Design and Synthesis of Selective Acetylcholinesterase Inhibitors: Arylisoxazole-Phenylpiperazine Derivatives

Article abstract of DOI:10.1002/cbdv.201800433

In this work, a novel series of arylisoxazole-phenylpiperazines were designed, synthesized, and evaluated toward acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Our results revealed that [5-(2-chlorophenyl)-1,2-oxazol-3-yl](4-phenylpiperazin

Full text of DOI:10.1002/cbdv.201800433

Title: Design and synthesis of selective acetylcholinesterase inhibitors:
arylisoxazole-phenylpiperazine derivatives
Authors: Mina Saeedi, Dorrin Mohtadi-Haghighi, Seyedeh Sara
Mirfazli, Mohammad Mahdavi, Roshanak Hariri, Hania Lotfian,
Najmeh Edraki, Aida Iraji, Omidreza Firuzi, and Tahmineh
Akbarzadeh
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.201800433
Link to VoR: http://dx.doi.org/10.1002/cbdv.201800433

10.1002/cbdv.201800433
Chemistry & Biodiversity
Design and synthesis of selective acetylcholinesterase inhibitors: arylisoxazole-
phenylpiperazine derivatives
c
d
e
Mina Saeedi a,b, Dorrin Mohtadi-Haghighi , Seyedeh Sara Mirfazli , Mohammad Mahdavi ,
b
c
f
f
f
Roshanak Hariri , Hania Lotfian , Najmeh Edraki , Aida Iraji , Omidreza Firuzi , Tahmineh
Akbarzadehc,b*
a
Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
b
Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences,
Tehran, Iran
c
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
d
Department of Medicinal Chemistry, School of Pharmacy-International Campus, Iran
University of Medical Sciences, Tehran, Iran
e
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical
Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
f
Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical
Sciences, Shiraz, Iran
This paper is dedicated to the memory of our unique teacher in Chemistry and Medicinal
Chemistry, Professor Abbas Shafiee (1937-2016) .
In this work, a novel series of arylisoxazole-phenylpiperazines 5a-k were designed, synthesized,
and evaluated toward acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Our
results revealed that (5-(2-chlorophenyl)isoxazol-3-yl)(4-phenylpiperazin-1-yl)methanone (5c)
1
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10.1002/cbdv.201800433
Chemistry & Biodiversity
was the most potent AChE inhibitor with IC50 of 21.85 µM. It should be noted that most of
synthesized compounds showed no BChE inhibitory activity and (5-phenylisoxazol-3-yl)(4-
phenylpiperazin-1-yl)methanone (5a) was the most active anti-BChE derivative (IC50 = 51.66
µM). Also, kinetic studies for the AChE and BChE inhibitory activity of compounds 5c and 5a
confirmed that they have simultaneously bound to the catalytic site (CS) and peripheral anionic
site (PAS) of both AChE and BChE. Furthermore, docking study of compound 5c showed
desired interactions of that compound with amino acid residues located in the active and
peripheral anionic sites. Compound 5c was also evaluated for its BACE1 inhibitory activity and
demonstrated IC50 = 76.78 µM. Finally, neuroprotectivity of compound 5c on Aβ-treated
neurotoxicity in PC12 cells depicted low activity.
Keyword: Arylisoxazole, Beta-secretase (BACE1), Cholinesterase, Docking, Kinetic study,
Neuroprotection, Phenylpiperazine
Introduction
Alzheimer's disease (AD) is known as a controversial neurodegenerative disorder. It is
the most common type of dementia among elderly people in such a manner that their quality of
life is directly affected by the various drawbacks.[1] As AD has depicted enormous effects on the
patients’ mental ability due to gradual death of neuronal cells, a wide range of difficulties such as
failure of memory and thinking skills have been emerged. In this respect, it has imposed a huge
burden on the societies and health care systems.
The exact origin of AD is not recognized and several factors are involved in the
pathogenesis of the diseases.[2] Among them, tau protein aggregation[3] and extracellular plaque
deposits of the β-amyloid peptide (Aβ)[4] are two factors have been recently discussed and
2
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10.1002/cbdv.201800433
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investigated. However, treatment strategies and drug discovery have been accomplished via the
cholinergic hypothesis[5] which is associated with the decreased level of acetylcholine (ACh) in
the brain.
