A multifunctional therapeutic approach: Synthesis, biological evaluation, crystal structure and molecular docking of diversified 1H-pyrazolo[3,4-b]pyridine derivatives against Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2019.04.038

2-(piperazin-1-yl)–N-(1H-pyrazolo[3,4-b]pyridin-3-yl)acetamides are described as a new class of selective and potent acetylcholinesterase (AChE) inhibitors and amyloid β aggregation inhibitors. Formation of synthesized compounds (P1–P9) was justified via H¹ NMR, C¹³ NMR, mass spectra and single crystal X-Ray diffraction study. All compounds were evaluated for their acetylcholinesterase and butyrylcholinesterase inhibitory activity, inhibition of self-mediated Aβ aggregation and Cu(II)-mediated Aβ aggregation. Also, docking study carried out was in concordance with in vitro results. The most potent molecule amongst the derivatives exhibited excellent anti-AChE activity (IC₅₀ = 4.8 nM). Kinetic study of P3 suggested it to be a mixed type inhibitor. In vitro study revealed that all the compounds are capable of inhibiting self-induced β-amyloid (Aβ) aggregation with the highest inhibition percentage to be 81.65%. Potency of P1 and P3 to inhibit self-induced Aβ_1-42 aggregation was ascertained by TEM analysis. Compounds were also evaluated for their Aβ disaggregation, antioxidation, metal-chelation activity.

Full text of DOI:10.1016/j.ejmech.2019.04.038

Accepted Manuscript
A multifunctional therapeutic approach: Synthesis, biological evaluation, crystal
structure and molecular docking of diversified 1H-pyrazolo[3,4-b]pyridine derivatives
against Alzheimer's disease
Tarana Umar, Shruti Shalini, Md Kausar Raza, Siddharth Gusain, Jitendra Kumar,
Prerna Seth, Manisha Tiwari, Nasimul Hoda
PII:
S0223-5234(19)30353-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.038
EJMECH 11273
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 March 2019
Revised Date: 3 April 2019
Accepted Date: 13 April 2019
Please cite this article as: T. Umar, S. Shalini, M.K. Raza, S. Gusain, J. Kumar, P. Seth, M. Tiwari, N.
Hoda, A multifunctional therapeutic approach: Synthesis, biological evaluation, crystal structure and
molecular docking of diversified 1H-pyrazolo[3,4-b]pyridine derivatives against Alzheimer's disease,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.04.038.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
A multifunctional therapeutic approach: Synthesis, biological evaluation, crystal structure
and Molecular docking of diversified 1H-pyrazolo[3,4-b]pyridine derivatives against
Alzheimer’s disease
a
‡b
‡c
b
d
Tarana Umar , Shruti Shalini , Md Kausar Raza , Siddharth Gusain , Jitendra Kumar , Prerna
b
b,
a,
Seth , Manisha Tiwari ∗ and Nasimul Hoda ∗
a
Department of Chemistry, Jamia Millia Islamia (Central University), New Delhi 110025, India
Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, New Delhi 110007, India
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560012, India
Department of Chemistry, Sardar Vallabhbhai Patel College, Bhabua, Kaimur- 821101, V. K. S. U., Ara, Bihar- 802301, India
b
c
d
‡
These authors contributed equally to this manuscript.
Abstract
-(piperazin-1-yl)-N-(1H-pyrazolo[3,4-b]pyridin-3-yl)acetamides are described as a new class of
selective and potent acetylcholinesterase (AChE) inhibitors and amyloid β aggregation
2
1
13
inhibitors. Formation of synthesized compounds (P1-P9) was justified via H NMR, C NMR,
mass spectra and single crystal X-Ray diffraction study. All compounds were evaluated for their
acetylcholinesterase and butyrylcholinesterase inhibitory activity, inhibition of self-mediated Aβ
aggregation and Cu(II)-mediated Aβ aggregation. Also, docking study carried out was in
concordance with in vitro results. The most potent molecule amongst the derivatives exhibited
excellent anti-AChE activity (IC50 = 4.8 nM). Kinetic study of P3 suggested it to be a mixed type
inhibitor. In vitro study revealed that all the compounds are capable of inhibiting self-induced β-
amyloid (Aβ) aggregation with the highest inhibition percentage to be 81.65%. Potency of P1
and P3 to inhibit self-induced Aβ1-42 aggregation was ascertained by TEM analysis. Compounds
were also evaluated for their Aβ disaggregation, antioxidation, metal-chelation activity.
