Exploration of multi-target potential of chromen-4-one based compounds in Alzheimer's disease: Design, synthesis and biological evaluations

Article abstract of DOI:10.1016/j.bmc.2017.09.012

A novel series of flavonoid based compounds were designed, synthesized and biologically evaluated for Acetylcholinesterase (AChE) inhibitory activity integrated with advanced glycation end products (AGEs) inhibitory and antioxidant potential. Most of the derivatives inhibited AChE in nanomolar IC₅₀ range along with good AGEs inhibitory and radical scavenging activity. Among them, 7m, strongly inhibited AChE (IC₅₀ = 5.87 nM) and found to be potent as compared to the reference drug donepezil (IC₅₀ = 12.7 nM). Its potent inhibitory activity has been justified by docking analysis that revealed its dual binding characteristic with both CAS (catalytic active site) and PAS (peripheral anionic site) of AChE, simultaneously. Additionally, this compound also displayed ability to prevent advanced glycation end products formation (IC₅₀ = 23.0 μM) with additional radical scavenging property (IC₅₀ = 37.12 nM). It (7m) also ameliorated scopolamine induced memory deficit in mice employing Morris water maze test. Thus, flavonoids might be the promising lead compounds as potential polyfunctional anti-Alzheimer's agents.

Full text of DOI:10.1016/j.bmc.2017.09.012

Accepted Manuscript
Exploration of chromen-4-one based scaffold’s potential in Alzheimer’s dis-
ease: Design, Synthesis and Biological evaluations
Manjinder Singh, Maninder Kaur, Nirmal Singh, Om Silakari
PII:
S0968-0896(17)30935-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.09.012
BMC 13972
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 May 2017
1 September 2017
9 September 2017
Please cite this article as: Singh, M., Kaur, M., Singh, N., Silakari, O., Exploration of chromen-4-one based
scaffold’s potential in Alzheimer’s disease: Design, Synthesis and Biological evaluations, Bioorganic & Medicinal
Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.09.012
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Exploration of chromen-4-one based scaffold’s potential in
Alzheimer’s disease: Design, Synthesis and Biological evaluations
Manjinder Singha, Maninder Kaura, Nirmal Singha, Om Silakaria,*
aDepartment of Pharmaceutical Sciences and Drug Research, Punjabi university, Patiala,
Punjab, India, 147002.
*E-mail: omsilakari@gmail.com, Telephone: +919501542696

ABSTRACT
A novel series of flavonoid based compounds were designed, synthesized and biologically
evaluated for Acetylcholinesterase (AChE) inhibitory activity integrated with advanced glycation
end products (AGEs) inhibitory and antioxidant ptential. Most of the derivatives inhibited AChE
in nanomolar IC50 range along with good AGEs inhibitory and radical scavenging activity.
Among them, 7m, strongly inhibited AChE (IC50=5.87 nM) and found to be potent as compared
to the reference drug donepezil (IC50=12.7 nM). Its potent inhibitory activity has been justified
by docking analysis that revealed its dual binding characteristic with both CAS (catalytic active
site) and PAS (peripheral anionic site) of AChE, simultaneously. Additionally, this compound
also displayed ability to prevent advanced glycation end products formation (IC50=23.0 µM) with
additional radical scavenging property (IC50=37.12 nM). It (7m) also ameliorated scopolamine
induced memory deficit in mice employing Morris water maze test. Thus, flavonoids might be
the promising lead compounds as potential polyfunctional anti-Alzheimer’s agents.
Keywords: Alzheimer’s disease, Acetylcholinesterase, Antioxidants, Flavonoids, AGEs, Morris
water maze

1. Introduction
Alzheimer’s disease (AD) is a complex neurodegenerative disorder among elderly people
characterized with gradual decline in various cognitive, executive and memory functions.1 Other
than Aβ (β-amyloid) deposits, τ-protein aggregation and low ACh level, oxidative damage is
eminently observed in AD brains.2 In cholinergic hypothesis, the acetylcholine, a
neurotransmitter responsible for behavior, memory, cognitive functions and emotions in the
brain areas is reduced because of its prompt hydrolysis by AChE enzyme. Additionally, it also
promotes the amyloid-β deposition, which further develop the senile plaques.3
Acetylcholinesterase inhibitors (AChEIs) could upsurge the ACh level through AChE inhibition
in patients of AD and, therefore, relieve some symptoms experienced by patients. Till date,
cholinergic hypothesis based therapeutic approach with AChEIs such as rivastigmine, donepezil
and galantamine has been used clinically for AD management.4 Therefore, inhibition of AChE
has been considered for the management of AD as it may increase acetylcholine level and
decrease Aβ deposition in the brain regions.
Furthermore, numerous evidences suggested the degrading influence of oxidative stress in
the AD pathophysiology and progression. Present reports specified that the oxidative impairment
may possibly endorse the amyloid plaques and neurofibrillary tangles formation in AD.5 The
increased level of reactive carbonyls and free radicals leads to form the AGEs, the
macroproteins, formed via Maillard reaction (non-enzymatic glycation) also complicated the AD
pathogenesis. It cross-linked and glycated the Aβ or tau proteins, cell death of neurons and glial
induction. Moreover, it interact with cell surface receptors RAGE (receptor for AGEs) that
provokes the generation of superoxide radicals, hydrogen peroxide, vascular inflammation etc.,
ultimately contribute to AD pathology.6 Consequently, drugs that have the capacity to prevent or
scavenge the free radicals generation and AGEs inhibitory potential could help for the
management of AD.
The complex nature of AD demands the designing of polyfunctional agents which could
simultaneously act on more than one pathway rather than only one. It also implemented diverse
biological properties with single bioavailability and pharmacokinetic metabolism. In the present
study, the well-established molecular pathways i.e. AChE, oxidative stress, and AGEs have been
explored for the designing of flavoinoid based polyfunctional agents for more effective therapy

of AD than existing one. Flavonoid was considered for designing as it possesses a broad
pharmacological properties range like anti-inflammatory, anti-oxidative, AGEs inhibitory effects,
and neuroprotective effects against AChE.7 Thus, the designing and synthesis of new
polyfunctional flavonoid derivatives is an interesting strategy for AD management.
The SAR of our previously reported flavonoids based novel AChE inhibitors,8 focused our
attention to design more flavonoid derivatives bearing a suitable tertiary amino group, which
characterizes the crucial requirement for good AChE inhibitory activity, linked with different
lengths of alkyl side chain with variedly substituted flavonoid scaffold. Therefore, we here
present work involving the synthesis of flavonoid based novel polyfunctional agents, which were
primarily evaluated for in vitro AChE inhibition, AGEs inhibition, antioxidant effect and
molecular modeling studies. Additionally, in vivo anti-amnestic and antioxidant activities of the
most active polyfunctional agent were determined.
2. Result and Discussion
2.1 Chemistry
Firstly, to evaluate the influence of different number of methoxy groups at ring-B with
hydroxyl group at 7th position of ring-A over the biological activities of flavonoids, compounds
5(a–c), were synthesized using well established Baker-Venkataraman rearrangement with slight
modifications is outlined in Scheme 1.9
Our research group previously reported various flavonoids having methoxy group on ring-B
and substituted cyclic amine on ring-A via two carbon spacer. Based on those observations, the
hydroxyl group of 5(a–c), was replaced by 4-methylpiperidine and 4-hydroxyethyl piperazine
amines linked via different carbon spacers of 4`-methoxy substituted flavonoids. The synthetic
methodologies employed to develop intermediates 6(a-e) and target compounds 7(a–j), are
outlined in scheme 2 and 3. Firstly, the 5(a-c) were alkylated with dibromoalkanes in acetone
that delivered intermediates 6(a-e) in satisfactory yields. Finally, the reaction of 6(a-e) with
commercially available secondary amines under reflux in acetonitrile in the presence of K2CO3
produced the final compounds 7(a–j) in 50–80% yields.

Further, synthesis of the compounds 7(k-n), with additional methoxy groups at ring-B and 4-
methylpiperidinoalkyloxy substituent attached at ring- B via two (7k and 7m) and three (7l and
7n) carbon spacer were accomplished using same procedures as shown in Scheme 3.
2.2 In vitro inhibition studies on AChE
To investigate the potential of synthesized compounds 5(a-c) and 7(a–n) for the management
of AD, their inhibition towards AChE (brain homogenate) was determined using donepezil as
standard compound.10 The results of all tested derivatives are summarized in Table 1. Among
the 7-hydroxyl derivatives 5(a-c), 5a with 4`-methoxy at ring-B was found to possess better IC50
value 35.0 nM than compounds 5b and 5c (IC50=37.3, 44.2 nM, respectively).
Another series 7(a-j), with a methoxy group at the ring-B and different cyclic amines having
varied alkyl side chains at ring-A, showed greater AChE inhibitory potential as compared to 5(a-
c). The alkylation of hydroxyl group at the ring-A in 5(a-c) with propyl to pentyl carbon chain
cyclic amino significantly increased AChE inhibitory potency (7(a-f), IC50 ranges from 7.7 to
17.3 nM) indicating the significance of the quaternary amine than the hydroxyl group. However
further increase in carbon spacer reduced the activity by 3-fold (7(g-j), IC50 ranges from 48.1 to
67.2 nM).
As presented in Table 1, among all (7a-7j), compounds 7a and 7b showed most potent
inhibitory activity against AChE having IC50 values of 7.7 and 8.2 nM, respectively. The effect
of different carbon chain linkers attached to aminoalkyl moiety on AChE inhibition were also
studied and observed that on increasing the chain length from four to eight carbons, the activity
was reduced (7(c-j), IC50 =13.53-67.2 nM) than compounds having two to three carbon (7(a-b)
and 7(k-n), IC50 =5.87-8.25 nM), thus ethyl to propyl alkyl chain length is optimum of cyclic
aminoalkyl groups for AChE inhibition. The substitution with additional –OCH3 group at ring-B
reliably enhanced the AChE inhibition with IC50 values ranges from 5.87-6.77 nM. The 3`,4`,5`-
Trimethoxy substituted compounds (7m and 7n, IC50 = 5.87 and 6.12 nM) showed good potencies
than 3`,4`-Dimethoxy-substituted derivatives (7l and 7k, IC50 = 6.48 and 6.77 nM).
Overall, the 3`,4`,5`-Trimethoxy-substituted flavone 7m was found to be most active
compound having IC50 value of 5.87 nM, which is 2-fold of donepezil activity (IC50=12.7 nM).
Therefore, compound 7m was selected for in vivo evaluation.

