New Flavone-Cyanoacetamide Hybrids with a Combination of Cholinergic, Antioxidant, Modulation of β-Amyloid Aggregation, and Neuroprotection Properties as Innovative Multifunctional Therapeutic Candidates for Alzheimer's Disease and Unraveling Their Mechanism of Action with Acetylcholinesterase

Article abstract of DOI:10.1021/acs.molpharmaceut.8b00041

In line with the modern multi-target-directed ligand paradigm of Alzheimer's disease (AD), a series of 19 compounds composed of flavone and cyanoacetamide groups have been synthesized and evaluated as multifunctional agents against AD. Biological evaluation demonstrated that compounds 7j, 7n, 7o, 7r, and 7s exhibited excellent inhibitory potency (AChE, IC₅₀ of 0.271 ± 0.012 to 1.006 ± 0.075 μM) and good selectivity toward acetylcholinesterase, significant antioxidant activity, good modulation effects on self-induced Aβ aggregation, low cytotoxicity, and neuroprotection in human neuroblastoma SK-N-SH cells. Further, an inclusive study on the interaction of 7j, 7n, 7o, 7r, and 7s with AChE at physiological pH 7.2 using fluorescence, circular dichroism, and molecular docking methods suggested that these derivatives bind strongly to the peripheral anionic site of AChE mostly through hydrophobic interactions. Overall, the multifunctional profiles and strong AChE binding affinity highlight these compounds as promising prototypes for further pursuit of innovative multifunctional drugs for AD.

Full text of DOI:10.1021/acs.molpharmaceut.8b00041

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Article
New flavone-cyanoacetamide hybrids with combination of cholinergic,
antioxidant, modulation #-amyloid aggregation and neuroprotection properties
as innovative multifunctional therapeutic candidates for Alzheimer’s
disease and unraveling their mechanism of action with acetylcholinesterase
Shaik Jeelan Basha, Penumala Mohan, Daniel Pushparaju Yeggoni, Zinka Raveendra Babu, Palaka
Bhagath Kumar, Ampasala Dinakara Rao, Rajagopal Subramanyam, and Amooru Gangaiah Damu
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00041 • Publication Date (Web): 10 May 2018
Downloaded from http://pubs.acs.org on May 13, 2018
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Molecular Pharmaceutics
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New flavoneꢀcyanoacetamide hybrids with combination of cholinergic,
antioxidant, modulation βꢀamyloid aggregation and neuroprotection
properties as innovative multifunctional therapeutic candidates for
Alzheimer’s disease and unraveling their mechanism of action with
acetylcholinesterase
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a
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Shaik Jeelan Basha , Penumala Mohan , Daniel Pushparaju Yeggoni , Zinka Raveendra Babu ,
c
c
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Palaka Bhagath Kumar , Ampasala Dinakara Rao , Rajagopal Subramanyam , Amooru
a,
Gangaiah Damu *
a
Department of Chemistry, Yogi Vemana University, Andhrapradesh, Kadapa, India
b
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad,
India
c
Centre for Bioinformatics, School of life Sciences, Pondicherry Central University, Puducherry,
India
*Corresponding author
Prof. Amooru Gangaiah Damu
Department of Chemistry
Yogi Vemana University
AndhrapradeshꢀKadapaꢀ516003 India
Tel: +91ꢀ9177888961
Email: agdamu01@gmail.com
KEYWORDS: Alzheimer’s disease, flavones, cyanoacetamide, Multiꢀtarget Directed Ligands,
binding studies, circular dichroism spectroscopy, fluorescence emission, molecular docking.
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ABSTRACT
In line with the modern multi targetꢀdirected ligand paradigm of Alzheimer’s disease (AD), a
series of nineteen compounds composed of flavone and cyanoacetamide groups have been
synthesized and evaluated as multifunctional agents against AD. Biological evaluation
demonstrated that compounds 7j, 7n, 7o, 7r and 7s exhibited excellent inhibitory potency
(AChE, IC50 0.271 ± 0.012 to 1.006 ± 0.075 ꢀM) and good selectivity toward
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acetylcholinesterase, significant antioxidant activity, good modulation effects on selfꢀinduced Aβ
aggregation, low cytotoxicity and neuroprotection in human neuroblastoma SKꢀNꢀSH cells.
Further, an inclusive study on the interaction of 7j, 7n, 7o, 7r and 7s with AChE at physiological
pH 7.2 using fluorescence, circular dichroism and molecular docking methods suggesting that
these derivatives bind strongly to peripheral anionic site of AChE mostly through hydrophobic
interactions. Overall, the multifunctional profiles and strong AChE binding affinity highlight
these compounds as promising prototypes for further pursuit of innovative multifunctional drugs
for AD.
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INTRODUCTION
Alzheimer’s disease (AD) is a progressive neurodegenerative disease that assaults the central
nervous system (CNS) leading to memory loss, cognitive impairment, behavioral disturbances
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and loss of intellectual ability . With more than 48 million people afflicted worldwide, AD has
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become one of the biggest global public health challenges. As a result of increasing incidence,
progressive clinical onset, mortality and unavailability of definitive treatments, AD has a severe
4
impact on patients’ and their families’ quality of life and its economic burden is massive.
Although the etiology of AD remain elusive, substantial evidences have revealed that AD is a
multifactorial syndrome derived from a complex array of neurochemical factors, such as low
levels of synaptic acetylcholine (ACh), oxidative stress and accumulation of neurotoxic amyloid
beta peptide, dyshomeostasis of biometals, the inflammation of neurons, and so on. Several
hypotheses based on these factors have been proposed to explain the mechanism of AD
5
, 6
pathogenesis.
Based on the ‘‘cholinergic hypothesis’’, AChE and BuChE inhibition have been documented as
critical targets for the effective management of AD by an increase the availability of ACh in the
7
brain regions. However, recently it has been reported that serious inhibition of BuChE also may
8
contribute to peripheral side effects. The tacrine a dual AChE and BuChE inhibitor, which
9
showed serious hepatotoxicity as well as other side effects was withdrawn from the market.
Therefore, the potential advantage of selective inhibition of AChE over BuChE may include
lesser degree of associated side effects. So it may be a good approach to expand selective AChE
3
inhibitors for the treatment of AD, with expectation for their fewer unfavorable effects.
According to “amyloid hypothesis”, the production and accumulation of oligomeric aggregates
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of amyloidꢀβ (Aβ) peptide in the brain are a central event in the pathogenesis of AD. Among
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the main isoforms, Aβ is a major component of the amyloid plaques and plays a critical role in
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the initiation of plaque formation and AD pathogenesis. The most toxic forms of Aβ including
senile plaques, fibrils, protofibrils, and oligomers are among the aggregates of amyloidogenic
peptides.
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However, it has been reported that among the toxic amyloidogenic peptides including oligomers
protofibrils, fibrils, and senile plaques, oligomers are more toxic to neurons in comparison with
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the fibril aggregates. It has also been reported that synaptic loss in the cerebral cortex is related
13, 14
to the concentration of soluble Aβ monomers and oligomers.
In support for this, a variety of
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molecules have been designed to modulate the aggregation of amyloidogenic peptides.
Therefore, finding molecules that ubiquitously modulate biological effects of the aggregates of
amyloidogenic peptides is a complementary approach and a matter of active research.
A wealth of experimental data shows that the reactive oxygen species (ROS) is another major
etiological factor of AD since amyloid plaques and neurofibrillary tangles along with ROS
overproduction are the main factors for the initiation of neuroinflammation that precede neuronal
16
degeneration in AD patients. Consequently, neuroprotection against oxidative damage in
neuronal cells has become highly beneficial and worthy strategy for either the prevention or the
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treatment of AD.
Therefore, the combination of selective AChE inhibition, Aβ aggregation modulation, reduction
of oxidative load and neuroprotection represents an additional rational approach for AD
management. Facing the complex etiology of AD, at present researchers are focused on
18, 19
developing new Multiꢀtarget Directed Ligands (MTLDs) to fight back against this disease.
In
this context, by exploiting MTDL approach, several hybrid molecules, which consist of two or
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more pharmacophores in one molecule and could simultaneously target different pathogenic
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factors of AD have been reported as successful multifunctional agents to treat AD.
Our group is also actively engaged to develop potent MTLDs against AD and recently reported
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tricyclic coumarin analogs as hybrids of coumarin and cyanoacetamides as potential
21, 22
multifunctional antiꢀAD.
Herein; we have developed a novel series of flavone derivatives to
investigate their multifunctional potential against AD. Flavones have been assessed for several
2
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therapeutic activities against AD like AChE inhibitory, neuroprotective, antioxidant, Aβ fibril
formation inhibitory, reduction effect of H O ꢀinduced ROS formation and GSK tau aggregation
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inhibitory activities. On the other hand the cyanoacetamide scaffold is widely used to develop
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numerous biologically active agents.
To proceed with our goal, flavone moiety was selected to combine with cyanoacetamide
fragment as well as variety of amides into single frame, to design a series of flavones derivatives
7a–s, to test their multifunctional activities. In this paper, a series of nineteen derivatives were
designed, synthesized and evaluated biological activities including AChE and BuChE inhibition,
the kinetics of enzyme inhibition, antiꢀoxidative effects, neuroprotective effects against H O ꢀ
2
2
induced SKSNH cell injury, amyloidꢀβ peptide (Aβ) modulating abilities based on the
multitargetꢀdirected ligands strategy (MTDLs). Further, we have used both biophysical and
computational approach to understand the binding mechanism of target compounds with AChE.
MATERIALS AND METHODS
Analytical grade starting materials and reagents were purchased from commercial sources
(Aldrich, Merck) and were used without purification. Prior the use, all the solvents were purified
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and dried by standard methods. Progress of the reactions were monitored using analytical thinꢀ
layer chromatography (TLC) on silica gel 60 F254 plates with a layer thickness of 0ꢀ20 mm from
sigma (USA), and visualized with an UV lamp (254 nm). Column chromatography was
performed on silica gel (Merck, 200ꢀ400 mm mesh or less than 200 mm). Melting points were
determined in open capillary tubes on a Fisher Scientific apparatus (India) and were uncorrected.
