.03
>10
3.51±0.03
1.60±0.03
2.77±0.03
5.32±0.04
>10
0.7
nd
nd
nd
nd
nd
nd
nd
nd
7.8
1.0
0.2
0.1
10.3
1.1
0.8
0.9
0.2
0.3
1.1
0.6
0.7
0.4
0.4
0.1
0.7
0.3
0.8
0.7
0.2
0.2
0.2
0.2
0.6
0.8
2.88±0.02
1.28±0.02
2.32±0.02
>10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2.78±0.03
1.04±0.02
3.36±0.03
0.72±0.01
3.03±0.04
>10
1.10±0.02
2.77±0.02
0.65±0.01
0.64±0.01
3.70±0.02
1.77±0.02
1.38±0.03
1.34±0.03
0.68±0.01
0.57±0.01
2.45±0.01
1.16±0.03
0.41±0.02
2.80±0.01
1.23±0.02
0.50±0.01
0.40±0.01
0.30±0.01
0.37±0.01
0.87±0.01
0.63±0.01
18.82±2.23
12.02±2.35
29
30
31
33
34
35
d
nd
d
1.59±0.03
2.18±0.03
1.85±0.03
1.92±0.02
1.54±0.03
>10
nd
d
nd
d
nd
14.46±2.15
21.17±1.21
36
37
38
39
40
41
42
43
44
45
46
47
d
nd
d
1.73±0.03
1.57±0.04
3.58±0.01
1.78±0.03
2.51±0.01
2.20±0.03
1.84±0.03
1.95±0.03
1.43±0.02
0.82±0.01
nd
16.56±1.75
d
nd
d
nd
16.17±1.96
26.35±2.46
27.65±2.91
25.79±3.53
23.26±2.51
19.39±2.65
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1
1
1
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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
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5
5
5
5
5
5
5
5
5
6
4
4
5
5
5
7
7
7
7
8
9
0
1
2
3
4
5
6
0.52±0.01
1.24±0.04
0.59±0.01
0.69±0.01
0.36±0.01
1.28±0.03
1.09±0.03
1.38±0.02
3.19±0.01
1.12±0.01
1.02±0.03
0.37±0.01
0.83±0.03
0.77±0.03
0.75±0.03
0.05±0.01
0.01±0.00
0.74±0.01
3.30±0.02
2.21±0.03
8.01±0.02
2.15±0.01
3.59±0.01
6.03±0.04
3.30±0.03
>10
0.7
15.00±2.13
d
0.4
0.3
0.1
0.2
0.4
0.2
0.4
0.3
0.7
0.6
0.2
0.4
0.3
0.4
4.9
0.01
nd
25.64±2.77
20.60±2.35
27.20±3.15
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
d
nd
d
nd
d
nd
d
nd
d
77
78
1.57±0.02
1.72±0.03
1.97±0.02
2.25±0.03
2.53±0.03
1.68±0.03
0.01±0.00
nd
d
nd
79
27.47±2.85
19.24±2.38
11.42±1.43
18.93±2.85
10.63±1.28
80
81
82
Tacrine
Donepezil
1.26±0.01
37.26±2.69
a
b
c
Results are the mean ± SEM of at least three determinations. Selectivity ratio = (IC of AChE)/(IC50 of BuChE). Inhibition of
AChEꢀinduced Aβ1ꢀ42 aggregation produced by the test compounds at 10 µM. Not determined.
50
d
Molecular docking studies on AChE and BuChE. All the compounds were docked in the
active site of AChE (PDB ID: 4EY7). Donepezil was used as a reference drug for the docking
7
1
studies showing similar binding interactions as reported in the literature. The most active
compound (44) in the series exhibited Gꢀscores of ꢀ10.80 for AChE (see supplementary
information, Table S1). The energy of the complex formed by compound (44) with AChE was
ꢀ57.22 kcal/mol indicating that compound (44) formed a stable complex with AChE. It was
observed that compound (44) exhibited a binding mode similar to that of donepezil. The
theoretical binding energy and the Gꢀscores were a pointer to a stronger binding affinity of the
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test compound (44) to AChE than the standard drug donepezil. Phenyl ring of the
benzylpiperidine group of compound (44) showed strong πꢀπ interactions with the anionic site
residue W86 (centroidꢀcentroid = 3.94 Å) similar to the benzylpiperidine tail of donepezil. The
piperidine ring of compound (44) packed against the hydrophobic portion of Y337, F338 and
H447 residues. The diphenyl thiazole scaffold of compound (44) was oriented towards the
entrance of the PAS gorge interacting favourably with Y72, Y124, W286, S293 and Y341
residues indicating strong binding interactions of the diphenyl thiazole ring system in the PAS.
