Design and Synthesis of Novel Arylisoxazole-Chromenone Carboxamides: Investigation of Biological Activities Associated with Alzheimer's Disease

Article abstract of DOI:10.1002/cbdv.201900746

A novel series of hybrid arylisoxazole-chromenone carboxamides were designed, synthesized, and evaluated for their cholinesterase (ChE) inhibitory activity based on the modified Ellman's method. Among synthesized compounds, 5-(3-nitrophenyl)-N-{4-[(2-oxo-

Full text of DOI:10.1002/cbdv.201900746

Title: Design and synthesis of novel arylisoxazole-chromenone
carboxamides: Investigation of biological activities associated
with Alzheimer’s disease
Authors: Mina Saeedi, Arezoo Rastegari, Roshanak Hariri, Seyedeh
Sara Mirfazli, Mohammad Mahdavi, Najmeh Edraki, Omidreza
Firuzi, and Tahmineh Akbarzadeh
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To be cited as: Chem. Biodiversity 10.1002/cbdv.201900746
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10.1002/cbdv.201900746
Chemistry & Biodiversity
Design and synthesis of novel arylisoxazole-chromenone carboxamides: Investigation of
biological activities associated with Alzheimer’s disease
Mina Saeedi,a,b Arezoo Rastegari,b,c Roshanak Hariri,c Seyedeh Sara Mirfazli,d Mohammad
Mahdavi,e Najmeh Edraki,f, Omidreza Firuzi,f Tahmineh Akbarzadeh*c,b
a Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences,
14155 Tehran, Iran
b Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences, 14155
Tehran, Iran
c Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences,
14155 Tehran, Iran
d Department of Medicinal Chemistry, School of Pharmacy-International Campus, Iran University
of Medical Sciences, 14665 Tehran, Iran
e
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical
Sciences Institute, Tehran University of Medical Sciences, 14155 Tehran, Iran
f
Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical
Sciences, 71348 Shiraz, Iran
This paper is dedicated to the memory of our unique teacher in Chemistry and Medicinal
Chemistry, Professor Abbas Shafiee (1937-2016).
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10.1002/cbdv.201900746
Chemistry & Biodiversity
Abstract: A novel series of hybrid arylisoxazole-chromenone carboxamides were designed,
synthesized, and evaluated for their cholinesterase (ChE) inhibitory activity based on the modified
Ellman's method. Among synthesized compounds, 5-(3-nitrophenyl)-N-(4-((2-oxo-2H-chromen-7-
yl)oxy)phenyl)isoxazole-3-carboxamide (11h) depicted the most acetylcholinesterase (AChE)
inhibitory activity (IC50 = 1.23 µM) and 5-(3-chlorophenyl)-N-(4-((2-oxo-2H-chromen-7-
yl)oxy)phenyl)isoxazole-3-carboxamide (11e) was found to be the most potent
butyrylcholinesterase (BChE) inhibitor (IC50 = 9.71 µM). Compound 11h was further investigated
for its BACE1 inhibitory activity as well as neuroprotectivity and metal chelating ability as
important factors involved in onset and progress of Alzheimer’s disease. It could inhibit BACE1 by
48.46% at 50 µM. Also, it showed 6.4% protection at 25 µM and satisfactory chelating ability toward
Zn2+, Fe2+, and Cu2+ ions. Also, docking studies of compounds 11h and 11e confirmed desired
interactions with those amino acid residues of the AChE and BChE, respectively.
Key words: Alzheimer's disease, BACE1, Cholinesterase, Chromenone, Coumarin, Docking,
Isoxazole, Metal chelating, Neuroprotectivity
Introduction
Alzheimer’s disease (AD) is a chronic neurodegenerative disorder, known as the most common
cause of dementia in elderly people leading to the various difficulties such as dependence,
disability, and mortality in patients.[1] It is estimated that 44 million people currently live with
dementia worldwide and is predicted to increase to more than triple by 2050.[2] In this regard, AD
has been recognized as a global public health priority by the World Health Organization due to the
versatile and controversial health and economic issues. AD is characterized by decline in memory,
language, problem solving and other cognitive skills that decrease the ability to perform daily
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activities due to destruction of neurons in the brain.[3] At the molecular level, AD is associated
with reduced synaptic levels of acetylcholine (ACh), abnormal deposits of β-amyloid peptide (Aβ),
and the presence of neurofibrillary tangles in the special areas of the brain such as the hippocampus
and cortex (cholinergic hypothesis) which are related to the memory and cognition.[4] Loss of pre-
cholinergic cells and decreased access to synaptic acetylcholine can lead to cognitive impairment
in the patients with AD.[5] AD is the first disease in which a broad erosion of the frontal cholinergic
system has been observed and cholinergic neurons may be lost up to 75% in the basal forehead
and septum area.[6-10] Therefore, one of the therapeutic approaches is to increase the level of
acetylcholine (ACh) in the brain by blocking the cholinesterase (ChE) enzymes responsible for the
ACh hydrolysis. Furthermore, several lines of recent evidence have suggested that when the level
of AChE gradually decreases the level of BChE significantly increases in the hippocampus and
temporal cortex of the brain of patients with AD.[11] Accordingly, the concurrent inhibition of both
AChE and BChE may improve AD signs and symptoms.[12] Due to the fact that AD is a multi-
factorial disease, design and synthesis of compounds affecting multiple pathological pathways are
highly in demand. A wide range of heterocyclic compounds possessing ChEI activity have been
documented in the literature.[13] Coumarine derivatives have been investigated for their
effectiveness on various pathological paths in AD. For example, various hydroxycoumarin
derivatives were synthesized and tested as AChE inhibitors and among them compound A (Fig. 1)
was found to be active toward AChE (IC50 = 4.5 μM).[14] Another study showed that compounds
possessing coumarin-amide moiety showed metal chelating abilities toward Cu2+ and Fe2+ ions as
well as neuroprotective properties on pc12 cell-line. In this respect, compound B depicted 84.9%
cell viability at the concentration of 50 μM (Fig. 1).[15] Also, we recently reported that compound
C (Fig. 1) demonstrated important selective BChEI activity (IC50 = 0.32 μM) comparing with
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AChEI activity (IC50 = 32.38 μM). Moreover, it could inhibit the BACE1 by 22%.[16] According
to our previous study, new derivatives of arylisoxazole-1,2,3-triazole showed moderate to good
anti-AChE activity. It was found that compound D (Fig.1) was the most potent AChE inhibitor
(IC50 = 0.90 μM), however, it inhibited BChE with IC50 value of 31.20 µM.[17] In addition, synthesis
and anti-ChE activity of arylisoxazole-N-phenylpiperazine hybrids were investigated and
compound E (Fig. 1) depicted good anti-AChE activity (IC50 = 21.85 μM).[18] Also, hybridization
of arylisoxazole-indole moieties (compound F) led to moderate ChEI activity and compound F
(Fig. 1) showed inhibitory activity toward AChE with IC50 value of 29.52 µM.[19] In continuation
of research on ChEI activity of arylisoxazole hybrids arylisoxazole-N-benzylpyridinium
derivatives were synthesized and among them compound G (Fig.1) was the most potent and
selective BChE inhibitor (IC50 = 0.32 μM); however it was less active toward AChE (IC50 = 16.82
μM).[20] It should be noted that the presence of the amide moiety played an important role in
inducing appropriate ChEI activity which has been considered in several studies (B-G). Herein, in
continuation of our studies on developing anti-ChE compounds (C-G) new arylisoxazole-
chromenone hybrids were designed, synthesized, and evaluated for biological tests involved in
onset and progress of AD (Fig. 1).
