Design, synthesis and biological evaluation of novel 2-phenyl-1-benzopyran-4-one derivatives as potential poly-functional anti-Alzheimer's agents

Article abstract of DOI:10.1039/c6ra17678j

The development of Multi-Target Directed Ligands (MTDLs) has emerged as a promising approach for targeting the complex etiology of Alzheimer's disease (AD). Following this approach, a new series of 2-phenyl-1-benzopyran-4-one derivatives was designed, synthesized and biologically evaluated as inhibitors of acetylcholinesterases (AChEs), advanced glycation end product (AGEs) formation and also for their radical scavenging activity. The in vitro studies showed that the majority of the synthesized derivatives inhibited acetylcholinesterase (AChE) with IC₅₀ values in the low-micromolar range. Among them, inhibitors 7a, 7h and 7k, strongly inhibited AChE, with IC₅₀ values of 6.33, 7.56 and 11.0 nM, respectively, and were more potent than the reference compound donepezil. Moreover, the molecular docking study showed that most potent compounds simultaneously bind to the catalytic active site and the peripheral anionic site of AChE. Additionally, these compounds exhibited a greater ability to inhibit advanced glycation end product formation with additional radical scavenging properties. Thus, 2-phenyl-1-benzopyran-4-one derivatives might be promising lead compounds for potential poly-functional anti-Alzheimer's agents.

Full text of DOI:10.1039/c6ra17678j

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Page 1 of 34
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Design, Synthesis and Biological Evaluation of Novel 2-phenyl-1-benzopyran-4-one
Derivatives as Potential Poly-functional Anti-Alzheimer’s Agents
Manjinder Singha, Om Silakaria,*
a Molecular Modeling Lab, Department of Pharmaceutical Sciences and Drug Research,
Punjabi university, Patiala, Punjab, India, 147002.
*Eꢀmail: omsilakari@gmail.com, Telephone: +919501542696
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Abstract
Development of MultiꢀTarget Directed Ligands (MTDLs) has emerged as a promising
approach for targeting complex etiology of Alzheimer’s disease (AD). Following this
approach, a new series of 2ꢀphenylꢀ1ꢀbenzopyranꢀ4ꢀone derivatives was designed, synthesized
and biologically evaluated as inhibitors of acetylcholinesterases (AChEs), advanced glycation
end products (AGEs) formation and also for their radical scavenging activity. The in vitro studies
showed that the majority of synthesized derivatives inhibited acetylcholinesterase (AChE) with
IC50 values in the lowꢀmicromolar range. Among them, inhibitors 7a 7h and 7k, strongly
inhibited AChE, with IC50 value of 6.33, 7.56 and 11.0 nM, respectively, and were more potent
than the reference compound donepezil. Moreover, the molecular docking study displayed that
most potent compounds simultaneously bind to catalytic active site and peripheral anionic site of
AChE. Besides, these compounds exhibited greater ability to inhibit advanced glycation end
products formation with additional radical scavenging property. Thus, 2ꢀphenylꢀ1ꢀbenzopyranꢀ4ꢀ
one derivatives might be the promising lead compounds as potential polyꢀfunctional antiꢀ
Alzheimer’s agents.
Keywords: Alzheimer’s disease, Acetylcholinesterase, Antioxidants, benzopyrans, Flavonoids,
AGEs
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1. Introduction
Alzheimer’s disease (AD) is the most common form of irreversible dementia in the ageing
patients. It is a neurodegenerative disorder associated with a selective loss of cholinergic neurons
and subsequent reduction of acetylcholine (ACh) in the brain leading to progressive decline in
cognitive, executive and memory functions and eventually to incapacitating dementia before
death.1,2 The prevalence of AD dramatically increases with age and it doubles for every fiveꢀyear
interval after the age of 65.3 Besides several diverse hallmarks such as βꢀamyloid (Aβ) deposits,
τꢀprotein aggregation and low levels of acetylcholine (ACh), AD brains display constant
evidence of oxidative damage.4ꢀ7 Cholinergic hypothesis represents one of the conventional
hypothesis which proposes that the extensive decrease of ACh leads to cognitive and memory
deficits in AD patients. Acetylcholinesterase (AChE) is the key enzyme, which hydrolysis
acetylcholine (ACh) at the cholinergic synapses. Acetylcholinesterase inhibitors (AChEIs) could
increase the level of ACh in AD patients through the inhibition AChE and, therefore, relieve
some symptoms experienced by patients.8,9 Till now, AChEIs are the only drugs used clinically
for the treatment of AD (Figure 1).10,11 Pathogenesis of AD is significantly impacted by
oxidative stress which may further promotes the formation of amyloid plaques and
neurofibrillary (NFT) tangles in AD.12,13 Oxidative stress particularly induces injury in the most
cellular macromolecules of AD brain including nucleic acids, proteins, and lipids. These findings
support the ‘oxidative stress hypotheses of AD, of which reactive oxygen species (ROS) play a
key role in AD onset and progression.14 Therefore, drugs aimed at clearing or preventing the
formation of the free radicals may be useful for the management of AD.
The advanced glycation endꢀproducts (AGEs) are senescent macroprotein derivatives, formed
through the nonꢀenzymatic glycation (called the “Maillard reaction”), involved in the
pathogenesis of AD.15ꢀ17 Glycation of Aβ or tau is reported to enhance its aggregation and
subsequent formation of senile plaques (SPs) and paired helical filaments in AD brain,
respectively. Moreover, AGEs interact with RAGE (receptor for AGEs) that provokes the
generation of ROS and vascular inflammation, and consequently alters the various gene
expressions in several types of cells, which could contribute to the pathological changes of AD
as well.18ꢀ20
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The current drugs for treatment of AD are limited to mainly four AChE inhibitors. However, due
to complexity of AD and involvement of various molecular pathways in its progression,
modulation of single pathway, might not be sufficient to produce desire efficacy of single target
therapy with AChE inhibitors.21ꢀ24 Various approaches can be used like hybrids, chimers, mutual
prodrugs, multiꢀtarget directed ligands (MTDLs), polyfunctional agents etc. for such type of
complex diseases. Thus, multifactorial nature of AD demands the designing of polyfunctional
agents simultaneously acting on more than one pathway rather than only one. Noticeably, drug
therapy of complex diseases with polyfunctional compounds embracing diverse biological
properties with single bioavailability and pharmacokinetic metabolism, will have pronounced
advantage over individualꢀtargeted drugs. In the present study, the wellꢀestablished molecular
pathways i.e. acetylcholinesterase (AChE), oxidative stress, and AGEs have been explored for
the designing of polyfunctional agents for effective therapy of AD than existing one.
Selection of appropriate privileged scaffold for designing of polyfunctional drug is a crucial
step in the search of clinical candidate for the treatment of AD. After critical literature review,
Flavonoid or 2ꢀphenylꢀ1ꢀbenzopyranꢀ4ꢀone as a privileged scaffold was considered for present
study as it possesses a broad range of pharmacological properties such as antiꢀinflammatory,25
antiꢀoxidative,26 AGEs inhibitory effects,27 and neuroprotective effects against AChE.
