4,6-Diphenylpyrimidine Derivatives as Dual Inhibitors of Monoamine Oxidase and Acetylcholinesterase for the Treatment of Alzheimer's Disease

Article abstract of DOI:10.1021/acschemneuro.8b00220

Alzheimer's disease (AD) is a neurodegenerative disorder with multifactorial pathogenesis. Monoamine oxidase (MAO) and acetylcholinesterase enzymes (AChE) are potential targets for the treatment of AD. A total of 15 new propargyl containing 4,6-diphenylpy

Full text of DOI:10.1021/acschemneuro.8b00220

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Article
4,6-Diphenylpyrimidine Derivatives as Dual Inhibitors of Monoamine
Oxidase and Acetylcholinesterase for the Treatment of Alzheimer’s Disease
Bhupinder Kumar, Ashish Ranjan Dwivedi, Bibekananda Sarkar, Sukesh Kumar
Gupta, Sairam Krishnamurthy, Anil K Mantha, Jyoti Prakash, and Vinod Kumar
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00220 • Publication Date (Web): 08 Oct 2018
Downloaded from http://pubs.acs.org on October 8, 2018
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4
,6ꢀDiphenylpyrimidine Derivatives as Dual Inhibitors of Monoamine Oxidase and
Acetylcholinesterase for the Treatment of Alzheimer’s Disease
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1
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3
Bhupinder Kumar , Ashish Ranjan Dwivedi , Bibekananda Sarkar , Sukesh Kumar Gupta ,
Sairam Krishnamurthy Anil K Mantha , Jyoti Prakash , and Vinod Kumar
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1*
,
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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-151001.
3
Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology
(Banaras Hindu University), Varanasi, India-221005.
Corresponding Author:
*
Dr. Vinod Kumar
Laboratory of Organic and Medicinal Chemistry, Department of Pharmaceutical Sciences and
Natural Products, Central University of Punjab, Bathinda, Punjab, Indiaꢀ151001.
Eꢀmail: vinod.kumar@cup.edu.in; vpathania18@gmail.com.
Phone No. +911642864214
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 neurodegenerative disorder with multifactorial pathogenesis.
Monoamine oxidase (MAO) and acetylcholinesterase enzymes (AChE) are potential targets for
the treatment of AD. A total of 15 new propargyl containing 4,6ꢀdiphenylpyrimidine derivatives
were synthesized and screened for the MAO and AChE inhibition activities along with ROS
production inhibition and metal chelation potential. All the synthesized compounds were found
selective and potent inhibitors of MAOꢀA and AChE enzymes at nano molar concentrations.
VB1 was found to be the most potent MAOꢀA and BuChE inhibitor with IC values of 18.34 ±
0
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.38 nM and 0.666 ± 0.03 ꢁM, respectively. It also showed potent AChE inhibition with IC50
value of 30.46 ± 0.23 nM. Compound VB8 was found to be the most potent AChE inhibitor with
IC value of 9.54 ± 0.07 nM and displayed IC value of 1010 ± 70.42 nM against MAOꢀA
50
50
isoform. In the cytotoxic studies, these compounds were found nonꢀtoxic to the human
neuroblastoma SHꢀSY5Y cells even at 25 ꢁM concentration. All the compounds were found
reversible inhibitors of MAOꢀA and AChE enzymes. In addition, these compounds also showed
good neuroprotective properties against 6ꢀOHDA and H O induced neurotoxicity in SHꢀSY5Y
2
2
cells. All the compounds accommodate nicely to the hydrophobic cavity of MAOꢀA and AChE
enzymes. In the molecular dynamics simulation studies, both VB1 and VB8 were found stable in
the respective cavities for 30 ns. Thus, 4,6ꢀdiphenylpyrimidine derivatives can act as promising
leads in the development of dual acting inhibitors targeting MAOꢀA and AChE enzymes for the
treatment of Alzheimer’s disease.
Key words: Alzheimer’s disease, MAO inhibitors, Diphenylpyrimidine, Acetylcholinesterase
inhibitors, Dual inhibitors, Neuroprotective agents
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Introduction
Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder characterized by the
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progressive memory loss, dementia and other cognitive impairments. It is estimated that one in
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every three senior persons dies with AD and one in ten persons over 65 years of age has AD.
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According to Alzheimer’s association, in recent years’ deaths due to stroke, heart disease, and
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HIV decreased 21%, 14%, and 54%, respectively, whereas deaths from AD increased 89%. The
exact molecular pathogenesis of AD is not clear however, in most of the cases the disease state is
linked with the degeneration of neurons and glial cells or inclined metabolism of signaling
neurotransmitters. Various biochemical and histopathological studies indicated that over
expressed monoamine oxidase (MAO) enzyme, low acetylcholine levels, amyloidꢀβ (Aβ)
3
deposits, hyperꢀphosphorylated tauꢀprotein aggregation and oxidative stress play crucial roles in
the pathophysiology of the disease. Depressive symptoms occur in patients suffering with AD
and these may be associated with the decreased serotonergic and noradrenergic transmission in
4, 5
the limbic system, Hyper activation of MAO enzyme has been observed in patients suffering
with AD that decreases the concentration of dopaminergic and serotoninergic neurotransmitters.
Some clinical trials have shown that deprenyl, a potential MAOꢀB inhibitor, alleviate the
6
ꢀ8
symptoms of AD. Acetylcholine is an important enzyme in the regulation of learning and
memory processes. It is hydrolyzed by acetylcholinesterase (AChE) and cholinesterase inhibitors
enhance the level of acetylcholine. Thus, cholinergic system has been explored as an important
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10
11
target for the treatment of AD. Currently, tacrine, donepezil, galantamine, and rivastigmine
are FDA (Food and Drug Administration US) approved drugs that improve AD symptoms by
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inhibiting AChE.
Apart from the beneficial palliative properties of AChE inhibitors in AD,
cholinergic drugs have shown little efficacy to prevent the progression of the disease.
Consequently, there is no efficient drug to cure, stop or even slow the progression of the disease
15, 16
therefore, effective therapeutics are sought for the permanent treatment of AD.
The pathogenesis of AD is complex and targeting single pathway may not be an effective
strategy for the complete treatment of the disease. Multiꢀtargeted ligands design strategy
involves the incorporation of two or more distinct pharmacophores of different drugs in the
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single structure to develop hybrid molecules.
simultaneously inhibit MAO, AChE and BuChE enzymes are being developed as drugs for the
Hence, the multitargeting ligands which can
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management/treatment of AD.
A number of multitargeting ligands have been reported as
21, 22
potential therapeutic agents for the treatment of AD.
Although many newly developed MAO,
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AChE and BuChE inhibitors are under different phases of clinical trials but none of these still
reached to clinical use because of the number of adverse effects. Recently, we have reported
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various phenylꢀ/benzhydrylpiperazine derivatives as selective ligands for MAOꢀA and MAOꢀB
isoforms. In another study, we have synthesized and screened a number of pyrimidine bridged
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biaryls for their MAO inhibition potential. Most of these compounds were found reversible and
selective inhibitors of the MAOꢀB isoform. There is a high structural resemblance between the
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two isoforms of MAO enzyme. The volume of entrance cavity of MAOꢀB is smaller (~300 Å )
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6, 21
as compared to the active cavity of MAOꢀA (~400 Å ).
Thus, MAOꢀB accommodates smaller
molecules while bulkier ligands selectively bind to the MAOꢀA isoform. A small alteration in the
structure of ligand can change its preference for either of the isoforms (MAOꢀA or MAOꢀB).
Taking leads from these studies, we got interested in the design and synthesis of dual acting
ligands that are equipotent to both MAO and AChE enzymes for the treatment of AD. From the
literature search, we noted that the propargylamino group play an important role in providing the
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neuronal and mitochondrial protective properties.
The propargyl group is also involved in
the crucial covalent bond formation with FAD coꢀfactor of MAO enzyme. Similarly, in the
literature survey it has been observed that most of the AChE inhibitors such as donepezil,
rivastigmine, pyridostigmine, phenserine galantamine etc. contained a tertiary amino group in the
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scaffold as a part of ring or open chain.
Hence, we presumed that a tertiary nitrogen atom
might be playing crucial role for the AChE inhibition activity and introduced a
piperidine/morpholine ring in the scaffold as potential pharmacophore for the AChE enzyme.
The chain length between the propargyl and piperidine moieties is reported to control the dual
interaction of these moieties with both the catalytic active site and peripheral anionic site of the
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AChE enzyme. It is envisaged that incorporation of a piperidine/morpholine ring in the scaffold
would make the molecules bulkier and these compounds might show selectivity towards MAOꢀA
isoform.