Results and discussion
Chemistry
Synthesis of required starting materials and target compounds 11a-m has been schematically
described in Schemes 1-3. Required precursors 3 were prepared by the reaction of diethyl oxalate
(1) and acetophenone derivatives 2 in the presence of sodium in dry ethanol. Then, reaction of the
later compounds and hydroxylamine hydrochloride (4) in ethanol under reflux conditions for 3 h
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gave related ethyl 5-arylisoxazole-3-carboxylate derivatives 5 which tolerated hydrolysis under
[21]
basic conditions leading to the formation of the corresponding carboxylic acid derivatives 6
(Scheme 1). On the other hand, 7-(4-aminophenoxy)-2H-chromen-2-one (10)[22] was prepared by
the reaction of 7-hydroxy-coumarin (7) and 1-fluoro-4-nitrobenzene (8) in DMF at 60-80 °C
following with the reduction of nitro group in the presence of Zn/NH4Cl in the mixture of
H2O/MeOH at room temperature (Scheme 2). Finally, the reaction of compounds 6 and 10 in the
presence of hydroxybenzotriazole (HOBT) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDCI) in dry acetonitrile at room temperature led to the formation of arylisoxazole- chromonenone
carboxamide hybrids 11a-m (Scheme 3).
Anticholinesterase activity
All synthesized compounds 11a-m were tested in vitro through the modified Ellman's method
comparing with donepezil as the reference drug. For each compound, 50% inhibitory concentration
(IC50) was determined and reported in Table 1. It is clear that electronic and steric properties of
substituents on the aryl ring connected to the isoxazole moiety as well as their position played
significant roles in the inhibitory activity.
In the case of AChEI activity, compound 11h possessing 3-nitroaryl group was found as the most
potent compound (IC50 = 1.23 µM). Interestingly, changing the position of nitro from 3- to 4-
(compound 11i) led to the deletion of activity (IC50 > 100 µM). It seems that the efficacy of strong
electron-withdrawing group (NO2) directly depended on the position of that group on the aryl ring.
Our data related to the AChEI activity also demonstrated that the presence of strong electron-
donating group (OMe) on the aryl ring completely removed anti-ChE activity in such a manner that
compounds 11j-l possessing OMe group either at 3- or 4- position did not show activity (IC50 > 100
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µM). It should be noted that the presence of moderate electron-donating group (Me) led to the
relatively weak inhibitory activity (IC50 = 71.55 µM). However, the absence of substituents on the
aryl ring (compound 11a) afforded moderate activity (IC50 = 44.94 µM). Another point comes back
to the derivatives 11b-g containing halogens. It was found that the presence of halogens (F, Cl, and
Br) led to good AChE activity; however, the activity was affected by their positions. Among
halogenated compounds 11b-g, compound 11e having 3-chloroaryl moiety showed the best
inhibitory activity (IC50 = 6.49 µM). Changing the position of Cl to 2- or 4- did not afford better
activity and IC50 values for compound 11d and 11f were calculated as 16.62 and 17.47 μM,
approximately 3 times weaker than compound 11e. The presence of bromine at 4- position of aryl
ring (compound 11g) led to the lower activity (IC50 = 20.14 µM) comparing with compound 11f at
the same position. Also, compound 11c having 4-fluoroaryl group showed similar activity (IC50 =
20.02 µM) to compound 11g. It seems that the efficacy of halogens can be considered as Cl>F~Br
for 4-halosubstituted derivatives. It is worth mentioning that compound 11b having F at 2- position
of aryl group (IC50 = 13.63 µM) was found to be more active than its analogue, compound 11c. In
addition, it demonstrated relatively higher activity than its counterpart, compound 11d.
In the case of BChEI activity, the best activity was related to compound 11e possessing 3-chloroaryl
group connected to isoxazole moiety (IC50 = 9.71 µM), however, the presence of chlorine at 2- and
4- position (compounds 11d and 11f) afforded no BChE activity (IC50 > 100 µM). Also, the same
result was obtained by compound 11g having 4-brominated moiety (IC50 > 100 µM). Unlike
compounds 11f and 11g, 4-fluorinated derivative 11c showed good BChEI activity (IC50 = 22.57
µM) but its analogue was not active and the calculated IC50 was more than 100 μM. Another point
was related to compound 11h (the most active AChE inhibitor) which was inactive towards BChE
and compound 11i was inversely active and IC50 value was calculated as 19.55 µM.
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It should be noted that in the case of electron-donating groups (Me and OMe) and lacking
substituents (H), no anti-BChE activity was observed. It can be concluded that the presence of
electron-donating group (Me and OMe) induced no ChEI activity neither anti-AChE nor anti-BChE
activity. The exception comes back to the compound 11m which depicted weak anti-AChE activity
(IC50 = 71.55 µM). On the contrary, strong electron-withdrawing group (NO2) and halogens (F, Cl,
and Br) were found to be beneficial for inducing ChE inhibitory activity, however, it should be
mentioned that most compounds were inactive toward BChE. Concisely, the presence of NO2 and
Cl groups at 3- position of aryl group connected to the isoxazole moiety played important roles in
the ChEI activity. Compound 11h selectively inhibited AChE (IC50 = 1.23 µM) while it showed no
activity against BChE and compound 11i lacking AChEI activity showed relatively good BChE
activity (IC50 = 19.55 µM).