Thus, the aromatic diarylthiazole scaffold of compound (44) was disposed in PAS gorge similar
to the dimethoxyindanone ring of donepezil. NH group of compound (44) formed a hydrogen
bond with Y124 residue, a part of PAS, and the fluorine on the phenyl ring in compound (44)
formed a hydrogen bond with the anionic site residue Y133 (Fig. 4). This interaction study
demonstrated that compound (44) was strongly bound to both the binding sites CAS as well as
PAS of AChE, supporting potentially high inhibitory activity of compound (44) which was
supported by the kinetic study exhibiting binding of the compound (44) to the dual sites.
4
5
6
7
8
9
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
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
Figure 4. Orientation of compound (44) in the active sites of AChE.
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2
2
2
2
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2
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6
Inhibition of AChEꢀinduced Aβ1ꢀ42 aggregation. There are reports suggesting that localized
AChE and Aβ promoted the assembly of Aβ into fibrils, and accelerated the deposition of Aβ
peptide in senile plaques. AChE directly binds to Aβ through its PAS to promote the aggregatory
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
72, 73
74
effect.
Blockade of PAS could attenuate the AChEꢀinduced Aβ peptide aggregation. Those
compounds which showed potent AChE inhibitory activity in the in vitro ChE inhibition studies
7
5, 76
were selected for thioflavin T (ThT) assay
(Table 1) to evaluate their potential to inhibit
AChEꢀinduced Aβ1ꢀ42 aggregation. Results of the study showed that three compounds (44, 52
and 79) among the series exhibited potent inhibition (~27 %) of AChEꢀinduced Aβ1ꢀ42
aggregation at 10 µM concentration. These compounds (44, 52 and 79) demonstrated potent
AChE inhibitory activity in submicromolar range in the earlier experiment also.
In vitro bloodꢀbrain barrier permeation assay. Successful crossing of blood−brain barrier
(BBB) by a drug is a preꢀrequisite for treatment of CNS disorders. To explore whether the
synthesized hybrid diarylthiazolꢀbenzylpiperidine derivatives have the ability to penetrate into
the brain, a parallel artificial membrane permeation assay of the bloodꢀbrain barrier (PAMPAꢀ
77ꢀ79
BBB) was performed as described by Di et al.
This model has the advantage of predicting
passive diffusion of a molecule through BBB with high accuracy. The in vitro permeability (Pe)
of the selected hybrid diarylthiazolꢀbenzylpiperidine derivatives (Table 2) and nine
commercially available drugs (see the supplementary information, Table S2) was determined
through a lipid extract of porcine brain lipid in PBS/ethanol (70:30). The assay was validated by
comparing the experimentally obtained permeability [P (exp)] of these nine drugs with the
e
reported values of permeation [P (ref)] offering a linear relationship i.e. P (exp.) = 1.171P (ref.)
e
e
e
2
+ 1.489 (R = 0.983) (see the supplementary information, Figure S1). From this equation and
77
considering the limits established by Di et al, for BBB permeation, it was concluded that
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ꢀ6
ꢀ1
compounds with permeability values (P ) greater than 5.9 × 10 cm s (see the supplementary
e
4
5
6
7
8
9
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
information, Table S3) were capable of crossing the BBB. All the tested compounds
demonstrated permeability values above this limit. Therefore, the experimentally determined
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
permeability values (P ) of the test compounds were a pointer towards their potential to
e
comfortably cross the BBB by passive diffusion.
ꢀ6
ꢀ1
Table 2. Permeability (P 10 cm s ) results from the PAMPAꢀBBB assay for some of the
e,
test compounds with their predictive penetration in CNS
ꢀ
6
ꢀ1 a
b
ꢀ6
ꢀ1 a
b
Compd
P (10 cm s )
Prediction
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
Compd
47
P (10 cm s )
Prediction
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
e
e
28
29
35
15.9±1.2
8.8±0.4
12.5±1.3
13.2±1.5
12.3±0.8
8.4±0.8
12.2±0.6
8.4±0.8
9.6±1.1
10.3±0.6
8.6±0.2
48
12.3±0.8
12.5±0.8
8.2±0.4
50
36
39
51
52
42
43
44
10.6±1.1
12.8±0.8
14.5±1.5
10.1±0.6
8.8±0.4
79
80
81
45
82
46
a
b
Data expressed as mean ± SEM of three independent experiments. CNS+ indicates good passive CNS permeation.
