Dipropargyl substituted diphenylpyrimidines as dual inhibitors of monoamine oxidase and acetylcholinesterase

Article abstract of DOI:10.1016/j.ejmech.2019.05.039

Alzheimer's disease (AD) is a multifactorial neurological disorder involving complex pathogenesis. Single target directed drugs proved ineffective and since last few years' different pharmacological strategies including multi-targeting agents are being ex

Full text of DOI:10.1016/j.ejmech.2019.05.039

Accepted Manuscript
Dipropargyl substituted diphenylpyrimidines as dual inhibitors of monoamine oxidase
and acetylcholinesterase
Bhupinder Kumar, Vijay Kumar, Vikash Prashar, Suresh Saini, Ashish Ranjan
Dwivedi, Binu Bajaj, Devashish Mehta, Jyoti Parkash, Vinod Kumar
PII:
S0223-5234(19)30449-0
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.039
EJMECH 11349
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 February 2019
Revised Date: 24 April 2019
Accepted Date: 13 May 2019
Please cite this article as: B. Kumar, V. Kumar, V. Prashar, S. Saini, A.R. Dwivedi, B. Bajaj, D. Mehta,
J. Parkash, V. Kumar, Dipropargyl substituted diphenylpyrimidines as dual inhibitors of monoamine
oxidase and acetylcholinesterase, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.05.039.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Multi-potent compounds targeting MAO and AChE enzymes

ACCEPTED MANUSCRIPT
Dipropargyl Substituted Diphenylpyrimidines as Dual Inhibitors of Monoamine Oxidase
and Acetylcholinesterase
1
1
2
1
1
Bhupinder Kumar , Vijay Kumar , Vikash Prashar , Suresh Saini , Ashish Ranjan Dwivedi ,
1
1
2*
1*
Binu Bajaj , Devashish Mehta , Jyoti Parkash and Vinod Kumar
1
Laboratory of Organic and Medicinal Chemistry, Department of Pharmaceutical Sciences and
Natural Products, Central University of Punjab, Bathinda, Punjab, India-151001.
2
Department of Animal Sciences, School of Basic and Applied Sciences, Central University of
Punjab, Bathinda, Punjab, India.
Corresponding Author:
*
Dr. Vinod Kumar
Laboratory of Organic and Medicinal Chemistry, Department of Pharmaceutical Sciences and
Natural Products, Central University of Punjab, Bathinda, Punjab, India-151001.
E-mail: vpathania18@gmail.com; vinod.kumar@cup.ac.in
Phone No. +911642864214
Co-corresponding Author:
Dr. Jyoti Parkash
Department of Animal Sciences, School of Basic and Applied Sciences, Central University of
Punjab, Bathinda, Punjab, India
E-mail: jtprksh.sharma@gmail.com
Conflict of Interest Statement: A provisional patent application has been filed containing these
compounds (Indian patent application number 201811008301).

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Abstract
Alzheimer’s disease (AD) is a multifactorial neurological disorder involving complex
pathogenesis. Single target directed drugs proved ineffective and since last few years’ different
pharmacological strategies including multi-targeting agents are being explored for the effective
drug development for AD. A total of 19 dipropargyl substituted diphenylpyrimidines have been
synthesized and evaluated for the monoamine oxidase (MAO) and acetylcholinesterase (AChE)
inhibition potential. All the compounds were found to be selective and reversible inhibitors of
MAO-B isoform. These compounds also displayed good AChE inhibition potential with IC50
values in low micromolar range. AVB4 was found to be the most potent MAO-B inhibitor with
IC50 value of 1.49 ± 0.09 µM and AVB1 was found to be the most potent AChE inhibitor with
IC50 value of 1.35 ± 0.03 µM. In the ROS protection inhibition studies, AVB1 and AVB4
displayed weak but interesting activity in SH-SY5Y cells. In the cytotoxicity studies involving
SH-SY5Y cells, both AVB1 and AVB4 were found to be non-toxic to the tissue cells. In the
molecular dynamic simulation studies of 30 ns, the potent compounds were found to be quite
stable in the active site of MAO-B and AChE. The results suggested that AVB1 and AVB4 are
promising dual inhibitors and have the potential to be developed as anti-Alzheimer’s drug.
Keywords: Alzheimer’s disease, MAO inhibitors, Acetylcholinesterase inhibitors
Diphenylpyrimidine, Dual inhibitors, Neurological disorders

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1
. Introduction
Alzheimer’s disease (AD) [1] is a multifactorial neurological disorder characterized by memory
loss and cognitive impairment. Pathophysiology of AD is highly complex and the disease state is
associated with number of factors such as over expressed monoamine oxidase enzyme, declined
levels of acetylcholine (ACh), hyperphosphorylation of tau proteins, amyloid-β aggregation and
oxidative stress [2]. Neurodegeneration in AD causes neuronal and synaptic impairment which
alters the concentration of various neurotransmitter systems in the brain that include cholinergic
systems, [3, 4] dopamine, serotonin [5] and glutamate [2]. Thus, multi targeting agents (MTA)
that can simultaneously modulate different cellular pathways are being explored as drug
candidates to achieve better therapeutic efficacy for AD.
Monoamine oxidase (MAO) enzyme is considered as an important target for the treatment of AD
[6]. It is involved in the oxidation of xenobiotic and endogenous monoamines including
monoamine neurotransmitters such as serotonin, adrenaline, noradrenaline and dopamine. The
oxidation reaction results in the formation of neurotoxic aldehydes and reactive oxygen species
(ROS) as end products [7]. MAO enzyme exists in two different isoforms, MAO-A and MAO-B
which differ in substrate and inhibitor specificity. MAO inhibitors have the potential to attenuate
the oxidative metabolism of monoamine neurotransmitters and improve their concentration in the
brain. MAO-A selective inhibitors are found to be effective against depression [8] while MAO-B
selective inhibitors are being explored for the treatment of Parkinson’s disease [9] and AD [10].
Recent studies have demonstrated that MAO-B inhibitors possess broader therapeutic effects
such as neuroprotection [11], neurorescue [12] and cognitive improvement [7] which are useful
for the treatment of AD.
Acetylcholine (ACh) is a neurotransmitter associated with cognitive functions and physical
awareness [13]. ACh is hydrolyzed by acetylcholine esterase (AChE) or butyrylcholinesterase
(BuChE) enzymes and imbalance in the dynamics is linked to the pathology of AD. The
concentration of ACh is found to be drastically low in the AD patients as compared to the
healthy persons. The peripheral anionic site (PAS) of AChE catalyzes amyloid-β aggregation
which leads to the formation of senile plaques in the brain causing neurodegeneration [14]. Thus,
AChE inhibitors can increase the cholinergic transmission in the synaptic cleft by inhibiting the
degradation of ACh. At present donepezil, galantamine, and rivastigmine are three AChE
inhibitors (Fig.1) in the clinical practice for the treatment of AD [15]. These cholinergic drugs

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are able to provide symptomatic relief but could not prevent the progression of disease. Hence,
using a single drug that act on a specific target may not be an efficient therapeutic strategy for
AD due to its complex etiology. Now the attention of many research groups is focused on the
multi-targeting agents (MTAs) to develop effective drug candidates for the treatment of AD.
MTAs have the ability to bind to two or more targets and intervene in the pathological events
underlying the etiology of the disease. The design strategy for MTA involves incorporation of
pharmacophoric groups of different drugs in the same scaffold. Thus, a ligand with MAO
inhibitory potential can exhibit more efficient therapeutic effect in AD when it can show
additional AChE inhibitory activity. Consequently, many MTAs have been developed and some
of them are in various stages of clinical development. Ladostigil (Fig.1), a dual inhibitor of
acetylcholinesterase and MAO-B, has completed phase II trial for AD (NCT01354691).
Rasagiline (Fig.1), is under phase-II clinical trials to determine its effect in the regional brain
metabolism in case of AD (NCT02359552, NCT00104273).
In our previous studies [15], we have developed novel pyrimidine derivatives as selective
inhibitors of MAO-B isoform. Most of these compounds were found to be reversible MAO-B
inhibitors and taking leads from this study, we have developed another series of 4,6-
diphenylpyrimidines as dual inhibitors of monoamine oxidase and acetylcholinesterase enzymes.
In both of the studies, propargyl group was incorporated in the main scaffold as potential
pharmacophore for MAO enzyme. Propargyl group is present in number of potent MAO
inhibitors [10, 16] such as clorgyline, selegiline, pargyline, rasagiline etc. and other
multitargeting agents [17-25]. It forms a crucial covalent bond with the FAD cofactor of MAO
enzyme [26]. Recently, Zindo et al. thoroughly reviewed the role of propargyl group as
functional moiety in the design and development of multifunctional drugs for various
neurological disorders [25]. Weinreb et al. [27] investigated the role of propargyl group in
rasagiline as a neuroprotective moiety. It has been concluded that neuroprotective effect of
rasagiline is associated with the propargyl moiety, which promotes mitochondrial viability and
stabilizes permeability transition by regulating Bcl-2 family proteins. Similarly, other research
groups [28, 29] also demonstrated that rasagiline and other propargyl containing ligands may
rescue degenerating dopamine neurons by inhibiting death signal transduction initiated by
mitochondrial permeability transition. Keeping in view the significance of propargyl moiety and
following our previous work, in the current study we have incorporated two propargyl groups in
the diphenylpyrimidine scaffold and investigated their MAO and AChE inhibition potential. To