Flavonoids or 2ꢀphenylꢀ1ꢀbenzopyranꢀ4ꢀone are also known for their inhibition of amyloid β
peptide aggregation, another key feature in AD. Aβ peptides can form insoluble plaques resulting
in the formation of senile plaques and then to neurofibrillary tangles, neuronal cell death, and
ultimately dementia. This privileged scaffold based single targeted molecules have previously
been reported for different pathways of AD. Thus, the designing and synthesis of new
polyfunctional flavonoid derivatives is an interesting strategy for the management of AD.28,29
Recently many studies revealed that tertiary amine at the terminal alkyl chain present in
flavonoid moiety are required for adequate AChE inhibition.30 To investigate the influence of
methoxy group on Bꢀring, with appropriate secondary amines using two carbon spacer on ringꢀA
of flavone, a new series of 2ꢀphenylꢀ1ꢀbenzopyranꢀ4ꢀone derivatives were synthesized in short
steps and newly synthesized derivatives were primarily evaluated for in vitro AChE inhibition.
Additionally, in vitro antiꢀoxidative and AGEs inhibitory potential of these compounds were also
determined.
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2. Result and Discussion
2.1. Chemistry
First, to assess the influence of introduction of the aminoalkyl chain, compounds 7(a–k), which
have a ꢀOCH3 group at 4th position of the ringꢀB and various aminoalkyl moieties at the 6ꢀ
position of the Aꢀring, were synthesized. The synthetic methodologies employed to develop
intermediates (5) and target compounds 7(a–k) are outlined in scheme S1 and 1, respectively.
The key intermediates were prepared according to widely used Bakerꢀvenkataraman
rearrangement with slight modifications.31,32 The alkylation of 5 with α, ωꢀdibromoalkanes in
acetone provided intermediate 6 in adequate yields. Finally, the reaction of 6 with commercially
available secondary amines under reflux in acetonitrile in the presence of K2CO3 produced the
final compounds 7(a–k) in 50–80% yields. The compounds were dried and then, purified on
silica columns using chloroform: methanol (9:1) as solvent. The structures and purities of the
target compounds were confirmed by IR, 1H NMR, 13C NMR, mass and elemental analysis.
2.2. In vitro inhibition studies on AChE
To determine the potential of target compounds 7(a–k) for the treatment of AD, their inhibition
towards AChE (brain homogenate) was determined by the Ellman method with slight
modifications using donepezil as reference compound.33 The IC50 values of all tested compounds
for AChE inhibition are summarized in Table 1. With the aim of improving the interaction of the
synthesized compounds with catalytic anionic site (CAS) and peripheral anionic site (PAS) of
AChE, different amines were selected and joined with 2ꢀphenylꢀ1ꢀbenzopyranꢀ4ꢀone scaffold by
a two carbon spacer exhibiting different levels of inhibitory activities against AChE. From Table
1, it could be seen that most of the tested compounds 7(a–k) had significant AChE inhibitory
activity. Compound 7h, 7k and 7a, with a 4ꢀmethylpiperidine, 4ꢀhydroxyethylpiperazine and
morpholino group combined with flavonoid using a two carbon spacer are the most potent
inhibitors for AChE with an IC50 value of 6.33, 7.56 and 11.0 nM, respectively.
The screening data exhibited that the terminal amino group of alkyl side chain at 7th position of
2ꢀphenylꢀ1ꢀbenzopyranꢀ4ꢀone moiety significantly influenced the inhibitory activities.
Compounds with different amines at two carbon spacer were compared for their ability to inhibit
AChE enzyme. The unsubstituted piperazine derivative (7c) has moderate activity (IC50=51 nM)
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whereas methyl substituted piperazine (7d) significantly increases the activity (IC50=31.0 nM).
Furthermore, the introduction of hydroxyethyl group (7k) in piperazine increases activity with
maximum IC50=7.56 nM, which is superior to reference drug donepezil (IC50=12.7 nM).
Ring size of terminal cyclic amine was also evaluated for its effect on AChE inhibitory activity.
A general observation was extracted out that six membered cyclic amine derivatives 7a, 7b, 7c,
7d, 7h, 7i and 7k (IC50 ranging from 6.33ꢀ51.0 nM) showed better activity than five membered
cyclic amine derivatives 7e and 7j (IC50 values 104.8 and 108.3 nM). This may be due to
improper fitting of five membered ring systems into the gorge of the enzyme.
Particularly 4ꢀmethylpiperidine derivative (7h) appears to be the most potent compound against
AChE, with an IC50 value of 6.33 nM. Contemporarily, this compound proves to be about 2ꢀfold
more potent than donepezil and 3.5ꢀfold more potent than unsubstituted piperidine containing
compound (7b, IC50=21.7 nM). Instead, compounds (7c and 7d) possessing additional nitrogen
atom in the terminal group, exhibited low potency than the compound having only one nitrogen
atom (7b and 7h). Moreover, morpholino derivative (7a) with IC50 value of 11.0 nM, also feature
good inhibitory activity against AChE. The diethyl amino derivative (7f) with IC50 value of 27.0
nM is about 3ꢀfold more potent than dimethyl amino derivative (7g, IC50=77.4 nM).
Interestingly, the diethylamine and dimethylamine derivatives are less effective than morpholino
and piperazine derivatives. Graph showing the comparison of AChE inhibitory potencies of
synthesized derivatives is displayed in Figure S2.
2.3. In vitro antioxidant activity
Oxidative stress plays an important role in neuronal degeneration in AD. Its reduction is another
decisive aspect in designing polyꢀfunctional agents for AD treatment. Synthesized compounds
7(a–k) were tested for their free radical scavenging activities using 1,1ꢀdiphenylꢀ2ꢀpicrylꢀ
hydrazyl (DPPH) assay at 517nm. The DPPH scavenging activities of test compounds are
summarized in Table 1 and displayed in Figure S2. Results clearly indicate that almost all
compounds exhibited radical scavenging activity, among these 7c, 7h, 7i & 7k are comparable to
ascorbic acid. The compound 7k was found to be the best radical scavenger (EC50=21.5 nM).
This might be due to radical scavenging property of hydroxyl group. Compounds 7h and 7c with
EC50 values 24.6 and 30.5, respectively also show good radical scavenging activity. The positive
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charge of free –NH groups intensify the free radical quenching. The flavonoid moiety due to
conjugation system appears to be crucial to the compound’s radical scavenging ability. It might
be attributed to the presence of electron rich benzopyran or flavone nucleus that can stabilize the
free radicals via resonance effect. The compounds 7d, 7b, 7f and 7a exhibited moderate activity
with EC50 values range from 45.4 to 54.3 nM.
2.4. Advanced glycation endꢀproduct formation inhibitory activity
All the synthesized compounds 7(a–k) were subjected to in vitro bioassay of AGEs formation
inhibitory activity using method reported by Matsuura et al with slight modification.34 The
potential of the compounds 7(a–k) to inhibit AGEsꢀformation is summarized in Table 1 and
displayed in Figure S3. All the synthesized compounds showed considerable AGEs formation
inhibitory activity as the fluorescence of AGEs was shown to be significantly inhibited by the all
the synthesized compounds. Among the various synthesized compounds, the compound 7k
(IC50=36.0 ꢁM) was found to be significantly potent than the reference drug aminoguanidine
(IC50=40.5 ꢁM). The compounds 7f, 7h, 7d and 7a (IC50 value ranging from 43.5 to 56.3 ꢁM)
exhibited moderate inhibitory activity as compared to the standard drug. However, the
compounds 7b, 7i, 7c, 7g, 7e and 7j exhibited lower antiꢀglycating activity than the standard
drug (IC50 value ranging from 64.0 to 113.1 ꢁM). As evident from the results, among all the
synthesized compounds, the compounds with hydroxyl substitution (7k) was found to be more
active than the derivatives that lack hydroxyl group. The compound with 4ꢀmethylpiperidine
moiety (7h) exhibited good antiꢀglycating activity than the compound with no substitution on
piperidine (7b). The six membered cyclic amines (7k, 7h, 7d, 7a, 7b and 7c) have good to
moderate antiꢀglycating activity as compared to five membered cyclic amines (7e and 7j). The
flavonoid with diethyl amine moiety (7f, IC50=50.3 ꢁM) exhibited good activity than
dimethylamine (7g, IC50=72.2 ꢁM).