2.3 Advanced glycation end-product formation inhibitory activity
All the synthesized compounds 5(a-c) and 7(a–n) were subjected to in vitro bioassay of
AGEs formation inhibitory activity summarized in Table 1.11 Among the various synthesized
compounds, the compound 7m (IC50=37.1 µM) was found to be comparable to the reference
drug aminoguanidine (IC50=40.0 µM). The compounds 7d, 7n, 7k, 7a, 5a and 7b (IC50 value
ranging from 38.4 to 43.1 µM) showed good to moderate inhibitory activity with respect to the
reference drug. However, the compounds 5b, 7l, 7c, 7f, 7h, 5c, 7j, 7e, 7g and 7i (IC50 value
ranging from 47.4 to 61.1 µM) displayed lower anti-glycating activity than the standard drug. As
evident from the results, the derivatives having alkyloxy linked to hydroxyethyl piperazine
substitution at 7th position of ring-A and 4’-methoxy ring-B of flavone (7b, 7d, 7f, 7h and 7j)
were found to be more active than the derivatives with 4-methylpiperidine alkyloxy at position
7th of ring-A (7c, 7e,7g and 7i). The compound with 4-methylpiperidine moiety at position 7th of
ring-A and 3’,4’,5’-trimethoxy on ring-B of flavone (7m) was found to be more active than the
compounds with 3’,4’-dimethoxy (7k and 7l, IC50=42.1, 47.7 µM, respectively) and mono
methoxy (7a, IC50=42.6 µM) on ring-B. The most active compound 7m (IC50=37.1 µM), 4-
methylpiperidine linked using two carbon spacer showed good activity as compared to
compound 7n (IC50=41.7 µM) having 4-methylpiperidine moiety linked using three carbon
spacer. The flavonoid 5a (IC50=42.0 µM) having hydroxyl at position 7th of ring-A and 4’-
methoxy at ring-B of flavone exhibited good activity than compounds 5b and 5c (IC50=47.4, 51.4
µM, respectively) having 3’,4’-dimethoxy and 3’,4’,5’-trimethoxy at ring-B of flavone,
respectively.
2.4 In vitro antioxidant activity
Synthesized compounds 5(a-c) and 7(a–n) were evaluated for in vitro antioxidant activity12 and
results are showed in Table 1. Results noticeably indicate that almost all the compounds
displayed radical scavenging activity. Among them, 5a, 5b, 7k, 7b & 7m (EC50=20.5-23.0 nM)
were comparable to ascorbic acid EC50=20.0 nM. The compound 5a was found to be the best
radical scavenger (EC50=20.5 nM), might be due to the hydroxyl group. The derivatives with 4-
(2-hydroxyethyl)piperazine-1-yl)alkoxy substitution at 7th position of ring-A (7b, 7d, 7f and 7j,
EC50 ranges from 22.7-35.7 nM ) were found to be more active than the derivatives with 4-

methylpiperidine alkyloxy at same position (7a, 7c, 7e and 7i, EC50 ranges from 26.5-42.6 nM).
The compounds with 2-(4-methylpiperidin-1-yl)ethoxy substituent showed good activity (7k and
7m, EC50=22.7, 23.0 nM, respectively) as compared to compounds with 3-(4-methylpiperidin-1-
yl)propoxy substituent (7l and 7n, EC50=24.1, 26.8 nM, respectively). Furthermore, increase in
the chain length s of alkyl spacer between ‘O’ and ‘N’ of substituents decrease the radical
scavenging activity by 1.5-2.0 fold. The double bond between C2=C3 in conjugation with a
carbonyl at C4 of flavonoid moiety provides electron delocalization from the ring-B, appears to
be crucial to scavenge the free radicals. Conjugation system of ring-A and B showed extended
resonance effect providing increased radical stability to the flavonoid nucleus.13
2.5 Kinetic characterization of AChE inhibition
Based on the in vitro results of AChE inhibition, the kinetic study was carried out on most
potent inhibitor 7m to determine the kind of enzyme inhibition. The Lineweaver–Burk plots
displayed both increased intercepts (higher Km) and slopes (decreased Vmax) at higher
concentration of compound 7m, indicating a mixed type of inhibition (Fig. 1). Thus, compound
7m have the ability to bind with both CAS as well as PAS of AChE enzyme.
2.6 Molecular docking studies
In order to discover the interaction patterns of flavonoids with AChE enzyme, docking studies
were carried out for the most potent inhibitor 7m with Torpedo californica AChE (TcAChE,
1EVE) crystal structure.14,15 Its 3D structure contains an active site situated at deep and narrow
gorge bottom comprising of Phe330 and Trp84 amino acid residues in the catalytic anionic site.
Additionally, aromatic residues Tyr70, Tyr121, Tyr334, Asp72 and Trp279 of peripheral anionic
site located near the gorge of active site.16 The compound 7m showed nice fit in active site by
binding with both CAS and PAS of the AChE enzyme simultaneously to afford maximal
inhibition. The binding pattern of compound 7m with AChE binding site is shown in Fig. 2.
As shown in Fig. 2, the compound 7m binds along the active-site gorge, extending from the
anionic subsite near Trp84 to the peripheral anionic site (PAS) near Trp279. The protonated
nitrogen atom the 4-methylpiperidine moiety of compound 7m, displayed cation-π interaction
with conserved amino acid residue Trp279, a major component of a peripheral “anionic” site
(PAS) which is 14 Å far from the active site and nearby to gorge top of the enzyme. It also

interacts with Ser286 of PAS via hydrogen bonding. Ring-B of flavonoidal scaffold showed π-π
stacking with Phe331, in acyl binding pocket. The ring-B also interacts with catalytic triad of
CAS, accountable for ACh ester bond hydrolysis, via π-π stacking with His440 amino acid and
hydrogen bonding of methoxy group at ring-B with Ser200 amino acid. The ‘O’ of –OCH3 group
also forms H-bond with peptidic amino groups of Gly118, another residue located in the esteratic
subsite. The docking study of 7m revealed the dual binding property as it interacted with both
catalytic as well as peripheral anionic site via hydrogen bond, π–π (aromatic) and hydrophobic
interactions. (Fig. 2)
2.7 Molecular Dynamic Simulations
Molecular dynamic simulation (MD) was undertaken to identify the ligand-protein interactions
in dynamic motion contributing for thermodynamic stability and to envisage the effect on
conformational changes induced by ligand binding at AChE pocket.17
The protein ligand binding complex of AChE and the most active compound 7m was used for
MD simulations. After MD, although the important interactions were conserved, some additional
interactions were also observed. After MD, compound 7m displayed cation-π interactions with
Trp279 amino acid of PAS. It also showed π-π stacking interactions with Trp84 amino acid of
CAS and strong hydrogen bonding with Ser81 (Fig. 3A). These interactions are summarized in
the protein-ligand contacts plot (Fig. 3B). After MD simulations, the RMSD (Root mean square
deviation) was calculated for trajectory of complex and plotted against time (ns). The plot of
RMSD showed, docked complex was quite stable all the way through with minor fluctuations in
the range of 1–1.5 Å (Fig. 3C). The stability of the simulated system indicates the inhibitory
nature of the 7m on AChE.
2.8 ADMET prediction
To envisage the drug likeness of the synthesized compounds, different indicators of
pharmacokinetic profile like Lipinski’s parameters, QPlogPo/w, Polar Surface Area (PSA),
QPlogKhsa, QPPCaco, QPlogBB, QPLogKhsa, QPlogBB, QPPMDCK etc. were predicted with
QikProp module.18,19 (Table 2)
The important features of drugs acting on CNS should have the ability to cross BBB (blood
brain barrier). The results showed that synthesized compounds 5(a-c) and 7(a–n) obey Lipinski’s

