Potent Acetylcholinesterase Selective and Reversible Homodimeric Agent Based on Tacrine for Theranostics

Article abstract of DOI:10.1021/acs.molpharmaceut.8b01058

Acetylcholinesterase (AChE) has been an important biomarker for diagnosing Alzheimer's disease (AD), due to reduction in AChE activity in post-mortem brains of AD patients. A potent, selective, and reversible homodimeric inhibitor of AChE, 5-amino-N¹,N³-bis(2-(1,2,3,4-tetrahydroacridin-9-ylamino)ethyl)isophthalamide (compound 4), was synthesized by using 9-alkyl(1,2,3,4-tetrahydroacridine) pharmacophore with appended functionality. In the present work, we report the synthesis of this bivalent inhibitor of AChE. The homodimeric ligand structure was designed and studied with molecular docking tools, which revealed its high affinity and interactions with active site gorge of AChE, which includes both catalytic active site (CAS) and peripheral active site (PAS). The IC₅₀ value of this bivalent inhibitor for AChE and BuChE were 0.54 ± 0.06 and 32.49 ± 1.2 nM, respectively, with a selectivity ratio of 60.16 toward AChE. The designed ligand also showed potent inhibitory properties on PAS activity as well as on AChE-induced amyloid aggregation with low cytotoxicity on rat hippocampal neurons. The AFM images further corroborated the Aβ_1-42 aggregation inhibition by compound 4 to an extent similar to bis(7)-tacrine. Moreover, the bivalent ligand was also proven to be of neurogenic potential due to its ability to induce S-phase post-treatment in rat hippocampal neuronal cells. On the basis of initial results, the agent could be further explored for its theranostic value clinically, which gives the possibility of tracing the AChE levels by molecular imaging techniques in correlation with progression of neurocognitive disorders like AD for better therapy response and patient management.

Full text of DOI:10.1021/acs.molpharmaceut.8b01058

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Article
A Potent Acetylcholinesterase Selective and Reversible
Homodimeric Agent based on Tacrine for Theranostics
Virendra Kumar Meena, Shubhra Chaturvedi, Rakesh
Kumar Sharma, Anil Kumar Mishra, and Puja Panwar Hazari
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01058 • Publication Date (Web): 06 May 2019
Downloaded from http://pubs.acs.org on May 7, 2019
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A Potent Acetylcholinesterase Selective and
Reversible Homodimeric Agent based on
Tacrine for Theranostics
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Virendra Kumar Meenaa,b, ShubhraChaturvedia, Rakesh Kumar Sharmab Anil Kumar Mishraa
,
,
Puja Panwar Hazaria,*
aDivision of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and
Allied Sciences, Brig S.K. Mazumdar Road, Delhi-110054, India.
bDepartment of Chemistry, University of Delhi, Delhi-110007, India
*Corresponding author
Dr. Puja PanwarHazari
Brig. S. K. Mazumdar Road, Delhi-110054, India
Phone: +91-11-23984480 Fax: +91-11-2391-9509
E-mail: puja.hazari@gmail.com
KEYWORDS: Alzheimer’s disease, Acetylcholinesterase, Tacrine, Multi-target directed ligands,
Amyloid β peptide, Theranostic Agent
Abstract
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Acetylcholinesterase (AChE) has been an important biomarker for diagnosing Alzheimer’s
disease (AD), due to reduction in AChE activity in postmortem brains of AD patients. A potent,
selective and reversible homodimeric inhibitor of acetylcholinesterase (AChE), 5-amino-N1,N3-
bis(2-(1,2,3,4-tetrahydroacridin-9-ylamino)ethyl)isophthalamide [Compound (4)] was
synthesized by using 9-alkyl(1,2,3,4-tetrahydroacridine) pharmacophore with appended
functionality. In the present work, we report the synthesis of this bivalent inhibitor of AChE.The
homodimeric ligand structure was designed and studied with molecular docking tools which
revealed its high affinity and interactions with active site gorge of AChE which includes both
catalytic active site (CAS) and peripheral active site (PAS).The IC50 value of this bivalent
inhibitor for AChE and BuChE were 0.54 ± 0.06 and 32.49 ± 1.2 nM respectively, with a
selectivity ratio of 60.16 towards AChE. The designed ligand also showed potent inhibitory
properties on PAS activity as well as on AChE-induced amyloid aggregation with low cytotoxicity
on rat hippocampal neurons. The AFM images further corroborated the Aβ1-42 aggregation
inhibition by compound (4) to extent similar like Bis(7)-tacrine. Moreover, the bivalent ligand was
also proven to be of neurogenic potential due to its ability to induce S-phase post-treatment in
rat hippocampal neuronal cells. On the basis of initial results, the agent could be further
explored for its theranostic value clinically, which gives the possibility of tracing the AChE levels
by molecular imaging techniques in correlation with progression of neurocognitive disorders like
Alzheimer’s disease for better therapy response and patient management.
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Abbreviations: Alzheimer’s disease (AD), Amyloid β Peptide (Aβ), Acetylcholinesterase
(AChE), Catalytic active site (CAS), Peripheral active site (PAS), Multi-target directed ligands
(MTDL)
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Introduction
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Alzheimer’s disease (AD) is the most common cause of dementia among the elderly people with
a multitude of multi-facet pathologies associated with neurodegeneration [1-2]. It is commonly
characterized by memory impairment and gradual deficits in various cognitive domains.[3] The
neuropathological hallmarks include aggregation of amyloid-beta 1-40/42 (Aβ) peptide inside
the soma of the neurons derived from the altered metabolism of APP protein by enzymes such
as α- and β-secretases, along with the appearance of neurofibrillary tangles (NFT) due to
aggregation of hyper phosphorylated tau proteins.[4-6] Aβ oligomers are implicated in many of the
molecular events during neurodegeneration which includes neurotoxicity, dysregulation of Ca+2
level homeostasis and chronic activation of microglial cells inside the brain which leads to
neuro-inflammation.[7-10] However, many of the causative pathways for this neurodegeneration
are still unclear, suggesting its multi-facet nature. Various theories have been forwarded to
explain the pathogenesis of Alzheimer’s disease including cholinergic hypothesis, the amyloid
cascade hypothesis and oxidative stress hypothesis with free radical generation depicting an
intertwined situation. There have been a number of diagnostic biomarkers used for detecting AD
condition. These biomarkers include neurochemical markers (i.e. Aβ peptides, total tau protein,
phosphorylated tau and AChE etc.) and neuroimaging markers (i.e. hippocampal volumetry,
entorhinal cortex volumetry etc.). Changes in various biochemical and histopathological
neurotransmitter markers results from use of cholinomimetic drugs speculate an association
between decline in learning and memory with deficit in excitatory amino acid (EAA)
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neurotransmission. Therefore, many therapeutic interventions has emerged aiming to restore
the loss of cholinergic neurotransmission by inhibiting AChE at cholinergic synapses in AD
brain. [11-13]Not surprising, AChE activity was assumed to be one of the major neurochemical
biomarker for degeneration of cholinergic neural network. [14]AChE activity can be considered as
the major therapeutic target as well as diagnostic biomarker for detecting AD condition. The
activity or density of cerebral AChE was thoroughly studied by PET imaging in various tissues in
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different disease states.[15-16] Previous studies has shown a decline in AChE activity in cerebral
cortex was reported in necropsy brain studies. Therefore, in vivo AChE activity measurement
could provide useful insight not only in disease diagnosis, monitoring disease progression but
also assist in therapeutic outcome of anti-dementia drugs. [17]
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Fig. 1 Different classes of 11C-labeled PET radiotracers for cerebral acetylcholinesterase (AChE).