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the best of our knowledge this is first report on the screening of compounds containing two
propargyl groups on a scaffold. The synthesized compounds were screened against MAO-A and
MAO-B isoforms and all the compounds were found to be selective and reversible inhibitors of
MAO-B isoform. These compounds also displayed good AChE inhibition potential with IC50
values in low micro molar range. In the series, AVB4 was found to be the most potent MAO-B
inhibitor with IC50 value of 1.49 ± 0.09 µM and AVB1 was found to be the most potent AChE
inhibitor with IC value of 1.35 ±0.03 µM. In the ROS protection inhibition studies, AVB1 and
5
0
AVB4 displayed weak but interesting activity in SH-SY5Y human neuroblastoma cells. The
most active compounds were also tested for neuroprotective potential in SH-SY5Y cells and
showed moderate activity. In the cytotoxicity studies involving SH-SY5Y cells, both AVB1 and
AVB4 were found to be non-toxic to the tissue cells.
O
N
H CO
3
O
HO
N
H CO
3
N
O
O
N
Rivastigmine
Galantamine OCH3
Donepezil
HN
HN
N
O
O
Rasagiline
Ladostigil
Fig 1. Drugs in market and under clinical trials for Alzheimer’s disease
2
2
. Results and discussions
.1 Chemistry
All the compounds were synthesized by following the reaction procedures described in Scheme-
1
13
1
. All the final products were characterized by H NMR, C NMR, ESI-MS and HRMS.

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Synthetic scheme-1:
O
3
O
a)
O
O
HO
R
O
1
O
5
b)
c)
R1
O
O
H
R
a)
R
H
HO
O
N
N
2
4
R
O
O
AVB1-19
Reagents and conditions: a) Propargyl bromide (1.2 eq), K CO (2.4 eq), acetone, reflux, 12h; b) CH OH as
2
3
3
solvent, 20% NaOH aq., rt, stirring 1h; c) Amidines (1.2 eq), Na CO (2.4 eq), CH CN as solvent, reflux, 24h
2
3
3
2
2
.2 Biological results
.2.1 hMAO and AChE inhibition studies
MAO and AChE inhibition potential of the synthesized compounds (AVB1−AVB19) was
evaluated through fluorimetric method using Amplex Red assay kits purchased from the
Molecular Probes, Inc. /Invitrogen. The results of MAO and AChE inhibition studies are
expressed in the form of IC50 values (µM) described in Table 1. Both the phenyl rings of
synthesized diphenylpyrimidine derivatives were substituted with propargyl groups at
1 2 3 6 5
ortho/meta/para positions. Different amidines were used with R H so
as −NH , or −CH or −C
as to develop structure activity relationship profile of this series of compounds. Clorgyline,
pargyline, and donepezil were used as standard inhibitors for MAO-A, MAO-B, and AChE
enzymes, respectively. All of the synthesized compounds were found to be potent inhibitors of
MAO-B isoform (Selective over MAO-A) and AChE with IC values in low micromolar range.
5
0
Compound AVB4 was found to be the most potent MAO-B inhibitor with IC50 value of 1.49 ±
.09 µM exhibiting 17-folds selectivity over MAO-A. It also showed potent inhibition of AChE
with IC value of 1.74 ± 0.07 µM. In the reported series, AVB1 was found to be the most potent
0
5
0
AChE inhibitor with an IC value of 1.35 ±0.03 µM and displayed MAO-B inhibitory activity
5
0
with an IC value of 3.30 ± 1.17 µM. Unfortunately, as evident from Table 1, all the compounds
5
0
were found to be unselective or weakly selective for MAO-B isoform.

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Table 1. Results of MAO-A, MAO-B and AChE inhibition studies of the synthesized
compounds
R1
N
N
B
R
C
O
A
O
AVB1-AVB19
Entry
Name
Propargyl Propargyl
R
R1
IC50 values (mean ± S.E. µM)
MAO
at ring A
at ring C
Selectivity
hMAO-A
hMAO-B
eeAChE
(
SI)
AVB1
AVB2
AVB3
AVB4
AVB5
AVB6
AVB7
AVB8
AVB9
AVB10
AVB11
AVB12
AVB13
AVB14
AVB15
AVB16
AVB17
C4
C4
C4
C4
C4
C4
C4
C4
C4
C3
C3
C2
C2
C4
C4
C4
C4
C4
C4
C4
C3
C3
C3
C2
C2
C2
C3
C3
C2
C2
C4
C4
C4
C4
H
H
NH
2
32.06 ± 1.19
34.15 ± 2.03
3.30 ± 1.17
7.83 ± 0.57
1.35 ±0.03
1.76 ± 0.06
7.53 ± 0.23
1.74 ± 0.07
5.88 ± 0.07
4.39 ± 0.16
1.81 ± 0.02
2.56 ± 0.13
7.25 ± 0.16
6.99 ± 0.09
6.73 ± 0.18
5.19 ± 0.21
8.12 ± 0.19
4.58 ± 0.08
5.92 ± 0.18
6.99 ± 0.16
7.05 ± 0.14
9.7
4.4
2.1
16.1
2.6
1.5
1.3
1.3
1.5
≥ 3
≥ 3
≥ 5
≥ 4
≥ 3
1.2
1.6
≥ 4
CH
3
H
C
6
H
25.57 ± 1.18 12.26 ± 0.99
5
H
NH
2
24.06 ± 0.71
21.14 ± 1.40
1.49 ± 0.09
8.09 ± 0.54
H
CH
3
H
C
6
H
25.83 ± 1.92 17.69 ± 1.13
28.55 ± 0.77 22.12 ± 1.68
23.19 ± 1.87 18.07 ± 1.75
22.02 ± 1.03 14.44 ± 0.78
5
H
NH
2
H
CH
3
H
C H
6 5
H
NH
***
***
***
***
***
22.09 ± 0.94
23.44 ± 1.17
11.13 ± 0.16
13.78 ± 0.22
20.03 ± 1.18
2
H
6 5
C H
H
NH
2
H
C H
6 5
OCH
OCH
OCH
NH
3
3
3
2
CH
3
24.99 ± 2.13 21.08 ± 1.83
24.93 ± 1.17 15.71 ± 0.95
C H
6 5
OCH
CH
NH
***
14.01 ± 1.07
2
3
2

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AVB18
AVB19
C4
C4
C4
C4
OCH
OCH
2
2
CH
3
3
CH
3
***
***
10.97 ± 0.82
29.03 ± 1.33
nd
6.38 ± 0.09
6.94 ± 0.16
nd
≥ 5
≥ 2
CH
C H
6 5
Clorgyline
4.39 ± 1.02
nM
Pargyline
Donepezil
nd
nd
0.15 ± 0.02
nd
nd
0.03 ± 0.001
*
**Inactive or showed less than 50% inhibitory activity at 50 µM concentration and precipitated
nd
at higher concentrations. not determined, SI for MAO-B: IC MAO-A/ IC MAO-B
5
0
50
2
.2.2 Reversibility inhibition studies
To determine whether the synthesized compounds are reversible or irreversible inhibitors of the
target enzymes, the most active compounds i.e. AVB1 and AVB4 were subjected to reversibility
inhibition studies [15]. Both the compounds were found to be reversible inhibitors of MAO-B
and AChE enzymes. At the IC50 concentration AVB1 showed 31.8 % and 42.4 % residual
activity for MAO-B and AChE respectively. Similarly, AVB4 displayed 28.7 % and 41.7 %
residual activity for MAO-B and AChE respectively. Compound AVB1 showed recovery in the
enzyme activity by up to 65.04 % and 66.45 % for MAO-B and AChE respectively at 0.1 X IC50
concentration, while compound AVB4 showed recovery in the enzyme activity up to 60.4% and
8
2.78% for MAO-B and AChE respectively at 0.1 X IC50 concentration (Fig. 2). At 1 X IC50
concentration of compound AVB1, enzymatic activity upto 31.84% and 42.4% was recovered
for MAO-B and AChE respectively, while for AVB4, enzymatic activity upto 28.7% and 41.7%
was recovered for MAO-B and AChE respectively. Thus, it can be concluded that the
synthesized compounds are weak reversible inhibitors of both MAO-B and AChE enzymes at 1
X IC50 concentration of these inhibitors.