Cell viability and neuroprotective studies. To check the therapeutic suitability of the selected
hybrid diarylthiazoleꢀbenzylpiperidine derivatives, their effect on cell viability and
neuroprotective potential against oxidative stress were evaluated using the human neuroblastoma
SHꢀSY5Y cell line. For the assessment of cytotoxicity of the test compounds, cells were exposed
to considerably high concentrations of the test compounds (40 µM and 80 µM) for 24 hr
followed by determination of the cell viability using 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
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diphenyltetrazolium bromide (MTT) assay. In this experiment, the selected test compounds
caused negligible cell death even at such high concentrations (Table 3).
In another set of experiments, neuroprotective potential of the selected hybrid
diarylthiazoleꢀbenzylpiperidine derivatives was assessed. Oxidative stressꢀlike condition in SHꢀ
0
1
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0
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8
9
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SY5Y cells was induced using H O as a toxic insult.
In this assay, addition of H O (100
2 2
2
2
µM) to the growth media caused significant cell death as evidenced by reduction in cell viability
52ꢀ56 %) compared to the control. To assess the neuroprotective potential of the test compounds
against the toxic onslaught of H O , the cells were treated for 2 hr with the test compounds (5
(
2
2
µM, 10 µM and 20 µM) prior to H O insult for 24 hr. At 5 µM concentration, the compounds
2
2
did not show significant protective effect (data not shown). However, as shown in (Table 3), the
selected derivatives exhibited significant neuroprotective effect at 10 µM and 20 µM
concentrations. Amongst all of the hybrid derivatives, compound (44) showed the highest
neuroprotection against the H O insult. The results revealed that compounds (28, 29, 35, 36, 39,
2
2
42ꢀ48, 50ꢀ52, 77 and 80ꢀ82) can protect neuronal cells against oxidative stressꢀassociated cell
death.
Table 3. Cell viability, neuroprotection and free radical scavenging activity of some of the
test compounds in the human neuroblastoma SHꢀSY5Y cell line and DPPH assay
a
b
c
Compound
Cell viability (%)
Neuroprotection (%)
RP of DPPH (%)
40 µM 80 µM
10 µM
20 µM
10 µM
20 µM
28
93.6±2.5
95.6±2.4
92.4±3.7
94.0±2.7
92.9±2.2
91.4±3.1
93.5±2.8
91.1±2.9
92.8±3.1
93.1±2.1
90.3±3.9
91.6±4.3
90.1±3.1
91.7±3.6
29.2±2.4
31.5±3.7
21.9±3.2
33.3±2.3
32.9±2.6
26.9±2.9
29.3±2.8
50.8±3.4
53.4±2.9
38.7±2.7
39.9±3.0
55.7±2.9
52.7±2.8
48.8±3.3
40.6±3.1
43.5±2.4
43.0±2.9
40.5±3.3
46.0±2.7
49.0±2.8
48.9±2.1
61.7±2.8
64.6±2.8
63.6±2.9
62.6±3.1
63.6±2.4
58.9±2.7
59.4±3.1
29
35
36
39
42
43
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95.2±2.6
91.8±2.0
95.4±1.9
92.4±3.7
92.7±2.6
91.6±3.3
94.2±3.6
94.2±2.2
94.2±3.2
95.4±2.7
92.1±3.6
94.5±2.6
89.4±2.3
96.9±1.4
nd
91.3±2.9
89.7±3.2
90.9±2.4
91.7±2.8
88.9±3.6
87.2±3.9
90.1±2.5
87.1±3.6
92.5±1.9
89.5±2.7
89.5±3.2
90.4±2.4
90.0±3.4
92.3±2.4
nd
39.6±2.3
28.8±1.4
23.6±2.8
29.8±2.2
31.5±2.7
34.2±2.1
33.2±3.1
28.8±2.4
32.0±3.3
29.9±2.1
25.5±2.8
30.4±2.4
44.7±3.3
48.2±2.4
nd
59.5±2.4
42.5±2.6
49.7±3.1
42.9±3.2
53.7±2.6
41.4±2.2
48.4±2.2
41.5±2.9
53.2±2.7
47.4±2.3
40.1±3.4
42.0±2.2
57.9±2.1
62.0±2.5
nd
54.6±2.7
51.2±2.7
45.4±2.9
44.8±2.2
46.3±3.2
42.5±3.1
51.8±2.8
48.3±2.5
42.2±2.4
47.2±3.1
49.3±2.2
50.0±2.9
54.9±2.1
59.9±2.6
56.4±2.7
70.5±2.6
56.5±3.3
63.4±2.4
60.9±2.2
57.4±2.3
63.4±2.7
66.9±2.6
57.1±2.8
63.2±3.2
63.4±2.3
65.5±2.9
66.2±2.4
68.8±3.7
75.5±2.9
67.7±2.7
45
46
47
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11
12