Keywords: N-(1H-pyrazolo[3,4-b]pyridin-3-yl)acetamides; AChE inhibitors; selectivity; amyloid
β aggregation inhibitors; docking
∗
∗
Corresponding author. Tel.: +0091-9910200655; fax: +0091-11-26985507; e-mail: nhoda@jmi.ac.in
Corresponding author. Tel.: +0-000-000-0000; fax: +0-000-000-0000; e-mail: mtiwari07@gmail.com
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1
. Introduction
Alzheimer's disease (AD) is the devastating disease that strongly correlates to morbidity and
mortality. AD is amongst the most predominant irreversible neurodegenerative disorders that
targets to cholinergic neurons of central nervous system (CNS) together with cognitive ability
and spatial awareness [1]. AD is recognized as the leading reason for dementia that mainly
troubles old age groups, although cases of age groups belonging 30s, 40s and 50s had started to
rise. The estimated figure of people suffering dementia in 2018 is 50 million worldwide and is in
progression [2].
Etiology of Alzheimer’s is not yet explained. However accumulation of amyloid beta [3],
shortage of neurotransmitter acetylcholine [4], tau- phosphorylation and oxidative stress [5] are
active participants in AD (pathophysiology) [6]. On account of its intricacy and as an answer to
this pluralism of causative factors and consequences, one feasible research strategy is to evolve a
multi targeted perspective, by which a unit molecule acts on separate targets of disease. Several
attractive targets present good opportunity towards the development of anti-AD agents, and its
multi-factorial nature demands multi-targeted molecules that may benefit AD treatment. It
further allows to overcome intrinsic disputes of the combination therapy, where exists possibility
of drug-drug interactions [7] and also to acquire superior therapeutic profile than single targeted
molecules. Recognition of multifactor nature of AD is widespread. Simultaneous targeting of AD
pathologies by exploiting independent and multiple activity drug therapies has come up as a very
strong strategy to address the multiplex, degenerative character of AD. Where from the
cholinergic and amyloid hypotheses are generally applied to originate reliable therapeutics.
The two capital modes have been described to be generously involved in disease growth: i)
accumulation of the amyloid β in extracellular region in fibrils, protofibrils and plaques and ii)
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continuous de-escalating of cholinergic neurons especially in cortex and hippocampus.
Amassment of Aβ due to evacuation of Aβ and/or, changed processing of APP happens
according to central AD hypothesis. The event leads to selective neuronal loss and widespread
synaptic dysfunction.
Anomalous assembling of misfolded Aβ peptides depicts a crucial characteristic and is possibly
the triggering mechanism for cognitive dysfunction in AD and neurodegeneration [8]. Amongst
the various forms of Aβ derived from APP, the most abundant one is Aβ1–42, having higher
tendency to congregate into different oligomeric and fibrillar complexes [9]. Aβ1–42 peptide is
believed to primarily comprise the predominant portion of Aβ plaques in AD patients [10,11].
Over a period of time the increasing accumulation of these toxic aggregates markedly contributes
towards downstream AD pathologies like oxidative damage, tangle formation, and neuronal
apoptosis [12,13]. A number of studies have confirmed pathophysiological consequences of its
neurotoxicity both by in vitro [14] and in vivo models [15].
On other part neurotransmitter acetylcholine (ACh) is critically important signaling molecule of
cholinergic transmission [16]. Breakdown of this neurotransmitter befalls via the terminator
enzyme of nerve impulse transmission, acetylcholinesterase at the cholinergic synapses. One
protein that appears to fill a prominent place within the multiplex pathological network of this
disease is AChE. It is a terminator enzyme of nerve impulse transmission at the cholinergic
synapses. Degeneration of cholinergic system in AD accounts for the contribution towards
cognitive impairment. This hypothesis asserts the declined ACh levels as it leads to cholinergic
failure and hence cognitive impairment. AChE has exceptionally great specific catalytic activity,
specifically for a serine hydrolase - each AChE molecule degrades approx. 25000 ACh
molecules per second [17,18]. Crystal structure of Torpedo californica AChE (TcAChE)
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indicated presence of a 200nm long, narrow gorge on enzyme surface. Its bottom is referred to as
catalytic active site, top as peripheral anionic site and there are five amino acid residues in
middle. Acetylcholinesterase inhibitors function by interaction with the PAS, CAS or both. For
molecule to actively bind to the gorge, it needs to have interacting groups at terminals that could
possess CAS/PAS binding. In relation to this AChE inhibition may provide higher level of
neurotransmitter in synaptic cleft, thereby upgrading cognition in mild-moderate level AD
patients [19]. For a period AChEIs were known to bring about only symptomatic revival, lacking
any influence on disease itself [20]. Nevertheless, other studies signify that AChE interacts with
amyloid β via hydrophobic environment in vicinity of PAS, thereby promoting Aβ fibril
formation [21,22]. Furthermore, the AChE-amyloid β complexes elevate the amyloid β
dependent neurotoxicity [23]. These reports recreate interest in AChEIs. Several clinical trials
revealed the effi cacy as well as safety of ChEIs in treating AD.
Oxidative stress is described amongst the early phenomena in AD pathogenesis [24].