Thus, on the basis of our previous experience and with the aim of developing dual inhibitors, a
series of 4,6ꢀdiphenylpyrimidine derivatives (VB1ꢀVB15) have been designed with a propargyl
group and a piperidine/morpholine moiety as potential pharmacophores for the MAO and AChE
enzymes. These compounds were found to be potent inhibitors of both MAO and AChE enzymes
with IC values in nano molar range. All the compounds were found reversible inhibitors of the
5
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MAOꢀA and AChE enzymes and displayed no toxicity to the human neuroblastoma SHꢀSY5Y
cells. Most of these compounds were also found to be inhibitors of BuChE in the micro molar
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range. In the molecular dynamics simulation studies of 30 ns, the most potent MAO inhibitor
(VB1) and most potent AChE inhibitor (VB8) were found quite stable in the active sites of the
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enzymes. These compounds also displayed neuroprotective potential against 6ꢀOHDA and H O2
induced neurotoxicity in SHꢀSY5Y cells.
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Results and discussion
Chemistry
All the compounds were synthesized as per reaction procedures described in Schemeꢀ1. Briefly,
Oꢀalkylated acetophenones (1) and benzaldehydes (2) were synthesized from corresponding
hydroxy acetophenones or benzaldehydes by refluxing these with suitable alkyl halides in the
presence of potassium carbonate and acetone as solvent. Oꢀalkylated acetophenones (1) and
benzaldehydes (2) were reacted through aldol condensation to get the intermediate chalcones (3).
In the final step chalcones (3) were reacted with various amidines in the presence of sodium
carbonate to obtain the target compounds 4,6ꢀdiphenylpyrimidine derivatives (VB1ꢀVB15). All
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the final products were characterized by IR, H NMR, C NMR, ESIꢀMS and HRMS.
Schemeꢀ1: Reaction scheme for the synthesis of target molecules
Reagents and Conditions: a) K CO , KI, acetone, reflux, 6 h; b) K CO , acetone, reflux, 6 h; c) CH OH, 10% NaOH aq., rt,
2
3
2
3
3
stirring, 3 h; and d) Na CO , CH CN, amidine/guanidine/benzamidine, reflux, 24h
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MAO, AChE and BuChE enzyme inhibition studies
MAO inhibition potential of the synthesized 4,6ꢀdiphenylpyrimidine derivatives (VB1ꢀVB15)
was evaluated through fluorimetric method using recombinant human MAOꢀA and MAOꢀB
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enzymes and Amplex® Red assay kit. Similarly, acetylcholinesterase inhibition activity was
determined using Amplex Red Acetylcholine/Acetylcholinesterase assay kit (A12217) purchased
from the Molecular probes Inc. Invitrogen. The results of MAO inhibition studies are described
in Table 1 in terms of IC50 values expressed in nano molar concentrations. Morpholine or
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piperidine ethyl chains (R ) were used as meta or para substituents at one of the phenyl ring. To
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develop structure activity relationship profile of 4,6ꢀdiphenylpyrimidines, three amidines were
used with R as ꢀNH , ꢀCH or ꢀC H . Similarly, second phenyl ring was substituted with a
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6
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propargyl group at ortho/meta/para positions. MAOꢀA, MAOꢀB and AChE inhibitory activities
of some of the intermediate chalcones were also evaluated to compare them with the final
products. Clorgyline, pargyline and donepezil were used as standard inhibitors for MAOꢀA,
MAOꢀB and AChE enzymes, respectively. All the compounds were found selective towards
MAOꢀA isoform with IC values varying from 18 nM to 2430 nM. MAOꢀB inhibition activities
5
0
of these compounds were found in the range of 7313 nM to 14850 nM. These compounds were
also found to be the potent inhibitors of AChE with IC values in nano molar range. However,
5
0
most of the compounds displayed BuChE inhibitory activity in the micro molar range (0.66 µM
to 161.6 µM . VB1 was found to be the most potent MAOꢀA inhibitor with IC value of 18.34
0.38 nM and selectivity index (SI) of 540 over MAOꢀB. VB1 was also found to be the most
potent BuChE inhibitor in the series with IC value of 0.666 ± 0.03 µM Similarly, VB8 was
found to be the most potent AChE inhibitor with IC value of 9.54 ± 0.07 nM. VB3 was also
)
5
0
±
.
5
0
5
0
found potent inhibitor of both AChE and MAOꢀA with IC values of 18.92 ± 0.29 nM and 28.33
5
0
±
3.22 nM, respectively. All the chalcone intermediates were found less active as compared to
the corresponding final products.
Table 1. Results of the MAO, AChE and BuChE inhibition studies of synthesized compounds
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Entry
Name
R1
R
Propargyl
group
X
IC values
IC values
MAO
SI
50
50
(mean ± S.E. nM)
(mean±S.E.
µM)
position
hMAOꢀA
hMAOꢀB
eeAChE
eqBuChE
0
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2
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0
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0
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0
VB1
VB2
VB3
C4
C4
C4
NH
2
C4
C4
C4
O
O
O
18.34 ± 0.38
86.35 ± 0.42
28.33 ± 3.22
9910.14 ± 32.83
30.46 ± 0.23
0.666 ± 0.03
14.84 ± 0.27
14.98 ± 0.31
540
80
C
6
H
6915.62 ± 26.72
8409.53 ± 25.69
39.83 ± 0.39
18.92 ± 0.29
5
CH
297
3
VB4
VB5
C4
C4
C
6
H
5
C4
C4
C
C
103.42 ± 11.43
752.63 ± 14.34
8376.28 ± 23.73
12140.12 ±
24.69 ± 0.76
765.14 ± 3.51
19.72 ± 0.27
54.46 ± 1.42
81
16
CH
3
1
00.64
VB6
C4
C
6
H
5
C3
O
2140.12± 50.43
14850.34 ±
80.62
1841.56 ±
27.67
8.216 ± 0.10
7
VB7
VB8
C4
C4
NH
CH
2
C3
C3
O
O
230.12 ± 7.22
1010 ± 70.42
7313.76 ± 65.32
20.45. ± 0.17
9.54 ± 0.07
16.65 ± 0.17
13.75 ± 0.23
32
12
12185.94 ±
3
1
90.86
VB9
VB10
VB11
VB12
C4
C4
C3
C3
NH
2
C2
C2
C4
C4
O
O
O
O
890.45 ± 8.32
690.34 ± 20.32
8875.37 ±
10.52
2390.23 ±
20.72
161.6 ± 5.88
22.68 ± 0.36
14.66 ± 0.20
23.48 ± 0.18
10
18
4
1
C
C
H
12305.63 ±
87.72
30105.12 ±
70.14
6
5
5
H
2430.23 ± 45.33 8974.73 ± 93.73
2269.82 ±
6
3
2.01
NH
360.33 ± 2.34
8738.97 ± 51.73
1438.54 ±
7.67
24
2
2
VB13
VB14
C3
C4
CH
3
C4
C3
O
C
450.44 ± 5.13
8469.63 ± 83.72 717.88 ± 7.54
15.27 ± 0.11
15.95 ± 0.12
19
7
C
6
H
1320.22 ± 30.22
9565.83 ±
120.53
2093.71 ±
15.69
5
VB15
C4
NH
2
C3
C
1040.12 ± 40.12 7706.23 ± 95.73
1441.42 ±
34.79 ± 0.27
7
2
0.33
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a
C4
ꢀꢀꢀꢀ
C4
O
260.12 ± 13.12
9320.12 ± 24.22
20427.18 ±
ꢀꢀꢀꢀ
36
2
2.15
b
C4
C3
ꢀꢀꢀꢀ
C2
C4
O
O
313.23 ± 17.22
532.42 ± 19.23
4.39 ± 1.02
8110.11 ± 40.11
7809.02 ± 35.13
ꢀꢀꢀꢀꢀ
**
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
26
15
3c
ꢀꢀꢀꢀ
**
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Clorgyline
ꢀꢀꢀꢀ
MAOꢀ
A
Pargyline
Donepezil
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
0.15 ± 0.02
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
MAOꢀ
B
ꢀꢀꢀꢀ
11.32 ± 0.001
1.28 ± 0.04
ꢀꢀꢀꢀ
*
*Inactive or showed less than 50% inhibitory activity at 50 µM concentration and precipitated at higher
concentrations. IC values of BuChE are expressed in µM. MAO SI= IC of MAOꢀB / IC of MAOꢀA.
5
0
50
50
Reversibility inhibition studies
First generation MAO inhibitors were irreversible in nature and associated with severe side
effects. Reversibility of the target compound is frequently considered in designing and
development of new class of inhibitors. Thus, to determine the reversible inhibition of the
enzyme by the most active and selective compounds i.e. VB1, VB3 and VB8, reversibility
2
3, 30, 31
inhibition studies were performed using earlier reported protocol by us and others.