2.5. Kinetic characterization of AChE inhibition
Based on the in vitro results of AChE inhibition, kinetic study was carried out on most
potent inhibitor 7h to investigate the type of enzyme inhibition. The Lineweaver–Burk plots
(Figure 2) displayed both increased slopes (decreased Vmax) and intercepts (higher Km) at higher
concentration of the inhibitor 7h, indicating a mixedꢀtype inhibition. Thus, compound 7h might
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be able to bind with both catalytic anionic site (CAS) as well as peripheral anionic site (PAS) of
AChE.
2.6. Molecular docking studies
In order to explore the binding patterns of 2ꢀphenylꢀ1ꢀbenzopyranꢀ4ꢀone derivatives in active
site of AChE, molecular docking studies were carried out for two most potent inhibitors 7h and
7k. Molecular docking study was carried out with the available crystal structure of Torpedo
californica AChE (TcAChE) (1EVE). The 3D structure of TcAChE enzyme revealed that the
active site is located at the bottom of a deep and narrow gorge comprising Trp84 and Phe330
residues in the CAS. Additionally, PAS is present near the active site gorge mainly comprising
of aromatic residues Tyr121, Tyr70, Trp279, Tyr334 and Asp72.35 These compounds showed
nice fit in the active site by interacting with both catalytic site and peripheral anionic site of the
enzyme simultaneously to provide maximal inhibitory profile. The position of compound 7h and
7k with respect to the key residues in the binding site is shown in Figure 3.
As shown in Figure 3, the compound 7h binds along the activeꢀsite gorge, extending from the
anionic subsite near Trp84 to the peripheral anionic site (PAS) near Trp279. The compound 7h
displayed πꢀπ stacking with conserved amino acid residue Trp279, a major component of a
peripheral “anionic” site (PAS). The quaternary amine of 4ꢀmethylpiperidine moiety showed πꢀ
cationic interactions with an aromatic residue, Phe330, a residue of hydrophobic site, and with
Tyr334 and Asp72 amino acid residue of PAS, 14 Å from the active site, near the top of the
gorge. It also showed one hydrogen bond between carbonyl oxygen of the ring C and Arg289,
acyl binding pocket. On the other hand, compound 7k showed πꢀπ stacking of ringꢀA and ringꢀB
with Phe331, acyl binding pocket and Trp279 amino acid residue of peripheral anionic site,
respectively. It also interacts with Phe288 and Arg289 amino acids, acyl binding pocket residue
of catalytic triad via hydrogen bonding. The hydroxyl oxygen of piperazine moiety forms
hydrogen bond with hydroxyl hydrogen of Tyr130 and Glu199, choline binding site (Figure 3).
The result of docking study revealed that the dual binding profile of selected compounds was
found due to hydrogen bond, π–π (aromatic) and hydrophobic interactions with both CAS and
PAS of AChE. Further, MMꢀGBSA binding energies, summarized in Table S1, were calculated
for all the synthesized compounds to estimate relative binding affinity.
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2.7. ADMET prediction
To predict the drugꢀlikeness of the designed compounds, a few indicators of their
pharmacokinetic profile like Lipinski’s parameters, QPlogPo/w, Polar Surface Area (PSA),
QPlogKhsa, QPPCaco, QPlogBB, QPLogKhsa, QPlogBB, QPPMDCK etc were predicted using
QikProp, v. 3.5 (Table S1).36 The important physiochemical features of CNS drugs are mostly
associated to their ability to cross the blood–brain barrier (BBB) and subsequent CNS activity.
The results showed that compounds 7(a–k) obey Lipinski’s rule of five (mol_MW <500,
QPlogPo/w <5, donorHB≤5, accptHB≤10) and therefore are drug like molecules. Based on the
predicted values for QPlogBB and CNS activity, all compounds might be able to penetrate into
the CNS. PSA is another important predictor for ability to cross Blood Brain Barrier (BBB). For
a compound to be CNS active, PSA should be lower and the value should range from 50 to 70
Å.37 The QPlogKhsa prediction value revealed about the protein binding amount of CNS active
drugs which is also a vital consideration and tends to be rather high. The predicted values of
QPlogKhsa for all compounds 7(a-k) showed their strong binding with plasma protein. All the
synthesized compounds PSA values are within acceptable limit that support their ability to
infiltrate the BBB. Among calculated parameters, QPPCaco, QPlogBB, QPLogKhsa, and
QPPMDCK primarily intemperate the capability of the compound’s distribution inside the body.
Most of the compounds exhibited moderate to significant penetrability for both in vitro MDCK
cells and in vitro Cacoꢀ2 cells except compound 7c. Furthermore, all compounds exhibited good
to significant oral absorption. The predicted ADME properties revealed that all compounds 7(a-
k), have drugꢀlike properties and could be considered as good drug candidate.
2.8. Determination of brain AChE activity
AntiꢀAlzheimer drug must be able to cross the blood brain barrier (BBB) efficiently and enter into
the brain. Thus, to determine whether the most active compound 7h as a representative could
penetrate the BBB, the brain AChE activity was measured as an indirect method.38,39 Various
concentrations of 7h (50, 100 and 200 mg/kg) was administered via intraperitoneal injection to the
mice (25ꢀ30 g). After 48 h of administration, mice were sacrificed and brain homogenates were
prepared. To determine the brain AChE activity, supernatant of brain homogenate was used. The rate
of hydrolysis of acetylthiocholine (ATCh) (15 µL of 1 mM) by brain AChE at 10, 20, 30, 40 and 50
µL was measured using Ellman method.33 The rate of hydrolysis of ATCh by brain AChE was
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decreaseed with increase in administered dose of 7h. Therefore, after treatment with 7h, the brain
AChE activity was decrease in dose dependent manner which is possible only if 7h had penetrated the
BBB and inhibit brain AChE activity (Figure S4).
2.9. Molecular Dynamic Simulations
Molecular dynamic simulations (MD) were undertaken to study proteinꢀligand interactions in
dynamic motion contributing for their thermodynamic stability and to visualize the effect of
ligand binding induced conformational changes at AChE pocket. The protein ligand binding
complex of AChE and most active compound 7h was used for MD simulations. The important
interactions after MD simulations were conserved although a slightly different interaction pattern
was observed. After MD, compound 7h displayed strong hydrogen bonding with Asp72, Phe288
and Arg289. Compound 7h also showed hydrophobic interactions and πꢀπ stacking interactions
with Tyr334 and Trp279 respectively (Figure 4). These interactions can be categorized by type
and summarized, as shown in the proteinꢀligand contacts plot (Figure S5). After MD simulations,
the Root mean square deviation (RMSD) was calculated for the trajectory of complex and plotted
against time (ns). The RMSD plot showed that the docked complex was quite stable throughout
with minor fluctuations in the range of 1–1.6 Å. Higher stability was observed from 2 to 5 ns
(Figure S5) and the stability of the simulated system indicate the inhibitory nature of the 7h on
AChE.