rule of five (mol_MW <500, QPlogPo/w <5, donorHB≤5, accptHB≤10) and therefore are drug
like molecules. The results for CNS activity and QPlogBB showed that all synthesized
compounds have the ability to enter into the brain. PSA (polar surface area) also predicted the
ability to pass through BBB. For a compound to be CNS active, PSA should be lower to
penetrate the BBB and the value should be less than 90 Å.20 The QPlogKhsa value determined
the protein binding amount of CNS active drugs which is also a vital consideration and be likely
to moderately high. The QPlogKhsa values for all compounds 5(a-c) and 7(a–n) showed their
strong plasma protein binding. All the synthesized compounds are able to cross the BBB though
their PSA values are within acceptable limits (56-85 Å). Among predicted parameters, QPlogBB,
QPLogKhsa, QPPMDCK and QPPCaco primarily reflect the capability of the compound’s
distribution in the body. Majority of the compounds exhibited moderate to significant
penetrability for in vitro MDCK cells as well as in vitro Caco-2 cells. The pharmacokinetic
parameters prediction disclosed that all synthesized compounds 5(a-c) and 7(a–n), have drug
like properties. Further, to estimate relative binding affinity of all the synthesized compounds
MM-GBSA binding energies were calculated as showed in Table 2. The energy of all the
synthesized derivatives ranges from –42.534 to –99.832, comparable to MM-GBSA energy of
donepezil –78.70, thus signifying durable ligand-enzyme binding interactions.
2.9 In vivo activity on scopolamine induced amnesia in mice assessed on Morris water maze.
The compound 7m was further assessed for memory restoration in scopolamine induced
amnesia in mice.21 Administration of scopolamine (0.5 mg/kg) significantly decreased ELT
(escape latency time) on day 4 and TSTQ (time spent in target quadrant) on day 5 signifying the
memory loss as evaluated using Morris water maze as compared to normal mice. Administration
with donepezil (2 mg/kg) significantly reversed amnesia induced by scopolamine in comparison
with scopolamine treated animals. Similarly, treatment with 7m (2, 5 and 10 mg/kg) significantly
attenuated the scopolamine induced decrease in ELT on day 4 and TSTQ on day 5 (Fig. 4 and
Fig. 5).
2.10 Effect of compound 7m on biochemical parameters
After behavioral studies, biochemical parameters were determined in brain homogenates, as
shown in Table 3, scopolamine administration significantly increased the TBARS levels and

AChE activity whereas endogenous antioxidant (reduced GSH) levels were significantly depleted
as compared to vehicle treated animals.22,23
The standard drug donepezil significantly reduced the TBARS levels and AChE activity
whereas increased the GSH levels. Similar effects were observed in mice treated with most
potent compound 7m (2, 5 and 10 mg/kg, i.p). In the present investigation, scopolamine
administration lead to enhanced oxidative stress showed by elevated TBARS levels and depleted
reduced GSH levels in homogenate of mice brain. The TBARS and reduced GSH levels were
restored with compound 7m treatment significantly. These results showed that compound 7m
may attenuate the cognitive deficit by preventing the oxidative damage and neurodegeneration
induced by scopolamine.
3. Experimental Section
3.1 Chemistry
All chemicals and solvents required for synthesis were obtained commercially from various
suppliers and were of LR grade, used without any purification. The solvents were dehydrated
according to standard methods. The synthesis was carried out using magnetic stirrer and hot plate
(Perfit), and solvents were recovered using rotary vacuum evaporator (Perfit). The completion of
each reaction was monitored by thin layer chromatography (DC-Alufolien (20x20 cm) Kieselgel
60 F254 chromato plates) using hexane:ethylacetate (6:4, v/v) and chloroform:methanol (9:1 v/v)
as a TLC development solvent system. Impure compounds and intermediates were purified on
silica columns from appropriate solvent. The melting points were recorded on an electronic
1
melting point apparatus. The purity of all organic compounds was confirmed by TLC, IR, H
NMR, 13C NMR and Mass spectrometry. IR spectra were recorded on a Bruker (Alpha E) FT/IR
1
spectrophotometer, H-NMR spectra were recorded on a Bruker Advance II 400 MHz NMR
spectrometer using chloroform (CDCl3) or dimethylsulfoxide (DMSO-d6) as solvent. The
chemical shifts are reported in parts per million (δ) downfield from TMS and coupling constants
are reported in hertz (Hz). Proton coupling patterns are abbreviated as single (s), double (d),
triplet (t) and multiple (m). Mass spectra (ESI-MS) were recorded with a Waters, Q-TOF
Micromass (LC-MS).

3.1.1 General procedures for the synthesis of intermediate o-Benzoyloxyacetophenone (3)
At first, the substituted o-Benzoyloxyacetophenone (3) was synthesized by stirring the
mixture of substituted o-Hydroxyacetophenone (0.1mol) and substituted benzoyl chlorides
(0.15mol) in dry pyridine. During the reaction, the mixture evolved spontaneous heat, and after
about 15 minutes when the temperature comes down to room temperature, the mixture was
poured, with constant stirring, into 3% hydrochloric acid containing crushed ice resulting in the
precipitation of solid residue. The solid residue was then filtered and washed with methanol
followed by water. The filtered residue was air dried, and re-crystallized from methanol resulting
in the white precipitates of substituted o-Benzoyloxyacetophenone, yield 90%.
3.1.2 General procedures for the synthesis of intermediate o-Hydroxydibenzoylmethane (4)
To a warm solution of substituted o-Benzoyloxyacetophenone (3) in dry pyridine, hot
pulverized 85% potassium hydroxide was added followed by mechanical stirring for 30 minutes
resulting in the gradual appearance of yellowish brown precipitates of potassium salt of
substituted o-Hydroxydibenzoylmethane (4). The reaction mixture was brought down to room
temperature, and subsequently acidified with 10% acetic acid to desalt the compounds. The light-
yellow precipitates of diketone (4) were filtered and dried, yield 85%.
3.1.3 General procedures for the synthesis of compounds 5(a-c).
The substituted o-Hydroxydibenzoylmethane (4), in the presence of glacial acetic acid and
conc. sulphuric acid, was refluxed on water bath for 1h, to achieve the cyclized product. Then,
the reaction mixture was poured onto crushed ice with vigorous stirring resulting in the
precipitation of the compounds 5(a-c). The product was filtered, thoroughly washed with water
to make it free from acid and dried. Then, purified on silica columns using hexane: ethyl acetate
(6:4%) as solvent [38, 39]. (Scheme 1)
3.1.4 General procedures for the synthesis of intermediates 6(a-g)
The synthetic protocol for compound 6(a-g) is outlined in Scheme 2. To synthesize the
intermediates 6(a-g), anhydrous K2CO3 (10.0 mmol) and different α,ω-dibromoalkanes (4.0
mmol) were added to a solution of substituted compounds 5(a-c) (2.0 mmol) in acetone (20 mL).
After reflux for 10 h until the starting material disappeared, the solvent was removed under

vacuum; the residue was then poured into water and extracted with ethyl acetate. The solution
was then dried over sodium sulphate and then concentrated. The intermediates 6(a-g) were
purified on silica columns using chloroform:methanol (9.5:0.5) [50]. (Scheme 2)
3.1.5 General procedures for the synthesis of final compounds 7(a-n).
To a solution of intermediates 6(a-g) (0.5 mmol) in acetonitrile (10 mL), different amines
(1.0 mmol) and anhydrous K2CO3 (2.5 mmol) were added. After stirring at 60°C for 12 h, the
solvent was removed under vacuum; the mixture was diluted with CHCl3 and then washed with
water and brine. The organic layer was dried over Na2SO4, filtered, concentrated in vacuo, and
the final compounds 7(a-n) were purified by column chromatography with CHCl3:MeOH (95:5)
elution [50]. (Scheme 3)
3.1.5.1. 7-hydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one (5a):
Brown black, amorphous solid, yield 91%, m.p.: 231-236°C; IR (KBr) (cm-1): (-OH) 3375
1
broad (s); (=C-H) str 3175 (m); (C=O) 1661 (s); 1626 Ar (C=C) str (m); H-NMR (400 MHz,
DMSO-d6, δ ppm): 10.62 (1H, s, -OH), 7.94-7.96 (2H, dd, J= 1.88, 7.0 Hz, ArH), 7.89 (1H, d, J=
8.68 Hz, ArH), 7.05-7.08 (2H, d, J= 2.96 Hz, ArH), 6.94 (1H, d, J= 2.16 Hz, ArH), 6.87-6.90
(1H, dd, J= 2.20, 8.68 Hz, ArH), 6.69 (1H, s, ArH), 3.87 (3H, s, -OCH3); (100 MHz, DMSO-d6,
δ ppm): 176.36, 162.54, 162.01, 161.77, 157.36, 127.69, 126.25, 123.45, 116.07, 114.65,
114.24, 105.03,102.34, 55.25; MS (ESI) m/z = 269.1 [M+H]+; Rf Value: 0.53 (Hexane: Ethyl
acetate, 6:4).
3.1.5.2 2-(3,4-Dimethoxyphenyl)-7-hydroxy-4H-chromen-4-one (5b):
Light Yellow, Amorphous solid, yield 60%;%; IR (KBr) (cm-1): 3375-3179 cm-1 (-OH,
broad (s); 1626 cm-1 (C=O, str, s); 1614 cm-1 (C=C, str, m); 1H-NMR (400 MHz, CDCl3, δ ppm):
10.51 (1H, s-broad, -OH), 7.87-7.90 (1H, d, J = 8.72 Hz, ArH), 7.60-7.62 (1H, d, J = 7.12 Hz,
ArH), 7.49 (1H, s, ArH), 7.06-7.08 (1H, d, J = 8.48 Hz, ArH), 6.95 (1H, s, ArH), 6.88-6.90 (1H,
d, J = 7.88 Hz, ArH), 6.76 (1H, s, ArH), 3.93 (3H, s, -OCH3), 3.90 (3H, s, -OCH3); 13C NMR
(100 MHz, DMSO-d6, δ ppm): 176.36, 162.54, 161.39, 157.48, 149.8, 148.7, 126.30, 123.45,
121.47, 116.67, 114.87, 111.74, 108.47, 105.47, 102.50, 56.36; MS (ESI) m/z = 299.47 [M+H]+;
Rf Value: 0.55 (Hexane:Ethyl acetate, 6:4).