All the
tested compounds were found reversible inhibitors of both MAOꢀA and AChE enzymes. Upon
treatment of MAOꢀA and AChE with the test compounds at concentrations of 10xIC50 and
1
00xIC activity was reduced to minimum. Upon 100 times dilution with the substrate solution,
50
recovery of more than 75% enzymatic activity was achieved as shown in Fig. 1A and Fig. 1B for
MAOꢀA and AChE respectively. Thus, it can be concluded that the tested compounds were
found to be reversible inhibitors of MAOꢀA and AChE.
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A enzyme; (B) Reversibility inhibition studies with AChE enzyme.
Intracellular ROS determination
It is a wellꢀknown fact that monoamine oxidase mediated oxidative metabolism of mono amines
3
2, 33
lead to the production of H O as a byproduct.
Subsequently, H O get converted to the free
2 2
2
2
radicals (ȮH, Ȯ ) through Fenton’s reaction which contribute to the oxidative stress.
2
Uncontrolled increase in the concentrations of the free radicals, initiate free radicalꢀmediated
chain reactions that causes oxidative damage to the cell membranes, lipid peroxidation and DNA
strand breakdown. Thus, prevention of ROS generation along with the MAO inhibition is
important strategy to reduce or eliminate neurotoxicity in neurodegenerative disorders.
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Fig. 2ꢀ ROS production inhibition studies of VB1, VB2 and VB3 against SHꢀSY5Y cell lines
Intracellular ROS level of SHꢀSY5Y cell lines was determined using nonꢀfluorescent compound
2
,7ꢀdichlorofluorescein diacetate (DCFꢀDA). It is permeable and oxidized by ROS to a
fluorescent compound 2,7ꢀDCF. VB1 was found to be the most potent ROS inhibitor and
reduced the intracellular ROS level to 35.36 % and 14.39 % at 1 ꢁM and 25 ꢁM concentration
respectively (Fig. 2). VB3 and VB8 also reduced the ROS levels to 42.25 % and 65.25 %
respectively at 25 ꢁM concentrations.
Neuroprotection studies
The most potent compounds (VB1, VB3 and VB8) were evaluated for their neuroprotective
potential against 6ꢀhydroxydopamine (6ꢀOHD) neurotoxin in the SHꢀSY5Y cell lines. But none
of the tested compounds showed promising neuroprotective potential at low concentrations (Fig.
3
). VB8 was found to be the most potent amongst the tested compounds and displayed recovery
of cells up to 61.77% at 25 ꢁM concentration as compared to the 6ꢀOHD which reduced the cell
viability to less than 50 % at 12.5 ꢁM.
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Cytotoxicity studies
The cytotoxic effects of the most active compounds (VB1, VB3 and VB8) were evaluated
against human neuroꢀblastome (SHꢀSY5Y) cell lines because of their similarity to the
34
dopaminergic neurons. The test compounds were incubated at 1 ꢁM, 5 ꢁM and 25 ꢁM
concentrations and were analyzed after 24h treatment time. The percentage cell viability was
measured using MTT assay. As depicted in Fig. 4, the compounds were found nonꢀtoxic against
the tested cells even at 25 ꢁM concentrations. It has been observed that as concentration of the
compounds increases, the percentage cell viability also increases. Least cell viability of 80% was
observed with VB8 at 1 ꢁM concentration. Thus, keeping in view the nano molar IC values
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obtained for enzyme inhibition, the current series of compounds were found to be nonꢀtoxic to
the tissue cells.
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Fig. 4ꢀ Cytotoxicity studies of VB1, VB3 and VB8 against SHꢀSY5Y cell lines at 1 ꢁM, 5 ꢁM
and 25 ꢁM.
Metal chelating studies
Metal chelating studies of the most potent compounds i.e. VB1, VB3 and VB8 were performed
with a UV−vis spectrophotometer. The absorption spectra of each compound (50 µM, final
concentration) alone or in the presence of CuSO , FeSO , and FeCl (50 µM, final concentration)
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3
was recorded. In metal chelating studies, compounds were found ineffective against metals and
does not form any chelates with the metal salts.
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 i.e. VB8 using eeAChE. The reciprocal LineweaverꢀBurk plots (Fig. 5)
illustrate increased slope (decreased Vmax) and higher intercepts (K ) with the increasing
m
concentration of VB8. The intersection point of the LineweaverꢀBurk reciprocal plots was
located in the second quadrant, which indicate that the inhibition mode of VB8 was mixedꢀtype
inhibition. Thus, it can be concluded that VB8 binds to both CAS and PAS of AChE
simultaneously.
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Fig. 5: Kinetic study on the mechanism of eeAChE inhibition by VB8. Overlaid
Lineweaver−Burk reciprocal plots of AChE initial velocity at increasing substrate concentration
(0.1−1 mM) in the absence or presence of VB8 are shown.
Molecular docking studies
The most potent compounds (VB1, VB3 and VB8) were subjected to molecular docking studies
to find the interaction pattern of the molecules with the amino acid lining and their orientation at
the active site of the receptors. Compounds were docked at the active site of hMAOꢀA (PDB IDꢀ
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5, 36
37
2
BXR and 2Z5X)
and AChE (PDB IDꢀ 1EVE). The Xꢀray crystal structures were imported
from the protein data bank using Maestro 11.1 (Schrödinger LLC). The docking procedure
employed was first validated by redocking the coꢀcrystallized ligands into the MAO and AChE
models and the process was found suitable for the current study. Three most potent compounds
(VB1, VB3 and VB8) were docked at the structure of MAOꢀA (2BXR and 2Z5X) enzymes coꢀ
crystallized with clorgyline and harmine.
All the compounds were found to accommodate nicely to the active site of MAOꢀA delineated
by hydrophobic residues Tyr69, Phe208, Arg296, Ile335, Leu337, Phe352, Tyr407, Trp441,
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Fig. 6ꢀ A) Binding pattern (3D) of VB1 with the amino acid residues at the active site of MAOꢀA
(2BXR) and B) Binding interactions (2D) of VB1 with various amino acids of MAOꢀA active cavity,
Tyr444 and FAD. Most of the interactions observed were hydrophobic and πꢀπ aromatic stacking
which showed hydrophobic nature of the MAOꢀA active site. Pyrimidine moiety was aligned
towards the FAD coꢀfactor while 4ꢀ(2ꢀphenoxyethyl)morpholine fragment was exposed to the
outer part of the active site cavity lined by the polar amino acid residues Thr205 and Arg206.
The (propꢀ2ꢀynꢀ1ꢀyloxy)benzene fragment of VB1 showed πꢀπ aromatic interaction with Phe352
while pyrimidine ring showed πꢀπ aromatic interaction with Tyr444. 4ꢀ(2ꢀ
phenoxyethyl)morpholine showed πꢀπ aromatic interaction with Trp441. VB3 and VB8 also
showed interactions with the same set of amino acids at the active cavity of the enzyme (Fig.
S1). The only difference observed in the conformation of VB8 was the orientation of propargyl
group. In case of VB1 and VB3 propargyl group was oriented towards rest of the molecule to the
inner side of the cavity while in VB8, it was directed away from the rest of the molecule towards
outer side of the cavity (Fig. 6 and Fig. S1). This difference in the conformation of VB8 might be
responsible for its lower activity towards MAOꢀA (Fig. 6 and S1). Propargylamino group of
clorgyline is reported to interact with FAD coꢀfactor and form strong covalent bond. These
strong interactions might be responsible for the irreversible nature of clorgyline. As evident from
Fig. 6 and Fig. S1, in VB1, VB3 and VB8 pyrimidine ring is aligned close to the FAD while
propargyl group is oriented away from the FAD coꢀfactor. These conformations of the ligands at
the active site negate any possibility of covalent bond formation between the propargyl group
and FAD. Thus, these ligands may not form strong covalent bond with the enzyme and hence
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display reversible inhibition activity. Son et al., reported a very high resolution structure of
human MAOꢀA coꢀcrystallized with a reversible MAOꢀA inhibitor harmine which was different
3
5
from the structure reported by Colibus et al. On the advice of one of the reviewer of this
manuscript, we have also performed docking studies of reversible MAOꢀA inhibitor VB1 with
PDBꢀ2Z5X (Fig. S2). In this case too, it has been observed that pyrimidine ring was aligned
towards the FAD coꢀfactor and the propargyl group was directed towards outer part of the cavity
aligned with amino acid resides Val484 and Thr487 (Fig. S2). These observations further
strengthen our claim that the propargyl group may not be able to form strong covalent bond with
the FAD and hence current series of compounds behave as reversible inhibitors.