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14
15
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18
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20
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28
29
30
31
32
33
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35
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
48
50
51
52
79
80
81
82
Tacrine
Donepezil
Ascorbic acid
a
Percentage cell viability of SHꢀSY5Y cells exposed at relatively high concentrations (40 µM and 80 µM) of test compounds.
Percentage neuroprotection of SHꢀSY5Y cells at relatively lower concentrations (10 µM and 20µM) of the test compounds
b
c
against H O (100 µM) insult. Results are the mean ± SEM of at least three independent experiments. RP of DPPH (%) =
2
2
reduction percentage of DPPH. Results are the mean ± SEM of at least three independent experiments. nd = not determined.
Antiꢀoxidant activity. Diphenylꢀ1ꢀpicrylhydrazyl (DPPH) is one of the few stable and
commercially available organic nitrogen radicals. DPPH assay is commonly used for preliminary
81
screening of compounds capable of scavenging activated oxygen species. The selected hybrid
diarylthiazoleꢀbenzylpiperidine derivatives were evaluated for their antiꢀoxidant potential using
DPPH assay. All of the test compounds were found to exhibit significant free radical scavenging
activity. At a concentration of 10 µM, all of the test compounds showed free radical scavenging
activity in the range of 40ꢀ55 % with compounds (44) showing the highest activity (55 %).
Moreover, at 20 µM concentration, the test compounds exhibited free radical scavenging activity
ranging from 56ꢀ70 % with compound (44) showing 70 % scavenging activity which was more
than what was shown by the standard drug ascorbic acid.
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ROS estimation in primary hippocampal neuronal culture. Hippocampal region of brain is
considered as the memory origin centre having ACh as the major neurotransmitter. Therefore,
degradation of hippocampal neurons causes impairment of memory and ultimately leads to
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degeneration of the neurons.
Elevated levels of ROS is a sign of heightened oxidative stress
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in various pathological conditions. Aβ
toxicity is known to induce oxidative stress in the
ꢀ42
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5
pathogenesis of AD. The antiꢀoxidant potential of the most potent test compound (44) was
evaluated by estimating intracellular levels of ROS in primary rat hippocampal neuronal cells
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using 2′,7′ꢀdichlorofluorescine diacetate (DCFHꢀDA) assay.
DCFHꢀDA crosses cell
membrane and gets readily hydrolyzed by intracellular esterases to nonfluorescent DCFH. In the
presence of ROS, DCFH is rapidly oxidized to the highly fluorescent 2′,7′ꢀdichlorofluorescine
(DCF). As shown in Figure 5A, Aβ1ꢀ42 insult elevated the ROS level significantly as compared
to the control cells (p<0.001). However, the test compound (44) (10 µM and 20 µM) (Fig. 5A,
p<0.001) like the standard drug donepezil (Fig. 5A, p<0.001) dose dependently attenuated the
ROS generation caused by Aβ1ꢀ42 toxicity. These results revealed excellent ROS scavenging
ability of compound (44).
Flow cytometry. Apoptosis is a key cellular event which plays an important role in the etiology
8
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of various neurodegenerative diseases.
It has been reported that Aβ1ꢀ42, a pathological
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hallmark of AD, causes apoptosis leading to neurodegeneration ultimately.