ACh is an essential neurotransmitter for the cognitive activities such as attention,
learning, memory, and motivation. There are two enzymes known as acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE) which catalyze the hydrolysis of ACh leading to the
reduction of synaptic availability of that substance in the brain. However, BChE plays a minor
role in regulating brain ACh as two enzymes differ in rate specificity, kinetics and activity in the
different parts of the brain.[6] Inhibition of AChE and BChE have been an important strategy for
the management of AD and AChE inhibitors have been found as the main treatment plan. Also,
non-cholinergic role of AChE and consequently AChE inhibitors (AChEIs) has attracted lots of
attention due to its participation in pro-aggregating activity of Aβ.[7] Amyloid fibril formation
can be achieved through various amino acid residues located in the PAS of AChE.[8]
Although AD has been characterized as a multifactorial disease, cholinesterase inhibitors
(ChEIs) are still the main target for the treatment of early and moderate stages of the disease
since FDA approved drugs including rivastigmine, galantamine, and donepezil are ChEIs[9] (Fig
1).
Heterocyclic compounds have been considered as the effective targets and versatile tools
in the enzymatic treatment of AD. In in this respect, design, synthesis and biological evaluation
of multi-target heterocycles have been the center of attention to develop potent and novel anti-
AD drugs[10-12] as well as diagnostic applications.[13,14] Focusing on the cholinesterase inhibitory
activity of heterocycles, design and synthesis of a wide range of compounds such as
coumarins,[15] 1,2,3-triazoles,[16-19] isoxazoles,[19,20] imidazoles,[21] pyrazoles,[22] and
3
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10.1002/cbdv.201800433
Chemistry & Biodiversity
quinazolines,[23] etc. have been developed. In this work, following anti-ChE activity of our
previously reported isoxazole derivatives A and B,[19,20] synthesis and biological evaluation of
novel isoxazoles connected to phenylpiperazine moiety was reported (Fig. 2).
Design of the target compounds is based on the potent inhibitory activity of isoxazoles
which can interact with the CAS and PAS of the AChE [19,20]. Also, the nitrogen atom from
phenylpiperazine group plays a key role in the inhibitory activity. It is the positive charge center
which is necessary in AChE inhibitory action to interact with the catalytic center of the AChE[24]
(Fig. 2).
Results and discussion
Chemistry
Synthesis of desired compounds 5a-k was carried out as depicted in Scheme 1. The
synthetic procedure was started from ethyl 2,4-dioxo-4-arylbutanoate 1 which was prepared
according to the literature.[19,20] The reaction of 1 and hydroxylamine hydrochloride in refluxing
ethanol afforded compounds 2. Hydrolysis of the ester group in the presence of potassium
hydroxide (KOH) in refluxing MeOH gave the corresponding carboxylic acid derivatives 3.
Finally, reaction of compound 3 and 1-phenylpiperazine 4 in the presence of 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDCI) and hydroxybenzotriazole (HOBt) in dry CH3CN at
room temperature yielded target arylisoxazole-phenylpiperazine derivatives 5.
AChE and BChE assay
In vitro AChE and BChE inhibitory activity of the synthesized compounds 5a-k was
evaluated using Ellman’s method comparing with donepezil and rivastigmine as the reference
4
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Chemistry & Biodiversity
drugs (Table 1).[25] The best anti-AChE activity was obtained by compound 5c possessing 2-
chlorophenylisoxazole group (IC50 = 21.85 µM). Changing the position of chlorine from ortho to
para position of aryl group (compound 5d) led to the reduction of AChEI activity (IC50 = 70.06
µM). However, compound 5e possessing two chlorine substituents showed lower activity (IC50 =
34.08 µM) than compound 5c but it was found to be stronger than compound 5d. Substitution of
different halogens such as fluorine and bromine showed versatile anti-AChE activity.