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Similarly, the most potent compounds (VB1, VB3 and VB8) were docked in the crystal structure
of AChE (1EVE) enzyme coꢀcrystallized with donepezil (Fig. 7 and S3). All the compounds
were accommodating nicely to the active site as well as the peripheral anionic site (PAS) of
AChE as done by the standard inhibitor donepezil. The AChE anionic site, composed of Trp84,
Tyr130, Phe330 and Phe331 residues while peripheral anionic site (PAS) consists of five amino
acidsꢀTyr70, Asp72, Tyr121, Trp279 and Tyr334. The increased chain length between the phenyl
and morpholine ring, as compared to the Nꢀbenzylpiperidine moiety of donepezil, pushed the 4ꢀ
(
2ꢀphenoxyethyl)morpholine fragment of VB1 and VB3 inside the CAS of AChE. The
morpholine moiety was aligned towards the narrowest part of the active site cavity. However,
for VB8, the binding alignment was inverted and 4ꢀ(2ꢀphenoxyethyl)morpholine fragment was
aligned towards the 2,3ꢀdihydroindenꢀ1ꢀone fragment of the donepezil (Fig. 8). Thus the (propꢀ2ꢀ
ynꢀ1ꢀyloxy)benzene fragment of VB8 was oriented towards CAS of AChE.
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Fig. 7 A) Binding pattern (3D) of VB8 with the amino acid residues at the active site of AChE (1EVE),
and B) Binding interactions (2D) of VB8 with various amino acids of AChE active cavity.
Comparison of the binding patterns (predicted) of donepezil and VB8 reveals the plausible
reason of its high potency for AChE. Donepezil showed π−π aromatic interactions of Nꢀ
benzylpiperidine fragment with Trp84 residue and cation−π interactions with Phe331 and Tyr334
residues. The 2,3ꢀdihydroindenꢀ1ꢀone fragment of the donepezil showed π−π stacking with
Trp279. VB1 and VB3 preserved most of the interactions of the donepezil with the same set of
amino acid residues. These compounds showed cation−π interactions with Trp84 and π−π
stacking with Trp279, Phe331 and Tyr334 residues. Similarly, pyrimidine fragment of VB8
showed π−π aromatic stacking with Phe331 and Tyr334. However, 4ꢀ(2ꢀ
phenoxyethyl)morpholine fragment was aligned in the opposite direction as compared to the
benzylpiperidine moiety of donepezil and displayed cation−π interactions with the Trp279
residue and hydrogen bonding interactions with the Phe288 residue through oxygen atom of the
morpholine ring (Fig. 7 and S3). Furthermore, the (propꢀ2ꢀynꢀ1ꢀyloxy)benzene fragment of VB8
showed additional aromatic π−π stacking with His440 residue, not observed for VB1, VB3 and
donepezil.
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(propꢀ2ꢀynꢀ1ꢀyloxy)benzene fragments of VB1 and VB3
were oriented towards 2,3ꢀ
dihydroindenꢀ1ꢀone fragment of the donepezil in PAS of AChE while 4ꢀ(2ꢀ
phenoxyethyl)morpholine fragment overlap with the Nꢀbenzylpiperidine moiety of donepezil
and oriented towards CAS. Pyrimidine rings of VB1 and VB3 showed good overlap with the
dihydroindenone part of donepezil. Surprisingly, VB8 showed reverse orientation and its (propꢀ
2ꢀynꢀ1ꢀyloxy)benzene fragment overlapped with the benzylpiperidine moiety of the donepezil
aligned towards CAS of AChE. The 4ꢀ(2ꢀphenoxyethyl)morpholine moiety of VB8 oriented
towards 2,3ꢀdihydroindenꢀ1ꢀone fragment of donepezil and accommodate in the PAS of AChE.
The pyrimidine ring of VB8 overlapped with the piperidine ring of donepezil while (propꢀ2ꢀynꢀ
1
ꢀyloxy)benzene fragment overlapped with the benzylic ring. Thus, altered orientation and
additional interactions at the active site might be responsible for the high potency of VB8
towards AChE enzyme.
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active site of AChE (1EVE).
Molecular dynamics simulation studies
Molecular dynamics simulation (MD) were performed to study the protein–ligand interactions
and to determine the thermodynamic stability of the docked compounds at the active pocket of
the enzymes. The protein–ligand docked complexes of the most active compounds VB1 with
MAOꢀA and VB8 with AChE were used for MD simulations. The MD simulation studies were
conducted for 30 ns and the interaction pattern of the test compound with different amino acids
was analyzed. MD simulations yielded stable trajectories for VB1 with MAOꢀA in the first 20
ns, as noted by the time evolution of the potential energy and the rootꢀmeanꢀsquare deviation
(RMSD) of the protein backbone, which ranged from 0.6 to 1.0 Å (Fig. 9). There were no major
structural alterations and the pyrimidine moiety adopted similar orientation in the active site of
MAOꢀA (Fig. 9) as obtained during the docking studies. In particular, the 4ꢀ(2ꢀ
phenoxyethyl)morpholine fragment of VB1 was stacked against Tyr444 and Arg206. After the
time interval of 22 ns, there was a sudden hike in the RMSD and thereafter it showed stable
trajectory up to 30 ns. RMSD oscillates in the range of 2 Å to 3 Å in this time interval. In the
MD simulations studies it has been observed that VB1 preserved most of the interactions
observed during the docking studies. In addition, some new interactions with various amino acid
residues were also observed (Fig 9). In the MD simulations, πꢀπ aromatic interaction with
Tyr444 was retained. The NH group present on the pyrimidine fragment move more towards
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Fig 9ꢀ A) RMSD graph of MD studies of VB1 with MAOꢀA for 30 ns, and B) interactions of
VB1 with the active site residues of MAOꢀA after 30 ns MD simulation studies
Tyr407 and formed a hydrogen bond with it. The nitrogen atom of the morpholine ring formed
hydrogen bonds with Pro72 and Arg206 through water bridge formation. It demonstrates the
structural integrity of the MAOꢀAꢀVB1 complex, and displayed similar binding features as done
by clorgyline.
The MD simulations of AChEꢀVB8 complex was performed to determine the binding stability of
VB8 at CAS and PAS of AChE. The complex yielded stable trajectories from 2 ns to 30 ns, as
noted by the time evolution of the potential energy and the rootꢀmeanꢀsquare deviation (RMSD)
of the protein backbone. The RMSD values for AChEꢀVB8 complex ranged from 1.6 to 2.0 Å
(Fig. 10). For the initial 2s, the RMSD value increase from 0.8 to 1.6 and there after it remained
stable up to 30 ns. Analyzing the MD trajectory of AChEꢀVB8 complex, it can be concluded that
VB8 showed strong tendency to be localized in the binding region of AChE. Nitrogen atom of
the pyrimidine ring form hydrogenꢀbond with Tyr121. In the PAS of AChE, the protonated
nitrogen atom of morpholine moiety maintained the initial cation−π interaction (from
comparison of Fig. 7 and Fig. 10) throughout the MD simulations interval, indicating stable
binding of VB8 with AChE. In addition, morpholine ring formed hydrogen bonding with the
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Glu278 residue through water bridge formation. The (propꢀ2ꢀynꢀ1ꢀyloxy)benzene fragment
aligned towards CAS of AChE and showed aromatic πꢀπ interaction of VB8 with Tyr330 and
Trp84. The MD simulations of VB8 does not indicate any abrupt local force that could
potentially break up the inhibitor or can even delocalize it from the binding site of AChE. Thus,
from the MD simulations studies of AChEꢀVB8 complex, it can be concluded that the complex is
stable and this stability might be responsible for the high in vitro potency of VB8 for AChE.
1
1
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0
Fig 10ꢀ A) RMSD graph of MD simulations studies of VB8 with AChE for 30 ns, and B)
interactions of VB8 with the active site residues of AChE during 30 ns MD simulation studies
ADME properties
To determine the drug like characteristics of the synthesized compounds, ADME parameters of
these compounds were determined using Qikprop application of Schrodinger. VB1 displayed
very good drugꢀlike profile with LogP value less than 5 and QPlogBB value as ꢀ0.65. In addition,
VB1 showed optimum oral absorption and bloodꢀbrain barrier permeability. VB3 and VB8 also
showed 100 % oral absorption and QPlogBB in the optimum range to cross the blood brain
barrier.