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Fig. 2- Reversibility inhibition studies of AVB1 and AVB4 with A) MAO-B enzyme and B)
AChE enzyme.
2
.2.3 Kinetic studies of AChE inhibition
To determine the mechanism of inhibition of AChE, kinetic study was carried out with the most
potent inhibitor of AChE (AVB4) using eeAChE. The reciprocal Lineweaver-Burk plots (Fig. 3)
m
illustrates the increased slope (decreased Vmax) and higher intercepts (K ) with the increasing
concentration of AVB4. The intersection point of the Lineweaver-Burk reciprocal plots was
located in the third quadrant, which indicate that AVB4 is mixed type inhibitor. AVB4 might be
binding to PAS and CAS of AChE simultaneously and inhibit the binding of substrate to active

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site. Binding of AVB4 with the PAS and CAS is further supported by docking and molecular
dynamics simulation studies.
Fig. 3- Kinetic study on the mechanism of eeAChE inhibition by AVB4. Overlaid
Lineweaver−Burk reciprocal plots of AChE initial velocity at increasing substrate concentration
(0.1−1 mM) in the absence or presence of AVB4 are shown.
2
.2.4 ROS inhibition studies
From literature survey, it is clear that MAO mediated metabolism of various monoamines leads
to the production of H O which further get converted into free radicals through Fenton’s
2
2
reaction and induces oxidative stress. Increased levels of these free radicals, initiate free radical-
mediated chain reactions that may cause oxidative damage to the cell membranes, DNA strand
breakdown and neuronal cell death. Thus, prevention of ROS generation along with MAO
inhibition is considered as an added advantage to prevent neurotoxicity in neurodegenerative
diseases. The most potent compounds in the series i.e. AVB1 and AVB4 were evaluated for their
ROS production inhibition potential against human neuroblastoma cells (SH-SY5Y) after 24h
interval treatment. For activity evaluation, 2,7-dichlorofluorescein diacetate (DCF-DA), a non-
fluorescent compound was used. DCF-DA in the presence of reactive oxygen species is oxidized
to 2,7-DCF which is a fluorescence emitting agent. Compound AVB1 reduced the ROS levels to
8
4
9.83% and 30.04 % at 1 µM and 5 µM concentrations, respectively in the SH-SY5Y cells (Fig.
). Similarly, compound AVB4 reduced the ROS levels up to 68.3% and 47.25 % at 1 µM and 5
µM concentrations, respectively in the SH-SY5Y cells. Thus, from these results, it is apparent

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that this series of compounds with two propargyl moieties may weakly protect the neuronal cells
from ROS.
Fig. 4- ROS inhibition studies of compound AVB1 and AVB4 on SH-SY5Y cells after treatment
for 24h.
2
.2.5 Neuroprotective effects
The most potent AChE and MAO-B inhibitors i.e AVB1 and AVB4 were evaluated for their
neuroprotective potential against 6-hydroxydopamine (6-OHDA) neurotoxin in SH-SY5Y cells.
These compounds showed mild neuroprotective potential against 6-OHDA. Maximum cell
viability of 66.6% was observed for compound AVB1 as compared to the 6-OHDA treated cells
(48.52%). As the concentration of the compounds increased from 5 µM to 25 µM, the cell
viability was further reduced as shown in Fig. 5.

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Fig. 5- Neuroprotective studies of AVB1 and AVB4 on SH-SY5Y cells.
2
.2.6 Metal chelation studies
Metal-chelating studies of AVB1 and AVB4 were performed with a UV−vis spectrophotometer.
The absorption spectra of both compounds (50 µM, final concentration) alone or in the presence
of CuSO , FeSO , and FeCl (50 µM, final concentration) were recorded. In metal chelating
4
4
3
studies, none of the compounds formed any metal chelate with any of the above metal salts.
Thus, these compounds are ineffective against metal salts and did not show any metal chelating
properties.
2
.2.7 Cytotoxicity studies
Cytotoxicity of the most potent inhibitors (AVB1 and AVB4) was determined against SH-SY5Y
cells using MTT assay. The test compounds at 1 µM, 5 µM and 25 µM concentrations were
incubated with SH-SY5Y cells and cell viability was determined after 24h time interval using
MTT assay. As depicted in Fig.6, the compounds were found non-toxic at the lower
concentrations. As the concentration of the compounds increased, there was a slight decrease in
the cell viability. Minimum cell viability of 69.17% was observed for AVB4 at 25 µM
concentration. At lower concentration of 1 µM, cell viability of 87.34% was observed as
compared to the control. Thus, keeping in view the IC values of the compounds which are in
5
0
low micromolar range, the current series of compounds were found to be nontoxic to the tissue
cells (Fig. 6).

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Fig. 6- Cytotoxicity studies of AVB1 and AVB4 on the SH-SY5Y after treatment for 24h.
2
.2.8 Molecular docking and simulation studies
Docking of the ligands and molecular dynamics simulations in the active site of the receptors
may provide crucial information about the orientations of the ligands and their interactions with
various amino acid residues at the receptor site. Most active compounds obtained in the series
were docked at the respective active sites of the receptor isoforms. Maestro 11.1 (Schrödinger
LLC) software was used for carrying out the docking studies and compounds AVB1 and AVB4
were docked at the hMAO-B (PDB ID-2BYB) [30] and AChE (PDB ID-1EVE) [31] and their
crystal structures were imported from the protein data bank. The docking procedures were first
validated by accurately redocking the co-crystallized ligands into the MAO models. The active
site of MAO-B is divided into two different compartments, an entrance cavity and a substrate
cavity separated by the side chain of Ile199 which serves as a “gate” between the two cavities.
The docking orientations of the compound AVB1 and AVB4 showed that the pyrimidine moiety
in both the compounds interact with the Ile199 and kept this residue in open gate conformation.
Phenyl ring with propargyl group at meta position in AVB4 is oriented towards FAD cofactor
and accommodated in the substrate cavity while the phenyl ring with para propargyl group is
aligned in the entrance cavity. In the enzyme-AVB1 complex, both the phenyl rings contain
propargyl groups at the para positions and one of the propargyl group is oriented towards the
FAD cofactor. The NH group of AVB1 showed hydrogen bonding with Phe168 and Cys172. In
2
the enzyme-AVB4 complex, pyrimidine ring remains close to the entrance cavity and NH group
2

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formed hydrogen bonds with Cys172 and Tyr188. The pyrimidine ring in AVB1 form π-π
stacking with Tyr326 while in AVB4 propargyl containing phenyl ring interacts with Tyr326 as
shown in Fig. 7. The inner phenyl ring of AVB4 in the substrate displayed π-π interactions with
Phe343. The increased MAO-B activity of AVB4 as compared to AVB1 might be due to the
additional π-π stacking of AVB4 with Tyr326 and Phe343.
Fig 7: A) Binding pattern (3D) of AVB1 with the amino acid residues at the active site of MAO-
B (2BYB), B) binding interactions (2D) of AVB1 with various amino acids of MAO-B active
cavity, C) Binding pattern (3D) of AVB4 with the amino acid residues at the active site of MAO-
B (2BYB), and D) binding interactions (2D) of AVB4 with various amino acids of MAO-B
active cavity.
Similarly, both of these compounds i.e. AVB1 and AVB4 were docked against AChE (PDB ID-
1
EVE co-crystallized with donepezil) to determine their binding alignment at the active site of
the enzyme. The active site of AChE is divided into two compartments; i) catalytic active site
(
CAS) composed of Trp84, Tyr130, Phe330, Phe331 residues and, ii) peripheral anionic site
PAS) which consists of amino acids-Tyr70, Asp72, Tyr121, Trp279, and Tyr334. The
(