Significance of oxidative damage in neuronal degeneration is also supported by the free radical
and oxidative stress theory of aging. Hence, protection of neurons against oxidative damage
could capably prevent AD. Recently molecules that modulate AChE inhibition, amyloid beta
disaggregation and inflammation altogether have been seen to show successful results for AD
[25]. With this frame of reference, the concept of multi-targeted agents exhibiting an integrated
action on more than one neurobiological target associated with origination and development of
the disease presently reassures the design of anti-Alzheimer’s agents [26-28].
1
H-Pyrazolo[3,4-b]pyridines are an eminent group of molecules owing to their variable
biological as well as pharmacological action. They have been demonstrated to possess, among
others, antimalarial [29], inhibition of cyclin-dependent kinases [30-35], GSK 3 inhibition [36],
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antiproliferative [37], antileishmanial [37], cardiovascular [38,39] and antiviral [40-42] activities
[43-45]. Investigation of molecules comprising 1H-Pyrazolo[3,4-b]pyridines has been developed
owed to their diverse effects in miscellaneous domains [29,36,37]. The 4-amino-5-carboxylates
of pyrazolo[3,4-b]pyridines; ethyl 1-ethyl-4-(2-(propan-2-ylidene)hydrazinyl)-1H-pyrazolo[3,4-
b]pyridine-5-carboxylate (Etazolate) is an anxiolytic drug that has also been known for its
neuroprotective nature [46]. A group of drugs from this scaffold; Tracazolate, Etazolate,
LASSBio-872, LASSBio-873, LASSBio-981, and LASSBio-982 have been used to treat anxiety
disorder related with GABA induced neuro-inhibition (Fig. 1).
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Fig. 1. Neurologically active drugs based on of pyrazolo[3,4-b]pyridines.
With present study we intend to develop potent neuroprotective agents against Alzheimer’s
disease derived from pyrazolopyridine and evaluate their biological activities. In this work we
report the first synthesis of pyrazolopyridine acetamide derivatives of this kind as well as the X-
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ray diffraction of two representative derivatives. In our preceding experimental project, a set of
quinoline derivatives was synthesized as amyloid β aggregation inhibitors, antioxidants and
metal chelators [47]. Thus, in the present study, further attempt has been made to search other
potential scaffolds and compounds that possess multi-targeting potential. To explore more anti-
Alzheimer’s agents, we present here a diversified synthesis of 1H-pyrazolo[3,4-b]pyridine
derivatives (P1-P9) as the new class in multi-functional Anti-Alzheimer’s agents integrating
anti-Aβ aggregation, AChE inhibition, radical scavenging and metal chelation activities in single
molecule.
2
2
. Results and discussion
.1. Chemistry
The development of the diversified pyrazolopyridine derivatives (P1-P9) was accomplished via
synthetic pathways shown in Scheme 1, 2 and 3. As a continuation of previous work [25] the
previously synthesized intermediates 6(a,b) were used to synthesize new pyrazolopyridine
compounds P1 and P2. Furthermore for some compounds we have tried to use the same
combination of piperazine with quinoline in order to achieve better results. 1-{5-methyl-
[1,2,4]triazolo[1,5-a]pyrimidin-7-yl}piperazine and ethyl 4-(piperazin-1-yl)quinoline-3-
carboxylate derivative were prepared via three step method described previously [25] as given in
Scheme 1 and 2. The 7-chloro-4-(piperazin-1-yl)quinolone [48] and 2-(piperazine-1-yl)pyridine-
3
N
-carbonitrile were prepared via nucleophilic aromatic substitution reaction (S Ar) of piperazine
on 4,7-dichloroquinoline and 2-chloro-3-cyanopyridine respectively. These piperazine
derivatives and other piperazine analogues were reacted with the 2-chloro-N-(1H-pyrazolo[3,4-
b]pyridin-3-yl)acetamide according to schemes 1, 2 and 3 to yield the final compounds P1-P9.
1
The compounds were stable in solid states at RT. The H NMR showed common signals around
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1
3.33, 11.05, 8.51, 8.37 and 7.17 of the N-(1H-pyrazolo[3,4-b]pyridin-3-yl)acetamide fragment
present in all the final compounds. The pyrazolopyridine NH was detected in a range of δ 13.34-
3.25, as singlet peak for all inhibitors. The NHCO proton in all inhibitors occurred as singlet in
range δ 10.81-10.26. The conversion of COCH Cl to COCH N shifted peak from more
1
2
2
deshielded region δ 4.37 to less deshielded region below δ 4.00. Other aromatic and/or aliphatic
substituents of compound were observed as expected. In a similar fashion common peaks for N-
1
3
(
1H-pyrazolo[3,4-b]pyridin-3-yl)acetamide fragment were observed in C spectra. The peak for
piperazine ring carbons was observed around 45 and 51 ppm. The different aromatic substituents
showed their respective peaks.
