In-vitro evaluation of antioxidant and anticholinesterase activities of novel pyridine, quinoxaline and s-triazine derivatives

Article abstract of DOI:10.1016/j.envres.2021.111320

Cholinesterase enzymes such as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) cause hydrolysis of acetylcholine (ACh), a neurotransmitter responsible for the cognitive functions of the brain such as acquiring knowledge and comprehension. Th

Full text of DOI:10.1016/j.envres.2021.111320

Environmental Research 199 (2021) 111320
Contents lists available at ScienceDirect
Environmental Research
journal homepage: www.elsevier.com/locate/envres
In-vitro evaluation of antioxidant and anticholinesterase activities of novel
pyridine, quinoxaline and s-triazine derivatives
,
,
M.V.K. Reddya 1, K.Y. Raob 1, G. Anushaa, G.M. Kumarc, A.G. Damub,
Kakarla Raghava Reddyd *, Nagaraj P. Shettie, Tejraj M. Aminabhavif ,
,
,*
Peddiahgari Vasu Govardhana Reddya
,
*
a Department of Chemistry, Organic and Biomolecular Chemistry Laboratories, Yogi Vemana University, Kadapa, 516005, Andhra Pradesh, India
b Department of Chemistry, Natural Products Laboratories, Yogi Vemana University, Kadapa, 516005, Andhra Pradesh, India
c Department of Biotechnology and Bioinformatics, Yogi Vemana University, Kadapa, 516005, Andhra Pradesh, India
d School of Chemical and Biomolecular Engineering, The University of Sydney, Sydney, NSW 2006, Australia
e School of Advanced Sciences, KLE Technological University, Vidyanagar, Hubballi, 580031, Karnataka, India
f Department of Chemistry, Karnatak University, Dharwad, 580 003, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Cholinesterase enzymes such as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) cause hydrolysis
of acetylcholine (ACh), a neurotransmitter responsible for the cognitive functions of the brain such as acquiring
knowledge and comprehension. Therefore, inhibition of these enzymes is an effective process to curb the pro-
gressive and fatal neurological Alzheimer’s disease (AD). Herein, we explored the potential inhibitory activities
of various pyridine, quinoxaline, and triazine derivatives (3a-k, 6a-j and 11a-h) against AChE and BuChE en-
zymes by following the modified Ellman’s method. Further, anti-oxidant property of these libraries was moni-
tored using DPPH (2,2′-diphenyl-1-picryl-hydrazylhydrate) radical scavenging analysis. From the studies, we
identified that compounds 6e, 6f, 11b and 11f behaved as selective AChE inhibitors with IC50 values ranging
Alzheimer’s disease
Antioxidant activity
Aza-heterocycles
Circular dichroism
Fluorescence assay
Molecular docking
from 7.23 to 10.35
μ
M. Further studies revealed good anti-oxidant activity by these compounds with IC50 values
M. The kinetic studies of the active analogues demonstrated mixed-type of inhi-
in the range of 14.80–27.22
μ
bition due to their interaction with both the catalytic active sites (CAS) and peripheral anionic sites (PAS) of the
AChE. Additionally, molecular simulation in association with fluorescence and circular dichroism (CD) spec-
troscopic analyses explained strong affinities of inhibitors to bind with AChE enzyme at the physiological pH of
7.2. Binding constant values of 5.4 × 104, 4.3 × 104, 3.2 × 104 and 4.9 × 104 Mꢀ 1 corresponding to free energy
changes ꢀ 5.593, ꢀ 6.799, ꢀ 6.605 and ꢀ 8.104 KcalMꢀ 1 were obtained at 25 ◦C from fluorescence emission
spectroscopic studies of 6e, 6f, 11b and 11f, respectively. Besides, CD spectroscopy deliberately explained the
secondary structure of AChE partly unfolded upon binding with these dynamic molecules. Excellent in vitro
profiles of distinct quinoxaline and triazine compounds highlighted them as the potential leads compared to
pyridine derivatives, suggesting a path towards developing preventive or therapeutic targets to treat the Alz-
heimer’s disease.
1. Introduction
worsening and or the death of the brain cells (Cavalli et al., 2008; Sal-
loway and Correia, 2015; Guzior et al., 2015). Approximately 5–20% of
Alzheimer’s disease (AD) is an age-related, multi-factorial neurode-
people of different ages all over the world suffer seriously from this
problem (Camps et al., 2000; He et al., 2013; Tsai et al., 2019; Liu et al.,
generative disorder shown by memory loss and cognitive impairments,
Abbreviations: ACh, acetylcholine; AChE, acetylcholinesterase; AD, Alzheimer’s disease; ATCh, S-acetylthiocholine iodide; BuChE, butyrylcholinesterase; BuTCh,
S-butyrylthiocholine iodide; CAS, catalytic active site; CD, circular dichroism; NHC–Cu, N-heterocyclic carbene-copper; PAS, peripheral anionic site; PB, phosphate
buffer; SAR, structure activity relationship.
* Corresponding authors.
E-mail addresses: reddy.chem@gmail.com (K.R. Reddy), aminabhavi@gmail.com (T.M. Aminabhavi), pvgr@yogivemanauniversity.ac.in (P.V.G. Reddy).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.envres.2021.111320
Received 15 March 2021; Received in revised form 6 May 2021; Accepted 9 May 2021
Available online 12 May 2021
0013-9351/© 2021 Elsevier Inc. All rights reserved.

M.V.K. Reddy et al.
Environmental Research 199 (2021) 111320
2020). The occurrence of AD is known to be originated from the various
environmental and genetic factors and is referred to as multi-factorial
disease (Cruz et al., 2017). The primary symptoms of AD are decline
in thinking, memory loss, forgetting recent things, and disturbances in
person’s capability to function independently, depression, apathy, social
withdrawal, mood swings, sleeping sickness, and change in lifestyle. The
main cause for AD is not fully traced till date, however the existing re-
ports suggest it probably due to multi-facet syndrome arising from the
intricate group of neurodegenerative elements including short levels of
synaptic acetylcholine (ACh) associated with the neurons dysfunction in
the brain, senile plaques or neurofibrillary tangles formation, increasing
levels of neurotoxic amyloid beta peptides, oxidative stress and others
(Salloway and Correia, 2015; Guzior et al., 2015; Das, 2020; Deshmukh,
2017; Roney et al., 2005; Aftab et al., 2018; Kulkarni, 2010; Ma et al.,
2015; Ma et al., 2016; Chaturvedi et al., 2019; Gulla et al., 2019).
In this respect, different hypotheses have been proposed to describe
the mechanism involved in Alzheimer’s pathogenesis (Corbett et al.,
2012) and assumed satisfactory results towards designing new drugs for
good governance of AD; according to cholinergic hypothesis, acetyl-
cholinesterase (AChE) enzyme degrades ACh (a neurotransmitter
responsible for memory and learning) in the pre-synaptic region that
causes dysfunction of the neurons featuring AD (Savini et al., 2003).
However, there are certain cases of AD where the AChE activity de-
creases and butyrylcholinesterase (BuChE) activity increases. The basic
difference reported in enzyme kinetics between AChE and BuChE ac-
tivity is the efficiency of ACh hydrolysis caused by the substrate con-
centration. In this concern, investigations also demonstrated the key role
of BuChE in ACh hydrolysis (Giacobini et al., 2002; Giacobini, 2004).
Therefore, the practical and flexible method to overcome this malfunc-
tion is by balancing AChE and/or BuChE inhibitions (Furukava-Hibi
et al., 2011). Nevertheless, it is difficult to maintain the proportion of
AChE/BuChE activity for the successful treatment of AD. Lately, it has
come to existence that the serious inhibition of BuChE may lead to
marginal side effects (Pacheco et al., 1995), and thus designing selective
inhibitory drugs with no adverse consequences is predicable. More
precisely, the current decade is witnessed with not many reports on the
design and development of structurally distinct nuclei that can battle
against AD (Basha et al., 2019; Yun et al., 2020).
et al., 2018b; Ghobadian et al., 2018; Kwong et al., 2019; Umar et al.,
2019).
Similarly, quinoxaline is another important class of active aza-
heterocycles present in many promising antibiotics such as actino-
leutin, echinomycin, levomycin, and other drug molecules such as clo-
fazimine (anti-leprosy) and grazoprevir (anti-HCV). Various quinoxaline
derivatives are well-known for their antiviral (Carta et al., 2018),
anti-tubercular (Wang et al., 2018), and several other properties (Tan-
gherlini et al., 2019; Karroum et al., 2019; Ali et al., 2020). Furthermore,
as a consequence of AD, different groups reported the development of
new quinoxaline hybrids for the cholinesterase inhibitory activity (Sagar
et al., 2019; Cevik et al., 2020; Almansour et al., 2020).
Triazines are yet another class of promising nitrogen hetero-
aromatics that are under constant study. Drugs such as altretamine (anti-
neoplastic), triethylenemelamine (chemotherapy), irsogladine (NSAID,
nonsteroidal anti-inflammatory drug) and ZSK474 (anti-tumor) possess
1,3,5-triazine nucleus are also available. These signify a broad spectrum
of pharmacological properties including anticancer (Srivatsava et al.,
2017),
α-glucosidase inhibition (Wang et al., 2017), anti-microbial
(Dinari et al., 2018), and anti-malarial (Xue et al., 2019) antibacterial
(Gunasekaran et al., 2019). Apart from these activities, sym-triazines
and other building blocks were also investigated for their cholines-
terase properties (Tripathi et al., 2019; Yazdani et al., 2019; Lolak et al.,
2020).
The majority of commercially available anti-AD drugs possess ni-
trogen heterocycle or side chain as a key pharmacophore unit (Fig. 1).
However, the synthesis of a few of them like Galantamine, Verubesce-
stat, Intepredine involves multisteps, laborious work-up procedures, and
long reaction times. By taking these into consideration, we have
demonstrated the biological importance of a few aza-heterocyclic li-
braries that were synthesized in our laboratory through simple routes in
short-times for anti-AChE and antioxidant properties.
2. Materials and methods
Reagents like acetylcholinesterase (EeAChE) enzyme abstracted
from electrophorus electricus, S-acetylthiocholine iodide (ATCh) sub-
strate, butyrylcholinesterase (EqBuChE) enzyme abstracted from equine
serum, S-butyrylthiocholine iodide (BuTCh), 5,5′-dithiobis-(2-nitro-
benzoic acid) (DTNB, Ellman’s reagent), Galantamine and Phosphate
buffer were procured from Sigma Aldrich company and used as such
without any further purification.
Some AD patients have been identified with disproportionate reac-
tive oxygen species that were suspected to induce the dysfunction of
mitochondria, leading to oxidative stress. These further bring about an
increase in Aβ production and aggregation, thereby promote the tau
protein phosphorylation persuading the vicious cycle of pathogenesis in
AD (Cheignon et al., 2018). Although several hypotheses have formu-
lated to manage AD, yet no perfect medication is brought into practice to
permanently treat this disorder. Hence, some researchers explored the
viable methods for an effective treatment of complex AD through “one
molecule-multiple targets” paradigm (Atwood et al., 2005; Zhang, 2005;
Dias and jr 2014). Of the different approaches established by various
researchers, the efficient multi-potent strategy identified for AD therapy
is the combined action of antioxidant and AChE activities (Guzior et al.,
2015; Dias and Junior, 2014).
2.1. In vitro assay of acetylcholinesterase and butyrylcholinesterase
inhibitory activities
Cholinesterase (AChE and BuChE) activities were investigated
following the Ellman’s spectrophotometry protocol employing EeAChE
The nitrogen heterocycles are ubiquitous in nature due to their
distinct biological properties that constitute a major fraction of drug
leads. Among them, pyridine component is more prevalently present in
many natural products and pharmaceutically active ingredients (Bagley
et al., 2000; Michael, 2005; Baumann and Baxendale, 2013; Vitaku
et al., 2014). Drugs like lavendamycin (anti-proliferative), streptonigrin
(anti-tumor), bay 60–5521 (anti-cholesteryl ester transfer protein) and
others possess this active pharmacophore unit. Functionalization/mo-
dification of pyridine with different substituents can extend the activ-
ities, including anti-oxidant (Hu et al., 2014), anti-inflammatory
(Yasodakrishna et al., 2016), anti-HIV (Podlekareva et al., 2016),
anti-microbial (El-Sayed et al., 2017) anti-cancer (Kumar et al., 2018a),
and anti-tubercular (Fumagalli et al., 2019). Efforts have also been made
towards the development of pyridine scaffolds for treating AD (Kumar
Fig. 1. Some of the commercially available anti-AD drugs containing N-het-
eroatoms/heterocycles.
2