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compounds were found to interact both with CAS as well as with PAS of AChE. Pyrimidine ring
in AVB1 and AVB4 accommodated in PAS and NH group formed hydrogen bonds with Asp72
2
(
Fig.8). In case of AVB1, one of the phenyl rings with a propargyl group is accommodated in the
CAS and showed π-π interactions with Trp84. In AVB4, phenyl ring with para propargyl group
is oriented towards PAS and displayed π-π stacking with Tyr334. An additional hydrogen bond
interaction of O-propargylated group with Phe288 in PAS was observed in AVB1 (Fig.8). Thus,
interactions of these compounds at CAS, as well as PAS, make them more effective against AD.
Fig 8: A) Binding pattern (3D) of AVB1 with the amino acid residues at the active site of AChE
(
1EVE), B) binding interactions (2D) of AVB1 with various amino acids of AChE active cavity,
C) Binding pattern (3D) of AVB4 with the amino acid residues at the active site of AChE
1EVE), and D) binding interactions (2D) of AVB4 with various amino acids of AChE active
cavity.
(
2
.2.9 Molecular dynamic simulation studies

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In order to investigate the binding stability of the potent inhibitors into the active site of the
MAO-B and AChE, molecular dynamic (MD) simulations studies were performed. For this
docking complexes of AVB1 and AVB4 with MAO-B and AChE were used. The MD
simulations studies were conducted for 30 ns and the interaction pattern of the test compounds
with different amino acids was analyzed. MD simulations studies of AVB4 with MAO-B yielded
the stable trajectory from 3 ns to 30 ns, oscillating in between 0.8 Å to 1.2 Å. For the initial 2 ns
time interval, a sharp hike was observed in RMSD from 0.4 Å to 1.6 Å, which stabilized to 1.2 Å
after 3 ns interval. The pyrimidine ring of AVB4 maintained its initial π-π interaction with
Tyr326 throughout MD time interval (Fig.9).
Fig 9: A) RMSD graph of MD simulations studies of AVB1 with MAO-B for 30 ns, B)
interactions of AVB1 with the active site residues of MAO-B during 30 ns MD simulations
studies, C) RMSD graph of MD simulations studies of AVB4 with MAO-B for 30 ns, and D)
interactions of AVB4 with the active site residues of MAO-B during 30 ns MD simulation
studies
Similarly, docking complexes of AVB1 and AVB4 with AChE were used for MD simulations to
determine their binding stability at the active site of the enzyme. In case of AVB4, a stable
trajectory was observed after the initial 3 ns interval which was determined through root-mean-
square-deviation (RMSD) of the protein backbone ranging from 0.8 Å to 1.2 Å (Fig.10). For the
first 3 ns time interval, a hike in RMSD trajectory was observed from 0.4 Å to 0.8 Å. In case of

ACCEPTED MANUSCRIPT
AVB1, RMSD kept oscillating from 0.4 Å to 1.2 Å throughout the MD simulations studies of 30
ns. Analyzing these MD trajectories of AChE-AVB1 and AChE-AVB4 complexes, it can be
concluded that these compounds (AVB1 and AVB4) had a strong tendency to be localized in the
binding region of AChE. One of the propargyl substituted ring of AVB1 maintained its initial π-
π interaction with Trp84. Similarly, for AVB4 the initial interactions such as hydrogen bond
formation of pyrimidine ring with Tyr121 and π-π interactions of pyrimidine ring with Trp84
were maintained throughout the MD simulations time interval (Fig. 10). Thus from these studies,
it can be concluded that MD simulations of AVB1 and AVB4 do not indicate any abrupt local
force that could potentially break up the inhibitors or can even delocalize these from the binding
site of AChE.
Fig 10: A) RMSD graph of MD simulations studies of AVB1 with AChE for 30 ns, B)
interactions of AVB1 with the active site residues of AChE during 30 ns MD simulation studies,
C) RMSD graph of MD simulations studies of AVB4 with AChE for 30 ns, and D) interactions
of AVB4 with the active site residues of AChE during 30 ns MD simulation studies
2
.2.10 Physicochemical properties
In order to assess the drug-like characteristics of the most active compounds (AVB1 and AVB4),
various physicochemical parameters of these compounds have been evaluated. Both the
compounds showed good drug-like properties and follow Lipinski’s rule of five. The logP value
of both the compounds is less than five and hydrogen bond donor and acceptor atoms are also

ACCEPTED MANUSCRIPT
less than five. Both the compounds showed 100% human oral absorption in Qikprop studies. The
QPlogBB values were also in the optimum range and hence these compounds are expected to
cross the blood-brain barrier. Thus, it can be concluded that both of these compounds have an
optimum chemical skeleton that can be developed as potential drug molecule.
Table 2. Physicochemical properties of some of the potent and selective MAO inhibitors.
Compound Mol.
Log
P
HB
HB
% human QPlogBB BBB
(Optimum permeability
absorption range -3.0 predicted
donor acceptor oral
number
Wt.
to 1.2)
AVB1
AVB4
355.39
355.39
4.52
4.51
3
3
4
4
100
100
-0.934
-0.93
+ve
+ve
+
ve = blood-brain barrier permeable; - ve = no permeability to the blood-brain barrier
2
.2.11 SAR studies
In the current studies, diphenylpyrimidines containing two propargyl groups were designed and
synthesized. A total of 19 compounds were synthesized and evaluated against MAO-A, MAO-B
and AChE enzymes. Most of the compounds of this series were found to be selective inhibitors
of MAO-B isoform and also displayed potent AChE inhibitory activities. As evident from Table
1
, AVB1 with propargyl groups at the para positions of the phenyl rings and a NH group on the
2
pyrimidine ring displayed IC values of 1.74 ± 0.07 µM and 1.35 ±0.03 µM for MAO-B and
5
0
AChE respectively. AVB1 was found to be the most potent AChE inhibitor in the reported series
of compounds. Replacement of NH with CH in AVB2 reduced MAO-B inhibitory activity by
2
3
2
more than 2-folds but retained AChE inhibition activity. Replacing NH with phenyl ring in
AVB3 further reduced the MAO-B inhibitory activity by almost 4-folds and AChE inhibition
activity by more than 5-folds. Shifting of one of the propargyl group from para (AVB1) to meta
position (AVB4) significantly increased MAO-B inhibitory activity by more than 2-folds and
retained AChE inhibition activity. AVB4 was found to be the most potent MAO-B inhibitor in
the series with an IC50 value of 1.49 ± 0.09 µM. Replacement of NH
2 3
group of AVB4 by CH
(AVB5) and phenyl (AVB6) groups reduced the MAO-B inhibitory activity by more than 5-folds

ACCEPTED MANUSCRIPT
and 12-folds respectively and AChE inhibitory activity was reduced by about 3-folds. Shifting of
propargyl group from meta (AVB4) to ortho position (AVB7 to AVB9) of the phenyl ring
proved detrimental and the MAO-B inhibitory activity was found to be reduced by more than 14-
folds. Similarly, reduced MAO-B inhibitory activity was observed when both the phenyl rings
were substituted by propargyl groups at the meta positions (AVB10 and AVB11) and at ortho
positions (AVB12 and AVB13). Presence of additional substituents at the phenyl ring (AVB14
to AVB19) was also found unfavorable for the MAO-B and AChE inhibitory activities.
3
. Conclusion
Multi-targeting agents can be an effective strategy for the treatment of multifactorial diseases
like AD. Multi-targeting ligands which can simultaneously target MAO and AChE enzymes are
being developed for the treatment of AD. In the current research work, a series of dipropargyl
substituted diphenylpyrimidines, have been synthesized and evaluated for MAO and AChE
inhibition activities. Most of the compounds were found to be selective inhibitors of MAO-B
isoform and potent AChE inhibitors. In the series, AVB1 and AVB4 were found to be the most
potent inhibitors of AChE and MAO-B with IC values of 1.35 ±0.03 µM and 1.49 ± 0.09 µM
5
0
respectively. In the reversible inhibition studies, the lead compounds were found to be reversible
inhibitors of MAO-B and AChE enzymes. In the ROS protection inhibition studies, AVB1 and
AVB4 displayed weak but interesting activity in SH-SY5Y cells and AVB1 reduced the ROS
levels up to 30 % at 5 µM. In the cytotoxicity studies involving SH-SY5Y cells, both AVB1 and
AVB4 were found to be non-toxic to the tissue cells. In the molecular dynamic simulation
studies, the lead compounds were found to be stable in the active site of MAO-B and AChE for
the time interval of 30 ns. The results suggested that AVB1 and AVB4 are promising dual
inhibitors and have the potential to be developed as anti-Alzheimer’s drug.
4
. Experimental
4
.1 Material and methods
All the reagents or chemicals used for the synthesis of intermediates or target compounds were
purchased from Avra synthesis, Sigma-Aldrich, Loba-Chemie Pvt. Ltd. and S.D. Fine Chemicals
and are used without further purification. Thin layer chromatography was done on glass silica

