Research Proposal for "CSIR - Senior Research Fellowship"

Name of the candidate: Mr. Deendyal

Name of the supervisor: Dr Sandip B. Bharate, Principal Scientist,
Medicinal Chemistry Division
Name of the institute: CSIR - Indian Institute of Integrative Medicine, Jammu

(A) Title of the Project: Medicinal chemistry of "chromones and chromone alkaloids" to
discover multi targeted anti-Alzheimer’s agents.

(B) Outline of the problem: Neurodegenerative diseases are slow progressive disorders
characterized by the loss of learning, memory and other cognitive functions. In particular, the
Alzheimer's disease (AD) is a multifactorial and fatal neurodegenerative disorder characterized
by dysfunction of cognitive functions leading to complete degradation of personality [1]. The
pathogenesis of AD involves the degeneration of of cholinergic, β-amyloid (Aβ) deposition, τ-
protein aggregation, oxidative stress and neuroinflammation [2]. The complicated pathogenesis
of AD makes it difficult to develop new drugs, and this has resulted in failures in clinical trials.
Despite these challenges, research of AD is still a hot topic, and the efforts and attempts of
scientific groups worldwide are continuing, with the aim of finding an effective treatment [3].
Currently, investigation of AD has led to finding of many targets that affects the
generation and exacerbation of the disease, such as AChE, BACE-1, GSK-3β, MAOs, metal ions
in the brain and NMDA receptor. A lots of potent and effective compounds for the treatment of
AD are discovered based on these targets, among which donepezil, galantamine, rivastigmine,
and memantine are the only four drugs approved by the FDA for the treatment of AD. Most
importantly, all these approved drugs for AD does not completely cure the disease. They provide
only symptomatic treatment of the disease, however progression of the disease cannot be halted.

Chromone is one of the most important oxygen containing heterocyclic class of moiety present in
the nature with wide range of biological activities, including antitumor, antimicrobial, antiviral,
anti-inflammatory, antioxidant, and so on [4]. Chromone scaffold [(4H)-1-benzopyran-4-one]
has been recognized as a pharmacophore of a large number of bioactive molecules of either
natural or synthetic origin [5]. Natural chromones (structures 1 and 2, shown below) have been
reported to possess neuroprotective effect against glutamate-induced neurotoxicity in primary
cultures [6]. Furthermore, this class of compounds are known to possess activity against MAOs,

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AChE and BChE (structures 3-5) [2, 7]. Thus, our work is focused on the design of new
multifunctional molecules favour with MAO-inhibiting, metal-chelating, AChE, BChE and
BACE-1 inhibition.

The recent failures of all single targeted compounds, has clearly indicated that agents with an
ability to engage more than one target of the disease pathway, would have better chances of
success at clinical stages. Thus, herein with the combined use of rational design, computational
techniques, present proposal aims to discover new chromone/ chromone alkaloid based multi-
targeted anti-Alzheimer agents.

(C) Preliminary work done: As a project fellow, I have been working on the synthesis of
chromones/chromone alkaloids and their derivatives for evaluation as potent anti-Alzheimer
agents. As of now we have prepared ~20 compounds. The preliminary screening against
cholinesterase enzymes has been performed on synthesized compounds, which has resulted in
identification of two interesting new hits. Compounds DD-30 and DD-120 showed 59 % and 61
% inhibition at 10 µM concentration against AChE, respectively. Since the identified hits brings
completely a new scaffold for this activity, this appears to be the promising potency for any new
scaffold. Further optimization efforts will be made under this proposal.

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(D) Objectives:
1. Design of new derivatives of chromones and chromone alkaloids class as potent anti-
Alzheimer’s agents using molecular modeling.
2. Synthesis of molecular libraries (series) in each scaffold.
3. Biological evaluation of synthesized compounds against various molecular targets od
Alzheimer’s disease.
4. Preclinical characterization of most active natural product/its analog.
5. In-vivo validation of most active lead in suitable animal model.

(E) Work of plan: The workplan for executing this proposal comprises step-wise activities as
described below.
a. Design of potential chromone based hybrid structures using rational drug design or
molecular docking. With the help of available literature knowledge and molecular modeling
studies, three series of compounds are proposed in this proposal. All proposed scaffolds are
expected to engage more than one molecular target. The proposed molecular targets comprises
AChE, BChE, BACE-1 and MAO-B. The proposed scaffolds are shown below, and the
representative proposed structures from series 1 and 2 are also shown.