3.1.5.3 7-hydroxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (5c)
Dark Brown, solid amorphous, yield 70%; IR (KBr) (cm-1): 3674 cm-1 (-OH broad, s);
1
2926 cm-1 (=C-H,) str, m); 1628 cm-1 (C=O, str, s); 1586 cm-1 (C=C, str, m); H-NMR (400
MHz, DMSO-d6, δ ppm): 10.68 (1H, s-broad, -OH), 7.87-7.90 (1H, d, J = 8.72 Hz, ArH), 7.25
(2H, s, ArH), 6.99 (1H, s, ArH), 6.89-9.62 (1H, m, ArH), 6.87 (1H, s, ArH) 3.93 (6H, s, -OCH3)
3.80 (3H, s, -OCH3); (100 MHz, DMSO-d6, δ ppm): 176.54, 162.36, 161.71, 157.31, 153.01,
141.11, 126.30, 123.42, 116.02, 114.75, 105.11, 102.42, 61.03, 56.23; MS (ESI) m/z = 329.1
[M+H]+; Rf Value: 0.60 (Hexane:Ethyl acetate, 6:4).
3.1.5.4. 7-(2-Bromopropoxy)-2-(4-methoxyphenyl)-4H-chromen-4-one (6a):
Light yellowish, solid amorphous, yield 55%; 1H-NMR (400 MHz, CDCl3, δ ppm): 8.10-
8.13 (1H, d, J = 9.40 Hz, ArH), 7.87 (1H, s, ArH), 7.85-7.86 (1H, d, J = 2.04 Hz, ArH), 7.01.-
7.03 (2H, m, ArH), 6.96-6.98 (2H, m, ArH), 6.68 (1H, s, ArH), 4.23-4.26 (2H, t, -CH2) 3.90-
3.91 (2H, d, -CH2), 3.89 (3H, s, -OCH3) 2.09-2.15 (2H, m, -CH2), Rf Value: 0.20 (CHCl3:
MeOH, 9:1).
3.1.5.5. 7-(4-Bromobutoxy)-2-(4-methoxyphenyl)-4H-chromen-4-one (6b):
Yellow, solid amorphous, yield 75%; 1H-NMR (400 MHz, CDCl3, δ ppm): 8.11-8.13
(1H, d, J = 8.64 Hz, ArH), 7.83-7.87 (2H, m, ArH), 6.99-7.03 (2H, m, ArH), 6.67 (1H, s, ArH),
4.10-4.13 (2H, t, -CH2), 3.88 (3H, s, -OCH3), 3.49-3.53 (2H, t, -CH2), 2.00-2.12 (4H, pentet, -
CH2). Rf Value: 0.65 (CHCl3: MeOH, 9:1).
3.1.5.6. 7-((5-Bromopentyl)oxy)-2-(4-methoxyphenyl)-4H-chromen-4-one (6c):
Yellowish cream, solid amorphous, yield 50%; 1H-NMR (400 MHz, CDCl3, δ ppm):
8.10-8.12 (1H, d, J = 8.72 Hz, ArH), 7.86-7.87 (1H, d, J = 2.08 Hz, ArH), 7.84-7.85 (1H, d, J =
2.0 Hz, ArH), 7.00-7.02 (2H, dd, , J = 6.92, 1.96 Hz, ArH), 6.93-6.97 (2H, m, ArH), 6.68 (1H, s,
ArH), 4.07-4.10 (2H, t, -CH2), 3.88 (3H, s, -OCH3), 3.44-3.47 (2H, t, -CH2), 1.86-1.98 (4H, m, -
CH2), 1.67-1.69 (2H, m, -CH2). Rf Value: 0.67 (CHCl3: MeOH, 9:1).
3.1.5.7. 7-((6-Bromohexyl)oxy)-2-(4-methoxyphenyl)-4H-chromen-4-one (6d):

1
Light yellowish brown, solid amorphous, yield 70%; H-NMR (400 MHz, DMSO-d6, δ
ppm): 8.00-8.02 (2H, d, J = 8.88 Hz, ArH), 7.92-7.94 (1H, d, J = 8.80 Hz, ArH), 7.25-7.26 (1H,
d, J = 2.28 Hz, ArH), 7.08-7.10 (2H, d, J = 8.88 Hz, ArH), 7.00-7.03 (1H, dd, J = 8.80 Hz,
ArH), 6.79 (1H, s, ArH), 4.22-4.25 (2H, t, -CH2) 3.87 (3H, s, -OCH3) 2.75-2.81 (5H, m, -CH2, -
NH), 2.52-2.53 (6H, t, -CH2) Rf Value: 0.70 (CHCl3: MeOH, 9:1).
3.1.5.8. 7-((8-Bromooctyl)oxy)-2-(4-methoxyphenyl)-4H-chromen-4-one (6e):
Light yellowish, solid amorphous, yield 75%; 1H-NMR (400 MHz, CDCl3, δ ppm): 8.03-
8.05 (1H, d, J = 8.76 Hz, ArH), 7.77.80 (2H, m, ArH), 6.95-6.96 (1H, t, ArH), 6.93-6.94 (1H, d, J
= 1.88 Hz, ArH ), 6.86-6.90 (2H, m, ArH), 6.60 (1H, s, ArH), 3.98-4.01 (2H, t, -CH2), 3.82 (3H,
s, -OCH3), 3.34-3.36 (2H, t, -CH2), 1.74-1.97 (4H, m, -CH2), 1.32-1.45 (8H, m, -CH2). Rf Value:
0.65 (CHCl3: MeOH, 9:1). Rf Value: 0.68 (CHCl3: MeOH, 9:1).
3.1.5.9. 7-(3-Bromopropoxy)-2-(3,4-dimethoxyphenyl)-4H-chromen-4-one (6f):
1
Yellow, solid amorphous, yield 55%; H-NMR (400 MHz, CDCl3, δ ppm): 8.12--8.15
(1H, d, J = 9.48 Hz, ArH), 7.53-7.56 (1H, dd, J = 8.52, 2.08 Hz, ArH), 7.37 (2H, t, J = 2.12 Hz,
ArH), 6.97-6.99 (2H, dd, J = 7.2, 1.04 Hz, ArH), 6.70 (1H, s, ArH), 4.23-4.26 (2H, t, -CH2), 3.99
(3H, s, -OCH3), 3.97 (3H, s, -OCH3), 3.62-3.66 (2H, t, -CH2), 3.38-3.41 (2H, pentet, -CH2). Rf
Value: 0.65 (CHCl3: MeOH, 9:1).
3.1.5.10. 7-(2-Bromoethoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (6g):
1
Light yellow, solid amorphous, yield 58%; H-NMR (400 MHz, CDCl3, δ ppm): 8.15--
8.17 (1H, d, J = 8.64 Hz, ArH), 7.11 (2H, s, ArH), 6.99-7.00 (2H, t, J = 2.48, 4.72 Hz, ArH), 6.72
(1H, s, ArH), 4.41-4.44 (2H, t, -CH2), 3.96 (6H, s, -OCH3), 3.93 (3H, s, -OCH3), 3.69-3.72 (2H,
t, -CH2). Rf Value: 0.67 (CHCl3: MeOH, 9:1).
3.1.5.11. 2-(4-Methoxyphenyl)-7-(3-(4-methylpiperidin-1-yl)propoxy)-4H-chromen-4-one (7a):
Greyish brown, solid amorphous, yield 55%; IR (KBr) (cm-1): 1625 cm-1 (C=O, str, s),
1
1595 cm-1 (C=C, s), 1250 cm-1 (C-O, asym., s), 1355 cm-1 (C-N, str, s); H-NMR (400 MHz,
CDCl3, δ ppm): 8.09-8.11 (1H, d, J = 9.4 Hz, ArH), 7.84-7.86 (2H, d, J = 10.4 Hz, ArH), 7.02
(1H, s, ArH), 7.00 (1H, s, ArH), 6.94-6.96 (2H, m, ArH), 6.67 (1H, s, ArH), 4.12-4.15 (2H, t, -