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plates with silica gel GF254 as the adsorbent. Ethyl acetate: petroleum ether (1:1, 1:2 and 1:3)
mixtures were used as a solvent system for the chromatographic purification of compounds using
column chromatography. Mass spectra were recorded on GC-MS (ESI), Central Instruments
1
13
Laboratory (CIL), Central University of Punjab, Bathinda. The H and C NMR of the
compounds were recorded on JEOL or Bruker Advance II instrument at 400 MHz frequency, in
3
CDCl or DMSO and TMS (δ=0) as an internal standard at IIT Ropar, and GJUS&T, Hisar. The
chemical shifts are reported in parts per million (δ) downfield from the signal of
tetramethylsilane added to the deuterated solvent. Melting points were recorded with Stuart
SMP30 melting point apparatus and are uncorrected. For MAO inhibition studies and
acetylcholinesterase inhibition studies, Amplex® Red MAO kit was purchased from Molecular
Probes (Invitrogen), Life technologies, India. Recombinant hMAO-A and hMAO-B enzymes
were purchased from Sigma-Aldrich. For absorption studies, UV-VIS spectrophotometer of
Shimadzu was used. Fluorescence studies were recorded using Biotek Microplate reader.
Molecular modelling studies were carried out using Maestro 11.1 (Schrödinger LLC) and
ChemBio Draw Ultra-12 installed on operating system centos 6.5 at HP-2800 workstation with
configuration of Intel (R) Xenon (R) X5660 @2.80 GHz, 2.789 GHz (2 processors).
4
4
.2 Chemistry
.2.1 General procedure for the synthesis of O-propargylated derivatives 3 and 4
To hydroxy substituted acetophenones (0.5g) or benzaldehydes (0.5g), Propargyl bromide
1.2eq) was added in the presence of potassium carbonate as a base (2.4eq.) and acetone (30 ml)
(
as solvent. The reaction mixture was refluxed overnight at 60˚ C. Progress of the reaction was
monitored via TLC. After completion of the reaction, excess of solvent was evaporated from the
mixture using vacuum rotary evaporator, water (10 mL) was added and aqueous phase was
extracted with ethyl acetate (10 mL × 3), and washed with brine, dried over anhydrous Na SO
2
4
and the organic solvent was concentrated under vacuum using rotary evaporator to obtain 3 and
4
.
4
.2.2 General procedure for the synthesis of 5
To a mixture of 3 (1 eq.) and 4 (1 eq.) in methanol (20 ml), aqueous sodium hydroxide (20 %)
was added slowly with continuous stirring. The reaction mixture was stirred for 3 h at room
temperature. The completion of reaction was monitored via TLC. After completion of reaction,

ACCEPTED MANUSCRIPT
excess of solvent was evaporated from the mixture using rotary evaporator. Chilled water was
poured into the reaction mixture and precipitates of 5 were filtered and dried. Further crude
product was recrystallized from ethanol or purified using column chromatography as per
requirement.
4
.2.3 General procedure for the synthesis of AVB1-19
To a mixture of 5 (500 mg), and amidine (1.2 eq.), anhydrous sodium carbonate (2.4 eq.) was
added in acetonitrile (10 mL) as solvent. The reaction mixture was refluxed for 24 h at 85˚ C.
The progress of the reaction was monitored via TLC. After completion of the reaction, excess of
solvent was evaporated under vacuum using rotary evaporator, and water (10 mL) was added
which was extracted with ethyl acetate (3×10 mL), washed with brine, dried over anhydrous
2 4
Na SO , and the organic solvent was concentrated under vacuum using rotary evaporator and
purified via column chromatography (EtOAc:Pet ether). The final products were characterized
by NMR spectroscopy and HRMS.
4
,6-bis(4-(prop-2-yn-1-yloxy)phenyl)pyrimidin-2-amine (AVB1): Yield: 55%; yellow solid,
1
mp 139-141 °C; H NMR (CDCl
3
, 400 MHz, δ with TMS = 0): 8.02 (4H, d,
= 8Hz), 5.17 (2H, s), 4.76 (4H, d, = 4Hz), 2.54 (2H, t,
CDCl , 100 MHz, δ with TMS = 0) δ: 165.40, 163.57, 159.49, 131.16, 128.66, 115.25, 103.02,
J = 8Hz), 7.34 (1H,
1
3
s), 7.06 (4H, d,
J
J
J = 4Hz), C NMR
(
3
+
+
7
3
17 3 2
6.81, 75.98, 55.92, HRMS: for C22H N O , calculated [M+H] : 356.1399; observed [M+H] :
56.1368
2
-methyl-4,6-bis(4-(prop-2-yn-1-yloxy)phenyl)pyrimidine (AVB2): Yield: 60%; blue solid,
1
mp 158-160 °C; H NMR (CDCl , 400 MHz, δ with TMS = 0): 8.10 (4H, d,
J
= 8Hz), 7.76 (1H,
3
1
3
s), 7.10 (4H, d,
J = 8Hz), 4.76 (4H, d, J = 4Hz), 2.81 (3H, s), 2.55 (2H, t, J = 4Hz), C NMR
(
CDCl , 100 MHz, δ with TMS = 0) δ: 168.34, 164.00, 159.59, 130.92, 128.71, 115.21, 108.55,
3
+
7
8.15, 75.90, 55.89, 26.49, HRMS: for C H N O , calculated [M+H] : 355.1447; observed
2
3
18
2
2
+
[
M+H] : 355.1402
2
-phenyl-4,6-bis(4-(prop-2-yn-1-yloxy)phenyl)pyrimidine (AVB3): Yield: 65%; greenish
1
solid, mp 148-150 °C; H NMR (CDCl , 400MHz, δ with TMS=0) 8.73 (2H, d,
J
J
= 8Hz), 8.30
= 4Hz), 2.59
3
(
4H, d,
2H, t,
J = 8Hz), 7.92 (1H, s), 7.55 (3H, m), 7.18 (4H, d, J = 8Hz), 4.82 (4H, d,
1
3
(
J
3
= 4Hz), C NMR (CDCl , 100 MHz, δ with TMS=0): 163.90, 159.70, 138.32, 130.98,