For studying the distribution pattern of AChE in various regions of brain, PET radiotracers for
AChE are available using two radiopharmaceutical approaches. The one uses radiolabeled
lipophilic acetylcholine analogs which acts as substrate for cerebral AChE and, second uses
radiolabeled AChE inhibitors as radiotracer which are presented in Fig. 1. Initially various
substrates based radioligands for AChE were developed, such as [11C]-N-methyl-4-piperidyl
acetate [11C]-MP4A and [11C]-MP4P which are analogs of acetylcholine. [11C]-MP4A, unlike
acetylcholine was able to cross blood brain barrier, and further cleaved by AChE into [11C]-N-
methyl-4-piperidinol, a hydrophilic metabolite which is trapped in the brain due to low blood
brain barrier permeability.[18] But the radiolabeled metabolite gave very diffuse radioactivity
signal inside the brain and failed to match the histochemical location of AChE inside the AD
brain.[19] Another approach uses radiolabeled inhibitors of AChE, i.e. [18F]-CP118 954, [11C]-
Sarin, [11C]-Physostigmine, [11C]-Donepezil, [11C]-Tacrine etc., which are reversible inhibitors of
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AChE showing excellent brain penetration, show longer retention time, appropriate
pharmacokinetics and match the histological location of AChE in AD brain.[20-24]
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Fig 2. Chemical structures of Tacrine (THA) and Bis(7)-tacrine.
Tacrine (THA) provides a scaffold to design and synthesizeAChE inhibitors with high specificity
and potency, which acts as multi-target directed ligands (MTDLs) with anti-Alzheimer properties.
[25-27]These ligands show selectivity towards AChE and also inhibit the aggregation of Aβ (1-
40/42) peptide in vitro and in vivo. [28] A wide spectrum of tacrine conjugates were made, but bis-
alkyl-tacrines are the most potent inhibitors reported of AChE activity with amyloid aggregation
inhibition. The mechanism of action of these bis-tacrine inhibitors via blocking the both active
sites of the AChE, CAS (Catalytic active site) which metabolizes the acetylcholine into acetyl
and choline at the site of action, and PAS (Peripheral active site) which facilitates the
aggregation of Aβ peptides. [29] With inhibition of AChE the levels of acetylcholine increase in
cholinergic system to improve the cholinergic neurotransmission as well as cognition function in
patients. Therefore, ligands with dual functionality as imaging tracer with therapeutic potential
for disorders involving AChE are much needed.
The present work has been directed for development of next generation of ligands which could
be used in nanomolar concentrations for probing the enzyme with therapeutic effects in same
concentration range. A homodimeric AChE targeted tracer was constructed using THA
pharmacophore with aromatic linker which has suitable site for [11C] radiolabeling without
affecting the specificity and affinity towards its target. Here, we report the synthesis of new
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homodimeric ligand 5-amino-N1,N3-bis(2-(1,2,3,4-tetrahydroacridin-9-
ylamino)ethyl)isophthalamide (4) and potential of this ligand as anti-Alzheimer’s
agent.Therefore, this ligand could be advantageous in critical patient management by
monitoring the disease progression and therapeutic outcome.
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Chemicals and Reagents
Anthranilic acid, Cyclohexanone, Phosphorous(V) oxychloride, Ethylene diamine, Phenol, 5-
Aminoisopthalic acid (5-AIA), 1-hydroxybenzotriazole (HOBt) and 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC) and Triethylamine (TEA) were
purchased from Merck Ltd (India). Reagents and chemicals were commercially purchased and
used without further purification unless mentioned. C-18 Sep-pak plus cartridges were procured
from Waters Corporation (Milford, MA, 120 USA)
Instrumentation
Thin layer chromatography (TLC) was carried using Merck Silica Gel 60 F254, 20x20 cm plates
and viewed under 254nm UV lamp. 1H and 13C NMR spectra were recorded on Bruker AVANCE
II 400 MHz NMR system (Ultra-shielded) using solvents D2O, CDCl3, CD3OD and DMSO-d6.
Agilent 6310 ion trap mass spectrometer was used for electrospray ionization mass
spectroscopy (ESI-MS) in positive and negative ion mode. High resolution mass spectroscopy
(QTOF) coupled with liquid chromatography mass spectrometry (HRMS-LCMS) was used to
obtain accurate mass of the compounds.
General methods for synthesis
Scheme 1. 9-Chloro-1,2,3,4-tetrahydroacridine(1). To a mixture of Anthranilic acid (1g, 7.283
mmol) and cyclohexanone (0.724mL, 6.986 mmol) on an ice bath, POCl3 (6.756mL) was
carefully and stirred for 15 min. The mixture was then heated under reflux (130oC) for 3 h, and
the reaction mixture was cooled to room temperature. The reaction mixture was then added to
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10% KOH solution in a beaker on ice bath with stirring. The mixture was further extracted with
ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated in
vacuo, affording an organish-yellow solid. It was further purified by silica gel chromatography,
and eluted with petroleum ether: ethyl acetate (8:2) to give the pale-yellow product. (1.54g,
94%): mp 68-70C; 1H NMR (400MHz, CDCl3, 25ºC) δ (ppm)1.97 (s, 4H, CH2-CH2), 3.0 (t, 2H,J
=6.4Hz, CH2), 3.1 (t, 2H, CH2), 7.55 (t, 1H,J = 8Hz, ArH), 7.68 (m, 1H, ArH), 8.00 (d, 1H,J =
8.4Hz, ArH), 8.17(d, 1H, J = 8.4Hz, ArH);13C NMR (400MHz, CDCl3, 25ºC) δ (ppm)22.64,
22.68, 27.52, 34.20, 123.71, 125.40, 126.50, 128.62, 128.89, 129.28, 141.49, 146.66, 159.52;
ESI-MS+: m/z calculated forC13H12Cl N [M + H]+, 218.69; found [M + H]+, 218.20; HRMS
(positive ions): m/z calculated for [M + H]+ 218.6941; found [M + H]+, 218.0734.
