Discovery of new phenyl sulfonyl-pyrimidine carboxylate derivatives as the potential multi-target drugs with effective anti-Alzheimer's action: Design, synthesis, crystal structure and in-vitro biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2021.113224

Alzheimer's disease (AD) is multifactorial, progressive neurodegeneration with impaired behavioural and cognitive functions. The multitarget-directed ligand (MTDL) strategies are promising paradigm in drug development, potentially leading to new possible therapy options for complex AD. Herein, a series of novel MTDLs phenylsulfonyl-pyrimidine carboxylate (BS-1 to BS-24) derivatives were designed and synthesized for AD treatment. All the synthesized compounds were validated by ¹HNMR, ¹³CNMR, HRMS, and BS-19 were structurally validated by X-Ray single diffraction analysis. To evaluate the plausible binding affinity of designed compounds, molecular docking study was performed, and the result revealed their significant interaction with active sites of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The synthesized compounds displayed moderate to excellent in vitro enzyme inhibitory activity against AChE and BuChE at nanomolar (nM) concentration. Among 24 compounds (BS-1 to BS-24), the optimal compounds (BS-10 and BS-22) displayed potential inhibition against AChE; IC₅₀ = 47.33 ± 0.02 nM and 51.36 ± 0.04 nM and moderate inhibition against BuChE; IC₅₀ = 159.43 ± 0.72 nM and 153.3 ± 0.74 nM respectively. In the enzyme kinetics study, the compound BS-10 displayed non-competitive inhibition of AChE with Ki = 8 nM. Respective compounds BS-10 and BS-22 inhibited AChE-induced Aβ_1-42 aggregation in thioflavin T-assay at 10 μM and 20 μM, but BS-10 at 10 μM and 20 μM concentrations are found more potent than BS-22. In addition, the aggregation properties were determined by the dynamic light scattering (DLS) and was found that BS-10 and BS-22 could significantly inhibit self-induced as well as AChE-induced Aβ_1-42 aggregation. The effect of compounds (BS-10 and BS-22) on the viability of MC65 neuroblastoma cells and their capability to cross the blood-brain barrier (BBB) in PAMPA-BBB were further studied. Further, in silico approach was applied to analyze physicochemical and pharmacokinetics properties of the designed compounds via the SwissADME and PreADMET server. Hence, the novel phenylsulfonyl-pyrimidine carboxylate derivatives can act as promising leads in the development of AChE inhibitors and Aβ disaggregator for the treatment of AD.

Full text of DOI:10.1016/j.ejmech.2021.113224

European Journal of Medicinal Chemistry 215 (2021) 113224
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of new phenyl sulfonyl-pyrimidine carboxylate derivatives
as the potential multi-target drugs with effective anti-Alzheimer’s
action: Design, synthesis, crystal structure and in-vitro biological
evaluation
Shoaib Manzoor a, Santosh Kumar Prajapati b, Shreyasi Majumdar b, Md Kausar Raza c,
Moustafa T. Gabr d, Shivani Kumar e, Kavita Pal a, Haroon Rashid a, Suresh Kumar e,
,
, *
Sairam Krishnamurthy b **, Nasimul Hoda a
a Drug Design and Synthesis Laboratory, Department of Chemistry, Jamia Millia Islamia, New Delhi, 110025, India
b Neurotherapeutics Laboratory, Department of Pharmaceutical Engineering and Technology, Indian Institute of Technology (Banaras Hindu University),
Varanasi, U.P, 221005, India
c Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore, 560012, India
d Department of Radiology, Stanford University, Stanford, CA, 94305, United States
e University School of Biotechnology Guru Gobind Singh Indraprastha University Dwarka, Sector 16C, New Delhi, 110078, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Alzheimer’s disease (AD) is multifactorial, progressive neurodegeneration with impaired behavioural and
cognitive functions. The multitarget-directed ligand (MTDL) strategies are promising paradigm in drug
development, potentially leading to new possible therapy options for complex AD. Herein, a series of
novel MTDLs phenylsulfonyl-pyrimidine carboxylate (BS-1 to BS-24) derivatives were designed and
synthesized for AD treatment. All the synthesized compounds were validated by 1HNMR, 13CNMR, HRMS,
and BS-19 were structurally validated by X-Ray single diffraction analysis. To evaluate the plausible
binding affinity of designed compounds, molecular docking study was performed, and the result revealed
their significant interaction with active sites of acetylcholinesterase (AChE) and butyrylcholinesterase
(BuChE). The synthesized compounds displayed moderate to excellent in vitro enzyme inhibitory activity
against AChE and BuChE at nanomolar (nM) concentration. Among 24 compounds (BS-1 to BS-24), the
optimal compounds (BS-10 and BS-22) displayed potential inhibition against AChE;
Received 2 October 2020
Received in revised form
13 January 2021
Accepted 20 January 2021
Available online 2 February 2021
Keywords:
Alzheimer’s disease
Acetylcholinesterase
Amyloid beta
Multitarget-directed ligand
Phenylsulfonyl-pyrimidine carboxylate
derivatives
IC50
¼
47.33
0.02 nM and 51.36
0.04 nM and moderate inhibition against BuChE;
IC50 ¼ 159.43 0.72 nM and 153.3 0.74 nM respectively. In the enzyme kinetics study, the compound
BS-10 displayed non-competitive inhibition of AChE with Ki ¼ 8 nM. Respective compounds BS-10 and
BS-22 inhibited AChE-induced Ab1-42 aggregation in thioflavin T-assay at 10 mM and 20 mM, but BS-10 at
10
mM and 20 mM concentrations are found more potent than BS-22. In addition, the aggregation
properties were determined by the dynamic light scattering (DLS) and was found that BS-10 and BS-22
could significantly inhibit self-induced as well as AChE-induced Ab1-42 aggregation. The effect of com-
pounds (BS-10 and BS-22) on the viability of MC65 neuroblastoma cells and their capability to cross the
blood-brain barrier (BBB) in PAMPA-BBB were further studied. Further, in silico approach was applied to
analyze physicochemical and pharmacokinetics properties of the designed compounds via the Swis-
sADME and PreADMET server. Hence, the novel phenylsulfonyl-pyrimidine carboxylate derivatives can
act as promising leads in the development of AChE inhibitors and Ab disaggregator for the treatment of
AD.
© 2021 Elsevier Masson SAS. All rights reserved.
* Corresponding author.
** Corresponding author.
E-mail addresses: ksairam.phe@iitbhu.ac.in (S. Krishnamurthy), nhoda@jmi.ac.in (N. Hoda).
https://doi.org/10.1016/j.ejmech.2021.113224
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

S. Manzoor, S.K. Prajapati, S. Majumdar et al.
European Journal of Medicinal Chemistry 215 (2021) 113224
1. Introduction
biological targets may not be sufficient to tackle the problem of
effective AD treatment. The medicines presently available which
were developed according to the “one-disease one-target” frame-
work are not fully effective, due to the complexity of AD and the
variety of pathogenic factors involved in it. Over the past several
years, a multitarget-directed ligands (MTDLs) approach has gained
much interest in developing potential inhibitors for the treatment
of complex AD [23]. MTDLs are designed by precisely and rationally
to modulate multiple targets simultaneously, resulting in greater
clinical therapy efficiency [24]. By considering all these facts, we
aimed to design and synthesize new MTDLs phenylsulfonyl-
pyrimidine carboxylate derivatives via Biginelli-reaction acting as
multifactorial activity against AD. The Biginelli-reaction is signifi-
cant multicomponent reactions used for the preparation of py-
rimidine derivatives [25,26]. Most of the Biginelli derivatives are
also well known for their biological and pharmacological applica-
tions [27,28], used for the treatment of different diseases like
antimalarial [29], anticancer [30], anti-inflammatory [31], anti-
Alzheimer’s disease [32,33], and other diseases [34e36].
Nitrogen-rich heterocycle pyrimidine derivatives has appeared in
promising anti-AD agents, which has been reported to show
Alzheimer’s disease (AD) is one of the prevalent progressive
neurodegenerative disorder in the elderly population. A case of
dementia is diagnosed in every 3 seconds. In 2018, approximately
50 million people were living with dementia, and are expected to
exceed 80 million in 2030 and 152 million in 2050 worldwide [1].
AD is one of the most common predominant irreversible neuro-
degenerative disorders that target cholinergic neurons of the cen-
tral nerve system (CNS) [2]. The exact triggers for AD are not still
understood but several concurrent abnormalities contribute to the
progression of AD, such as low levels of the neurotransmitter
acetylcholine (ACh), amyloid-beta (A
phosphorylated tau-protein ( ) deposition, oxidative stress and
b) aggregation, hyper-
t
activation of monoamine oxidase (MAO) [3e5]. Due to AD com-
plexities and its development by various potential factors, a lot of
attention has been devoted to multitarget-directed ligand (MTDL)
strategies, used as a therapeutic method for AD treatment [6,7].
Acetylcholinesterase (AChE) is a ubiquitous serine hydrolase
enzyme that ceases nerve impulses by hydrolyzing the neuro-
transmitter acetylcholine (Ach) to choline and acetate. Its
numerous crystal structures are available, which reveals that AChE
can control the concentration of Ach at the synapse. AChE plays a
significant role in learning ability, memory and different cognitive
processes [8,9]. Both catalytic and peripheral sites are available in
AChE enzyme to which inhibitors can bind and inhibit its activity
[10]. In the forebrain, AChE inhibitors increase cholinergic neuro-
transmission by reducing the dissociation of acetylcholine. Two
major types of cholinesterase’s (ChEs) in the mammal brain are
AChE and BuChE (butyrylcholinesterase). The enzyme BuChE just
like AChE is also involved in the hydrolysis neurotransmitter ACh. In
AD affected brain, the BuChE level and activity gradually increases,
while the activity of AChE decreases. AChE and BuChE both come
together to regulate the central Ach levels, making them a pursu-
able target. To date, some AChE inhibitors which are commercially
available like donepezil, tacrine, rivastigmine, and galantamine are
FDA (Food and Drug Administration, USA) approved drugs for the
treatment of AD [11e13].
cholinesterase ChE inhibitory and A
[37,38]. Therefore, multitargeted therapeutics with inhibition of
AChE, BuChE and A aggregation and also with significant neuro-
b anti-aggregatory activity
b
protection and BBB permeation may prove to be fruitful in modi-
fying the AD progression, instead than offering only symptomatic
relief. To extend our research work on MTDLs, herein, we reported
new phenylsulfonyl-pyrimidine carboxylate derivatives with
inhibitory activity against AChE, BuChE and
Ab aggregation
endowed with an effective anti-Alzheimer’s disease activity.
2. Result and discussion
2.1. Designing strategy of novel phenylsulfonyl-pyrimidine
carboxylate derivatives
To combat against AD disease having complicated etiology,
MTDLs development was identified as the most effective approache
to drug discovery. Despite significant research on new available
target for the treatment AD, inhibitors of cholinesterase enzymes
are even now the drugs of choice, even though they provide tem-
porary and symptomatic benefits to the AD patients and inhibition
of BuChE induces the level of Ach neurotransmitters. Another
Among the several aspects that increase AD, the production and
accumulation of extracellular amyloid-beta (A
neurofibrillary intracellular tangles are the two prominent neuro-
pathological hallmarks of AD. A protein are disordered peptides
(range 36e43 amino acids) resulted from amyloid precursor pro-
tein (APP) through sequential cleavages by -and -secretase. In
general, the prevalent species found in plaques are amylodogenic
b) plaques and
b
b
g
pathogenesis of AD is Ab aggregation plays very critical role in the
progression of AD [39,40]. However, inhibitors for single patho-
genesis may not be adequate to treat such extremely complex
disease. So that MTDLs may increase the probability of combating
AD successfully.
A
b1ꢀ42 (Ab42) are much more toxic [14e16]. Biochemical studies
show that A
number of A
b
peptides develop monomers, oligomers, and a large
plaques [17]. It may cause mitochondrial injury, an
b
overload of calcium neuronal apoptosis [18]. Accumulation and
overproduction of A in brain activates subsequent pathological
events like tau hyperphosphorylation, synaptic degeneration,
neuroinflammation, oxidative stress and ultimately death of neu-
rons. Among two major isoforms of Ab peptides, Ab1-42 is the most
critical one, having a crucial role of accumulating different fibrillar
complex and oligomers [19,20]. It has also been shown that AChE
plays a critical role in accelerating Ab aggregation [21]. Moreover,
oxidative damage recommended by oxidative stress and free
radical ageing theory plays a vital role in neuronal degeneration.
Targeting oxidative stress could possibly prevent AD by defending
neuronal cells during oxidative damage [22]. The identification of
the most appropriate therapeutic targets for AD is not yet clarified.
In this scenario, for the clinical treatment of AD, the discovery of
effective therapeutic scaffolds is a key point that can modulate all
factors simultaneously, which induces AD.
To design new MTDLs phenylsulfonyl-pyrimidine carboxylate
derivatives against Alzheimer’s disease, based on different phar-
macophore combination strategy, displayed in Fig. 1. The design
strategies were used to design a series of inhibitors that could
simultaneously bind to the PAS (peripheral anionic site) and CAS
(catalytic anionic site) of AChE and BuChE enzyme. Donepezil is
FDA approved drug inhibiting AChE used as a therapeutic target for
Alzheimer’s disease [41]. The choice of selecting pyrimidine core
moiety was due to its different biological properties and are widely
used in the medicinal world [42,43]. Moreover, pyrimidine de-
rivatives are extensively used as a drug for the treatment of AD-
b
related deposition of A
b in brain [44,45]. The groove of AChE
enzyme is lined with significant number of aromatic residues and
Nitrogen-rich aromatic phenyl-pyrimidine derivatives were pre-
dicted to promote the non-covalent interactions like H-bonding
and
p-p interaction with the essential residues of AChE. The
Searching for a drug that can work on only one of the possible
phenyl-pyrimidine fragment is considered to be a pharmacophore
2