M.V.K. Reddy et al.
Environmental Research 199 (2021) 111320
(enzyme abstracted from electrophorus electricus) and EqBuChE
(BuChE enzyme abstracted from equine serum) (Ellman et al., 1961;
Nadri et al., 2010). Phosphate buffer solution (pH 7.7) was freshly
prepared just before the start of the experiment. Individual test com-
pounds were dissolved in required volume of dimethylsulfoxide (DMSO)
and diluted using phosphate buffer solution (PB) (pH = 7.7, 200 mM) for
the final range of concentrations. A common chromogenic agent, DTNB
was used for AChE or BuChE catalyzed hydrolysis. In fresh 96-well
obtained IC50 values were expressed as mean ± SEM.
2.4. Statistical analyses
All the assays were performed in triplicate and the values were
expressed as mean ± standard error of the mean (SEM). The concen-
tration giving 50% inhibition (IC50) was calculated with Excel Microsoft
Office Professional Plus 2017. Statistical analysis of the results was
performed using Graph Pad Prism (Graph Pad Software 7).
microplates, each well was filled with 145
test sample solution, 80 L of DTNB solution (prepared by dissolving
18.5 mg reagent in 10 mL of PB solution), and 10 L of AChE or BuChE
solution (prepared by dissolving 0.8U/mL enzyme in 15 mL PB solution)
μL of PB solution, 10 μL of
μ
μ
2.5. Fluorescence spectroscopy
in the same order. Finally, 15 μL of 1 mM ATCh or BuTCh was added as a
The potential interactions of the active compounds (6e, 6f, 11b &
11f) with AChE were determined quantitatively using the Perkin Elmer-
LSS fluorescence spectrometer equipped with a 1.0 cm quartz cell. The
fluorescence emission spectra of the inhibitor molecules were docu-
mented at concentrations of 0.001–0.009 mM by keeping the AChE
value i.e., 0.001 mM as constant. After the addition of AChE to each test
sample concentration, they were allowed to incubate for 5 min and the
results were analyzed within 300–500 nm wavelength range at 285 nm
excitation wavelength. For both excitation and emission, scanning was
repeated at 100 nm per min by adjusting the slit width to 5 nm. To
obtain the concrete data, samples 6e, 6f, 11b and 11f were prepared and
measured the spectral values in triplicates.
substrate to the respective samples and assayed after pre-incubation for
5 min at 25 ◦C. The enzymatic reactions proceeded to completion in the
plate within 5 min of incubation time at 37 ◦C. Sample concentrations
ranging from 2.0 to 22.0 μM were analyzed in triplicates. Absorbance
data of the mixtures were collected at 517 nm using a micro plate reader
(Bio-Rad). The same method was employed for the control using gal-
antamine instead of a test compound. Blank assessments were per-
formed with the reaction solutions in the absence of inhibitors following
a similar method to afford 100% AChE or BuChE activity yields. Finally,
IC50 values were obtained by interpolation from the linear regression
analysis and expressed as mean ± S.E.M.
2.2. Kinetics studies of AChE inhibition
2.6. Circular dichroism spectroscopy (CD)
The mode of inhibition of highly potential compounds against AChE
enzyme was confirmed by performing the kinetic assessments following
the protocol similar to that of cholinesterases inhibition study (Ellman’s
method). Rate of enzyme activity of highly potent compounds at con-
centrations ranging from 1 mM, 10 mM, and 50 mM were measured
using ATCh (0.1–0.5 mM) keeping AChE value as constant (0.8 U/mL in
15 mL of phosphate buffer solution). Stock solutions of the inhibitors 6e,
6f, 11b and 11f, substrate (ATCh) and Ellman’s reagent were prepared
using PB solution (pH = 7.7). The 96-well microplates were loaded with
To confirm the activity of potent analogues, CD spectrometer (JASCO
J-1500) with a quartz cuvette of path length 0.1 cm was operated under
nitrogen atmosphere. 1 μL of each sample with various concentrations
(0.002 mM, 0.006 mM, 0.009 mM) of inhibitors and 0.001 mM AChE
were incubated for 5 min and scanned at a scan speed of 100 nm per
minute within wavelength range of 190–280 nm and each experiment
was performed in triplicates. CDNN 2.1 web-based software was used to
predict the percentages of α-helix, β-sheets, and random coils of AChE
and AChE-test sample complexes.
145
concentrations (1 mM, 10 mM, and 50 mM), 10
mL) and 80
L of DTNB solution and stored in dark for 5 min at 25 ◦C.
Following this, 15 L of substrate (ATCh) was added to the test samples
μ
L of phosphate buffer solution, 10
μ
L of target compounds at three
μL of enzyme (0.8 U/
μ
2.7. Molecular docking studies
μ
at five different concentrations (0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM and
0.5 mM) to initiate enzymatic reactions. Simultaneously, control ana-
lyses were also performed in the absence of any inhibitors. After
administering all the reagents, the reaction mixture was incubated at
25 ◦C for 5 min and tested the reactivity by using Elisa microplate reader
(Bio-Rad) at 517 nm. Velocities were calculated at various concentra-
tions of the substrates (S), and velocity reciprocals [V] were plotted
against the reciprocals of [ATCh] employing a graph pad prism.
Most of the drugs for anti-AD possess nitrogen atoms and reveal high
binding capacity. It was for this reason that we aimed at exploring the
plausible interactions and binding mechanisms of the active analogues
(6e, 6f, 11b and 11f) with the active sites of the protein. In order to this,
we extracted the crystal structure of EeAChE (PDB: 1C20) from Broo-
khaven National Laboratory protein bank for molecular simulation
studies of these compounds by binding to AChE using autodock program
of version 4.2.3. Lamarckian genetic algorithm (LGA) has high accuracy
in predicting the structure and flexibility of compounds (Morris et al.,
2009), from where the possible conformations of the inhibitor molecules
can be obtained upon binding to a protein via protein-inhibitor com-
plexes formation (Yeggoni et al., 2016). Geometries of all these active
complexes were optimized using Discovery Studio 3.5 software by
building 3D packages from two-dimensional (2D) structures. For
attaining appropriate geometry of protein structure with active sites, we
detached the water molecules and other ions, and supplemented
hydrogen atoms to the functional groups. 1.0 Å spacing with 126 × 126
x 126 dimensions along x-, y-, and z-axis was selected as a docking area.
Apart from these, several other factors such as 150 individual pop-
ulations, 1 maximum number of top individuals that survive automati-
cally, 30 genetic algorithm (GA) runs, 27000 maximum numbers of
generations were utilized and the output was chosen as Lamarckian GA
(4.2). Thirty conformations were taken out for each compound and the
best conformers with least free energies closer to investigational free
energy values were regarded for binding modes and binding sites
analysis (Malleda et al., 2012).
2.3. DPPH radical scavenging assay for antioxidant property
The method reported by Liyana-Pathiranan and Shahidi (Liyana--
Pathirana et al., 2005) was exploited with minor changes to analyze the
antioxidant activity of three different libraries. Test samples of different
concentrations were prepared by dissolving samples in 80% MeOH.
Later, 2.45 mg of DPPH was dissolved in 50 mL of 80% methanol, made
to 125 mM DPPH stock solution, and incubated at 25 ◦C for few minutes.
Ascorbic acid solution (reference material) was prepared by diluting
ascorbic acid in 80% methanol just before use. A 10
samples with concentrations ranging from 2.0 to 10.0
μ
L each of test
μ
M were mixed
with 290 μL of DPPH in a 96-well ELISA microplate, vortexed and stored
in dark for 30 min at 25 ◦C before analysis. Absorbencies of the mixtures
were determined at 517 nm using a microplate reader (Bio-Rad). The
same method was employed for ascorbic acid control and phosphate
buffer solution (pH 7.4). Scavenging assay experiments of the test
compounds towards DPPH radical were repeated in triplicates and the
3