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NH2
O
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Cl
HN
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NH2
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N
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Anthranilic
acid
Cyclohexanone
(1)
(2)
Scheme 1.Synthesis of N1-(1,2,3,4-tetrahydroacridin-9-yl)ethane-1,2-diamine (2). Reagents and
conditions: i) POCl3, Reflux, 3h; ii) NH2(CH2)2NH2, Phenol, NaI, 180˚C, 3h.
N1-(1,2,3,4-tetrahydroacridin-9-yl)ethane-1,2-diamine (2). To the mixture of intermediate (1)
(0.50g, 2.296mmol), phenol (1.0g), ethylenediamine (5.0mL, 65.90mmol) and NaI (0.05g) was
carefully added and heated to 180°C under inert nitrogen conditions for 3 h. The mixture was
then cooled to room temperature and diluted with dichloromethane, then this mixture was made
basic with 10% KOH solution. The organic layer was washed with water and brine, and dried
over anhydrous sodium sulfate. The residue was further purified with silica gel chromatography,
and eluted with dichloromethane: methanol (9.0:1.0) with 7mL (aq. NH3, 30%) per liter of
solution to give product as pale yellow solid. (0.48g, 88%); 1H NMR (400MHz, CD3OD, 25ºC) δ
(ppm) 1.98-2.00 (t, 4H, CH2-CH2), 2.8 (s, 2H, CH2), 3.0 (s, 2H, CH2), 3.4 (t, 2H, CH2), 4.2 (t,
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2H,J = 6.4Hz, CH2), 7.6 (t, 1H,J = 8Hz, ArH), 7.8 (d, 1H,J = 8Hz, ArH), 7.8 (d, 1H, ArH), 8.3(d,
1H,J = 10Hz, ArH);13C NMR (400MHz, CD3OD, 25ºC) δ (ppm) 20.29, 21.52, 23.73, 27.99,
38.97, 44.61, 112.66, 115.94, 118.95, 124.54, 125.49, 132.82, 138.24, 151.33, 156.72; ESI-
MS+: m/z calculated forC15H19N3 [M + H]+, 242.33; found [M + H]+, 242.20; HRMS (positive ions):
m/z calculated for [M + H]+ 242.3315; found [M + H]+, 242.1654
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Scheme 2. 5-amino-N1,N3-bis(2-(1,2,3,4-tetrahydroacridin-9-ylamino)ethyl)isophthalamide(4).
To a solution of 5-Aminoisophthalic acid (100mg, 0.552 mmol) in 10 mL anhydrous DMF with N2
supply on ice bath were added 1-hydroxybenzotriazole (HOBt,) (164 mg, 1.2144 mmol) and 1-
ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC,) (232.8 mg, 1.2144 mmol)
and kept on stirring for another 15 min. The amine intermediate (2)(293.1 mg, 1.2144mmol) was
added in a dropwise manner to the above resulting solution on ice bath. The mixture solution
was allowed to stir for another 30 min and trimethylamine (TEA) was added with continued
stirring overnight (12h-18h). Next day, the mixture was evaporated in vacuo and the residue
was washed twice with mixture of DCM with water. The organic fraction was again washed with
saturated brine to yield the final product. The DCM was dried in vacuo to afford the final product
(4) in form as pale white solid. This residue was further purified by silica gel column
chromatography, and eluted with mixture of dichloromethane: methanol (8:2) with 7mL (aq.
NH3, 30% solution) per liter of the mixture to give product as white solid. (230 mg, 30.2%), 1H
NMR (400MHz, CD3OD, 25ºC) δ (ppm)1.9 (m, 8H, 2 X CH2-CH2), 2.8 (s, 4H, 2 X CH2), 3.0 (s,
4H, 2 X CH2), 3.6 (t, 4H, 2 X CH2), 3.7 (t, 4H,J = 5.6Hz, 2 X CH2), 7.3 (t, 2H, J = 8Hz, 2 X ArH),
7.6 (t, 2H,J = 8Hz, 2 X ArH), 7.8 (d, 2H, 2 X ArH), 8.1 (d, 2H,J = 8Hz, 2 X ArH);13C NMR
(400MHz, CD3OD, 25ºC) δ (ppm) 22.34, 22.68, 24.43, 32.91, 50.46, 61.12, 116.27, 120.21,
123.07, 123.47, 126.73, 128.30, 146.69, 151.81, 157.98, 160.05; ESI-MS+: m/z calculated
forC15H19N3 [M + H]+, 628.78; found [M + H]+, 628.6; [M/2Z + H]+, 314.8; HRMS (positive ions):
m/z calculated for [M + H]+ 627.7778; found [M + H]+, 628.3380; [M/2Z + H]+, 314.6727.
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NH2
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NH2
O
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NH2
NH
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EDC, HOBt
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DMF, RT, 12h
OH
OH
(2)
(3)
(4)
Scheme 2.Synthesis scheme of 5-amino-N1,N3-bis(2-(1,2,3,4-tetrahydroacridin-9-
ylamino)ethyl)isophthalamide (4) by condensation of 5-Aminoisophthalic acid (3) and 9-aminoalkylamino-
1,2,3,4-tetrahydroacridine (2). Reagents and conditions: EDC, HOBt, anhydrous DMF, DIPEA, RT, 12h.
AChE and BuChE inhibition studies
AChE human (E.C.3.1.1.7, recombinant protein expressed in HEK293 cells), BuChE human
(E.C.3.1.1.8, from human serum), 5,5’-Dithiobis(2-nitrobenzoic acid) (DTNB, Ellman’s reagent),
Acetylthiocholine (ATC), Butyrylthiocholine (BTC) were purchased from Sigma-Aldrich
(Germany). OD and kinetic OD measurement were done in 24-well plate on Biotek H4
Hybridmulti-plate reader system, USA. The reference inhibitory drugs Tacrine (THA) was
purchased from Sigma-Aldrich (Germany) and Bis(7)-tacrine from Tocris, Bristol, UK.