ACCEPTED MANUSCRIPT
1
20 2 2
30.51, 128.75, 128.40, 115.17, 108.78, 78.17, 75.92, 55.90, HRMS: for C28H N O , calculated
+
+
[
M+H] : 417.1603; observed [M+H] : 417.1549
4
-(3-(prop-2-yn-1-yloxy)phenyl)-6-(4-(prop-2-yn-1-yloxy)phenyl)pyrimidin-2-amine
1
(
AVB4): Yield: 65%; white solid, mp 139-141 °C; H NMR (CDCl , 400 MHz, δ with TMS =
3
0
): 8.02 (2H, d,
m), 5.25 (2H, s), 4.75 (4H, d,
with TMS = 0) δ: 165.66, 163.57, 159.59, 158.02, 139.47, 130.98, 129.90, 128.69, 120.45,
17.15, 115.11, 113.52 103.82, 78.49, 75.81, 56.04, 55.93, HRMS: for C22 , calculated
J
= 8Hz), 7.69-7.63 (2H, m), 7.42 (1H, d,
J = 8Hz),7.37 (1H, s), 7.11 - 7.04 (3H,
1
3
J
= 4 Hz), 2.54 (2H, t, = 4 Hz), C NMR (CDCl , 100 MHz, δ
J
3
1
17 3 2
H N O
+
+
[
M+H] : 356.1399; observed [M+H] : 356.1350
2
-methyl-4-(3-(prop-2-yn-1-yloxy)phenyl)-6-(4-(prop-2-yn-1-yloxy)phenyl)pyrimidine
1
(
AVB5): Yield: 63%; white solid, mp 116-118 °C; H NMR (CDCl , 400MHz, δ with TMS=0)
3
8
4
.15 (2H, d,
.83 (4H, d,
J
J
= 8Hz), 7.83 (1H, s), 7.78 (2H, d,
= 4Hz), 2.86(3H, s), 2.58 (2H, t,
J
= 8Hz), 7.47 (1H, t,
J
= 8Hz), 7.15 (3H, m),
1
3
J
= 4Hz), C NMR (CDCl3, 100 MHz, δ with
TMS=0): 164.27, 158.10, 139.22, 129.98, 128.75, 120.49, 117.14, 115.24, 113.73, 109.44, 78.37,
+
7
8.19, 75.92, 75.72, 56.04, 55.88, 26.48, HRMS: for C23
H
18
N
2
O
2
, calculated [M+H] : 355.1447;
+
observed [M+H] : 355.1390
2
-phenyl-4-(3-(prop-2-yn-1-yloxy)phenyl)-6-(4-(prop-2-yn-1-yloxy)phenyl)pyrimidine
1
(
AVB6): Yield: 60%; white solid, mp 147-148 °C; H NMR (CDCl , 400 MHz, δ with TMS =
3
0
): 8.72-8.70 (2H, m), 8.2 (2H, d,
8Hz), 4.83 (4H, d, = 2.4 Hz), 2.59 (2H, m); C NMR (CDCl , 100 MHz, δ with TMS = 0) δ:
64.38, 164.18, 164.11, 159.84, 158.18, 139.28, 138.20, 130.82, 130.72, 130.02, 128.87, 128.52,
20 2 2
20.50, 117.18, 115.24, 113.96, 109.70, 78.50, 76.06, 56.13, 55.95, , HRMS: for C28H N O ,
J = 8 Hz), 7.97-7.85 (3H, m), 7.54-7.48 (4H, m), 7.15 (3H, d, J
1
3
=
J
3
1
1
+
+
calculated [M+H] : 417.1603; observed [M+H] : 417.1553
4
-(2-(prop-2-yn-1-yloxy)phenyl)-6-(4-(prop-2-yn-1-yloxy)phenyl)pyrimidin-2-amine
1
(
AVB7): Yield: 64%; green solid, mp 134-136 °C; H NMR (CDCl , 400MHz, δ with TMS=0)
3
8
.07 (2H, d,
J
= 8Hz), 7.90 (1H, d,
J
= 8Hz), 7.66 (1H, s), 7.45 (1H, t,
J
= 8Hz), 7.18 (2H, d, J
1
3
=
8Hz), 7.09 (2H, d,
J
=8Hz), 5.15 (2H, bs), 4.79 (4H, t,
J
= 4Hz), 2.57 (2H, t, J = 4Hz), C
NMR (CDCl , 100 MHz, δ with TMS=0): 164.54, 164.29, 163.34, 159.39, 155.67, 131.21,
3
1
30.87, 128.72, 128.21, 122.15, 114.95, 113.54, 108.57, 78.46, 78.23, , 76.70, 75.83, 56.66,

ACCEPTED MANUSCRIPT
+
+
5
3
5.85, 29.70, HRMS: for C22
H
17
N
3
O
2
, calculated [M+H] : 356.1399; observed [M+H] :
56.1372
2
-methyl-4-(2-(prop-2-yn-1-yloxy)phenyl)-6-(4-(prop-2-yn-1-yloxy)phenyl)pyrimidine
1
(
AVB8): Yield: 58%; green solid, mp 132-134 °C; H NMR (CDCl , 400MHz, δ with TMS=0)
3
8
4
1
1
.13 (3H, t,
J
= 8Hz), 7.99 (1H, d,
J = 8Hz), 7.47 (1H, t, J = 8Hz),7.17 (4H, m), 4.80 (4H, d, J =
1
3
Hz), 2.86 (3H, s), 2.58 (2H, t ,
J
= 4Hz), C NMR (CDCl3, 100 MHz, δ with TMS=0): 168.04,
63.14, 162.88, 159.49, 155.73, 131.22, 131.12, 131.08, 130.77, 128.85, 127.88, 122.30, 115.12,
14.64, 114.25, 113.34, 78.37, 78.19, 75.91, 75.86, 56.62, 55.86, 26.53, HRMS: for
+
+
C H N O , calculated [M+H] : 355.1447; observed [M+H] : 355.1405
23
18
2
2
2
-phenyl-4-(2-(prop-2-yn-1-yloxy)phenyl)-6-(4-(prop-2-yn-1-yloxy)phenyl)pyrimidine
1
(
AVB9): Yield: 65%; cream solid, mp 89-91 °C; H NMR (CDCl , 400 MHz, δ with TMS = 0):
3
8
.67 (2H, dd, J13 = 8 Hz; J12 = 4 Hz), 8.29-8.25 (4H, m), 7.52 - 7.46 (4H, m), 7.24 - 7.19 (1H,
1
3
m), 7.15-7.11 (3H, m); 4.77 (4H, d,
J = 4 Hz), 2.54 (2H, t , J = 4Hz), C NMR (CDCl , 100
3
MHz, δ with TMS = 0) δ: 164.18, 163.16, 162.83, 159.66, 156.18, 138.51, 131.61, 131.22,
30.47, 128.98, 128.48, 127.86 122.34, 115.14, 114.86, 113.49, 78.48, 76.82, 56.73, 55.94,
1
+
+
HRMS: for C H N O , calculated [M+H] : 417.1603; observed [M+H] : 417.1555
2
8
20
2
2
4
,6-bis(3-(prop-2-yn-1-yloxy)phenyl)pyrimidin-2-amine (AVB10): Yield: 57%; yellowish
1
brown, mp 146-148 °C; H NMR (CDCl , 400 MHz, δ with TMS = 0): 7.72 (2H, s), 7.69 (2H, d,
3
J
= 8Hz), 7.45 (3H, t,
J
= 8Hz), 7.14 (2H, d,
J = 8Hz), 5.21 (2H, bs), 4.81 (4H, d, J = 4 Hz), 2.58
1
3
(
2H, bs), C NMR (CDCl , 100 MHz, δ with TMS = 0) δ: 165.84, 163.50, 157.98, 139.23,
3
1
29.86, 120.39, 117.22, 113.46, 104.53, 78.40, 75.74, 55.99, 29.86, HRMS: for C H N O ,
22 17 3 2
+
+
calculated [M+H] : 356.1399; observed [M+H] : 356.1358
2
-phenyl-4,6-bis(3-(prop-2-yn-1-yloxy)phenyl)pyrimidine (AVB11): Yield: 59%; greenish
1
yellow solid, mp 118-120 °C; H NMR (CDCl
3
, 400MHz, δ with TMS=0) 8.75 (2H, d,
J =
8
7
Hz), 8.45 (1H, s) 8.01 (2H, d,
J
= 8Hz), 7.59 (1H, s), 7.57 (2H, d,
J
= 8Hz), 7.50-7.59 (4H, m),
1
3
.20 (2H, d, = 8Hz), 4.86 (4H, d,
J
J
= 4Hz), 2.62 (2H, t, = 4Hz), C NMR (CDCl3, 100
J
MHz, δ with TMS=0): 164.43, 164.35, 158.15, 139.05, 137.98, 130.74, 129.98, 128.48, 120.48,
+
1
4
17.28, 113.94, 110.51, 78.40, 75.86, 56.08, 29.71, HRMS: for C H N O , calculated [M+H] :
2
8
20
2
2
+
17.1603; observed [M+H] : 417.1602