M.V.K. Reddy et al.
Environmental Research 199 (2021) 111320
3. Results and discussion
3.2. Biology
3.1. Chemistry
3.2.1. Cholinesterases inhibition studies
The ability of ab initio synthesized aza-heterocycle derivatives (3a-k,
6a-j, 11a-g) to exhibit cholinesterase inhibitory potential was assessed
by adopting the Ellman’s method with slight modifications employing
electrophorus electricus derived acetylcholinesterase (EeAChE) and
equine serum-derived butyrylcholinesterase (EqBuChE) (Ellman et al.,
1961; Nadri et al., 2010). The obtained inhibitory activities of all the
synthetic congeners were displayed in Tables 1–3 in terms of IC50 values
and a conclusive structure-activity relationship (SAR) was discussed
based on their efficacies. Results indicate that most of the analogues
have excelled at low micromolar range of inhibitory activities against
AChE, being highly selective over BuChE.
Previously, we have paid much interest in developing new method-
ologies for the preparation of distinct heterocycle motiffs (Reddy et al.,
2016a,b,c; Reddy et al., 2019a,b; Anusha et al., 2020; Reddy et al.,
2020). In this consequence, a series of substitution reactions were car-
ried out on 3-chloro pyridine (1), o-phenylenediamines (4) and 2,
4-dichloro-6-morpholino-1,3,5-triazine (7) with different coupling
partners such as aryl/heteroaryl amines, boronic acids, and acetylenes
using different reagents and conditions as shown below in Scheme 1.
In one series, 3-chloropyridine was amalgamated with various 1◦/2◦
amines through carbon-nitrogen bond formation via Buchwald-Hartwig
amination in the presence of PEPPSI-Pd (pyridine enhanced pre-catalyst
preparation stabilization and initiation-palladium) complexes and
KOtBu base in toluene at 90 ◦C for 3 h to afford pyridine-3-amines (3a-k)
(Reddy et al., 2020). In another case, in situ generated NHC–Cu
(N-heterocyclic carbene-copper) catalysts were utilized for reacting
o-phenylenediamines and alkynes in EtOH:H2O at 80 ◦C for two to 3 h
through carbon-carbon, carbon-nitrogen bond formations and cycliza-
tion reactions to result in 2-aryl-3-arylethynyl quinoxalines (6a-i)
(Reddy et al., 2019a). Similarly, 4-(4,6-dichloro-1,3,5-triazin-2-yl)
morpholine was reacted with alkyl/aryl/heteroarylboronic acids and
aryl alkynes in the presence of PEPPSI–SONO–SP2 catalyst in ethanolic
solution of K2CO3 or Cs2CO3 at 80 ◦C for 1–2h via Suzuki-Miyaura and
Sonogashira cross-couplings respectively to accomplish the 2,4,6-trisub-
stituted-1,3,5-triazine targets (11a-h) (Reddy et al., 2016b). Interest-
ingly, most of these synthesized compounds are new and their
therapeutic activity have not been studied before.
In the case of pyridine amine derivatives (Table 1), those with simple
phenyl (3a) and benzyl moieties (3k) executed greater IC50 values of
38.61 ± 3.57 μM and 39.94 ± 1.04 μM, respectively. Compound with
electron-donating group like –CH3 (3b, IC50 35.13 ± 5.23
less influence compared to that containing electron-withdrawing group
like –CF3 (3d, IC50 21.65 ± 3.30 M). In the case of 3-chlorophenyl- (3f)
μM) showed
μ
and 4-chlorophenyl- (3g) groups coupled compounds, the former
molecule demonstrated good inhibitory activity than the latter (IC50
22.48 ± 2.92 and 25.54 ± 3.97, respectively). Considering the different
halogen substituents bearing a bromo substituent (3e) marked a little
higher activity than that with chloro substituents (3g). Specifically, it
can be noticed that the analogues with heterocycles such as pyridine and
pyrimidine (3hꢀ 3j) displayed good activities with IC50 values of 18.77
± 1.60, 14.22 ± 0.95, 21.24 ± 1.09
chloropyridine (3i) analog experienced the highest inhibitory potency
against AChE (with IC50 value 14.22 ± 0.95 M) in this series. On the
other side, these entities displayed greater IC50 values ranging from
μM, respectively. Superficially, 2-
μ
63.35 ± 3.03 to >100 μM and explained their lower inhibitory activities
against BuChE enzyme. Therefore, good inhibition selectivity
(3.968–4.454) of AChE over BuChE was noted for the analogues (3d, 3h-
j), which is comparable with that of galantamine (4.192).
With regard to quinoxaline series (Table 2), 6c-g and 6i behaved as
more potent AChE inhibitors than the rest that showed moderate ac-
tivity. Derivative 6f with two trifluoromethyl groups (-CF3) at positions
3- and 5- on phenyl ring presented a magnificent inhibition with IC50
value 7.23 ± 0.01 μM. A noticeable fact is that change in the position of
methyl group (–CH3) from C-3 (6a) to C-4 (6b) of phenyl ring led to a
decrease in the AChE inhibitory activity. When substituent on phenyl
ring was replaced from methyl (6c) to n-propyl (6d), a slight lowering in
activity was observed. Contrarily, replacing the –CH3 group at C-4 of
phenyl moiety (6c) with strong electron-withdrawing atom such as –F
(6e) found to be beneficial for inducing AChE inhibitory activity.
Furthermore, when –CH3 group was present on the phenyl ring, inhib-
itory activity was improved from 21.91 ± 2.83 μM (6h) to 14.66 ± 0.37
μ
M (6i). As far as BuChE is concerned, among the quinoxaline de-
rivatives, 6e and 6f displayed better inhibitory activities while 6b, 6c,
and 6g inhibited moderately. Taken together, most of the active com-
pounds (6c, 6e-g) had better AChE inhibition selectivity (5.048–6.737)
over BuChE than that of galantamine (4.192).
In the next category of molecules, i.e., triazine library (Table 3), four
compounds (11b, 11c, 11d and 11f) executed attractive results for
AChE inhibition with better IC50 values ranging between 8.49 ± 0.01 to
14.49 ± 0.05
μM. More curiously, 11b and 11f compounds with
methoxy phenyl and trifluoromethyl pyridine substituents provided the
best IC50 values (10.02 ± 0.40 and 8.49 ± 0.01, respectively) and were
suspected as the most active compounds amongst all against the AChE.
For a comparative study, when –CH3 group was introduced on the
phenyl ring from 11c to 11d and from 11g to 11h, enhancement in
activities was detected. Similar to AChE inhibitory activities, molecules
11b, 11d, and 11f also showed good BuChE inhibitory potentials and
Scheme 1. Reagents and conditions: a) 3-chloropyridine 1 (1.0 mmol),
aliphatic/aromatic/heteroaromatic amine 2 (1.0 mmol), PEPPSI-Pd catalyst
(0.5 mol%), KOtBu (3.0 mmol), toluene (3 mL), 90 ◦C, 3h; b) o-phenylenedi-
amine 4 (1.0 mmol), alkyne 5 (2.0 mmol), Cu(OAc)2 (5 mol%), NHC (5 mol%),
Cs2CO3 (3.0 mmol), EtOH:H2O (1:1, 3 mL), 80 ◦C and 2–3h; c) Step i: 4-(4,6-
dichloro-1,3,5-triazin-2-yl)morpholine 7 (1.0 mmol), R1-B(OH)2 (1.0 mmol),
K2CO3 (3.0 mmol), PEPPSI-SONO-SP2 (0.1 mol%), EtOH:H2O (1:1, 3 mL),
80 ◦C, 1–2h. Step ii: compound 9 (1.0 mmol), alkyne 10 (1.0 mmol), Cs2CO3
(1.0 mmol), PEPPSI-SONO-SP2 (0.1%), EtOH: H2O (1:1, 3 mL), 80 ◦C, 1–2h.
the activity of 11f (IC50 value 24.79 ± 0.28
μM) was comparable to
galantamine. With respect to selectivity, compounds 11b and 11d
4