All the stocks of AChE (5U/mL), BuChE (5U/mL), DTNB (10mM), Acetylthiocholine (ATC, 3mM),
and Butyrylthiocholine (BTC, 3mM) were prepared in 0.1M phosphate buffer (pH-7.0). The
stocks of the test compounds were prepared in 10% ethanol (C2H5OH) in same 0.1M phosphate
buffer (pH-7.0) in final volume of 1mL, and various concentrations of the test compounds
ranging from 10-4M – 10-15 M were prepared and tested in the following mentioned procedure by
Ellman’s method. [30]
In vitro enzyme inhibitor potency of the synthesized molecules THA,(2), Bis(7)-tacrine and
(4),and all intermediate derivatives were evaluated by Ellman’s method. The enzymatic reaction
mixture was prepared in a 24 well plate system, which constitutes AChE (0.0788U/mL), DTNB
(315.45µM) and final volume of the reaction mixture was 634 µL. The enzymatic reaction was
initiated by addition of ATC in mixture at final concentration of 473.18 µM at 37±0.5˚C in 0.1M
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phosphate buffer (pH-8.0). The optical density (O.D) was measure at 412 nm in 15 s interval for
total time period of 5 min in Synergy HT hybrid multi-plate reader. The initial enzymatic rate (∆
O.D/min) was determined by linear regression analysis of the absorbance vs time curve in more
than triplicate (n ≥ 3). The initial enzymatic rate without inhibitor (R0) corresponds to 100%
enzymatic activity, and initial enzymatic rate in presence of various concentrations of inhibitor
(Ri) were determined. The percentage enzyme activity in absence or presence of inhibitor was
calculated by expression of % enzyme activity = (Ri/R0) x 100. In case of determining initial
enzymatic rate with known standard inhibitor, the above reaction mixture was mixed with
standard inhibitor at various concentrations and allowed to stand the mixture at room
temperature (RT) for 4.5 min. Similarly, enzymatic reaction was initiated by adding ATC to the
mixture at concentrations mentioned above. All readings of initial rate in presence of standard
inhibitor were taken in more than triplicate (n ≥ 3). For determining the background non-
enzymatic rate, the mixture follows the same above-mentioned procedure while replacing the
enzyme solution by buffer solution. The enzyme activity was plotted against the log
concentration values of the inhibitor. Non-linear regression analyses were done on the curve to
determine the IC50 values of the various synthesized ligands by using GraphPadPrism software
(version 5.0).
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BuChE inhibition studies were also performed by same procedure reported above, while using
BuChE and BTC at same concentration at place of enzyme and substrate respectively.
Similarly, the enzyme activity was plotted against log concentration values of inhibitors, and
non-linear regression analysis was done to determine the IC50 values of various synthesized
ligands by using GraphPadPrism software (version 5.0).
AChE and BuChE kinetic analysis
The kinetic measurement of the enzyme was evaluated at different concentrations of substrate
were: 15.6 µM, 31.25 µM, 62.5 µM, 125 µM, 250 µM, 500 µM. The absorbance was measured
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at different substrate concentrations in presence of inhibitor at concentration range (0–80 nM) to
determine the Km and Vmax by non-linear regression analysis of the substrate-velocity curves.
The double reciprocal plots or Lineweaver-Burk plots were made between inverse of substrate
concentration [1/S] and inverse of initial reaction rate [1/V0] for better illustration purposes,
accurate values of Km and Vmax can also be calculated by double reciprocal plots. All the data
processing was done using GraphPadPrism 5 software (version 5.0).
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Thioflavin T competition assay
Thioflavin T and hAChE (Recombinant expressed in HEK293 cells) were purchased from
Sigma-Aldrich (Germany). Fluorescence readings were recorded at room temperature (22.0 ±
2.0˚C) using Synergy H4 Hybrid multi-plate reader (Biotek, US). The fluorescence readings of
mixture solutions were made at 448nm and 488nm for excitation and emission respectively.
The PAS inhibitory activity of compound (4) was evaluated by a procedure described previously.
[31]A solution of recombinant hAChE (500µM) in 50 mM glycine-NaOH buffer (pH-8.5) was
incubated at room temperature for 24 h on stirring. After incubation, an aliquot of enzyme was
added to a solution of Thioflavin T to a final concentration of 180 µM and 15 µM respectively in
50 mM glycine-NaOH buffer (pH-8.5). The solution was further incubated at room temperature
with stirring for another 1 h and fluorescence was recorded (F1). To evaluate the potency of
inhibitors to inhibit PAS, a solution of hAChE, Thioflavin T and inhibitor was incubated to a final
concentration of 180 µM, 15 µM and 15 µM respectively in 50 mM glycine-NaOH buffer (pH-8.5)
at room temperature for 1 h. And the corresponding fluorescence was measured (F2). Finally,
fluorescence of control solution containing Thioflavin T to a final concentration of 15µM was
recorded after incubation for 1 h in 50mM glycine-NaOH buffer (pH-8.5) on stirring (F0). The
percentage reduction of Thioflavin T fluorescence was calculated as
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AChE induced Aβ aggregation inhibition
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Human amyloid β protein fragment 1-42 trifluoroacetate salt (0.1mg), 1,1,1,3,3,3-hexafluoro-2-
propanol (HFIP) and absolute DMSO were purchased from Sigma-Aldrich (Germany).Human
amyloid β protein fragment 1-42 trifluoroacetate salt was (0.1 mg, Sigma-Aldrich, USA)
dissolved in HFIP at a concentration of 1 mg/mL.The amyloid aggregation inhibition properties
of compound (4) and standard inhibitors were tested as per procedure described previously.[31]
The peptide suspension was vortexed at 4ºC for 2 h, followed by sonicated for 15 mins creating
a clear suspension of peptide in HFIP solution. The mixture was sealed and left overnight at
4ºC, next day the HFIP was removed by bubbling of nitrogen gas in the vial leaving a thin clear
film on the wall of the vial. The sample was dissolved in DMSO, and aliquots of 4µL each were
stored at -20ºC until needed for experiment. The peptide aliquot 4µL was further reconstituted in
100mM phosphate buffer (pH-8.0) at a final concentration of 230µM. For co-incubation
experiments, 16µL of the hAChE with Aβ (final concentration of 2.30µM, Aβ/AChE molar ratio of
100:1) and hAChE with Aβ in presence of 2µL of synthesized inhibitor (final concentration of
100µM) in 100mM phosphate buffer (pH-8.0) were added. The final volume of the reaction
mixture was 20µL, for measurement of amyloid fibril formation Thioflavin T (ThT) was added at
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a final concentration of 2.5µM in total volume (120µL) of the solution. Blanks containing Aβ1-42
,
hAChE alone and Aβ1-42 with tested inhibitor (final concentration 100µM) were taken separately.
The fluorescence intensities were taken at λex = 446nm &λem= 490nm which are characteristic of
ThT bound to β-sheet of amyloid fibrils. The percent inhibition of the AChE-induced aggregation
inhibition by tested compounds were calculated according to the following expression: 100 –
[(IFi/IF0) x 100], where IFi and IF0 are fluorescence intensities obtained for Aβ1-42 with AChE in
presence and absence of the tested inhibitor respectively.