Comparing our results from anti-AChE activity of arylisoxazole-coumarine hybrids with previous
studies on arylisoxazole hybrids (E-G) showed that compound 11h (IC50 = 1.23 µM) was stronger
than arylisoxazole-N-phenylpiperazine E (IC50 = 21.85 µM), arylisoxazole-indole F (29.52), and
arylisoxazole-N-pyridinium G (IC50 = 16.82) hybrids, but it was not as active as compound E,
arylisoxazole-1,2,3-triazole hybrid (IC50 = 0.90 µM).[18] BChEI activity of newly synthesized
compound 11 was comparable to those compounds D-F, however, they were much less active than
arylisoxazole-N-pyridinium hybrid G (IC50 = 0.32) and coumarin-N-pyridinium hybrid C (IC50 =
0.32).[20]
Kinetic Studies
Kinetic studies were performed to investigate the mechanism of inhibition by compounds 11h and 11e
against AChE and BChE, respectively. Graphical analysis of the reciprocal Lineweaver–Burk plot
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related to compound 11h described a competitive inhibition (Fig. 2) which prevented the substrate from
binding to the active site. Also, the Ki value was calculated using the secondary plot as Ki = 2.28 µM.
In the case of compound 11e, the same study demonstrated mixed type inhibition against BChE
confirming that it could simultaneously bind to the CAS and PAS of the enzyme. It should be noted
that Ki = 9.11 µM was calculated for the corresponding inhibition.
2.4. Metal chelating Activity
Compound 11h was tested for its metal chelating ability towards Fe2+, Cu2+, and Zn2+ ions (Fig. 4)
The UV spectrum of methanolic solution (20 µM) of that compound showed two characteristic
absorption peaks at 316.3 and 273.6 nm. After interaction of compound 11h with Zn2+ ions for 30
min, increase of absorption at the same wavelengths was observed. Interaction of compound 11h
with Fe2+ ions demonstrated three absorption peaks at 318.4, 312.0 and 273.6 nm indicating two red
and blue shifts which are associated with the formation of different complexes. Also, similar results
were obtained in the case of interaction of compound 11h with Cu2+ ions which showed three
absorption peaks at 320.5, 312.0, and 273.6 nm.
The stoichiometry of complex 11h-Cu2+ was also studied (Fig. 5). The concentration of the test
compound 11h was 20 μM and the final concentration of Cu2+ ranged from 0-40 μM with 4 μM
intervals at 273.6 nm. The plot was obtained by the corresponding absorption against the mole
fraction of Cu2+ to ligand 11h. According to the plot, the ratio 1:1 complexation ration of 11h-
Cu2+ can be seen at the fracture point of the plot with the mole fraction of 0.6.
BACE1 enzymatic assay
β-Secretase (BACE-1) inhibitors have been emerged as the important approach to the treatment of
AD due to the role of BACE1 in the proteolytic cleavage of the amyloid protein precursor (APP).[23]
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Accordingly, the BACE-1 inhibitory activity of the most potent AChE inhibitor, compound 11h was
evaluated via a fluorescence resonance emission transfer (FRET) method. The used kit consisted of
BACE1 enzyme and APP peptide based substrate (Rh-EVNLDAEFK- quencher) comparing with
OM99-2 (IC50 = 0.014 μM) as the reference drug. It was found that compound 11h induced moderate
inhibitory activity; 48.46% and 31.66% inhibition at the concentrations of 50 and 10 µM,
respectively.
Neuroprotective effect against Aβ-Induced Damage measured in PC12 Cells
Neuroprotective effect of compound 11h against damage induced by Aβ25–35 was investigated in
PC12 cells by MTT assay.[24] This compound showed 6.4% protection at the concentration of 25
µM comparing with rutin with 13.4% protection at the same concentration.
Molecular docking study
The observed AChE and BChE inhibitory activity of the target arylisoxazole-chromonenone was
confirmed through active site-docking simulation of compounds 11h and 11 j into 1EVE and 11e,
into 1P0P active sites (Figs. 6-9). The corresponding protein crystal structures for AChE (1EVE)
and BChE (1P0P) were retrieved from the protein data bank and co-crystal ligand donepezil was
re-docked using smina in Linux platform.[25] The range of minimized affinity values of the poses
of ligands 11h, 11j and 11e were −12.82 to −11.73 , −12.50 to −11.10 and −11.1 to −10.4
kcal/mol, respectively.
Binding interactions mode of compounds
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The interactions of the best-docked conformation of the most potent AChE inhibitor (11h) with
the active site residues was presented in Fig. 6. According to the 1EVE simulated inhibition
docking evaluation, compound 11h constructed two hydrogen bonds with the active site. The
nitrogen of the isoxazole ring formed a hydrogen bond with Gly123 residue and the oxygen of
nitroaryl moiety also formed a hydrogen bond with His440 residue of the 1EVE which confirmed
the efficacy of the isoxazole and 3-nitroaryl moieties for inducing the desired bioactivity.
Furthermore, on the other side of the molecule, chromenone ring formed pi-anion, pi-cation and
pi-pi stacking interactions with Trp272, Asp72 and Tyr334, respectively which fixed compound
11h in the active site. Hydrophobic interactions well stabilized the compound–1EVE complex into
the active site (Fig. 6). This cooperative binding might be beneficial to increase the inhibitory
activity of the compound–1EVE complex to describe the promising efficacy of compound 11h
against AChE.[26]
The proposed binding mode of compound 11j is illustrated in Fig. 7. The oxygen of methoxy group
formed a hydrogen bond with Gly118 residue of the enzyme, however, it was not apparently
beneficial to induce desired AChE inhibitory activity comparing with compound 11h. In another
docking study (Fig. 8.), both compounds 11j and 11h as well as native ligand were superimposed
into the active site. It was found that the orientation of 3-nitroaryl moiety of compound 11h
resembled that of the native ligand, donepezil. The proposed binding was thus very similar to that
of donepezil. The interactions were dominated in the region of His440 and Trp84 amino acid
residues due to pronouncing the existence of hydrogen bonds and pi-pi stacking interaction at the
catalytic anionic site. However, 3-methoyaryl moiety of compound 11j represented perpendicular
orientation into the active site which misled proper interactions.
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Fig. 9. represented binding interactions of compound 11e over 1P0P. Targeted compound
produced conventional H-bonding interaction with the backbone of Pro285 via NH of amide
moiety. Furthermore, the aryl group was stabilized at specific pocket by side chain of Ala277 via
pi-alkyl interaction. Also, formation of hydrophobic interactions through π-anion and π-π stack
interactions of chromenone moiety of compound 11e with Gly115 as well as Glu197 and His438
from different position were observed.[27] The aromatic linker between arylisoxazole and
chromenone moieties was completely fixed through π-alkyl and π-π stack interactions with Ala328
and Tyr332 residues in the protein’s active site.