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Scheme 1. Synthesis of 2-(4-substituted piperazin-1-yl)-N-(1H-pyrazolo[3,4-b]pyridin-3-
yl)acetamide derivatives (P1, P2, P3). Reagents and conditions: (i) Hydrazine hydrate, EtOH, 60
°
C; (ii) glac.AcOH, NaOAc, chloroacetyl chloride, RT; (iii) Benzene, 83 °C; (iv) POCl , 110 °C;
3
(v) Piperazine, MeOH, reflux; (vi) TEA, THF, reflux; (vii) piperazine, TEA, THF, 60 °C.
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Scheme 2. Synthesis of 2-(4-substituted piperazin-1-yl)-N-(1H-pyrazolo[3,4-b]pyridin-3-
yl)acetamide derivatives (P4, P5, P6) linked to 1-{5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-
yl}piperazine (10), 7-chloro-4-(piperazin-1-yl)quinoline (11), and 2-(piperazine-1-yl)pyridine-3-
carbonitrile (12) respectively. Reagents and conditions: (viii) Acetic acid, 110 °C; (ix) POCl3,
1
10 °C; (x) piperazine, MeOH, reflux (xi) TEA, THF, reflux.
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O
R'
O
X
R'
X
HN
HN
Cl
N
HN
P7: X = CH; R' = H
P8: X = O
P9: X = N; R' = CH3
N
N
(xii)
N
N
N
N
H
H
3
P7-P9
Scheme 3. Synthesis of 2-(4-substituted piperazin-1-yl)-N-(1H-pyrazolo[3,4-b]pyridin-3-
yl)acetamide derivatives (P7-P9). Reagents and conditions: (xii) piperazine analogue, THF, 65
°
C.
Fig. 2. Molecular strategy for pyrazolopyridine hybrid molecules; 2-( piperazin-1-yl)-N-(1H-
pyrazolo[3,4-b]pyridin-3-yl)acetamide with triazolopyrimidine, pyrazolopyridine, quinoline and
pyridine.
Aromatic residues cover the deep narrow gorge of the AChE active site. Thus using piperazine
linker could increase molecule length and associate with π-cation interaction with aromatic
gorge. Combination with scaffolds such as, pyrazolopyridine, quinoline, pyridine and
1
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triazolopyrimidine was preferred choice having various valuable properties (see Fig. 2). Since
pyrazolopyridine possesses a planar structure it is expected to favour intercalation between A
fibrils and inhibit aggregation. In AChE it is expected to engage with the CAS and PAS leading
to preferred enzyme inhibition. Quinoline has a great biological background with neuroprotective
effects, AChE inhibition, free radical scavenging and chelation. Quinoline was supposed to bind
β
with CAS of AChE by π- π interaction as well as disaggregate Aβ sheets owing to its planarity.
Incorporating various combinations of these scaffolds in one candidate as anti-Alzheimer’s
agent, we carried out a detailed in vitro study of these compounds on different hypothesis factors
of AD.
2
.2. Single crystal structure
X-ray crystallographic determination of P6 and P9 was carried out (Fig. 3, Table 1, and Fig.
S29-S34, Supporting Information). P6 crystallizes in P121/c1 space group of monoclinic (Fig.
3
1
a) and P9 in P212121 of the orthorhombic crystal system (CCDC number: 1870982 and
870983) (Fig. 3b). The C-O bond distance of carbonyl group in both the compounds P6 and P9
was found to in between 1.2214(16) and 1.2155(19) Å respectively. The bond lengths of N(2)-
N(3) in P6 and P9 were 1.3639(16) and 1.3599(17) Å, respectively. For compound P6, the N(2)-
C(6), N(4)-C(6) and N(1)-C(1) bonds were of 1.3168(17), 1.4009(16) and 1.3260(20) Å whereas
for compound P9 it was 1.3134(18), 1.4098(17) and 1.334(20) Å, respectively. The bond angles
between N(2)-C(6)-N(4) for both P6 and P9 were 116.48(11)º and 119.64(13)º whereas O(1)-
C(7)-N(4) were 125.23(12)º and 123.70(13)º, respectively. The pi-pi stacking and H-bonding
interactions along with short contact bonding for both the P6 and P9 compounds with their
adjacent atoms were observed. The unit cell packing diagram of P6 and P9 was given in
supporting information (Fig. S29-S34).
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Fig. 3. ORTEP diagram of compounds P6 (a) and P9 (b) (50% probability level of thermal
ellipsoids). Color codes: Carbon, black; Nitrogen, green and Oxygen, red. Hydrogen atoms are
omitted for clarity.
Table 1. Crystal data and structure refinement for 2-(4-(3-cyanopyridin-2-yl)piperazin-1-yl)-N-
(1H-pyrazolo[3,4-b]pyridin-3-yl)acetamide
(P6)
and
(4-methylpiperazin-1-yl)-N-(1H-
pyrazolo[3,4-b]pyridin-3-yl)acetamide (P9) respectively.