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Table 2: Physiochemical properties of the most potent and selective MAOꢀA and AChE
inhibitors
Name Mol. Wt. Log P HB
donor
HB
% human QPlogBB
(Optimum
BBB
acceptor oral
permeability
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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7
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9
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1
2
3
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9
0
absorption range ꢀ3.0 – predicted
.2)
1
VB1
VB3
VB8
3.94
5.06
5.09
3
8
7
7
96
ꢀ0.65
0.22
0.28
+ve
+ve
+ve
4
30.505
429.518
429.518
1
1
100
100
+
ve = high bloodꢀbrain barrier permeability, HB = Hydrogen bond, QPlogBB = qualitatively predicted logarithmic
ratio between the concentration of a compound in the brain and blood, LogP = partition coefficient of a molecule
between an aqueous and lipophilic phase (octanol and water)
SAR studies
In the current studies, three different amidines have been used with R as ꢀNH , ꢀCH or ꢀC H
5
2
3
6
(Table 1). One of the ring of diphenylpyrimidines is optionally substituted with the morpholine
or piperidine ethyl chain while other phenyl ring is functionalized with a propargyl group at
ortho, meta or para positions. The effect of different substituents on the activity is analyzed as
reported in the Table 1. The compounds were evaluated for MAOꢀA, MAOꢀB, eeAChE and
eqBuChE inhibitory activities using enzymatic assays. All the compounds were found selective
for MAOꢀA isoform with IC values in nano molar range and with moderate to very high
5
0
selectivity index. It has been observed that the compounds with morpholine ethyl chain and
propargyl group at para positions of both the phenyl rings showed high potency for MAOꢀA
isoform and eeAChE. VB1 with R as ꢀNH was found to be the most potent MAOꢀA inhibitor
2
with IC value of 18.34 nM and it also showed potent eeAChE inhibitory activity with IC
50
5
0
value of 30.46 nM. VB1 was also found to be the most potent BuChE inhibitor in the series with
IC value of 0.666 ± 0.03 µM. VB3 with R as ꢀCH3 also showed strong inhibition potential
50
against MAOꢀA and eeAChE with IC50 values of 28.33 nM and 18.92 nM respectively.
Replacement of ꢀNH with C H in VB2 reduces MAOꢀA activity by 5ꢀfolds. Shifting of
2
6
5
propargyl group from para position to meta position (VB7) reduces MAOꢀA inhibitory activity
by more than 12ꢀfolds but there was no effect on the eeAChE inhibitory activity. Similarly, in
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VB8 meta propargyl group reduces MAOꢀA inhibitory activity by 35ꢀfolds when compared to
VB3. However, VB8 was found to be the most potent eeAChE inhibitor in the series with IC50
values of 9.54 nM. In general, replacement of morpholine ring with the piperidine ring reduces
MAOꢀA inhibitory activity (VB4 and VB5). Shifting of morpholine substituent from para (VB1)
to meta position (VB12) decreases MAOꢀA activity by 19ꢀfolds and eeAChE activity by 47ꢀ
folds. It has been found that compounds with a propargyl substitution at the ortho and meta
positions were less active towards MAOꢀA as compared to the para substituents. The current
series of compounds displayed high selectivity index towards AChE as compared to BuChE
1
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(Table 1). VB1 was found to be the most potent BuChE inhibitor however, it showed SI of 22ꢀ
3
folds for AChE. In the current series of compounds, VB8 displayed highest SI of about 10 for
AChE. Replacement of amino group with methyl or phenyl groups (VB2 and VB3) decreases
BuChE inhibitory activity by about 22ꢀfolds. Shifting of propargyl group from para (VB1) to
ortho (VB9) position drastically reduced the activity by about 240ꢀfolds. Similarly, shifting of
morpholine ring from para (VB1) to meta position (VB12) decreases BuChE activity by 35ꢀ
folds.Thus, VB1, VB3 and VB8 were found to be the most promising compounds amongst the
reported series of compounds.
Conclusion
AD is multifactorial in nature and different enzymes including MAO, AChE and amyloid beta
are implicated in its pathogenesis. Pathomechanism of AD is complex in nature and single target
drugs proved ineffective for the treatment of the disease. Thus, multiꢀtarget directed approach is
being explored for the development of effective drug candidates for the treatment of AD. A drug
active on multiple targets may be characterized by an improved efficacy when compared to a
highly selective pharmacological agent. Multiꢀtarget activities may potentiate efficacy either
additively or synergistically and be less prone to the drug resistance. In the current study, a series
of 4,6ꢀdiphenylpyrimidines has been rationally designed that can simultaneously target MAO
enzymes and acetylcholinesterases for the treatment of AD. The phenyl rings were optionally
substituted with a morpholine or piperidine ethyl chain and Oꢀpropargylated groups at ortho,
meta and para positions and structure activity relationship profile has been generated. Most of
the compounds were found potent dual inhibitors of MAOꢀA isoform and AChE with IC values
5
0
in nano molar range. In the current series, VB1 was found to be the most potent MAOꢀA and
BuChE inhibitor with IC values of 18.34 ± 0.38 nM and 0.66 ± 0.03 µM. It also displayed
50
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potent AChE inhibitory activity with IC value of 30.46 ± 0.23 nM. VB3 was another promising
5
0
compound in the series with IC values of 28.33 ± 3.22 nM and 18.92 ± 0.29 nM against MAOꢀ
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A and AChE respectively. VB8 was found to be the most potent AChE inhibitor with IC value
50
3
of 9.54 ± 0.07 nM and displayed very high SI (10 ) for AChE over BuChE. In the reversibility
0
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inhibition studies, VB1, VB3 and VB8 were found reversible inhibitors of MAOꢀA and AChE.
These compounds also displayed neuroprotective potential against 6ꢀOHDA and H O induced
2
2
neurotoxicity in SHꢀSY5Y cells. Cytotoxicity studies were also performed with the three lead
compounds using SHꢀSY5Y cell lines and the compounds were found nonꢀtoxic even at 25 ꢁM
concentration. In the MD simulation studies of 30 ns, VB1 and VB8 were found stable in the
active site of MAOꢀA and AChE respectively. In the physicochemical evaluation studies, the
most active compounds were found to possess drug like characteristics with optimum oral
absorption and good permeability to blood brain barrier. Thus, some of these dual acting
3
0
compounds have the potential to be developed as drug candidates for the treatment of AD.
Experimental
General procedure for the synthesis of 1 and 2
To hydroxy substituted acetophenone (0.5g) or benzaldehydes (0.5g), morpholine or piperidine
ethylchloride (1.2eq) was added in the presence of potassium carbonate as base (2.4eq.) and
acetone (30 ml) as solvent. The reaction mixture was refluxed for 12 h at 60˚ C. The progress of
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 and the organic solvent was concentrated under vacuum using rotary
2
4
evaporator.
General procedure for the synthesis of 3
To a mixture of 1 (1 eq.) and 2 (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,
excess of solvent was evaporated from the mixture using rotary evaporator. Chilled water was
poured into the reaction mixture and precipitates of 3 were filtered and dried.
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General procedure for the synthesis of VB1ꢀVB15
To a mixture of 3 (500 mg), and amidine (1.2 eq.), anhydrous sodium carbonate (2.4 eq.) was
added in acetonitrile (5 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, water (10 mL) was added
aqueous phase was extracted with ethyl acetate (3×10 mL), washed with brine, dried over
anhydrous Na SO , and the organic solvent was concentrated under vacuum using rotary
1
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2
4
evaporator and purified via column chromatography (EtOAc:Pet ether). The final products were
characterized by NMR spectroscopy and HRMS.