Conclusions
In conclusion, different and novel arylisoxazole-chromonenone carboxamides were designed,
synthesized, and evaluated for their ChEI activity. Among them, compounds 11h (IC50 = 1.23 μM)
and 11e (IC50 = 9.71 μM) respectively possessing 3-nitroaryl and 3-chloroaryl groups connected to
the arylisoxazole moiety, were found to be the most potent AChE and BChE inhibitors. Furthermore,
evaluation of BACE1 inhibitory activity, neuroprotectivity, and metal chelating ability of compound
11h demonstrated good results which make the compound worthy to be considered for further
biological activities associated with AD.
Experimental
All chemicals and reagents were purchased from Merck and Aldrich. Melting points were
determined using Kofler hot stage apparatus and are uncorrected. The IR spectra were obtained on
a Nicolet Magna FTIR 550 spectrometer (potassium bromide disks). NMR spectra were recorded
on a Bruker 500 spectrometer and chemical shifts were expressed as δ (ppm) with tetramethylsilane
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as internal standard. Mass spectra were determined on an Agilent Technology (HP) mass
spectrometer operating at an ionization potential of 70 eV.
Chemistry
Synthesis of 2,4-dioxo-4-aryl butanoates 3
Initially, sodium (1 mol) was added to dry ethanol (to 250 ml) and the solution was stirred in an ice
bath while a mixture of diethyl oxalate (1) (1 mol) and the acetophenone derivative 2 (1 mol) was
added dropwise to the solution. The yellow pasty material was left at room temperature for 15 h and
after that, it was stirred at 80 °C in water bath for 90 min. The crude product was treated with diluted
sulfuric acid to adjust pH = 2. Then, the oily product was extracted using chloroform and washed
using saturated NaHCO3 solution and brine. After drying the organic phase using sodium sulfate,
the solvent was removed under vacuum to obtain 2,4-dioxo-4-aryl butanoate 3 which was
recrystallized from chloroform and hexane.[21]
4.1.2. Synthesis of ethyl 5-arylisoxazole-3-carboxylates 5
The mixture of hydroxylamine hydrochloride (4) (0.23 mol) and 2,4-dioxo-4-aryl butanoate
derivative 3 (0.08 mol) was heated at reflux in ethanol (220 ml) for 3 h. After the completion of the
reaction (checking by TLC), half of the solvent was removed and the mixture was poured onto the
ice and the precipitate was extracted by dichloromethane and washed with water, NaOH solution
(4%), and brine, respectively. The organic phase was dried and the solvent was removed under
vacuum to afford ethyl 5-arylisoxazole-3-carboxylate 5.[21]
4.1.3. Synthesis of ethyl 5-arylisoxazole 3-carboxylic acids 6
Ethyl 5-arylisoxazole-3-carboxylate 5 (1 mmol) was hydrolyzed in the presence of KOH (3 mmol)
in methanol (15 ml) under reflux conditions for 3 h. After the completion of the reaction (checking
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by TLC), the mixture was poured into the water and ice, and the concentrated HCl was added drop
wise to form a white precipitated product. The mixture was stirred at room temperature for 20 min
and the precipitate was filtered off resulting in the corresponding acid 6.
4.1.4. Synthesis of 7-(4-nitrophenoxy)-2H-chromen-2-one (9)
The mixture of 7-hydroxy-coumarin (7) (10 mmol) and potassium carbonate (K2CO3, 10 mmol) in
DMF (10 ml) was stirred at 60-80 °C for 1 h. Then, 1-fluoro-4-nitrobenzene (8) (10 mmol) was
added and the reaction mixture was stirred at 60-80 °C for 24 h. After the completion of the reaction
(checking by TLC), the mixture was added to ice and water, the precipitated product was filtered
off, and washed thoroughly with water; resulting in 7-(4-nitrophenoxy)-2H-chromen-2-one (9).
4.1.5. Synthesis of 7-(4-aminophenoxy)-2H-chromen-2-one (10)
The mixture of compound 9 (5 mmol) and ammonium chloride (30 mmol) in the mixture of methanol
and water (80 ml, 7:1) was stirred at room temperature for 5 min. Then, zinc powder (100 mmol)
was gradually added within 10 min and the reaction was stirred for 40 min. The reaction was checked
by TLC and at the end of reaction, the mixture was filtered using sintered glass funnel and celite,
and the filtered solution was added onto the ice. The resulting precipitated product was filtered off
to give 7-(4-aminophenoxy)-2H-chromen-2-one (10).
4.1.6. General procedure for the preparation of chromonen-3-aryl isoxazole-5-carboxamide
derivatives 11 a-m
The mixture of 5-arylisoxazole-3- carboxylic acid derivative 6 (1 mmol), HOBT (1 mmol) and EDCI
(1 mmol) was stirred in dry acetonitrile at room temperature for 1 h, then compound 10 (1 mmol)
was added to the mixture and the reaction was continued overnight. After the completion of the
reaction (checking by TLC), the solvent was evaporated under vacuum and the crude was extracted
using chloroform and washed with water and solutions of NaHCO3 10%, NaCl and citric acid,
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respectively. After drying the organic phase over sodium sulfate, the solvent was removed and the
solid product was recrystallized from ethyl acetate and n-hexane.
N-(4-((2-Oxo-2H-chromen-7-yl)oxy)phenyl)-5-phenylisoxazole-3-carboxamide (11a)
Yield: 67.1%; M.p. = 215-225 °C; IR (KBr): 3330, 3150, 1732, 1672, 1657, 1541 cm-1; 1H NMR
(DMSO-d6, 500 MHz): δ = 10.92 (s, 1H, NH), 8.04 (d, J = 9.5 Hz, 1H, H4), 7.98 (d, J = 6.4 Hz,
2H, H2", H6"), 7.90 (d, J = 8.8 Hz, 1H, H3’, H5’), 7.71 (d, J = 8.6 Hz, 1H, H5), 7.58-7.55 (m, 3H,
H3”, H4”, H5”), 7.52 (s, 1H, isoxazole), 7.19 (d, J = 8.8 Hz, 1H, H2’, H6’), 6.96 (dd, J = 8.6, 2.2
13
Hz, 1H, H6), 6.93 (d, J = 2.0 Hz, H8), 6.37 (d, J = 9.5 Hz, 1H, H3) ppm. C NMR (DMSO-d6,
125 MHz): δ = 170.6, 160.6, 160.0, 159.9, 157.2, 155.1, 151.0, 144.1, 135.0, 131.0, 130.1, 129.4,
126.3, 125.9, 122.4, 120.5, 114.2, 114.0, 113.8, 104.5, 100.2 ppm. MS: m/z (%) = 424 [M]+ (100),
306 (95), 279 (79), 251 (58), 172 (20), 105 (53), 89 (41), 77 (32). Anal. calcd. for C25H16N2O5:
C, 70.75; H, 3.80; N, 6.60. Found: C, 70.51; H, 3.62; N, 6.83.