AFM imaging of AChE induced amyloid aggregation inhibition
The peptide aliquot 4µL was reconstituted in 100mM phosphate buffer (pH-8.0) at a final
concentration of 230µM. For AFM analysis of AChE induced amyloid aggregation, 16µL of the
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hAChE with Aβ (final concentration of 2.30µM, Aβ/AChE molar ratio of 100:1) and hAChE with
Aβ in presence of 2µL of synthesized inhibitor (final concentration of 100µM) in 100mM
phosphate buffer (pH-8.0) were added. The final volume of reaction mixture was 20µL. The
samples for AFM imaging were prepared by loading a droplet of mixture solution (10-15µL)
directly on fresh mica and left for incubation for 15-20 mins in a small container chamber, gently
rinsed with buffer (10mM Tris, 5mM EDTA, 10mM KCl, 0.05% NaN3, pH-7.4) and transferred to
AFM microscope for image acquisition was done BioScope Catalyst Atomic Force Microscope
(AFM) machine by Bruker.
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Cytotoxicity studies
DMEM-F12 powdered media, Neuronal Media, N2 supplement, Propidium iodide (PI), cytosine
arabinofuranoside (AraC), 4’6-diamidino-2-phenylindole (DAPI), Triton X-100, Trypsin and
Diastase were purchased from Sigma-Aldrich (Germany). Horse serum and fetal bovine serum
were purchased from Gibco, USA. Poly-L-lysine coated 6-well plates and 40µ cell strainer were
purchased from BD.In vitro viability assay was performed on hippocampal neurons isolated from
adult mice brain and which were grown in poly-L-lysine coated 6-well plates with neuronal
culture media with 10% horse serum, 5% fetal bovine serum and N2 supplement. Initially, mice
were anaesthetized and sacrificed by as per IAEC norms and approved protocols. Later on,
brain were excised and both hippocampi from 4 adult mice were isolated under a
stereomicroscope. Isolated hippocampus were immediately transferred to 4ºC in artificial CSF
solution. Later to collection, all the hippocampi were subjected to diastase treatment and kept at
37˚C for 15 min to release individual cells from the hippocampus tissue. This resulting solution
was further pipetted to make single cell suspension and passed through a 40µ cell strainer. Cell
strainer passed solution was centrifuged and cells were washed twice with neuronal media and
resuspended in complete neuronal media containing N2 supplement. These resuspended cells
were then plated onto the poly-L-lysine coated 6-well plates at a density of 10000 cells/well in
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3mL of media. For next 21 days, daily attached cells were washed twice with Mg+2 and Ca+2 free
artificial CSF and consumed media was discarded and new media was replenished (2mL fresh
+ 1mL consumed). Once the cultures become mature, they were treated with 1µMcytosine
arabinofuranoside (AraC) overnight to remove all the non-neuronal cells. The viability of the
cells were determined on the basis on PI (propidium iodide) exclusion assay. The neuronal cells
were treated with different concentrations of tested inhibitor in triplicate for 24h. After 24h, cells
were incubated with PI (10µg/mL) and 300nM 4’6-diamidino-2-phenylindole (DAPI) as a counter
stain to stain nuclei of the cells. The cells were incubated with both the dyes for 30min, and
were fixed with 4% paraformaldehyde solution and permeabilized by 0.1% Triton X-100 solution.
Fluorescence reading were taking in Synergy HT hybrid multi-plate reader with excitation at
544nm and emission 612nm for PI. The healthy cells which has intact plasma membrane were
able to exclude PI, while their nuclei were stained blue by DAPI. The non-viable cells were
positive for PI while retaining the DAPI in their nuclei, this indicate more PI uptake by the dead
cells as compared to live cells. The viability was also monitored morphologically, where dead
cells become rounded in shape as compared to viable cells which retain their neuronal
phenotype.
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Cell cycle analysis
Cell cycle analysis was performed on the neural tissue cells isolated from adult mice brain
suspended in neuronal culture media with 10% horse serum, 5% fetal bovine serum and N2
supplement by using flow cytometry technique. Briefly, hippocampi from four adult mice brain
were isolated, the tissue was subjected to diastase and trypsin treatment which was further
converted to single cell suspension by gentle pipetting.The single cell suspension were passed
through a cell strainer (40µ pore size). The collected suspension was centrifuged, washed twice
with PBS and cells were resuspended in fresh media at a density of 5 x 105 cells/vial containing
desired concentration of the inhibitor tested. All the cells treated with different concentrations of
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inhibitor were incubated for 3h at 37˚C with humidified 5% CO2 environment in incubator. Post
3h, the cells were again centrifuged, washed twice with PBS and were resuspended in fresh
complete media without inhibitor. Cells were then acquired for cell cycle analysis in flow
cytometer and analyzed by BD Accuri C6 system with software.
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Molecular Docking studies
All the computational studies were performed using Schrödinger Software; Maestro 9.7
(Schrödinger, LLC, New York, NY, 2014); maestro 9.5 Prime version 2.3, Ligprep Version 2.9,
Glide version 6. The crystal structure of Torpedo Californica Acetylcholinesterase
Complexed With Alkylene-Linked Bis-Tacrine Dimer (7 Carbon Linker) Resolution of 2.15 Å,
Species Torpedo Californica; PDB: 2CKM was subjected to Protein Preparation Wizard which
includes pre-processing by assigning bond orders, addition of hydrogen and deletion of water
molecules beyond 5 Å from hetero (het) groups and further minimization and optimization of
protein structure was performed in the presence of force field OPLS2005.[32] Docking were
performed on the crystal structure data of torpedo californica acetylcholinesterase, the docking
was compared between the compound (4) and Bis(7)-tacrine for dual site binding in the enzyme
pocket sites[33]. The grid was prepared according to the Bis(7)-tacrine binding site coordinates in
the torpedo acetylcholinesterase and subjected to docking with analysis done with help of
Schrodinger software. The grid formation was chosen in the area encompassing CAS (Trp 84,
Phe 330, His 440) and PAS (Trp 279, Tyr 70, Ile 275). And for all the docking procedure, default
settings were used for the all evaluated molecules.
Results and Discussion
Synthesis. Synthesis of compound (4) was carried out in three-step procedure. In the first step,
Anthranilic acid was condensed with cyclohexanone at high temperature to yield 9-chloro-
1,2,3,4-tetrahydroacridine (1) with 94% yield. Second step involves preparation of 9-
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aminoalkylamino-1,2,3,4-tetrahydroacridine (2) was prepared by amino alkylation of 9-Chloro-
1,2,3,4-tetrahydroacridine (1) in presence of phenol and sodium iodide as previously reported
in the literature.[34] In the last step, final product (4) was efficiently synthesized by the
condensation of 9-aminoalkylamino-1,2,3,4-tetrahydroacridine with (3) (5-Aminoisophthalic acid,
5-AIA) via EDC/HOBt coupling procedure with 30.2% yield as previously reported procedure.[35]
The compound (4) can further provide site for functionalization with reporter groups as well for
various applications.
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AChE and BuChE inhibition
The inhibitory potential of various tacrine derivatives were evaluated by ellman’s colorimetric
assay. The catabolic reaction of acetylthiocholine (ATC) was intiated by AChE in the presence
of a chromogenic substrate,5’5-dithiobis (2-nitrobenzoic acid) (DTNB).