CH2), 3.88 (3H, s, -OCH3) 3.00-3.03 (2H, t, -CH2), 2.59-2.63 (2H, t, -CH2), 2.03-2.11 (4H, m, -
CH2), 1.65-1.68 (2H, t, -CH2), 1.32-1.41 (3H, m, -CH, -CH2) 1.22-1.28 (4H, m, -CH2), 0.93-0.95
(3H, d, -CH3); 13C NMR (100 MHz, CDCl3): 177.91, 163.40, 163.08, 162.25, 157.87, 127.86,
126.96, 124.15, 117.73, 114.58, 106.09, 101.91, 66.95, 55.21, 53.87, 33.70, 30.55, 26.26, 21.73;
m/z = 408.45 [M+H]+; Rf Value: 0.23 (CHCl3: MeOH, 9:1).
3.1.5.12. 7-(3-(4-(2-Hydroxyethyl)piperazin-1-yl)propoxy)-2-(4-methoxyphenyl)-4H-chromen-
4-one (7b):
Light yellowish brown, solid amorphous, yield 78%; IR (KBr) (cm-1): 3299 cm-1 (OH,
str, s), 1630 cm-1 (C=O, str, s), 1597 cm-1 (C=C, str, s), 1252 cm-1 (C-O, asym., s), 1375 cm-1 (C-
1
N, str, s); H-NMR (400 MHz, CDCl3, δ ppm): 8.02-8.04 (1H, d, J = 9.4 Hz, ArH), 7.77-7.79
(2H, d, J = 8.88 Hz, ArH), 6.92-6.95 (2H, s, ArH), 6.90 (1H, d, J = 2.28 Hz, ArH ), 6.60 (1H, s,
ArH), 4.52 (1H, s-broad, -OH), 4.05-4.08 (2H, t, -CH2), 3.81 (3H, s, -OCH3), 3.54-3.57 (2H, m, -
CH2), 2.46-2.50 (8H, m, -CH2), 2.19-2.21 (4H, t, -CH2), 1.94-1.98 (2H, pentet, -CH2); 13C NMR
(100 MHz, CDCl3): 177.94, 163.49, 163.09, 162.26, 157.90, 127.86, 126.97, 124.14, 117.70,
114.55, 114.43, 106.09, 100.94, 66.85, 59.25, 57.73, 55.52, 53.23, 52.83, 26.51; MS (ESI) m/z =
439.42 [M+H]+; Rf Value: 0.35 (CHCl3: MeOH, 9:1).
3.1.5.13. 2-(4-Methoxyphenyl)-7-(4-(4-methylpiperidin-1-yl)butoxy)-4H-chromen-4-one (7c):
Greyish brown, solid amorphous, yield 55%;; IR (KBr) (cm-1): 1629 cm-1 (C=O, str, s),
1
1597 cm-1 (C=C, s), 1252 cm-1 (C-O, asym., s), 1354 cm-1 (C-N, str, s); H-NMR (400 MHz,
CDCl3, δ ppm): 8.10-8.12 (1H, d, J = 8.76 Hz, ArH), 7.84-7.87 (2H, m, ArH), 7.00-7.03 (2H, m,
ArH), 6.93-6.97 (2H, m, ArH), 6.67 (1H, s, ArH), 4.08-4.11 (2H, t, -CH2), 3.88 (3H, s, -OCH3)
2.94-2.91 (2H, t, -CH2), 2.39-2.42 (2H, t, -CH2), 1.62-1.98 (8H, m, -CH2) 1.21-1.34 (3H, m, -
CH2, -CH), 0.91-0.93 (3H, d, -CH3); 13C NMR (100 MHz, CDCl3): 177.94, 163.50, 163.06,
162.22, 157.89, 127.85, 126.92, 124.12, 117.62, 114.57, 114.41, 106.04, 100.84, 68.41, 58.45,
55.50, 53.94, 34.03, 30.72, 27.15, 23.36, 21.83; MS (ESI) m/z = 422.51 [M+H]+; Rf Value: 0.28
(CHCl3: MeOH, 9:1).
3.1.5.14. 7-(4-(4-(2-Hydroxyethyl)piperazin-1-yl)butoxy)-2-(4-methoxyphenyl)-4H-chromen-4-
one (7d):

Light yellowish grey, solid amorphous, yield 70%; IR (KBr) (cm-1): 3356 cm-1 (OH, str,
s), 1627 cm-1 (C=O, str, s), 1599 cm-1 (C=C, str, s), 1253 cm-1 (C-O, asym., s), 1370 cm-1 (C-N,
str, s); 1H-NMR (400 MHz, CDCl3, δ ppm): 8.10-8.12 (1H, d, J = 8.72 Hz, ArH), 7.86-7.87 (1H,
d, J = 2.09 Hz, ArH), 7.85 (1H, d, J = 2.08 Hz, ArH), 7.01-7.03 (2H, m, ArH), 6.93-6.97 (2H, m,
ArH ), 6.68 (1H, s, ArH), 4.27 (1H, s-broad, -OH), 4.08-4.12 (2H, t, -CH2), 3.89 (3H, s, -OCH3),
3.60-3.62 (2H, t, -CH2), 2.53-2.56 (8H, m, -CH2), 2.41-2.45 (2H, t, -CH2) 1.86-1.90 (2H, t, -
CH2), 1.68-1.74 (4H, pentet, -CH2); 13C NMR (100 MHz, CDCl3): 176.38, 162.99, 162.29,
161.87, 157.34, 127.70, 126.02, 123.32, 117.00, 114.42, 114.23, 105.02, 100.93, 68.02, 56.75,
55.25, 52.13, 51.39, 26.18, 22.21; ; MS (ESI) m/z = 453.44 [M+H]+; Rf Value: 0.37 (CHCl3:
MeOH, 9:1).
3.1.5.15. 2-(4-Methoxyphenyl)-7-((5-(4-methylpiperidin-1-yl)pentyl)oxy)-4H-chromen-4-one
(7e):
Brown, solid amorphous, yield 60%; IR (KBr) (cm-1): 1625 cm-1 (C=O, str, s), 1597 cm-1
1
(C=C, s), 1245 cm-1 (C-O, asym., s), 1366 cm-1 (C-N, str, s); H-NMR (400 MHz, CDCl3, δ
ppm): 8.10-8.12 (1H, d, J = 8.76 Hz, ArH), 7.78-7.80 (2H, d, J = 8.8 Hz, ArH), 7.93-7.95 (2H, d,
J = 8.8 Hz, ArH), 6.86-6.88 (2H, d, J = 6.92 Hz, ArH), 6.65 (1H, s, ArH), 4.08-4.11 (2H, t, -
CH2), 3.88 (3H, s, -OCH3) 3.45-3.48 (2H, t, -CH2), 2.83-2.87 (2H, t, -CH2), 2.49-2.51 (2H, t, -
CH2), 1.73-1.83 (6H, m, -CH2) 1.45-1.61 (5H, m, -CH2), 0.91-0.93 (3H, d, -CH3); 13C NMR (100
MHz, CDCl3): 177.94, 163.32, 163.18, 162.29, 157.91, 127.89, 126.94, 124.02, 117.71, 114.59,
114.41, 105.98, 100.86, 67.93, 56.71, 55.51, 50.14, 34.07, 29.69, 28.40, 23.48, 21.17; MS (ESI)
m/z = 436.48 [M+H]+; Rf Value: 0.27 (CHCl3: MeOH, 9:1).
3.1.5.16.
7-((5-(4-(2-Hydroxyethyl)piperazin-1-yl)pentyl)oxy)-2-(4-methoxyphenyl)-4H-
chromen-4-one (7f):
Light grey, solid amorphous, yield 50%; IR (KBr) (cm-1): 3401 cm-1 (OH, str, s), 1628
cm-1 (C=O, str, s), 1599 cm-1 (C=C, str, s), 1252 cm-1 (C-O, asym., s), 1360 cm-1 (C-N, str, s); 1H-
NMR (400 MHz, CDCl3, δ ppm): 8.09-8.11 (1H, d, J = 8.72 Hz, ArH), 7.84-7.86 (2H, d, J = 8.92
Hz, ArH), 7.02 (1H, s, ArH), 7.02 (1H, s, ArH), 7.00 (1H, s, ArH), 6.92-6.96 (2H, m, ArH), 6.67
(1H, s, ArH), 4.32 (1H, s-broad, -OH), 4.05-4.08 (2H, t, -CH2), 3.88 (3H, s, -OCH3), 3.60-3.62
(2H, t, -CH2), 2.53-2.56 (2H, t, -CH2), 2.37-2.40 (3H, t, -CH2,), 1.70-1.90 (8H, t, -CH2), 1.51-

1.59 (6H, m, -CH2); 13C NMR (100 MHz, CDCl3): 177.92, 163.55, 163.06, 162.24, 157.90,
127.84, 126.93, 124.13, 117.62, 114.54, 114.41 106.06, 100.86, 68.48, 59.27, 58.23, 57.60,
55.50, 52.61, 52.30, 29.68, 28.89, 27.11; MS (ESI) m/z = 467.47 [M+H]+; Rf Value: 0.32
(CHCl3: MeOH, 9:1).
3.1.5.17.
(7g):
2-(4-Methoxyphenyl)-7-((6-(4-methylpiperidin-1-yl)hexyl)oxy)-4H-chromen-4-one
Grey, solid amorphous, yield 55%; IR (KBr) (cm-1): 1628 cm-1 (C=O, str, s), 1598 cm-1
1
(C=C, s), 1251 cm-1 (C-O, asym., s), 1365 cm-1 (C-N, str, s); H-NMR (400 MHz, CDCl3, δ
ppm): 8.09-8.11 (1H, d, J = 8.56 Hz, ArH), 7.84-7.87 (2H, d, J = 8.76 Hz, ArH), 7.00-7.02 (2H,
m, ArH), 6.93-6.95 (2H, m, ArH), 6.67 (1H, s, ArH), 4.04-4.07 (2H, t, -CH2), 3.88 (3H, s, -
OCH3) 3.20-3.22 (2H, t, -CH2), 2.61-2.65 (2H, t, -CH2), 2.23-2.28 (2H, t, -CH2), 1.82-1.86 (2H,
13
t, -CH2), 1.70-1.72 (3H, m, -CH2, -CH), 1.22-1.44 (8H, m, -CH2), 0.95-0.97 (3H, d, -CH3); C
NMR (100 MHz, CDCl3): 177.92, 163.52, 163.07, 162.25, 157.92, 127.86, 126.93, 124.14,
117.65, 114.57, 114.43, 106.06, 100.85, 68.40, 57.51, 55.51, 32.10, 29.88, 29.71 28.80, 26.95,
25.05, 21.26; MS (ESI) m/z = 450.49 [M+H]+; Rf Value: 0.28 (CHCl3: MeOH, 9:1).
3.1.5.18.
7-((6-(4-(2-Hydroxyethyl)piperazin-1-yl)hexyl)oxy)-2-(4-methoxyphenyl)-4H-
chromen-4-one (7h):
Greyish brown, solid amorphous, yield 60%; IR (KBr) (cm-1): 3328 cm-1 (OH, str, s),
1623 cm-1 (C=O, str, s), 1594 cm-1 (C=C, str, s), 1252 cm-1 (C-O, asym., s), 1356 cm-1 (C-N, str,
1
s); H-NMR (400 MHz, CDCl3, δ ppm): 8.08-8.10 (1H, d, J = 8.72 Hz, ArH), 7.82-7.86 (2H, m,
ArH), 6.99-7.02 (2H, m, ArH), 6.91-6.96 (2H, m, ArH), 6.66 (1H, s, ArH), 4.35 (1H, s-broad, -
OH), 4.03-4.07 (2H, t, -CH2), 3.87 (3H, s, -OCH3), 3.63-3.66 (2H, t, -CH2), 2.59-2.66 (8H, m, -
CH2), 2.42-2.46 (2H, t, -CH2), 1.80-1.85 (2H, pentet, -CH2), 1.47-1.59 (8H, m, -CH2); 13C NMR
(100 MHz, CDCl3): 177.90, 163.48, 163.03, 162.21, 157.97, 127.87, 126.90, 124.18, 117.67,
114.58, 114.44, 106.02, 100.89, 68.53, 59.30, 58.28, 57.48, 55.52, 52.71, 52.12, 29.74, 28.82,
27.10, 25.42; MS (ESI) m/z = 481.49 [M+H]+; Rf Value: 0.31 (CHCl3: MeOH, 9:1).
3.1.5.19.
(7i):
2-(4-Methoxyphenyl)-7-((8-(4-methylpiperidin-1-yl)octyl)oxy)-4H-chromen-4-one