S. Manzoor, S.K. Prajapati, S. Majumdar et al.
European Journal of Medicinal Chemistry 215 (2021) 113224
Fig. 1. Design Strategy for new phenylsulfonyl-pyrimidine carboxylate derivatives (MTDLs) as Anti-Alzheimer’s agents.
that can bind to the PAS site of AChE [46]. A hit structure selected
from the study (4-dihydropyrimidine-2-thione (I) derivatives,
(Fig. 1) has displayed promising AChE inhibition with IC50 values
ranging from 8.23 0.2 to 0.07 0.01 mM and hence was reported
effective therapeutic option for the treatment of AD [47]. Piper-
azinyl pyrimidine (II) derivatives are found promising AChE in-
dual AChE and BuChE inhibitory activity. In addition to that they
will also hold Ab disaggregation ability. Furthermore, in silico study
suggest that the inhibitors will have excellent blood brain-barrier
(BBB) permeability and favorable interaction with AChE and
BuChE active site. Furthermore, physicochemical and pharmaco-
kinetics properties was performed to analyze the oral bioavail-
ability of the leading candidates.
hibitors and A
pyrimidines are found to be
in A disaggregation for the treatment of AD [48]. As represented in
b
disaggregators. The derivatives of piperazinyl
b
-secretase modulators for possible use
b
2.2. Chemistry
Fig. 1, the pharmacophore fragment phenyl-pyrimidine derivatives
are conjugated with phenylsulfonyl derivatives through a pipera-
zine linker. Phenylsulfonyl scaffold is another essential structural
unit which is present in different biologically active compounds.
The significance of phenylsulfonyl derivatives in neuropharma-
cology was discovered gradually for their potential anti-AD activity.
The compound 2-(2,4-dichloro-6-((4-(furan-2-carbonyl)piperazin-
1-yl)sulfonyl)phenoxy)-N-(4-ethylphenyl)acetamide (III) showed
significant inhibitory activity against AChE and BuChE with
The synthetic route for the designed dual inhibitor
phenylsulfonyl-pyrimidine carboxylate derivatives via multiple
steps as displayed in Schemes 1e3. In Scheme 1, the intermediate
ethyl 6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-
carboxylate derivatives 4(a-c) were synthesized via Biginelli-
reaction three components in one pot, between substituted alde-
hyde 1(a-c), ethylacetoacetate (2) and urea (3) in ethanol in the
presence of HCl as a catalyst. To obtain ethyl 4-methyl-6-oxo-2-
phenyl-5,6-dihydropyridine-3-carboxylate derivatives 5(a-c), the
intermediates 4(a-c) were oxidized with nitric acid at 0 ꢁC to room
temperature. Derivative 5(a-c) were refluxed in POCl3 to obtain
ethyl-2-chloro-6-methyl-4-phenylpyrimidine-5-carboxylate de-
rivatives 6(a-c). The 6(a-c) were reacted with piperazine in CHCl3
solvent in presence of base K2CO3 to obtain ethyl 4-methyl-6-
phenyl-2-(piperazin-1-yl) pyrimidine-5-carboxylate derivatives
7(a-c). In Scheme 2, the 4-acetamidobenzene-1-sulfonyl chloride
derivatives 10(a-d) were synthesized from different anilines 8(a-d)
through intermediate N-phenylacetamide derivatives 9(a-d). The
different anilines 8(a-d) were reacted with acetyl chloride in DCM
in presence of Et3N as a base to afford intermediate N-phenyl-
acetamide derivatives 9(a-d). The 4-acetamidobenzene-1-sulfonyl
chloride derivatives 10(a-d) was obtained from N-phenyl-
acetamide derivatives 9(a-d) treated with HSO3Cl at different
temperatures. In Scheme 3, the first 12 target compounds BS(1e12)
IC50 ¼ 5.54
0.03 and 9.15
0.01 mM, respectively [49]. BMS-
708163 (Avagacestaf) (IV) progressed into various stages of clin-
ical trials as a potent therapeutic treatment in AD, which is a po-
tential
g-secretase inhibitor, reduces amyloid-beta (Ab40
IC50 ¼ 0.30 nM) exhibiting 193-fold selectivity against Notch/A
b
precursor protein. Furthermore, a study performed by Gillman et al.
reported that the oral treatment of BMS-708163 significantly
decreased the level of Ab40 for sustained periods in cerebrospinal
fluid, plasma, and brain in rats as well as in beagle dogs [50,51].
Piperazine is widely used for designing and synthesizing of bio-
logically active compounds and its derivatives have found potential
AD therapeutic effect by strongly inhibiting AChE, BuChE and
amyloid-beta aggregations [52,53].
The current study comprises of design, synthesis, and biological
evaluation of a series of new phenylsulfonyl-pyrimidine carbox-
ylate derivatives. These new derivatives are expected to possess
3

S. Manzoor, S.K. Prajapati, S. Majumdar et al.
European Journal of Medicinal Chemistry 215 (2021) 113224
Scheme 1. Reagents and conditions (i) EtOH, cat. HCl, reflux (5e6 h); (ii) HNO3 (68e70%),ꢁC to rt; (iii) POCl3, Reflux, 3e4 h (iv) piperazine, K2CO3, CHCl3, reflux (4e5 h).
Scheme 2. Reagents and conditions (i) Acyl Chloride, Et3N, DCM; (ii) ClSO3H.
Scheme 3. Reagents and conditions (i) K2CO3, CHCl3, reflux (2e3 h); (ii) 6N HCl, 1,4-Dioxane 80 ꢁC (2e3 h).
were obtained by the reaction of 7(a-c) and 4-acetamidobenzene-
1-sulfonyl chloride derivatives 10(a-d) in presence of anhydrous
K2CO3 in CHCl3 solvent at 60e65 ꢁC for 3e4 h. Finally, the other 12
target compounds BS(13e24) were obtained from BS (1e12)
treated with 6N HCl in 1,4 dioxane, neutralized with NaOH solution.
All the target compounds were purified by column chromatography
and characterized by 1HNMR, 13CNMR and mass spectrometry. In
addition, BS-19 was further characterized by X-ray crystallography.
Thin-layer chromatography and spectral analysis assist the com-
pound purity. The purity of all target compounds was determined
by UPLC. With the support of the enzyme model and crystal
structure data in PDB, we also analyze the computational survey,
rationalizing enzyme-binding molecular modes.
information). The compound BS-19 crystallized in P121/n1 space
group of monoclinic crystal system. The unit-cell packing diagram
of BS-19 displayed the hydrogen bonding interaction between the
hydrogen atoms of aromatic phenyl ring with the Carbon atom of
phenyl ring. The distance between the H-atom of C20 of phenyl ring
and C1 of the phenyl ring is 2.837 Å. The unit cell packing diagrams
of BS-19 showed short contact bonding with their adjacent atoms.
The unit cell packing diagram of BS-19 compounds were given in
supporting
information
(Figures
S73eS74,
Supporting
Information).
2.4. Molecular docking studies
To demonstrate molecular docking and scoring of synthesized
compounds with AChE and BuChE was done with the respective
ParDock module of Sanjeevini (SCFBIO, IITD) [54] and AutoDock
Vina program [55] (version 1.5.6), a complete drug design suite.
Pymol software was used to develop ligand-receptor interaction
2.3. Crystallography study
Compound BS-19 were structurally validated by single crystal X-
ray crystallography (Fig. 2 and Table 1 and Fig. S73eS74 supporting
4

S. Manzoor, S.K. Prajapati, S. Majumdar et al.
European Journal of Medicinal Chemistry 215 (2021) 113224
Fig. 2. ORTEP diagram of compounds BS-19 (50% probability level of thermal ellipsoids). Color codes: Carbon, light blue; Nitrogen: lavender; Oxygen: red; S, light yellow and
Fluorine: green. Hydrogen atoms are hidden for clarity.
Table 1
simultaneously. As presented in Fig. 3, compound BS-10 were
stacked very nicely into the active site groove of the AChE enzyme.
Selected crystal data and structure refinement for ethyl 2-(4-((4-amino-3-
fluorophenyl)sulfonyl)piperazin-1-yl)-4-methyl-6-phenylpyrimidine-5-
carboxylate (BS-19).
The result of docking possesses both H-bonding and
teractions, depicted with red dotted line in Fig. 3. The aromatic ring
of the 3,4-dimethoxyphenyl group of BS-10 is engaged in p-p
p-p in-
BS-19
C24H27FN5O4S
Empirical formula
Fw/g Mꢀ1
Crystal system
Space group
a/Å
stacking interaction with Trp279 residues (distance 3.8 Å) at PAS.
The aromatic ring of the 4-acetamido-3-chlorobenzenesulfonyl
500.56
monoclinic
P121/n1
10.488(11)
10.488(11)
26.79(3)
90
90
90
2458(5)
4
298(2)
group of BS-10 compound is involved in sandwich-type
p-p
stacking with residue Trp84 (distance 4.1 Å) at CAS. The Tyr121
formed a H-bond with the nitrogen of planar pyrimidine ring of
compound BS-10 at distance 3.1 Å. Arg289 exhibited strong H-
bonding with a methoxy group of BS-10 compound at distance
1.8 Å. In addition, Ser286 is also involved in H-bonding with the
methoxy group of BS-10 at distance 3.2 Å. Carbonyl group of BS-10
is also engaged in forming H-bond with respective Gly123 and
serine124 at bond distance 2.7 Å and 3.3 Å. The molecular docking
suggests that the selected compound reveal dual binding-activity
inhibiting AChE by targeting PAS and CAS may be due to H-
b/Å
c/Å
ꢁ
ꢁ
a
/
b
/
ꢁ
g
/
V/Å3
Z
T, K
rcalcd/Mg mꢀ3
1.353
l/Å (Mo-K
a
)
0.71073 Å
5041/3/319
1052
Data/restraints/param
bonding and aromatic p-p interactions.
F(000)
GOF
0.983
Similarly, the molecular docking study of synthesized com-
pounds was further extended with the crystal structure of hBuChE
(PDB code: 4TPK) by using AutoDock software. The docking study of
potent compound (BS-10) with BuChE is depicted in Fig. 4. The
most effective compound BS-10 (ꢀ8.7 kcal/mol as per AutoDock)
displayed better interaction with active site receptors of BuChE. The
R(Fo),a I > 2
s
(I) [wR(Fo)b]
0.0813 [0.2162]
0.1479 [0.2521]
0.292, ꢀ0.288
A ¼ 0.1429
B ¼ 0.0000
R (all data) [wR (all data)]
Largest diff peak, hole (e Åꢀ3
)
w ¼ 1/[ (
s
Fo)2 þ (AP)2 þ (BP)]
P
P
a
R ¼ ׀׀Fo׀ - ׀Fc׀ /׀׀Fo׀.
P
P
b
wR ¼ { [w(F2o - F2c)2]/ [w(Fo)2]}1/2, where P ¼ (Fo2 þ 2F2c)/3.
aromatic ring of potent compound is engaged in
p-p stacking
interaction with Trp82 residue at CAS, shown by the red dotted line
in Fig. 4 (A). Further, the methoxy group of potent BS-10 compound
is stabilized with Ser70 by H-bonding and eNH group on the ligand
exhibited the strongest bond with Glu115 residue. By molecular
docking study, it may be suggested that novel phenylsulfonyl-
pyrimidine carboxylate derivatives inhibit both AChE and BuChE
by targeting PAS and CAS in both enzymes.
image. All the target compounds were docked with the crystal
structure of enzyme AChE (PDB Code: 1EVE co-crystal with done-
pezil) to explore binding free energy (-DGb). Investigation of the
binding mode of designed compounds was performed by using
ParDock software. All the target compounds were adapting effec-
tively to the active site as well as PAS of AChE as achieved by
reference donepezil drug. These derivatives displayed stability in
CAS of AChE (binding free energy ranges from ꢀ9.69 kcal/mol
2.5. In vitro biological studies
to ꢀ12.38 kcal/mol) through different H-bonding and
pꢀp in-
2.5.1. hAChE and hBuChE inhibition assays
teractions. The most potent compound BS-10 (ꢀ12.29 kcal/mol as
per ParDock) were imperiled to molecular docking studies to find
out the interaction pattern of the target molecule with amino acid
lining and orientation with receptors of the active site (Fig. 3). As
depicted in Fig. 3, BS-10 were able to bind with both CAS and PAS
The inhibitory activities of novel 24 synthesized phenylsulfonyl-
pyrimidine carboxylate derivatives against AChE and BuChE was
assessed in vitro using an Ellman’s spectrophotometric method
[56], where donepezil (FDA approved drug) was used as the stan-
dard compound. The AChE inhibitory and BuChE inhibitory
5