(a)
(b)
Fig.3 Enzyme inhibition curve of THA, TEDA, Bis(7)-tacrine and compound (4) on AChE (a) and BuChE
(b) in concentration range ( 100µM – 1fM) of inhibitor tested.
For measuring potency of inhibitor, enzymatic activity was evaluated at various concentrations
in the presence or absence of the inhibitor. The initial reaction rate were determined by using
∆OD412/min from optical density versus time curves for various concentrations in prescence or
absence of inhibitor respectively. All the IC50 values were calculated from non-linear regression
analysis of the % AChE/BuChE activity versus concentration of inhibitor using graphpad prism
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5.0. All the tacrine derivatives synthesized were shown to be very potent inhibitors of the AChE
and BuChE in subnanomolar concentrations in vitroexperiments with IC50 values ranging from
100 nM to 0.2 nM were represented in Fig.3. The dimeric molecule (4) exhibited more potency
for AChE and BuChE as compared to THA, (2) but similar to Bis(7)-tacrine. The IC50 values for
different derivatives synthesized along with THA and Bis(7)-tacrine were depicted in Table 1.
The AChE IC50 value of THA, TEDA, Bis(7)-tacrineand compound (4) were determined
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Table 1. Results of inhibition assay of reference and synthesized derivatives of tacrine and their
selectivities are expressed as the ratios of IC50 values.
as 184.6 ± 1.2, 229.1 ± 1.2, 0.33 ± 0.04 and 0.54 ± 0.06 nM, while on BuChE were6.57 ± 1.2,
59.84 ± 1.1, 15.78 ± 1.2 and 32.49 ± 1.2 nM respectively. The selective inhibition of AChE was
also calculated for reference and tacrine derivative compounds by ratios of IC50 of
BuChE/AChE. THA, TEDA, Bis(7)-tacrine and compound (4) showed selective ratio of 0.035,
0.261, 47.81 and 60.16 respectively, this suggest the highest selectivity of compound (4)
towards AChE. Being the homodimeric inhibitor, we also evaluated the hill slope of the AChE
and BuChE inhibition mechanism. THA, TEDA, Bis(7)-tacrine and (4) shows hill cofficient of
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0.70, 0.78, 1.24 and 1.42, this suggests the higher binding and cooperative binding of the
dimeric inhibitors as compared to monovalent inhibitors.
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To study the inhibitory mechanism of prepared tacrine derivative (4), kinetic inhibition studies
were performed with varying substrate and inhibitor concentrations, and substrate-velocity curve
or Lineweaver-Burk plots were generated.The Lineweaver-Burk curve for bivalent inhibitor (4)
was represented by Fig.4 (a) and (b), this curve gives Vmax value (i.e. reciprocal of the Y
intercept) and Km value (i.e. negative reciprocal of X intercept), but Vmax cannot be calculated
(a)
(b)
Fig. 4 The Lineweaver-Burk plot of compound (4) on AChE (a) and BuChE (b) in a concentration range of
compound (4) was ranged (0-80 nM) and substrate concentration (ATC) was ranged (15.625-500 µM).
due to non-extrapolation of graph to infinity in Michaelis-Menton curves. So for better clarity and
illustration reciprocals of velocity and substrate were plotted in the form of Lineweaver-Burk
plots shown in Fig. 4. The Lineweaver-Burk plot gives Vmax and Kmax values for compound (4)
which were found to be 0.3594 µM sec-1 and 106.47 µM for AChE, while Vmax and Km for BuChE
were 0.0481 µM sec-1 and 74.23 µM respectively. It was also seen from observed data that, with
increase in concentration of compound (4) the Vmax value decreases and Km value remains
approximately the same, this suggests the mechanism of inhibition of AChE and BuChE was
non-competitive type.
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Thioflavin T competition assay
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The previous data about compound (4) were suggestive of cooperative binding with both the
available site in the gorge of AChE, i.e. CAS and PAS. It was also assumed that only AChE
have PAS, while BuChE lacks this additional binding site. Therefore, compound (4) also
explored forits ability to inhibit the PAS, which is the site reported to accelerate the amyloid
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Fig. 5Peripheral active site (PAS) inhibition potential of various tacrine derivatives was evaluated by
releasing ThT from PAS of AChE. Results were expressed as percentage inhibition of PAS activity, the
values represents mean value with standard error mean of more than triplicate (n ≥ 3).
Peptideaggregation supposed to be one of the major event in AD pathology. PAS inhibition
assay for THA, Bis(7)-tacrine and compound (4) was performed on human AChE. ThT reported
to bind to PAS site of the AChE which leads to increase in fluorescence of ThT in the bound
form. A reduction in ThT fluorescence was measured as the parameter for replacing ThT from
the PAS of AChE, which would give the measure of inhibition of PAS activity. THA, Bis(7)-
tacrine and compound (4) showed 88 ± 1.4, 26.65 ± 1.3 and 36.19 ± 6.3 percent of PAS activity
respectively shown in Fig 5. The maximumreduction in PAS activity was achieved by Bis(7)-
tacrine, while that of compound (4) was slightly lesser then Bis(7)-tacrine. These data suggests
that compound (4) has shown equally potent dual binding site inhibitorwhich can inhibit PAS site
of the enzyme effectively. Compound (4) presumably inhibit the CAS as well as PAS both the
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sites simultaneously, which was expected from molecular modelling data of compound (4) with
AChE.
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As previously stated in many reports that, PAS of AChE facilitates and accelerate the
aggregation of the amyloid peptide in AD brain. This event could be considered as the core
mechanism by which amyloid peptide leads to multifactorial disease condition. Therefore, we
evaluated its potency in inhibiting the AChE induced amyloid peptide aggregation in vitro. In this
assay, ThT was used as native dye which binds to aggregated form of amyloid peptide and
shows enhanced fluorescence as compared to unbound ThT. A decrease in ThT fluorescence
was considered as parameter for inhibiting AChE induced amyloid aggregation of the tested
compound. A single concentration of 100µM was selected for better comparison in inhibiting
potential of all the reference drugs and prepared compounds. THA, Bis(7)-tacrine and
compound(4)were tested for, their ability to inhibit amyloid aggregation.
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Fig.6 Amyloid aggregation inhibition potential of tacrine derivatives were evaluated. Potency were
measured as parameter of reducing fluorescence of bound ThT. All derivatives were evaluated at a
concentration of 100µM. Aggregation inhibition expressed as percentage compared to control values, the
values represents mean value with standard error mean of more than triplicate(n ≥ 3).