Introduction of fluorine into ortho position of aryl group connected to isoxazole moiety
(compound 5a) deleted anti-AChE activity (IC50 > 100 µM) and compound 5b possessing 4-
fluorophenyl group showed lower activity than compound 5c almost similar AChEI activity to its
counterpart, compound 5d having 4-chlorophenyl group. Increasing the size of halogen atom in
compound 5f possessing 4-bromophenyl led to lower activity comparing with other 4-
halosubstituted compounds (5b and 5d).
As can be seen in Table 1, introduction of electron-donating groups including methyl and
methoxy groups (compounds 5g and 5h) as well as removal of substituents from aryl group
(compound 5k) eliminated inhibitory activity (IC50 > 100 µM). In the case of derivatives
possessing electron-withdrawing groups (NO2, compounds 5i and 5j) the position of nitro group
was significant in inducing anti-AChE activity in such a manner that compound 5i having 3-
nitrophenyl group connected to isoxazole moiety depicted no activity (IC50 > 100 µM) whereas
compound 5j containing 4-nitrophenyl showed moderate inhibitory activity (IC50 = 44.14 µM).
It can be concluded that replacement of halogen substituents especially chlorine would be
suitable for inducing better AChEI activity in the series of arylisoxazole-phenylpiperazines 5a-k.
Finally, comparing our results with those reported in our previous studies on anti-AChE
activity of isoxazoles A and B[19,20] revealed that the synthesized compounds 5 were found to be
5
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lower active than 1,2,3-triazole-isoxazole hybrids A confirming that replacement of 1,2,3-
triazole by phenylpiperazine did not afford higher activity. However, the presence of
phenylpiperazine led to higher anti-AChE activity in comparison to compound B possessing
tryptamine moiety. Also, compound 5c similar to compound B possessed 2-chlorophenyl group
connected to isoxazole moiety.
In the case of BChEI activity, most of synthesized compounds were inactive toward
BChE. Among them, compounds 5a and 5k possessing 2-fluorophenyl and phenyl groups were
found to be moderate BChE inhibitors with IC50s of 51.66 and 72.27 µM, respectively. It should
be noted these compounds were not AChEIs. Also, BChEI activity of series of compounds 5a-k
was lower than compounds A and B.
Kinetic study
The most active anti-AChE and anti-BChE compounds 5c and 5a, respectively were
candidate for kinetic studies of enzymes inhibition using Lineweaver-Burk plots in the presence
(three concentrations) and absence of those inhibitors. As indicated in Fig. 3 and 4, graphical
analysis of the reciprocal Lineweaver-Burk plots depicted both increasing slopes and intercepts
at increasing concentration of both compounds 5c and 5a (Fig. 3 and 4, respectively). The
reciprocal plots confirmed mixed type of inhibition for both AChE and BChE suggesting that
compounds 5c and 5a were able to bind both CAS and PAS of AChE and BChE. As shown in
Fig. 3 and 4, the inhibition constants Ki were calculated for 5c and 5a from the secondary plots
of the slope versus the concentration of inhibitors (38.13 µM and 18.19 µM, respectively).
6
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BACE1 enzymatic assay
Beta-secretase 1 (BACE1) is an enzyme which catalyzes beta-site amyloid precursor
protein (APP) cleaving enzyme1.[31] In this respect, development of BACE1 inhibitors has been
emerged as the versatile therapeutic strategy. Compound 5c, the most potent anti-AChE
derivative was evaluated for its BACE1 inhibitory activity. The calculated IC50 indicated 76.78
µM ± 2.65 comparing with OM-99-2 (IC50 = 14.70 nM ±2.83) as the reference inhibitor.
Neuroprotective effect against Aβ-induced damage measured in PC12 cells
Neuroprotective effect of compound 5c against damage induced by Aβ25-35 was
investigated in PC12 cells by MTT assay.[31] This compound showed negligible activity up to the
concentration of 25 µM.