S. Manzoor, S.K. Prajapati, S. Majumdar et al.
European Journal of Medicinal Chemistry 215 (2021) 113224
Fig. 3. Docking interaction of BS-10 (A) in active site of AChE (PDB code 1EVE). The inhibitor is shown in yellow color stick structure. The residues are depicted by different colors
like Phe330, Phe331 (yellow orange); Tyr70, Tyr121, Tyr130, Tyr334 (green); Trp84, Trp279 (magenta); Ser122, Ser124, Ser200, Ser286 (blue); Gly123(hot pink); His (red); Glu199
(wheat) and Arg289 (lemon). The nitrogen and oxygen in complexes are represented by respective blue and red color. H-bonding and
line. (B) Represents surface view of BS-10 into the active groove site of the AChE enzyme.
p-p Stacking are represented by red dashed
Fig. 4. Docking interaction of BS-10 (A) in active site of BuChE (PDB code 4TPK). The inhibitor is shown in yellow color stick structure. The residues are depicted by different colors
like Tyr (green), Trp (magenta), Ser (blue), His (red), Gly (cynas), Glu (wheat), Ala (orange), Asn (cynas teal), Asp (firebrick) and Phe (light blue). H-bonding and
p-p Stacking are
represented by red dashed line. (B) Represents surface view of BS-10 into the active groove site of the BuChE enzyme.
activities of synthesized target compounds BS-1 to BS-24 are
depicted as IC50 values in Table 2. The synthesized lead compounds
displayed significant inhibitory activity towards AChE. Almost all
the target compounds show high selective for AChE over BuChE.
The result demonstrated mild to excellent inhibition of AChE in
nanomolar (nM) to sub-nanomolar concentration except for the
compounds BS-17 and BS-19 (IC50 > 200 nM). Similarly, BS-1, BS-2,
BS-5, BS-17, BS-19, and BS-24 displayed poor inhibitory activity
against BuChE. However, amongst all synthesized moiety, com-
pound BS-10 and BS-22 showed excellent inhibition of AChE
(IC50 ¼ 47.33 nM and 51.36 nM respectively). Interestingly, com-
pound BS-10 and BS-22 are more than 3.5 and 3-fold selective for
AChE than for BuChE, respectively. It is observed that most of the
compounds without substituted electron-donating group (-OCH3),
i.e., BS-5 to BS-8 and BS-17 to BS-20 has poor AChE inhibitory ac-
tivity than their respective substituted electron-donating group
(-OCH3) compounds. However, monosubstituted electron-donating
group (-OCH3) at para-position of aromatic phenyl ring improved
the inhibitory activity against AChE (compounds BS-1 to BS-4 and
BS-13 to BS-16). While, 3,4-disubstituted electron-donating groups
(-OCH3) greatly improved the inhibitory activity against AChE
(compounds BS-9 to BS-12 and BS-21 to BS-24). Further, the 2,4-
dimethoxy substitution with groups (-H, eF, and -OCH3) (BS-9,
IC50 ¼ 88.79 nM; BS-11, IC50 ¼ 76.52 nM; B-12,IC50 ¼ 79.16 nM; BS-
IC50 ¼ 85.58 nM) showed almost similar AChE inhibitory activity
against mono-methoxy substituted compounds (BS-1,
IC50 ¼ 132.05 nM; BS-3, IC50 ¼ 91.9 nM; B-4, IC50 ¼ 79.25 nM; BS-
13,IC50 71.89 nM; BS-15 IC50 100.6 nM and BS-16,
¼
¼
IC50 ¼ 68.5 nM). The 2,4-dimethoxy substitution with electron-
withdrawing chloro-group (BS-10 IC50 ¼ 47.33 nM and BS-22,
IC50 ¼ 51.36 nM) showed excellent AChE inhibitory activity than
their
mono-methoxy
substituted
compounds
(BS-2,
IC50 ¼ 87.62 nM and BS-14, IC50 ¼ 77.91 nM) with electron-
withdrawing chloro-group. The 2,4-dimethoxy substituted with
chloro electron-withdrawing group (BS-10, IC50 ¼ 47.33 nM and BS-
22, IC50 ¼ 51.36 nM) exhibited prominent AChE inhibitory activity
at low nanomolar range close to donepezil (IC50 ¼ 26.55 nM).
Moreover, the IC50 of donepezil for BuChE was significantly higher
(IC50 ¼ 1210 nM) (shown in Table S78 supporting information) as
observed previously [57]. Therefore, this result implicated that the
compound BS-10 and BS-22 showed highest AChE inhibitory ac-
tivity, which could be due to the addition of electron-donating
dimethoxy and electron-withdrawing chloro group that increases
the binding affinity towards AChE. This finding was further
confirmed by the molecular docking study of potent compound
(BS-10), which is engaged significantly with CAS and PAS residues
of AChE.
The enzyme kinetics study for the most potent compound BS-10
on AChE was performed by the Lineweaver-Burk method to
21, IC50
¼
68.02 nM, BS-23, IC50
¼
66.07 nM and BS-24,
6

S. Manzoor, S.K. Prajapati, S. Majumdar et al.
European Journal of Medicinal Chemistry 215 (2021) 113224
Table 2
Structure, binding free energy values (Kcal/mol), in vitro inhibitory activity of AChE and BuChE, and AChE selective index of evaluated compounds.
Compounds
R1
R2
R3
free energy (Kcal/mol) AChE
IC50 (nM)a SEM
AChE
AChE Selectivity ratiob
BuChE
BS-1
BS-2
BS-3
BS-4
BS-5
BS-6
BS-7
BS-8
OCH3
OCH3
OCH3
OCH3
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
OCH3
OCH3
OCH3
OCH3
e
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
e
H
Cl
F
OCH3
H
Cl
F
OCH3
H
Cl
F
OCH3
H
Cl
F
OCH3
H
Cl
F
OCH3
H
Cl
ꢀ11.37
ꢀ11.84
ꢀ11.02
ꢀ11.60
ꢀ10.27
ꢀ11.77
ꢀ10.34
ꢀ10.47
ꢀ11.89
ꢀ12.39
ꢀ12.35
ꢀ12.26
ꢀ10.76
ꢀ11.53
ꢀ11.33
ꢀ11.26
ꢀ9.69
132.05 0.025
87.62 0.027
91.9 0.030
79.25 0.026
117.8 0.21
143.51 0.09
97.43 0.045
78.99 0.023
88.79 0.05
47.33 0.02
76.52 0.034
79.16 0.08
71.89 0.089
77.91 0.06
100.6 0.13
68.5 0.03
>200
79.12 0.027
>200
84.65 0.031
68.02 0.062
51.36 0.04
66.07 0.032
85.58 0.012
26.55 0.04
>200
>200
0.42
0.4
0.42
0.4
167.9 0.70
194.25 0.66
>200
155.51 0.9
194.3 0.5
184.3 0.73
167.5 0.65
159.43 0.72
186.2 0.84
>200
191.9 0.05
178.16 0.5
156.6 0.37
187.5 0.92
>200
137.32 0.73
>200
184.5 0.73
>200
153.3 0.74
176.5 0.67
>200
0.54
0.61
0.35
0.42
0.35
0.23
0.41
0.38
0.33
0.43
0.64
0.36
e
BS-9
BS-10
BS-11
BS-12
BS-13
BS-14
BS-15
BS-16
BS-17
BS-18
BS-19
BS-20
BS-21
BS-22
BS-23
BS-24
DZP
ꢀ10.19
ꢀ9.90
0.57
e
ꢀ10.06
ꢀ11.44
ꢀ12.37
ꢀ10.96
ꢀ11.46
ꢀ7.84
0.29
0.31
0.28
0.37
0.4
F
OCH3
e
1210 0.35
0.02
a
Values are expressed as mean SD of three independent experiments.
Selectivity index for AChE is defined as IC50(BuChE)/IC50(AChE).
b
determine the mechanism of inhibition of cholinesterase enzyme
[58]. The Lineweaver- Burk plot and Michaelis-Menten graph
showed decrease in Vmax, while increase in concentration of
substrate Km remained constant as illustrated in Fig. 5. The
Lineweaver-Burk plots suggested that the compound BS-10 fol-
lowed a non-competitive type of enzyme inhibition on both
enzyme-substrate complex and free enzyme. The intersection
points were found to be greater than zero on both 1/[S] and 1/Vmax
axes, implying that the inhibitor with free enzyme displayed strong
affinity instead of the enzyme-substrate complex.
In addition, three concentrations including 25, 50, and 100 nM
along with control (0 nM) of inhibitors were used to plot the Dixon
plot and the result indicated that compound BS-10 has Ki ¼ 8 nM.
Among these compounds, further kinetic measurement of com-
pound BS-10 was selected for its most potent AChE inhibitor. The
Michaelis e Mental’s linear Lineweaver e Burk equation was used
to measure the inhibition profile. In Fig. 5, compound BS-10 dis-
played the graphical study of steady-state inhibition, which dis-
played different slopes and intercepts followed by increasing
compound BS-10 concentration. Results suggested that increased
Km value with concentration however Vmax remains constant for
BS-10 compound implicating mixed-type inhibition. Further, it is
indicated that the compound BS-10 may be able to bind with the
enzyme-substrate complex and free enzyme with specific bind
equilibrium constants. The free enzyme inhibition constants (E), Ki,
and that one enzyme-substrate (ES) complex, Ki, are derived from
re-plots of the concentration of compound BS-10 versus respective
slope and intercept, both of which are linear Fig. 5.
compounds BS-10 and BS-22 were able to bind both PAS and CAS of
AChE. Thus, the ability of compound BS-10 and BS-22 to inhibit
hAChE-induced Ab1-42 aggregation was evaluated by Thioflavin T
(ThT) assay. A fixed amount of A
trations of donepezil drug, compounds BS-10 and BS-22 (5, 10 and
20 M) were used in the Thioflavin T-assay (Fig. 6). A with AChE
were used as a negative control. At 5 M concentration, donepezil,
BS-10, and BS-22 did not produce much significant results. Com-
pound BS-10 at 10:10 and 10:20 M concentrations showed 35.5
and 51.3% inhibition, respectively. Further, BS-22 at 10:10 M and
10:20 concentration showed 22.2 and 35.3% inhibition,
respectively. Finding results indicated that BS-10 at 10 M and
20 M concentrations showed more potent that BS-22. Interest-
b (10 mM) and variable concen-
m
b
m
m
m
mM
m
m
ingly, both the compounds (BS-10 and BS-22) showed dose-
dependent effects on thioflavin T-assay aggregation, indicating
the direct effect on AChE-induced Ab aggregation.
2.5.3. Dynamic light scattering studies
Dynamic light scattering (DLS) technique are used to monitor
the rate of A aggregation and the size of aggregates. Moreover, the
b
ability of BS-10 and BS-22 to inhibit self-induced as well as AChE-
induced amyloid aggregation was evaluated using dynamic light
scattering (DLS) studies. As shown in Figs. 7 and 8, BS-10 and BS-22
(10
mM) resulted in effective reduction in the average particle size of
Ab42 aggregates as determined by DLS.
2.5.4. Transmission electron microscopy (TEM) assay
TEM was further used to confirm the ability of BS-10 and BS-22
compounds to inhibit the Ab1e42 aggregation. As shown in (Fig. 9b)
2.5.2. AChE-induced A
The AChE was also illustrated to be involved in the development
of amyloid fibrils due to the interaction of AChE with A by a hy-
drophobic region close to PAS. Therefore, compounds binding to
the PAS of AChE can decrease AChE-accelerated A aggregation
[59]. As described in molecular docking study, the designed
b
aggregation assay (thioflavin T-assay)
A
b1e42 alone after incubation at 37 ꢁC for 24 h aggregated into long
and network fibril as compared to Ab1e42 alone at 0 ꢁC for 0 h
(Fig. 9a). Whereas, disaggregation effects were significantly visible
b
upon addition of compounds BS-10, BS-22 and Donepezil (22
each), as sparse A fibrils were observed (Fig. 9c, d and e), respec-
tively, as compared to Ab1e42 alone (Fig. 9b). These results indicate
mM
b
b
7

S. Manzoor, S.K. Prajapati, S. Majumdar et al.
European Journal of Medicinal Chemistry 215 (2021) 113224
Fig. 5. Lineweaver-Burk plot on three distinct concentration of BS-10 compound for AChE: Vmax, Km and Vmax/Km at 25 nM, 50 nM and 100 nM are found to be 7.2 0.83 U/min,
6.52 0.66 U/min, 5.54 0.52 U/min and 77.24 9.6 nM, 85.23 8.3 nM, 96.5 9.44 nM and 0.094, 0.076, 0.056 respectively. The Km of the enzyme kinetics suggests that a >1
(aKi).
that the compounds BS-10 and BS-22 significantly inhibited and
slowed down the aggregation of A at physiological temperature.
2.5.6. Bloodꢀbrain barrier penetration
b
The ability of small molecules to cross the blood-brain barrier
(BBB) is essential for their development into therapeutics for
neurodegenerative diseases. Thus, we performed parallel artificial
membrane permeability assay for BBB (PAMPA-BBB) [61] for BS-10
and BS-22 to estimate their ability to cross the BBB. The perme-
ability (Pe) values of 24.3 ꢂ 10ꢀ6 and 23.7 ꢂ 10ꢀ6 cm/s for BS-10
and BS-22, respectively, indicates high potential for BBB perme-
ability (Table 3). Donepezil as a high passive permeability drug and
Caffeine as a low passive permeability drug were used as controls
for the permeability assay as shown in Table 3.
2.5.5. Neurotoxicity studies on MC-65 cell lines
Human neuroblastoma cell line MC-65 generates Ab1-40 by
conditional expression of protein C-terminal APP. It facilitates
cytotoxicity of cells by fragmentation of DNA generated by ROS
[60]. The cell line in tetracycline removal condition (-TC) is asso-
ciated with oxidative stress and A
b stimulated cellular toxicity.
MMT assay were used to determine the therapeutic viability and
effectiveness of candidate molecules to combat oxidative stress.
Based on the analysis of cytotoxicity and neuroprotection, the most
active compounds BS-10 and BS-22 (1e80
mM) was chosen in
2.6. Physicochemical properties and ADME analysis
Fig. 10. The ability of neuroprotective of compound BS-10 is iden-
tical to tetracycline (TCþ) as compared to BS-22. The cell viability
percentage of compound BS-10 was estimated to be 99.41 in rela-
tion to þ TC (Fig. 10). In the MTT assay, it was observed that the BS-
10 molecule had the capability to compensate oxidative stress
induced by the unavailability of TC.
To determine the in silico physicochemical and pharmacokinetic
properties of synthesized compounds is the most important
method to develop a new drug molecule. To examine the possible
drug-likeness of compounds by two different methods of com-
pounds BS-10, BS-22 and DZP, physicochemical properties such as
8