5-(2-Fluorophenyl)-N-(4-((2-oxo-2H-chromen-7-yl)oxy)phenyl)isoxazole-3-carboxamide (11b)
Yield: 53.9%; M.p. = 240-244 °C; IR (KBr): 3315, 3184, 1726, 1666, 1620, 1535, 1495 cm-1; 1H
NMR (DMSO-d6, 500 MHz): δ = 10.91 (s, 1H, NH), 8.04-8.02 (m, 2H, H4, H6”), 7.90 (d, J = 8.4
Hz, 1H, H3’, H5’), 7.71 (d, J = 8.4 Hz, 1H, H5), 7.64-7.63 (m, 1H, H4”), 7.48 (t, J = 9.5 Hz, 1H,
H5”), 7.45-7.42 (m, 1H, H3”), 7.30 (s, 1H, isoxazole), 7.19 (d, J = 8.4 Hz, 1H, H2’, H6’), 6.96 (d,
13
J = 8.4 Hz, 1H, H6), 6.92 (s, 1H, H8), 6.36 (d, J = 9.4 Hz, 1H, H3) ppm. C NMR (DMSO-d6,
125 MHz): δ = 165.0, 160.6, 160.0, 159.8, 158.5 (d, JC-F = 251.2 Hz), 156.9, 155.1, 151.1, 144.1,
135.0, 133.3, 130.1, 128.0, 125.5, 122.5, 120.5, 116.8 (d, JC-F = 20.9 Hz), 114.4 (d, JC-F = 11.8
Hz), 114.2, 114.0, 113.8, 104.6, 103.0 (d, JC-F = 8.8 Hz) ppm. MS: m/z (%) = 442 [M]+ (85), 401
(17), 279 (23), 251 (25), 190 (20), 135 (100), 123 (26), 89 (21), 77 (14). Anal. calcd. for
C25H15FN2O5: C, 67.87; H, 3.42; N, 6.33. Found: C, 67.95; H, 3.12; N, 6.60.
14
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5-(4-Fluorophenyl)-N-(4-((2-oxo-2H-chromen-7-yl)oxy)phenyl)isoxazole-3-carboxamide (11c)
Yield: 44.5%; M.p.> 250 °C; IR (KBr): 3369, 3149, 1732, 1673, 1617, 1537, 1507 cm-1; 1H NMR
(DMSO-d6, 500 MHz): δ = 10.92 (s, 1H, NH), 8.06-8.03 (m, 2H, H4, H5), 7.89 (d, J = 8.7, 2H,
H3’, H5’), 7.73-7.68 (t, J = 8.7 Hz, 2H, H2”, H6”), 7.51 (s, 1H, isoxazole), 7.43 (t, J = 8.7 Hz, 2H,
H3”, H5”), 7.19 (d, J = 8.7 Hz, 2H, H2’, H6’), 6.96 (d, J = 8.6 Hz, 1H, H6), 6.93 (s, 1H, H8), 6.37
(d, J = 9.6 Hz, 1H, H3) ppm. 13C NMR (DMSO-d6, 125 MHz): δ = 170.0, 166.1 (d, JC-F = 250.0
Hz), 160.6, 160.4, 160.0, 157.1, 155.1, 151.0, 144.1, 136.8, 135.0, 131.8 (d, JC-F = 12.5 Hz), 130.1,
128.5 (d, JC-F = 11.2 Hz), 122.4, 120.5, 116.6 (d, JC-F = 22.4 Hz), 114.1 (d, JC-F = 17.2 Hz), 113.8,
104.5, 100.4 (d, JC-F = 10.1 Hz) ppm. MS: m/z (%) = 442 [M]+ (10), 401 (26), 191 (13), 135 (100),
121 (13), 92 (17), 77 (17), 55 (21), 41 (25). Anal. calcd. for C25H15FN2O5: C, 67.87; H, 3.42; N,
6.33. Found: C, 67.54; H, 3.65; N, 6.11.
5-(2-Chlorophenyl)-N-(4-((2-oxo-2H-chromen-7-yl)oxy)phenyl)isoxazole-3-carboxamide (11d)
Yield: 63.6%; M.p. = 210-215 °C; IR (KBr): 3330, 3094, 1732, 1676, 1675, 1544, 1480 cm-1; 1H
NMR (CDCl3, 500 MHz): δ = 8.62 (s, 1H, NH), 7.95 (m, 1H, H4”), 7.75-7.73 (m, 3H, H3”, H6”,
H5), 7.65 (d, J = 9.5 Hz, 1H, H4), 7.55 (t, J = 4.5 Hz, 1H, H5”), 7.46-7.43 (t, J = 4.5 Hz, 2H, H3’,
H5’), 7.41 (s, 1H, isoxazole), 7.12 (d, J = 8.0 Hz, 1H, H2’, H6’), 6.91 (d, J = 9.0 Hz, 1H, H6), 6.89
(s, 1H, H8), 6.30 (d, J = 9.5 Hz, 1H, H3) ppm. 13C NMR (DMSO-d6, 125 MHz): δ = 167,5, 160,6,
160.0, 159.6, 157.0, 155.0, 151.1, 144.1, 135.0, 132.4, 131.1, 131.0, 130.1, 130.0, 128.1, 125.0,
122.5, 120.5, 114.2, 114.0, 113.8, 104.6, 104.1 ppm. MS: m/z (%) = 460 [M+2]+ (31), 458 [M]+
(93), 279 (100), 251 (77), 222 (18), 206 (20), 139 (90), 111 (35), 89 (55), 75 (23), 63 (37), 50 (17).
Anal. calcd. for C25H15ClN2O5: C, 65.44; H, 3.29; N, 6.11. Found: C, 65.27; H, 3.48; N, 5.97.