IR spectra were measured as KBr discs with a Perkin Elmer Spectrum two spectrophotometer
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ꢀ1
ꢀ1
(
Singapore) and reported the frequency of absorption (cm ) in the range from 4000 to 400 cm .
1
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H NMR (400 MHz) and C NMR (100 MHz) were recorded at room temperature in CDCl3
solution using Bruker Avance II (Switzerland) spectrometer. NMR spectra of representative
compounds are shown in the Supplementary Information. Chemical shifts are reported in parts
per million (δ ppm) relative to tetramethylsilane (TMS), an internal standard. The peak
multiplicity was indicated by abbreviations viz.: s = singlet; d = doublet; t = triplet; q = quartet;
m = multiplet; bs = broad singlet. Mass spectra were obtained on an Agilent LC/MSD trap SL
1
100 series mass spectrometer with a 70 eV (ESI probe) in positive mode and highꢀresolution
mass spectra were obtained by using ESIꢀQTOF mass spectrometry in positive mode.
Preparation of resacetophenone (1)
Freshly fused 33 g of ZnCl was dissolved in 32mL of acetic acid while heating; 22 g of
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resorcinol was added. The reaction mixture was heated to 140ꢀ150 C and stirred for 15 min.
After being settled for 1h, the mixture was treated with 100 mL of 1:1 HCl to break the zinc
0
chloride complex. The reaction was cooled to 5 C and the resulted solid was filtered. The
2
7,28
product was further crystallized from 20% HCl to get 90% yield of compound 1.
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Preparation of 1ꢀ(4ꢀbenzoyloxyꢀ2ꢀhydroxyphenyl)ꢀ3ꢀphenylꢀ1,3ꢀpropanedione (2)
To a mixture of resacetophenone 1 (4.56 g, 30 mmol) and powdered potassium carbonate (30.36
g, 220 mmol) in 150 mL of anhydrous acetone, benzoyl chloride (7 mL, 60 mmol) was added
drop wise with stirring at room temperature. The resulting mixture was refluxed for 20 h. After
being cooled to room temperature, the deposits were filtrated off. The filtrate then poured into
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30 mL of 10% glacial acetic acid and formed yellow solid was collected by filtration and
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recrystallized from acetone to give 51 % yield of desired product 2.
Preparation of 7ꢀhydroxyflavone (3)
A mixture of 100 mL of concentrated sulphuric acid and compound 2 was stirred for 4 h keeping
the temperature of the reaction mixture not beyond 5 °C. The liquid was poured into 500 g of ice
under vigorous stirring to give a white solid, which was filtered, washed thoroughly with water
and dried. To this solid, 250 mL of 5% potassium carbonate solution was added. The mixture
was heated to 100 °C and stirred for 1 h. To the reaction mixture was acidified with concentrated
hydrochloric acid by adding drop wise to pH = 5 and white solid thus formed was collected by
filtration. After drying, the raw material was recrystallized from absolute ethanol to give 82 %
3
1
yield of 7ꢀhydroxyflavone.
Preparation of 8ꢀformylꢀ7ꢀhydroxyflavone (4)
Under microwave irradiation at 300 W for 7 min, hexamethylenetetramine (HMTA) (5.6 g, 40
mmol) was added to a solution of 7ꢀhydroxyflavone (2.52 g, 10 mmol) in glacial acetic acid (50
mL). The reaction mixture was treated with 20% HCl (50mL) and further irradiated for another 4
min with MW at 200 W. After cooling to room temperature, the reaction solution was poured
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into crushed ice. The acidic solution was extracted with ether (500 mL × 1, 250 mL × 2). The
combined organic phases were washed three times with 10% sodium bicarbonate solution (100
mL ×3), dried over Na SO and filtered. The solvent was evaporated to dryness under reduced
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pressure to get the pale yellow solid. The afforded solid was recrystallized from ethanol to get
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5% yield of compound 4.
General procedure for the preparation of cyanoacetamides 6(aꢀs)
To a solution of ethylcyanoacetate 5 (1.2 mmol) in ethanol (10 mL), the appropriate amine (1
mmol) was added in an easily available screw cap bottle. The mixture was stirred at room
temperature for 5ꢀ8 h and monitored by TLC. Once the reaction had completed, the reminder
was cooled down to 0ꢀ5 °C in an ice bath. In most cases, the obtained amide, typically
21, 22
precipitated after some minutes to hours.
The deposit was filtered off and washed several
times with ether to obtain pure Nꢀsubstituted cyanoacetamide derivatives.
General synthetic procedure for preparation of final products 7(aꢀs)
To a solution of compound 4 (1.5 mmol) and appropriate Nꢀsubstituted cyanoacetamide (6aꢀs)
(
3.1 mmol) in ethanol (10 mL), was added Et N (0.1 mmol) drop wise at room temperature. The
3
resulting mixture was refluxed for 2ꢀ3 h. After that, the reaction mixture was allowed to cool to
room temperature. The precipitated product was collected by filtration and washed with cold
33
methanol (2ꢀ3 mL) to yield corresponding final products (7aꢀs).
8
ꢀiminoꢀNꢀmethylꢀ4ꢀoxoꢀ2ꢀphenylꢀ4H,8Hꢀpyrano[2,3ꢀf]chromeneꢀ9ꢀcarboxamide (7a)
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Off white solid; yield 67%; mp: 213–215°C; IR (KBr) vmax: 3436, 3239, 3068, 2925, 2854, 1730,
ꢀ1
1
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677, 1643, 1606, 1555, 1437, 1351, 1199, 1078, 827, 774, 686 cm ; H NMR (400 MHz,
CDCl ) δ: 10.02 (br s, 1H, 12ꢀNH), 9.05 (s, 1H,10ꢀH), 8.28 (d, 1H, J = 8.8 Hz, 5ꢀH), 7.95 (d, 2H,
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J = 6.0 Hz , 2'ꢀH and 6'ꢀH), 7.81 (s, 1H, 8ꢀNH), 7.55 (m, 3H, 3'ꢀH, 4'ꢀH and 5'ꢀH), 7.16 (d, 1H, J
1
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=
8.8 Hz, 6ꢀH), 6.85 (s, 1H, 3ꢀH), 3.01 (d, 3H, J = 4.4 Hz, 1''ꢀCH ); C NMR (100 MHz, CDCl )
3 3
δ: 176.9 (Cꢀ4), 163.6 (Cꢀ2), 162.3 (Cꢀ6a), 157.2 (Cꢀ8), 156.4 (Cꢀ11), 153.2 (Cꢀ1a), 134.2 (Cꢀ1'),
1
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32.2 (Cꢀ10), 131.0 (Cꢀ4'), 129.8 (Cꢀ5), 129.5 (Cꢀ3', 5'), 126.4 (Cꢀ2', 6'), 121.0 (Cꢀ4a), 120.0 (Cꢀ
+
), 113.4 (Cꢀ10a), 109.2 (Cꢀ6), 108.2 (Cꢀ3), 26.6 (Cꢀ1''); ESIꢀMS m/z: 347.4 [M+H] ; HRMS
calcd. For C H O N : 347.3876, found: 347.3885.
2
0
15
4
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Nꢀethylꢀ8ꢀiminoꢀ4ꢀoxoꢀ2ꢀphenylꢀ4H,8Hꢀpyrano[2,3ꢀf]chromeneꢀ9ꢀcarboxamide (7b)
Off white solid; yield 68%; mp: 176–178°C; IR (KBr) vmax: 3442, 3252, 3075, 2972, 2922, 1680,
ꢀ1
1
1
645, 1587, 1556, 1436, 1383, 1352, 1198, 1031, 830, 771, 685 cm ; H NMR (400 MHz,
CDCl ) δ: 10.10 (br s, 1H, 12ꢀNH), 9.06 (s, 1H, 10ꢀH), 8.28 (d, 1H, J = 8.8 Hz, 5ꢀH), 7.95 (m,
3
2H, 2'ꢀH and 6'ꢀH), 7.81 (s, 1H, 8ꢀNH), 7.55 (m, 3H, 3'ꢀH, 4'ꢀH and 5'ꢀH), 7.16 (d, 1H, J = 8.8
1
3
Hz, 6ꢀH), 6.85 (s, 1H, 3ꢀH), 3.49 (m, 2H, 1''ꢀCH ), 1.27 (t, 3H, J = 7.6 Hz, 2''ꢀCH ) ; C NMR
2
3
(
100 MHz, CDCl ) δ: 176.9 (Cꢀ4), 163.6 (Cꢀ2), 161.5 (Cꢀ6a), 157.2 (Cꢀ8), 156.4 (Cꢀ11), 153.2
3
(Cꢀ1a), 134.2 (Cꢀ1'), 132.2 (Cꢀ10), 131.0 (Cꢀ4'), 129.8 (Cꢀ5), 129.4 (Cꢀ3', 5'), 126.4 (Cꢀ2', 6'),
1
21.1 (Cꢀ4a), 120.0 (Cꢀ9), 113.4 (Cꢀ10a), 109.2 (Cꢀ6), 108.2 (Cꢀ3), 34.9 (Cꢀ1''), 14.7 (Cꢀ2'');
+
ESIꢀMS m/z: 361.4 [M+H] ; HRMS calcd. For C H O N : 361.3786, found: 361.3791.