Light grey, solid amorphous, yield 65%; IR (KBr) (cm-1): 1625 cm-1 (C=O, str, s), 1598
cm-1 (C=C, s), 1254 cm-1 (C-O, asym., s), 1368 cm-1 (C-N, str, s); 1H-NMR (400 MHz, CDCl3, δ
ppm): 8.09-8.11 (1H, d, J = 8.68 Hz, ArH), 7.86 (1H, d, J = 1.96 Hz, ArH), 7.84 (1H, d, J = 1.92
Hz, ArH),7.00-7.02 (2H, m, ArH), 6.93-6.96 (2H, m, ArH), 6.67 (1H, s, ArH), 4.04-4.07 (2H, t, -
CH2), 3.88 (3H, s, -OCH3) 3.14-3.17 (2H, d, -CH2), 2.54-2.58 (2H, t, -CH2), 2.21-2.24 (2H, t, -
CH2), 1.90 (1H, m, -CH), 1.81-1.84 (2H, m, -CH2), 1.63-1.71 (4H, m, -CH2), 1.32-1.36 (10H, m,
13
-CH2), 0.94-0.96 (3H, d, -CH3); C NMR (100 MHz, CDCl3): 177.92, 163.60, 163.03, 162.22,
157.91, 127.84, 126.90, 124.17, 117.60, 114.57, 114.41, 106.07, 100.83, 68.59, 58.26, 55.49,
53.35, 32.51, 31.63, 29.69, 29.35, 28.92, 27.24, 25.47, 21.36; MS (ESI) m/z = 478.50 [M+H]+;
Rf Value: 0.25 (CHCl3: MeOH, 9:1).
3.1.5.20.
7-((8-(4-(2-Hydroxyethyl)piperazin-1-yl)octyl)oxy)-2-(4-methoxyphenyl)-4H-
chromen-4-one (7j):
Light greyish, solid amorphous, yield 70%; IR (KBr) (cm-1): 3328 cm-1 (OH, str, s), 1622
cm-1 (C=O, str, s), 1597 cm-1 (C=C, str, s), 1252 cm-1 (C-O, asym., s), 1357 cm-1 (C-N, str, s); 1H-
NMR (400 MHz, CDCl3, δ ppm8.09-8.11 (1H, d, J = 8.76 Hz, ArH), 7.86 (1H, d, J = 1.96 Hz,
ArH), 7.84 (1H, d, J = 1.96 Hz, ArH), 6.99-7.02 (2H, m, ArH), 6.92-6.97 (2H, m, ArH), 6.67
(1H, s, ArH), 4.04-4.07 (2H, t, -CH2), 3.88 (3H, s, -OCH3), 3.60-3.63 (2H, t, -CH2), 3.10-3.13
(2H, t, -CH2), 2.55-2.57 (8H, t, -CH2), 2.34-2.38 (2H, t, -CH2) 1.81-1.85 (2H, pentet, -CH2),
1.31-1.36 (10H, m, -CH2); 13C NMR (100 MHz, CDCl3): 177.96, 163.63, 163.07, 162.25,
157.93, 127.85, 126.94, 124.16, 117.59, 114.59, 114.42, 106.07, 100.84, 68.65, 59.28, 58.60,
57.68, 55.50, 52.98, 52.62, 29.70, 29.52, 28.98, 27.47, 25.92; MS (ESI) m/z = 509.50 [M+H]+;
Rf Value: 0.34 (CHCl3: MeOH, 9:1).
3.1.5.21.
(7k):
2-(3,4-Dimethoxyphenyl)-7-(2-(4-methylpiperidin-1-yl)ethoxy)-4H-chromen-4-one
Light yellow, solid amorphous, yield 50%; IR (KBr) (cm-1): 1627 cm-1 (C=O, str, s),
1
1596 cm-1 (C=C, s), 1259 cm-1 (C-O, asym., s), 1366 cm-1 (C-N, str, s); H-NMR (400 MHz,
CDCl3, δ ppm): 8.15-8.18 (1H, d, J = 8.76 Hz, ArH), 7.36-7.37 (1H, d, J = 1.68 Hz, ArH), 7.19
(1H, s, ArH), 6.90-6.92 (3H, m, ArH), 6.62 (1H, s, ArH), 4.07-4.09 (2H, t, -CH2), 3.91 (3H, s, -
OCH3), 3.90 (3H, s, -OCH3), 2.69-2.73 (2H, t, -CH2), 1.87-2.01 (4H, m, -CH2), 1.19-1.31 (5H,

13
m, -CH2, -CH), 0.84-0.86 (3H, d, -CH3); C NMR (100 MHz, CDCl3): 177.91, 163.40, 162.93,
157.87, 149.81, 148.06, 127.09, 122.9, 121.07, 117.83, 114.81, 111.9, 107.06, 103.10, 101.17,
66.71, 58.77, 56.40, 54.53, 34.12, 31.81, 21.85; MS (ESI) m/z = 424.49 [M+H]+; Rf Value: 0.22
(CHCl3: MeOH, 9:1).
3.1.5.22.
(7l):
2-(3,4-Dimethoxyphenyl)-7-(3-(4-methylpiperidin-1-yl)propyl)-4H-chromen-4-one
Light yellow, solid amorphous, yield 50%; IR (KBr) (cm-1): 1623 cm-1 (C=O, str, s),
1
1592 cm-1 (C=C, s), 1257 cm-1 (C-O, asym., s), 1362 cm-1 (C-N, str, s); H-NMR (400 MHz,
CDCl3, δ ppm): 8.03-8.06 (1H, d, J = 9.4 Hz, ArH), 7.46-7.48 (1H, dd, J = 8.44, 2.12 Hz, ArH),
7.30 (1H, d, J = 2.12 Hz, ArH), 6.89-6.92 (3H, m, ArH), 6.62 (1H, s, ArH), 4.06-4.09 (2H, t, -
CH2), 3.92 (3H, s, -OCH3), 3.90 (3H, s, -OCH3), 2.44-2.48 (2H, t, -CH2), 1.96-2.00 (2H, t, -
CH2), 1.87-1.92 (5H, t, -CH2), 1.18-1.30 (5H, m, -CH2, CH), 0.85-0.87 (3H, d, -CH3); 13C NMR
(100 MHz, CDCl3): 177.79, 163.30, 162.97, 157.98, 149.52, 148.25, 127.03, 122.2, 121.05,
117.87, 114.72, 112.6, 107.31, 103.72, 100.96, 66.76, 58.71, 56.43, 53.51, 32.81, 31.96, 25.63,
21.45; MS (ESI) m/z = 438.55 [M+H]+; 478.62 [M+K]+; Rf Value: 0.26 (CHCl3: MeOH, 9:1).
3.1.5.23. 7-(2-(4-Methylpiperidin-1-yl)ethoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
(7m):
Light grey, solid amorphous, yield 55%; IR (KBr) (cm-1): 1625 cm-1 (C=O, str, s), 1595
cm-1 (C=C, s), 1248 cm-1 (C-O, asym., s), 1357 cm-1 (C-N, str, s); 1H-NMR (400 MHz, CDCl3, δ
ppm): 8.10-8.13 (1H, d, J = 9.62 Hz, ArH), 7.11 (2H, s, ArH), 7.00 (1H, d, J = 9.56 Hz, ArH),
6.98 (1H, s, ArH), 6.71 (1H, s, ArH), 4.23-4.26 (2H, t, -CH2), 3.96 (6H, s, -OCH3), 3.93 (3H, s, -
OCH3), 2.86-2.89 (2H, t, -CH2), 2.13-2.19 (2H, t, -CH2), 1.31-1.41 (6H, m, -CH2), ), 0.93-0.94
(3H, d, -CH3) 0.86-0.89 (1H, m, -CH); 13C NMR (100 MHz, CDCl3): 177.81, 163.35, 162.89,
157.89, 153.56, 141.04, 127.09, 127.02, 117.79, 114.85, 107.03, 103.60, 101.11, 66.67, 61.05,
57.12, 56.36, 54.47, 34.01, 30.45, 21.80; MS (ESI) m/z = 454.56 [M+H]+; Rf Value: 0.22
(CHCl3: MeOH, 9:1).
3.1.5.24.
one (7n):
7-(3-(4-Methylpiperidin-1-yl)propoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-