S. Manzoor, S.K. Prajapati, S. Majumdar et al.
European Journal of Medicinal Chemistry 215 (2021) 113224
molecular weight (MW), hydrogen bond acceptors (HBA), number
of hydrogen bond donors (HBD), number of rotatable bonds (RB),
lipophilicity (LogP), topological polar surface area (TPSA) was pre-
dicted from the server Swiss ADME (http://www.swissadme.ch/)
and blood-brain barrier permeability (BBB) predicted from Pre-
ADMET server (http://preadmet.bmdrc.org) (Table 4). The ability to
penetrate the BBB is a requirement for neurotherapeutic drugs. The
online BBB permeability prediction of BS-10 and BS-22 yielded
score 0.05 for both, which are significantly higher than minimum
required for BBB permeation (0.02) [62]. It can be seen that the
compounds BS-10 and BS-22 obey most rules of Lipinski’s rule of
five [63] and veber’s rule of 3 [64]. Such results suggest that the
desired compounds BS-10 and BS-22 are worthy to be orally active
drug candidate for the CNS.
In addition, all the synthesized target compounds (BS-1 to BS-24
and DZP) were screened against online PreADMET server to
determine pharmacokinetic properties (Table S75 and S76, Sup-
porting Information). The program determines pharmacokinetic
properties like human intestinal absorption (HIA%), MDCK cell
permeability, Plasma Protein Binding (%), skin permeability (log
kp), BBB penetration and CaCo-2 permeability. This study revealed
that all the target compounds (BS-1 to BS-24) except DZP displayed
moderate permeability for Caco-2 cells. The human intestinal ab-
sorption (HIA) exhibited that all the target compounds (BS-1 to BS-
24 and DZP) exhibited high HIA% which revealed better absorption
to human Intestine. All the target compounds (BS-1 to BS-24) dis-
played moderately strong binding with Plasma Protein Binding (%)
and revealed very moderate permeability values for MDCK
permeability. All the synthesized target compounds (BS-1 to BS-24)
are expected to exhibit moderate penetration through BBB
(BloodeBrain Barrier) to the central nerve system (CNS) and may be
less likely to create neurotoxicity. Among all these compounds,
potent compounds (BS-10 and BS-22) with good anti-Alzheimer’s
activity may reveal more penetration through BBB to the brain.
The metabolic properties of target compounds were predicted that
all compounds (BS-1 to BS-24) demonstrated no inhibition for
CYP2C19 and CYP2D6 but all molecules inhibit CYP2C9 and CYP3A4
which implies high risk for interaction with drugs which are
metabolized by same enzyme CYP2C9. In addition, all the target
compounds demonstrated inhibitors of P-gp (P-glycoprotein).
Fig. 6. Effect of Donepezil (DZP), compounds BS-10 and BS-22 on AChE induced A
b
aggregation: DZP, compound BS-10 and BS-22 displayed a concentration-dependent
decrease in the A aggregation. AChE-induced the A aggregation, and it is reduced
b
b
by the DZP, compound BS-10 and BS-22 (One-way ANOVA followed by one-way
analysis of variance***p < 0.0001), error bars represent the standard deviation (SD)
of the normalized fluorescence intensity (NFI).
3. Conclusion
Fig. 7. Changes in the average particle size of self-induced Ab42 (100
m
M) aggregation
based on DLS measurements in the presence and absence of 10
and BS-22. Error bars represent standard deviation (n ¼ 3).
mM of Donepezil, BS-10,
In summary, this work presented the computer-based design,
synthesis, characterized by spectroscopic techniques (HRMS, 1H
NMR, 13CNMR) and pharmacological evaluation of new series of
phenylsulfonyl-pyrimidine carboxylate hybrids as multifunctional
agents for AD therapy. The ParDock and AutoDock were used for
docking and scoring purposes. The molecular docking study of
designed compounds was achieved with the target enzyme to
elucidate binding affinity and these binding displayed better
ligand-receptor complexes of most potent compounds. The syn-
thesized hybrids exhibited moderate to excellent in vitro inhibitory
activity against AChE and BuChE at nanomolar concentration.
Among 24 compounds (BS-1 to BS-24), BS-10 and BS-22 com-
pounds
IC50 ¼ 47.33 0.02, 51.36 0.04 and BuChE, IC50 ¼ 159.43 0.72,
153.3 0.74 (nM), respectively. Compounds BS-10 and BS-22
exhibited
excellent
inhibition
against
AChE,
exhibited the highest AChE inhibitory activity, which is due to the
addition of electron-donating dimethoxy and chloro group which
increases the binding affinity towards AChE. By molecular docking,
these derivatives were able to bind with both CAS and PAS of AChE
simultaneously. In the enzyme kinetics study, molecule BS-10
exhibited non-competitive inhibition of AChE with Ki ¼ 8 nM.
The lead compounds BS-10 and BS-22 inhibited AChE-induced Ab1-
Fig. 8. Changes in the average particle size of AChE-induced Ab42 (100
m
M) aggregation
based on DLS measurements in the presence and absence of 10
mM of Donepezil, BS-10,
and BS-22. Error bars represent standard deviation (n ¼ 3).
9

S. Manzoor, S.K. Prajapati, S. Majumdar et al.
European Journal of Medicinal Chemistry 215 (2021) 113224
Fig. 9. TEM image analysis of Ab1e42 aggregation with or without of compounds BS-10, BS-22 and Donepezil; (a) Ab1e42 (44
m
M), 0 ꢁC, 0 h; (b) Ab1e42 alone (44
mM) and BS-10 (22 m mM) and BS-22 (22 m
37 ꢁC for 24 h; (c) Ab1e42 (44 M) were co-incubated at 37 ꢁC for 24 h; (d) Ab1e42 (44 M) were co-incubated at 37 ꢁC for 24 h; (e) Ab1e42
mM) was incubated at
(44
m
M) and Donepezil (22
m
M) were co-incubated at 37 ꢁC for 24 h. Scale bar ¼ 500 nm.
Fig. 10. MC65 neuroprotection with compound BS-10 and BS-22: In the absence of TC, MC65 cells were treated with BS-10 and BS-22 compounds at specified concentrations. TCþ
was carried as control kg (One-way ANOVA followed by multiple comparison Newman-Keulstest evaluate all pair of column ***p < 0.0001).
Table 3
PAMPA-BBB permeability (Pe ꢂ 10ꢀ6 cm/s) of BS-10 and BS-22 and controls expressed as Pe and their predictive penetration to the CNS.
Comp.
Pe ( ꢂ 10ꢀ6 cm/s)
Predication of CNS penetration
BS-10
BS-22
Donepezil
Caffeine
24.3
23.7
22.8
2.18
High
High
High
Low
Table 4
Physiochemical properties of compounds BS-10, BS-22, and donepezil (DZP).
Compound
MWa
HBAa
HBDa
rot. bondsa
tPSAa
cLogPa
LogBBb
BS-10
BS-22
618.10
576.08
379.4
ꢃ500
10
9
4
ꢃ10
1
1
0
ꢃ5
9
8
6
ꢃ10
148.6
145.5
38.7
ꢃ140
3.3
3.1
4.0
2e5
0.05
0.05
0.18
>0.02
DZP
crequired parameters
a
Calculated using Swiss ADME online server: MW ¼ molecular weight; HBD ¼ number of hydrogen donors; HBA ¼ number of hydrogen acceptors; rot. bonds ¼ number of
rotatable bonds; cLogP ¼ log octanol/water partition coefficient; tPSA ¼ total polar surface area.
b
calculated using PreADMET online server: LogBB ¼ brain/blood partition coefficient.
c
Required parameters necessary to achieve suitable physiochemical properties mostly important for BBB permeation.
10

S. Manzoor, S.K. Prajapati, S. Majumdar et al.
European Journal of Medicinal Chemistry 215 (2021) 113224
42 aggregation in thioflavin T-assay. The respective compounds BS-
water (3 ꢂ 50 ml) and dried under vaccum to afford pure inter-
mediate 6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-
5-carboxylate derivatives 4(a-c) as a white solid in 60e70% yield.
10 at 10:10 and 10:20
inhibition, while BS-22 at 10:10 and 10:20
revealed 22.2 and 35.3%, results that BS-10 at 10
m
M concentration exhibited 35.5 and 51.3%
M concentration
M and 20
m
m
mM
concentration revealed more potent than BS-22. Moreover, DLS
studies exhibited that BS-10 and BS-22 inhibit self-induced as well
4.1.2. General procedure for the synthesis of ethyl 6-methyl-2-oxo-4-
phenyl-1,2-dihydropyridine-5-carboxylate derivatives 5(a-c)
To a solution of 68e70% nitric acid (140 mL) in an open vessel
as AChE-induced Ab aggregation. The compound BS-10 and BS-22
(10 M) resulted in effective reduction in the average particle size
of Ab42 aggregates as determined by DLS. Importantly, compounds
BS-10 and BS-22 showed remarkable neuroprotection in MC65 cell
m
was
added
portion-wise
1,2,3,4-tetrahydropyrimidine-5-
carboxylate derivative 4(a-c) (0.0275 mol) at 0 ꢁC. After stirring
30 min at 0 ꢁC, the solution was left to reach room temperature and
stirred for an additional 2 h. The reaction mixture was poured into
crushed ice, neutralized (pH 7e8) with Na2CO3, and extracted with
CHCl3 (3 ꢂ 300 mL). Combined organic layers were washed with 3
times water, brine, dried over Na2SO4, filtered and concentrated in
vacuo to afford the desired solid ethyl 6-methyl-2-oxo-4-phenyl-
1,2-dihydropyridine-5-carboxylate derivatives 5(a-c) in 60e65%
yield and used directly in the next steps.
line assay at 80
mM. Compound BS-10 estimated to be 99.41 in
relation to þ TC was found to be a more significant neuroprotective
agent as compared to BS-22. The lead compounds, BS-10 and BS-22
with permeability (Pe) values of 24.3 ꢂ 10e6 and 23.7 ꢂ 10-6 cm/s
respectively, indicates high potential for BBB permeability. The in
silico physicochemical and pharmacokinetics properties indicates
that lead compounds predict low toxicity with proper absorption,
penetration, and permeability abilities in human body. All the
target compounds (BS-1 to BS-24) are expected to exhibit moderate
permeability for CaCO-2 cells, high human intestinal absorption
(HIA%), strong binding with Plasma Protein Binding (%), moderate
MDCK permeability. Overall, results suggest that BS-10 and BS-22
were the most effective MTDLs and could be regarded as a prom-
ising “lead” for AD therapy.
4.1.3. General procedure for the synthesis of ethyl-2-chloro-6-
methyl-4-phenylpyrimidine-5-carboxylate derivatives 6(a-c)
1,2-dihydropyridine-5-carboxylate derivatives 5(a-c) (0.02 mol)
was refluxed in phosphorus oxychloride (0.21 mol) at 105 ꢁC for
3e4 h. The resulting mixture was cooled to room temperature and
excess phosphorus oxychloride was reduced under reduced pres-
sure. The reaction mass was poured into crushed ice, extracted with
ethyl acetate (3 ꢂ 100 mL). The combined ethyl acetate layers were
washed with water, brine and dried over anhydrous Na2SO4,
filtered and evaporated under reduced pressure. Crude products
were purified by column chromatography using Ethyl acetate/pe-
troleum ether (20:80) as the mobile phase to obtain pure ethyl-2-
4. Materials and methods
4.1. Chemistry
All the reagents and chemicals used during synthesis and
characterization of the compounds without further purifications
were brought from commercial suppliers like Alfa-Aesar, Spec-
trochem Pvt. Ltd. (India), Sigma Aldrich and SRL (Sisco Research
Laboratories Pvt. Ltd.). The solvents were dried by using standard
methods. The melting points were recorded using Tanco PLT-276
apparatus and are uncorrected. The NMR (Nuclear Magnetic Reso-
nance) spectral data were recorded with Bruker Avance NMR
spectrometer, 1H NMR at 400 and 500 MHz and 13C NMR at 101 and
chloro-6-methyl-4-phenylpyrimidine-5-carboxylate
6(a-c) in 70e80% yield.
derivatives
4.1.4. General procedure for the synthesis of ethyl 4-methyl-6-
phenyl-2-(piperazin-1-yl)pyrimidine-5-carboxylate derivatives 7(a-
c)
In
a
round bottom flask ethyl-2-chloro-6-methyl-4-
phenylpyrimidine-5-carboxylate derivatives 6(a-c) (0.018 mol)
was added to a solution of piperazine (0.09 mol) and K2CO3
(0.06 mol) in CHCl3 (60 mL) at room temperature. The reaction
mixture was heated at 70e80 ꢁC for 4e5 h. The progress of reaction
was monitored through TLC. The reaction was then diluted with
CHCl3 (400 mL) and washed with water. Water layer was also
washed with CHCl3 (2 ꢂ 150 mL). Combined organic layers were
washed with water, brine and dried over anhydrous Na2SO4,
filtered and evaporated under reduced pressure. Crude products
were purified by column chromatography using MeOH/CHCl3
(30:70) as mobile phase to obtain pure ethyl 4-methyl-6-phenyl-2-
(piperazin-1-yl)pyrimidine-5-carboxylate derivatives 7(a-c) in
70e75% yield.
126 MHz. The chemical shift values were revealed in
d ppm and
tetramethylsilane (TMS) was used as internal standard. The signals
of NMR multiplicities are labelled as s (singlet), d (doublet), dd
(double doublet), t (triplet), q (quartet), m (multiplet). Mass spectra
of target compounds were acquired with an Agilent 6538 Ultra
High-Definition Accurate Mass-Q-TOF (LC-HRMS) instrument.
Analytical thin-layer chromatography was conducted using pre-
coated aluminum sheets with silica gel 60e120 F254 (Merck)
thickness (0.25 mm). The TLC plates were visualized in the Iodine
gas chamber and in long-wavelength (365 nm) UV and in short-
wavelength (254 nm) UV lights and flash column chromatog-
raphy was conducted on silica gel (SRL No. 63025 mesh 200e400).
The purity of target compounds was examined using Aquity UPLC
united with Column X-bridge C-18 as well as PDA detector. Samples
was dissolved in DMSO, acetonitrile and water, and an injection of
4.1.5. General procedure for synthesis of N-phenylacetamide
derivatives 9(a-d)
1
m
L was used. DMSO, acetonitrile and water used as gradient
To a 250 mL flask equipped with a stir-bar was added aniline
derivative (0.04 mol), triethylamine (0.06 mmol) in 100 mL of
CH2Cl2. Add a solution of acyl chloride (0.05 mol) in dry CH2Cl2
mobile phase and flow rate was 1 mL/min. All the target com-
pounds reported here displayed more than 93% UPLC purity.
(50 mL) dropwise to the reaction mixture for a period of 20e30 min
ꢁ
4.1.1. General procedure for synthesis of ethyl 6-methyl-2-oxo-4-
phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate derivatives 4(a-
c)
at 0 C. The reaction mixture was continued to stir to reach room
temperature for 1e2 h. The reaction was monitored through TLC
using 30% ethyl acetate in pet ether as a mobile phase. After
completion of reaction, the reaction mixture was then diluted with
water and extracted with CH2Cl2 (100 mL x 2). The combined
organic layers were washed with water, brine, dried over Na2SO4,
filtered and concentrated under reduced pressure to obtain the
solid product N-phenylacetamide derivatives 9(a-d) in 80e90%
To a stirred solution of substituted aldehydes 1(a-c) (0.094 mol),
Ethyl acetoacetate (0.10 mol) and urea (0.094 mol) in ethanol
(40 mL) was treated with 4e5 drops of concentrated hydrochloric
acid and heated to reflux for 5e6 h. The reaction mixture was
cooled to 0 ꢁC, and the obtained solid was filtered, washed with cold
11