P6
P9
Empirical formula
Formula weight
Crystal system
Space group
a/Å
C
18
H
18
N
8
O
1
13 16 6 1
C H N O
362.40
274.33
monoclinic
P121/c1
orthorhombic
P212121
8.8090(10)
10.3642(6)
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b/Å
c/Å
26.311(3)
7.8339(9)
90
10.3714(6)
13.0071(7)
90
α
/°
β
/
°
110.743(3)
90
90
γ
/
°
90
3
V/Å
Z
1698.0(3)
4
1398.15(14)
4
T, K
273 (2)
273 (2)
1.303
-3
ρcalcd/g cm
1.4175
λ
/Å (Mo-K
α
)
0.71073
3920/0/244
760
0.71073
3234/0/183
584
Data/restraints/param
F(000)
GOF
1.0566
1.110
a
b
R(F ), I > 2
σ
(I) [wR(F ) ]
0.0430 [0.1082]
0.0503 [0.1138]
0.2864, -0.2429
0.0345 [0.1019]
0.0370 [0.1037]
0.178, -0.157
o
o
R (all data) [wR (all data)]
-3
Largest diff peak, hole (e Å )
2
2
o
w = 1/[ (σF ) + (AP) + (BP)] A = 0.0547, B = 0.5255 A = 0.0592, B = 0.1561
a
b
2
2 2
2
1/2
R
=
Σ
|
F
2o | - |Fc|/
Σ|Fo|. wR = {Σ[w(Fo - Fc ) ]/Σ[w(Fo) ]} ; where P =
2
(
Fo +2Fc )/3
Table 2. Selected bond distance (Å) and bond angle (°) for complex P6 and P9 with estimated
standard deviations in parentheses.
P6
P9
O(1)
-
C(7)
1.2214(16)
1.3376(16)
1.4009(16)
1.3168(17)
1.3468(17)
1.3260(20)
1.2155(19)
1.3439(19)
1.4098(17)
1.3134(18)
1.3524(19)
1.334(20)
N(4)-C(7)
N(4)-C(6)
N(2)-C(6)
N(3)-C(5)
N(1)-C(1)
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N(4)-C(5)
N(2)-N(3)
1.3455(17)
1.3639(16)
1.145(2)
1.4046(19)
1.3599(17)
-
N(8)-C(18)
N(8)-C(18)-C(17)
O(1)-C(7)-N(4)
O(1)-C(7)-C(8)
N(2)-C(6)-N(4)
N(3)-C(5)-N(1)
C(1)-N(1)-C(5)
174.18(16)
125.23(12)
119.95(12)
116.48(11)
125.53(12)
113.50(12)
-
123.70(13)
120.58(14)
119.64(13)
111.20(11)
112.96(15)
2
2
.3. Molecular docking studies
.3.1. Molecular docking of P1, P2 and P3 with amyloid
β
Neurotoxicity of amyloid peptide is connected to Aβ fibril generation. Development of a β-
sheet pattern may assist aggregation of A 1-42.
β
Hence, entities which hold the capacity to
-sheet
restrain -sheet formation were helpful in A aggregation inhibition. The action of β
β
β
conformational change during fibrillogenesis is majorly stabilized by a Asp23/Glu22 and
Lys28 residue salt bridge in Aβ1–42 [49] and hydrophobic interactions. To elaborate the
possible binding modes of inhibitors with Aβ, P1, P2 and P3 were chosen for computational
study with the help of Auto Dock software and PyMOL to develop the images. PDB:1IYT
was selected for the docking. From Fig. 4, all of these three compounds are situated near the
vicinity of Aβ C-terminus and majorly stabilized through the hydrophobic bonding with
His13, Lys16, Leu17, Ala21, Phe20, Glu22, Asp23, Val24, Lys28, Gly29, Ile31, Ile32,
Leu34 and Met35 residues. In both Fig. 4 (a) and 4 (b), a strong intermolecular H-bonding
existed between oxygen of carbonyl of carboxylate and hydrogen of N-H of Lys16 with
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average distance of 1.8 Å. An intramolecular H-bonding existed between hydrogen of amide
of N-H and oxygen of carbonyl of carboxylate at a closest distance of 2.3 Å and 2.8 Å
respectively. In addition, three strong H-bondings existed between the hydrogen of N-H of
amide and oxygen of hydroxyl of Glu22 (Fig. 4 (c)), another hydrogen of N-H of amide and
oxygen of carbonyl of carboxylic acid, and hydrogen of N-H of pyrazolo ring and oxygen of
hydroxyl of carboxyl group of Asp23 (average distance 2.1 Å, 2.3 Å and 2.6 Å respectively).