Apoptosis was
assessed using flow cytometry by staining the primary rat hippocampal neurons with annexin Vꢀ
FITC and propidium iodide (PI). Annexin V enters into the cells in both, the early and the late
stages of apoptosis while the PI stains the cells only in the late stage of apoptosis or the necrosis
stage. Aβ1ꢀ42 (10 µM) toxicity increased the percentage of early apoptotic cells significantly (Fig.
5B, 38.80 %) as compared to the control cells (Fig. 5B, 1.78 %). Compound (44) (20 µM) (Fig.
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B, 13.40 %) significantly abrogated the percentage of early apoptotic cells induced by Aβ1ꢀ42
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insult in a manner similar to donepezil (Fig. 5B, 7.84 %). The results demonstrated antiꢀ
apoptotic potential of compound (44) against Aβ1ꢀ42ꢀinduced toxicity which further supported its
antiꢀAlzheimer’s potential.
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Figure 5. In vitro ROS scavenging and antiꢀapoptotic potential of compound (44) against Aβ1ꢀ42 (10 µM) insult in primary rat
hippocampal neuronal culture. (A) Percentage inhibition of ROS generation as assessed by DCFHꢀDA assay. (B) Flow
cytometric assessment of apoptosis using Annexin VꢀFITC and PI staining. Cells in the lower left quadrant are viable (Annexin
VꢀFITCꢀ/PIꢀ). Cells in the lower right quadrant are early apoptotic (Annexin VꢀFITC+/PIꢀ) and those in the upper right quadrant
###
are late apoptotic or necrotic (Annexin VꢀFITC+/PI+). Data are expressed as mean ± SEM. p<0.001 vs. control cells. ***
p<0.00 vs. Aβ1ꢀ42ꢀtreated control cells. C=control cells.
Behavioural studies. To determine the in vivo antiꢀChE activity of the most potent compound
9
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(44) in the series, scopolamine model was adopted.
Scopolamineꢀinduced amnesia in rodents
is an accepted standard model in behavioural pharmacology for evaluation of ChE inhibiting
potential of drug candidates to be developed for the antiꢀAlzheimer’s therapy. Scopolamine
blocks the muscarinic cholinergic receptors distinctly, that lead to impairment of the cognitive
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functions. To evaluate the antiꢀamnesic effect of compound (44), Morris Water Maze (MWM)
test was utilized. During the last five days of the treatment period, escape latency time (ELT) and
number of platform area crossings were recorded for the animals of different experimental
groups. The escape latencies were significantly prolonged (Fig. 6A, p<0.001) while the number
of platform area crossings (Fig. 6B, p<0.001) were significantly reduced by the scopolamine
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ꢀ1
ꢀ1
treatment (1.4 mg kg , i.p.). Donepezil (at 5 mg kg , p.o.) significantly reduced ELT (Fig. 6A,
p<0.001) and increased platform area crossings (Fig. 6B, p<0.01) as compared to the
scopolamineꢀtreated control group. At an equivalent dose corresponding to donepezil, compound
(44) significantly shortened ELT (Fig. 6A, p<0.001) and increased the number of platform area
crossings (Fig. 6B, p<0.05) as compared to the scopolamineꢀtreated control group. The results
revealed antiꢀamnesic effect of compound (44) against the scopolamineꢀinduced ADꢀlike
phenotype.
In further experimentation, the effect of compound (44) on the AChE and BuChE levels
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64
of brain was estimated in mice using Ellman’s method.
As described in our previous report,
scopolamine treatment significantly elevated AChE (Fig. 6C, p<0.001) and BuChE (Fig. 6D,
p<0.001) levels compared to the vehicleꢀtreated control group. However, the elevated levels of
AChE (Fig. 6C, p<0.001) and BuChE (Fig. 6D, p<0.001) were significantly attenuated by
compound (44) at a dose equivalent to that of donepezil.
In order to further elucidate the mechanism of antiꢀamnesic effect of compound (44),
brain levels of malondialdehyde (MDA ꢀ a byproduct of lipid peroxidation) and catalase (CAT ꢀ
which catalyzes H O decomposition) were determined following completion of the MWM test.
2
2
Scopolamine treatment significantly elevated the MDA level (Fig. 6E, p<0.001) while reduced
the CAT level in the brains of the treated animals (Fig. 6F, p<0.001) compared to the vehicleꢀ
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treated control group animals. However, treatment of the amnesic mice with compound (44)
significantly attenuated the MDA level (Fig. 6E, p<0.001) while elevated the CAT level (Fig.