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Results have shown that amyloid aggregation was inhibited by THA, Bis(7)-tacrine and
compound (4) at 100µM was 8.3 ± 0.32, 86.8 ± 3.45 and 80.16 ± 2.94 percentage as compared
to the control respectively shown in Fig. 6. However, these results indicate compound (4) as
equally potent inhibitor as compared to Bis(7)-tacrine in inhibiting AChE induced amyloid
aggregation.
AFM imaging of AChE induced amyloid aggregation inhibition:
As indicated from previous data, that selected inhibitors Bis(7)-tacrine and compound (4) were shown to
the most potent inhibitors for AChE induced amyloid aggregation. Therefore, AFM image acquisition were
done on mixtures containing Bis(7)-tacrine and compound (4) while comparing to the control aggregation
process Fig 7. The AFM images reveals and correlated with the data obtained using decrease in ThT
fluorescence in the previous experiments. The compound (4) shows aggregation inhibition to extent
similar like Bis(7)-tacrine.
A
C
Fig 7 A) Atomic Force Microscopy (AFM) of amyloid aggregation Aβ1-42 fibrils B) Inhibtion of aggregation
by Bis(7)-tacrine C) Inhibtion of aggregation by Compound (4)
Cytotoxicity studies
From in vitro data of previous studies suggest compound (4) as equally potential inhibitor of
AChE/BuChE activity, PAS activity and AChE induced amyloid aggregation, but it is very much
needed to evaluate the overall toxicity of this compound on cells of neuronal tissue. Therefore,
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cytotoxicity of the Bis(7)-tacrine and compound (4) was evaluated by PI exclusion assay on
hippocampal neurons at various concentration of treatment (Fig 8). Both Bis(7)-tacrine and
compound (4) were non-toxic up to concentration of 200µM, but as the concentration increases
further Bis(7)-tacrine started to show cytotoxicity at 300µM while compound (4) started to show
cytotoxicity at 400µM. The percentage cellular viability at 300µM of bis(7)-tacrine treatment
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(a)
(b)
Fig. 8 (a) Isolation and culturing of primary hippocampal neuronal cultures from adult mice brain. (b)
Cellular cytotoxicity of compound (4) on primary hippocampal neuronal cultures was evaluated with the
reference inhibitor Bis(7)-tacrine. Percentage of control cell growth was plotted against concentration
range (0 – 700 µM). The the values represents mean value with standard error mean of more than
triplicate(n ≥ 3).
was 65%, while of compound (4) was 90%. The IC50 values for Bis(7)-tacrine and compound (4)
on hippocampal neurons were ≈320µM and ≈385µM. These findings suggests the non-toxic
nature of the compound (4) to further explore this ligand as therapeutic ligand without causing
significant cytotoxicity on neuronal tissue, therefore both the ligands could be used as potential
inhibitors for treatment of AD pathology in clinics.
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Cell cycle analysis
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In continuation to cytotoxicity evaluation, we also tried to check the effect of compound (4) on
cell cycle. Most of anti-Alzheimer’s drugs having neurogenic potential as they not only mitigate
the AD insult but also induce cellular multiplication. Therefore, we also explored the neurogenic
potential of compound (4) as compared to Bis(7)-tacrine. In the present assay, fraction of cells
present in different phases of cell cycle were measured, more fraction of cells in S-phase
represents more amount of cellular synthetic activities leads to cellular multiplication. Fig. 8
represents the results of cell cycle analysis, which reveals cell cycle variations at different
concentration of the inhibitor tested. Upper panel represents cells treated with different
concentrations of Bis(7)-tacrine, while the lower shows the cells treated with compound (4).
Theupper control panel shows percent of total cells in G1, S and G2/M phases were 53.5%,
16.7% and 20.7% respectively, while the lower control panel shows 56.8%, 13.1% and 19.9%
respectively. Comparison of both control panel suggest that approximately equal fraction of cells
in all three phases.But when Bis(7)-tacrine and compound (4) were introduced at 150µM
concentration, the cells in G1 phase increases to 62.7% in Bis(7)-tacrine, while in case of
compound (4) it remains similar to control at 51.0%. The S-phase in Bis(7)-tacrine reduces to
10.9%, whereas in compound (4) it increases to 15.4%, and cells in G2/M phase decreased
to8.6% in Bis(7)-tacrine whereas it remains same in compound (4) at 19.9%. When both Bis(7)-
tacrine and compound (4) introduced at 300µM, the G1, S and G2/M phases were 67.1%, 6.6%,
8.2% and 63.4%, 14.4%, 9.1% respectively,it is worthwhile to note that there was more than 2-
fold increase in S-phase population at higherconcentration of compound (4) as compared to
Bis(7)-tacrine. At concentration of 800µM, G1, S and G2/M phases were 80.0%, 6.6%, 3.1%
and 77.7%, 14.0%, 2.8% in case of Bis(7)-tacrine and compound (4) respectively.
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Fig. 9 Comparison profile of cell cycle analysis by flow cytometry. Upper Panel shows cells in different
cell cycle phases after Bis(7)-tacrine treatment, While lower panel shows fraction of cells after treatment
with compound (4) at different concentrations.
These observations suggest induction of more population of cells in S-phase when subjected to
pharmacologically active concentration of the compound (4) as compared to Bis(7)-tacrine
Molecular Docking studies
Three-dimensional molecular modelling was performed to assess the binding affinity of the
newly synthesized compound (4) and compared with Bis(7)-tacrine and other reported potent
inhibitors on the active site of the AChE.[32,34] Both static and dynamic molecular docking was
performed using 2CKM (Torpedo Californica Acetylcholinesterase complexed with alkylene-
linked Bis-Tacrine Dimer (7 Carbon Linker) Resolution of 2.15 Å, Species Torpedo Californica).
Bivalent ligand approach have been known for AChE inhibitors because of the favorable
(A)
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Fig. 10 The two dimensional interaction pattern of Bis(7)-tacrine and compound (4)on the amino acid
residues of AChE of Torpedo californica where green colored are hydrophobic, cyan are polar, red are
negative and purple are positive amino acid residues. Pink lines shows the hydrogen bonds while green
lines represents π – cation interactions.
Geometric consideration of the active site gorge of AChE where two binding site were present at
the extreme ends in the form of CAS and PAS. In the present docking study, the docking pattern
of Bis(7)-tacrine was compared with compound (4), which showed more number of interactions
due to aromatic linker in compound (4) as compared to alkyl linker in Bis(7)-tacrine. The tricyclic
pharmacophores in both ligands resides in the expected location of CAS and PAS. The green
lines show the hydrophobic interactions including π-π and stacking interactions, while the pink
line shows the hydrogen bond interactions. The pharmacophores in both theligands interacts
with the key residues of CAS were Trp84, Phe330 and His440, the aromatic cationic nitrogen
also forms hydrogen bonds with Phe330 and His440. Similarly, if we analyzethe interaction of
anotherpharmacophore which resides in the PAS of AChE in both the ligands,this shows that
Bis(7)-tacrinehaving hydrophobic interactions with Tyr70 and Trp279 and aromatic cationic
nitrogen having hydrogen bond with Tyr70. But if we see the interaction profile of PAS residing
pharmacophore in compound (4) reveals additional interaction with Ile275. In the compound
(4)the aromatic linker was introduced to provide an additional site for radiolabeling, but the
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aromatic and two amide bonds provides additional set of interactions which would lead to
enhanced docking and glide scoreFig. 10.Two amide bonds were involved in formation of
hydrogen bonds with Tyr121 and Trp279, while the aromatic ring made π-π and stacking
interactions with Tyr334. We have also compared the docking and glide scores of other reported
ligands with compound (4) given in the Table 2.