Docking study
1EVE was selected among 100 crystal structures of acetylcholinesterase complexed with
different ligands in PDB. The most promising anti-AChE inhibitor 5c was subjected to dock with
1EVE by using smina in Linux platform. The range of minimized affinity values of the poses of
ligand is −11.42 to −9.59 kcal/mol. The Interactions of the best-docked conformation of ligand
with the active site residues of acetylcholinesterase (1EVE) is depicted in Fig. 5. Target
compound strongly positioned in AChE peripheral anionic site (PAS) to form hydrogen bond
with Tyr121 and pi-anion interaction with Asp72 through isoxazole moiety. 2-Chlorophenyl ring
surrounded by Phe330, Trp84, and Phe331 near the wall of the active gorge is responsible for the
extra activities. Regarding to docking results and inhibitory activity of synthesized compounds,
7
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10.1002/cbdv.201800433
Chemistry & Biodiversity
aromatic ring of the molecule having lipophilic withdrawing group at ortho position possessed
an optimum fit into the CAS and PAS.[27,28]
As demonstrated in docking study there was no interaction between an electron-rich
moiety of compound 5c and Trp279 of the PAS which is important for the inhibition of AChE-
induced Aβ aggregation.[7,8,29]
Molecular docking study of compound 5c on BACE1 (2qp8) also demonstrated -8.55 to -
7.71 kcal/mol as the range of minimized affinity values of that derivative. The ligand formed
hydrogen bond with Thr292 through oxygen atom of carbonyl group. Also, Pi-alkyl interaction
with Pro131 and Pi-Pi interaction with Tyr132 residues were observed (Fig. 6).
Conclusion
In conclusion, a novel series of arylisoxazole-phenylpiperazines were designed,
synthesized, and evaluated as anti-AChE agents. Among them, (5-(2-chlorophenyl)isoxazol-3-
yl)(4-phenylpiperazin-1-yl)methanone (5c) showed the most potent AChEI activity (IC50 = 21.85
µM). However, they generally depicted no significant BChEI activity. Only, compounds 5a and
5k showed moderate to low activity (IC50s = 51.66 and 72.27 µM, respectively) which were not
active toward AChE. Kinetic as well as docking studies of compounds 5a and 5c confirmed their
dual inhibitory activity since they could simultaneously bind to amino acid residues located at
the CAS and PAS of both the AChE and BChE.
Experimental
1
Melting points were determined on a Kofler hot stage apparatus and are uncorrected. H
and 13C NMR spectra were recorded on a Bruker FT-500, using TMS as an internal standard. IR
8
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10.1002/cbdv.201800433
Chemistry & Biodiversity
spectra were obtained on a Nicolet Magna FTIR 550 spectrophotometer (KBr disks). MS were
recorded on an Agilent Technology (HP) mass spectrometer operating at an ionization potential
of 70 eV. Elemental analysis was performed on an Elementar Analysensystem GmbH VarioEL
CHNS mode. All chemicals were obtained from Merck and compounds 1, 2, and 3 were
synthesized according to our previous reports.[30]
General procedure for the synthesis of arylisoxazole-phenylpiperazines 5a-k
A mixture of compound 3 (1 mmol), EDCI (1.1 mmol), and HOBT (1 mmol) in dry
CH3CN (10 mL) was stirred for 1 h at room temperature. Then, 1-phenylpiperazine 4 (1 mmol)
was added to the reaction mixture and stirred for 24-48 h. After completion of reaction as
indicated by TLC, the solvent was evaporated under vacuum, dichloromethane (50 mL) was
added and the organic layer was washed with saturated NaHCO3 solution (2×50 mL) following
with brine (2×50 mL). The organic layer was separated, dried over Na2SO4, and concentrated
under vacuum to yield the crude product which was purified by recrystallization from petroleum
ether/ethyl acetate.