3. Conclusion
In the present study, a new series of 2ꢀphenylꢀ1ꢀbenzopyranꢀ4ꢀone derivatives have been
developed as potential polyꢀfunctional antiꢀAlzheimer’s agents. These compounds were
evaluated in vitro and in silico against important biological targets and molecular processes
including AChE enzyme, AGEs formation with free radical scavenging activity. Among the
synthesized compounds most of the compounds showed the AChE inhibitory activity with good
radical scavenging and AGEs product formation inhibitory activity. The structure activity
relationship studies revealed that the extent of AChE inhibition was mainly influenced by the
presence of the electron releasing group on ringꢀC and substituted cyclic amine on ringꢀA of
flavonoid moiety via two carbon spacer. The compounds 7h and 7k (IC50 = 6.33 nM and 7.53
nM, respectively) displayed about 2ꢀ and 1.5ꢀfold higher potency for AChE than reference
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compound donepezil. Moreover, the in silico pharmacokinetic profiles were predicted and
revealed that all compounds have drugꢀlike properties with good penetration in brain and good
oral absorption. Further investigation of molecular modeling study indicated that compound 7h
and 7k simultaneously bind with both CAS and PAS of active site gorge. Additionally, these
compounds had significant capacity to absorb free radicals and inhibit the AGEs product
formation. The synthesized 2ꢀphenylꢀ1ꢀbenzopyranꢀ4ꢀone derivatives 7h and 7k have been found
to be maximally potent polyꢀfunctional molecules. This polyꢀfunctional attribute, make them
potential candidates for the development of drugs for Alzheimer’s disease. In summary, these
preliminary findings suggested that designed new compounds could be further investigated for
pharmacological development in AD therapy.
4. Experimental Section
4.1. Chemistry
All chemicals and solvents required for synthesis were procured 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 (DCꢀAlufolien (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 spectroscopy. IR spectra were recorded on a Bruker (Alpha E) FT/IR
1
spectrophotometer, HꢀNMR spectra were recorded on a Bruker Advance II 400 MHz NMR
spectrometer using chloroform (CDCl3) or dimethylsulfoxide (DMSOꢀd6) 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 (ESIꢀMS) were recorded with a Waters, QꢀTOF
Micromass (LCꢀMS).
4.1.1. General procedures for the synthesis of intermediate oꢀBenzoyloxyacetophenone (3)
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At first, the substituted oꢀbenzoyloxyacetophenone (3) was synthesized by stirring the mixture of
substituted oꢀhydroxyacetophenone (0.1mol) and substituted benzoyl chloride (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 reꢀcrystallized from methanol resulting in the white
precipitates of substituted oꢀbenzoyloxyacetophenone, yield 90%.
4.1.2. General procedures for the synthesis of intermediate oꢀHydroxydibenzoylmethane (4)
To a warm solution of substituted oꢀbenzoyloxyacetophenone (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 oꢀ
hydroxydibenzoylmethane (4). The reaction mixture was brought down to room temperature, and
subsequently acidified with 10% acetic acid to desalt the compounds. The lightꢀyellow
precipitates of diketone (4) were filtered and dried, yield 85%.
4.1.3. General procedures for the synthesis of intermediate 7ꢀhydroxyꢀ2ꢀ(4ꢀmethoxyphenyl)ꢀ4Hꢀ
chromenꢀ4ꢀone (5)
The substituted oꢀhydroxydibenzoylmethane (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 the7ꢀhydroxyꢀ2ꢀ(4ꢀmethoxyphenyl)ꢀ4Hꢀchromenꢀ4ꢀone (5). 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. (Scheme S1)
4.1.4. General procedures for the synthesis of intermediate 7ꢀ(2ꢀbromoethoxy)ꢀ2ꢀ(4ꢀ
methoxyphenyl)ꢀ4Hꢀchromenꢀ4ꢀone (6)
The synthetic protocol for intermediate 7ꢀ(2ꢀbromoethoxy)ꢀ2ꢀ(4ꢀmethoxyphenyl)ꢀ4Hꢀchromenꢀ4ꢀ
one (6) is outlined in Scheme 1. Anhydrous K2CO3 (10.0 mmol) and α,ωꢀdibromoalkane (4.0
mmol) were added to a solution of intermediate 5 (2.0 mmol) in acetone. After reflux for 10h
until the starting material 5 disappeared, the solvent was removed under vacuum; the residue was
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poured into water and extracted with EtOAc, the solution was dried over Na2SO4 and then
concentrated. Then, the intermediate 6 was purified on silica columns using CHCl3:MeOH
(9:1%) as solvent.40
4.1.4. General procedures for the synthesis of final compounds 7(a-k).
To a solution of intermediate 6 (0.5 mmol) in CH3CN (10 mL) was added different amines (1.0
mmol) and anhydrous K2CO3 (2.5 mmol). 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
compounds 7(a-k) were purified by column chromatography with CHCl3:MeOH (95:5%)
elution.40 (Scheme 1)
7ꢀhydroxyꢀ2ꢀ(4ꢀmethoxyphenyl)ꢀ4Hꢀchromenꢀ4ꢀone (5):
1
Brown black, amorphous solid, yield 91%, m.p.: 231ꢀ236°C; HꢀNMR (400 MHz, DMSOꢀd6, δ
ppm): 3.87 (3H, s, ꢀCH3), 6.69 (1H, s), 6.87ꢀ6.90 (1H, dd, J= 2.20, 8.68 Hz, ArH), 6.94 (1H, d,
J= 2.16 Hz, ArH), 7.05ꢀ7.08 (2H, d, J= 2.96 Hz, ArH), 7.89 (1H, d, J= 8.68 Hz, ArH), 7.94ꢀ7.96
(2H, dd, J= 1.88, 7.0 Hz, ArH), 10.62 (1H, s, ꢀOH). IR: (C=O) 1652 cmꢀ1 (s) (OꢀH) 3701 cmꢀ1,
MS (ESI) m/z = 269.1 [M+H]+. Elemental Anal. Calcd for C16H12O4: C, 71.64; H, 4.51. Found:
C, 71.51; H, 4.82; Rf Value: 0.53 (Hexane: Ethyl acetate, 6:4).
7ꢀ(2ꢀbromoethoxy)ꢀ2ꢀ(4ꢀmethoxyphenyl)ꢀ4Hꢀchromenꢀ4ꢀone (6):
1
Light Yellow, Amorphous, yield 60%; HꢀNMR (400 MHz, CDCl3, δ ppm): 8.14ꢀ8.16 (1H, d, J
= 8.72 Hz, ArH), 7.85ꢀ7.87 (2H, dd, J = 6.88 Hz, ArH), 6.97ꢀ7.03 (4H, m, ArH), 6.69 (1H, s,
ArH), 4.40ꢀ4.43 (2H, t, J = 6.28 Hz, ꢀCH2) 3.89 (3H, s, ꢀOCH3) 3.69ꢀ3.72 (2H, t, J = 6.28 Hz, ꢀ
CH2). Anal. Calcd for C18H15BrO4: C, 57.62; H, 4.03. Found: C, 58.51; H, 5.31; Rf Value: 0.65
(CHCl3: MeOH, 9:1).