Upon analyzing the outcomes of molecular modeling, we propose that inhibitors P1, P2 and
P3 bind strongly with A
aggregation. Molecular docking studies showed these test inhibitors to effectively bind with
, inhibit the toxic conformation of Aβ1–42 and stabilize the -helical content. Inhibition
assays revealed a lead compound with tight binding in the nanomolar range.
β1-42 and potentially interfere with β-sheets formation and inhibit Aβ
Aβ
α
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Fig. 4(a). Association of P1 (oxygen as red, nitrogen coloured blue and carbon coloured green)
and Aβ1-42 C-terminus via molecular docking; (b) Association of P2 (oxygen as red, nitrogen
coloured blue and carbon coloured magenta) and A 1
β
C-terminus via docking calculations; (c)
-42
Association of P3 (oxygen as red, nitrogen coloured blue and carbon coloured yellow) and Aβ
1-42
C-terminus via molecular docking. The H-bonding interactions are indicated with red dashed
lines. Plot (D, E, F) represents the 2D view of docking interactions for inhibitors (P1, P2, P3)
respectively by Ligplot. Green dashed line represents the H-bonds.
2
.3.2. Free energy calculation by molecular docking of inhibitors with AChE
Synthesized inhibitors were docked with AChE (PDB Code 1EVE) in the active site. The
positive control taken here for this computational study was Donepezil, displaying similar
interactions as given in literature. Position of compounds P3 corresponding to the binding site
key residues is given in Fig. 5. Each of the two pyrazolopyridine moieties present in compound
P3 are involved in
π-π stacking with Trp84 and Trp279 at distance of 3.3 Å and 5.1 Å
respectively. In addition, there are three intermolecular hydrogen bonding. Firstly, an
intermolecular hydrogen bonding is observed between carbonyl group of carboxyl of Asp72 with
hydrogen of N-H of 1H- Pyrazolo[3,4]pyridine at average distance of 2.3 Å. Another two
intermolecular hydrogen bondings are shown among oxygen of carbonyl group of amide bond
with hydrogen of hydroxyl of Tyr121 and carbonyl group of Asp285 with hydrogen of N-H of
1
H- Pyrazolo[3,4]pyridine having average distance of 2.4 Å and 1.7 Å respectively. The high
binding affinity indicates that the selected inhibitors possess dual binding affinity owing to
stacking, hydrophobic binding and H-bonding.
π-π
Table 2. The free energy values of P1-P9 intermolecular docking using ParDOCK
Inhibitors
Calculated free energy (Kcal/mol)
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AChE (PDB ID: 1EVE)
P1
P2
-7.81
-9.11
-9.32
-5.58
-7.41
-7.47
-4.31
-4.18
-4.58
-6.96
-4.90
P3
P4
P5
P6
P7
P8
P9
Donepezil
Tacrine
Fig. 5. The inhibitor is shown in ball and stick model while the key residues are shown in stick
model. Different residues are represented by individual colors like Glu199 (red), Ser200 (Cyan),
and His440 (Orange), Phe330, Phe331 (firebrick), Tyr70, Tyr121, Tyr334 (Blue), Trp84, Trp279
(Hot-pink), Asp72, Asp284 (Yellow). Other residues are shown in stick representation with
1
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carbons coloured pink. Oxygen and nitrogen atoms in the complexes are coloured red and blue
respectively. Plot (A) depicts interaction of P3 with active site key residue of AChE and plot (B)
Ligplot representation. π-π stacking is displayed as yellow dashed lines and intermolecular
hydrogen bonding are shown by red broken lines. Hydrogen atoms removed for clarity. Structure
is drawn through PyMol.
2
2
.4. Pharmacological evaluation
.4.1. Cholinesterase inhibitory activity (AChE and BuChE)
The inhibition potencies of the target inhibitors against both cholinesterase enzymes were
evaluated via Ellman’s method having Donepezil used as the reference molecule. Here, the
AChE enzyme hydrolyses acetylthiocholine giving acetate and thiocholine. The thiocholine thus
produced reduces the Dithiobis-Nitrobenzoic acid producing nitrobenzoate that absorbs at 412
nm. In a similar fashion, BuChE hydrolyses butyrylthiocholine giving butyrate and thiocholine.
The potency and selectivity of the nine test inhibitors are summarized in Table 2. Most of the
synthesized inhibitors showed very good inhibition potential in comparison to Donepezil (49
nM) with IC50 ranging from 4.8 nM to 113 nM. Compound P7 displayed the maximum
inhibition of AChE with IC50 of 4.8 nM followed by compound P9 with an IC50 of 4.9 nM.
However no significant inhibition of BuChE was observed by the compounds up to 100 µM
concentration, suggesting the extremely selective nature of the test compounds towards AChE.