(5-(2-Fluorophenyl)isoxazol-3-yl)(4-phenylpiperazin-1-yl)methanone (5a)
1
Yield = 75%, mp 76-78 ºC. IR (KBr, cm-1): 2971, 2914, 284, 1625, 1597. H NMR (500 MHz,
CDCl3): 7.97 (t, J = 8.0 Hz, 1H, H4′), 7.47 (dd, J = 13.7, 8.0 Hz, 1H, H3′), 7.30 (t, J = 7.5 Hz,
2H, Ph), 7.26 (s, 1H, H4), 7.22 (t , J = 8.0 Hz, 1H, H5′), 7.03-7.04 (m, 1H, H6′), 6.95 (d, J = 7.5
Hz, 2H, Ph), 6.92 (t, J = 7.5 Hz, 1H, Ph), 4.08 (t, J = 5.0 Hz, 2H, piperazine), 3.99 (t, J = 5.0 Hz,
13
2H, piperazine), 3.30 (t, J = 5.0 Hz, 2H, piperazine), 3.26 (t, J = 5.0 Hz, 2H, piperazine). C
NMR (125 MHz, CDCl3): 166.5, 162.5 (d, JC-F = 250.0 Hz), 159.4, 159.3, 150.9, 132.1, 129.3,
127.5, 124.8, 122.8, 120.7, 116.8, 116.4 (d, JC-F = 21.2 Hz), 104.7 (d, JC-F = 11.2 Hz), 50.1, 49.5,
46.9, 42.6. MS (m/z, %): 351 (M+, 88), 334 (20), 228 (64), 200 (55), 119 (52), 159 (100), 132
9
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Chemistry & Biodiversity
(100), 104 (100), 91 (53), 56 (100), 42 (53).Anal. Calcd for C20H18FN3O2: C, 68.36; H, 5.16; N,
11.96. Found: C, 68.51; H, 5.30; N, 11.76.
(5-(4-Fluorophenyl)isoxazol-3-yl)(4-phenylpiperazin-1-yl)methanone (5b)
1
Yield = 85%, mp 155-157 ºC. IR (KBr, cm-1): 3138, 3018, 2917, 2837, 1621, 1503. H NMR
(500 MHz, CDCl3): 7.80 (dd, J = 8.5, 5.2 Hz, 2H, H2′, H6′) 7.30 (t, J = 7.5 Hz, 2H, Ph), 7.19 (t,
J = 8.5 Hz, 2H, H3′, H5′), 6.97-6.92 (m, 3H, Ph), 6.82 (s, 1H, H4), 3.99-4.12 (m, 4H,
piperazine), 3.26-3.31 (m, 4H, piperazine). 13C NMR (125 MHz, CDCl3): 169.4, 164.0 (d, JC-F
=
250.6 Hz), 159.2, 159.1, 150.2, 129.2, 128.0, 123.0, 121.6, 116.8, 116.3 (d, JC-F = 21.9 Hz),
100.6, 50.1, 49.5, 46.8, 42.5. MS (m/z, %): 351 (M+, 11), 228 (15), 190 (23), 159 (50), 132 (88),
104 (100), 77 (96), 56 (80). Anal. Calcd for C20H18FN3O2: C, 68.36; H, 5.16; N, 11.96. Found:
C, 68.10; H, 4.89; N, 12.18.
(5-(2-Chlorophenyl)isoxazol-3-yl)(4-phenylpiperazin-1-yl)methanone (5c)
1
Yield = 84%, mp 103-105 ºC. IR (KBr, cm-1): 2913, 2847, 1636, 1598. H NMR (500 MHz,
CDCl3): 7.96 (dd, J = 7.5, 3.5 Hz, 1H, H6′), 7.53 (dd, J = 7.5, 3.5 Hz, 1H, H3′), 7.43-7.41 (m,
2H, H4′, H5′), 7.31-7.28 (m, 3H, Ph, H4), 6.95 (d, J = 7.5 Hz, 2H, Ph), 6.92 (t, J = 7.5 Hz, 1H,
Ph), 4.10 (t, J = 5.0 Hz, 2H, piperazine), 3.99 (t, J = 5.0 Hz, 2H, piperazine), 3.30 (t, J = 5.0 Hz,
2H, piperazine), 3.27 (t, J = 5.0 Hz, 2H, piperazine). 13C NMR (125 MHz, CDCl3): 166.8, 159.3,
159.0, 150.9, 132.1, 131.3, 131.0, 129.4, 129.3, 127.2, 125.6, 120.7, 116.8, 105.6, 50.1, 49.5,
46.9, 42.6. MS (m/z, %): 369 ([M+2]+., 20), 367 (M+, 60), 228 (73), 200 (61), 159 (90), 132
(100), 104 (97), 77 (82), 56 (100), 42 (47).