2ꢀ(4ꢀmethoxyphenyl)ꢀ7ꢀ(2ꢀmorpholinoethoxy)ꢀ4Hꢀchromenꢀ4ꢀone (7a):
Intermediate 6 was treated with morpholine according to general procedure to give the desired
product 7a as light yellow, solid crystalline, yield 65%; IR (KBr) (cmꢀ1): 1650 cmꢀ1 (C=O, str, s),
1
1605 cmꢀ1 (C=C, s), 1263 cmꢀ1 (CꢀO, asym., s), 1373 cmꢀ1 (CꢀN, str, s); HꢀNMR (400 MHz,
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CDCl3, δ ppm): 8.11ꢀ8.13 (1H, d, J = 8.68 Hz, ArH), 7.84ꢀ7.87 (2H, dd, J = 6.84, 2.08 Hz, ArH),
6.96ꢀ7.03 (4H, m, ArH), 6.68 (1H, s, ArH), 4.21ꢀ4.24 (2H, t, J = 5.60 Hz, ꢀCH2) 3.89 (3H, s, ꢀ
OCH3) 3.74ꢀ3.76 (4H, t, J = 4.72 Hz, ꢀCH2) 2.85ꢀ2.88 (2H, t, J = 5.72 Hz, ꢀCH2) 2.59ꢀ2.62 (4H,
t, J = 5.72 Hz, ꢀCH2); 13C NMR (100 MHz, CDCl3 ): 177.84, 163.10, 162.28, 157.83, 127.85,
127.07, 124.13, 117.93, 114.47, 114.45, 106.14, 101.12, 66.92, 66.53, 57.35, 55.52, 54.13; MS
(ESI) m/z = 382.31 [M+H]+. Anal. Calcd for C22H23NO5: C, 69.28; H, 6.08; N, 3.67. Found: C,
69.21; H, 5.95; N, 3.26; Rf Value: 0.30 (CHCl3: MeOH, 9:1).
2ꢀ(4ꢀmethoxyphenyl)ꢀ7ꢀ(2ꢀ(piperidinꢀ1ꢀyl)ethoxy)ꢀ4Hꢀchromenꢀ4ꢀone (7b):
Intermediate 6 was treated with piperidine according to general procedure to give the desired
product 7b as light yellow, solid amorphous, yield 60%; IR (KBr) (cmꢀ1): 1630 cmꢀ1 (C=O, str,
s), 1601 cmꢀ1 (C=C, s), 1258 cmꢀ1 (CꢀO, asym., s), 1359 cmꢀ1 (CꢀN, str, s); 1HꢀNMR (400 MHz,
CDCl3, δ ppm): 8.10ꢀ8.12 (1H, d, J = 8.64 Hz, ArH), 7.84ꢀ7.87 (2H, dd, J = 7.00, 1.88 Hz, ArH),
7.00ꢀ7.03 (2H, dd, J = 7.08, 1.80 Hz, ArH), 6.98ꢀ6.99 (1H, d, J = 2.36 Hz, ArH), 6.96 (1H, s,
ArH), 6.68 (1H, s, ArH), 4.21ꢀ4.24 (2H, t, J = 5.92 Hz, ꢀCH2) 3.89 (3H, s, ꢀOCH3) 2.84ꢀ2.87
(2H, t, J = 5.92 Hz, ꢀCH2), 2.56 (2H, m, ꢀCH2) 1.61ꢀ1.66 (4H, m, ꢀCH2) 1.45ꢀ1.48 (2H, m, ꢀ
13
CH2); C NMR (100 MHz, CDCl3 ): 177.86, 163.20, 163.07, 162.25, 157.84, 127.85, 126.91,
124.12, 117.82, 114.60, 114.43, 106.08, 101.06, 66.57, 57.52, 55.50, 55.02, 29.70, 29.37, 25.76;
MS (ESI) m/z = 380.20 [M+H]+. Anal. Calcd for C23H25NO4: C, 72.80; H, 6.64; N, 3.69. Found:
C, 72.29; H, 7.47; N, 3.48; Rf Value: 0.35 (CHCl3: MeOH, 9:1).
2ꢀ(4ꢀmethoxyphenyl)ꢀ7ꢀ(2ꢀ(piperazinꢀ1ꢀyl)ethoxy)ꢀ4Hꢀchromenꢀ4ꢀone (7c):
Intermediate 6 was treated with piperazine according to general procedure to give the desired
product 7c as light yellow, solid amorphous, yield 55%; IR (KBr) (cmꢀ1): 1651 cmꢀ1 (C=O, str,
s), 1606 cmꢀ1 (C=C, s), 1263 cmꢀ1 (CꢀO, asym., s), 1374 cmꢀ1 (CꢀN, str, s); 1HꢀNMR (400 MHz,
DMSOꢀd6, δ ppm): 8.00ꢀ8.02 (2H, d, J = 8.88 Hz, ArH), 7.92ꢀ7.94 (1H, d, J = 8.80 Hz, ArH),
7.25ꢀ7.26 (1H, d, J = 2.28 Hz, ArH), 7.08ꢀ7.10 (2H, d, J = 8.88 Hz, ArH), 7.00ꢀ7.03 (1H, dd, J =
8.80, 2.28 Hz, ArH), 6.79 (1H, s, ArH), 4.22ꢀ4.25 (2H, t, J = 5.64 Hz, ꢀCH2) 3.87 (3H, s, ꢀ
OCH3) 2.75ꢀ2.81 (5H, m, ꢀCH2, ꢀNH), 2.52ꢀ2.53 (6H, t, J = 1.64 Hz, ꢀCH2); 13C NMR (100
MHz, CDCl3 ): 177.87, 163.10, 162.28, 157.88, 127.86, 127.05, 124.09, 117.89, 114.50, 114.44,
106.09, 101.11, 66.52, 57.27, 55.51, 53.81, 45.29; MS (ESI) m/z = 381.34 [M+H]+. Anal. Calcd
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for C22H24N2O4: C, 69.46; H, 6.36; N, 7.36. Found: C, 69.11; H, 6.92; N, 7.49; Rf Value: 0.25
(CHCl3: MeOH, 9:1).
2ꢀ(4ꢀmethoxyphenyl)ꢀ7ꢀ(2ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)ethoxy)ꢀ4Hꢀchromenꢀ4ꢀone (7d):
Intermediate 6 was treated with 4ꢀmethylpiperazine according to general procedure to give the
desired product 7d as brown black, solid amorphous flakes, yield 78%, mp 231ꢀ236°C; IR (KBr)
(cmꢀ1): 1652 cmꢀ1 (C=O, str, s), 1610 cmꢀ1 (C=C, s), 1258 cmꢀ1 (CꢀO, asym., s), 1370 cmꢀ1 (CꢀN,
str, s); 1HꢀNMR (400 MHz, CDCl3, δ ppm): 8.10ꢀ8.12 (1H, d, J = 8.64 Hz, ArH), 7.85ꢀ7.87 (2H,
dd, J = 6.8, 2.08 Hz, ArH), 7.01ꢀ7.03 (2H, m, ArH), 6.96ꢀ7.03 (2H, dd, J = 10.84, 1.84, ArH),
6.68 (1H, s, ArH), 4.20ꢀ4.23 (2H, t, J = 6.00 Hz, ꢀCH2) 3.89 (3H, s, ꢀOCH3) 2.83ꢀ2.86 (2H, t, J =
6.00 Hz, ꢀCH2), 2.09ꢀ2.15 (2H, m, ꢀCH2) 1.64ꢀ1.69 (6H, m, ꢀCH2) 1.25ꢀ1.30 (3H, m, ꢀOCH3); 13C
NMR (100 MHz, CDCl3 ): 177.94, 163.35, 163.11, 162.25, 157.91, 127.91, 127.01, 124.21,
117.85, 114.68, 114.49, 106.15, 101.12, 66.89, 57.33, 55.56, 54.63, 30.62; MS (ESI) m/z =
432.25 [M+K]+. Anal. Calcd for C23H26N2O4: C, 70.03; H, 6.64; N, 7.10. Found: C, 70.45; H,
6.80; N, 6.91; Rf Value: 0.36 (CHCl3: MeOH, 9:1).