2
1
17
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Nꢀdodecylꢀ8ꢀiminoꢀ4ꢀoxoꢀ2ꢀphenylꢀ4H,8Hꢀpyrano[2,3ꢀf]chromeneꢀ9ꢀcarboxamide (7c)
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Off white solid; yield 67%; mp: 115–117°C; IR (KBr) vmax: 3447, 3318, 3223, 3064, 2921, 2850,
ꢀ1
1
1
729, 1680, 1651, 1624, 1559, 1543, 1438, 1384, 1353, 1199, 1077, 827, 774, 686 cm ; H
NMR (400 MHz, CDCl ) δ: 10.12 (br s, 1H, 12ꢀNH), 9.03 (s, 1H, 10ꢀH), 8.26 (d, 1H, J = 8.8 Hz,
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0
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ꢀH), 7.94 (d, J = 6.4 Hz, 2H, 2'ꢀH and 6'ꢀH), 7.81 (s, 1H, 8ꢀNH), 7.54 (m, 3H, 3'ꢀH, 4ꢀH' and 5'ꢀ
H), 7.15 (d, 1H, J = 8.8 Hz, 6ꢀH), 6.84 (s, 1H, 3ꢀH), 3.43 (m, 2H, 1''ꢀCH ), 1.62 (m, 2H, 2''ꢀCH ),
2
2
1
3
1.37 (br s, 18H, 3''–11''ꢀCH ), 0.85 (t, 3H, J= 6.4 Hz, 12''ꢀCH ); C NMR (100 MHz, CDCl ) δ:
2
3
3
1
1
76.9 (Cꢀ4), 163.5 (Cꢀ2), 161.5 (Cꢀ6a), 157.1 (Cꢀ8), 156.4 (Cꢀ11), 153.2 (Cꢀ1a), 134.2 (Cꢀ1'),
32.2 (Cꢀ10), 131.0 (Cꢀ4'), 129.7 (Cꢀ5), 129.4 (Cꢀ3', 5'), 126.4 (Cꢀ2', 6'), 121.0 (Cꢀ4a), 120.0 (Cꢀ
9), 113.3 (Cꢀ10a), 109.2 (Cꢀ6), 108.1 (Cꢀ3), 40.1 (Cꢀ1''), 32.0 (Cꢀ2''), 29.7 (Cꢀ3''), 29.7 (Cꢀ4''),
2
9.6 (Cꢀ5''), 29.4 (Cꢀ6'',7''), 29.4 (Cꢀ8'',9''), 27.2 (Cꢀ10''), 22.7 (Cꢀ11''), 14.2 (Cꢀ12''); ESIꢀMS
+
m/z: 510.3 [M+H] ; HRMS calcd. For C H O N : 510.7753, found: 510.7761.
3
1
37
4
2
Nꢀcyclopropylꢀ8ꢀiminoꢀ4ꢀoxoꢀ2ꢀphenylꢀ4H,8Hꢀpyrano[2,3ꢀf]chromeneꢀ9ꢀcarboxamide (7d)
Off white solid; yield 75%; mp: 204–206°C; IR (KBr) vmax: 3437, 3250, 3065, 2925, 2853, 1730,
ꢀ1
1
1
670, 1646, 1606, 1580, 1480, 1350, 1199, 1075, 830, 775, 687 cm ; H NMR (400 MHz,
CDCl ) δ: 10.13 (br s, 1H, 12ꢀNH), 9.06 (s, 1H, 10ꢀH), 8.27 (d, 1H, J = 8.8 Hz, 5ꢀH), 7.95 (d,
3
2
H, J = 6.4 Hz, 2'ꢀH and 6'ꢀH), 7.77 (s, 1H, 8ꢀNH), 7.54 (m, 3H, 3'ꢀH, 4'ꢀH and 5'ꢀH), 7.15 (d,
1H, J = 8.8 Hz, 6ꢀH), 6.85 (s, 1H, 3ꢀH), 2.95 (m, 1H, 1''ꢀH), 0.86 (m, 2H, 2''ꢀCH ), 0.64 (m, 2H,
2
1
3
3
1
''ꢀCH ); C NMR (100 MHz, CDCl ) δ: 176.9 (Cꢀ4), 163.5 (Cꢀ2), 163.0 (Cꢀ6a), 157.1 (Cꢀ8),
2
3
56.3 (Cꢀ11), 153.2 (Cꢀ1a), 134.2 (Cꢀ1'), 132.2 (Cꢀ10), 131.0 (Cꢀ4'), 129.8 (Cꢀ5), 129.4 (Cꢀ3',
5'), 126.3 (Cꢀ2', 6'), 120.8 (Cꢀ4a), 120.0 (Cꢀ9), 113.3 (Cꢀ10a), 109.2 (Cꢀ6), 108.1 (Cꢀ3), 23.0 (Cꢀ
+
1
''), 6.58 (Cꢀ2'', 3''); ESIꢀMS m/z: 373.5 [M+H] ; HRMS calcd. For C H O N : 374.0125,
2
2
17
4
2
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Nꢀcyclohexylꢀ8ꢀiminoꢀ4ꢀoxoꢀ2ꢀphenylꢀ4H,8Hꢀpyrano[2,3ꢀf]chromeneꢀ9ꢀcarboxamide (7e)
Off white solid; yield 71%; mp: 190–192°C; IR (KBr) vmax: 3437, 3240, 3066, 2924, 2853, 1728,
ꢀ1 1
1
1
1
1
1
1
1
1
1
1
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2
2
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6
0
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7
8
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0
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0
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0
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7
8
9
0
1
676, 1648, 1620, 1585, 1542, 1437, 1385, 1352, 1195, 1067, 828, 775, 687 cm ; H NMR (400
MHz, CDCl ) δ: 10.12 (d, 1H, J = 7.2 Hz, 12ꢀNH), 9.04 (s, 1H, 10ꢀH), 8.26 (d, 1H, J = 8.8 Hz,
3
5ꢀH), 7.94 (d, 2H, J = 6.4 Hz, 2'ꢀH and 6'ꢀH), 7.81 (s, 1H, 8ꢀNH), 7.54 (m, 3H, 3'ꢀH, 4'ꢀH and 5'ꢀ
H), 7.15 (d, 1H, J = 8.8 Hz, 6ꢀH), 6.84 (s, 1H, 3ꢀH), 3.97 (m, 1H, 1''ꢀH), 1.97 (m, 2H, 2'ꢀCH2),
13
1
.73 (m, 2H, 6'ꢀCH ), 1.61ꢀ1.27 (m, 6H, 3',4',5'ꢀCH ); C NMR (100 MHz, CDCl ) δ: 176.9 (Cꢀ
2
2
3
4), 163.5 (Cꢀ2), 160.5 (Cꢀ6a), 157.1 (Cꢀ8), 156.3 (Cꢀ11), 153.1 (Cꢀ1a), 134.1 (Cꢀ1'), 132.2 (Cꢀ
0), 131.0 (Cꢀ4'), 129.7 (Cꢀ5), 129.4 (Cꢀ3', 5'), 126.3 (Cꢀ2', 6'), 121.2 (Cꢀ4a), 120.0 (Cꢀ9), 113.3
1
(Cꢀ10a), 109.2 (Cꢀ6), 108.1 (Cꢀ3), 48.8 (Cꢀ1''), 32.6 (Cꢀ2'',6''), 25.7 (Cꢀ4''), 24.6 (Cꢀ3'',5''); ESIꢀ
+
MS m/z: 415.6 [M+H] ; HRMS calcd. For C H O N : 415.6650, found: 415. 6704.
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4
21
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2
8
ꢀiminoꢀ2ꢀphenylꢀ9ꢀ(pyrrolidineꢀ1ꢀcarbonyl)pyrano[2,3ꢀf]chromenꢀ4(8H)ꢀone (7f)
Off white solid; yield 65%; mp: 210–212°C; IR (KBr) vmax: 3436, 3228, 3064, 2925, 2885, 1739,
ꢀ1 1
1
646, 1631, 1431, 1385, 1191, 1066, 775, 685 cm ; H NMR (400 MHz, CDCl ) δ: 8.40 (d, 1H,
3
J = 8.8 Hz, 5ꢀH), 7.88 (m, 3H, 10ꢀH, 2'ꢀH and 6'ꢀH), 7.83 (s, 1H, 8ꢀNH), 7.56 (m, 3H, 3'ꢀH, 4'ꢀH
and 5'ꢀH), 7.15 (d, 1H, J = 8.8 Hz, 6ꢀH), 6.82 (s, 1H, 3ꢀH), 3.68 (m, 2H, 2''ꢀCH ), 3.53 (m, 2H,
2
1
3
5
1
''ꢀCH ) 1.95 (m, 4H, 3''ꢀH and 4''ꢀH); C NMR (100 MHz, CDCl ) δ: 177.2 (Cꢀ4), 164.0 (Cꢀ2),
2
3
63.4 (Cꢀ6a), 157.1 (Cꢀ8), 156.9 (Cꢀ11), 152.6 (Cꢀ1a), 134.2 (Cꢀ1'), 132.1 (Cꢀ10), 131.3 (Cꢀ4a),
129.4 (Cꢀ3', 5'), 128.5 (Cꢀ4'), 126.6 (Cꢀ5), 126.3 (Cꢀ2', 6'), 119.8 (Cꢀ9), 113.8 (Cꢀ10a), 109.2 (Cꢀ
+
6
), 108.3 (Cꢀ3), 46.1(Cꢀ2'', 5''), 24.4 (Cꢀ3'', 4''); ESIꢀMS m/z: 387.5 [M+H] ; HRMS calcd. For
C H O N : 387.6650, found: 387. 6704.