Light Brown, solid amorphous, yield 50%; IR (KBr) (cm-1): 1627 cm-1 (C=O, str, s),
1
1595 cm-1 (C=C, s), 1241 cm-1 (C-O, asym., s), 1350 cm-1 (C-N, str, s); H-NMR (400 MHz,
CDCl3, δ ppm): 8.09-8.11 (1H, d, J = 8.4 Hz, ArH), 7.10 (2H, s, ArH), 6.94-6.96 (2H, d, J = 8.76
Hz, ArH), 6.98 (1H, s, ArH), 6.70 (1H, s, ArH), 4.02-4.21 (2H, t, -CH2), 3.95 (6H, s, -OCH3),
3.91 (3H, s, -OCH3), 2.73-2.71 (2H, t, -CH2), 2.20-2.23 (4H, t, -CH2), 1.23-1.71 (6H, m, -CH2, -
CH), 0.90-0.95 (3H, d, -CH3); 13C NMR (100 MHz, CDCl3): 177.83, 163.34, 162.95, 157.92,
153.58, 141.06, 127.05, 117.81, 114.78, 107.29, 103.61, 100.99, 66.74, 61.06, 56.38, 54.97,
53.49, 32.75, 31.92, 25.56, 21.81; MS (ESI) m/z = 468.55 [M+H]+; Rf Value: 0.25 (CHCl3:
MeOH, 9:1).
3.2 Biological activity
3.2.1. In vitro inhibition of Acetylcholinesterase Enzyme
The inhibitory potency of target compounds on brain AChE was determined using
spectroscopic method at 450 nm by the method of Ellman et al. with slight modification [40].
The potency of synthesized compounds was expressed as IC50 with donepezil as standard
acetylcholinesterase inhibitor. Each concentration was analyzed in triplicate. Data from
concentration–inhibition experiments of the inhibitors were calculated by nonlinear regression
analysis, using the GraphPad Prism 5 program.
3.2.2. In vitro advanced glycation end-product (AGEs) formation inhibitory activity
The assay for the ability of the flavone to inhibit the glucose-mediated protein glycation and
the development of fluorescent AGEs was performed. Different concentrations of various
compounds were prepared by dissolving in DMSO. Anti-glycation assay was performed
according to the methods reported by Matsuura and colleagues with slight modification.11 The
Fluorescence was measured spectrofluorometerically (LS-55 (Perkin Elmer) at 370 nm
(excitation) and 440 nm (emission). Aminoguanidine was used as a positive control. All
experiments were performed in triplicate.
3.2.3. DPPH radical scavenging activity method
The stable 1, 1-diphenyl-2-picryl hydrazyl radical (DPPH) was used for determination of free
radical-scavenging activity of the test compounds by widely used Blois et.al. method.12 The

absorbance was recorded at 517 nm spectrophotometerically. Ascorbic acid was used as
standard. IC50 values denote the concentration of sample, which is required to scavenge 50% of
DPPH free radicals.
3.2.4. Kinetic characterization of AChE inhibition
The kinetics studies were performed using the Ellman’s method with three different
concentrations (0.01, 0.05 and 0.1 µM) of compound 7m. The Lineweaver–Burk reciprocal plots
were constructed by plotting the inverse initial velocity (1/V) as a function of the inverse of the
substrate concentration (1/[S]) at varying concentrations of the substrate acetylthiocholine (0.05–
0.5 mM). The parallel control experiments were achieved without inhibitor in the assay. The
double reciprocal plots were evaluated by a weighted least-squares analysis. Each experiment
was performed in triplicate.
3.2.5. Molecular docking studies
To understand the binding interactions and selectivity of our synthesized compounds against
AChE, molecular docking simulation was carried out with Glide program of Schrödinger
software. The compounds showing good anti-cholinesterase activity were sketched and cleaned
in maestro molecular modeling workspace followed by energy minimization in ‘ligprep’
program of Schrödinger software using OPLS_2005 force field at pH of 7.4.14 The X-ray
crystallographic structure of AChE complex with donepezil (PDB code 1EVE) was obtained
from the Protein Data Bank (http://www.rcsb.org/pdb) and optimized for docking analysis.15 The
optimization protocol includes addition of hydrogen atoms, deletion of water molecules,
completion of bond orders, assignment of hydrogen bonds and complex minimization to RMSD
of 0.20Å using OPLS_2005 force field. The active molecules were docked into the active site of
the protein using extra precision (XP) docking mode of ‘glide’ program.16
3.2.6. Molecular Dynamic Simulations
The molecule with the highest inhibitory activity docked with the AChE protein was used in
molecular dynamics simulations (10 ns) using Desmond molecular dynamics module of
Schrodinger.17 The simulations aided to stabilize the protein-ligand complex and analyze the
most probable interaction by studying its simulation–interaction diagram. From this analysis, the
important interactions for ligand were assessed and can be utilized for further identification. For

molecular dynamics simulations, the system was first built using the TIP3P solvent model with
orthorhombic box shape; the pH was adjusted by adding Na+ ions, and the salt concentration was
set at 0.15 M. The simulation was carried out using the NPT ensemble and a time step of 1.0 fs;
the temperature was fixed at 310 K using the Nose-Hoover Chain method as the thermostat and
pressure of 1.01325 bar using Martyn–Tobias–Klein as the barostat.
3.2.7. ADME property prediction
To investigate the potential of the new compounds as anti-AD agents, their ADME
(Absorption, Distribution, Metabolism and Excretion) properties were predicted using
QikProp program, v. 3.5 of the Schrödinger software.18 This provided an estimate of the
physicochemical properties and the bioavailability of the compounds. Various parameters such
as polar surface area (PSA), solvent accessible surface area (SASA), QPPCaco (predicted
apparent Caco-2 cell permeability in nm/s, CNS activity (predicted central nervous system
activity on a -2 (inactive) to +2 (active) scale). QPlogBB (predicted brain/blood partition
coefficient), Caco-2 cells is a model for the gut blood barrier), QPPMDCK (predicted apparent
MDCK cell permeability in nm/s. MDCK cells are considered to be a good mimic for the blood–
brain barrier), QPlogS (predicted aqueous solubility), QPlogKhsa (prediction of binding to
human serum albumin), and percent human oral absorption (predicted human oral absorption
on 0–100% scale) were calculated. The acceptability of the compounds based on the Lipinski’s
rule of five was also estimated from the results.19
3.3. In vivo assay
3.3.1. Experimental Animals
Swiss albino mice of either sex, weighing 20–25 g (procured from Central Research Institute,
Kasauli, Himachal Pradesh, India) were used for in vivo evaluation of learning and memory. The
experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC). The
animals were housed in the Animal house facility, Punjabi University Patiala, India and the care
of the animals was carried out as per the guidelines of the Committee for the Purpose of Control
and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest,
Government of India (Reg. No. 107/1999/CPCSEA).

3.3.2. Drugs and chemicals
Scopolamine bromide was dissolved in normal saline and test drug was dissolved in 10%
dimethylsulfoxide (DMSO). All other reagents used in the present study were of analytical grade
and freshly prepared.
3.3.3. Assessment of learning and memory by Morris water maze
Morris water maze is one of the most commonly used animal models to test memory.21 It
consists of large circular pool and is divided in to four quadrants (Q1, Q2, Q3, and Q4). Each
animal was subjected to trial of 120 s in the water maze for five consecutive days and the
memory was assessed in terms of: (i) escape latency time (ELT), that is, the time taken by the
animal to locate the hidden platform in the target quadrant (Q4) for the first 4 days of training,
(ii) time spent in target quadrant (Q4) on fifth day of trial, that is, the day of retrieval.
3.3.4. Experimental protocol
Six groups, each group comprising five Swiss albino mice, were employed in the present
study.
3.3.4.1. Group I: normal control.
Normal mice, without any treatment, were subjected to trials on the water maze for 5 days to
note escape latency time (ELT) for first 4 days (an index of learning) and time spent in target
quadrant (TSTQ) on fifth day of trial (an index of retrieval).
3.3.4.2. Group II: scopolamine in vehicle treated control.
Scopolamine (0.5 mg/kg) in 10% DMSO, vehicle treated control was administered in
scopolamine treated mice (30 min prior to scopolamine administration) to each mouse before
subjecting to first four trials and rest of the procedure was same as described in group I.
3.3.4.3. Group III: Donepezil in scopolamine treated control.
Donepezil (2 mg/kg) was administered in scopolamine treated mice (30 min prior to
scopolamine administration) to each.

3.3.4.4. Group IV, V and VI: test compound (7m) (2, 5, and 10 mg/kg) in scopolamine treated
control.
The test compound with most potent acetylcholinesterase inhibitory activity (7m) was
administered in scopolamine treated mice (30 min prior to scopolamine administration) to each
mouse before subjecting to first four trials and rest of the procedure was same as described in
group I.
3.4. Biochemical estimations
At the end of the protocol, animals were euthanized by cervical dislocation and brains were
removed carefully. The different parts of the brain i.e. cortex and hippocampus were separated.
Isolated parts were homogenized in ice cold phosphate buffer pH 7.4. The homogenate was
centrifuged at 14,500 rpm for 15 min at 4 °C. The clear supernatant was used for estimation of
thiobarbituric acid reactive substance (TBARS), reduced glutathione (GSH), and brain AChE
activity.
3.4.1. Estimation of thiobarbituric acid reactive substance (TBARS)
The TBARS level was estimated in mice brain homogenates, respectively, as an index of lipid
peroxidation by the method of Ohkawa, et al.22
3.4.2. Estimation of reduced glutathione (GSH) level
The GSH level was estimated in mice brain homogenates by the method reported by Beutler,
et al.23
3.4.3. Estimation of brain AChE activity
The brain AChE activity was measured by following method.10
4. Statistical analysis
The results were expressed as mean standard deviation. The data obtained from various
groups was statistically analyzed by two-way ANOVA followed by Tukey’s multiple
comparison tests. A value of p<0.05 was considered to be statistically significant.