Spectral analysis
4
-(4-(2-morpholinoethoxy)phenyl)-6-(4-(prop-2-yn-1-yloxy)phenyl)pyrimidin-2-amine (VB1):
1
Yield 59%, H NMR (CDCl , 400 MH , δ with TMS=0): 8.05ꢀ8.00 (4H, m), 7.35 (1H, s), 7.07
3
Z
(2H, d, J= 8Hz), 6.99 (2H, d, J= 8Hz), 5.16 (2H, s), 4.76 (2H, d, J= 2.4Hz), 4.18 (2H, t, J= 4Hz),
13
3
.75 (4H, t, J= 4Hz), 2.83 (2H, t, J= 4Hz), 2.60 (4H, t, J= 4Hz), 2.55 (1H, t, J= 2.4Hz), C NMR
(
CDCl , 100 MH , δ with TMS=0) δ: 165.40, 165.28, 163.49, 160.71, 159.41, 131.13, 130.36,
3 Z
128.36, 128.58, 115.00, 114.70, 102.86, 78.19, 75.89, 66.93, 65.89, 57.58, 55.85, 54.13 HRMS:
+
+
for C H N O , calculated [M+H] : 431.2083; observed [M+H] : 431.2060
2
5
26
4
3
4-(2-(4-(2-phenyl-6-(4-(prop-2-yn-1-yloxy)phenyl)pyrimidin-4-yl)phenoxy)ethyl)morpholine
1
(
VB2): Yield 63%, H NMR (CDCl , 400 MH , δ with TMS=0): 8.68 (2H, d, J= 8Hz), 8.24 (4H,
3
Z
t, J= 8Hz), 7.86 (1H, s), 7.53ꢀ7.49 (3H, m), 7.12 (2H, d, J= 8Hz), 7.04 (2H, d, J= 8Hz), 4.77 (2H,
d, J= 2.4Hz), 4.19 (2H, t, J= 4Hz), 3.75 (4H, t, J= 4Hz), 2.84 (2H, t, J= 4Hz), 2.60 (4H, t, J=
13
4Hz), 2.56 (1H, t, J= 2.4Hz), C NMR (CDCl , 100 MH , δ with TMS=0) δ: 164.01, 163.89,
3
Z
1
6
61.02, 159.73, 138.40, 130.59, 130.28, 128.83, 128.48, 115.20, 114.89, 108.75, 78.19, 75.89,
+
6.99, 65.93, 57.66, 55.95, 54.20 HRMS: for C H N O , calculated [M+H] : 492.2287;
3
1
29
3
3
+
observed [M+H] : 492.2271
4
-(2-(4-(2-methyl-6-(4-(prop-2-yn-1-yloxy)phenyl)pyrimidin-4-yl)phenoxy)ethyl)morpholine
1
(
VB3): Yield 58%, H NMR (CDCl , 400 MH , δ with TMS=0): 8.11ꢀ8.07 (4H, m), 7.76 (1H, s),
3
Z
7.10 (2H, d, J= 8Hz), 7.02 (2H, d, J= 8Hz), 4.77 (2H, d, J= 2.4Hz), 4.19 (2H, t, J= 4Hz), 3.75
(4H, t, J= 4Hz), 2.84 (2H, t, J= 4Hz), 2.81 (3H, s), 2.61 (2H, t, J= 4Hz), 2.56 (1H, t, J= 2.4Hz),
(
CDCl , 100 MH , δ with TMS=0) δ: 168.32, 164.07, 163.96, 160.85, 159.55, 130.94, 130.17,
3
Z
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
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1
28.72, 115.18, 114.87, 108.49, 78.13, 75.93, 66.91, 65.90, 57.57, 55.87, 54.13, 26.53 HRMS:
+
+
for C H N O , calculated [M+H] : 430.2131; observed [M+H] : 430.2079
2
6
27
3
3
2
-phenyl-4-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-6-(4-(prop-2-yn-1-yloxy)phenyl)pyrimidine
1
(VB4): Yield 61%, H NMR (CDCl , 400 MH , δ with TMS=0): 8.69 (2H, dd, J = 8Hz, J =
3
Z
1
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
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4Hz), 8.23 (4H, t, J= 8Hz), 7.84 (1H, s), 7.52ꢀ7.42 (3H, m) 7.11 (2H, d, J= 8Hz), 7.03 (2H, d, J=
8
Hz), 4.76 (2H, d, J= 2.4Hz), 4.19 (2H, t, J= 4Hz), 2.82 (2H, t, J= 4Hz), 2.57ꢀ2.55 (5H, m),
.66ꢀ1.60 (4H,m) 1.46 (2H, bd), (CDCl , 100 MH , δ with TMS=0) δ: 164.26, 164.04, 163.82,
1
3
Z
161.12, 159.70, 138.43, 131.04, 130.56, 130.11, 128.80, 128.71, 128.48, 115.18, 114.89, 108.71,
8.26, 76.04, 66.02, 57.83, 55.94, 55.14, 29.81, 25.88, 24.19 HRMS: for C H N O , calculated
7
32 31
3
2
+
+
[
M+H] : 490.2495; observed [M+H] : 490.2470
2-methyl-4-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-6-(4-(prop-2-yn-1-yloxy)phenyl)pyrimidine
1
(VB5): Yield 67%, H NMR (CDCl , 400 MH , δ with TMS=0): 8.07 (4H, t, J= 8Hz), 7.74 (1H,
3
Z
s), 7.08 (2H, d, J= 8Hz), 7.00 (2H, d, J= 8Hz), 4.75 (2H, d, J= 4Hz), 4.17 (2H, t, J= 4Hz), 2.82ꢀ
2.79 (5H, m), 2.53 (4H, b), 1.61 (4H, b), 1.44 (2H, b), 1.23 (1H, b), (CDCl , 100 MH , δ with
3
Z
TMS=0) δ: 168.35, 164.18, 163.98, 161.03, 159.59, 131.01, 130.79, 130.35, 130.11, 130.06,
1
29.47, 128.77, 115.33, 115.23, 114.95, 108.53, 78.21, 76.01, 66.07, 57.85, 55.92, 55.13, 29.79,
+
+
2
5.89, 24.17, HRMS: for C H N O , calculated [M+H] : 428.2338; observed [M+H] :
27 29 3 2
428.2293
4
-(2-(4-(2-phenyl-6-(3-(prop-2-yn-1-yloxy)phenyl)pyrimidin-4-yl)phenoxy)ethyl)morpholine
1
(VB6): Yield 62%, H NMR (CDCl , 400 MH , δ with TMS=0): 8.70 (2H, dd, J = 8Hz, J =
3
Z
1
2
4
7
4
Hz), 8.25 (2H, d, J= 8Hz), 7.97 (1H,s), 7.91 (1H,s), 7.86 , (1H, d, J= 8Hz), 7.54ꢀ7.56 (4H, m),
.15 (1H, dd, J = 8Hz, J = 4Hz), 7.06 (2H, d, J= 8Hz), 4.82 (2H, d, J= 4Hz), 4.20 (2H, t, J=
1
2
Hz), 3.76 (4H, t, J= 4Hz), 2.85 (2H, t, J= 4Hz), 2.60 (5H, m), (CDCl , 100 MH , δ with
3
Z
TMS=0) δ: 164.37, 164.27, 164.06, 161.13, 158.17, 139.32, 138.25, 130.70, 130.10, 130.01,
1
5
28.88, 128.51, 120.50, 117.11, 114.93, 113.99, 109.61, 78.50, 75.94, 67.01, 65.98, 57.65, 56.13,
+
+
4.21, HRMS: for C H N O , calculated [M+H] : 492.2287; observed [M+H] : 492.2256
3
1
29
3
3
4
-(4-(2-morpholinoethoxy)phenyl)-6-(3-(prop-2-yn-1-yloxy)phenyl)pyrimidin-2-amine (VB7):
1
Yield 59%, H NMR (CDCl , 400 MH , δ with TMS=0): 8.00 (2H, d, J= 8Hz), 7.90 (1H, dd, J =
3
Z
1
8
Hz, J = 4Hz), 7.65 (1H, m), 7.39 (1H, t, J= 8Hz), 7.36 (1H, s), 7.08 (1H, dd, J = 8Hz, J = 4Hz),
2 1 2
6.98 (2H, d, J= 8Hz), 6.90 (1H, t, J= 8Hz), 5.22 (2H, s), 4.77 (2H, d, J= 4Hz), 4.16 (4H, t, J=
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Hz), 3.73 (2H, t, J= 4Hz), 2.82 (4H, t, J= 4Hz), 2.53 (2H, b), (CDCl , 100 MH , δ with
3
Z
TMS=0) δ: 165.71, 163.56, 160.86, 158.01, 139.49, 130.69, 129.89, 128.70, 120.44, 117.08,
14.79, 114.48, 114.29, 113.53, 103.73, 78.49, 75.82, 66.97, 65.93, 57.63, 56.04, 54.18, HRMS:
1
+
+
for C H N O , calculated [M+H] : 431.2083; observed [M+H] : 431.2060
2
5
26
4
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
4-(2-(4-(2-methyl-6-(3-(prop-2-yn-1-yloxy)phenyl)pyrimidin-4-yl)phenoxy)ethyl)morpholine
1
(VB8): Yield 59%, H NMR (CDCl , 400 MH , δ with TMS=0): 8.08 (2H, d, J= 8Hz), 7.78 (1H,
3
Z
m), 7.72ꢀ7.69 (2H, m), 7.43 (1H, t, J= 8Hz), 7.10 (1H, d, J= 8Hz), 7.01 (2H, t, J= 8Hz), 4.78
(2H, d, J= 4Hz), 4.18 (2H, t, J= 4Hz), 3.74 (4H, t, J= 4Hz), 2.83 (2H, t, J= 4Hz), 2.83 (3H, s),
2
1
7
.59 (4H, t, J= 4Hz), 2.54 (1H, t, J= 4Hz), (CDCl , 100 MH , δ with TMS=0) δ: 168.50, 164.37,
3 Z
64.29, 161.01, 158.14, 139.31, 131.03, 130.07, 128.83, 120.56, 117.13, 114.97, 113.77, 109.45,
8.44, 75.84, 66.98, 65.97, 57.64, 56.08, 54.20, 29.79, HRMS: for C H N O , calculated
2
6
27
3
3
+
+
[M+H] : 430.2131; observed [M+H] : 430.2086