The results of compound P7, P8, and P9 suggest that the N-(pyrazolo[3,4-b]pyridinyl)acetamide
clubbed with piperazine analogues possesses satisfactory inhibitory potential. The results of
other compounds suggest that the inhibitory action of synthesized molecules was highly
influenced with substitutions in N-(pyrazolo[3,4-b]pyridinyl)acetamide. SAR studies of the
compounds revealed that the cholinesterase inhibition is minimal for compounds having an
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adjacent electron withdrawing substituent at the deactivated aryl ring as in compounds P1, P2
and P6 (IC (AChE) >100 µM). Most likely the ester substituent on the quinoline ring and cyano
5
0
substituent on pyridine ring laid their lower inhibitory activity. On comparing P1 and P2 with
analogous compound 10a [25,] we observe a decrease in cholinesterase activity. Reasonably
good activity was found for compounds with sterically favorable and active side groups like P3,
P4 and P5. Maximal activity was found for the compounds with small molecules as substituents
on the piperazine ring (compound P9). However, presence of morpholine ring in compound P8
contributed to quite lower inhibitory activity than P9.
Table 3. Table for AChE and BuChE inhibition by synthesized inhibitors and the selectivity
index for AChE.
IC50 ± SD(
µ
M)
c
Selectivity
Compounds
a
b
AChE
BuChE
BuChE/AChE
(R=)
P1
>
100
>100
-
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P2
>
100
>100
-
P3
P4
0
0
.045 ±0.010
.022 ±0.009
>100
>100
>
>
2222.22
4545.45
P5
P6
0
.012 ±0.006
>100
>
8333.33
>100
-
>
100
P7
P8
0
.0048 ±0.001
>100
>100
>
20833.33
0
.113 ±0.052
>
884.955
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P9
0
.0049 ±0.001
>100
>
20408.163
Donepezil
-
0
.049 ± 0.05
8.71 ± 1.36
1
66.7
a
AChE; inhibitor concentration (mean ± SD for these experiments) corresponding to 50%
inhibition of AChE.
b
BuChE; inhibitor concentration (mean ± SD for these experiments) corresponding to 50%
inhibition of BuChE.
c
Selectivity for AChE: IC50 (BuChE)/IC50 (AChE)
.
2
.4.2. Kinetic study of AChE inhibition
Study for mechanism was carried out with the active multifunctional compound P3. A
Lineweaver–Burk double reciprocal plot was generated and is shown in Fig. 6. The graphical
analysis shows the interception of lines in the plot above the x-axis with rise in slope (decreased
Vmax) and increase in intercepts (higher Km) at elevating concentrations of compound. This
indicates a mixed-type inhibition, verifying that P3 might interact with both the CAS and PAS of
AChE.
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Fig. 6. Kinetic study of AChE inhibition mechanism with compound P3. Lineweaver–Burk
reciprocal plot of AChE initial velocity with increasing substrate concentrations.
2
.4.3. Self-mediated Aβ1-42 aggregation inhibition study
A significant pathological characteristic linked with AD is amyloid plaques. Amyloid assembly
initiates from disordered amyloidogenic protein monomers that are incorporated into a cascade
of protein interactions to form a critical nucleus which later commences to adopt a cross β sheet
of amyloid [50]. Aβ is produced via enzymatic cleavage of amyloid precursor protein which is
usually present in the synapses of neuronal cells by α-, β-, and γ-secretases. The Aβ40, variant of
length 40 residues is the most prevalent form however, the 42-residue variant is more
hydrophobic and aggregation prone. This Aβ is the predominant one in the amyloid plaques.
4
2
Targeted compounds were tested for their Aβ1-42 aggregation inhibition potential via the ThT
fluorescence method. ThT is a fluorophore which displays an elevation in fluorescence on
binding to amyloid fibrils.
Most inhibitors displayed a very good inhibition ranging from 50.60% to 81.64% in comparison
to curcumin’s 52.70 %. Compounds P4, P5 and P6 showed lower inhibition against amyloid
2
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aggregates. This may be due to the presence of aromatic rings that provides rigidity, reduced
flexibility and less favourable structures to intercalate between the amyloid fibrils. However the
data for compounds P1, P2, P3 and P8 exhibiting aggregation inhibition of 81.65%, 69.31%,
7
9.47% and 74.62% respectively, suggests that both the presence of a sterically favourable
structure and a substitution having interactive sites is important. On comparing compounds P1
and P2 with analogous compound 10a [25] we observe an increase in inhibition percentage
which is probably due to increase in interactive groups which bind with fibrils to disturb the
aggregation. Most of our compounds showed higher inhibition with respect to the standard
curcumin. Presence of a diamide linker on both head and tail regions in P3 increased polarity and
provided flexibility similar to Curcumin and contributed towards its activity. Increased
hydrophobicity in tail region of compounds P1 and P2 resulted in their high inhibitory potency.