2ꢀ(4ꢀmethoxyphenyl)ꢀ7ꢀ(2ꢀ(pyrrolidinꢀ1ꢀyl)ethoxy)ꢀ4Hꢀchromenꢀ4ꢀone (7e):
Intermediate 6 was treated with pyrrolidine according to general procedure to give the desired
product 7e as light brownish, solid amorphous, yield 50%; %; IR (KBr) (cmꢀ1): 1627 cmꢀ1
1
(C=O, str, s), 1602 cmꢀ1 (C=C, s), 1258 cmꢀ1 (CꢀO, asym., s), 1360 cmꢀ1 (CꢀN, str, s); HꢀNMR
(400 MHz, DMSOꢀd6, δ ppm): 8.10ꢀ8.12 (1H, d, J = 8.76 Hz, ArH), 7.84ꢀ7.88 (2H, m, ArH),
6.96ꢀ7.03 (4H, m, ArH), 6.68 (1H, s, ArH), 4.21ꢀ4.24 (2H, t, J = 5.80 Hz, ꢀCH2) 3.89 (3H, s, ꢀ
OCH3) 2.96ꢀ2.99 (2H, t, J = 5.84 Hz, ꢀCH2), 2.65ꢀ2.68 (4H, m, ꢀCH2) 1.81ꢀ1.86 (4H, m, ꢀCH2);
13C NMR (100 MHz, CDCl3 ): 177.94, 163.27, 163.10, 157.86, 127.88126.99, 124.14, 117.81,
114.63, 114.43, 106.10, 101.02, 67.74, 55.52, 54.76, 54.73, 23.50; MS (ESI) m/z = 366.30
[M+H]+. Anal. Calcd for C22H23NO4: C, 72.31; H, 6.34; N, 3.83. Found: C, 72.84; H, 5.67; N,
3.22; Rf Value: 0.32 (CHCl3: MeOH, 9:1).
7ꢀ(2ꢀ(diethylamino)ethoxy)ꢀ2ꢀ(4ꢀmethoxyphenyl)ꢀ4Hꢀchromenꢀ4ꢀone (7f):
Intermediate 6 was treated with diethylamino according to general procedure to give the desired
product 7f as light yellow, solid amorphous flakes, yield 55%; IR (KBr) (cmꢀ1): 1627 cmꢀ1
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1
(C=O, str, s), 1597 cmꢀ1 (C=C, s), 1246 cmꢀ1 (CꢀO, asym., s), 1363 cmꢀ1 (CꢀN, str, s); HꢀNMR
(400 MHz, CDCl3, δ ppm): 8.10ꢀ8.13 (1H, d, J = 8.56 Hz, ArH), 7.85ꢀ7.86 (2H, dd, J = 6.92,
1.96 Hz, ArH), 7.01ꢀ7.03 (2H, m, ArH), 6.99.ꢀ7.00 (1H, d, J = 2.32 Hz, ArH), 6.97 (1H, s, ArH),
6.68 (1H, s, ArH), 4.16ꢀ4.19 (2H, t, J = 6.04 Hz, ꢀCH2) 3.89 (3H, s, ꢀOCH3) 2.94ꢀ2.97 (2H, t, J =
13
6.04 Hz, ꢀCH2), 2.66ꢀ2.71 (4H, q, ꢀCH2), 1.09ꢀ1.12 (6H, t, J = 7.16 Hz, ꢀCH3); C NMR (100
MHz, CDCl3 ): 177.89, 163.32, 163.07, 162.26, 157.88, 127.86, 127.00, 124.20, 117.83, 114.58,
114.45, 106.15, 101.04, 67.36, 55.51, 51.52, 47.92, 11.83; MS (ESI) m/z = 368.34 [M+H]+.
Anal. Calcd for C22H25NO4: C, 71.91; H, 6.86; N, 3.81. Found: C, 71.50; H, 6.54; N, 3.50; Rf
Value: 0.28 (CHCl3: MeOH, 9:1).
7ꢀ(2ꢀ(dimethylamino)ethoxy)ꢀ2ꢀ(4ꢀmethoxyphenyl)ꢀ4Hꢀchromenꢀ4ꢀone (7g):
Intermediate 6 was treated with dimethylamino according to general procedure to give the
desired product 7g as light yellowish brown, solid amorphous flakes, yield 60%; IR (KBr) (cmꢀ
1): 1637 cmꢀ1 (C=O, str, s), 1596 cmꢀ1 (C=C, s), 1252 cmꢀ1 (CꢀO, asym., s), 1361 cmꢀ1 (CꢀN, str,
s); 1HꢀNMR (400 MHz, CDCl3, δ ppm): 8.11ꢀ8.13 (1H, d, J = 8.60 Hz, ArH), 7.85ꢀ7.87 (2H, dd,
J = 6.88, 2.04 Hz, ArH), 6.97.ꢀ7.03 (4H, m, ArH), 6.68 (1H, s, ArH), 4.18ꢀ4.21 (2H, t, J = 5.56
Hz, ꢀCH2) 3.89 (3H, s, ꢀOCH3) 2.82ꢀ2.84 (2H, t, J = 5.60 Hz, ꢀCH2), 2.39 (6H, s, ꢀCH3); 13C
NMR (100 MHz, CDCl3 ): 177.89, 163.18, 163.08, 162.25, 127.87, 127.01, 124.12, 117.87,
114.59, 106.11, 101.02, 66.54, 57.90, 55.52, 45.82; MS (ESI) m/z = 340.34 [M+H]+. Anal. Calcd
for C20H21NO4: C, 70.78; H, 6.24; N, 4.13. Found: C, 70.50; H, 6.50; N, 3.43; Rf Value: 0.24
(CHCl3: MeOH, 9:1).
2ꢀ(4ꢀmethoxyphenyl)ꢀ7ꢀ(2ꢀ(4ꢀmethylpiperidinꢀ1ꢀyl)ethoxy)ꢀ4Hꢀchromenꢀ4ꢀone (7h):
Intermediate 6 was treated with 4ꢀmethylpiperidine according to general procedure to give the
desired product 7h as brownish black, solid amorphous sticky, yield 40%; IR (KBr) (cmꢀ1): 1632
1
cmꢀ1 (C=O, str, s), 1362 cmꢀ1 (CꢀN, str, s); HꢀNMR (400 MHz, CDCl3, δ ppm): 8.10ꢀ8.12 (1H,
dd, J = 6.80, 2.72 Hz, ArH), 7.86ꢀ7.84 (2H, d, J = 8.8 Hz, ArH), 7.02 (1H, s, ArH), 7.00 (1H, s,
ArH), 6.98ꢀ6.96 (2H, m, ArH ) 6.68 (1H, s, ArH), 4.27ꢀ4.24 (2H, t, J = 5.76 Hz, ꢀCH2), 3.89 (3H,
s, ꢀOCH3) 2.92ꢀ2.89 (2H, t, J = 5.76 Hz, ꢀCH2), 1.25ꢀ1.42 (8H, m, ꢀCH2), 0.95ꢀ0.94 (3H, d, J =
13
6.0 Hz, ꢀCH2), 0.92ꢀ0.86 (1H, q, ꢀCH2); C NMR (100 MHz, CDCl3 ): 177.91, 163.14, 163.00,
162.27, 157.84, 127.88, 127.01, 124.07, 117.89, 114.61, 114.44, 106.07, 101.08, 66.29, 57.03,
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55.52, 52.40, 33.69, 30.29, 29.72, 29.38, 21.73; MS (ESI) m/z = 394.26 [M+H]+. Anal. Calcd for
C24H27NO4: C, 73.26; H, 6.92; N, 3.56. Found: C, 72.68; H, 7.26; N, 3.53; Rf Value: 0.12
(CHCl3: MeOH, 9:1).