5-(3-Chlorophenyl)-N-(4-((2-oxo-2H-chromen-7-yl)oxy)phenyl)isoxazole-3-carboxamide (11e)
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1
Yield: 44%; M.p.= 242-244°C; IR (KBr): 3340, 3078, 1724, 1671, 1620, 1544, 1508 cm-1; H
NMR (DMSO-d6, 500 MHz): δ = 10.83 (s, 1H, NH), 8.02 (m, 2H, H5”, H5), 7.93-7.89 (m, 3H,
H6”, H3’, H5’), 7.70 (d, J = 8.6 Hz, 1H, H4”), 7.60-7.59 (m, 3H, H4, H2”, Isoxazole), 7.18 (d, J
= 8.9 Hz, 2H, H2’, H6’), 6.95 (dd, J = 2.0, 8.5 Hz 1H, H6), 6.91 (d, J = 2.2 Hz, 1H, H8), 6.35 (d,
J = 9.5 Hz, 1H, H3) ppm. 13C NMR (DMSO-d6, 125 MHz): δ = 169.5, 161.0, 160.4, 160.4, 157.4,
155.5, 151.5, 144.5, 135.4, 134.6, 131.7, 131.1, 130.5, 128.6, 126.0, 124.9, 122.9, 120.9, 114.6,
114.5, 114.2, 105.0, 101.8 (d, JC-F = 8.8 Hz) ppm. MS: m/z (%) = 460 [M+2]+ (33), 458 [M]+ (100),
279 (27), 252 (39), 206 (21), 139 (37), 111 (20), 89 (38). Anal. calcd. for C25H15ClN2O5: C, 65.44;
H, 3.29; N, 6.11. Found: C, 65.62; H, 3.08; N, 6.36.
5-(4-Chlorophenyl)-N-(4-((2-oxo-2H-chromen-7-yl)oxy)phenyl)isoxazole-3-carboxamide (11f)
1
Yield: 36.5%; M.p. >250 °C; IR (KBr): 3327, 3093, 1726, 1669, 1611, 1538 cm-1; H NMR
(CDCl3, 500 MHz): δ = 8.58 (s, 1H, NH), 7.77 (d, J = 8.0 Hz, 2H, H3”, H5”), 7.73 (d, J = 8.0 Hz,
2H, H2”, H6”), 7.65 (d, J = 9.5 Hz, 1H, H4), 7.50 (d, J = 8.0 Hz, 2H, H3’, H5’), 7.42 (d, J = 8.5
Hz, 1H, H5), 7.13 (d, J = 8.0 Hz, 2H, H2’, H6’), 7.05 (s, 1H, isoxazole), 6.92 (d, J = 8.5 Hz, 1H,
13
H6), 6.89 (s, 1H, H8), 6.31 (d, J = 9.5 Hz, 1H, H3) ppm. C NMR (DMSO-d6, 125 MHz): δ =
169.5, 160.8, 160.0, 159.0, 157.2, 155.1, 151.2, 144.2, 135.8, 135.1, 132.7, 130.2, 129.5, 127.7,
122.4, 120.6, 114.2, 114.0, 113.9, 104.5, 101.1 ppm. MS: m/z (%) = 460 [M+2]+ (19), 458 [M]+
(57), 279 (100), 251 (54), 139 (52), 111 (24), 89 (40), 75 (14), 63 (20). Anal. calcd. for
C25H15ClN2O5: C, 65.44; H, 3.29; N, 6.11. Found: C, 65.15; H, 3.13; N, 5.80.
5-(4-Bromophenyl)-N-(4-((2-oxo-2H-chromen-7-yl)oxy)phenyl)isoxazole-3-carboxamide (11g)
1
Yield: 42%; M.p. = 235-240 °C; IR (KBr): 3332, 1728, 1671, 1615, 1535, 1502 cm-1; H NMR
(CDCl3, 500 MHz): δ = 8.58 (s, 1H, NH), 7.74-7.65 (m, 6H, H3”, H5”, H2”, H6”, H3’, H5’), 7.56
(d, J = 8.0 Hz, 1H, H5), 7.42 (d, J = 8.5 Hz, 1H, H4), 7.12 (d, J = 8.5 Hz, 2H, H2’, H6’), 7.07 (s,
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1H, isoxazole), 6.92 (d, J = 8.0 Hz, 1H, H6), 6.89 (s, 1H, H8), 6.30 (d, J = 8.5 Hz, 1H, H3) ppm.
13C NMR (DMSO-d6, 125 MHz): δ = 169.9, 162.4, 160.3, 159.4, 155.8, 150.5, 149.6, 143.2, 135.2,
131.1, 128.3, 126.0, 123.7, 123.0, 121.7, 120.9, 116.5, 114.4, 113.9, 106.1, 101.5 ppm. MS: m/z
(%) = 504 [M+2]+ (80), 502 [M]+ (80), 279 (100), 251 (76), 222 (15), 234 (12), 196 (10), 185 (53),
155 (25), 133 (14), 115 (10), 89 (53), 76 (21), 63 (25), 50 (10). Anal. calcd. for C25H15BrN2O5: C,
59.66; H, 3.00; N, 5.57. Found: C, 59.47; H, 3.24; N, 5.73.
5-(3-Nitrophenyl)-N-(4-((2-oxo-2H-chromen-7-yl)oxy)phenyl)isoxazole-3-carboxamide (11h)
Yield: 55.6%; M.p. = 230-243 °C; IR (KBr): 3327, 3090, 1711, 1662, 1614, 1522, 1554 and 1354
cm-1; 1H NMR (CDCl3, 500 MHz): δ = 8.70 (s, 1H, NH), 8.58 (S, 1H, H2”), 8.36 (d, J = 8.0 Hz,
1H, H6”), 8.15 (d, J = 8.0 Hz, 1H, H4”), 7.75-7.34 (m, 3H, H5”, H5’, H3’), 7.65 (d, J = 9.5 Hz,
1H, H4), 7.42 (d, J = 8.0 Hz, 1H, H5), 7.22 (s, 1H, isoxazole), 7.13 (d, J = 8.0 Hz, ٢H, H2’, H6’),
6.92 (d, J = 8.0 Hz, 1H, H6), 6.89 (s, 1H, H8), 6.30 (d, J = 9.5 Hz, 1H, H3) ppm. 13C NMR (DMSO-
d6, 125 MHz): δ = 169.2, 161.0, 160.5, 160.0, 157.3, 155.5, 151.5, 148.8, 144.5, 135.4, 132.3,
131.5, 130.5, 127.8, 125.7, 122.9, 121.8, 120.8, 115.3, 114.4, 114.2, 104.9, 103.2 ppm. MS: m/z
(%) = 469 [M]+ (1), 279 (100), 251 (37), 222 (10), 150 (53), 104 (14), 89 (20), 76 (17), 63 (19),
50 (13). Anal. calcd. for C25H15N3O7: C, 63.97; H, 3.22; N, 8.95. Found: C, 64.22; H, 3.01; N,
8.73.