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Scheme 1: Synthesis of novel Rohitukine-N-oxide (RHK-N-oxide) derivatives

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Scheme 2: Synthesis of khellin derivatives

b. Synthesis of designed compounds. All designed compounds will be synthesized using

suitable synthetic paths. Some of the paths are shown below.

c. Biological evaluation of synthesized compounds for AChE, BChE, BACE-1, MAO-A,

MAO-B; and GSK-3β inhibition. All in-vitro target assays will be performed using established
protocols. The protocols of various enzymatic assays are provided below.
Ellman assay (AChE, BChE inhibition). Stock solutions of test compounds (10 mM) will be
prepared in DMSO and further dilutions with 0.1 M phosphate buffer (PBS, pH 7.2). 20 μL
enzyme solution (AChE or BChE with 2.5 units/mL enzyme activity), 20 μL test compound
solution (inhibitor) and 140 μL 0.3 mM 5,5-dithiobis-(2-nitrobenzoic acid) DTNB solution will
be added into the 96-well plate. Assay solutions with and without inhibitor will be pre-incubated

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at 25 °C for 20 min, followed by the addition of 20 μL 10 mM substrate (AtChI or BTChI)
solution. The final concentration of DMSO will be kept at 0.1%. Absorbance of the assay
solutions will be measured kinetically at a wavelength of 412 nm. All assays will be done in
triplicate. The % inhibition will be calculated by the following expression: 100 × (1- Ai/Ao)
where Ai and Ao are the respective absorbance values in the presence and in the absence of
inhibitor (Positive control: donepezil).
FRET assay (BACE-1 inhibition). The BACE-1 FRET assay kit supplied by Sigma-Aldrich
(Product No. CS0010, Saint Louis, USA) will be used for performing the assay according to the
protocol provided by the supplier. Stock solutions of the test compounds (10 mM) will be
prepared using DMSO and desired concentrations by further dilutions with Fluorescent Assay
Buffer (FAB, 50mM sodium acetate buffer, pH 4.5) supplied in the kit. 10 μL test compound, 20
μL of 50 μM substrate, 68 μL FAB and 2 μL BACE-1(0.3 unit/μL) will be added into 96 well
black polystyrene microplate. The final concentration of DMSO will be kept at 0.1%.
Fluorescence will be measured (excitation at 320 nm and emission at 405 nm) at time zero and
after incubation for 2 h at 37 °C. All assays will be done in triplicate. The background signal
containing all the reagents, except BACE-1, will be subtracted. Difference between the
fluorescence readings (‘time zero’ and 2 h) will be used for calculations. The % inhibition will
be calculated by the following expression: 100 − (FIi/FIo × 100) where FIi and FIo are the
respective fluorescence intensities obtained in the presence and in the absence of inhibitor
(Positive control: β-secretase inhibitor IV, Calbiochem).
GSK-3β assay. The GSK-3β activity assay kit (product no CS0990) from Sigma will be used.
The assay [ADP‐Glo™ Kinase Assay] is based on immuno-precipitation of GSK-3β using a
specific anti-GSK-3β antibody. The immuno-precipitated kinase will be incubated with γ−32P-
ATP and the incorporation of 32P into the substrate is measured. This assay will be performed
using the protocol as described in our earlier publication (Chem. Biol. Drug. Des. 2017, 89,
964−971). Briefly, the GSK-3β (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01%
Brij35, 5% glycerol, 0.1% β-mercaptoethanol, 1 mg/ml BSA) will be assayed against phospho-
GS2 peptide (YRRAAVPPSPSLSRHSSPHQS(PO4)EDEEE) in a final volume of 25.5 µl
containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 20 µM Phospho GS2 peptide, 10 mM
magnesium acetate and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min
at room temperature. Assays will be stopped by addition of 5 µl of 0.5 M (3%) orthophosphoric
acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric
acid. In-vitro % inhibition values will be calculated as an average of three independent
observations. In-vitro IC50 values represent mean and standard deviations from two independent
experiments.
MAO-B inhibition assay. Monoamine Oxidase Activity Assay Kit (product no: MAK136) from
Sigma will be used for initial screening, to know the general MAO inhibition activity. In this
assay, MAO reacts with p-tyramine, a substrate for both MAO-A and MAO-B, resulting in the
formation of H2O2, which is determined by a fluorimetric method (λex = 530/λem = 585 nm).
Amyloid-beta aggregtion. The effect of test compounds will also be studied for their ability to
stop or reverse the amyloid-beta aggregation, using a standard thioflavin T assay (Eur. J. Med.
Chem. 2019, 166,11-21).
BBB permeability using PAMPA assay. This assay will be performed using the BBB-Pampa
Explorer kit (pION Inc., Woburn, MA, USA) using standard protocol. Briefly, compound stocks