Anal. Calcd for C20H18ClN3O2: C, 65.31; H, 4.93; N, 11.42. Found: C, 65.51; H, 5.24; N, 11.58.
(5-(4-Chlorophenyl)isoxazol-3-yl)(4-phenylpiperazin-1-yl)methanone (5d)
10
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Chemistry & Biodiversity
1
Yield = 62 %, mp 168-170 ºC. IR (KBr, cm-1): 3025, 2912, 2846, 1632, 1598. H NMR (500
MHz, CDCl3): 7.74 (d, J = 8.5 Hz, 2H, H3′, H5′), 7.47 (d, J = 8.5 Hz, 2H, H2′, H6′), 7.29 (t, J =
8.5 Hz, 2H, Ph), 6.95 (d, J= 8.0 Hz, 2H, Ph), 6.91 (t, J= 8.0 Hz, 1H, Ph), 6.85 (s, 1H, H4), 4.11
(t, J = 5.0 Hz, 2H, piperazine), 3.98 (t, J = 5.0 Hz, 2H, piperazine), 3.30 (t, J = 5.0 Hz, 2H,
13
piperazine), 3.26 (t, J = 5.0 Hz, 2H, piperazine). C NMR (125 MHz, CDCl3): 169.0, 159.2,
158.8, 150.8, 136.8, 129.5, 129.3, 127.2, 125.2, 120.7, 116.8, 101.2, 50.1, 49.5, 46.9, 42.7. MS
(m/z, %): 369 ([M+2]+., 9), 367 (M+, 27), 228 (29), 200 (25), 159 (58), 132 (97), 119 (35), 104
(74), 77 (70), 56 (100), 42 (53). Anal. Calcd for C20H18ClN3O2: C, 65.31; H, 4.93; N, 11.42.
Found: C, 65.11; H, 5.14; N, 11.24.
(5-(2,4-Dichlorophenyl)isoxazol-3-yl)(4-phenylpiperazin-1-yl)methanone (5e)
1
Yield = 85%, mp 110-112 ºC. IR (KBr, cm-1): 3127, 2907, 2811, 1632, 1603. H NMR (500
MHz, CDCl3): 7.90 (d, J = 8.5 Hz, 1H, H6′), 7.56 (d, J = 2.0 Hz, 1H, H3′), 7.40 (dd, J = 8.5, 2.0
Hz, 1H, H5′), 7.29 (t, J = 8.0 Hz, 2H, Ph), 7.28 (s, 1H, H4), 6.95 (dd, J = 8.0, 1.0, 2H, Ph), 6.92
(td, J = 8.0, 1.0 Hz, 1H, Ph), 4.10 (t, J = 5.2 Hz, 2H, piperazine), 3.98 (t, J = 5.2 Hz, 2H,
piperazine), 3.30 (t, J = 5.2 Hz, 2H, piperazine), 3.26 (t, J = 5.2 Hz, 2H, piperazine). 13C NMR
(125 MHz, CDCl3): 165.8, 159.3, 159.1, 150.8, 136.9, 132.7, 130.9, 130.1, 129.3, 127.8, 124.1,
120.7, 116.8, 105.8, 50.1, 49.4, 46.9, 42.7. MS (m/z, %): 405 ([M+4]+., 1.5), 403 ([M+2]+., 10),
401 (M+, 15), 228 (21), 200 (20), 173 (14), 159 (44), 132 (85), 104 (62), 77(62), 55 (100), 42
(41). Anal. Calcd for C20H17Cl2N3O2: C, 59.71; H, 4.26; N, 10.45. Found: C, 59.57; H, 4.41; N,
10.61.