4
-(4-(2-morpholinoethoxy)phenyl)-6-(2-(prop-2-yn-1-yloxy)phenyl)pyrimidin-2-amine (VB9):
1
Yield 55%, H NMR (CDCl , 400 MH , δ with TMS=0): 8.00 (2H, d, J= 8Hz), 7.85 (1H, dd, J =
3
Z
1
8
Hz, J = 4Hz), 7.60 (1H, m), 7.40 (1H, dd, J = 8Hz, J = 4Hz), 7.12 (2H, t, J= 8Hz), 6.97 (2H, d,
2 1 2
J= 8Hz), 5.12 (2H, s), 4.75 (2H, d, J= 4Hz), 4.16 (2H, t, J= 4Hz), 3.73 (4H, t, J= 4Hz), 2.82 (2H,
t, J= 4Hz), 2.59 (4H, b), 2.52 (1H, t, J= 4Hz), (CDCl , 100 MH , δ with TMS=0) δ: 164.69,
3
Z
164.30, 163.39, 160.70, 155.69, 130.94, 128.81, 122.22, 114.70, 114.16, 113.54, 109.19, 108.56,
+
7
8.53, 75.92, 66.99, 65.92, 57.66, 56.69, 54.18, HRMS: for C H N O , calculated [M+H] :
2
5
26
4
3
+
4
31.2083; observed [M+H] : 431.2036
4
-(2-(4-(2-phenyl-6-(2-(prop-2-yn-1-yloxy)phenyl)pyrimidin-4-yl)phenoxy)ethyl)morpholine
1
(VB10): Yield 63%, H NMR (CDCl , 400 MH , δ with TMS=0): 8.67 (1H, dd, J = 8Hz, J =
3
Z
1
2
4
Hz), 8.27 (1H, s), 8.26 (2H, d, J= 8Hz), 7.53ꢀ7.44 (4H, m), 7.21 (1H, t, J= 8Hz), 7.13 (1H, d, J=
8Hz), 7.04 (2H, d, J= 8Hz), 4.80 (2H, d, J= 4Hz), 4.19 (2H, t, J= 4Hz), 3.75 (4H, t, J= 4Hz),
2
1
1
.84 (2H, t, J= 4Hz), 2.60 (4H, b), 2.56 (1H, t, J= 4Hz), (CDCl , 100 MH , δ with TMS=0) δ:
3 Z
64.19, 163.29, 162.79, 160.97, 156.23, 138.60, 131.59, 131.33, 130.56, 130.40, 128.97, 128.44,
28.41, 126.56, 122.35, 114.86, 114.72, 113.53, 78.50, 76.00, 67.02, 66.05, 57.68, 56.82, 54.22,
+
+
HRMS: for C H N O , calculated [M+H] : 492.2287; observed [M+H] : 492.2256
3
1
29
3
3
4
-(2-(3-(2-phenyl-6-(4-(prop-2-yn-1-yloxy)phenyl)pyrimidin-4-yl)phenoxy)ethyl)morpholine
1
(VB11): Yield 62%, H NMR (CDCl , 400 MH , δ with TMS=0): 8.68 (2H, dd, J = 8Hz, J =
3
Z
1
2
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2
2
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Hz), 8.26 (2H, dd, J = 8Hz, J = 4Hz), 7.90 (1H, d, J= 8Hz), 7.85 (1H, s), 7.81 (1H, d, J= 8Hz),
1
2
7.54ꢀ7.50 (3H, m), 7.44 (1H, d, J= 8Hz), 7.13 (2H, dd, J = 8Hz, J = 4Hz), 7.01 (1H, dd, J =
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2
1
8
4
1
Hz, J = 4Hz), 4.78 (2H, d, J= 4Hz), 4.26 (2H, t, J= 4Hz), 3.77 (4H, t, J= 4Hz), 2.90 (2H, t, J=
2
Hz), 2.67 (4H, b), 2.56 (1H, t, J= 4Hz), (CDCl , 100 MH , δ with TMS=0) δ: 166.67, 163.80,
3
Z
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
61.95, 160.94, 147.89, 138.06, 130.75, 129.60, 128.88, 128.62, 128.42, 119.49, 116.86, 114.91,
114.58, 112.04, 110.66, 80.96, 76.80, 71.58, 66.75, 65.72, 57.56, 54.09, HRMS: for
+
+
C H N O , calculated [M+H] : 492.2287; observed [M+H] : 492.2256
31
29
3
3
4-(3-(2-morpholinoethoxy)phenyl)-6-(4-(prop-2-yn-1-yloxy)phenyl)pyrimidin-2-amine (VB12):
1
Yield 72%, H NMR (CDCl , 400 MH , δ with TMS=0): 8.01 (2H, d, J= 8Hz), 7.60 (1H, s), 7.57
3
Z
(
1H, d, J= 8Hz), 7.36 (2H, t, J= 8Hz), 7.04 (2H, d, J= 12Hz), 7.00 (1H, d, J= 8Hz), 5.35 (2H, s),
.73 (2H, d, J= 2.4Hz), 4.18 (2H, t, J= 4Hz), 3.73 (4H, t, J= 4Hz), 2.82 (2H, t, J= 4Hz), 2.59
(4H, t, J= 4Hz), 2.54 (1H, t, J= 2.4Hz), (CDCl , 100 MH , δ with TMS=0) δ: 165.71, 163.56,
4
3
Z
1
1
60.86, 158.01, 139.49, 130.69, 129.89, 128.70, 120.44, 117.08, 114.79, 114.48, 114.29, 113.53,
03.73, 78.49, 75.82, 66.97, 65.93, 57.63, 56.04, 54.18, HRMS: for C H N O , calculated
2
5
26
4
3
+
+
[
M+H] : 431.2083; observed [M+H] : 431.2018
4
-(2-(3-(2-methyl-6-(4-(prop-2-yn-1-yloxy)phenyl)pyrimidin-4-yl)phenoxy)ethyl)morpholine
1
(VB13): Yield 54%, H NMR (CDCl , 400 MH , δ with TMS=0): 8.09 (2H, d, J= 8Hz), 7.78
3
Z
(1H, s), 7.64 (1H, d, J= 8Hz), 7.51ꢀ7.47 (1H, m), 7.38 (1H, t, J= 8Hz), 7.07 (2H, d, J= 12Hz),
7
4
1
.02 (1H, dd, J = 8Hz, J = 4Hz), 4.74 (2H, d, J= 4Hz), 4.28 (2H, t, J= 4Hz), 4.19 (2H, t, J=
1 2
Hz), 3.72 (4H, t, J= 4Hz), 2.81 (3H, s), 2.59 (4H, t, J= 4Hz), 2.54 (1H, t, J= 2.4Hz), (CDCl3,
00 MH , δ with TMS=0) δ: 168.47, 164.44, 164.25, 159.72, 159.33, 139.14, 131.02, 130.02,
Z
128.91, 128.83, 118.83, 116.96, 115.27, 113.29, 109.49, 78.18, 76.07, 66.98, 65.88, 65.66, 57.75,
+
+
5
4
5.92, 54.17, HRMS: for C H N O , calculated [M+H] : 430.2131; observed [M+H] :
26 27 3 3
30.2101
2
-phenyl-4-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-6-(3-(prop-2-yn-1-yloxy)phenyl)pyrimidine
1
(VB14): Yield 53%, H NMR (CDCl , 400 MH , δ with TMS=0): 8.70 (2H, dd, J = 8Hz, J =
3
Z
1
2
4
Hz), 8.24 (2H, d, J= 8Hz), 7.96 (1H, t, J= 8Hz), 7.91 (1H, s), 7.85 (1H, dd, J = 8Hz, J = 4Hz),
1 2
7.53ꢀ7.45 (4H, m), 7.13 (1H, dd, J = 8Hz, J = 4Hz), 7.05 (2H, d, J= 8Hz), 4.81 (2H, d, J= 8Hz),
1
2
4
.20 (2H, t, J= 4Hz), 2.83 (2H, t, J= 4Hz), 2.58 (4H, t, J= 4Hz), 1.66ꢀ1.60 (4H, m), 1.47ꢀ1.40
(3H, m), (CDCl , 100 MH , δ with TMS=0) δ: 164.35, 164.37, 164.05, 161.16, 158.17, 139.36,
3
Z
138.29, 130.70, 130.01, 128.85, 128.51, 120.52, 117.11, 114.56, 113.96, 109.61, 78.39, 75.83,
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+
5
7.92, 56.16, 75.83, 29.72, 25.86, 24.13, HRMS: for C H N O , calculated [M+H] : 490.2495;
32 31 3 2
+
observed [M+H] : 490.2480
4
-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-6-(3-(prop-2-yn-1-yloxy)phenyl)pyrimidin-2-amine
1
(
VB15): Yield 58%, H NMR (CDCl , 400 MH , δ with TMS=0): 7.99 (2H, d, J= 8Hz), 7.65ꢀ
3
Z
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7.62 (2H, m), 7.39 (1H, t, J= 8Hz), 7.35 (1H, s), 7.08 (1H, dd, J = 8Hz, J = 4Hz), 6.97 (2H, d, J=
1
2
8
Hz), 5.26 (2H, s), 4.75 (2H, d, J= 4Hz), 4.16 (2H, t, J= 4Hz), 2.80 (2H, t, J= 4Hz), 2.53 (5H, b),
.62ꢀ1.58 (4H, m), 0.97ꢀ0.92 (2H, m), (CDCl , 100 MH , δ with TMS=0) δ: 165.75, 165.55,
1
3
Z
163.60, 160.96, 158.07, 139.53, 131.02, 129.87, 128.67, 120.45, 117.08, 114.79, 113.50, 103.68,
8.51, 75.83, 66.01, 57.85, 56.03, 55.11, 25.87, 34.17, HRMS: for C H N O , calculated
7
26 28
4
2
+
+
[
M+H] : 429.2291; observed [M+H] : 429.2238
(E)-1-(4-(2-morpholinoethoxy)phenyl)-3-(4-(prop-2-yn-1-yloxy)phenyl)prop-2-en-1-one (3a):
1
Yield 85%, H NMR (CDCl , 400 MH , δ with TMS=0):8.01 (2H, d, J= 8Hz), 7.66 (1H, d, J=
3
Z
1
6Hz), 7.60 (2H, d, J= 8Hz), 7.43 (1H, d, J= 16Hz), 7.02ꢀ6.95 (4H, m), 4.73 (2H, d, J= 4Hz),