M.V.K. Reddy et al.
Environmental Research 199 (2021) 111320
Table 1
Inhibitory activity of pyridine-3-amine analogues.
Compound
R1
H
R2
EeAChE IC50
(μ
M)a
EqBuChE IC50
(
μ
M)a
S.I.b
DPPH radical scavenging activity IC50
(
μ
M)c
3a
38.61 ± 3.57
>100
–
>50
3b
H
35.13 ± 5.23
>100
–
>50
3c
3d
H
H
29.83 ± 1.04
21.65 ± 3.30
>100
–
49.63 ± 5.20
>50
85.91 ± 0.23
3.968
3e
3f
H
H
24.68 ± 7.01
22.48 ± 2.92
>100
>100
–
–
>50
>50
3g
3h
H
H
25.54 ± 3.97
18.77 ± 1.60
>100
–
>50
77.43 ± 9.81
4.125
37.84 ± 5.12
3i
H
14.22 ± 0.95
63.35 ± 3.03
4.454
43.27 ± 6.44
3j
H
21.24 ± 1.09
39.94 ± 1.04
91.67 ± 1.30
>100
4.315
41.62 ± 1.11
>50
3k
CH3
–
Gal
Asc
–
–
–
–
5.0 ± 0.471
20.96 ± 0.45
4.192
–
–
–
–
10.74 ± 0.36
a,cIC50 values were expressed as mean ± standard error of the mean (S.E.M.) of at least three experiments. bS.I. - Selectivity index: IC50 EqBuChE/IC50 EeAChE. Gal =
Galantamine. Asc = Ascorbic Acid.
disclosed superior inhibition selectivity towards AChE than that of
clinically approved galantamine.
complex. Similarly, Ki1 and Ki2 values deliberated to compounds 6f, 11b
& 11f were 27.34 & 27.89 μM, 33.33 & 31.72 μM, 37.16 & 41.8 μM,
Conclusively, it can be noticed that quinoxaline and triazine de-
rivatives produced better AChE and BuChE activities, and inspite of the
higher activity of 6e, 6f, 11b and 11f, additional investigations like
kinetics of inhibition, fluorescence, CD, and docking experiments were
implemented. The selectivity profile disclosed by the active analogues
may be advantageous to reduce the peripheral cholinergic side effects
associated with BuChE inhibition and to provide low toxic nature. In yet
another point, compounds 6e, 6f, 11b and 11f exemplified dual inhib-
itory nature against AChE and BuChE enzymes.
respectively (Fig. 2). Considerably, in both the cases, the values
increased with increasing inhibitor’s concentrations.
3.2.3. In vitro antioxidant evaluation
DPPH radical scavenging assay is the extensively used method for
measuring an anti-oxidant property of the active components. Using this
strategy, we examined the captivity of compounds (3a-k, 6a-j, and 11a-
h) to shift the labile H-atoms to the DPPH free radical and their abilities
to scavenge DPPH radical was expressed as IC50 values in Tables 1–3.
From the data, it was noted that most of the tested aza-heterocycle de-
rivatives exhibited poor to good anti-oxidant property. Amongst
pyridine-3-amine analogues, 3c, 3h, 3i and 3j had moderate scavenging
capabilities with IC50 values of 49.63 ± 5.20, 37.84 ± 5.12, 43.27 ±
3.2.2. Kinetic study of AChE inhibition
In order to probe and understand the mechanism involved in AChE
inhibition, kinetic assessments were carried out for the compounds that
showed higher inhibition potency (6e, 6f, 11b, and 11f) and results are
summarized in Fig. 2. Steady-state inhibitions (Lineweaver-Burk plot
graphical analysis of velocity) were obtained by plotting their reciprocal
velocities against reciprocal concentrations [ATCh]ꢀ 1. From Fig. 2, it
can be observed that the double reciprocal graphical analyses presented
both increasing slopes (lessen Vmax) and intercepts (higher Km) with
increasing inhibitor concentration. Lines were intersected in the left
quadrant of the graph and indicated mixed-type inhibitions in the case of
inhibitors. This arouse due to binding of inhibitors to AChE in both the
CAS and PAS regions. Inhibitory constants, Ki1 and Ki2 were calculated
from the slopes and intercepts by using Microsoft excel software. The Ki1
6.44, 41.62 ± 1.11 μM, respectively. With respect to 2-aryl-3-aryle-
thynyl quinoxalines, compounds 6c and 6i with –CH3 group on ben-
zene ring showed moderate antioxidant activity while 6e and 6f with
electron-withdrawing substituents displayed better scavenging prop-
erty. From the triazine library, 11b and 11f behaved as the blessed
antioxidants with IC50 values of 14.80 ± 1.73 and 16.72 ± 1.39 μM,
respectively, which are nearer to the positive control, ascorbic acid (IC50
value 10.74 ± 0.36 M). More importantly, analogues 11a, 11d, 11g
and 11h experienced better DPPH scavenging abilities with IC50 values
in the range of 34–49 M.
μ
μ
value (40.54
μ
M) obtained for 6e corresponds to free enzyme binding
3.2.4. Fluorescence emission assay
and Ki2 value (37.85
μ
M) related to binding to enzyme substrate
According to Lakowicz (2006), fluorescence spectroscopy is the more
5

M.V.K. Reddy et al.
Environmental Research 199 (2021) 111320
Table 2
Inhibitory activity of 2-aryl-3-arylethynyl quinoxalines.
Compound
R1
R2
H
R3
EeAC hE IC50
(μ
M)a
EqBuChE IC50
(μ
M)a
S.I.b
DPPH radical scavenging activity IC50
(
μ
M)c
6a
CH3
29.50 ± 2.73
>100
–
>50
6b
6c
6d
CH3
CH3
CH3
H
33.83 ± 1.35
15.67 ± 2.39
18.00 ± 2.13
92.54 ± 0.02
79.11 ± 2.73
>100
2.735
5.048
–
>50
CH3
CH3
48.66 ± 1.03
>50
6e
6f
CH3
CH3
CH3
CH3
10.35 ± 0.60
7.23 ± 0.01
57.05 ± 7.26
48.71 ± 2.81
5.512
6.737
21.56 ± 5.23
27.22 ± 1.27
6g
6h
CH3
F
CH3
H
C7H11
13.70 ± 1.51
21.91 ± 2.83
77.35 ± 3.92
>100
5.645
>50
>50
–
6i
F
H
14.66 ± 0.37
>100
–
42.78 ± 0.03
6j
F
–
–
H
–
–
C7H11
29.27 ± 6.92
5.0 ± 0.295
–
>100
–
–
Gal
-
-
20.96 ± 0.45
4.192
–
Asc
–
–
10.74 ± 0.36
a,cIC50 values were expressed as mean ± standard error of the mean (S.E.M.) of at least three experiments. bS.I. - Selectivity index: IC50 EqBuChE/IC50 EeAChE. Gal =
Galantamine. Asc = Ascorbic Acid.
Table 3
Inhibitory activity of 4-(4-aryl-6-(arylethynyl)-1,3,5-triazin-2-yl morpholines.
Compound
R1
R2
EeAChE IC50
(μ
M)a
EqBuChE IC50
(μ
M)a
S.I.b
DPPH radical scavenging activity IC50
(
μ
M)c
11a
CH3
18.46 ± 1.09
>100
–
49.57 ± 0.72
11b
11c
10.02 ± 0.40
14.49 ± 0.05
68.12 ± 0.02
>100
6.798
14.80 ± 1.73
>50
–
11d
11e
12.38 ± 0.51
26.95 ± 7.48
83.43 ± 7.05
>100
6.739
47.54 ± 0.07
>50
–
11f
8.49 ± 0.01
24.17 ± 4.12
19.26 ± 1.88
24.79 ± 0.28
>100
2.919
16.72 ± 1.39
41.28 ± 5.41
34.13 ± 4.22
11g
11h
–
–
>100
Gal
Asc
–
–
–
–
5.0 ± 0.295
20.96 ± 0.45
4.192
–
–
–
–
10.74 ± 0.36
a,cIC50 values were expressed as mean ± standard error of the mean (S.E.M.) of at least three experiments. bS.I. - Selectivity index: IC50 EqBuChE/IC50 EeAChE. Gal =
Galantamine. Asc = Ascorbic Acid.
6

M.V.K. Reddy et al.
Environmental Research 199 (2021) 111320
Fig. 2. (A) Lineweaver-Burk plots of inhibition kinetics of compounds, 3i, 6e, 6f, 11b and 11f in the order from top to bottom. ATCh = acetylthiocholine iodide. V =
initial velocity rate. (B) Lineweaver-Burk secondary plots - Slope Vs concentrations. (C) Intercept Vs concentrations.
reliable technique to identify the binding interactions, binding sites,
binding constants, free energies, and intermolecular distances. It was
known that AD drugs have a greater affinity towards binding with AChE
enzyme and hence, to get further insights on the mode of action of active
compounds 6e, 6f, 11b, and 11f, it was imperative need to comprehend
the protein drug interactions. Therefore, the quinoxaline and triazine
analogues potential interactions (6e, 6f, 11b and 11f) with AChE were
determined quantitatively by fluorescence spectroscopic analysis. Fig. 3
represents the fluorescence emission spectra without and with an
assortment of concentrations (0.001–0.009 mM) of the target com-
pounds by keeping AChE value (0.001 mM) constant. Strong
fluorescence emission absorption maximum was obtained for AChE at a
wavelength of 350 nm (λmax = 285 nm). After incubation of samples
with increasing inhibitor and standard AChE concentrations, fluores-
cence spectroscopy was recorded and observed quenching of fluores-
cence emission of AChE by suppressing the intensity of maximum peak
without any change in its position. This can be attributed to the reduc-
tion in quantum yield of fluorescence on account of molecular in-
teractions of AChE with the quencher molecule. It is evident that the
tyrosine component caused quenching of fluorescence emission under
ionization, phenylalanine offered only a limited quantum yield and the
intrinsic fluorescence of AChE was mainly by the tryptophan residue
7