ACCEPTED MANUSCRIPT
4
,6-bis(2-(prop-2-yn-1-yloxy)phenyl)pyrimidin-2-amine (AVB12): Yield: 64%; semi-solid;
1
H NMR (CDCl , 400MHz, δ with TMS=0) 7.83 (2H, d,
J = 8Hz), 7.68 (1H, s), 7.44 (2H, t, J =
3
1
3
8
1
1
Hz), 7.13-7.19 (4H, m), 5.47 (2H, bs), 4.80 (4H, d, J = 4Hz), 2.53 (2H, bs), C NMR (CDCl3,
00 MHz, δ with TMS=0): 163.79, 163.19, 155.55, 131.04, 130.80, 128.25, 122.00, 113.73,
+
13.56, 78.58, 75.86, 56.47, 29.72, HRMS: for C H N O , calculated [M+H] : 356.1399;
2
2
17
3
2
+
observed [M+H] : 356.1368
2
-phenyl-4,6-bis(2-(prop-2-yn-1-yloxy)phenyl)pyrimidine (AVB13): Yield: 68%; semi-solid;
1
H NMR (CDCl
Hz), 7.49-7.54 (5H, m), 7.22-7.27 (4H, m), 4.87 (4H, d,
CDCl3, 100 MHz, δ with TMS=0): 164.08, 162.32, 156.01, 138.53, 131.70, 131.10, 130.27,
28.38, 128.29, 128.22, 122.15, 120.46, 113.56, 78.57, 75.95, 56.48, 29.74, HRMS: for
3
, 400MHz, δ with TMS=0) 8.69 (2H, d, J = 8Hz), 8.49 (1H, s), 8.24 (2H, d, J =
1
3
8
J = 4Hz), 2.55 (2H, bs), C NMR
(
1
+
+
C H N O , calculated [M+H] : 417.1603; observed [M+H] : 417.1552
28
20
2
2
4
-(3-methoxy-4-(prop-2-yn-1-yloxy)phenyl)-6-(4-(prop-2-yn-1-yloxy)phenyl)pyrimidin-2-
1
amine (AVB14): Yield: 60%; light brown solid, mp 118-120 °C; H NMR (CDCl , 400 MHz, δ
3
with TMS = 0): 8.04 (2H, d,
J
= 8Hz), 7.69 (1H, d,
= 4Hz), 4.76 (2H, d,
2H, t, J = 4Hz), C NMR (CDCl , 100 MHz, δ with TMS = 0) δ: 165.41, 163.47, 159.49,
J
= 8Hz), 7.60-7.58 (1H, m), 7.35 (1H, s),
7
.12-7.06 (3H, m), 5.29 (2H, S), 4.83 (2H, d,
J
J
= 4Hz), 3.99 (3H, s), 2.55
1
3
(
3
1
7
49.78, 148.82, 131.73, 131.01, 128.63, 119.84, 115.04, 113.65, 110.48, 103.17, 78.19, 76.73,
+
5.09, 56.68, 56.06, 55.87, HRMS: for C H N O , calculated [M+H] : 386.1505; observed
2
3
19
3
3
+
[
M+H] : 386.1508
4
-(3-methoxy-4-(prop-2-yn-1-yloxy)phenyl)-2-methyl-6-(4-(prop-2-yn-1-
1
3
yloxy)phenyl)pyrimidine (AVB15): Yield: 60%; white solid, mp 132-134 °C; H NMR (CDCl ,
4
00 MHz, δ with TMS = 0): 8.10 (2H, d,
Hz), 7.15 – 7.09 (3H, m), 4.84 (2H, d, = 4Hz), 4.77 (2H, d,
= 4Hz), C NMR (CDCl , 100 MHz, δ with TMS = 0) δ: 168.34, 164.05,
J
= 8H), 7.77 (2H, s), 7.66 (1H, dd, J13 = 8Hz, J12
=
4
J
J
= 4Hz), 4.01 (3H, s), 2.82 (3H,
1
3
s), 2.56-2.53 (2H, t,
J
3
1
7
64.03, 157.60, 149.97, 148.97, 131.62, 130.88, 128.72, 119.93, 115.22, 113.83, 110.56, 108.74,
8.16 78.14, 76.18, 75.92, 56.71, 56.12, 55.80, 26.50, HRMS: for C24
H
20
N
2
O
3
, calculated
+
+
[
M+H] : 385.1552; observed [M+H] : 386.1555
4
-(3-methoxy-4-(prop-2-yn-1-yloxy)phenyl)-2-phenyl-6-(4-(prop-2-yn-1-
1
yloxy)phenyl)pyrimidine (AVB16): Yield: 70%; greenish white solid, mp 160-162 °C; H

ACCEPTED MANUSCRIPT
NMR (CDCl
3
and DMSO, 400 MHz, δ with TMS = 0): 8.64 (2H, d,
J
= 8Hz), 8.45 (2H, d,
= 4 Hz),
.00 (3H, s), 3.36 (2H, t, J = 4Hz), C NMR (CDCl and DMSO, 100 MHz, δ with TMS = 0) δ:
J = 8
Hz), 8.29 (1H, s), 8.05 - 8.01 (2H, m), 7.58 - 7.52 (3H, m), 7.18 (3H, m), 4.88 (4H, d,
J
1
3
4
3
1
63.39, 163.31, 163.03, 159.47, 149.32, 148.96, 137.74, 130.39, 130.37, 129.81, 128.76, 128.36,
27.86, 120.19, 114.85, 113.59, 110.62, 108.89, 79.88, 78.67, 78.58, 77.84, 56.11, 55.72, 55.55,
1
+
+
22 2 3
HRMS: for C29H N O , calculated [M+H] : 447.1709; observed [M+H] : 447.1709
4
-(3-ethoxy-4-(prop-2-yn-1-yloxy)phenyl)-6-(4-(prop-2-yn-1-yloxy)phenyl)pyrimidin-2-
1
amine (AVB17): Yield: 65%; pale yellow solid, mp 162-164 °C; H NMR (CDCl
with TMS=0) 8.05 (2H, d, = 8Hz), 7.71 (1H, s), 7.62 (1H, d, = 8Hz), 7.38 (1H, s), 7.16 (1H,
= 8Hz), 5.21 (2H, bs), 4.87 (2H, d, = 4Hz), 4.80 (2H, d, = 4Hz),
= 4Hz), C NMR (CDCl3, 100 MHz, δ with
3
, 400MHz, δ
J
J
d,
J
= 8Hz), 7.13 (2H, d,
J
J
J
1
3
4
.26 (2H, q,
J
= 8Hz), 2.57 (2H, m), 1.53 (3H, t, J
TMS=0): 165.51, 165.36, 163.42, 159.47, 149.27, 149.12, 131.83, 131.05, 128.60, 119.83,
1
1
15.03, 114.42, 112.00, 103.20, 78.43, 78.17, 75.99, 75.66, 64.64, 56.85, 55.87, 30.27, 29.67,
+
+
4.86, HRMS: for C H N O , calculatSed [M+H] : 400.1661; observed [M+H] : 400.1665
2
4
21
3
3
4
-(3-ethoxy-4-(prop-2-yn-1-yloxy)phenyl)-2-methyl-6-(4-(prop-2-yn-1-
1
yloxy)phenyl)pyrimidine (AVB18): Yield: 60%; greenish yellow solid, mp 108-110 °C; H
NMR (CDCl , 400MHz, δ with TMS=0) 8.13 (2H, d, = 8Hz), 7.79 (2H, s), 7.67 (1H, d,
= 8Hz), 4.87 (2H, d, = 4Hz), 4.80 (2H, d, = 4Hz),
= 4Hz), 2.85 (3H, s), 2.57 (2H, m), 1.56 (3H, t, = 4Hz), C NMR (CDCl3, 100
J
J =
3
4
4
Hz), 7.17 (1H, d,
.28 (2H, q,
J
= 4Hz), 7.13 (2H, d,
J
J
J
1
3
J
J
MHz, δ with TMS=0): 168.33, 164.13, 164.01, 159.59, 149.45, 149.27, 131.68, 130.91, 128.72,
1
1
19.94, 115.22, 114.56, 112.09, 108.78, 78.39, 78.14, 76.05, 75.91, 64.71, 56.85, 55.89, 26.50,
+
+
4.86, HRMS: for C H N O , calculated [M+H] : 399.1709; observed [M+H] : 399.1709
2
5
22
2
3
4
-(3-ethoxy-4-(prop-2-yn-1-yloxy)phenyl)-2-phenyl-6-(4-(prop-2-yn-1-
1
yloxy)phenyl)pyrimidine (AVB19): Yield: 67%; greenish yellow solid, mp 138-140 °C; H
NMR (CDCl , 400MHz, δ with TMS=0) 8.72 (2H, d, = 8Hz), 8.30 (2H, d, = 8Hz), 7.97 (1H,
= 8Hz), 7.57 (3H, m), 7.21 (1H, d, = 8Hz), 7.19 (2H, d,
= 4Hz), 4.83 (2H, d, = 4Hz), 4.32 (2H, q, = 12Hz), 2.65 (2H, dd,
J
J
3
d,
J
= 4Hz), 7.91 (1H, s), 7.82 (1H, d,
J
J
J
4
1
1
= 8Hz), 4.90 (2H, d,
J
J
J
J =
1
3
Hz), 1.65 (3H, m) C NMR (CDCl3, 100 MHz, δ with TMS=0): 164.06, 159.72, 149.44,
49.41, 138.29, 131.75, 130.94, 130.54, 128.76, 128.42, 128.39, 120.03, 115.18, 114.52, 112.27,
09.02, 78.40, 78.16, 76.07, 75.93, 64.79, 56.86, 55.90, 14.88, HRMS: for C H N O ,
3
0
24
2
3
+
+
calculated [M+H] : 461.1865; observed [M+H] : 461.1866