7ꢀ(2ꢀ(benzyl(methyl)amino)ethoxy)ꢀ2ꢀ(4ꢀmethoxyphenyl)ꢀ4Hꢀchromenꢀ4ꢀone (7i):
Intermediate 6 was treated with Nꢀbenzylmethylamino according to general procedure to give the
desired product 7i as greyish brown, solid amorphous, yield 55%; IR (KBr) (cmꢀ1): 1624 cmꢀ1
1
(C=O, str, s), 1595 cmꢀ1 (C=C, s), 1252 cmꢀ1 (CꢀO, asym., s), 1355 cmꢀ1 (CꢀN, str, s); HꢀNMR
(400 MHz, DMSOꢀd6, δ ppm): 7.99ꢀ7.97 (2H, dd, J = 7.0, 1.96 Hz, ArH), 7.95ꢀ7.93 (1H, d, J =
8.84 Hz, ArH), 7.34ꢀ7.29 (4H, m, ArH), 7.23 (1H, s, ArH), 7.19ꢀ7.18 (1H, d, J = 11.12 Hz, ArH)
7.08 (1H, d, J = 1.96 Hz, ArH), 7.06 (1H, s, ArH), 6.98ꢀ7.01 (1H, dd, J = 8.8, 2.32 Hz, ArH),
6.75 (1H, s, ArH), 4.27ꢀ4.24 (2H, t, J = 5.76 Hz, ꢀCH2), 3.87 (3H, s, ꢀOCH3) 3.62 (2H, s, ꢀCH2),
13
2.86ꢀ2.83 (2H, t, J = 5.76, 5.64 Hz, ꢀCH2), 2.31 (3H, m, ꢀCH3); C NMR (100 MHz, CDCl3 ):
177.95, 163.21, 163.16, 162.33, 157.91, 129.26, 128.48, 127.93, 127.47, 127.07, 124.20, 117.92,
114.62, 114.50, 106.17, 101.11, 66.90, 62.67, 55.58, 55.37, 42.93; MS (ESI) m/z = 416.34
[M+H]+. Anal. Calcd for C26H25NO4: C, 75.16; H, 6.06; N, 3.37. Found: C, 75.06; H, 6.49; N,
2.62; Rf Value: 0.20 (CHCl3: MeOH, 9:1).
7ꢀ(2ꢀ(1Hꢀimidazolꢀ1ꢀyl)ethoxy)ꢀ2ꢀ(4ꢀmethoxyphenyl)ꢀ4Hꢀchromenꢀ4ꢀone (7j):
Intermediate 6 was treated with 4ꢀimidazoline according to general procedure to give the desired
product 7j as light yellowish, solid amorphous powder, yield 65%; IR (KBr) (cmꢀ1): 1625 cmꢀ1
1
(C=O, str, s), 1598 cmꢀ1 (C=C, s), 1256 cmꢀ1 (CꢀO, asym., s), 1367 cmꢀ1 (CꢀN, str, s); HꢀNMR
(400 MHz, CDCl3, δ ppm): 8.13ꢀ8.11 (1H, d, J = 8.6 Hz, ArH), 7.84ꢀ7.82 (2H, d, J = 8.88 Hz,
ArH), 7.63 (1H, s, ArH), 7.10 (1H, s, ArH), 7.07 (1H, s, ArH), 7.02ꢀ7.00 (2H, d, J = 8.88 Hz,
ArH), 6.96ꢀ6.95 (1H, d, , J = 2.24 Hz, ArH ), 6.94ꢀ6.91 (1H, m, ArH), 6.67 (1H, s, ArH), 4.43ꢀ
13
4.40 (2H, t, J = 4.84 Hz, ꢀCH2), 4.34ꢀ4.32 (2H, t, J = 5.04 Hz ꢀCH2), 3.88 (3H, s, ꢀOCH3); C
NMR (100 MHz, CDCl3 ): 177.73, 163.24, 162.34, 162.14, 157.65, 129.81, 127.88, 127.37,
123.91, 118.42, 114.46, 113.97, 106.11, 101.41, 67.71, 55.53, 46.22; MS (ESI) m/z = 363.38
[M+H] +, 385.35 [M+Na]+. Anal. Calcd for C21H18N2O4: C, 69.60; H, 5.01; N, 7.73. Found: C,
69.83; H, 5.44; N, 7.51; Rf Value: 0.20 (CHCl3: MeOH, 9:1).
7ꢀ(2ꢀ(4ꢀ(hydroxyethyl)piperazinꢀ1ꢀyl)ethoxy)ꢀ2ꢀ(4ꢀmethoxyphenyl)ꢀ4Hꢀchromenꢀ4ꢀone (7k):
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Intermediate 6 was treated with 4ꢀhydroxymethylpiperazine according to general procedure to
give the desired product 7k as dark brown, solid amorphous, yield 45%; IR (KBr) (cmꢀ1): 1630
1
cmꢀ1 (C=O, str, s), 1365 cmꢀ1 (CꢀN, str, s); HꢀNMR (400 MHz, CDCl3, δ ppm): 8.13ꢀ8.11 (1H,
d, J = 8.6 Hz, ArH), 7.88ꢀ7.85 (2H, d, J = 6.52 Hz, ArH), 7.09ꢀ7.01 (2H, m, ArH), 7.00ꢀ6.96
(2H, m, ArH ) 6.69 (1H, s, ArH), 4.24ꢀ4.21 (2H, t, J = 5.24 Hz, ꢀCH2), 3.89 (3H, s, ꢀOCH3) 3.87ꢀ
3.86 (1H, tꢀbroad, ꢀOH) 3.64ꢀ3.61 (2H, m, ꢀCH2) 2.90ꢀ2.87 (2H, t, J = 5.68 Hz ꢀCH2), 2.65ꢀ2.55
13
(8H, m, ꢀCH2); C NMR (100 MHz, CDCl3 ): 177.94, 163.11, 163.00, 162.20, 157.87, 127.84,
127.06, 124.07, 116.80, 114.69, 106.07, 101.04. 66.29, 59.54, 58.01, 56.51; MS (ESI) m/z =
425.47 [M+H] +, 363.56 [M+K]+. ]+. Anal. Calcd for C24H28N2O5: C, 67.91; H, 6.65; N, 6.60.
Found: C, 67.50; H, 6.30; N, 6.82; Rf Value: 0.15 (CHCl3: MeOH, 9:1).
4.2. Biological activity
4.2.1. In vitro inhibition of AChE
The inhibitory potency of target compounds on AChE was determined using spectroscopic
method of Ellman et al. with slight modification and was expressed as IC50 , that is, inhibitory
concentration that reduces the cholinesterase activity by 50%.; rat cortex homogenate was used
as the resource of AChE. 5,5`ꢀdithiobisꢀ(2ꢀnitrobenzoic acid) (Ellman’s reagent, DTNB),
acetylthiocholine iodide (ATC), donepezil was purchased from Sigma Aldrich. For assay of
AChE activity, a reaction mixture containing 100ꢁl acetylthiocholine iodide 0.075M/L, 100ꢁl
sodium phosphate buffer (0.1M/L, pH 7.4), 20ꢁl homogenate or serum and different
concentrations of test compounds 20ꢁl was incubated at 37° C for 15 min. The reaction was
terminated by adding 50ꢁl 3% sodium lauryl sulfate, then, 50ꢁl, 0.2% of 5,5’–dithioꢀbisꢀ(2ꢀ
nitrobenzoic acid) was added to produce the yellow anion of 5ꢀthioꢀ2ꢀnitroꢀbenzoic acid. Assays
were carried out with a blank containing all components except AChE in order to account for
nonꢀenzymatic reaction. The values of IC50 were calculated by UV spectroscopy from the
absorbance changes at 450 nm. A positive control of donepezil was used in the same range of
concentrations. 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.