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(20 mM) in DMSO will be diluted to 100 μM with pH 7.4 Prisma HT buffer. 200 μL of this
solution will be added to each well of the donor plate (n = 6). 5 μL of the BBB-1 lipid
formulation will be painted to filter membrane of each well of the acceptor plate, and 200 μL of
brain sink buffer will be added. The acceptor filter plate will be placed on top of the donor plate
to form a “sandwich”. The sandwich will be incubated for 4 h at room temperature without
stirring. After incubation, UV-visible spectra will be measured in the reference acceptor and
donor plates using a microplate reader. The −log Pe will be calculated for each compound using
the PAMPA Explorer software v. 3.7 (pION). (Positive control: theophylline).
Preformulation parameters (aqueous solubility, Log P, Log D, pKa, plasma stability). As per
our published protocols.

In-vivo study will be carried out in suitable ant-dementia memory model.

(F) Timeline:
Work Plan Year 1 Year 2 Year 3
Task 1: Design of compounds
Task 2: Synthesis of designed compounds
Task 3: In-vitro screening against proposed
molecular targets
Task 4: To study best compounds for
preformulation parameters
Task 5: PAMPA assay of best compounds
Task 6: Scale up of most active compounds for in-
vivo studies
Task 7: PK and in-vivo study

(G) References cited

[1] G.F. Makhaeva, N.P. Boltneva, S.V. Lushchekina, E.V. Rudakova, O.G. Serebryakova, L.N.
Kulikova, A.A. Beloglazkin, R.S. Borisov, R.J. Richardson, Synthesis, molecular docking,
and biological activity of 2-vinyl chromones: Toward selective butyrylcholinesterase
inhibitors for potential Alzheimer's disease therapeutics, Bioorg Med Chem, 26 (2018) 4716-
4725.
[2] F. Li, J.-J. Wu, J. Wang, X.-L. Yang, P. Cai, Q.-H. Liu, L.-Y. Kong, X.-B. Wang, Synthesis
and pharmacological evaluation of novel chromone derivatives as balanced multifunctional
agents against Alzheimer's disease, Bioorg Med Chem, 25 (2017) 3815-3826.
[3] C.A. Lane, J. Hardy, J.M. Schott, Alzheimer's disease, Eur J Neurol, 25 (2018) 59-70.
[4] I.C.H. Castaneda, S.E. Ulic, C.O.D. Vedova, N. Metzler-Nolte, J.L. Jios, One-pot synthesis
of 2-trifluoromethylchromones, Tetrahedron Lett, 52 (2011) 1436-1440.

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[5] A.M. Helguera, A. Pérez-Garrido, A. Gaspar, J. Reis, F. Cagide, D. Vina, M.N.D.S.
Cordeiro, F. Borges, Combining QSAR classification models for predictive modeling of
human monoamine oxidase inhibitors, Euro J Med Chem, 59 (2013) 75-90.
[6] J.S. Yoon, M.K. Lee, S.H. Sung, Y.C. Kim, Neuroprotective 2-(2-Phenylethyl)chromones of
Imperata cylindrica, J Nat Prod, 69 (2006) 290-291.
[7] J. Reis, F. Cagide, M.E. Valencia, J. Teixeira, D. Bagetta, C. Pérez, E. Uriarte, P.J. Oliveira,
F. Ortuso, S. Alcaro, M.I. Rodríguez-Franco, F. Borges, Multi-target-directed ligands for
Alzheimer's disease: Discovery of chromone-based monoamine oxidase/cholinesterase
inhibitors, Euro J Med Chem, 158 (2018) 781-800.

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