S. Manzoor, S.K. Prajapati, S. Majumdar et al.
European Journal of Medicinal Chemistry 215 (2021) 113224
yield.
NMR (400 MHz, CDCl3)
d
8.59 (t, J ¼ 7.6 Hz, 1H), 7.65 (d, J ¼ 3.2 Hz,
1H), 7.56e7.50 (m, 3H), 7.48 (d, J ¼ 2.0 Hz, 1H), 6.92 (d, J ¼ 8.8 Hz,
2H), 4.12 (q, J ¼ 7.2 Hz, 2H), 4.08e4.04 (m, 4H), 3.85 (s, 3H),
3.14e3.03 (m, 4H), 2.43 (s, 3H), 2.25 (s, 3H), 1.07 (t, J ¼ 7.2 Hz, 3H);
4.1.6. General procedure for the synthesis of 4-acetylamino
benzenesulfonyl chloride derivatives 10(a-d)
In a round bottom flask equipped with a stir-bar was added
chlorosulfonic acid (0.13 mol) in a drop wise fashion in respective
N-phenylacetamide 9(a-d) (0.025 mol) at 0 ꢁC. The resulted reaction
mixture was heated to the appropriate temperature and the stirring
13C NMR (101 MHz, CDCl3)
d 169.72, 168.98, 167.20, 165.13, 161.47,
160.13, 152.73, 150.27, 132.53, 131.66, 130.67, 130.14, 125.20, 121.67,
114.84, 114.11, 61.62, 55.81, 46.41, 43.43, 25.22, 23.41, 14.21; HRMS
in MeOH for C27H30FN5O6S (Mol. wt ¼ 571.1901 g molꢀ1) (ESI, m/z):
þ
was continued until the gas evolution ceased almost completely
observed [MþH]þ: 572.1786; calculated [M H]þ: 572.1979. UPLC
(25 ꢁC/2 h for 9a; 70 ꢁC/3.5 h for 9b; 70 ꢁC/4 h for 9c; 25 C/2 h for
purity: 96.60%.
ꢁ
9d). Then the reaction mixture was cooled and poured on crushed
ice in a beaker with vigorous shaking in order to make free the
adhered sulfonyl chlorides on the walls of the beaker and then
aqueous phase was discarded. After washing the solid with ice-cold
water several times, the respective crude 4-acetylamino benzene-
sulfonyl chloride derivatives 10(a-d) (75e85% yield) was separated,
dried and used directly in the next steps.
4.1.7.4. Ethyl
2-(4-((4-acetamido-3-methoxyphenyl)sulfonyl)
piperazin-1-yl)-4-(4-methoxyphenyl)-6-methylpyrimidine-5-
carboxylate (BS-4). White solid; yield 87%; mp 194e196 ꢁC. 1H
NMR (400 MHz, CDCl3)
d
8.78 (d, J ¼ 2.0 Hz, 1H), 7.79 (s, 1H), 7.52 (d,
J ¼ 8.8 Hz, 2H), 7.47 (dd, J ¼ 8.8, 2.4 Hz, 2H), 7.01e6.87 (m, 2H), 4.11
(q, J ¼ 7.2 Hz, 2H), 4.08e4.02 (m, 4H), 3.95 (s, 3H), 3.84 (s, 3H),
3.11e3.06 (m, 4H), 2.42 (s, 3H), 2.21 (s, 3H), 1.06 (t, J ¼ 6.8 Hz, 3H);
4.1.7. General procedure for the synthesis of ethyl 2-(4-((4-
acetamido-3-substitutedphenyl)sulfonyl)piperazin-1-yl)-4-(4,5-
substitutedphenyl)-6-methylpyrimidine-5-carboxylate BS(1e12)
In a 100 mL round bottom flask 4-acetylamino benzenesulfonyl
chloride derivatives 10(a-d) (0.0028 mol) was added to a solution of
13C NMR (101 MHz, CDCl3)
d 169.79, 168.80, 167.15, 165.06, 161.43,
160.15, 151.29, 131.73, 130.17, 128.67, 127.92, 124.37, 119.21, 114.81,
113.98, 110.03, 61.57, 56.60, 55.81, 46.56, 43.44, 25.28, 23.41, 14.21;
HRMS in MeOH for C28H33N5O7S (Mol. wt ¼ 583.2101 g molꢀ1) (ESI,
m/z): observed [MþH]þ: 584.1867; calculated [MþH]þ: 584.2179.
UPLC purity: 97.11%.
ethyl
4-methyl-6-phenyl-2-(piperazin-1-yl)pyrimidine-5-
carboxylate derivatives 7(a-c) (0.0028 mol) and K2CO3
(0.003 mol) in CHCl3 (50 ml) at room temperature. The reaction
mixture was refluxed at 70e80 ꢁC for 2e3 h. TLC was used to
monitor the progress of reaction. After completion of reaction, the
reaction mixture was diluted with water and extracted with CHCl3
(3 ꢂ 70 mL). The organic layers were combined, washed with water,
brine, dried over Na2SO4 and concentrated under reduced pressure.
Crude products were purified by column chromatography using
Ethyl acetate/Pet. Ether as mobile phase to obtain pure BS (1e12)
compounds in 80e90% yield.
4.1.7.5. Ethyl 2-(4-((4-acetamidophenyl)sulfonyl)piperazin-1-yl)-
4-methyl-6-phenylpyrimidine-5-carboxylate (BS-5). White solid;
yield 85%; mp 159e161 ꢁC. 1H NMR (500 MHz, CDCl3)
d 7.70e7.65
(m, 4H), 7.60 (s, 1H, eNH), 7.52e7.49 (m, 2H), 7.41e7.38 (m, 3H),
4.15e3.94 (m, 4H, -Pip-H, 2H, eOCH2), 3.06e3.02 (m, 4H), 2.44 (s,
3H), 2.19 (s, 3H), 0.95 (t, J ¼ 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3)
d
168.92, 168.59, 167.11, 165.65, 159.74, 142.29, 139.08, 129.98,
129.57, 129.05, 128.20, 128.03, 119.31, 114.94, 61.11, 45.99, 42.98,
24.69, 23.07, 13.55; HRMS in MeOH for
C26H29N5O5S (Mol.
wt ¼ 523.1889 g molꢀ1) (ESI, m/z): observed [MþH]þ: 524.1966;
4.1.7.1. Ethyl 2-(4-((4-acetamidophenyl)sulfonyl)piperazin-1-yl)-
4-(4-methoxyphenyl)-6-methylpyrimidine-5-carboxylate (BS-1).
yellow solid; yield 90%; mp 123e125 ꢁC. 1H NMR (400 MHz, CDCl3)
calculated [MþH]þ: 524.1968. UPLC purity: 96.96%.
4.1.7.6. Ethyl 2-(4-((4-acetamido-3-chlorophenyl)sulfonyl)piper-
d
7.80 (s, 1H), 7.57 (s, 4H), 7.45e7.36 (m, 2H), 6.81 (d, J ¼ 8.8 Hz, 2H),
azin-1-yl)-4-methyl-6-phenylpyrimidine-5-carboxylate
(BS-6).
4.02 (q, J ¼ 7.1 Hz, 2H), 3.96e3.92 (m, 4H), 3.74 (s, 3H), 2.97e2.92
White solid; yield 83%; mp 177e179 ꢁC. 1H NMR (400 MHz, CDCl3)
(m, 4H), 2.32 (s, 3H), 2.07 (s, 3H), 0.97 (t, J ¼ 6.8 Hz, 3H); 13C NMR
d
8.64 (d, J ¼ 8.8 Hz,1H), 7.81 (s,1H), 7.78 (d, J ¼ 2.0 Hz,1H), 7.65 (dd,
(101 MHz, CDCl3)
d
169.81, 169.25, 167.14, 165.13, 161.45, 160.14,
J ¼ 8.8, 2.0 Hz, 1H), 7.53 (dd, J ¼ 7.6, 1.6 Hz, 2H), 7.44e7.39 (m, 2H),
4.12e4.03 (m, 4H, -Pip-H, 2H, eOCH2), 3.09e3.06 (m, 4H), 2.46 (s,
3H), 2.28 (s, 3H), 0.96 (t, J ¼ 6.8 Hz, 3H); 13C NMR (101 MHz, CDCl3)
142.91, 131.66, 130.66, 130.13, 129.41, 119.78, 115.06, 114.10, 61.63,
55.80, 46.42, 43.41, 25.04, 23.42, 14.20; HRMS in MeOH for
C
27H31N5O6S (Mol. wt ¼ 553.1995 gmol-1) (ESI, m/z): observed
d 169.29, 168.87, 167.58, 166.10, 160.16, 139.50, 139.21, 131.14, 130.02,
[MþH]þ: 554.2135; calculated [MþH]þ: 554.2073. UPLC purity:
128.84, 128.65, 128.49, 127.99, 122.93, 121.36, 115.48, 61.55, 46.43,
43.42, 25.44, 23.49, 13.99; HRMS in MeOH for C26H28ClN5O5S (Mol.
wt ¼ 557.1500 g molꢀ1) (ESI, m/z): observed [MþH]þ: 558.1382;
calculated [MþH]þ: 558.1578. UPLC purity: 99.56%.
95.89%.
4.1.7.2. Ethyl 2-(4-((4-acetamido-3-chlorophenyl)sulfonyl)piper-
azin-1-yl)-4-(4-methoxyphenyl)-6-methylpyrimidine-5-
carboxylate (BS-2). White solid; yield 80%; mp 138e139 ꢁC. 1H
4.1.7.7. Ethyl 2-(4-((4-acetamido-3-fluorophenyl)sulfonyl)piper-
NMR (400 MHz, CDCl3)
d
8.62 (d, J ¼ 8.8 Hz, 1H), 7.79 (s, 1H), 7.76 (d,
azin-1-yl)-4-methyl-6-phenylpyrimidine-5-carboxylate
(BS-7).
J ¼ 2.0 Hz, 1H), 7.63 (dd, J ¼ 8.8, 2.0 Hz, 1H), 7.51 (d, J ¼ 8.8 Hz, 2H),
6.90 (d, J ¼ 8.8 Hz, 2H), 4.10 (q, J ¼ 6.8 Hz, 2H), 4.07e4.03 (m, 4H),
3.83 (s, 3H), 3.08e3.03 (m, 4H), 2.41 (s, 3H), 2.26 (s, 3H), 1.04 (t,
White solid; yield 80%; mp 189-180 ꢁC. 1H NMR (500 MHz, CDCl3)
d
8.54 (t, J ¼ 8.0 Hz, 1H), 7.56e7.41 (m, 5H), 7.40e7.30 (m, 3H),
4.14e4.83 (m, 4H, -Pip-H, 2H, eOCH2), 3.17e2.85 (m, 4H), 2.40 (s,
3H), 2.20 (s, 3H), 0.90 (t, J ¼ 7.5 Hz, 3H); 13C NMR (126 MHz, CDCl3)
J ¼ 7.2 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 169.70, 168.88, 167.22,
165.13, 161.47, 160.14, 139.21, 131.69, 131.13, 130.14, 128.83, 127.99,
122.93, 121.35, 115.16, 114.12, 61.60, 55.81, 46.43, 43.42, 25.43,
d 167.85, 167.44, 166.11, 164.62, 164.57, 158.71, 138.02, 130.03,
128.56, 127.19, 127.01, 123.73, 120.18, 114.01, 113.58, 113.40, 60.10,
45.02, 44.95, 41.97, 23.78, 22.03, 12.53; HRMS in MeOH for
23.43, 14.20; HRMS in MeOH for
C27H30ClN5O6S (Mol.
wt ¼ 587.1605 g molꢀ1) (ESI, m/z): observed [MþH]þ: 588.1481:
C
26H28FN5O5S (Mol. wt ¼ 541.1795 g molꢀ1) (ESI, m/z): observed
calculated [MþH]þ: 588.1684. UPLC purity: 93.08%.
[MþH]þ: 542.1925; calculated [MþH]þ:542.1873. UPLC purity:
99.79%.
4.1.7.3. Ethyl 2-(4-((4-acetamido-3-fluorophenyl)sulfonyl)piper-
azin-1-yl)-4-(4-methoxyphenyl)-6-methylpyrimidine-5-
carboxylate (BS-3). White solid; yield 85%; mp 157e159 ꢁC. 1H
4.1.7.8. Ethyl
2-(4-((4-acetamido-3-methoxyphenyl)sulfonyl)
piperazin-1-yl)-4-methyl-6-phenylpyrimidine-5-carboxylate (BS-
12