4.2.2. In vitro Advanced glycation endꢀproduct (AGEs) formation inhibitory activity
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The assay for the ability of the flavone to inhibit the glucoseꢀmediated protein glycation and the
development of fluorescent AGEs was performed. Different concentrations of various compounds
were prepared by dissolving in DMSO. Antiꢀglycation assay was performed according to the methods
reported by Matsuura and colleagues with slight modification. In all experiments, about 500 µl of
Bovine serum albumin (1mg/ml final concentration) was incubated with 400 µl of glucose (500
mM) in the presence of 100 µl of test compounds, aminoguanidine or PBS as control buffer at
different concentrations, The reaction was allowed to proceed at 60°C for 24hrs and thereafter
reaction was stopped by adding 10 µl of 100% (w/v) trichloroacetic acid (TCA). Then the mixture
was kept at 4°C for 10 min before subjected to centrifugation at 15,000 rpm. The precipitate was
redissolved in 1ml alkaline PBS (pH 10) and immediately quantified for relative amount of glycated
BSA based on fluorescence intensity by spectrofluorometer LSꢀ55 (Perkin Elmer) at 370 nm
(excitation) and 440 nm (emission). Aminoguanidine was used as a positive control. Percentage
inhibition was calculated. All experiments were performed in triplicate.41
4.2.3. DPPH radical scavenging activity method
The stable 1, 1ꢀdiphenylꢀ2ꢀpicryl hydrazyl radical (DPPH) was used for determination of free radicalꢀ
scavenging activity of the test compounds. The 0.1 mM solution of DPPH in methanol (39.4 mg in
1000 ml) was freshly prepared. Different concentrations of test compounds were added with an equal
volume to methanol solution of DPPH. After 30 min at room temperature, the absorbance was
recorded at 517 nm. Ascorbic acid was used as standard. IC50 values denote the concentration of
sample, which is required to scavenge 50% of DPPH free radicals. IC50 value was determined from
the plotted graph of scavenging activity against the different concentrations of test compounds.
Ascorbic acid was applied as positive drug.42
4.2.4. 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 7h. 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 leastꢀsquares analysis. Each experiment was performed in triplicate.
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4.2.5. 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 antiꢀcholinesterase 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 [43]. The Xꢀray
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 [44].
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.45,46
4.2.6. ADME property prediction
To investigate the potential of the new compounds as antiꢀAD agents, their ADME
(Absorption, Distribution, Metabolism and Excretion) properties were predicted using QikProp
program, v. 3.5 of the Schrödinger software.34 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 Cacoꢀ2 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), Cacoꢀ2 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.47
4.2.7. In vivo brain AChE activity assay
4.2.7.1. Animals
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Swiss albino mice of either sex weighing 20–25 g (procured from Central Research Institute, Kasauli,
Himachal Pradesh, India) were used for this study. 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).
4.2.7.2. Drug administration
Compound 7h was dissolved in 10% dimethylsulfoxide (DMSO) and administered through
intraperitoneal injection at doses ranging from 50 to 200 mg/kg. All other reagents used in the present
study were of analytical grade and freshly prepared.
4.2.7.3 Tissue preparation for bioassay
After 48 h of intraperitoneal injection, animals were sacrificed by cervical dislocation and their brains
were taken out quickly and perfused with 0.15 M NaCl until free of blood. Dissected brain was
homogenized in 200 mM phosphate buffer (pH 7.7) to get 5% (w/v) homogenate. The homogenate
ᵒ
was centrifuged at 10,000 rpm for 10 min at 4 C to separate the nuclear debris. The supernatant thus
obtained was used for the estimation of activity of acetylcholinesterase. The brain acetylcholinesterase
activity at different volumes of supernatant (10, 20, 30, 40 and 50 µL) with 15 µL of 1 mM ATCh was
determined using Ellman method reported in section 4.2.1.
4.2.8. 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.48 The
simulations aided to stabilize the proteinꢀligand 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
NoseꢀHoover Chain method as the thermostat and pressure of 1.01325 bar using Martyn–Tobias–Klein
as the barostat.
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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 (ICMRꢀSRF); Award No. BIC/11(11)/2014. Dr. Balraj
Saini, Sophisticated Instrument Centre (SIC), Punjabi University, Patiala, and SAIF, Punjab
University, Chandigarh is acknowledged for providing access to spectrofluorometer and spectral
facilities.
Conflict of interest
We declare that we have no conflict of interest.
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Figure Captions
Figure 1: FDA approved drugs for the treatment of Alzheimer’s disease.
Figure 2: 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 7h at 2, 5 and 10 µM.
Figure 3: Docked pose of interactions of compounds 7k and 7h with AChE active site
(1EVE) using Glide. (A) 2D representation of different interactions of 7k with
TcAChE. (B) 3D representation of different interactions of compound 7k 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 7k as stick in the binding pocket. (D) 2D
representation of different interactions of 7h with TcAChE generated using Glide.
(B) 3D representation of different interactions of compound 7h with residues in the
binding sites of TcAChE. (C) The TcAChE enzyme is depicted in surface view and
compound 7h as stick in the binding pocket.
Figure 4: postꢀMD hydrogen bonds and hydrophobic interactions of 7h with AChE
Scheme 1: Synthetic scheme for the of 2ꢀphenylꢀ1ꢀbenzopyranꢀ4ꢀone derivatives 7(a–k).
Table 1: The AChE inhibition, Radical scavenging activities and advanced glycation end
products (AGEs) formation inhibitory activity data 7(a-k).
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Table 1: The AChE inhibition, Radical scavenging activities and advanced glycation end products (AGEs)
formation inhibitory activity 7(a-k).
C. No.
-NR1R2
AChE Inhibitory
Activity
Radical scavenging activity
AGEs Inhibitory Activity
(EC50c± SDa, nM)
(IC50d± SDa, µM)
(IC50b ±SDa, nM)
11.0±0.30
7a
7b
7c
7d
7e
7f
54.33±1.13
48.43±0.83
30.5±2.19
45.46±0.96
69.46±2.05
49.1±1.44
92.26±1.46
24.66±1.12
34.2±1.57
56.33±0.94
64.03±1.90
72.92±1.25
51.16±1.40
80.23±2.21
50.36±0.65
75.23±1.02
43.53±0.85
69.33±2.04
21.76±0.70
51.00±1.40
31.06±2.87
104.83±2.74
27.1±1.16
77.46±1.06
6.33±0.98
36.5±0.92
7g
7h
7i
7j
108.3±3.99
7.56±1.17
87.13±1.58
21.56 ±1.20
113.14±1.05
36.76±1.51
7k
Std.
Std.
Std.
Donepezil
Ascorbic acid
Aminoguanidine
(AG)
12.7±0.20
ꢀ
ꢀ
ꢀ
20.0± 0.54
ꢀ
40.06±1.78
a
b
c
d
SD: Data are expressed as mean ± SD (n=3). Data were statistically analyzed by one way ANOVA; *P<0.05 vs Std.
IC50 , inhibitor concentration (means ± SD of three experiments) for 50% inactivation of AChE.
EC50 was defined as the effective concentration resulting in 50% scavenging activity.
IC50 was defined as the concentration resulting in 50% inhibition of AGEs product formation.
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FDA approved drugs for the treatment of Alzheimer’s disease.
122x90mm (300 x 300 DPI)