5. Conclusion
Novel flavonoids based potential polyfunctional agents were synthesized to manage AD.
Most of the compounds showed the AChE inhibitory with good radical scavenging and AGEs
product formation inhibitory activity. The SAR revealed the influence of varied methoxy groups
on ring-B and length of alkyl chain of cyclic amino alkyloxy group on ring-A of flavonoid on
AChE inhibition. The compound 7m (IC50 = 5.87 nM) was 2-fold more potent than donepezil for
AChE inhibition. Moreover, the in silico pharmacokinetic profiles revealed that all compounds
have drug like properties. The molecular modeling study indicated that compound 7m
simultaneously bind with both PAS and CAS of active site gorge. Additionally, the compound
had significant capacity to absorb free radicals and inhibit the AGEs product formation. The
compound 7m also ameliorated scopolamine induced amnesia in mice in terms of restoration of
ELT and TSTQ. Thus, the polyfunctional attribute of these flavonoids make them potential
candidates for the AD management.
Acknowledgments
I am grateful to my colleagues and assistants for this study and editorial assistance with the
manuscript. We wish to thank the ‘Indian Council of Medical Research (ICMR)’, New Delhi, for
providing us a Senior Research Fellowship (ICMR-SRF); Award No. BIC/11(11)/2014.
Sophisticated Instrument Centre (SIC), Punjabi University, Patiala, Punjab, India and SAIF,
Punjab University, Chandigarh, Punjab, India is acknowledged for providing access to
spectrofluorometer and spectral facilities.
Disclosure statement
The authors declare that they have no conflict of interests.
References
(1) Cardozo MG, Kawai T, Iimura Y, Sugimoto H, Yamanishi Y, Hopfinger A, Sugimoto H.
Yamanishi Y, Hopfinger AJ. Conformational analyses and molecular-shape comparisons of a
series of indanone-benzylpiperidine inhibitors of acetylcholinesterase. J Med Chem
1992;35:590-601.

(2) Singh M, Kaur M, Chadha N, Silakari O. Hybrids: a new paradigm to treat Alzheimer’s
disease. Mol Divers 2016;20:271-297.
(3) (a) Bartolini M, Bertucci C, Cavrini V, Andrisano V. β-Amyloid aggregation induced by
human acetylcholinesterase: inhibition studies. Biochem Pharmacol 2003;65:407-416; (b)
Rees T, Hammond PI, Soreq H, Younkin S, Brimijoin S. promotes beta-amyloid plaques in
cerebral cortex. Neurobiol Aging 2003;24:777-787.
(4) Singh M, Kaur M, Kukreja H, Chugh R, Silakari O, Singh D. Acetylcholinesterase inhibitors
as Alzheimer therapy: from nerve toxins to neuroprotection. Eur J Med Chem 2013;70:165-
188.
(5) He Y, Yao PF, Chen SB, Huang ZH, Huang SL, Tan JH, Li D, Gu LQ, Huang ZS. Synthesis
and evaluation of 7, 8-dehydrorutaecarpine derivatives as potential multifunctional agents for
the treatment of Alzheimer's disease. Eur J Med Chem 2013;63:299-312.
(6) Münch G, Thome J, Foley P, Schinzel R, Riederer P. Advanced glycation end products in
ageing and Alzheimer’s disease. Brain Res Rev 1997;23:134-147.
(7) (a) Singh M, Kaur M, Silakari O. Flavones: an important scaffold for medicinal chemistry.
Eur J Med Chem 2014;84:206-239; (b) Solanki I, Parihar P, Mansuri ML, Parihar MS.
Flavonoid-based therapies in the early management of neurodegenerative diseases. Adv Nutr
2015;6:64-72.
(8) Singh M, Silakari O. Design, synthesis and biological evaluation of novel 2-phenyl-1-
benzopyran-4-one derivatives as potential poly-functional anti-Alzheimer's agents. RSC Adv
2016;6:108411-108422.
(9) (a) Baker W. Molecular rearrangement of some o-acyloxyacetophenones and the mechanism
of the production of 3-acylchromones. J Chem Soc 1933;10:1381-1389; (b) Mahal HS,
Venkataraman K. Synthetical experiments in the chromone group. Part XIV. The action of
sodamide on 1-acyloxy-2-acetonaphthones. J Chem Soc 1934;56:1767-1769.
(10) Ellman GL, Courtney KD, Andres V, Featherstone RM. A new and rapid colorimetric
determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88-95.
(11) Matsuura N, Aradate T, Sasaki C, Kojima H, Ohara M, Hasegawa J, Ubukata M. Screening
system for the Maillard reaction inhibitor from natural product extracts. J Health Sci
2002;48:520-526.

(12) Blois MS. Antioxidant determinations by the use of a stable free radical. Nature
1958;181:1199-1200.
(13) Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidant activity relationships of
flavonoids and phenolic acids. Free Rad Bio Med 1996;20:933-956.
(14) Ligprep, version 2.5; Schrodinger, LLC, New York, 2011.
(15) Kryger G, Silman I, Sussman JL. Structure of acetylcholinesterase complexed with E2020
(Aricept®): implications for the design of new anti-Alzheimer drugs. Structure 1999;
7:297-307.
(16) Glide, version 5.6; Schrodinger, LLC, Newyork, NY, 2010.
(17) Bowers KJ, Chow E, Xu H, et al. Scalable algorithms for molecular dynamics simulations
on commodity clusters, Proceedings of the ACM/IEEE. IEEE, 2006, pp 43.
(18) QikProp, Version 3.5; Schrödinger, LLC: New York, NY, 2012.
(19) Lipinski CA, Lombardo F, Dominy BW, Dror RO, Eastwood MP, Gregersen BA, Klepeis
JL, Kolossvary I, Moraes MA, Sacerdoti FD, Salmon JK. Experimental and computational
approaches to estimate solubility and permeability in drug discovery and development
settings. Adv Drug Deliv Rev 1997;46:3-26.
(20) Kelder J, Grootenhuis PD, Bayada DM, Delbressine LP, Ploemen JP. Polar molecular
surface as a dominating determinant for oral absorption and brain penetration of
drugs. Pharm Res 1999;16:1514-1519.
(21) Morris R. Developments of a water-maze procedure for studying spatial learning in the
rat. J Neurosci Methods 1984;11:47-60.
(22) Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric
acid reaction. Anal Biochem 1979;95:351-358.
(23) Beutler E, Duron O, Kelly BM. Improved method for the determination of blood
glutathione. J Lab Clin Med 1963;61:882-888.

Figures Legends
Fig. 1. Lineweaver–Burk plot from the substrate–velocity curves of the AChE activity with
different substrate concentrations (0.05–0.5 mM) in the absence and presence of
compound 7m at 2, 5 and 10 µM.
Fig. 2. Docked pose of interactions of compound 7m with AChE active site (1EVE) using
Glide. (A) 2D representation of different interactions of 7m with TcAChE. (B) 3D
representation of different interactions of compound 7m with residues in the
binding sites of TcAChE. The compound is rendered in green stick model and
the residues are rendered in blue sticks. Hydrogen bonds are indicated with green
dashed lines. Residues involved in hydrophobic interactions with the ligand are
shown with dashed lines. (C) The TcAChE enzyme is depicted in surface view and
compound 7m as stick in the binding pocket.
Fig. 3. post-MD hydrogen bonds and hydrophobic interactions of 7m with AChE. (A) Protein
interactions fractions with the ligand (7m) plot throughout the simulation. (B)
protein-ligand contacts plot of compound 7m with AChE (C) RMSD trajectory plot
for compound 7m.
Fig. 4. Effect different interventions on escape latency time (ELT) at day 1 and day 4 using
Morris water maze for memory evaluation. Data was presented as Mean S.D. and
analyzed by two way ANOVA followed by Tukey’s multiple range test;
b
c
ap < 0.05 vs day 1 ELT in normal; p < 0.05 vs day 4 ELT in normal; p < 0.05 vs
d
day 4 ELT in vehicle + scopolamine; p < 0.05 vs day 4 ELT in donepezil +
scopolamine.

Fig. 5. Effect of different interventions on time spent in target quadrant (TSTQ), that is, Q4
in Morris water maze test for memory evaluation. Values are expressed as
mean S.D. and analyzed using two way ANOVA followed by Tukey’s multiple
range test.
b
ap <0.05 versus time spent in other quadrants (Q1, Q2, and Q3) in normal; p <0.05
versus TSTQ in normal; cp <0.05 versus TSTQ in vehicle + scopolamine treated; dp
< 0.05 vs TSTQ in donepezil + scopolamine.
Scheme 1: The synthetic methodology employed to develop compound 5(a-c).
Scheme 2: Synthetic scheme for the synthesis of intermediates 6(a–g) of flavonoid
derivatives
Scheme 3: Synthetic scheme for final compounds 7(a–n).
Table 1: The AChE inhibition, Radical scavenging activities and advanced glycation end
products (AGEs) formation inhibitory activity of compounds 5(a-c) and 7(a-n).
Table 2: Predicted ADMET properties of the target compounds
Table 3: Effect of most active compound 7m, on AChE activity and oxidative stress
(TBARS and GSH) in rat brain.