S. Manzoor, S.K. Prajapati, S. Majumdar et al.
European Journal of Medicinal Chemistry 215 (2021) 113224
8). White solid; yield 85%; mp 148e151 ꢁC. 1H NMR (400 MHz,
111.80, 111.20, 110.03, 61.60, 56.60, 56.43, 46.54, 43.48, 25.24, 23.34,
CDCl3)
d
8.79 (d, J ¼ 1.6 Hz, 1H), 7.79 (s, 1H), 7.53 (dd, J ¼ 7.6, 1.6 Hz,
14.23;
HRMS
in
MeOH
for
C
29H35N5O8S
(Mol.
2H), 7.48 (dd, J ¼ 8.6, 2.4 Hz, 1H), 7.42e7.39 (m, 3H), 6.95 (d,
J ¼ 8.8 Hz, 1H), 4.11e4.01 (m, 4H, -Pip-H, 2H, eOCH2), 3.95 (s, 3H),
3.13e3.07 (m, 4H), 2.45 (s, 3H), 2.22 (s, 3H), 0.96 (t, J ¼ 6.4 Hz, 3H);
wt ¼ 613.2206 g molꢀ1) (ESI, m/z): observed [MþH]þ: 614.1949;
calculated [MþH]þ: 614.2285. UPLC purity: 96.33%.
13C NMR (101 MHz, CDCl3)
d
169.38, 168.78, 167.51, 166.03, 160.21,
4.1.8. General procedure for the synthesis of ethyl 2-(4-((4-
amino-3-substitutedphenyl)sulfonyl)piperazin-1-yl)-4-(4-
substitutedphenyl)-6-methylpyrimidine-5-carboxylate
BS(13e24)
151.30, 139.56, 129.96, 128.68, 128.62, 128.51, 128.04, 124.39, 119.27,
115.16, 110.03, 61.50, 56.60, 46.56, 43.45, 25.26, 23.48, 13.99; HRMS
in MeOH for C27H31N5O6S (Mol. wt ¼ 553.1995 g molꢀ1) (ESI, m/z):
observed [MþH]þ: 554.2118; calculated [MþH]þ: 554.2073. UPLC
purity: 99.36%.
Compounds BS(1e12) (0.0008 mol) was suspended in a mixture
of 6N HCl (4 mL) and 1,4 Dioxane (10 mL) and was stirred at 80 ꢁ
C
for 2e3 h. The reaction mixture was cooled, poured into crushed ice
with vigorous stirring, neutralized with solution of sodium hy-
droxide. The precipitate settled down was filtered to dryness and
purified by column chromatography using Ethyl acetate/Pet. Ether
as mobile phase to obtain pure BS(13e24) compounds in 70e85%
yield.
4.1.7.9. Ethyl 2-(4-((4-acetamidophenyl)sulfonyl)piperazin-1-yl)-
4-(3,4-dimethoxyphenyl)-6-methylpyrimidine-5-carboxylate (BS-
9). White solid; yield 90%; mp 180e182 ꢁC. 1H NMR (500 MHz,
DMSO- d6)
d
10.36 (s, 1H), 7.81 (d, J ¼ 8.8 Hz, 2H), 7.66 (d, J ¼ 8.8 Hz,
2H), 7.09 (dd, J ¼ 5.5, 2.0 Hz, 2H), 7.03 (d, J ¼ 8.8 Hz, 1H), 4.09 (q,
J ¼ 7.2 Hz, 2H), 3.93 (s, 4H), 3.80 (s, 3H), 3.76 (s, 3H), 2.93 (s, 4H),
2.34 (s, 3H), 2.08 (s, 3H), 1.01 (t, J ¼ 7.2 Hz, 3H); 13C NMR (126 MHz,
4.1.8.1. Ethyl
2-(4-((4-aminophenyl)sulfonyl)piperazin-1-yl)-4-
DMSO‑d6)
d
169.03, 168.33, 165.80, 163.72, 159.23, 150.37, 148.33,
(4-methoxyphenyl)-6-methylpyrimidine-5-carboxylate(BS-13).
143.56, 130.54, 128.77, 128.08, 121.93, 118.61, 114.11, 111.38, 60.85,
55.60, 45.68, 42.44, 24.09, 22.57, 13.55; HRMS in MeOH for
Pale yellow; yield 80%; mp 120e122 ꢁC. 1H NMR (400 MHz, CDCl3)
d
7.53 (dd, J ¼ 8.8, 2.8 Hz, 4H), 6.92 (d, J ¼ 8.8 Hz, 2H), 6.74e6.61 (m,
C
28H33N5O7S (Mol. wt ¼ 583.2101 g molꢀ1) (ESI, m/z): observed
4H), 4.12 (q, J ¼ 7.2 Hz, 2H), 4.07e4.02 (m, 4H), 3.85 (s, 3H),
[MþH]þ: 584.1809; calculated [MþH]þ: 584.2179. UPLC purity:
3.06e3.01 (m, 4H), 2.43 (s, 3H), 1.06 (t, J ¼ 7.2 Hz, 3H); 13C NMR
98.27%.
(101 MHz, CDCl3) d 169.75, 167.20, 165.15, 161.43, 160.22, 151.31,
131.82, 130.74, 130.36, 130.15, 123.72, 114.48, 114.10, 61.56, 55.81,
46.49, 43.43, 23.46, 14.20; HRMS in MeOH for C25H29N5O5S (Mol.
wt ¼ 511.1889 g molꢀ1) (ESI, m/z): observed [MþH]þ: 512.2023;
calculated [MþH]þ: 512.1968. UPLC purity: 95.73%.
4.1.7.10. Ethyl
2-(4-((4-acetamido-3-chlorophenyl)sulfonyl)
piperazin-1-yl)-4-(3,4-dimethoxyphenyl)-6-methylpyrimidine-5-
carboxylate (BS-10). White solid; yield 85%; mp 157e159 ꢁC. 1H
NMR (400 MHz, CDCl3)
d
8.62 (d, J ¼ 8.8 Hz,1H), 7.82 (s, 1H), 7.77 (d,
J ¼ 2.0 Hz, 1H), 7.64 (dd, J ¼ 8.8, 2.0 Hz, 1H), 7.20e7.08 (m, 2H), 6.89
(d, J ¼ 8.0 Hz, 1H), 4.11 (q, J ¼ 7.2 Hz, 2H), 4.08e4.05 (m, 4H), 3.92 (s,
3H), 3.89 (s, 3H), 3.09e3.05 (m, 4H), 2.43 (s, 3H), 2.28 (s, 3H), 1.06 (t,
4.1.8.2. Ethyl 2-(4-((4-amino-3-chlorophenyl)sulfonyl)piperazin-
1-yl)-4-(4-methoxyphenyl)-6-methylpyrimidine-5-carboxylate
(BS-14). Pale yellow solid; yield 85%; mp 177e179 ꢁC. 1H NMR
J ¼ 7.2 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d
169.73, 168.89, 167.14,
(400 MHz, CDCl3)
d
7.65 (d, J ¼ 2.0 Hz, 2H), 7.53 (d, J ¼ 8.8 Hz, 2H),
165.15, 160.14, 151.01, 149.23, 139.21, 131.95, 131.17, 128.84, 127.97,
122.95, 121.67, 121.30, 115.40, 111.81, 111.22, 61.65, 56.43, 46.41,
43.46, 25.42, 23.35, 14.23; HRMS in MeOH for C28H32ClN5O7S (Mol.
wt ¼ 617.1711 g molꢀ1) (ESI, m/z): observed [MþH]þ: 618.1596;
calculated [MþH]þ: 618.1789. UPLC purity: 98.20%.
7.43 (dd, J ¼ 8.4, 2.0 Hz, 2H), 6.92 (d, J ¼ 8.8 Hz, 2H), 6.78 (d,
J ¼ 8.8 Hz, 1H), 4.12 (q, J ¼ 7.2 Hz, 2H), 4.08e4.04 (m, 4H), 3.85 (s,
3H), 3.07e3.02 (m, 4H), 2.44 (s, 3H), 1.06 (t, J ¼ 7.1 Hz, 3H); 13C NMR
(101 MHz, CDCl3)
d 169.74, 167.22, 165.15, 161.45, 160.17, 147.65,
131.75, 130.76, 130.15, 129.84, 128.31, 124.47, 118.98, 115.11, 114.11,
61.58, 55.81, 46.48, 43.39, 23.45, 14.20; HRMS in MeOH for
4.1.7.11. Ethyl 2-(4-((4-acetamido-3-fluorophenyl)sulfonyl)piper-
azin-1-yl)-4-(3,4-dimethoxyphenyl)-6-methylpyrimidine-5-
carboxylate (BS-11). White solid; yield 80%; mp 140e142 ꢁC. 1H
C
25H28ClN5O5S (Mol. wt ¼ 545.1500 g molꢀ1) (ESI, m/z): observed
[MþH]þ: 546.1639; calculated [MþH]þ: 546.1578. UPLC purity:
93.21%.
NMR (400 MHz, CDCl3)
d
8.58 (t, J ¼ 8.0 Hz, 1H), 7.68 (d, J ¼ 3.2 Hz,
1H), 7.53 (d, J ¼ 8.8 Hz, 1H), 7.49 (dd, J ¼ 10.0, 2.0 Hz, 1H), 7.16 (d,
J ¼ 2.0 Hz, 1H), 7.13 (dd, J ¼ 2.8, 1.6 Hz, 1H), 6.89 (d, J ¼ 8.0 Hz, 1H),
4.11 (q, J ¼ 7.2 Hz, 2H), 4.08e4.03 (m, 4H), 3.92 (s, 3H), 3.89 (s, 3H),
3.16e3.04 (m, 4H), 2.43 (s,3H), 2.25 (s, 3H), 1.06 (t, J ¼ 7.2 Hz, 3H);
4.1.8.3. Ethyl 2-(4-((4-amino-3-fluorophenyl)sulfonyl)piperazin-
1-yl)-4-(4-methoxyphenyl)-6-methylpyrimidine-5-carboxylate
(BS-15). White solid; yield 75%; mp 193e195 ꢁC. 1H NMR
(400 MHz, CDCl3)
d
7.53 (d, J ¼ 8.8 Hz, 2H), 7.41e7.32 (m, 1H), 7.16
13C NMR (101 MHz, CDCl3)
d
169.76, 169.01, 168.90, 167.14, 167.11,
(d, J ¼ 7.6 Hz, 2H), 7.09 (d, J ¼ 8.4 Hz, 2H), 6.92 (d, J ¼ 8.4 Hz, 2H),
4.12 (q, J ¼ 7.2 Hz, 2H), 4.08e4.03 (m, 4H), 3.85 (s, 3H), 3.09e3.04
(m, 4H), 2.44 (s, 3H), 1.06 (t, J ¼ 7.2 Hz, 3H); 13C NMR (101 MHz,
165.13, 160.17, 151.00, 149.22, 131.95, 131.53, 131.30, 121.68, 115.48,
115.07, 114.85, 111.81, 111.22, 61.66, 56.43, 46.40, 43.46, 25.18, 23.35,
14.23; HRMS in MeOH for C28H32FN5O7S (Mol. wt ¼ 601.2006
g molꢀ1) (ESI, m/z): observed [MþH]þ: 602.2084; calculated
[MþH]þ: 602.2085. UPLC purity: 96.64%.
CDCl3) d 169.71, 167.23, 165.15, 161.45, 160.17, 155.42, 152.96, 136.13,
131.76,130.68, 130.15, 125.68, 118.56, 116.06, 114.11, 61.59, 55.81,
46.49, 43.43, 23.45, 14.20; HRMS in MeOH for C25H28FN5O5S (Mol.
wt ¼ 529.1795 g molꢀ1) (ESI, m/z): observed [MþH]þ: 530.1656;
calculated [MþH]þ: 530.1873. UPLC purity: 94.92%.
4.1.7.12. Ethyl
2-(4-((4-acetamido-3-methoxyphenyl)sulfonyl)
piperazin-1-yl)-4-(3,4-dimethoxyphenyl)-6-methylpyrimidine-5-
carboxylate (BS-12). White solid; yield 80%; mp 123e125 ꢁC. 1H
4.1.8.4. Ethyl
2-(4-((4-amino-3-methoxyphenyl)sulfonyl)piper-
NMR (400 MHz, CDCl3)
d
8.77 (d, J ¼ 2.0 Hz, 1H), 7.79 (s, 1H), 7.47
azin-1-yl)-4-(4-methoxyphenyl)-6-methylpyrimidine-5-
(dd, J ¼ 8.8, 2.4 Hz, 1H), 7.15 (d, J ¼ 2.0 Hz, 1H), 7.13 (s, 1H), 6.93 (d,
J ¼ 8.8 Hz, 1H), 6.88 (d, J ¼ 8.8 Hz, 1H), 4.11 (q, J ¼ 6.8 Hz, 2H),
4.06e4.02 (m, 4H), 3.94 (s, 3H), 3.91 (s, 3H), 3.89 (s, 3H), 3.12e3.07
(m, 4H), 2.41 (s, 3H), 2.21 (s, 3H), 1.05 (t, J ¼ 7.2 Hz, 3H); 13C NMR
carboxylate(BS-16). Pale yellow solid; yield 80%; mp 183e185 ꢁC.
1H NMR (400 MHz, CDCl3)
d
7.53 (d, J ¼ 8.8 Hz, 2H), 7.13 (dd, J ¼ 8.4,
2.0 Hz, 1H), 7.05 (d, J ¼ 2.0 Hz, 2H), 6.92 (d, J ¼ 8.8 Hz, 2H), 6.83 (d,
J ¼ 8.4 Hz, 2H), 4.12 (q, J ¼ 7.2 Hz, 2H), 4.07e4.03 (m, 4H), 3.90 (s,
3H), 3.85 (s, 3H), 3.07e3.03 (m, 4H), 2.43 (s, 3H), 1.06 (t, J ¼ 7.2 Hz,
(101 MHz, CDCl3)
d 169.83, 168.80, 167.07, 165.06, 160.21, 151.31,
150.96, 149.21, 132.02, 128.66, 128.00, 124.37, 121.68, 119.24, 115.08,
3H); 13C NMR (101 MHz, CDCl3)
d 170.41, 167.88, 165.83, 162.10,
13