23
19
4
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ꢀiminoꢀ2ꢀphenylꢀ9ꢀ(piperidineꢀ1ꢀcarbonyl)ꢀ4H,8Hꢀpyrano[2,3ꢀf]chromenꢀ4ꢀone (7g)
Off white solid; yield 71%; mp: 210–212°C; IR (KBr) vmax: 3454, 3228, 3064, 2937, 2885, 1732,
ꢀ1 1
1640, 1592, 1431, 1385, 1194, 1067, 861, 776, 685 cm ; H NMR (400 MHz, CDCl ) δ: 8.21 (d,
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H, J = 8.8 Hz, 5ꢀH), 7.88 (m, 2H, 2'ꢀH and 6'ꢀH), 7.83 (s, 1H, 10ꢀH), 7.56 (m, 3H, 3'ꢀH, 4'ꢀH
and 5'ꢀH), 7.40 (s, 1H, 8ꢀNH), 7.15 (d, 1H, J = 8.8 Hz, 6ꢀH), 6.81 (s, 1H, 3ꢀH), 3.74 (br s, 2H, 2''ꢀ
1
3
CH ), 3.40 (br s, 2H, 6''ꢀCH ) 1.70 (br s, 6H, 3''ꢀH, 4''ꢀH and 5''ꢀH); C NMR (100 MHz, CDCl )
2
2
3
δ: 177.0 (Cꢀ4), 164.1 (Cꢀ2), 163.4 (Cꢀ6a), 157.2 (Cꢀ8), 156.9 (Cꢀ11), 152.5 (Cꢀ1a), 134.2 (Cꢀ1'),
32.2 (Cꢀ10), 131.3 (Cꢀ4a), 129.4 (Cꢀ3', 5'), 128.5 (Cꢀ4'), 126.5 (Cꢀ5), 126.3 (Cꢀ2', 6'), 119.8 (Cꢀ
1
9), 113.8 (Cꢀ10a), 108.6 (Cꢀ6), 108.2 (Cꢀ3), 47.3 (Cꢀ2'', 6''), 25.5 (Cꢀ3'', 5''), 24.4 (Cꢀ4'') ; ESIꢀ
+
MS m/z: 401.6 [M+H] ; HRMS calcd. For C H O N : 401.6120, found: 401. 6151.
2
4
21
4
2
8ꢀiminoꢀ9ꢀ(morpholineꢀ4ꢀcarbonyl)ꢀ2ꢀphenylꢀ4H,8Hꢀpyrano[2,3ꢀf]chromenꢀ4ꢀone (7h)
Off white solid; yield 68%; mp: 218–220°C; IR (KBr) vmax: 3445, 3220, 3064, 2981, 2924, 2856,
ꢀ1
1
651, 1593, 1484, 1470, 1448, 1388, 1355, 1252, 1218, 1194, 1068, 1111, 987, 776, 683 cm ;
1
H NMR (400 MHz, CDCl ) δ: 8.24 (d, 1H, J = 8.8 Hz, 5ꢀH), 7.89 (m, 3H, 10ꢀH, 2'ꢀH and 6'ꢀH),
3
7
.81 (s, 1H, 8ꢀNH), 7.58 (m, 3H, 3'ꢀH, 4'ꢀH and 5'ꢀH), 7.15 (d, 1H, J = 8.8 Hz, 6ꢀH), 6.83 (s, 1H,
13
3
ꢀH), 3.81 (br s, 4H, 3''ꢀH and 5''ꢀH), 3.72 (m, 4H, 2''ꢀH and 6''ꢀH); C NMR (100 MHz,
CDCl ) δ: 176.9 (Cꢀ4), 164.4 (Cꢀ2), 163.4 (Cꢀ6a), 157.1 (Cꢀ8), 156.9 (Cꢀ11), 152.6 (Cꢀ1a), 134.2
3
(Cꢀ1'), 132.2 (Cꢀ10), 131.3 (Cꢀ4a), 129.4 (Cꢀ3', 5'), 128.9 (Cꢀ4'), 127.8 (Cꢀ5), 126.3 (Cꢀ2', 6'),
1
19.9 (Cꢀ9), 113.8 (Cꢀ10a), 108.5 (Cꢀ6), 108.3 (Cꢀ3), 66.7 (Cꢀ3'', 5''), 47.3 (Cꢀ2'', 6''); ESIꢀMS
+
m/z: 403.5 [M+H] ; HRMS calcd. For C H O N : 404.0220, found: 404. 0251.
2
3
19
5
2
Nꢀbenzylꢀ8ꢀiminoꢀ4ꢀoxoꢀ2ꢀphenylꢀ4H,8Hꢀpyrano[2,3ꢀf]chromeneꢀ9ꢀcarboxamide (7i)
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Off white solid; yield 80%; mp: 220–222°C; IR (KBr) vmax: 3448, 3305, 2925, 1730, 1675, 1650,
ꢀ1
1
1
620, 1587, 1548, 1438, 1386, 1353, 1190, 1066, 848, 775, 687 cm ; H NMR (400 MHz,
CDCl ) δ: 10.56 (br s, 1H, 12ꢀNH), 9.08 (s, 1H, 10ꢀH), 8.28 (d, 1H, J = 8.8 Hz, 5ꢀH), 7.94 (m,
3
1
1
1
1
1
1
1
1
1
1
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2
2
2
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2
2
2
2
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3
4
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5
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5
5
5
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0
1
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7
8
9
0
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7
8
9
0
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0
2
H, 2'ꢀH and 6'ꢀH), 7.81 (s, 1H, 8ꢀNH), 7.55 (m, 3H, 3'ꢀH, 4ꢀH' and 5'ꢀH), 7.36 (m, 5H, 2''ꢀH, 3''ꢀ
H, 4''ꢀH, 5''ꢀH and 6''ꢀH), 7.15 (d, 1H, J = 8.8 Hz, 6ꢀH), 6.84 (s, 1H, 3ꢀH), 4.67 (d, 2H, J = 5.6
13
Hz, 13ꢀH); C NMR (100 MHz, CDCl ) δ: 176.8 (Cꢀ4), 163.6 (Cꢀ2), 161.7 (Cꢀ6a), 157.2 (Cꢀ8),
3
1
1
56.2 (Cꢀ11), 153.2 (Cꢀ1a), 138.2 (Cꢀ 1''), 134.5 (Cꢀ1'), 132.2 (Cꢀ10), 131.0 (Cꢀ4'), 129.9 (Cꢀ5),
29.4 (Cꢀ3', 5'), 128.8 (Cꢀ3'', 5''), 127.6 (Cꢀ2'', 6''), 127.4 (Cꢀ 4''), 126.4 (Cꢀ2', 6'), 120.9 (Cꢀ4a),
+
120.1 (Cꢀ9), 113.4 (Cꢀ10a), 109.2 (Cꢀ6), 108.2 (Cꢀ3), 44.0 (Cꢀ13); ESIꢀMS m/z: 423.2 [M+H] ;
HRMS calcd. For C H O N : 423.5110, found: 423. 5143.
2
6
20
4
2
8ꢀiminoꢀ4ꢀoxoꢀ2ꢀphenylꢀNꢀ(1ꢀphenylethyl)ꢀ4H,8Hꢀpyrano[2,3ꢀf]chromeneꢀ9ꢀcarboxamide
7j)
Off white solid; yield 71%; mp: 210–212°C; IR (KBr) vmax: 3447, 3329, 3250, 3061, 2964, 2926,
(
ꢀ1 1
1
728, 1681, 1653, 1622, 1586, 1543, 1436, 1382, 1193, 1060, 836, 774, 686 cm ; H NMR (400
MHz, CDCl ) δ: 10.65 (d, 1H, J = 7.2 Hz, 12ꢀNH), 9.04 (s, 1H, 10ꢀH), 8.28 (d, 1H, J = 8.8 Hz,
3
5
ꢀH), 7.92 (d, J = 6.8 Hz, 2H, 2'ꢀH and 6'ꢀH), 7.86 (s, 1H, 8ꢀNH), 7.55 (m, 3H, 3'ꢀH, 4ꢀH' and 5'ꢀ
H), 7.36 (m, 5H, 2''ꢀH, 3''ꢀH, 4''ꢀH, 5''ꢀH and 6''ꢀH), 7.16 (d, 1H, J = 8.8 Hz, 6ꢀH), 6.84 (s, 1H, 3ꢀ
1
3
H), 5.26 (q, J = 7.0 Hz 1H, 13ꢀH), 1.60 (d, 3H, J = 6.8 Hz, 14ꢀCH ); C NMR (100 MHz,
3
CDCl ) δ: 176.8 (Cꢀ4), 163.5 (Cꢀ2), 160.8 (Cꢀ6a), 157.1 (Cꢀ8), 156.4 (Cꢀ11), 153.2 (Cꢀ1a), 143.7
3
(Cꢀ 1''), 134.5 (Cꢀ1'), 132.2 (Cꢀ10), 130.9 (Cꢀ4'), 129.9 (Cꢀ5), 129.4 (Cꢀ3', 5'), 128.8 (Cꢀ 4''),
1
27.3 (Cꢀ2'', 6''), 126.3 (Cꢀ2', 6'), 126.1 (Cꢀ3'', 5''), 120.9 (Cꢀ4a), 120.0 (Cꢀ9), 113.4 (Cꢀ10a),
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09.2 (Cꢀ6), 108.1 (Cꢀ3), 50.0 (Cꢀ13), 22.8 (Cꢀ14); ESIꢀMS m/z: 437.3 [M+H] ; HRMS calcd.
For C H O N : 437.5010, found: 437. 5021.
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Nꢀbenzhydrylꢀ8ꢀiminoꢀ4ꢀoxoꢀ2ꢀphenylꢀ4H,8Hꢀpyrano[2,3ꢀf]chromeneꢀ9ꢀcarboxamide (7k)
Off white solid; yield 63%; mp: 230–232°C; IR (KBr) vmax: 3447, 3309, 2924, 2824, 1728, 1683,
ꢀ
1 1
1
641, 1624, 1541, 1437, 1384, 1352, 1206, 1183, 1130, 1068, 829, 774, 702 cm ; H NMR (400
MHz, CDCl ) δ: 11.20 (br s, 1H, 12ꢀNH), 9.11 (s, 1H, 10ꢀH), 8.31 (d, 1H, J = 8.8 Hz, 5ꢀH), 7.94
3
(
d, J = 6.0 Hz, 2H, 2'ꢀH and 6'ꢀH), 7.88 (s, 1H, 8ꢀNH), 7.56 (m, 3H, 3'ꢀH, 4ꢀH' and 5'ꢀH), 7.36
(br s, 10H, 2''ꢀH, 3''ꢀH, 4''ꢀH, 5''ꢀH, 6''ꢀH and 2'''ꢀH, 3'''ꢀH, 4'''ꢀH, 5'''ꢀH, 6'''ꢀH), 7.19 (d, 1H, J =
1
3
8
.8 Hz, 6ꢀH), 6.86 (s, 1H, 3ꢀH), 6.41 (d, J = 7.2 Hz,13ꢀH); C NMR (100 MHz, CDCl ) δ: 176.8
3
(
Cꢀ4), 163.5 (Cꢀ2), 160.8 (Cꢀ6a), 157.1 (Cꢀ8), 156.4 (Cꢀ11), 153.2 (Cꢀ1a), 143.7 (Cꢀ 1'',1'''),
134.5 (Cꢀ1'), 132.2 (Cꢀ10), 130.9 (Cꢀ4'), 129.9 (Cꢀ5), 129.4 (Cꢀ3', 5'), 128.8 (Cꢀ 4'', 4'''), 127.3
(
Cꢀ2'', 6'' and Cꢀ2''', 6'''), 126.3 (Cꢀ2', 6'), 126.1 (Cꢀ3'', 5'' and Cꢀ3'', 5'''), 120.9 (Cꢀ4a), 120.0 (Cꢀ
+
9
), 113.4 (Cꢀ10a), 109.2 (Cꢀ6), 108.1 (Cꢀ3), 52.0 (Cꢀ13); ESIꢀMS m/z: 499.7 [M+H] ; HRMS
calcd. For C H O N : 499.9110, found: 499.9081.