5-(4-Nitrophenyl)-N-(4-((2-oxo-2H-chromen-7-yl)oxy)phenyl)isoxazole-3-carboxamide (11i)
Yield: 32.1%; M.p. >250 °C; IR (KBr): 3729, 3327, 1726, 1675, 1617, 1529, 1578 and 1383 cm-
1; 1H NMR (CDCl3, 500 MHz): δ = 8.59 (s, 1H, NH), 8.40 (d, J = 8.0 Hz, 2H, H3”, H5”), 8.04 (d,
J = 8.0 Hz, 2H, H2”, H6”), 7.75 (d, J = 8.0 Hz, 2H, H3’, H5’), 7.66 (d, J = 9.0 Hz, 1H, H4), 7.42-
7.40 (m, 2H, H5, isoxazole), 7.14 (d, J = 8.0 Hz, 2H, H2’, H6’), 6.93 (d, J = 8.0 Hz, 1H, H6), 6.88
(s, 1H, H8), 6.31 (d, J = 9.0 Hz, 1H, H3) ppm. 13C NMR (DMSO-d6, 125 MHz): δ = 169.3, 161.8,
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160.4, 160.0, 154.9, 151.2, 150.7, 146.8, 143.4, 132.6, 131.6, 129.1, 125.1, 124.1, 122.9, 120.8,
116.0, 114.0, 113.5, 107.0, 101.7 ppm. MS: m/z (%) = 469 [M]+ (100), 279 (73), 251 (59), 217
(17), 150 (27), 104 (10), 89 (25), 63 (10). Anal. calcd. for C25H15N3O7: C, 63.97; H, 3.22; N, 8.95.
Found: C, 63.72; H, 3.54; N, 9.16.
5-(3-Methoxyphenyl)-N-(4-((2-oxo-2H-chromen-7-yl)oxy)phenyl)isoxazole-3-carboxamide (11j)
1
Yield: 60%; M.p.= 225-229 °C; IR (KBr): 3348, 3120, 1735, 1681, 1602, 1541, 1492 cm-1; H
NMR (CDCl3, 500 MHz): δ = 8.61 (s, 1H, NH), 7.74 (d, J = 8.5 Hz, 2H, H3’, H5’), 7.65 (d, J =
9.5 Hz, 1H, H4), 7.43-7.41 (m, 3H, H6”, H5”, H5), 7.36 (s, 1H, isoxazole), 7.13 (s, 1H, H2”), 7.12
(s, 1H, H4”), 7.04 (d, J = 8.5 Hz, 2H, H2’, H6’), 6.91 (d, J = 9.0 Hz, 1H, H6), 6.89 (s, 1H, H8),
6.30 (d, J = 9.5 Hz, 1H, H3), 3.90 (s, 3H, OCH3) ppm. 13C NMR (DMSO-d6, 125 MHz): δ = 169.0,
165.2, 161.0, 160.3, 157.7, 155.5, 151.6, 148.6, 144.6, 135.3, 131.2, 130.6, 129.8, 123.0, 120.9,
118.5, 117.4, 115.2, 114.6, 114.2, 111.4, 105.0, 100.8, 55.9 ppm. MS: m/z (%) = 454 [M]+ (100),
279 (47), 252 (56), 222 (11), 202 (23), 135 (55), 107 (20), 89 (47), 77 (23), 63 (24). Anal. calcd.
for C26H18N2O6: C, 68.72; H, 3.99; N, 6.16. Found: C, 68.53; H, 3.71; N, 6.35.
5-(4-Methoxyphenyl)-N-(4-((2-oxo-2H-chromen-7-yl)oxy)phenyl)isoxazole-3-carboxamide (11k)
Yield: 44.4%; M.p. >250 °C; IR (KBr): 3411, 2925, 1733, 1672, 1617, 1537, 1507 cm-1; 1H NMR
(DMSO-d6, 500 MHz): δ = 10.85 (s, 1H, NH), 8.02 (d, 2H, J = 10.0 Hz, H4), 7.90-7.87 (m, 4H,
H3’, H5’, H2”, H6”), 7.71 (d, J = 8.5 Hz, 1H, H5), 7.31 (s, 1H, isoxazole), 7.18 (d, J = 6 Hz, 2H,
H3”, H5”), 7.11 (d, J = 7.0 Hz, 2H, H2’, H6’), 6.96 (d, J = 8.5 Hz, 1H, H6), 6.91 (s, 1H, H8), 6.36
(d, J = 10.0 Hz, 1H, H3), 3.83 (s, 3H, OCH3) ppm. 13C NMR (DMSO-d6, 125 MHz): δ = 168.4,
162.1, 161.7, 161.6, 158.7, 155.3, 153.2, 151.5, 144.6, 130.5, 128.1, 125.8, 122.9, 120.9, 119.2,
115.3, 114.2, 113.5, 112.8, 109.6, 98.9, 55.9 ppm. MS: m/z (%) = 454 [M]+ (100), 279 (25), 252
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(27), 135 (50), 89 (23), 77 (10). Anal. calcd. for C26H18N2O6: C, 68.72; H, 3.99; N, 6.16. Found:
C, 68.94; H, 3.67; N, 6.21.
5-(3,4-Dimethoxyphenyl)-N-(4-((2-oxo-2H-chromen-7-yl)oxy)phenyl)isoxazole-3-carboxamide
(11l)
Yield: 54.4%; M.p.>250 °C; IR (KBr): 3322, 2931, 1731, 1672, 1617, 1538, 1511 cm-1; 1H NMR
(DMSO-d6, 500 MHz): δ = 10.86 (s, 1H, NH), 8.02 (d, J = 9.6 Hz, 1H, H4), 7.88 (d, J = 8.5 Hz,
2H, H3’, H5’), 7.70 (d, J = 8.6 Hz, 1H, H6”), 7.52 (d, J = 8.5 Hz, 1H, H5), 7.48 (s, 1H, isoxazole),
7.40 (s, 1H, H2”), 7.18 (d, J = 8.5 Hz, 2H, H2’, H6’), 7.11 (d, J = 8.6 Hz, 1H, H5”), 6.95 (d, J =
8.5 Hz, 1H, H6), 6.90 (s, 1H, H8), 6.35 (d, J = 9.6 Hz, 1H, H3), 3.86 (s, 3H, OCH3), 3.82 (s, 3H,
OCH3) ppm. 13C NMR (DMSO-d6, 125 MHz): δ = 171.3, 161.0, 160.2, 157.8, 155.5, 151.5, 151.4,
149.7, 149.6, 144.5, 135.4, 130.5, 123.1, 123.0, 120.9, 119.4, 114.5, 114.2, 112.5, 109.7, 104.9,
103.4, 99.3, 56.3, 56.1 ppm. MS: m/z (%) = 484 [M]+ (100), 279 (43), 251 (43), 205 (21), 176 (15),
165 (83), 133 (15), 89 (43), 77 (17), 63 (21). Anal. calcd. for C27H20N2O7: C, 66.94; H, 4.16; N,