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4
4
.3 Biological studies
.3.1 Determination of hMAO inhibition activity
The effects of the test compounds on hMAO-A and hMAO-B enzymes were evaluated by using
Amplex® Red assay kit based on detection of fluorescence [32, 33].
Briefly, 100 µL of sodium phosphate buffer (0.05 M, pH 7.4) containing the test drugs or
reference inhibitors, in various concentrations along with adequate amounts of recombinant
hMAO (hMAO-A: 1.1 µg protein; specific activity: 150 nmol of p-tyramine oxidized to p-
hydroxyphenylacetaldehyde/min/mg protein; hMAO-B: 7.5 µg protein; specific activity: 22 nmol
of p-tyramine transformed/min/mg protein) enzyme, were incubated for 30 min at 37 °C in a flat-
black-bottom 96-well plate (Tarsons) in incubator. After this incubation period, the reaction was
started by adding (final concentrations) 200 µM Amplex® Red reagent, 1 U/mL horseradish
2 2
peroxidase and 1 mM p-tyramine. After 30 min incubation in the dark, the production of H O
HI
was quantified at 37 °C in a multi-detection microplate fluorescence reader (Synergy , Bio-
Tek® Instruments) based on the fluorescence generated at the excitation wavelength of 545 nm,
and emission wavelength of 590 nm. Control experiments were carried out simultaneously by
replacing the test drugs with the vehicle. To minimize the possibility of any interference of test
drugs to the non-enzymatic fluorescence generated through drug and Amplex® Red reagent
interactions blank reading was taken with drug and Amplex® Red reagent without adding MAO
enzyme in a sodium phosphate buffer. No fluorescence could be observed in the absence of
MAO enzyme thus eliminating the possibility of any false reading. The specific final
fluorescence emission was calculated after subtraction of the background activity, determined
from vials containing all components except the hMAO enzyme replaced by a sodium phosphate
buffer solution.
4
.3.2 Acetylcholinesterase inhibition assay
Acetylcholinesterase inhibition activity
was
determined
using
Amplex
Red
Acetylcholine/Acetylcholinesterase assay kit (A12217) purchased from the Molecular Probes
Inc. Invitrogen [15, 34]. Briefly, 100 µl of Tris-HCl buffer (0.05 M, pH 8.0) containing the
synthesized test drugs and reference inhibitors, in various concentrations along with adequate
amounts of recombinant AChE (0.2U/mL) enzyme, were incubated for 15 min at 37 °C in a flat
bottom 96-well plates (Tarsons). Reaction was started by adding 100 µl of working solution of

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4
00 µM Amplex Red reagent containing 2 U/mL horseradish peroxidase, 0.2 U/mL choline
oxidase and 100 µM acetylcholine. After 30 min incubation in dark, the production of H O and
2
2
subsequent formation of resorufin from Amplex red dye was quantified at 37 °C in a multi-
HI
detection microplate fluorescence reader (Synergy , Bio-Tek® Instruments) based on the
fluorescence generated at excitation wavelength of 545 nm, and emission wavelength of 590 nm.
Positive control experiment was carried out simultaneously by replacing the test drugs with the
vehicle. Second positive control was carried out by using 20 mM H O . 1X reaction buffer
2
2
without acetylcholinesterase was used as negative control. The specific final fluorescence
emission was calculated after subtraction of the background activity, determined from wells
containing all components except the AChE replaced by a 1X buffer solution. Each experiment
was performed in triplicate (n=3).
4
.3.3 Reversibility studies
For reversibility studies, dilution protocol was adopted as reported in the literature [35, 36].
Briefly, the test inhibitors were incubated with the MAO enzyme at concentrations of 10 × IC50
and 100 × IC50 at 37 °C for of 30 min (negative control performed in the absence of inhibitor),
and 4% DMSO was added as co-solvent to all incubations. After 30 min incubation period, the
samples were subsequently diluted to 100–fold with the addition of tyramine substrate to achieve
final inhibitor concentrations of 0.1 × IC50 and 1 x IC50 value, respectively. As positive control
MAO-B was incubated with the irreversible inhibitor pargyline at 10 × IC concentrations and
50
then diluted 100-fold to achieve final inhibitor concentrations of 0.1 × IC50. The residual MAO-B
activity was measured (n=3) after dilutions and enzyme activities were expressed as mean ± SD.
4
.3.4 Kinetic studies
To determine the mechanism of action of the most potent inhibitor of AChE i.e. AVB4, kinetic
study was performed using eeAChE with the help of earlier reported protocols [18, 37].
Lineweaver-Burk double reciprocal graph was plotted at different concentrations of substrate
ACh (0.1 mM- 1 mM) by using same methodology reported for the in vitro inhibition study of
AChE. Progress curves were analyzed by steady-state turnover of substrate and values of linear
regression were fitted according to Lineweaver-Burk replots using excel software (2016). Three
concentrations of AVB4 (1 µM, 2 µM and 5 µM) were used for the kinetic study. The plots were
assessed by weighted least-square analysis that assumed the variance of ν to be constant for

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whole data set. Slopes of the reciprocal of ν were then plotted against the reciprocal of the
substrate concentration.
4
.3.5 Cell culture and treatment
The human neuroblastoma (SH-SY5Y) cells were obtained from National Centre for Cell
Science (Pune, India). The cells were routinely grown in DMEM supplemented with 10% FBS
(Biological Industries) and 1XxPSN mix (Invitrogen) at 37°C in a humidified atmosphere
containing 5% CO . Cells were subcultured by trypsinization and seeded in 96 and 24 well plates
2
according to the requirement of the experiments.
4
.3.6 ROS inhibition studies
Intracellular levels of ROS were determined by using the protocol described elsewhere [15],
using non-fluorescent compound 2,7-dichlorofluorescein diacetate (DCF-DA) that is permeable
to the cell membrane where it is hydrolyzed by intracellular esterases and oxidized by ROS to a
fluorescent compound 2,7-DCF. The human neuroblastoma (SH-SY5Y) cells (approximately
1
0,000 cells/well) were seeded of 96 well plate in DMEM/F-12 media containing 10% FBS and
horse serum supplemented 1% penicillin antibiotic solution for 24h in a humidified atmosphere
containing 5% CO . The media was removed, washed with PBS and cells were treated with test
2
compounds (without FBS) for 24h at different concentrations (1 µM, 5 µM and 25 µM).
Thereafter, cells were rinsed with PBS three times and treated with H DCF-DA (50µM) and
2
incubated for 30 min at 37 ºC. Following incubation, cells were rinsed with PBS and
fluorescence was detected at a wavelength of 478 nm excitation and 518 nm emission.
4
.3.7 Neuroprotective studies
The neuroprotective potential of compounds was determined against 6-OHDA neurotoxin using
4
MTT assay [38]. For this SH-SY5Y cells were plated in 96 wells, at 10 cells/well density. The
cells were cultured for 24h in DMEM/F-12 media containing 10% FBS and horse serum
supplemented 1% penicillin antibiotic solution. Then cells were treated with the target
compounds (at concentrations of 1-25 µM), 4h before 6-OHDA (10 µM). After 24 h incubation
in the oven, at 37 °C and a 5% CO , 95% O atmosphere, the tested compounds were replaced
2
2
with 100 µL of MTT in media (5 mg/mL, final concentration). The cells were incubated for
another 3h. After the removal of MTT, the formazan crystals were dissolved in DMSO. The