32 23
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ꢀiminoꢀNꢀ(1ꢀ(4ꢀmethoxyphenyl)ethyl)ꢀ4ꢀoxoꢀ2phenylꢀ4H,8Hꢀpyrano[2,3ꢀf]chromeneꢀ9ꢀ
carboxamide (7l)
Off white solid; yield 74%; mp: 218–220°C; IR (KBr) vmax: 3445, 3309, 2924, 2823, 1730, 1683,
ꢀ1
1
1
641, 1624, 1541, 1437, 1384, 1352, 1206, 1183, 1130, 1068, 829, 774, 687 cm ; H NMR (400
MHz, CDCl ) δ: 10.57 (d, J = 5.6 Hz, 1H, 12ꢀNH), 9.02 (s, 1H, 10ꢀH), 8.25 (d, 1H, J = 8.4 Hz,
3
5ꢀH), 7.91 (d, J = 6.0 Hz, 2H, 2'ꢀH and 6'ꢀH), 7.85 (s, 1H, 8ꢀNH), 7.53 (br s, 3H, 3'ꢀH, 4ꢀH' and
5
'ꢀH), 7.32 (m, 2H, 2''ꢀH and 6''ꢀH), 7.14 (d, 1H, J = 8.4 Hz, 6ꢀH), 6.88 (m, 2H, 3''ꢀH and 5''ꢀH),
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.82 (s, 1H, 3ꢀH), 5.20 (m, 1H, 13ꢀH), 3.78 (s, 3H, 4''ꢀOCH ), 1.57 (d, 3H, J = 6 Hz, 14ꢀCH );
3
3
1
3
C NMR (100 MHz, CDCl ) δ: 176.8 (Cꢀ4), 163.5 (Cꢀ2), 160.6 (Cꢀ6a), 158.7 (Cꢀ 4''), 157.1(Cꢀ
3
8), 156.3 (Cꢀ11), 153.1 (Cꢀ1a), 135.8 (Cꢀ 1''), 134.3 (Cꢀ1'), 132.2 (Cꢀ10), 130.9 (Cꢀ4'), 129.8 (Cꢀ
1
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0
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), 129.4 (Cꢀ3', 5'), 127.3 (Cꢀ2'', 6''), 126.3 (Cꢀ2', 6'), 120.9 (Cꢀ4a), 120.0 (Cꢀ9), 114.1 (Cꢀ3'', 5''),
13.3 (Cꢀ10a), 109.1 (Cꢀ6), 108.1 (Cꢀ3), 55.3 (4''ꢀOCH ), 49.4 (Cꢀ13), 22.7 (Cꢀ14); ESIꢀMS m/z:
3
+
465.1 [MꢀH] ; HRMS calcd. For C H O N : 465.0180, found: 465.0186.
2
8
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Nꢀ(2,4ꢀdimethoxybenzyl)ꢀ8ꢀiminoꢀ4ꢀoxoꢀ2ꢀphenylꢀ4H,8Hꢀpyrano[2,3ꢀf]chromeneꢀ9ꢀ
carboxamide (7m)
Off white solid; yield 69%; mp: 213–215°C; IR (KBr) vmax: 3449, 3309, 3066, 2926, 1728, 1683,
ꢀ1
1
1
649, 1624, 1508, 1438, 1385, 1353, 1206, 1154, 1130, 1071, 1036, 825, 774, 687 cm ; H
NMR (400 MHz, CDCl ) δ: 10.47 (br s, 1H, 12ꢀNH), 9.07 (s, 1H, 10ꢀH), 8.28 (d, 1H, J = 8.8 Hz,
3
5
ꢀH), 7.95 (d, 2H, J = 6.4 Hz, 2'ꢀH and 6'ꢀH), 7.81 (s, 1H, 8ꢀNH), 7.56 (m, 4H, 3'ꢀH, 4ꢀH', 5'ꢀH
and 6''ꢀH), 7.15 (d, 1H, J = 8.8 Hz, 6ꢀH), 6.85 (s, 1H, 3ꢀH), 6.45 (m, 2H, 3''ꢀH and 5''ꢀH), 4.58 (d,
1
3
2
H, J = 5.2 Hz, 13ꢀCH ), 3.86 (s, 3H, 2''ꢀOCH ), 3.79 (s, 3H, 4''ꢀOCH ); C NMR (100 MHz,
2 3 3
CDCl ) δ: 177.0 (Cꢀ4), 163.6 (Cꢀ2), 161.4 (Cꢀ6a), 160.5 (Cꢀ4''), 158.7 (Cꢀ2''), 157.2 (Cꢀ8), 156.2
3
(Cꢀ11), 153.2 (Cꢀ1a), 134.2 (Cꢀ1'), 132.2 (Cꢀ10), 131.0 (Cꢀ4'), 130.1 (Cꢀ6''), 129.7 (Cꢀ5), 129.4
(Cꢀ3', 5'), 126.4 (Cꢀ2', 6'), 121.3 (Cꢀ4a), 120.0 (Cꢀ9), 118.9 (Cꢀ1''), 113.4 (Cꢀ10a), 109.2 (Cꢀ6),
1
08.2 (Cꢀ3), 103.9 (Cꢀ3''), 98.7 (Cꢀ6''), 55.5 (2'', 4''ꢀOCH ), 39.3 (Cꢀ13) ; ESIꢀMS m/z: 483.7
3
+
[
M+H] ; HRMS calcd. For C H O N : 484.0040, found: 484.0051.
2
8
23
6
2
Nꢀ(2ꢀbromobenzyl)ꢀ8ꢀiminoꢀ4ꢀoxoꢀ2ꢀphenylꢀ4H,8Hꢀpyrano[2,3ꢀf]chromeneꢀ9ꢀcarboxamide
7n)
(
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Off white solid; yield 75%; mp: 211–213°C; IR (KBr) vmax: 3436, 3328, 3058, 2925, 2854, 1729,
ꢀ1 1
1
680, 1651, 1622, 1436, 1385, 1350, 1353, 1195, 1069, 1028, 822, 774, 683 cm ; H NMR (400
MHz, CDCl ) δ: 10.70 (br s, 1H, 12ꢀNH), 9.05 (s, 1H, 10ꢀH), 8.27 (d, 1H, J = 8.8 Hz, 5ꢀH), 7.93
3
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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8
9
0
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9
0
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5
6
7
8
9
0
(d, 2H, J = 6.4 Hz, 2'ꢀH and 6'ꢀH), 7.81 (s, 1H, 8ꢀNH), 7.55 (m, 4H, 3'ꢀH, 4ꢀH' 5'ꢀH and 3''ꢀH),
7
.45 (d, 1H, J = 7.6 Hz, 4''ꢀH), 7.29 (m, 1H, 5''ꢀH), 7.14 (m, 2H, 6ꢀH and 6''ꢀH), 6.84 (s, 1H, 3ꢀ
1
3
H), 4.71 (d, 2H, J = 5.6 Hz, 13ꢀH); C NMR (100 MHz, CDCl ) δ: 176.8 (Cꢀ4), 163.6 (Cꢀ2),
3
1
1
61.8 (Cꢀ6a), 157.2 (Cꢀ8), 156.2 (Cꢀ11), 153.2 (Cꢀ1a), 137.2 (Cꢀ 1''), 134.6 (Cꢀ1'), 132.9 (Cꢀ10),
32.2 (Cꢀ3''), 130.9 (Cꢀ4'), 130.0 (Cꢀ6''), 129.9 (Cꢀ5), 129.4 (Cꢀ3', 5'), 129.1 (Cꢀ 4''), 127.7 (Cꢀ
5''), 126.4 (Cꢀ2', 6'), 123.8 (Cꢀ2''), 120.8 (Cꢀ4a), 120.0 (Cꢀ9), 113.4 (Cꢀ10a), 109.1 (Cꢀ6), 108.2
+
(
Cꢀ3), 44.4 (Cꢀ13); ESIꢀMS m/z: 499.3 [M+H] ; HRMS calcd. For C H BrO N : 499.0140,
26
18
4
2
found: 499.0151.