5.78. Found: C, 66.75; H, 3.98; N, 5.54.
N-(4-((2-Oxo-2H-chromen-7-yl)oxy)phenyl)-5-(p-tolyl)isoxazole-3-carboxamide (11m)
Yield: 55%; M.p. >250 °C; IR (KBr): 3416, 3340, 1729, 1673, 1617, 1536, 1506 cm-1; 1H NMR
(DMSO-d6, 500 MHz): δ = 10.90 (s, 1H, NH), 8.01-7.70 (m, 6H, H2”, H6”, H3’, H5’, H4, H5),
7.38 (m, 3H, H3”, H5”, isoxazole), 7.18 (d, J = 8.4 Hz, 2H, H2’, H6’), 6.92 (m, 2H, H6, H8), 6.36
(d, J = 9.5 Hz, 1H, H3), 2.37 (s, 3H, CH3) ppm. 13C NMR (DMSO-d6, 125 MHz): δ = 170.8, 164.9,
160.2, 159.6, 155.4, 151.4, 149.5, 144.4, 130.4, 129.3, 128.5, 127.9, 126.2, 123.0, 122.9, 120.9,
117.9, 114.2, 114.1, 106.0, 99.8, 22.6 ppm. MS: m/z (%) = 438 [M]+ (100), 279 (41), 252 (58),
222 (11), 186 (24), 133 (11), 119 (75), 89 (47), 63 (17). Anal. calcd. for C26H18N2O5: C, 71.23; H,
4.14; N, 6.39. Found: C, 71.52; H, 4.33; N, 6.12.
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Biological Activity
Anticholinesterase activity
The in vitro anticholinesterase activity of all synthesized compounds 11a-m was assayed using
modified Ellman's method, exactly according to our previous study.[16, 28]
Kinetic Studies
The kinetic study for the inhibition of AChE and BChE by compounds 11h and 11e was carried out
according to the Ellman's method used for the inhibition assay[28] using four different concentrations
of inhibitors. For the kinetic study of AChE, compound 11h was used at the concentrations of 0,
0.43, 3.19, and 6.39 µM. The Lineweaver–Burk reciprocal plot was constructed by plotting 1/V
against 1/[S] at variable concentrations of the substrate acetylthiocholine (187.5, 750, 1500, 3000
µM). The inhibition constant Ki was achieved by the plot of slopes versus the corresponding
concentrations of the compound 11h. The same method was performed for the kinetic study of BChE
using four different concentrations of compound 11e (0, 5.45, 21.79, and 43.59 µM) and
butyrylthiocholine (187.5, 750, 1500, 3000 µM).
Metal chelating Activity
To study the metal binding ability of compound 11h, a mixture of methanolic solutions of that
compound (1 mL) and the related metal (1 mL) with the same final concentrations (20 µM) in a
quartz cuvette was incubated at room temperature for 30 min. Then, the absorption spectra were
recorded in the range of 200–600 nm. The stoichiometry of complex 11h-Cu2+ was also studied
using the molar ratio method.[29] The concentration of compound 11h was 20 μM and the final
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Chemistry & Biodiversity
concentration of Cu2+ ranged from 0-40 μM with 4 μM intervals at 273.6 nm. The plot was obtained
by the corresponding absorption versus mole fraction of Cu2+ to ligand 11h.
BACE1 enzymatic assay
A FRET-based BACE1 enzyme assay kit was used to evaluate the inhibitory activity of compound
11h against BACE1. The kit was purchased from Invitrogen (former Pan Vera Corporation,
Madison, WI) and evaluation procedure was conducted according to the manufacturer’s
instructions.[23]
Neuroprotection effect against Aβ-induced damage
The neuroprotective effect of compound 11h in protecting neuronal PC12 cells against damage
induced by Aβ25–35 was examined according to our previous report.[24]
Molecular docking study
A docking study was performed using Auto Dock Tools (version 1.5.6) and the pdb structures of
1EVE (AChE) and 1P0I (BChE) were taken from the Brookhaven protein database
(http://www.rcsb.org). The 3D structures of the selected compounds were created by MarvinSketch
5.8.3, 2012, ChemAxon (http://www.chemaxon.com) and by Auto Dock Tools converted to pdbqt
coordinates. Moreover, the pdbqt coordinates of the enzymes were prepared using the Auto Dock
Tools. Before preparation of the pdbqt form of the enzymes, the water molecules and inhibitors were
removed. Then, by using Auto Dock Tools, polar hydrogen atoms were added, Kollman charges
were assigned, and the obtained protein structures were used as input files for the AUTOGRID
program. In AUTOGRID for each atom type in the inhibitors, maps were calculated with 0.375 Å
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spacing between grid points, and the center of the grid box was placed at x=2.023, y=63.295, and
z=67.062 for AChE and x=137.985, y=122.725, and z=38.78 for BChE. The dimensions of the
active site box were set at 40×40×40 Å for AChE and 55×55×55 Å for BChE. Flexible ligand
dockings were accomplished for the selected compounds. Each docked system was carried out by
50 runs of the AUTODOCK search by the Lamarckian genetic algorithm (LGA). The best position
of each compound was selected for analyzing the interactions between AChE or BChE and the
inhibitor, and the results were visualized using Discovery Studio 4.0 Client.
Acknowledgments
This work was supported by grants from the Research Council of Tehran University of Medical
Sciences with project No. 98-3-104-46061.
Author Contribution Statement
Mina Saeedi contributed to the design of compounds and preparation of manuscript. Arezoo
Rastegari and Mohammad Mahdavi contributed to the synthesis and characterization of
compounds. Seyedeh Sara Mirfazli performed the docking study. Roshanak Hariri performed the
biological tests. Najmeh Edraki and Omidreza Firuzi supervised biological tests. Tahmineh
Akbarzadeh supervised all phases of the study.
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Chemistry & Biodiversity
Fig. 1. The structure of ChE inhibitors A-G and designed compounds. Compounds A-G contain
coumarin, arylisoxazole, and amide moieties and designed compounds are new coumarin-
arylisoxazole hybrids.
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10.1002/cbdv.201900746
Chemistry & Biodiversity
Scheme 1. Synthesis of 5-arylisoxazole 3-carboxylic acids 6.
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10.1002/cbdv.201900746
Chemistry & Biodiversity
Scheme 2. Synthesis of 7-(4-aminophenoxy)-2H-chromen-2-one (10).
29
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