M.V.K. Reddy et al.
Environmental Research 199 (2021) 111320
Fig. 3. Fluorescence emission spectra of AChE-6e (A), AChE-6f (B), AChE-11b (C) and AChE-11f (D) at room temperature in phosphate buffer solution. Inserts shown
correspond to modified stern-volmer plots; log(dF/F) Vs log[Q] at λex = 285 nm and λem = 360 nm. For every compound, three independent experiments were
repeated and attained identical spectra at each time.
(Basha et al., 2019). This lowering of fluorescence intensity of AChE was
log [F0 ꢀ F / F] = log Ks + n log[Q]
suspected due to the formation of complexes with inhibitors in a
concentration-dependent manner and the binding of these entities
(where Ks corresponds to binding constant, n reads slope corresponding
resulted in conformational rearrangements within the pocket of binding
to a number of binding sites and Q relates to quencher concentration).
sites, thereby induced change in the environment around the tryptophan
The obtained ‘n’ values for 6e, 6f, 11b and 11f were 0.942, 1.364
residue. Thus, AChE-inhibitors complexes formation was quantified
and 1.194, 1.537, respectively which were nearer to “one” for almost all
upon increasing the concentrations of inhibitors. These caused
compounds and left an idea of interaction with AChE in a one-to-one
increasing absorbencies of excitations, and/or the emission radiations
ratio. Similarly, binding constants measured from the intercepts of 6e,
leading to the inner filter effect and extended the nonlinear relationships
6f, 11b and 11f, were 5.4 × 104, 4.3 × 104, 3.2 × 104 and 4.9 × 104 Mꢀ 1
,
between fluorescence intensities and concentrations of inhibitor mole-
cules. The effect was corrected using the following fluorescence
equation:
respectively which demonstrated a strong correlation ship with
computationally obtained binding constant values, 2.78 × 104, 1.79 ×
104, 5.65 × 104 and 1.89 × 104 Mꢀ 1
.
Fobs10(Aex + Aem
)
Fcor
=
3.2.4.1. Free energy calculations. Standard free energy changes were
calculated and evaluated for highly potential compounds following the
equation, ΔG◦ = -RTlnk (where ΔGo represents free energy change, R
stands for gas constant at room temperature T, and k correspond to
binding constant). The binding constant (k) values received from fluo-
rescence emission were utilized to predict electrostatic forces, van der
Waals forces, and hydrogen bonds between AChE and potent molecules
(6e, 6f, 11b and 11f). Free energy changes, ΔG◦ received for 6e, 6f, 11b
and 11f were ꢀ 5.593, ꢀ 6.799, ꢀ 6.605 and ꢀ 8.104 KcalMꢀ 1, respec-
tively. Signs and magnitudes of ΔGo confirmed the key binding forces
involved in inhibitor-AChE binding and stabilization of complexes. The
negative values due to hydrophobic interactions explained the
exothermic nature of the reactions besides the easy formation of in-
hibitor moleculeꢀ AChE complexes.
2
where Fcor correspond to fluorescence intensity corrected, Fobs relate to
observed fluorescence, Aex and Aem are the excited (nm) and emitted
(nm) fluorescence wavelength absorbencies of AChE, respectively at 285
and 360 nm. With respect to quenching mechanisms of fluorophore,
they may be either dynamic or static types depending upon the collisions
or complex formation with AChE. Herein, the collisions between
quencher and fluorophore were dynamic type while the complexes
formations were of a static kind. To distinguish the type of fluorescence
quenching mechanism (static or dynamic) between AChE and the active
probes (6e, 6f, 11b and 11f), log F0/F Vs log Q graphs were plotted and
obtained linear plots (inserts) representing the static quenching by the
complex formation in all the cases (Fig. 3). The modified Stern-Volmer
equation was adopted for calculating the number of binding constants
and binding sites with respect to static quenching (Min et al., 2004).
3.2.5. Circular dichroism analysis
Circular dichroism (CD) can be used to understand the structural and
8

M.V.K. Reddy et al.
Environmental Research 199 (2021) 111320
conformational analysis of a compound during its interaction with
protein. Therefore, CD experiments were performed for AChEꢀ 6e, 6f,
11b and 11f complexes by incubating compounds 6e, 6f, 11b and 11f at
different concentrations of 0.001, 0.005 and 0.009 mM with AChE and
analyzed the absorbencies at a wavelength region of 190ꢀ 280 nm.
Interestingly, these inhibitors showed partial distortions in the second-
ary structures of AChE absorption bands due to the chromophore
property which appeared at 208 nm and 218 nm (negative absorption
bands) and 193 nm (positive absorption band). This was ascribed by the
trivial unfoldings of these complexes attained on account of changes in
microenvironment of the tryptophan residues upon binding to AChE.
This further substantiated the quenching mechanism of fluorescence
emission analysis. The conformational changes of AChE thus elucidated
the actuality of binding inhibitor molecules to the enzyme. Results dis-
closed a steady decrease of peak intensities in a concentration-
dependent pattern. This opened the conformational modifications of
most active molecules with AChE. It is the most commonly used
computational approach capable of recognizing the binding interactions
and binding sites of ligands (inhibitors) with protein (AChE) and thereby
helps in understanding the therapeutic efficacy or potency of new
chemical entities.
To get an acquaintance on the mode of action of enzyme catalysis,
essential structural elements of protein were acquired from x-ray crys-
tallography data. The representative protein had 20 Å deep hydrophobic
gorge (Botti et al., 1999) containing Trp286, Tyr72, Asp74 and Tyr124
amino acids in PAS region and a catalytic triad, His447, Ser203, and
Glu334 at the bottom of the CAS region and bound with the cationic
substrate, ACh. Besides these, the anionic binding sites contained some
amino acid residues such as His441, Glu199, His444, Trp84, Tyr130,
and Tyr337 in the surrounding of catalytic triad and were able to bind
with quaternary ammonium functionality of the ligands (6e, 6f, 11b and
11f). Particularly, Phe330 present in the mid gorge was responsible for
the recognition of ligands. Therefore, to understand the binding in-
teractions, molecular docking studies were characterized for the active
sites of EeAChE with potent inhibitors (6e, 6f, 11b and 11f). For this
analysis, we initially retrieved 3D structure of AChE from PDB (PDB ID:
IC20) and the consequences were portrayed on the basis of G scores
received from GLIDE docking (Grid based Ligand Docking with Ener-
getics) (Jones et al., 1997). Results disclosed that compounds 6e and 6f
oriented in one fashion, in a way different from 11b and 11f. Binding
poses of the potential inhibitors in the active sites of AChE were repre-
sented in Fig. 5.
protein (AChE) by the virtue of lowering
enhancing β-sheets and random coils formation. From Gor IV data bank
report, we identified that free AChE has -helix, β-sheets and random
coils in 30.61%, 17.07%, and 52.32%, respectively (Garnier et al.,
α-helical content and
α
̴
̴
1996). In the case study, we observed that the secondary structures of
AChE have undergone marginal variations at low concentrations and
significant changes at high concentrations of 6e, 6f, 11b and 11f when
tested individually. Here, the protein’s (AChE) α-helical content was
decreased to 29.45%, 24.00%, 27.25% and 29.38%, while β-sheet
increased to 17.98%, 22.17%, 20.91% and 18.92% and random coil
increased to 54.23%, 55.00, 53.12% and 53.76%, respectively for 6e, 6f,
11b and 11f (Fig. 4). These changes in secondary structures certainly
explained the potential binding of 6e, 6f, 11b and 11f to AChE.
In the 3D orientation, docking pose of 6e placed quinoxaline core in
the PAS region sandwiched between the amino acid residues Tyr341 and
Trp286 through π-π stacking so that the 4-fluorophenyl acetylene scaf-
fold leaned towards CAS region and likely induced hydrophobic in-
teractions with Gln 291, Phe 297, Tyr124, Ser 293, Phe 338 and Tyr 337
residues. Specifically, 4-fluorophenyl ring stacked against the Tyr 72
3.2.6. Molecular docking analysis
Docking analysis was carried out to corroborate the binding poses of
Fig. 4. Circular dichroism (CD) spectra of AChE-6e (A), AChE-6f (B), AChE-11b (C) and AChE-11f (D) complexes.
9

M.V.K. Reddy et al.
Environmental Research 199 (2021) 111320
Fig. 5. Docking studies of 6e (A), 6f (B), 11b (C) and 11f (D) in the vicinity of AChE employing autodock 4.2.3 package. Active molecules and amino acids were
shown in respective green and grey colors of ball and stick modular representations. (For interpretation of the references to color in this figure legend, the reader is
referred to the Web version of this article.)
and Trp 286 via T-shape (edge-to-face)
π
ꢀ
π
interactions. Compound 6e
inhibitory ability.
also possessed two hydrogen bond interactions with Tyr 72 and Tyr 124
at a bond distance of 3.49 Å and 3.31 Å, respectively.
More importantly, compounds 6e, 6f, 11b and 11f bound with AChE
active sites with free energies of binding, ¡10.31, ¡13.3, ꢀ 11.25 and
ꢀ 11.9 kcalMꢀ 1, respectively (obtained computationally for the low
energy conformers). Interestingly, molecular docking studies also
inferred the strong interactions of compounds 6e, 6f, 11b and 11f with
Similarly, 6f accommodated quinoxaline ring in PAS region so that
both phenyl rings inclined towards CAS region of AChE and the 3,5-tri-
fluoromethyl phenylacetylene ring was sandwiched between Trp 286
and Tyr 72 residues by
πꢀ π
stacking and stabilized by hydrophobic in-
AChE through their binding constant values of 2.78 × 104, 1.79 × 104
,
teractions with Ile 294, Leu 289, Gln 291, Ser 293, Tyr 124 and Phe 297
residues. It was also observed that 6f borne six hydrogen bonding in-
teractions with Tyr 124, Leu 289, Ser 293, His 287 and Thr 318 at a bond
distance of 3.03 Å, 3.14 Å, 3.50 Å, 2.77 Å, 3.35 Å, 3.35 Å and 3.64 Å
which indicated its strong binding efficiency with AChE, and hence
potent inhibition of AChE.
5.65 × 104 and 1.89 × 104 Mꢀ 1, respectively which were close to those
of experimentally determined values and supported their potential na-
ture to exhibit therapeutic effect against AD.
4. Conclusions
In triazine series, compound 11b well occupied both the PAS and
In summary, the activities of three series of synthesized pyridine,
quinoxaline and triazine analogues were evaluated by multiple biolog-
ical studies including AChE/BuChE inhibition, antioxidant property,
kinetics of inhibition, molecular interaction, and molecular docking
studies to assess their anti-Alzheimer’s properties. Among the tested
compounds, 6e, 6f, 11b and 11f replicated more potent and selective
AChE inhibitory activities in micromolar range along with excellent
antioxidant properties. Kinetic and molecular docking analysis proved
the mixed-type of inhibition exhibited by compounds 6e, 6f, 11b and
11f upon binding to CAS as well as PAS regions of AChE. Fluorescence
emission studies demonstrated that, these analogues bind to AChE
spontaneously via the static mechanism. Further, conformational anal-
ysis from the CD spectroscopic studies revealed partial unfolding of the
secondary structure of AChE due to the complex formation between 6e,
6f, 11b, 11f and AChE, however, it was not significant enough to
damage protein’s stability and function. Put together, these findings
provide an ample scope towards the development of quinoxaline and
triazine-based scaffolds as new anti-Alzheimer’s candidates and these
prove worthy for further biological activities associated with AD.
CAS regions in the binding sites of AChE. The triazine core showed πꢀ π
stacking with Trp 286, whereas 4-methoxyphenyl ring was sandwiched
between Tyr 241 and Trp 286 via πꢀ π stackings and surrounded by Tyr
124, Ser 293, Phe 338 and Phe 294 residues. Morpholine group showed
hydrophobic interactions with Phe 297, Leu 289, and Arg 296 residues.
Phenylacetylene portion of 11b was stacked against the Tyr 72 and Leu
76 via edge-to-face πꢀ π interactions and encircled by Asp 74 and Tyr 75.
Furthermore, 11b¡AChE complex was stabilized by two hydrogen
bonds with Ser 293 and Tyr 124 present at bond distances of 3.20 Å and
3.55 Å, respectively.
Similar to 11b, different parts of compound 11f were also well
spread into the binding pocket of AChE covering both PAS and CAS
regions. Specifically, this trifluoromethyl pyridine moiety was sur-
rounded by the amino acids Tyr 341, Phe 338, Tyr 337, and Ser 293
forming hydrophobic interactions. Triazine ring formed the πꢀ π stack-
ing with Trp 286 whereas morpholine protruded into deep active site
and covered by Phe 297, Gln 291 and Leu 289 residues with hydro-
phobic interactions. Further, phenylacetylene part exhibited πꢀ π in-
teractions with Tyr 72. Moreover, 11f¡AChE complex was well
stabilized by six hydrogen bond formations with Tyr 72, Tyr 124, Ser
293 and Trp 286 amino acid residues with bond distances of 3.79 Å,
3.45 Å, 3.16 Å, 3.43 Å, respectively which supported its potential AChE
Credit author statement
M. V. K. Reddy: Conceptualization, Investigation, Writing - Original
10