S. Manzoor, S.K. Prajapati, S. Majumdar et al.
European Journal of Medicinal Chemistry 215 (2021) 113224
160.87, 151.62, 137.94, 132.51, 130.82, 128.19, 119.87, 115.68, 114.77,
114.11, 110.81, 62.23, 56.86, 56.49, 47.24, 44.12, 24.13, 14.88; HRMS
in MeOH for C26H31N5O6S (Mol. wt ¼ 541.1995 g molꢀ1) (ESI, m/z):
observed [MþH]þ: 542.2215; calculated [MþH]þ: 542.2073. UPLC
purity: 95.77%.
148.76, 131.65, 130.01, 123.15, 121.25, 114.73, 114.09, 111.24, 110.72,
61.28, 56.06, 56.02, 46.12, 43.04, 23.06, 13.89; HRMS in MeOH for
C
26H31N5O6S (Mol. wt ¼ 541.1995g molꢀ1) (ESI, m/z): observed
[MþH]þ: 542.2075; calculated [MþH]þ: 542.2068. UPLC purity:
97.52%.
4.1.8.5. Ethyl 2-(4-((4-aminophenyl)sulfonyl)piperazin-1-yl)-4-
methyl-6-phenylpyrimidine-5-carboxylate (BS-17). Light brown
solid; yield 70%; mp 196e198 ꢁC. 1H NMR (400 MHz, CDCl3)
4.1.8.10. Ethyl
2-(4-((4-amino-3-chlorophenyl)sulfonyl)piper-
azin-1-yl)-4-(3,4-dimethoxyphenyl)-6-methylpyrimidine-5-
carboxylate (BS-22). Light brown solid; yield 80%; mp 121e123 ꢁC.
d
7.59e7.49 (m, 5H), 7.48e7.36 (m, 4H), 6.68 (d, J ¼ 8.4 Hz, 2H),
1H NMR (400 MHz, CDCl3)
d
7.65 (d, J ¼ 2.0 Hz, 1H), 7.44 (dd, J ¼ 8.8,
4.22e3.92 (m, 4H, -Pip-H, 2H, eOCH2), 3.06e3.02 (m, 4H), 2.46 (s,
2.0 Hz, 1H), 7.20e7.10 (m, 3H), 6.89 (d, J ¼ 8.0 Hz, 2H), 6.78 (d,
J ¼ 8.4 Hz, 1H), 4.12 (q, J ¼ 7.2 Hz, 2H), 4.08e4.04 (m, 4H), 3.92 (s,
3H), 3.90 (s, 3H), 3.08e3.03 (m, 4H), 2.44 (s, 3H), 1.06 (t, J ¼ 7.2 Hz,
3H), 0.96 (t, J ¼ 7.2 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 169.33,
167.56, 166.12, 160.24, 151.27, 139.63, 130.38, 129.96, 128.63, 128.49,
123.82, 115.25, 114.49, 61.50, 46.49, 43.44, 23.51, 13.99; HRMS in
MeOH for C24H27N5O4S (Mol. wt ¼ 481.1784 g molꢀ1) (ESI, m/z):
observed [MþH]þ: 482.1901; calculated [MþH]þ: 482.1862. UPLC
purity: 97.56%.
3H); 13C NMR (101 MHz, CDCl3)
d 169.78, 167.15, 165.17, 160.20,
150.98, 149.22, 147.62, 132.03, 129.86, 128.33, 124.55, 121.67, 119.00,
115.26, 115.10, 111.81, 111.21, 61.64, 56.44, 46.47, 43.43, 23.39, 14.24;
HRMS in MeOH for C26H30ClN5O6S (Mol. wt ¼ 575.1605 g molꢀ1
)
(ESI, m/z): observed [MþH]þ: 576.1393; calculated [MþH]þ:
4.1.8.6. Ethyl 2-(4-((4-amino-3-chlorophenyl)sulfonyl)piperazin-
576.1684. UPLC purity: 96.58%.
1-yl)-4-methyl-6-phenylpyrimidine-5-carboxylate
Light brown solid; yield 70%; mp 185e187 ꢁC. 1H NMR (400 MHz,
CDCl3)
(BS-18).
4.1.8.11. Ethyl 2-(4-((4-amino-3-fluorophenyl)sulfonyl)piperazin-
1-yl)-4-(3,4-dimethoxyphenyl)-6-methylpyrimidine-5-
carboxylate (BS-23). Light brown solid; yield 85%; mp 134e136 ꢁC.
d
7.66 (d, J ¼ 2.0 Hz, 2H), 7.54 (dd, J ¼ 7.6, 1.6 Hz, 2H), 7.45 (d,
J ¼ 2.0 Hz, 1H), 7.43e7.40 (m, 3H), 6.79 (d, J ¼ 8.4 Hz, 2H), 4.19e4.01
(m, 4H, -Pip-H, 2H, eOCH2), 3.08e3.03 (m, 4H), 2.47 (s, 3H), 0.96 (t,
1H NMR (400 MHz, CDCl3)
d 7.40e7.32 (m, 1H), 7.18e7.14 (m, 3H),
J ¼ 7.2 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d
169.33, 167.58, 166.13,
7.09 (dd, J ¼ 4.0, 2.0 Hz, 2H), 6.89 (d, J ¼ 8.0 Hz, 2H), 4.12 (q,
J ¼ 7.2 Hz, 2H), 4.07e4.04 (m, 4H), 3.92 (s, 3H), 3.90 (s, 3H),
3.09e3.03 (m, 4H), 2.43 (s, 3H), 1.06 (t, J ¼ 7.2 Hz, 3H); 13C NMR
160.20, 147.63,139.57, 130.00, 129.86, 128.64,128.50, 128.33,124.52,
119.01, 115.33, 115.11, 61.54, 46.49, 43.40, 23.52, 14.00; HRMS in
MeOH for C24H26ClN5O4S (Mol. wt ¼ 515.1394 g molꢀ1) (ESI, m/z):
observed [MþH]þ: 516.1541; calculated [MþH]þ: 516.14.72. UPLC
purity: 93.03%.
(101 MHz, CDCl3)
d 169.77, 167.15, 165.16, 160.21, 155.41, 152.95,
150.98, 149.22, 140.06, 132.03, 125.66, 123.98, 121.67, 118.57, 116.21,
111.81, 111.21, 61.64, 56.43, 46.48, 43.45, 23.38, 14.24; HRMS in
MeOH for C26H30FN5O6S (Mol. wt ¼ 559.1901 g molꢀ1) (ESI, m/z):
observed [MþH]þ: 560.2042; calculated [MþH]þ: 560.2042. UPLC
purity: 98.14%.
4.1.8.7. Ethyl 2-(4-((4-amino-3-fluorophenyl)sulfonyl)piperazin-
1-yl)-4-methyl-6-phenylpyrimidine-5-carboxylate
Light brown solid; yield 80%; mp 158e160 ꢁC. 1HNMR (500 MHz,
CDCl3)
7.45 (d, J ¼ 6.5 Hz, 2H), 7.36e7.28 (m, 5H), 7.07 (d,
(BS-19).
d
4.1.8.12. Ethyl 2-(4-((4-amino-3-methoxyphenyl)sulfonyl)piper-
azin-1-yl)-4-(3,4-dimethoxyphenyl)-6-methylpyrimidine-5-
carboxylate (BS-24). Off white solid; yield 85%; mp 141e143 ꢁC. 1H
J ¼ 7.0 Hz, 1H), 7.01 (d, J ¼ 9.0 Hz, 2H), 4.17e4.92 (m, 4H, -Pip-H, 2H,
eOCH2), 3.14e2.96 (m, 4H), 2.45 (s, 3H), 0.94 (t, J ¼ 7.0 Hz, 3H); 13
C
NMR (126 MHz, CDCl3)
d
168.88, 168.84, 167.20, 167.15, 165.68,
NMR (400 MHz, CDCl3)
d
7.17 (d, J ¼ 2.0 Hz, 1H), 7.14 (s, 2H), 7.12 (d,
159.77, 159.75, 139.12, 129.56, 128.20, 128.05, 125.23, 118.22, 115.62,
115.39, 61.09, 46.05, 42.99, 23.07, 13.55; HRMS in MeOH for
J ¼ 2.4 Hz, 1H), 7.04 (d, J ¼ 2.4 Hz, 2H), 6.89 (d, J ¼ 8.0 Hz, 1H), 6.83
(d, J ¼ 8.4 Hz, 1H), 4.11 (q, J ¼ 7.2 Hz, 2H), 4.07e4.03 (m, 4H), 3.92 (s,
3H), 3.90 (s, 3H), 3.90 (s, 3H), 3.08e3.03 (m, 4H), 2.43 (s, 3H),1.06 (t,
C
24H26FN5O4S (Mol. wt ¼ 499.1690 g molꢀ1) (ESI, m/z): observed
[MþH]þ: 500.1802; calculated [MþH]þ: 500.1768. UPLC purity:
J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 169.76, 167.12, 165.16,
98.40%.
160.20, 150.95, 149.21, 137.25, 132.10, 127.56, 127.51, 121.66, 119.20,
115.16, 113.44, 111.84, 111.21, 110.12, 61.59, 56.43, 56.18, 46.54, 43.48,
4.1.8.8. Ethyl
2-(4-((4-amino-3-methoxyphenyl)sulfonyl)piper-
23.38, 14.23; HRMS in MeOH for
C27H33N5O7S (Mol.
azin-1-yl)-4-methyl-6-phenylpyrimidine-5-carboxylate (BS-20).
wt ¼ 571.2101 g molꢀ1) (ESI, m/z): observed [MþH]þ: 572.1994;
Light brown solid; yield 75%; mp 139e141 ꢁC. 1H NMR (400 MHz,
calculated [MþH]þ: 572.2179. UPLC purity: 97.17%.
CDCl3)
d
7.48 (dd, J ¼ 7.6, 1.6 Hz, 2H), 7.39e7.34 (m, 4H), 7.08 (dd,
J ¼ 8.4, 2.0 Hz, 1H), 6.99 (d, J ¼ 2.0 Hz, 2H), 6.78 (d, J ¼ 8.4 Hz, 1H),
4.05e3.94 (m, 4H, -Pip-H, 2H, eOCH2), 3.86 (s, 3H), 3.02e2.97 (m,
4H), 2.41 (s, 3H), 0.90 (t, J ¼ 6.8 Hz, 3H); 13C NMR (101 MHz, CDCl3)
4.2. Docking protocol
The 3D crystal structure of AChE (PDB code:1eve) and BuChE
(PDB code: 4TPK) protein were obtained from the RCSB Protein
Data Bank (https://www.rcsb.org/) [65]. Molecular docking and
scoring determination of ligands with protein was performed by
using the AutoDock vina 1.5.6 [55] and Pardock [54] module of
Sanjeevini, a complete suite for drug design, based on physico-
chemical properties. Molecular docking of ligands with AChE pro-
tein were performed by using Pardock. The docked structures are
energy minimized by using AMBER sander module and Bappl
scoring function [66], based on all atom scoring function,
comprising electrostatic, hydrophobicity, van der Waals, loss of the
protein side chains conformation entropy after complexation. In
addition, Autodock vina were also used to perform molecular
docking studies of ligands with BuChE. The downloaded protein
from the protein data bank (PDB) were prepared by addition of
d
169.28,167.58,166.11,160.21,160.19,155.42,152.96,149.20,139.57,
135.99, 132.00, 129.99, 125.66, 118.58, 115.33, 61.53, 46.49, 43.43,
31.34, 23.51, 13.99; HRMS in MeOH for
C25H29N5O5S (Mol.
wt ¼ 511.1889 g molꢀ1) (ESI, m/z): observed [MþH]þ: 512.2007;
calculated [MþH]þ: 612.1968. UPLC purity: 95.82%.
4.1.8.9. Ethyl 2-(4-((4-amino-3-chlorophenyl)sulfonyl)piperazin-
1-yl)-4-(3,4-dimethoxyphenyl)-6-methylpyrimidine-5-
carboxylate (BS-21). Light brown solid; yield 85%; mp 133e135 ꢁC.
1H NMR (400 MHz, CDCl3)
d
7.49 (d, J ¼ 8.4 Hz, 2H), 7.19e7.02 (m,
3H), 6.86 (d, J ¼ 8.0 Hz, 2H), 6.64 (d, J ¼ 8.4 Hz, 2H), 4.08 (q,
J ¼ 7.2 Hz, 2H), 4.03e3.99 (m, 4H), 3.89 (s, 3H), 3.86 (s, 3H),
3.02e2.98 (m, 4H), 2.40 (s, 3H), 1.02 (t, J ¼ 6.8 Hz, 3H); 13C NMR
(101 MHz, CDCl3)
d 169.44, 166.78, 164.79, 159.82, 150.93, 150.50,
14