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Table 2. Molecular docking analysis of various reference tacrine derivatives with compound (4). The
results are expressed here as percentange of scores of tacrine derivatives mentioned here with respect to
Bis(7)-tacrine.
Therefore, the study conclude that compound (4) has highest binding affinity towards AChE in
molecular docking studies which in turn lead us to synthesize compound (4) and evaluate its
potential as AChE inhibitor.
Discussion
In the present study, we described the design, synthesis and its biological evaluation of
compound (4) as a potential inhibitor of AChE, which further could be utilized as a tracer
molecule to detect fluctuation of AChE expression levels in different regions of the brain in
disorders involving AChE. In the current work, we merged two different approaches for making a
successful tracer molecule which should retain its therapeutic potential as well while used for
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molecular imaging applications. [33] According to cholinergic hypothesis, enhancement of
acetylcholine at junctional synapses significantly improves the cognitive functions, this fact
further lead to synthesis of various classes of AChE inhibitors for treatment of AD. THA was the
first drug approved by FDA for treatment of AD, so many more THA based derivatives were
synthesized and evaluated. But due to low specificity THA was having multiple side effects.
Therefore, Pang et al, (1996) designed and synthesized a novel catalytic active site structure
based inhibitor Bis(7)-tacrine with subnanomolar potency[25]. Many reports also suggests that,
Bis(7)-tacrine was the most potent inhibitor with high specificity towards AChE known to us till
date. Therefore, we considered to make a homodimeric inhibitorhaving equal potency based
imaging agent for tracing AChE in neurological disorders.
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Many attempts were made to visualize AChE with the help of radiotracers in PET/SPECT
imaging modalities. As far as radiotracers of AChE are concerned, they can be classified into
two categories 1) AChE substrate based tracer and 2) AChE inhibitor based tracers. AChE
substrate based tracers were not successful due to diffusion of radio labeled products inside the
brain area, which in turn not suggestive of accurate location of AChE in different brain
regions.[18] While the AChE inhibitor based radiotracers were much more stable and accurate in
locating AChE in brain imaging.[22,23] So, here we tried to use homodimeric inhibitor of AChE
coupled with radiotracer ability to design a theranostic ligand for treatment of AD which could
lead to better patient management and monitoring the progression of the disease.
In the current synthesis, we have inserted an aromatic linker (5-aminoisophthalic acid) in a
dimeric tacrine based inhibitor of AChE, which could provide an additional site to radiolabeling
for imaging applications retaining the same subnanomolar potency of inhibiting AChE. The
coupling procedure reported here is most convenient due to water solubility of side products
which can be easily separated from the products by extraction procedure.[35] 5-AIA is often used
as linker for making dimeric molecules, which contains two similar moieties connected to
isophthalate linker. 5-AIA was previously employed in attaching two chelating moieties to the
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biotin derivative for various applications including radio imaging. The presence of 5-amino group
in the isophthalate linker provide an extra site for further functionalization with an optical or
radioactive reporter (i.e. FITC or 11C chemistry).
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The enzyme inhibition and enzyme kinetics data suggests inhibitor properties for compound (4)
which was having IC50 value in subnanomolar concentration with non-competitive kind of
enzyme inhibition, which is very much similar to Bis(7)-tacrine mode of inhibition. Compound (4)
also showed more positive cooperative binding of tacrine pharmacophore to active sites of
AChE more than Bis(7)-tacrine. The PAS inhibition and amyloid aggregation data also reveals
its potency for inhibiting the PAS site of AChE was equivalent to Bis(7)-tacrine. Evaluation of
cytotoxic potential was also checked with comparison to Bis(7)-tacrine, compound (4) comes
out to have less cytotoxicity effect on the hippocampal neurons. Apart from cytotoxicity,
compound (4) was also found out to be of neurogenic potential through flow cytometry analysis.
Compound (4) treated cells exhibited more fraction of cell population in the S-phase of cell cycle
as compared to Bis(7)-tacrine, these data proves the neurogenic potential of compound (4).
Molecular docking analysis showed higher affinity of compound (4) for CAS and PAS as
compared to Bis(7)-tacrine and other reported ligands. The preliminary evaluation of the
compound (4) with its theranostic potential makes this ligand a promising PET imaging tracer for
imaging AChE in vivo, which could open new avenues for diagnosis and therapy in Alzheimer’s
disease.
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ASSOCIATED CONTENT
Supporting Information available: [pp. 1S-8S] NMR and Mass Spectra of compounds,
Enzyme kinetic curves (Michaelis-Menton curve) of compound (4) on AChE and BuChE. This
material is available free of charge via the internet at http://pubs.acs.org.
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AUTHOR INFORMATION
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Corresponding author
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*E-mail: puja.hazari@gmail.com Tel: +91-01123905244 Fax: +91-01123919509
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
The present work was supported by Project INM 319, Defence Research and Development
Organization, Ministry of Defence and CSIR, Delhi. The authors also extend their thank to the
Director, INMAS and Prof. Ramesh Chandra, HOD, Chemistry Dept., Delhi University for
providing necessary facilities.
References
1. Gauthier, S.; Reisberg, B.; Zaudig, M.; Petersen, R. C.; Ritchie, K.; Broich, K.;
Belleville, S.; Brodaty, H.; Bennett, D.; Chertkow, H.; Cummings, J. L.; Leon, M. D.;
Feldman, H.; Ganguli, M.; Hampel, H.; Scheltens, P.; Tierney, M. C.; Whitehouse, P.;
Winblad, B. Mild cognitive impairment. Lancet2006, 367, 1262–1270.
2. Mucke, L. Neuroscience: Alzheimer’s disease. Nature2009, 461,895 − 897.
3. Salmon, D. P.; Lange, K. L. Cognitive screening and neuropsychological assessment
in early Alzheimer’s disease. Prog. Neurobiol.2001, 60, 139-165.
4. Glenner, G.G. The pathobiology of Alzheimer’s disease. Annu. Rev. Med.1989, 40:45–
51.
5. Selkoe, D. J. Alzheimer’s disease: genes, proteins, and therapy. Physiol. Rev.2001,
81, 741–766.
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