M.V.K. Reddy et al.
Environmental Research 199 (2021) 111320
Corbett, A., Pickett, J., Burns, A., Corcoran, J., Dunnett, S.B., Edison, P., Ballard, C.,
2012. Drug repositioning for Alzheimer’s disease. Nat. Rev. Drug Discov. 11,
833–846. https://doi.org/10.1038/nrd3869.
Draft. K. Y. Rao: Methodology, Writing - Reviewing & Editing. G.
Anushaa: Methodology. G. M. Kumar: Formal analysis. A. G. Damu:
Validation. Kakarla Raghava Reddy: Supervision, Writing - Reviewing
& Editing. Nagaraj P. Shetti: Software. Tejraj M. Aminabhavi: Su-
pervision, Writing - Reviewing & Editing. Peddiahgari Vasu Govard-
hana Reddy: Funding acquisition, Supervision, Writing - Reviewing &
Editing.
Chaturvedi, K., Ganguly, K., More, U.A., Reddy, K.R., Dugge, T., Naik, B., Aminabhavi, T.
M., Noolvi, M.N., 2019. Sodium Alginate in Drug Delivery and Biomedical Areas,
Chapter 3, pp. 59–100.
Cheignon, C., Tomas, M., Bonnefont-Rousselot, D., Hurreau, C., Collin, F., 2018.
Oxidative stress and the amyloid beta peptide in Alzheimer’s disease. Redox Biol 14,
450–464. https://doi.org/10.1016/j.redox.2017.10.014.
Cruz, I., Puthongking, P., Cravo, S., Palmeira, A., Cidade, H., Pinto, M., Sousa, E., 2017.
Xanthone and flavone derivatives as dual agents with acetylcholinesterase inhibition
and antioxidant activity as potential anti-Alzheimer agents. Hindawi J. Chem. 1–16.
https://doi.org/10.1155/2017/8587260, 2017.
Declaration of competing interest
Das, S.S., Alkahtani, S., Bharadwaj, P., Ansari, M.T., Alkahtani, M.D., Pang, Z.,
Hasnain, M.S., Nayak, A.K., Aminabhavi, T.M., 2020. Molecular insights and novel
approaches for targeting tumour metastasis. Int. J. Pharm. 585, 119556. https://doi.
org/10.1016/j.ijpharm.2020.119556.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Deshmukh, A.S., Chauhan, P.N., Noolvi, M.N., Chaturvedi, K., Ganguly, K., Shukla, S.S.,
Nadagouda, M.N., Aminabhavi, T.M., 2017. Polymeric micelles: basic research to
clinical practice. Int. J. Pharm. 532, 249–268. https://doi.org/10.1016/j.
ijpharm.2017.09.005.
Acknowledgments
Dias, K.S.T., Junior, C.V., 2014. Multi-target directed drugs: a modern approach for
design of new drugs for the treatment of Alzheimer’s disease. Curr. Neuropharmacol.
12, 239–255. https://doi.org/10.2174/15701591203140511153200.
Dinari, M., Gharahi, F., Asadi, P., 2018. Synthesis, spectroscopic characterization,
antimicrobial evaluation and molecular docking study of novel triazine-
quinazolinone based hybrids. J. Mol. Struct. 1156, 43–50. https://doi.org/10.1016/
j.molstruc.2017.11.087.
This work was financially supported by the Department of Atomic
Energy (DAE)-Board of Research in Nuclear Sciences (BRNS), Mumbai,
India (2012/37C/33/BRNS) and Council of Scientific and Industrial
Research (CSIR), Pusa, New Delhi, India (No. 02(0248)/15/EMR-II).
The research fellow M. V. K. Reddy is grateful to the Council of Scientific
and Industrial Research (CSIR) for supporting with Senior Research
Fellowship (No. 09/1076 (0002)/2018-EMR-I, Ack No.121252/2k17/
1). The authors would like to thank Prof. Rajagopal Subramanyam,
Department of Plant Sciences, University of Hyderabad, Hyderabad,
Telangana State, India for his assistance with fluorescence and CD
spectroscopy studies.
Ellman, G.L., Courtney, K.D., jr, V.A., Featherstone, R.M., 1961. A new and rapid
colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7,
88–95. https://doi.org/10.1016/0006-2952(61)90145-9.
El-Sayed, K.F., Moustafa, A.H., El-Torky, A.E., Abd El-Salam, E.A., 2017. A series of
pyridines and pyridine based sulfa drugs as antimicrobial agents: design, synthesis
and antimicrobial activity. Russ. J. Gen. Chem. 87, 2401–2408. https://doi.org/
10.1016/j.bmcl.2019.02.019.
Fumagalli, F., de Melo, S.M.G., Ribeiro, C.M., Solcia, M.C., Pavan, F.R., Emery, F.S.,
2019. Exploiting the furo[2,3-b]pyridine core against multidrug resistant
Mycobacterium Tuberculosis. Bioorg. Med. Chem. Lett 29, 974–977. https://doi.
org/10.1016/j.bbr.2011.07.035.
References
Furukawa-Hibi, Y., Alkam, T., Nitta, A., Matsuyama, A., Mizoguchi, H., Suzuki, K.,
Yamada, K., 2011. Butyrylcholinesterase inhibitors ameliorate cognitive dysfunction
induced by amyloid-beta peptide in mice. Behav. Brain Res. 225, 222–229. https://
doi.org/10.1016/j.bbr.2011.07.035.
Aftab, S., Shah, A., Nadhman, A., Kurbanoglu, S., Ozkan, S.A., Dionysiou, D.D., Shukla, S.
S., Aminabhavi, T.M., 2018. Nanomedicine: an effective tool in cancer therapy. Int.
J. Pharm. 540, 132–149.
Garnier, J., Gibrat, J.F., Robson, B., 1996. GOR secondary structure prediction method
version IV. Methods Enzymol. 266, 540–553.
Ali, K., Mahsa, F., Saghi, S., 2020. 1,2,3-Triazoles based 3-substituted 2-thioquinoxalines:
synthesis, anti-bacterial activities, and molecular docking studies. J. Mol. Struct.
1202, 127262. https://doi.org/10.1016/j.molstruc.2019.127262.
Almansour, A.I., Arumugam, N., Kumar, R.S., Kotresha, D., Manohar, T.S., Venketesh, S.,
2020. Design, synthesis and cholinesterase inhibitory activity of novel
spiropyrrolidine tethered imidazole heterocyclic hybrids. Bioorg. Med. Chem. Lett
30, 126789. https://doi.org/10.1016/j.bmcl.2019.126789.
Ghobadian, R., Mahdavi, M., Nadri, H., Moradi, A., Edraki, N., Akbarzadeh, T.,
Amini, M., 2018. Novel tetrahydrocarbazole benzyl pyridine hybrids as potent and
selective butryl cholinesterase inhibitors with neuroprotective and β-secretase
inhibition activities. Eur. J. Med. Chem. 155, 49–60. https://doi.org/10.1016/j.
ejmech.
Giacobini, E., 2004. Cholinesterase inhibitors: new roles and therapeutic alternatives.
Pharmacol. Res. 50, 433–440. https://doi.org/10.1016/j.phrs.2003.11.017.
Giacobini, E., Spiegel, R., Enz, A., Veroff, A.E., Cutler, N.R., 2002. Inhibition of acetyl-
and butyryl-cholinesterase in the cerebrospinal fluid of patients with Alzheimer’s
disease by rivastigmine: correlation with cognitive benefit. J. Neural. Transm. 109,
1053–1065. https://doi.org/10.1007/s007020200089.
Anusha, G., Reddy, M.V.K., Reddy, P.V.G., 2020. Benzimidazole bearing Pd-PEPPSI
complexes catalysed direct C2-arylation/heteroarylation of N-substituted
benzimidazoles. Appl. Organomet. Chem. 34, 1–12. https://doi.org/10.1002/a
oc.5869 e5869.
Atwood, C.S., Robinson, S.R., Smith, M.A., 2005. Amyloid-beta: redox-metal chelator
and antioxidant. J. Alzheim. Dis. 4, 203–214. https://doi.org/10.3233/jad-2002-
4310.
Gulla, S., Lomada, D., Srikanth, V.V.S.S., Reddy, K.R., Soni, S., Reddy, M.C., 2019. Recent
advances in nanoparticles-based strategies for cancer therapeutics and antibacterial
applications. Methods Microbiol. 46, 255–293.
Bagley, M.C., Bashford, K.E., Hesketh, C.L., Moody, C.J., 2000. Total synthesis of the
thiopeptide promothiocin A. J. Am. Chem. Soc. 122, 3301–3313. https://doi.org/
10.1021/ja994247b.
Gunasekaran, P., Rajasekaran, G., Han, E.H., Chung, Y.-H., Choi, Y.-J., Yang, Y.J.,
Bang, J.K., 2019. Cationic amphipathic triazines with potent anti-bacterial, anti-
inflammatory and anti-atopic dermatitis properties. Sci. Rep. 9, 1292. https://doi.
org/10.1038/s41598-018-37758-z.
Basha, S.J., Pushparaju, Y.D., Rao, K.Y., Mohan, P., Babu, Z.R., Viswanath, K.K.,
Damu, A.G., 2019. Synthesis and biological evaluation of flavone-8-acrylamide
derivatives as potential multi-target-directed anti Alzheimer agents and
investigation of binding mechanism with acetylcholinesterase. Bioorg. Chem. 88,
102960. https://doi.org/10.1016/j.bioorg.2019.102960.
Guzior, N., Więckowska, A., Panek, D., Malawska, B., 2015. Recent development of
multifunctional agents as potential drug candidates for the treatment of Alzheimer’s
disease. Curr. Med. Chem. 22, 373–404. https://doi.org/10.2174/
0929867321666141106122628.
Baumann, M., Baxendale, I.R., 2013. An overview of the synthetic routes to the best
selling drugs containing 6-membered heterocycles. Beilstein J. Org. Chem. 9,
2265–2319. https://doi.org/10.3762/bjoc.9.265.
He, Y., Yao, P.-F., Chen, S.-B., Huang, Z.-B., Huang, S.-L., Tan, J.-H., Huang, Z.S., 2013.
Synthesis and evaluation of 7,8-dehydrorutaecarpine derivatives as potential
multifunctional agents for the treatment of Alzheimer’s disease. Eur. J. Med. Chem.
63, 299–312. https://doi.org/10.1016/j.ejmech.2013.02.014.
Botti, S.A., Felder, C.E., Lifson, S., Sussmann, J.L., Silman, I., 1999. A modular treatment
of molecular traffic through the active site of cholinesterase. Biophys. J. 77,
2430–2450. https://doi.org/10.1016/S0006-3495(99)77080-3.
Hu, Y., Zhang, J., Yu, C., Li, Q., Dong, F., Wang, G., Guo, Z., 2014. Synthesis,
characterization, and antioxidant properties of novel inulin derivatives with
aminopyridine group. Int. J. Biol. Macromol. 70, 44–49. https://doi.org/10.1016/j.
ijbiomac.
Camps, P., El Achab, R., Morral, J., Munoz-Torrero, D., Badia, A., Banos, J.E., Luque, F.J.,
2000. New tacrine-huperzine A hybrids (huprines): highly potent tight- binding
acetylcholinesterase inhibitors of interest for the treatment of Alzheimer’s disease.
J. Med. Chem. 43, 4657–4666. https://doi.org/10.1021/jm000980y.
Carta, A., Sanna, G., Briguglio, I., Madeddu, S., Vitale, G., Piras, S., Giliberti, G., 2018.
Quinoxaline derivatives as new inhibitors of coxsackie virus B5. Eur. J. Med. Chem.
145, 559–569. https://doi.org/10.1016/j.ejmech.2017.12.083.
Jones, G., Willett, P., Glen, R.C., Leach, A.R., Taylor, R., 1997. Development and
validation of a genetic algorithm for flexible docking. J. Mol. Biol. 267, 727–748.
https://doi.org/10.1006/jmbi.1996.0897.
Karroum, N.B., Moarbess, G., Guichou, J.-F., Bonnet, P.-A., Patinote, C., Tayoun, H.-B.,
Masquefa, C.D., 2019. Novel and selective TLR7 antagonists among the imidazo[1,2-
a]pyrazines, imidazo[1,5-a]quinoxalines, and pyrazolo[1,5-a]quinoxalines series.
J. Med. Chem. 62, 7015–7031. https://doi.org/10.1021/acs.jmedchem.9b00411.
Kulkarni, P.V., Roney, C.A., Antich, P.P., Bonte, F.J., Raghu, A.V., Aminabhavi, T.M.,
2010. Quinoline-n-Butylcyanoacrylate-based nanoparticles for brain targeting for
the diagnosis of Alzheimers Disease. John Wiley & Sons Inc., New York,
WIREscNanomedicine and Nanotechnology 2, 35–47.
Cavalli, A., Bolognesi, M.L., Minarini, A., Rosini, M., Tumiatti, V., Recanatini, M.,
Melchiorre, C., 2008. Multi-target directed ligands to combat neurodegenerative
diseases. J. Med. Chem. 51, 347–372. https://doi.org/10.1021/jm7009364.
Cevik, U.A., Osmaniye, D., Saglik, B.N., Cavusoglu, B.K., Levent, S., Karaduman, A.B.,
Turan, G., 2020. Multifunctional quinoxaline-hydrazone derivatives with
acetylcholinesterase and monoamine oxidases inhibitory activities as potential
agents against Alzheimer’s disease. Med. Chem. Res. 29, 1000–1011. https://doi.
org/10.1007/s00044-020-02541-4.
11