S. Manzoor, S.K. Prajapati, S. Majumdar et al.
European Journal of Medicinal Chemistry 215 (2021) 113224
hydrogen, removal of water and removal of original ligand from
PDB file. The files of designed ligand and prepared protein were
converted to pdbqt file by AutoDock. A grid box was constructed
around the docking area and docking procedure was carried out. In
last, Pymol visualizing software were used to visualize the inter-
action results of docking. Weak non-covalent interactions such as
experiment was carried out in triplicates, and the IC50 values of all
the assessed compounds were evaluated graphically by using log
concentration-percentage inhibition curves (Graph Pad Prism
5.01).
Enzyme kinetics experiment was conducted to understand the
mechanism of hAChE inhibition action by the most potent com-
pound BS-10. The enzyme kinetic parameters, Km and Vmax were
evaluated by determining the enzymatic study at three different
concentration of compound BS-10 range of 25, 50 and 100 nM. Each
concentration of compound BS-10 was evaluated with six separate
concentration of ATCI. The activity was evaluated for 10 min in the
presence and absence of compound BS-10 at an interval of 2 min.
The product developed during the 10 min time frame was
measured by the Beer-Lambert law. The values of Vmax and Km
were determined by using the Michaelis-Menten non-linear
regression graph, and the enzyme inhibition mechanism was
calculated by Lineweaver-Burk reciprocal linear regression plots
[58] by Graph Pad Prism 5.01. In addition, Dixon plot was used to
calculate the Ki value as a function of four separate concentrations
for inhibitor [70]. The enzyme-kinetic assay was carried out in
triplicate.
p-p stacking interaction and H-bonding between ligand and pro-
tein were observed.
4.3. X-ray crystallographic procedure
The Crystal structure of compound BS-19 was procured using
single-crystal X-ray diffraction method. The colorless rectangular
block crystals of complex BS-19 were acquired from mixture of
chloroform and methanol on slow evaporation. These crystals were
mounted on loops with mineral oil. All the geometric data and
intensity data were revealed by an automated Bruker SMART APEX
CCD diffractometer equipped with
(1.75 kW) Mo K X-ray source (
¼ 0.71073 Å) with increasing
(per frame width 0.3ꢁ) at 5 s frameꢀ1 scanning speed.
-2 scan
a fine focus sealed tube
a
l
u
u
q
mode were utilized for collecting intensity data and then corrected
for Lorentz-polarization as well as absorption effects [67]. The
crystal structures were solved and refined with WinGx suit of
programs (version 1.63.04a) via SHELXL-2013 method [68]. All non-
hydrogen atoms were refined with coefficients of anisotropic
displacement and non-hydrogen atoms coordinates were allowed
to be on their respective carbon atoms. The atomic position of all
atoms, isotropic thermal parameters for hydrogen atoms, and
anisotropic thermal parameters for non-hydrogen were implicated
in the final refinement. The structural view of crystallized mole-
cules (BS-19) were obtained using ORTEP [69]. Furthermore, the
details like crystallographic parameters; angles data and bond
distances, are depicted in Table 1. The CCDC deposition numbers for
BS-19 is 2032914.
4.4.2. AChE induced-A
In 1% v/v ammonium hydroxide solution was dissolved Ab1-42
M stock solution and was preserved
b inhibition thioflavin T-assay
(Sigma,ꢁIndia) to obtain 2000
m
at ꢀ80 C. At first, potent test compounds, BS-10 and BS-22 were
dissolved in DMSO, and further diluted with PBS (pH 7.4). For the
experiment of AChE-induced Ab1-42 aggregation inhibition, the Ab1-
42 (final concentration 10
(final concentration 230
for 48 h with or without tested compounds (5
20 M; 2 l) and were tested by thioflavin T-assay [71e73]. The test
solution was diluted with the addition of 50 mM glycine-NaOH
buffer (pH 8.0) containing 5 M thioflavin T to the final volume of
200 l. The results with respect to control were recorded as
m
M, 2
m
L) and human erythrocytes AChE
l) mixture were incubated at 37 ꢁC
M, 10 M, and
m
M, 16
m
m
m
m
m
m
m
4.4. Biological studies
normalized fluorescence intensity. All the tests were performed in
three separate experiments.
4.4.1. In vitro hAChE and hBuChE inhibition studies
The procedure of Cholinesterase (hAChE and hBuChE) inhibition
assay of synthesized compounds was performed spectrometrically
by using Elmann et al. method [56]. Both enzymes hAChE (CAS N0.
9000-81-1) and hBuChE (CAS N0. 9001-08-5) were purchased from
sigma Aldrich. Hydrolysis of thiolated substrates are mostly cata-
lyzed by cholinesterase to produce the thiocholine, which de-
creases the 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) to give
product in yellow color that can be analyzed calorimetrically at
412 nm. AChE from human erythrocytes (EC No. 3.1.1.7, buffered
aqueous solution, ꢄ500 units/mg protein) was dissolved in 20 mM
HEPES buffer pH 8, 0.1% Triton X-100 and BuChE from human
serum (EC No. 3.1.1.8, vial of ꢄ4 units, Sigma Aldrich) was dissolved
in 0.1% aqueous solution of gelatine to obtain the 0.25U/ml enzyme
solutions. The stock solutions of inhibitors have been prepared in
dimethyl sulfoxide (DMSO), and five increasing inhibitors concen-
trations have been used that could induce 20e80% in range of
enzyme inhibition. The last test solution composed of 0.1 M
4.4.3. Monitoring amyloid aggregation using DLS
For self-induced amyloid aggregation, a stock solution of Ab1-42
(100
mM) in PBS (pH 7.4) was incubated with BS-10, BS-22, and
donepezil (10
m
M). The mixtures were incubated in the dark for
48 h at 37 ꢁC. Particle size distribution analysis was measured by
DLS experiments using a Malvern Zetasizer Nano ZS instrument.
For AChE-induced amyloid aggregation, the procedure above was
adopted in the presence of to 10
mL of AChE (10 unit/mL).
4.4.4. Transmission electron microscopy (TEM) assay
The morphology of A
pounds and donepezil was visualized using TEM. Ab1e42
(44 M) and with compounds BS-10, BS-22, and Donepezil (22
was incubated at 37 ꢁC for 24 h. After 24 h of incubation, aliquots of
10 L samples were placed on carbon-coated copper grid. The
b
fibrils with and without the test com-
controls
M)
A
b
m
m
m
samples were stained with 1% uranyl acetate and were placed on a
clean paper for removing excess staining solution and dried for
15 min at room temperature. Images from each sample were taken
by CRYO-TEM (TALOS S) transmission electron microscope.
phosphate-buffered saline (PBS) pH 7.4, 340 mM DTNB, 550 mM ATCI
or BTCI for hAChE or hBChE, respectively. The test solutions were
pre-incubated for 10 min and the substrate was added. The
increasing rate of absorbance was spectrometrically calculated at
37 ꢁC for 6 min at 412 nm with Multimode Microplate Reader
(BioTek Synergy H1M, USA). The blank measurements were also
performed by analyzing the solution for all components except for
AChE or BuChE to calculate the non-enzymatic reaction rate. The
reaction rates of all compounds were compared, and the inhibition
percentage was determined due to each tested compound. The
4.4.5. MC65 neuroprotection assay
MC65 cell was acquired as a kind gift from Dr. George M. Martin
(University of Washington) [74,75]. The kit of MTT assay was
bought from Himedia. The MC65 cells were cultivated in MEM,
carefully washed with PBS and were resuspended in MEM. More-
over, 5 ꢂ 104 cells/well were placed in 96 well plates. The cells were
15

S. Manzoor, S.K. Prajapati, S. Majumdar et al.
European Journal of Medicinal Chemistry 215 (2021) 113224
incubated in CO2 incubator (Heal force, Germany) with þTC and
Acknowledgments
-TC. In the assessed group (80-1
mM) TC was absent (TC-). Test
compounds BS-10 and BS-22 were added, and cells were incubated
Shoaib Manzoor (S M) is extremely thankful to University Grants
Commission, Government of India for financial assistance through
Central University Ph.D. Students Fellowship. S M thanks A. Q. Mir
and V Lakshmi to carry out HRMS data during revision of the
manuscript.
for 12
h
at 37 ꢁC. 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) was added (20 mL, 5 mg/ml)
at the end of 12 h and the cells were again incubated for the next
4 h. The medium was eliminated, and the formazan crystals formed
were dissolved in 150 mL of solvent (DMSO:H2O, 1:1). The absor-
bance was recorded immediately at 570 nm and the response was
conveyed in cell viability percentage relative to þ TC as control. The
cell assay was carried out in triplicate and in three distinct runs.
Abbreviations
AD
Alzheimer’s disease
multitarget-directed ligands
acetylcholinesterase
butyrylcholinesterase
acetylcholine
MTDLs
AChE
BuChE
Ach
4.4.6. Parallel artificial membrane permeation assay (PAMPA)
Donepezil and caffeine were used as controls in the experiment.
96-well donor microplates and 96-well acceptor microplates were
A
b
amyloid beta
purchased from Millipore. 300
the acceptor microplates. The filter membrane was filled with 10
of porcine brain lipid (PBL) in dodecane (20 mg/mL). Stock solutions
of BS-10 and BS-22 were diluted with PBS/EtOH (7:3) until 100 g/
mL concentration. The donor wells were then filled with 200 L of
MG-6267 solution and 300 L of PBS/EtOH (7:3). Donor filter plate
mL of PBS/EtOH (7:3) was added to
CNS
HIA
BBB
PPB
PAS
CAS
TLT
DLS
PAMPA
NMR
DMSO
CHCl3
POCl3
MeOH
K2CO3
rt
central nerve system
human intestinal absorption
blood-brain barrier
Plasma Protein Binding
peripheral anionic site
catalytic anionic site
transfer latency time
Dynamic light scattering
parallel artificial membrane permeation assay
Nuclear Magnetic Resonance
dimethyl sulfoxide
chloroform
phosphorus oxychloride
methanol
potassium carbonate
room temperature
mL
m
m
m
was placed on the acceptor plate and incubated for 16 h at 25 ꢁC.
After removal of the donor plate, the concentrations of BS-10 and
BS-22 were detected in the acceptor, donor and reference wells
using a plate reader. Samples were analyzed three independent
runs in four wells.
4.5. Physicochemical properties and ADME analysis
The physicochemical and pharmacokinetics properties of the
selected synthesized compounds were estimated by using online
server SwissADME (http://www.swissadme.ch/) [76] and Pre-
ADMET online tool (http://preadmet.bmdrc.org) [77]. The predic-
tion of physicochemical properties of BS-10 and BS-22 compounds
like molecular weight (MW), hydrogen bond acceptors (HBA),
number of hydrogen bond donors (HBD), number of rotatable
bonds (RB), lipophilicity (LogP), topological polar surface area
(TPSA) was done by SwissADME online tool. In silico ADME analysis
exhibiting the behavior of synthesized compounds within an or-
ganism by determining essential pharmacokinetics parameters like
distribution, metabolism, absorption, and excretion. The pharma-
cokinetic study of novel synthesized compounds (BS-1 to BS-24)
was predicted by using the PreADMET online tool. The PreADMET
online program describes pharmacokinetic properties like skin
permeability, Human intestinal absorption (HIA %), Plasma Protein
Binding, colon adenocarcinoma (Caco-2) cell permeability,
bloodebrain barrier (BBB) penetration, Madin-Darby canine kidney
(MDCK) cell permeability.
Mp
melting point
UV
ultra violet
TLC
ppm
thin layer chromatography
parts per million
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113224.
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Author contributions
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
Declaration of competing interest
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