Table 4. Percent inhibition of Aβ1-42 aggregation, percent disaggregation and ORAC trolox
equivalents of synthesized inhibitors.
b
c
Compounds Self Aβ1-42 aggregation
Aβ1-42 disaggregation (%)
Trolox equiv
a
inhibition (%)
P1
P2
P3
P5
P4
P6
P7
P8
81.646 ±3.06
69.306 ±1.84
79.472 ±2.65
23.600 ±0.32
43.433 ±0.41
39.976 ±0.27
50.607 ±0.78
74.619 ±2.54
61.59 ±2.16
52.13 ±1.24
53.80 ±1.82
0.274
0.271
0.283
0.216
0.286
0.163
0.650
0.192
±
±
±
±
±
±
±
±
0.08
0.06
0.07
0.07
0.10
0.08
0.10
0.06
d
nd
d
nd
d
nd
d
nd
47.36 ±1.62
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d
P9
50.848 ±0.83
52.70 ± 1.67
-
nd
0.362 ± 0.17
Curcumin
Resveratrol
-
n.d
-
e
61.0
a
The Th-T fluorescence assay was employed. Percent inhibition of aggregation (means ± SD)
taken at inhibitor concentration of 25 µM.
c
Percent disaggregation of Aβ1-42 (mean+ S.D) exhibited by compounds
c
Data are expressed as µM of trolox equivalent/µM of tested compound (mean ± SD of three
experiments).
d
nd, not determined.
e
taken from reference of Chuanjun Lu et al. [51]
Fig. 7. Fluorescence intensity of Aβ1-42 alone (dark blue), Aβ1-42 with P1 (light blue), Aβ 1-42
with P3 (red), Aβ1-42 with P8 (pink) measured using ThT assay.
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2
.4.4. Disaggregation study of self-mediated Aβ Aggregation
The Amyloid plaques result from increase in Aβ1-42 peptide production and accumulation. This
Aβ1-42 peptide oligomerizes and gets deposits as senile plaques. The compounds possessing a
higher potential to inhibit self-induced Aβ aggregation were tested for their disaggregation
potential against self-mediated Aβ1-42 aggregation fibrils using a ThT assay. Compounds P1, P2,
P3 and P8 were taken based on their high potency to inhibit self-mediated Aβ aggregation. Fresh
Aβ was incubated for 48 h at 37° to enable the formation of fibrils. Then selected inhibitor was
added to the sample and incubated for additional 24 h. Disaggregation of Aβ was measured via
ThT assay, where the fluorescence intensity indicates degree of Aβ fibrillar aggregation. ThT
stains amyloid deposits. Table 4 demonstrates the Aβ1-42 disaggregation inhibition (%) of the
compounds and all four compounds exhibited a considerably high disaggregation percent.
Compound P1 and P3 revealed the most favorable disaggregation results with 61.43% and
5
3.63% respectively, followed by P2 with 51.95% and P8 with 47.17%. High disaggregation by
P1 and P3 is in concordance with the inhibition of self-induced Aβ fibril aggregation results.
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Fig. 8. Fluorescence intensity of Aβ1-42 alone (dark blue), Aβ1-42 with P1 (light blue), Aβ1-42 with
P2 (green), Aβ1-42 with P3 (red), Aβ1-42 with P8 (pink) measured using ThT assay.
2
.4.5. Concentration dependent self Aβ aggregation inhibition study
Table 5. Percent inhibition of self- mediated Aβ aggregation by different concentrations of drug
a
Inhibitors
Aβ1-42 aggregation inhibition (%)
1
0µM
15µM
43.79
39.85
20µM
84.10
58.04
25µM
81.64
79.4
IC50
15.72µM
17.89µM
P1
P3
28.62
3.9
a
%
inhibition of self-mediated Aβ aggregation (mean + S.D) after incubation of 25 µM Aβ1-42
with 10, 15, 20 and 25 µM compounds as indicated.
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Fig. 9. Percent inhibition of self-mediated Aβ aggregation at four concentrations of drug (10, 15,
2
0 and 25 µM). The bar graph depicts a proportional relation between increase in concentration
and % inhibition for compounds P1 (a) and P3 (b).
Concentration dependent self Aβ aggregation inhibition study was carried out in order to find the
concentration-Aβ aggregation inhibition pattern. Compounds having the highest self Aβ
aggregation inhibition and Aβ disaggregation were tested in this study. Inhibition of compounds
P1 and P3 towards Aβ aggregation was observed at four different concentrations 10 µM, 15 µM,
2
0 µM and 25 µM. Sample of each concentration were incubated with fresh Aβ1-42 for 48 h at 37
°
C, and inhibition was measured using ThT fluorescence assay. A general trend of increase in Aβ
aggregation inhibition with increase in concentration was observed for compound P3, whereas
compound P1 showed the highest inhibition at 20 µM. Compound P1 exhibits high inhibition
percentage at both 20 µM (84.10%) and 25 µM (81.64%). Altogether the results indicated a high
inhibition for both compounds at 25 µM. Table 5 summarizes the inhibitory activity of both
2
8