M.V.K. Reddy et al.
Environmental Research 199 (2021) 111320
Kumar, G.S., Poornachandra, Y., Kumar, G.S., Reddy, K.R., Jaheer, M., Kamal, S.,
Kumar, C.G., Narsaiah, B., 2018a. Synthesis of novel hetero ring fused pyridine
derivatives; Their anticancer activity, CoMFA and CoMSIA studies. Bioorg. Med.
Chem. Lett 28, 2328–2337. https://doi.org/10.1016/j.bmcl.2018.04.031.
Kumar, J., Gill, A., Shaikh, M., Singh, A., Shandilya, A., Jameel, E., Jayaram, B., 2018b.
Pyrimidine-triazolopyrimidine and pyrimidine-pyridine hybrids as potential
acetylcholinesterase inhibitors for alzheimer’s disease. ChemistrySelect 3, 736–747.
https://doi.org/10.1002/slct.201702599.
Reddy, S.M., Reddy, B.N., Reddy, M.V.K., Anusha, G., Pathak, M., Reddy, P.V.G., 2019b.
Pd-NHC catalyzed Suzuki–Miyaura couplings on 3-bromo-9H-pyrido[2,3- b]indole-
6-sulfonamide. Synth. Commun. 49, 1987–1996. https://doi.org/10.1080/
00397911.2019.1614194.
Roney, C., Kulkarni, P.V., Arora, V., Antich, P., Bonte, F., Wu, A., Mallikarjuna, N.N.,
Manohar, S., Liang, H.F., Kulkarni, A.R., Sung, H.W., Sairam, M., Aminabhavi, T.M.,
2005. Targetted nanoparticles for drug delivery through blood brain barrier.
J. Contr. Release 108, 193–214.
Kwong, H.C., Chidan Kumar, C.S., Mah, S.H., Mah, M.L., Chia, T.S., Quah, C.K.,
Chandraju, S., 2019. Crystal correlation of heterocyclic imidazo[1,2-a]pyridine
analogues and their anticholinesterase potential evaluation. Sci. Rep. 9, 926. https://
doi.org/10.1038/s41598-018-37486-7.
Sagar, S.R., Singh, D.P., Das, R.D., Panchal, N.B., Sudarsanam, V., Nivsarkar, M., Vasu, K.
K., 2019. Pharmacological investigation of quinoxaline-bisthiazoles as multitarget-
directed ligands for the treatment of Alzheimer’s disease. Bioorg. Chem. 89, 102992.
https://doi.org/10.1016/j.bioorg.2019.102992.
Lakowicz, J.R., 2006. Principles of Fluorescence Spectroscopy, vol. 8. Springer, Berlin,
pp. 278–292.
Salloway, S., Correia, S., 2015. Alzheimer disease: time to improve its diagnosis and
treatment. Cleve. Clin. J. Med. 76, 49–58. https://doi.org/10.3949/cjm.76a.072178.
Savini, L., Gaeta, A., Fattorusso, C., Catalanotti, B., Campiani, G., Chiasserini, L.,
Saxena, A., 2003. Specific targeting of acetylcholinesterase and
Liu, J., Ghastine, L., Um, P., Rovit, E., Wu, T., 2020. Environ. Res. 110406 https://doi.
org/10.1016/j.envres.2020.110406.
Liyana-Pathirana, C.M., Shahidi, F., 2005. Antioxidant activity of commercial soft and
hard wheat (Triticum aestivum L) as affected by gastric pH conditions. J. Agric. Food
Chem. 53, 2433–2440. https://doi.org/10.1021/jf049320i.
butyrylcholinesterase recognition sites. Rational design of novel, selective, and
highly potent cholinesterase inhibitors. J. Med. Chem. 46, 1–4. https://doi.org/
10.1021/m0255668.
Lolak, N., Boga, M., Tuneg, M., Karakoc, G., Akocak, S., Supuran, C.T., 2020.
Sulphonamides incorporating 1,3,5-triazine structural motifs show antioxidant,
acetylcholinesterase, butyrylcholinesterase, and tyrosinase inhibitory profile.
J. Enzym. Inhib. Med. Chem. 35, 424–431. https://doi.org/10.1080/
14756366.2019.1707196"7196.
Srivastava, J.K., Pillai, G.G., Bhat, H.R., Verma, A., Sing, U.P., 2017. Design and
discovery of novel monastrol-1,3,5-triazines as potent anti-breast cancer agent via
attenuating epidermal growth factor receptor tyrosine kinase. Sci. Rep. 7, 5851.
https://doi.org/10.1038/s41598-017-05934-5.
Tsai, T., Lin, Y., Hwang, B., Nakayama, S., Tsai, C., Sun, X., Ma, C., Jung, C., 2019.
Environ. Res. 177, 108638. https://doi.org/10.1016/j.envres.2019.108638.
Tangherlini, G., Kalinin, D.V., Schepmann, D., Che, T., Mykicki, N., Stander, S.,
Wunsch, B., 2019. Development of novel quinoxaline-based κ-opioid receptor
agonists for the treatment of neuro inflammation. J. Med. Chem. 62, 893–907.
https://doi.org/10.1021/acs.jmedchem.8b01609.
Ma, J., Porter, A.L., Aminabhavi, T.M., Zhu, D., 2015. Nano-enabled drug delivery
systems for brain cancer and Alzheimer’s Disease: research patterns and
opportunities. Nanomed. Nanotechnol. Biol. Med. 11, 1763–1771.
Ma, J., Porter, A.L., Aminabhavi, T.M., 2016. Nanoenabled drug delivery in cancer
therapy: in identifying latent relationships in biomedical literature using the MeSH
system. Pharm. Nanotechnol. 4, 1–15.
Tripathi, P.N., Srivastava, P., Sharma, P., Tripathi, M.K., Seth, A., Tripathi, A.,
Shrivastava, S.K., 2019. Biphenyl-3-oxo-1,2,4-triazine linked piperazine derivatives
as potential cholinesterase inhibitors with anti-oxidant property to improve the
learning and memory. Bioorg. Chem. 85, 82–96. https://doi.org/10.1016/j.
bioorg.2018.12.017.
Malleda, C., Ahalawat, N., Gokara, M., Subramanyam, R., 2012. Molecular dynamics
simulation studies of betulinic acid with human serum albumin. J. Mol. Model. 18,
2589–2597. https://doi.org/10.1007/s00894-011-1287-x.
Michael, J.P., 2005. Quinoline, quinazoline and acridone alkaloids. 2005. Nat. Prod. Rep.
22, 627–646. https://doi.org/10.1039/B509528J.
Umar, T., Shalini, S., Raza, M.K., Gusain, S., Kumar, J., Seth, P., Hoda, N., 2019.
A multifunctional therapeutic approach: synthesis, biological evaluation, crystal
structure and molecular docking of diversified 1H-pyrazolo[3,4-b]pyridine
derivatives against Alzheimer’s disease. Eur. J. Med. Chem. 175, 2–19. https://doi.