4.18 (2H, t, J= 4Hz), 3.73 (4H, t, J= 4Hz), 2.83 (2H, t, J= 4Hz), 2.58 (2H, t, J= 4Hz), 2.55 (1H, t,
13
J= 4Hz), C NMR (CDCl , 100 MH , δ with TMS=0): 188.72, 162.50, 159.37, 143.68, 131.45,
3
Z
1
30.80, 130.12, 128.68, 120.01, 115.34, 114.42, 77.46, 76.12, 66.99, 66.09, 57.55, 55.90, 54.19
+
+
MS: for C H NO , calculated [M] : 391.17; observed [M] : 391
2
4
25
4
(
E)-1-(4-(2-morpholinoethoxy)phenyl)-3-(2-(prop-2-yn-1-yloxy)phenyl)prop-2-en-1-one (3b):
1
Yield 83%, H NMR (CDCl , 400 MH , δ with TMS=0): 8.02ꢀ8.09 (3H, m), 7.63ꢀ7.69 (2H, m),
3
Z
7.34 (1H, t, J= 8H ), 7.03 (2H, t, J= 8H ), 6.96 (2H, d, J= 8H ), 4.79 (2H, d, J= 2.4 H ), 4.18
Z
Z
Z
Z
(
2H, t, J= 4H ), 3.73 (4H, t, J= 4H ), 2.82 (2H, t, J= 4H ), 2.56 (4H, t, J= 4H ), 2.55 (1H, t, J=
Z Z Z Z
13
4
H ), C NMR (CDCl , 100 MH , δ with TMS=0) δ: 189.25, 162.48, 156.72, 139.33, 131.39,
Z 3 Z
1
30.93, 129.57, 124.76, 123.17, 121.77, 114.38, 112.80, 78.14, 76.07, 66.99, 66.06, 57.55, 56.26,
+
+
54.18 MS: for C H NO , calculated [M] : 391.17; observed [M] : 391
2
4
25
4
(
E)-1-(3-(2-morpholinoethoxy)phenyl)-3-(4-(prop-2-yn-1-yloxy)phenyl)prop-2-en-1-one (3c):
1
Yield 87%, H NMR (CDCl , 400 MH , δ with TMS=0): 7.76 (1H, d, J= 16Hz), 7.58 (3H, t, J=
3
Z
8
4
4
1
5
Hz), 7.52 (1H, s), 7.40ꢀ7.36 (2H, m), 7.11 (1H, d, J= 8Hz), 7.00 (2H, dd, J = 8Hz, J = 4Hz),
1 2
.72 (2H, t, J= 4Hz), 3.72 (4H, t, J= 4Hz), 2.81 (2H, t, J= 4Hz), 2.57 (4H, b), 2.54 (1H, t, J=
1
3
Hz), C NMR (CDCl , 100 MH , δ with TMS=0) δ: 190.25, 159.57, 159.07, 144.61, 139.86,
3
Z
30.27, 129.66, 128.47, 121.23, 120.23, 119.70, 115.39, 113.67, 78.06, 76.15, 66.97, 65.95,
+
+
7.67, 55.91, 54.15 MS: for C H NO , calculated [M] : 391.17; observed [M] : 391
2
4
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Biological studies
Determination of hMAO inhibition activity
The synthesized compounds were evaluated against MAOꢀA and MAOꢀB isoforms for their
inhibitory potential using Amplex® Red assay kit through fluorimetric method described by us
0
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23
and others.
Briefly, 100 µl of sodium phosphate buffer (0.05 M, pH 7.4) containing the test drug and
reference inhibitors, in various concentrations along with adequate amounts of recombinant
hMAO (hMAOꢀA: 1.1 ꢁg protein; specific activity: 150 nmol of pꢀtyramine oxidized to pꢀ
hydroxyphenylacetaldehyde/min/mg protein; hMAOꢀB: 7.5 ꢁg protein; specific activity: 22 nmol
of pꢀtyramine transformed/min/mg protein) enzyme, were incubated for 15 min at 37 °C in a flatꢀ
blackꢀbottom 96ꢀwell plates (Tarsons) in a incubator. After the incubation period, the reaction
was started by adding (final concentrations) 200 ꢁM Amplex® Red reagent, 1 U/mL horseradish
peroxidase and 1 mM pꢀtyramine. After 30 min incubation in dark, the production of H O was
2
2
HI
quantified at 37 °C in a multiꢀdetection microplate fluorescence reader (Synergy , BioꢀTek®
Instruments) based on the fluorescence generated at excitation wavelength of 545 nm, and
emission wavelength of 590 nm. Control experiments were carried out simultaneously by
replacing the test drug with the vehicle. A direct reaction of test drug with the Amplex® Red
reagent in a sodium phosphate buffer was performed to minimize the possibility of the test drugs
to influence the fluorescence generated in the reaction mixture due to nonꢀenzymatic inhibition.
However, no significant modulation in the fluorescence was observed. The specific final
fluorescence emission was calculated after subtraction of the background activity determined
from vials containing all the components except the hMAO enzymes replaced by a sodium
phosphate buffer solution.
Acetylcholinesterase inhibition assay
Acetylcholinesterase
inhibition
activity
was
determined
using
Amplex
Red
Acetylcholine/Acetylcholinesterase assay kit (A12217) purchased from the Molecular probes
38
Inc. Invitrogen. Briefly, 100 µl of TrisꢀHCl 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 flatꢀblackꢀ
bottom 96ꢀwell plates (Tarsons). Reaction was started by adding 100 µl of working solution of
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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 multiꢀ
HI
detection microplate fluorescence reader (Synergy , BioꢀTek® Instruments) based on the
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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).
BuChE inhibition assay
The procedure described by Ellman et al. was used for BuChE inhibition assays with minor
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9
modifications.
BuChE was purchased from sigma Aldrich (CAS No. 9001ꢀ80ꢀ5).
’
Butyrylthiocholine iodide (BTCI), 5.5 ꢀdithiobis (2ꢀnitrobenzoic acid) (DTNB ꢀ Ellman’s
reagent) were purchased from Himedia. The assays were performed trisꢀHCl buffer (pHꢀ8) and
donepezil was used as standard compound. Six different concentrations of 20 µM, 10 µM, 1µM,
ꢀ
0
.1 µM, 0.01 µM, 0.001 µM of test compound were used to determine the IC . 50 µL (0.6 Uml
50
1
)
and 20 µL of test or standard compounds were incubated in 96 well plates for 30 min. 100 µL
(1.5 mM) of DTNB was added in the above solution. The substrate i.e. BTCI (30 mM, 10 µL)
was added into it and absorbance was recorded immediately at 415 nm for 20 min at 1 min
interval using Biotek well plate reader. The IC50 values were calculated using absorbance
obtained from the test and standard compounds. The assays were performed in triplicate and in
three independent runs.
Reversibility inhibition studies
3
0, 31
For reversibility inhibition studies protocol was adopted from the literature.
inhibitors were incubated with the MAO enzymes at concentrations of 10 × IC and 100 × IC
50
Briefly, the test
5
0
at 37 °C for 30 min (negative control performed in the absence of inhibitor), and 4% DMSO was
added as coꢀsolvent 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 × IC and 1 x IC value, respectively. As positive controls, MAOꢀA and
50
50
ACS Paragon Plus Environment