(5-(4-Bromophenyl)isoxazol-3-yl)(4-phenylpiperazin-1-yl)methanone (5f)
1
Yield = 90%, mp 170-172 ºC. IR (KBr, cm-1): 3126, 2973, 2903, 2807, 1631, 1602. H NMR
(500 MHz, CDCl3): 7.67 (d, J = 8.0 Hz, 2H, H3′, H5′), 7.63 (d, J = 8.0 Hz, 2H, H2′, H6′), 7.29 (t,
11
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Chemistry & Biodiversity
J = 7.5 Hz, 2H, Ph), 6.95 (d, J = 7.5 Hz, 2H, Ph), 6.90 (t, J = 7.5 Hz, 1H, Ph), 6.87 (s, 1H, H4),
4.11 (t, J = 5.0 Hz, 2H, piperazine), 3.98 (t, J = 5.0 Hz, 2H, piperazine), 3.30 (t, J = 5.0 Hz, 2H,
piperazine), 3.26 (t, J = 5.0 Hz, 2H, piperazine). 13C NMR (125 MHz, CDCl3): 165.1, 159.2,
159.1, 150.8, 132.4, 129.3, 127.3, 125.6, 125.1, 120.7, 116.7, 101.3, 50.1, 49.4, 46.9, 42.6. MS
(m/z, %): 413 ([M+2]+., 14), 411 (M+, 14), 228 (29), 200 (22), 159 (55), 132 (92), 104 (72), 91
(40), 77 (69), 56 (100), 42 (45). Anal. Calcd for C20H18BrN3O2: C, 58.26; H, 4.40; N, 10.19.
Found: C, 58.42; H, 4.21; N, 10.33.
(4-Phenylpiperazin-1-yl)(5-(p-tolyl)isoxazol-3-yl)methanone (5g)
1
Yield = 65%, mp 110-112 ºC. IR (KBr, cm-1): 3030, 2921, 2833, 1649, 1602. H NMR (500
MHz, CDCl3): 7.83 (d, J = 7.5 Hz, 2H, H2′, H6′), 7.26-7.29 (m, 4H, H3′, H5′, Ph), 6.95-6.92 (m,
3H, Ph), 6.60 (s, 1H, H4), 3.88-3.85 (m, 4H, piperazine), 3.26-3.24 (m, 4H, piperazine). 13C
NMR (125 MHz, CDCl3): 164.5, 159.5, 158.4, 150.5, 132.1, 129.6, 129.3, 127.4, 123.9, 120.8,
116.8, 95.9, 50.1, 49.4, 46.1, 42.0, 21.7. MS (m/z, %): 347 (M+, 8), 322 (21), 161 (20), 132 (42),
119 (55), 104 (32), 91 (100), 77 (44), 56 (38). Anal. Calcd for C21H21N3O2: C, 72.60; H, 6.09; N,
12.10. Found: C, 72.84; H, 5.82; N, 12.38.
(5-(3-Methoxyphenyl)isoxazol-3-yl)(4-phenylpiperazin-1-yl)methanone (5h)
1
Yield = 70%, mp 110-112 ºC. IR (KBr, cm-1): 3035, 2966, 2906, 2809, 1630, 1574. H NMR
(500 MHz, CDCl3): 7.41-7.39 (m, 2H, H5′, H6′), 7.33 (s, 1H, H2′), 7.30 (t, J = 7.5 Hz, 2H, Ph),
7.01 (d, J = 7.5 Hz ,1H, H4′), 6.95 (d, J = 7.5 Hz, 2H, Ph), 6.92 (t, J = 7.5 Hz, 1H, Ph), 6.85 (s,
1H, H4), 4.11 (t, J = 5.0 Hz, 2H, piperazine), 3.98 (t, J = 5.0 Hz, 2H, piperazine), 3.30 (t, J = 5.0
13
Hz, 2H, piperazine), 3.26 (t, J = 5.0Hz, 2H, piperazine). C NMR (125MHz, CDCl3): 165.0,
159.4, 159.1, 154.9, 150.9, 130.3, 129.3, 128.2, 120.7, 118.5, 116.8, 116.7, 111.1, 101.1, 55.4,
50.1, 49.5, 46.9, 42.7. MS (m/z, %): 363 (M+, 12), 229 (17), 200 (16), 159 (38), 132 (78), 104
12
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Chemistry & Biodiversity
(81), 91 (46), 77 (100), 56 (92), 42 (18). Anal. Calcd for C21H21N3O3: C, 69.41; H, 5.82; N,