Nꢀ(3ꢀbromobenzyl)ꢀ8ꢀiminoꢀ4ꢀoxoꢀ2ꢀphenylꢀ4H,8Hꢀpyrano[2,3ꢀf]chromeneꢀ9ꢀcarboxamide
7o)
Off white solid; yield 78%; mp: 192–194°C; IR (KBr) vmax: 3437, 3319, 3061, 2926, 2854, 1726,
(
ꢀ1
1
687, 1643, 1625, 1580, 1549, 1437, 1389, 1350, 1354, 1215, 1195, 1072, 824, 773, 686 cm ;
1
H NMR (400 MHz, CDCl ) δ: 10.63 (br s, 1H, 12ꢀNH), 9.05 (s, 1H, 10ꢀH), 8.28 (d, 1H, J = 8.8
3
Hz, 5ꢀH), 7.93 (m, 2H, 2'ꢀH and 6'ꢀH), 7.84 (s, 1H, 8ꢀNH), 7.53 (m, 3H, 3'ꢀH, 4ꢀH' and 5'ꢀH),
7
.50 (br s, 1H, 2''ꢀH), 7.39 (d, 1H, J = 7.6 Hz, 6''ꢀH), 7.29 (d, 1H, J = 7.6 Hz, 4''ꢀH), 7.20 (t, 1H,
J = 8.0 Hz, 5''ꢀH), 7.15 (d, 1H, J = 8.8 Hz, 6ꢀH), 6.83 (s, 1H, 3ꢀH), 4.62 (d, 2H, J = 6 Hz, 13ꢀH);
1
3
C NMR (100 MHz, CDCl ) δ: 176.8 (Cꢀ4), 163.6 (Cꢀ2), 161.9 (Cꢀ6a), 157.2 (Cꢀ8), 156.2 (Cꢀ
3
1
1), 153.2 (Cꢀ1a), 140.6 (Cꢀ 1''), 134.7 (Cꢀ1'), 132.9 (Cꢀ10), 132.2 (Cꢀ2''), 130.9 (Cꢀ4'), 130.5 (Cꢀ
''), 130.3 (Cꢀ4''), 130.0 (Cꢀ5), 129.4 (Cꢀ3', 5'), 126.4 (Cꢀ2', 6'), 126.2 (Cꢀ6''), 122.8 (Cꢀ3''), 120.7
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(
Cꢀ4a), 120.0 (Cꢀ9), 113.4 (Cꢀ10a), 109.1 (Cꢀ6), 108.2 (Cꢀ3), 43.4 (Cꢀ13); ESIꢀMS m/z: 499.3
+
[
M+H] ; HRMS calcd. For C H BrO N : 499.0171, found: 499.0176.
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Nꢀ(2ꢀchlorobenzyl)ꢀ8ꢀiminoꢀ4ꢀoxoꢀ2ꢀphenylꢀ4H,8Hꢀpyrano[2,3ꢀf]chromeneꢀ9ꢀcarboxamide
7p)
Off white solid; yield 80%; mp: 211–213°C; IR (KBr) vmax: 3438, 3303, 3292, 3058, 2924, 2853,
(
ꢀ1
1
728, 1677, 1648, 1621, 1585, 1539, 1437, 1386, 1353, 1194, 1071, 1053, 830, 774, 686 cm ;
1
H NMR (400 MHz, CDCl ) δ: 10.68 (t, 1H, 12ꢀNH), 9.04 (s, 1H, 10ꢀH), 8.27 (d, 1H, J = 8.8 Hz,
3
5ꢀH), 7.92 (dd, 2H, J = 6.0, 1.6 Hz, 2'ꢀH and 6'ꢀH), 7.88 (br s, 1H, 8ꢀNH), 7.54 (m, 3H, 3'ꢀH, 4ꢀ
H' and 5'ꢀH), 7.45 (dd, 1H, J = 6.8, 1.6 Hz, 3''ꢀH), 7.38 (dd, 1H, J = 6.8, 1.6 Hz, 6''ꢀH), 7.23 (m,
2
H, 4''ꢀH, 5''ꢀH), 7.14 (d, 1H, J = 8.8 Hz, 6ꢀH), 6.83 (s, 1H, 3ꢀH), 4.73 (d, 2H, J =5.6 Hz, 13ꢀH);
1
3
C NMR (100 MHz, CDCl ) δ: 176.8 (Cꢀ4), 163.6 (Cꢀ2), 161.8 (Cꢀ6a), 157.2 (Cꢀ8), 156.2 (Cꢀ
3
1
6
1), 153.2 (Cꢀ1a), 135.6 (Cꢀ 1''), 134.6 (Cꢀ1'), 133.7 (Cꢀ2''), 132.2 (Cꢀ10), 130.9 (Cꢀ4'), 130.0 (Cꢀ
''), 129.7 (Cꢀ5), 129.6 (Cꢀ3''), 129.4 (Cꢀ3', 5'), 128.9 (Cꢀ 4''), 127.1 (Cꢀ5''), 126.3 (Cꢀ2', 6'), 120.8
(Cꢀ4a), 120.0 (Cꢀ9), 113.4 (Cꢀ10a), 109.1 (Cꢀ6), 108.2 (Cꢀ3), 42.0 (Cꢀ13); ESIꢀMS m/z: 457.5
+
[
M+H] ; HRMS calcd. For C H ClO N : 458.0141, found: 458.0146.
2
6
18
4
2
Nꢀ(3ꢀchlorobenzyl)ꢀ8ꢀiminoꢀ4ꢀoxoꢀ2ꢀphenylꢀ4H,8Hꢀpyrano[2,3ꢀf]chromeneꢀ9ꢀcarboxamide
(7q)
Off white solid; yield 75%; mp: 184–186°C; IR (KBr) vmax: 3436, 3301, 3304, 2924, 2854, 1727,
ꢀ1 1
1
686, 1658, 1626, 1553, 1449, 1437, 1385, 1348, 1196, 1069, 827, 772, 683 cm ; H NMR (400
MHz, CDCl ) δ: 10.54 (br s, 1H, 12ꢀNH), 8.97 (s, 1H, 10ꢀH), 8.19 (d, 1H, J = 8.4 Hz, 5ꢀH), 7.84
3
(d, 2H, J = 6.0 Hz, 2'ꢀH and 6'ꢀH), 7.76 (s, 1H, 8ꢀNH), 7.41 (m, 3H, 3'ꢀH, 4'ꢀH and 5'ꢀH), 7.17
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m, 4H, 2''ꢀH, 4''ꢀH, 5''ꢀH and 6''ꢀH), 7.09 (d, 1H, J = 8.8 Hz, 6ꢀH), 6.75 (s, 1H, 3ꢀH), 4.54 (d,
13
2
H, J =5.2 Hz, 13ꢀH); C NMR (100 MHz, CDCl ) δ: 176.8 (Cꢀ4), 163.6 (Cꢀ2), 161.9 (Cꢀ6a),
3
157.2 (Cꢀ8), 156.3 (Cꢀ11), 153.2 (Cꢀ1a), 140.3 (Cꢀ 1''), 134.8 (Cꢀ1'), 134.6 (Cꢀ10), 132.2 (Cꢀ3''),
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7
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0
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3
4
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6
7
8
9
0
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6
7
8
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8
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0
1
6
30.9 (Cꢀ4'), 130.1 (Cꢀ5''), 130.0 (Cꢀ5), 129.4 (Cꢀ3', 5'), 127.6 (Cꢀ4''), 127.6 (Cꢀ2''), 126.4 (Cꢀ2',
'), 125.7 (Cꢀ6''), 120.6 (Cꢀ4a), 120.0 (Cꢀ9), 113.4 (Cꢀ10a), 109.1 (Cꢀ6), 108.2 (Cꢀ3), 43.4 (Cꢀ
+
13); ESIꢀMS m/z: 457.5 [M+H] ; HRMS calcd. For C H ClO N : 458.0243, found: 458.0247.
2
6
18
4
2
Nꢀ(4ꢀ(tertꢀbutyl)benzyl)ꢀ8ꢀiminoꢀ4ꢀoxoꢀ2ꢀphenylꢀ4H,8Hꢀpyrano[2,3ꢀf]chromeneꢀ9ꢀ
carboxamide (7r)
Off white solid; yield 61%; mp: 210–212°C; IR (KBr) vmax: 3439, 3363, 2958, 2925, 2855, 1729,
ꢀ1
1
1
681, 1644, 1548, 1436, 1384, 1352, 1195, 1073, 828, 774, 685 cm ; H NMR (400 MHz,
CDCl ) δ: 10.41 (t, 1H, 12ꢀNH), 8.95 (s, 1H, 10ꢀH), 8.15 (d, 1H, J = 8.8 Hz, 5ꢀH), 7.81 (d, 2H, J
3
=
5.6 Hz, 2'ꢀH and 6'ꢀH), 7.70 (s, 1H,8ꢀNH), 7.42 (m, 3H, 3'ꢀH, 4ꢀH' and 5'ꢀH), 7.25 (d, 2H, J =
8
1
.0 Hz, 2''ꢀH, 6''ꢀH), 7.17 (d, 2H, J = 8.0 Hz, 3''ꢀH, 5''ꢀH ), 7.02 (d, 1H, J = 8.8 Hz, 6ꢀH), 6.72 (s,
1
3
H, 3ꢀH), 4.51 (d, 2H, J = 5.6 Hz, 13ꢀH), 1.17 (s, 9H, 4''ꢀC(CH ) ); C NMR (100 MHz, CDCl )
3 3
3
δ: 176.9 (Cꢀ4), 163.6 (Cꢀ2), 161.7 (Cꢀ6a), 157.2 (Cꢀ8), 156.2 (Cꢀ11), 153.2 (Cꢀ1a), 150.4 (Cꢀ 4''),
35.1 (Cꢀ 1''), 134.5 (Cꢀ1'), 132.2 (Cꢀ10), 131.0 (Cꢀ4'), 129.9 (Cꢀ5), 129.4 (Cꢀ3', 5'), 127.4 (Cꢀ2'',
1
6''), 126.4 (Cꢀ2', 6'), 125.7 (Cꢀ3'', 5''), 120.9 (Cꢀ4a), 120.0 (Cꢀ9), 113.4 (Cꢀ10a), 109.2 (Cꢀ6),
+
1
08.2 (Cꢀ3), 43.70 (Cꢀ13), 34.6 (3'ꢀC(CH ) ), 31.4 (3'ꢀC(CH ) ); ESIꢀMS m/z: 480.4 [M+H] ;
3
3
3 3
HRMS calcd. For C H O N : 480.3840, found: 480. 3843.