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6F, p<0.01). The results therefore revealed the in vivo ChE inhibitory and antiꢀoxidant activities
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of compound (44) similar to donepezil, which further substantiated antiꢀAlzheimer’s potential of
this most promising test compound (44).
Figure 6. Compound (44) improved spatial learning and memory in scopolamineꢀtreated mice through antiꢀChE and antiꢀoxidant
ꢀ
1
activities. In MWM test, scopolamine (1.4 mg kg , i.p.) increased (A) ELT while (B) reduced number of platform area crossings.
ꢀ
1
This pattern was significantly reversed by 44 similar to donepezil at 5 mg kg . (C) AChE and (D) BuChE levels elevated by
scopolamine treatment were significantly attenuated by 44. Oxidative stress parameters are represented as (E) MDA and (F)
CAT levels. Altered MDA and CAT levels after scopolamine treatment were significantly reversed by treatment with compound
#
##
##
#
(
44). Data are expressed as mean ± SEM (n=6). p<0.001, p<0.01, p<0.05 vs. vehicleꢀtreated control group.*** p<0.001, **
p<0.01, * p<0.05 vs. scopolamineꢀtreated control group. C=vehicleꢀtreated control group.
Y maze test. To develop ADꢀlike phenotype, animals were subjected to intracerebroventricular
(ICV) injection of Aβ1ꢀ42 in the hippocampal region of brain. This is a promising model to
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evaluate the neuroprotective potential of any drug candidate in ADꢀlike condition.
Impairment of the working memory in the animals was assessed using Y maze test.
“Spontaneous alteration” is indicative of the working memory. Aβ1ꢀ42ꢀtreated control animals
exhibited significantly reduced percentage of “spontaneous alterations” throughout the
experimental session as compared to the vehicleꢀtreated control animals (Fig. 7A, p<0.001).
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Animals treated with compound (44) displayed increased percentage of “spontaneous
alterations” (Fig. 7A, p<0.01). However, the mean number of arm entries remained unchanged
across all the experimental groups which indicated that the general locomotor activity was not
hampered by Aβ1ꢀ42 toxicity (Fig. 7B). Thus, Aβ1ꢀ42ꢀworsened hippocampalꢀdependant working
memory was improved by the treatment with compound (44).
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Figure 7. Compound (44) improved immediate working memory in rats which received ICV injection of Aβ1ꢀ42 in Y maze test.
(A) Percentage of spontaneous alterations and (B) total number of arm entries, as recorded by a blind observer. Data are
#
##
##
#
expressed as mean±SEM (n=6). p<0.001, p<0.01, p<0.05 vs. vehicleꢀtreated control group.*** p<0.001, ** p<0.01, *
p<0.05 vs. scopolamineꢀtreated control group. C=vehicleꢀtreated control group.
Western blot analysis. The in vivo neuroprotective potential of compound (44) was assessed by
determining the levels of pathological hallmarks of AD in the hippocampal region of rat brain
which was subjected to ICV injection of Aβ1ꢀ42. Elevated levels of Aβ1ꢀ42 and pꢀtau are the key
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indicators of AD progression. At the end of the ICV experimental model, animals from
different groups were scarified to estimate Aβ1ꢀ42 and pꢀTau levels using Western blot analysis
(Fig. 8A). A significant rise in the levels of Aβ1ꢀ42 and pꢀTau was observed in the Aβ1ꢀ42ꢀtreated
control group as compared to the vehicleꢀtreated control group. Supporting the results of the in
vitro Aβ1ꢀ42 aggregation inhibition (ThT) assay, treatment of the animals with compound (44)
significantly attenuated the levels of Aβ1ꢀ42 (Fig. 8B, p<0.01) and pꢀTau (Fig. 8C, p<0.05)
similar to donepezil. The results confirmed the antiꢀAβ1ꢀ42 aggregatory and antiꢀAlzheimer’s
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potential of compound (44). Additionally, to assess the in vivo antiꢀapoptotic potential of
compound (44), cleavedꢀcaspaseꢀ3 and cleavedꢀPARP levels were also determined in the
hippocampal region of the rat brain which received Aβ1ꢀ42 insult. It has been well documented
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that cleavage/activation of caspaseꢀ3 is a key event in cell apoptosis. It has been demonstrated
that caspaseꢀ3 is either partially or fully responsible for the proteolytic cleavage of poly(ADPꢀ
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ribose) polymeraseꢀ1 (PARP), a nuclearꢀDNA binding protein
involved in numerous cellular
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events including DNA repair.