org/10.1016/j.ejmech.2019.04.038.
Min, J., Meng-Xia, X., Dong, Z., Yuan, L., Xiao-Yu, L., Xing, C., 2004. Spectroscopic
studies on the interaction of cinnamic acid and its hydroxyl derivatives with human
serum albumin. J. Mol. Struct. 692, 71–80. https://doi.org/10.1016/j.
molstruc.2004.0101.003.
Morris, G.M., Huey, R., Lindstrom, W., Sanner, M.F., Belew, R.K., Goodsell, D.S.,
Olson, A.J., 2009. AutoDock 4 and AutoDock Tools 4: automated docking with
selective receptor flexibility. J. Comput. Chem. 30, 2785–2791. https://doi.org/
10.1002/jcc.21256.
Vitaku, E., Smith, D.T., Njardarson, J.T., 2014. Analysis of the structural diversity,
substitution patterns, and frequency of nitrogen heterocycles among U.S. FDA
approved pharmaceuticals. J. Med. Chem. 57, 10257–10274. https://doi.org/
10.1021/jm501100b.
Nadri, H., Pirali-Hamedani, M., Shekarchi, M., Abdollahi, M., Sheibani, V.M.,
Amanlou, M., Foroumadi, A., 2010. Design, synthesis and anticholinesterase activity
of a novel series of 1-benzyl-4-((6-alkoxy-3-oxobenzofuran-2(3H)ylidene)methyl)
pyridinium derivatives. Bioorg. Med. Chem. 18, 6360–6366. https://doi.org/
10.1016/j.bmc.2010.07.012.
Wang, G., Li, X., Wang, J., Xie, Z., Li, L., Chen, M., Peng, Y., 2017. Synthesis, molecular
docking and α-glucosidase inhibition of 2-((5,6-diphenyl-1,2,4-triazin-3-yl)thio)-N-
arylacetamides. Bioorg. Med. Chem. Lett 27, 1115–1118. https://doi.org/10.1016/j.
bmcl.2017.01.094.
Wang, T., Tang, Y., Yang, Y., Ana, Q., Sang, Z., Yang, T., Luo, Y., 2018. Discovery of
novel anti-tuberculosis agents with pyrrolo[1,2-a]quinoxaline based scaffold.
Bioorg. Med. Chem. Lett 28, 2084–2090. https://doi.org/10.1016/j.
bmcl.2018.04.043.
Pacheco, G., Palacios-Esquivel, R., Moss, D.E., 1995. Cholinesterase inhibitors proposed
for treating dementia in Alzheimer’s disease: selectivity toward human brain
acetylcholinesterase compared with butyrylcholinesterase. J. Pharmacol. Exp.
Therapeut. 274, 767–770.
Xue, L., Shi, D.-H., Harjani, J.R., Huang, F., Beveridge, J.G., Dingjan, T., Baell, J.B., 2019.
3,3′-Disubstituted 5,5′-bi(1,2,4-triazine) derivatives with potent in Vitro and in Vivo
antimalarial activity. J. Med. Chem. 62, 2485–2498. https://doi.org/10.1021/acs.
jmedchem.8b01799.
Podlekareva, D.N., Efsen, A.M., Schultze, A., Post, F.A., Skrahina, A.M., Panteleev, A.,
Kirk, O., 2016. Tuberculosis-related mortality in people living with HIV in Europe
and Latin America: an international cohort study. Lancet HIV 3, 120–131. https://
doi.org/10.1016/S2352-3018(15)00252-0.
Yasodakrishna, S., Hanmanth Reddy, V., Rajashaker, B., Lingaiah, N., Sathish Babu, V.,
Srigiridhar, K., Jagadeesh Babu, N., 2016. Three-component, one-pot synthesis of
benzo[6,7]cyclohepta[1,2-b]pyridine derivatives under catalyst free conditions and
evaluation of their anti-inflammatory activity. Bioorg. Med. Chem. Lett 26, 858–863.
https://doi.org/10.1016/j.bmcl.2015.12.078.
Reddy, B.R.P., Reddy, M.V.K., Reddy, P.V.G., Kumar, D.P., Shankar, M.V., 2016a.
Protonated trititanate nanotubes: an efficient catalyst for one-pot three-component
coupling of benzothiazole amines, heterocyclic aldehydes, and dialkyl/diaryl
phosphites with a greener perspective. Tetrahedron Lett. 57, 696–702. https://doi.
org/10.1016/j.tetlet.2016.01.001.
Yazdani, M., Edraki, N., Badri, R., Firuzi, O., 2019. 5,6-Diphenyl triazinethio methyl
triazole hybrid as a new Alzheimer’s disease modifying agents. Mol. Divers. 1–14
https://doi.org/10.1007/s11030-019-09970-3.
Reddy, M.V.K., Anusha, G., Reddy, P.V.G., 2019a. Cu-N-heterocyclic carbene-catalysed
synthesis of 2-aryl-3-(arylethynyl)quinoxalines from one-pot tandem coupling of o-
phenylenediamines and terminal alkynes. Appl. Organomet. Chem. 33, 1–12.
https://doi.org/10.1002/aoc.5188 e5188.
Yeggoni, D.P., Rachamallu, A., Subramanyam, R., 2016. A comparative binding
mechanism between human serum albumin and α-1-acid glycoprotein with
Reddy, M.V.K., Anusha, G., Reddy, P.V.G., 2020. Sterically enriched bulky 1,3- bis(N,N’-
aralkyl)benzimidazolium based Pd-PEPPSI complexes for Buchwald-Hartwig
Amination reactions. New J. Chem. 44, 11694–11703. https://doi.org/10.1039/
D0NJ01294G.
corilagin: biophysical and computational approach. RSC Adv. 6, 40225–40237.
https://doi.org/10.1039/C6RA06837E.
Yun, Y., Yang, J., Miao, Y., Wang, X., Sun, J., 2020. Synthesis and biological evaluation
of 4-arylcoumarins as potential anti-Alzheimer’s disease agents. Bioorg. Med. Chem.
Lett 30, 126900. https://doi.org/10.1016/j.bmcl.2019.126900.
Zhang, H.-Y., 2005. One-compound-multiple-targets strategy to combat Alzheimer’s
disease. FEBS (Fed. Eur. Biochem. Soc.) Lett. 579, 5260–5264. https://doi.org/
10.1016/j.febslet.2005.09.006.
Reddy, M.V.K., Reddy, P.V.G., Reddy, C.S., 2016b. PEPPSI-SONO-SP2: a new highly
efficient ligand-free catalyst system for the synthesis of tri-substituted triazine
derivatives via Suzuki–Miyaura and Sonogashira coupling reactions under a green
approach. New J. Chem. 40, 5135–5142. https://doi.org/10.1039/C5NJ03299G.
Reddy, P.S., Reddy, M.V.K., Reddy, P.V.G., 2016c. Camphor-derived thioureas: synthesis
and application in asymmetric Kabachnik-Fields reaction. Chin. Chem. Lett. 27,
943–947. https://doi.org/10.1016/j.cclet.2016.01.046.
12