3
0
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2
8ꢀiminoꢀNꢀ(2ꢀ(2ꢀmethoxyphenoxy)ethyl)ꢀ4ꢀoxoꢀ2ꢀphenylꢀ4H,8Hꢀpyrano[2,3ꢀf]chromeneꢀ9ꢀ
carboxamide (7s)
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Off white solid; yield 71%; mp: 178–180°C; IR (KBr) vmax: 3446, 3310, 3066, 2926, 1730, 1685,
ꢀ1 1
1
649, 1624, 1580, 1438, 1384, 1352, 1255, 1221, 1195, 1124, 830, 774, 687 cm ; H NMR (400
MHz, CDCl ) δ: 10.53 (br s, 1H, 12ꢀNH), 9.05 (s, 1H, 10ꢀH), 8.28 (d, 1H, J = 8.4 Hz, 5ꢀH), 7.95
3
1
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0
(
d, 2H, J = 6.0 Hz, 2'ꢀH and 6'ꢀH), 7.85 (s, 1H, 8ꢀNH), 7.56 (br s, 3H, 3'ꢀH, 4'ꢀH and 5'ꢀH), 7.16
d, 1H, J = 8.4 Hz, 6ꢀH), 6.91 (m, 5H, 3ꢀH, 3''ꢀH, 4''ꢀH, 5''ꢀH, 6''ꢀH), 4.23 (br s, 2H, 13ꢀCH2),
(
1
3
3.87 (br s, 5H, 14ꢀCH , 2''ꢀOCH ); C NMR (100 MHz, CDCl ) δ: 176.8 (Cꢀ4), 163.6 (Cꢀ2),
2
3
3
1
1
62.0 (Cꢀ6a), 157.2 (Cꢀ8), 156.3 (Cꢀ11), 153.2 (Cꢀ1a), 150.0 (Cꢀ1''), 148.1 (Cꢀ2''), 134.4 (Cꢀ1'),
32.2 (Cꢀ10), 131.0 (Cꢀ4'), 130.0 (Cꢀ5), 129.4 (Cꢀ3', 5'), 126.4 (Cꢀ2', 6'), 122.0 (Cꢀ5''), 121.1 (Cꢀ
4a), 120.0 (Cꢀ9), 114.9 (Cꢀ4''), 113.4 (Cꢀ10a), 112.4 (Cꢀ6''), 110.4 (Cꢀ3''), 109.1(Cꢀ6), 108.2 (Cꢀ
+
3
), 68.1(Cꢀ14), 56.1(2''ꢀOCH ), 39.6 (Cꢀ13); ESIꢀMS m/z: 483.7 [M+H] ; HRMS calcd. For
3
C H O N : 484.0120, found: 484. 0123.
28
23
6
2
BIOLOGICAL EVALUATION
Inhibition assays of AChE and BuChE
To assess the inhibition potency of compounds against cholinesterases (ChE), all target
3
4, 35
compounds were subjected to spectrophotometric method of Ellman et al.
The assay for
inhibition of AChE and BuChE was performed as described in the following procedure; in 96ꢀ
well plate, 10 ꢀL of test sample , 145 ꢀL phosphate buffer 200 mM (pH 7.7), 80 ꢀL of DTNB
(18.5 mg of DTNB dissolved in 10 mL phosphate buffer pH 7.7), and 10 ꢀL of enzyme (0.4
U/mL) was pre incubated for 5 minutes at 37 °C before adding substrate. Subsequently, by
addition of 15 ꢀL of 1 mM of ATCh or BTCh (according to the respective enzyme) the
enzymatic reaction was developed for 5 min at 37 °C. In order to obtain inhibition curve, at least
five different concentrations of the test compounds were assayed at 412 nm, each concentration
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in triplicate. Serving as a blank, an identical reaction solution without the inhibitor was processed
using the same protocol to yield 100% of AChE or BChE activities. IC values were determined
5
0
graphically by plotting the percentage enzyme activity (100% for the reference) versus logarithm
of test compound concentration.
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Kinetic characterization of AChE inhibition
For the determination of inhibition model and inhibition constant Ki, for compound 7s, kinetic
36
characterization of AChE inhibition was performed based on a reported method. The
LineweavereBurk plots were generated by using Graph pad prism version 5 at fixed amount of
AChE and varying amounts of the substrate ATCh (0.1ꢀ0.5 mM) and in the absence or presence
of different inhibitor concentrations of 7s (0.1, 0.4 and 1 ꢀM). The kinetic experiments were
performed similar to enzyme inhibition assay. The assay solution consists of 145 ꢀL of 200 mM
phosphate buffer (pH 7.7), 80 ꢀL of DTNB (18.5 mg of DTNB dissolved in 10 mL phosphate
buffer pH 7.7) and 10 ꢀL of 0.4 units/mL AChE. Three different concentrations of inhibitor 7s
0
was added to the assay solution and preꢀincubated for 5 min at 25 C. The enzymatic reaction
was initiated by the addition of ATCh (15 ꢀL) in different concentrations and the mixture was
again incubated for 5 min. Kinetic characterization of hydrolysis was monitored at 412 nm over
6
min. The parallel control experiments were carried out without inhibitor. The reꢀplots of the
slopes and intercepts of the double reciprocal plots against inhibitor concentrations gave the
inhibitor constants (Ki1 and Ki2, for the binding to free enzyme and enzyme substrate complex)
as the intercepts on the negative xꢀaxis. Data analysis was performed using Microsoft Excel.
Antioxidant Activity Assay
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The ABTS scavenging activity assay was performed to assess the compounds for their
3
7
antioxidant activity. ABTS stock solution (8mM) was prepared by dissolving 44mg ABTS in
•+
10mL Millipore water. ABTS radical cation (ABTS ) was generated by adding 3mM potassium
1
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persulfate to the ABTS stock solution in equal volumes and keeping it in the dark at room
•
+
temperature for 12ꢀ18 h. ABTS work solution was prepared freshly before the experiment in
1
:29 ratio with methanol. The 10 ꢀL of the test compounds were allowed to react with 290 ꢀL of
•
+
ABTS solution. The plate was placed in the wells of a 96 well plate microplate reader and the
absorbance was recorded 30 min after initial mixing at 734 nm. The plate was automatically
shaken prior to each reading. Trolox was used as a standard. A blank using phosphate buffer
instead of antioxidant and trolox was carried out in each assay. To obtain IC data each
5
0
experiment was performed in thrice, averaged and reported in the form of mean ± S.E.M.
ThT fluorescence assay
Thioflavin Tꢀbased flurometric assay (ThT) was performed to identify Aβ aggregates with high
22, 38
sensitivity.
The βꢀAmyloid1–42 (Aβ1–42) and Thioflavin T were purchased from sigma USA.
Then the Aβ1–42 was solubilized in dry DMSO to a final stock concentration of 200 ꢁM. The
stock solution was centrifuged at the speed of 12,000 rpm for 10 min and supernatant that
obtained was kept frozen at ꢀ80 °C until use for experiments. The solutions of 2.0 mM target
flavone analogs were prepared in DMSO for storage and diluted at in phosphateꢀbuffered saline
(PBS at pH 7.4) before use. The reaction mixture was prepared at fixed concentration of 2 ꢀL of
2
00 mM Aβ1−42 and 2 ꢀL of target flavone analogs at different concentrations (0.02 ꢀ 0.1mM) in
6 ꢀL of phosphateꢀbuffered saline (PBS at pH 7.4). After the incubation for 24 h at room
7
temperature, 80 ꢀL of 5 ꢀM ThT solution (in 50 mM glycineꢀNaOH at pH 8.5) was added to the
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reaction solution. The potential interaction between target flavone analogs with Aβ1–42 was
performed by LSꢀ55 spectrofluorimeter (PerkinElmer, USA), with 1.0 cm quartz cells.
o
Fluorescence emission spectra were recorded at 25 C with a wavelength range of 410–600 nm
0
1
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3
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and excited at 390nm. The bandwidth was fixed to 5.0 nm for both excitation and emission. The
independent experiments were performed thrice and each time identical spectra were obtained.
Cell Viability Assay
Human neuroblastoma (SKꢀNꢀSH) cells were obtained from National Centre for Cell Sciences
(NCCS), Pune, India. The SKꢀNꢀSH cells were cultured in minimal essential medium (MEM)
containing 0.5 mM Lꢀglutamine, 0.1 mM sodium pyruvate and 1 mM nonꢀessential amino acids
0
and 10% Fetal bovine serum (FBS) and maintained at 37 C in humidified incubator under 5%
CO / 95% air. When SKꢀNꢀSH cells’ reached 80% confluence, and then were used in the
2
following in vitro experiments. SKꢀNꢀSH cells’ were seeded in 96ꢀwell plates at the quantity of
6
0
.2 × 10 cells per well. The cells were treated with test compounds or galantamine in increasing
concentrations of 40, 80 and 120 ꢀM for 24 h in humidified CO incubator with 5% CO at 37 °C
2
2
for 24 h. At the end, 20 ꢀL of MTT at a final concentration of 5 mg/mL was supplemented and
incubated for an additional 4 h in humidified atmosphere followed by the addition of 200 ꢀL of
DMSO to the wells to dissolve the MTT formazan crystals. In a control experiment, cells were
grown in a same media without test compounds. Absorbance was recorded at 570 nm
immediately after the development of purple colour. The formazan generated in the control cells
was considered to represent 100% viability. Relative cell viability was evaluated according to the
39
quantity of MTT converted into insoluble formazan salt. Three selfꢀdetermining experiments
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were carried out and mean ± S.E.M was calculated and reported as (%) of cell viability vs
concentration (ꢀM).
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Protection against H O induced cell death in SKꢀNꢀSH cells
2
2
To measure neuroprotective effect of selected test compounds against H O induced oxidative
2
2
injury in SKꢀNꢀSH cells,
3
9
MTT assay was performed as described earlier. Thus, SKꢀNꢀSH cells were preꢀtreated with
different concentrations of test compounds 40, 80 and 120 ꢀM and galantamine for 3 h before
treatment with H O in a humidified CO incubator with 5% CO . To induce oxidative stress, 1.0
2
2
2
2
mM H O was added to the medium and incubated for 24 h in humidified atmosphere. Further
2
2
the cell viability was measured by MTT colorimetry as described above.