PARP overactivation is associated with various
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neurodegenerative conditions including AD. It has been reported earlier that Aβ peptide caused
100, 101
induction of PARP activity in the hippocampal region of adult rat brain.
Cleavage of PARP
is followed by the caspaseꢀ3 activation which subsequently leads to apoptosis and ultimately
neuronal cell death. Thus, cleavedꢀcaspaseꢀ3 and cleavedꢀPARP are the two significant key
102
markers of apoptosis. In this study, the levels of cleavedꢀcaspaseꢀ3 (Fig. 8D, p<0.001) and
cleavedꢀPARP (Fig. 8E, p<0.001) were found to be significantly elevated in the hippocampal
region of the brains of Aβ1ꢀ42ꢀtreated control animals as compared to the vehicleꢀtreated control
group. Supporting the in vitro results, compound (44) demonstrated significant antiꢀapoptotic
potential by lowering the levels of cleavedꢀcaspaseꢀ3 (Fig. 8D, p<0.001) and cleavedꢀPARP
(Fig. 8E, p<0.05) in the treated group similar to donepezil. In conclusion, compound (44) has
exhibited multiꢀtargetꢀdirected actions highlighting its potential as a possible therapeutic
alternative for the treatment of AD.
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Figure 8. In vivo neuroprotective and antiꢀapoptotic potential of compound (44). (A) The expression of Aβ1ꢀ42, pꢀ
Tau, cleavedꢀcaspaseꢀ3 and cleavedꢀPARP was assessed by Western blot analysis in the hippocampal region of rat
brain which was given ICV injection of Aβ1ꢀ42. Densitometric analysis revealed attenuation of (B) Aβ1ꢀ42, (C) pꢀTau,
(D) cleavedꢀcaspaseꢀ3 and (E) cleavedꢀPARP levels by compound (44) which were elevated by Aβ toxicity. Data
are expressed as mean±SEM (n=6). p<0.001, p<0.01, p<0.05 vs. vehicleꢀtreated control group.*** p<0.001, ** p<0.01,
* p<0.05 vs. Aβ1ꢀ42ꢀtreated control group. C=vehicleꢀtreated control group. D=donepezilꢀtreated group.
1ꢀ42
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Acute toxicity study. For the development of a compound as a new drug, determination of its
acute toxicity profile is considered to be an important criterion. Acute toxicity of compound (44),
the most promising candidate of the current study was determined in male Swiss Albino mice at
ꢀ1
doses of 0, 677, 1333 and 2000 mg kg (n = 5 per group) by oral administration. The animals
were regularly observed for 14 days after treatment. After the observation period of 14 days, all
the animals remained alive and appeared healthy in terms of fur sleekness, water and food
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consumption and body weight. On the 15 day, all the animals were sacrificed for macroscopic
examination of the heart, liver and kidneys for any damage. No damage was observed to these
organs. The results of the study showed that mice treated with compound (44) did not produce
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any acute toxicity or mortality immediately or during the postꢀtreatment period. Therefore,
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compound (44) can be considered to be nonꢀtoxic and well tolerated at doses up to 2000 mg kg .
Pharmacokinetic analysis. Preliminary pharmacokinetic analysis of compound (44) was
performed in male Wistar rats. Compound (44) was administered orally to the animals at a dose
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of 5 mg kg followed by blood sampling at the indicated time points (see experimental section).
Pharmacokinetic parameters summarized in Table 4 were calculated from the concentrationꢀtime
profile using noncompartmental extravascular analysis. The mean concentrationꢀtime course of
compound (44) after single oral administration is shown in Figure 9. An asymmetric curve of the
compound (44) deviating from the Guaussian distribution curve suggested a prolonged
elimination phase as compared to the absorption phase. Peak plasma concentration (Cmax) of the
compound (44) was achieved approximately at 3.66±0.57 hr after single dose oral
administration. The terminal elimination halfꢀlife (t ) was observed to be 19.42±4.69 hr.
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Overall, the results suggested that the compound (44) exhibited good oral absorption and was
eliminated at a rate which is moderate as compared to the absorption phase. However, in vivo
human clinical trials are required to be performed to prove the actual utility of the test compound
(44).
Table 4. Pharmacokinetic parameters of compound (44) after administration of single oral
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dose (5 mg kg ).
Pharmacokinetic
parameters
Compound
a
(44)
Cmax (ng/ml)
21.20±1.99
tmax (hr)
3.66±0.57
AUC(0ꢀ8) (ng.hr/ml)
587.16±62.09
19.42±4.69
t (hr)
1/2
a
Date are expressed as mean±SD (n=4).
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Figure 9. Mean plasma concentration vs. time curve of single oral dose (5 mg kg ) of compound (44) in rats. Error bars
represents the standard deviation of the mean (n=4).
CONCLUSION
A novel series of hybrid molecules have been synthesized by combining the benzylpiperidine
fragment of donepezil and vicinal diarylthiazole as a privileged scaffold to obtain chemical
entities exhibiting antiꢀAlzheimer’s potentials. The synthesized hybrids were evaluated for their
in vitro AChE and BuChE inhibitory activities. Some of the test compounds (28, 29, 35, 36, 39,
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2ꢀ48, 50ꢀ52, 79, 81 and 82) showed moderate to high AChE inhibitory activity. These
derivatives also exhibited good binding affinity for the active sites of AChE in the molecular
modeling studies. Among the series, compound (44) exhibited the most promising inhibitory
potential for AChE (IC = 0.30 ± 0.01 ꢁM) and BuChE (IC = 1.84 ± 0.03 ꢁM). Enzyme
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kinetics proved that compound (44) caused a mixed type of AChE inhibition by binding to both
the active sites (PAS and CAS) of AChE. Those derivatives which showed good AChE
inhibitory activities also exhibited reasonably good inhibition of AChEꢀinduced Aβ1ꢀ42
aggregation. Among these, three compounds (44, 52 and 79) demonstrated the highest level of
antiꢀamyloid activity (~ 27 % inhibition) at a concentration of 10 ꢁM. Although these molecules
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(44, 52 and 79) showed submicromolar AChE inhibitory potencies and their concurrent Aβ1ꢀ42
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aggregation inhibitory activities were in the singleꢀdigit micromolar range, they exhibited
noticeable neuroprotective activities in vitro and in vivo. In particular, compound (44) showed
ROS scavenging and antiꢀapoptotic properties against Aβ1ꢀ42 insult in the primary rat
hippocampal neuron cultures. In the Aβ1ꢀ42ꢀinduced Alzheimer’s rat model, compound (44)
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improved the spatial learning and memory. Compound (44) significantly reversed the Aβ1ꢀ42
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induced enhancement of ChEs levels and favourably altered the oxidative stress parameters
(MDA and CAT) in the hippocampal region of rat brain similar to the standard drug donepezil.
Results of the in vivo studies revealed the antiꢀChE and antiꢀoxidant potential of compound (44)
which further substantiated the results of the in vitro experiments. Compound (44) also exhibited
significant neuroprotection in rats subsequent to intraꢀhippocampal injection of Aβ1ꢀ42 by
attenuating the abnormal levels of Aβ1ꢀ42, pꢀTau, cleavedꢀcaspaseꢀ3 and cleavedꢀPARP proteins
as assessed by Western blot analysis. Over and above, compound (44) was found to be well
tolerated and nontoxic up to 2000 mg/kg, p.o. dose. Pharmacokinetic analysis revealed that
compound (44) exhibited good oral absorption, and was eliminated at a relatively moderate rate
compared to the absorption phase. Thus, compound (44) has shown its real potential as a multiꢀ
targetꢀdirected ligand by interacting with different pathophysiological targets of AD
demonstrating significant multiple effects like antiꢀChE, antiꢀAβ aggregatory, neuroprotective,
ROS scavenging, antiꢀoxidant and antiꢀapoptotic activities in different in vitro and in vivo
experiments. All put together, the beneficial effects of the novel diarylthiazoleꢀbenzylpiperidine
hybrid molecules qualified them as potential lead candidates to be developed as new drugs for
the treatment of AD, and the most promising multiꢀtargetꢀdirected ligand (44) amongst them
could be considered as a potential drug molecule for further development.
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