Fluorescence spectroscopy
4
0
All fluorescence measurements were carried out with Perkin Elmer LS55 fluorescence
spectrometer using 1.0 cm quartz cell to determine quantitatively potential interactions between
o
flavones analog and AChE. The fluorescence emission spectra were recorded at 25 C, in the
wavelength range from 300 to 500 nm, with an excitation wavelength of 285 nm. The slit width
of 5.0 nm is set for both excitation and emission. The scan speed was adjusted to100 nm/min.
The experiments were carried at the fixed AChE concentration (0.001 mM) and different
concentrations of flavone derivatives from 0.001 to 0.009 mM in 0.1M phosphate buffer solution
at the physiological pH of 7.4. The samples were allowed to equilibrate for 5 min after each
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addition. The binding constant were calculated using the maximum fluorescence value at 350 nm
for AChE. Three independent experiments were performed at each time and identical spectra
were recorded.
0
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Circular dichroism (CD)
Determination of conformational changes of AChE with different concentrations of flavone
derivatives was carried out on JASCO Jꢀ1500 CD spectrometer using a quartz cell with a path
length of 0.1 cm cell in a nitrogen atmosphere. Three scans were accumulated at a scan speed of
ꢀ1
1
00 nm min , with data being collected at room temperature for every 0.5 nm in the range of 190
4
1
to 260 nm. All the parameters were followed according to our previous procedures. For CD
studies, the final concentration of 0.001 mM AChE and 1ꢀL of test compounds with varied
concentrations (0.001, 0.005 and 0.009 mM) were used. After 5 min incubation, spectra was
recorded. Each experiment is repeated thrice and the result was averaged. The percentage of
relative contents of secondary structure; αꢀ helix, βꢀsheets and random coils were calculated
using CDNN 2.1, a web based software.
Autodock analysis
Since the experimental evidence showed flavone derivatives binding to AChE and also superior
at inhibiting the AChE, we were fascinated in exploratory the probable binding mechanism and
important interactions in atomic details in the binding site via molecular docking. The Autodock
program (version 4.2.3) was used to perform the molecular docking of flavone derivatives
4
2, 43
binding to AChE according to earlier studies.
To obtain the binding site and type of
interactions involved in the formation of Ligandꢀprotein complexes; in the present study,
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Lamarckian genetic algorithm (LGA) was implemented to calculate the possible conformation of
4
4
the drug that binds to the proteins.
Many reports discussed that, LGA is to be the best
performing method of docking in terms of its ability in finding the least energy and also showed
4
2
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the accuracy in the structure prediction; it also includes the ligand flexibility. The known
crystal structure of electric eel acetylcholinesterase (EeAChE, PDB Id: 1C2O) was retrieved
from the Brookhaven Protein Data Bank. The threeꢀdimensional structure (3D) of flavones
derivatives were built from its twoꢀdimensional structure (2D), and its geometry was optimized
using Discovery studio 3.5 software in the InsightꢀII/Builder program. In Auto Dock (4.2.3)
program water molecules and ions were removed (including ordered water molecules) and
hydrogen atoms were added to functional groups with the appropriate geometry within the
protein, in order to get proper protonation state of active site of the protein which was ionized as
required at physiological pH. To describe all binding sites and to have structural efforts, blind
docking was carried out using a grid based procedure. Now the output was saved as a PDBQT.
Grid box is set and output is saved as a .gpf file. The docking area was defined by a grid spacing
of 1.00 Å and dimensions of (126 X 126 X 126) with points along the x, y and z axes. The
docking parameters used were; number of genetic algorithm (GA) runs: 30, individual
population size: 150, maximum number of energy evaluations: 2500000, maximum number of
generations: 27000, maximum number of top individuals that automatically survive: 1, rate of
genetic mutation: 0.02, rate of crossover: 0.8, GA crossover mode: two points, output is selected
as Lamarckian GA (4.2) and file is saved as .dpf. Among 30 conformations the conformer with
the least free energy which is close to the experimental free energy was considered for the
4
5
analysis of binding site and binding mode.
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RESULTS AND DISCUSSION
Chemistry
The synthetic route to target flavone derivatives (7a–s) starting from commercially available
0
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resorcinol is shown in Scheme 1. Resacetophenone (1) was easily achieved by acetylation of
27, 28
readily available precursors, such as resorcinol, ZnCl and acetic acid,
which was on reaction
2
with benzoyl chloride afforded 1ꢀ(4ꢀbenzoyloxyꢀ2ꢀhydroxyphenyl)ꢀ3ꢀphenylꢀ1,3ꢀpropanedione
2
9, 30
(2).
Subsequently, treatment of 2 with Conc. H SO followed by 5% K CO gave the
2 4 2 3
31
desired 7ꢀhydroxyflavone backbone (3) at a yield of 67%. Next, 8ꢀformylꢀ7ꢀhydroxyflavone (4)
was accomplished by Duff formylation in an acidic medium between 7ꢀhydroxyflavone (3) and
3
2
hexamethylenetetramine (HMTA) under microwave irradiation at 300 W for 7 min. A series of
Nꢀsubstituted cyanoacetamide derivatives (6a–s) were readily prepared by treating different
2
1,
amines with equivalent amount of ethylcyanoacetate (5) as described in our recent publication.
2
2
Finally, the key intermediate 8ꢀformylꢀ7ꢀhydroxyflavone (4) was reacted with the
corresponding Nꢀsubstituted cyanoacetamides (6a–s) in the presence of Et N to provide the
3
33
1
target products (7a–s). All new compounds showed analytical and spectroscopic data (IR, H
1
3
NMR, C NMR and Mass) in good agreement with their structures, as detailed in the
experimental section and then submitted to biological evaluation.
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HO
OH
HO
OH
ii
O
OH
O
i
O
Resorcinol
1
O
2
O
1
1
1
1
1
1
1
1
1
1
2
2
2
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2
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4
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0
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7
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9
0
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CHO
HO
O
HO
O
O
iii
iv
O
4
3
R1
N
v
OEt
NC
HO
NC
R2
O
O
6
(aꢀs)
5
R1
N
1
2
O
O
R2
11
31
9
HN
CHO
21
41
51
1
0
8
R1
1
11
O
O
10a
vi
7
1a
O
2
N
6
a
61
NC
R2
+
4
a
3
O
6(aꢀs)
6
4
4
5
O
7
(aꢀs)
0
Scheme 1. Reagents and conditions: (i) ZnCl , GAA, 140ꢀ150 C; (ii) K CO , dry
acetone, benzoyl chloride, 20h, reflux (iii) a. H SO , 4h, 5 C; b. 5% K CO , con HCl (iv)
HMTA, GAA; MW, 300W, for 7min, 20% HCl, 200W for 4min; (v) corresponding
amine, EtOH (vi) Et N, EtOH.
2
2
3
0
2
4
2
3
3
H
R
1
R
2
N =
ꢀNHCH3
ꢀNHC H
ꢀNH(CH ) CH3
2
5
2 11
NH
N
N
N
b
a
c
d
e
g
f
OCH3
N
N
O
N
H
H
N
N
H
h
i
N
H
H
l
OCH
3
OCH3
OCH3
j
m
k
Cl
Br
Cl
O
Br
N
N
H
N
H
N
N
N
H
H
H
H
p
q
r
n
o
s
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Molecular Pharmaceutics
Page 28 of 64
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9
1
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1
1
1
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1
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2
2
2
2
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6
BIOLOGY
In vitro cholinesterase inhibition activity
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
To evaluate the potential of the target flavone derivatives 7a–s for the management of AD, their
cholinesterase inhibitory activities were evaluated by following method of Ellman et al with
minor changes using AChE from Electrophorus electricus
(
E
AChE) ans BuChE from Equine
3
4, 35
serum (BuChE),
with commercially available Galantamine, Tacrine, Donepezil and
Rivastigmine as reference standards. Table 1 illustrates the anticholinesterase potency of
compounds as expressed in IC values and selectivity index.
5
0
Table 1. An in vitro AChE and BuChE inhibitory activities and ABTS radical scavenging
capacities by the test compounds, and their selectivity index.
a
c
IC (
ꢀ
M) ± S.E.M.
IC (ꢀM) ± S.E.M.
50
5
0
Compounds
AChE
BuChE
Selectivity
ABTS radical
b
for AChE
scavenging activity
7
7
7
a
b
c
0.418 ± 0.019
0.338 ± 0.005
0.344 ± 0.013
>100
>100
>100
–
–
–
>50
>50
>50
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Page 29 of 64
Molecular Pharmaceutics
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d
e
0.337 ± 0.022
0.438 ± 0.042
0.305 ± 0.011
0.375 ± 0.023
0.469 ± 0.033
0.581 ± 0.028
0.273 ± 0.002
1.006 ± 0.075
0.694 ± 0.077
0.811 ± 0.097
0.286 ± 0.010
0.280 ± 0.003
0.406 ± 0.023
0.547 ± 0.04
0.291 ± 0.007
0.271 ± 0.012
80.0 ± 5.78
>100
237.38
>50
–
29.34 ± 1.2
>50
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7f
90.0 ± 9.89
NA
295.08
–
7
7
7
7
7
7
g
>50
h
i
NA
–
>50
35.0 ± 2.45
35.84 ± 3.46
NA
60.24
131.28
–
16.24 ± 1.59
26.40 ± 1.21
44.81 ± 0.77
>50
j
k
l
>100
–
7m
25.37 ± 3.46
87.43 ± 9.81
77.36 ± 6.65
57.30 ± 4.21
17.93 ± 1.29
57.93 ± 1.81
>100
31.28
305.69
276.28
141.13
32.77
221.95
–
>50
7
7
n
o
28.35 ± 0.75
38.46 ± 3.48
31.80 ± 3.55
42.34 ± 4.80
49.84 ±5.2
>50
7p
7
7
7
q
r
s
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