11.56. Found: C, 69.61; H, 5.56 N, 11.31.
5-(3-Nitrophenyl)isoxazol-3-yl)(4-phenylpiperazin-1-yl)methanone (5i)
1
Yield = 82%, mp 170-172 ºC. IR (KBr, cm-1): 3133, 2913, 2829, 1639, 1597, 1528, 1348. H
NMR (500 MHz, CDCl3): 8.66 (s, 1H, H2′), 8.33 (d, J = 8.0 Hz, 1H, H4′), 8.13 (d, J = 8.0
Hz,1H, H6′), 7.72 (t, J = 8.0 Hz,1H, H5′), 7.30 (t, J = 7.5 Hz, 2H, Ph), 7.04 (s, 1H, H4), 6.96 (d,
J = 7.5 Hz, 2H, Ph), 6.93( t, J = 7.5 Hz, 1H, Ph), 4.13 (t, J = 5.0 Hz, 2H, piperazine), 3.99 (t, J =
5.0 Hz, 2H, piperazine), 3.31 (t, J = 5.0 Hz, 2H, piperazine), 3.28 (t, J = 5.0 Hz, 2H, piperazine).
13C NMR (125 MHz, CDCl3): 167.8, 159.5, 158.8, 150.8, 148.8, 131.4, 130.4, 129.3, 128.3,
125.1, 121.0, 120.8, 116.8, 102.8, 50.1, 49.5, 47.0, 42.8. MS (m/z, %): 378 (M+, 6), 217 (18),
200 (12), 161 (33), 132 (56), 119 (24), 104 (100), 91 (38), 77 (69), 56 (69), 42 (35). Anal. Calcd
for C20H18N4O4: C, 63.48; H, 4.79; N, 14.81. Found: C, 63.22; H, 4.52; N, 15.12.
(5-(4-Nitrophenyl)isoxazol-3-yl)(4-phenylpiperazin-1-yl)methanone (5j)
Yield = 78%, mp 201-203 ºC. IR (KBr, cm-1): 3123, 3087, 2899, 2821, 1630, 1600, 1516, 1340.
1H NMR (500 MHz, CDCl3): 8.40 (d, J = 8.3 Hz, 2H, H3′, H5′), 8.22 (d, J = 8.3 Hz, 2H, H2′,
H6′), 7.61 (s, 1H, H4), 7.24 (t, J = 7.5 Hz, 2H, Ph), 6.98 (d, J = 7.5, 2H, Ph), 6.83 (t, J = 7.5 Hz,
1H, Ph), 3.83 (t, J = 5.0 Hz, 4H, piperazine), 3.79 (t, J = 5.0 Hz, 4H, piperazine). 13C NMR (125
MHz, CDCl3): 167.8, 159.5, 158.7, 150.8, 148.8, 132.0, 129.3, 126.8, 124.5, 120.8, 116.8, 103.7,
50.1, 49.5, 47.0, 42.8. MS (m/z, %): 378 (M+, 31), 228 (14), 200 (12), 159 (35), 132 (88), 104
(71), 91 (32), 77 (60), 56 (100), 42 (29). Anal. Calcd for C20H18N4O4: C, 63.48; H, 4.79; N,
14.81. Found: C, 63.60; H, 4.92; N, 14.92.
(5-Phenylisoxazol-3-yl)(4-phenylpiperazin-1-yl)methanone (5k)
13
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10.1002/cbdv.201800433
Chemistry & Biodiversity
1
Yield = 65 %, mp 118-120 ºC. IR (KBr, cm-1): 3060, 3016, 2916, 2840, 1623, 1597. H NMR
(500 MHz, CDCl3): 7.81 (d, J = 7.0 Hz, 2H, H2′, H6′), 7.48 -7.49 (m, 3H, H3′, H4′, H5′), 7.30
(t, J = 7.5 Hz, Hz, 2H, Ph), 6.96 (d, J = 7.5 Hz, 2H, Ph), 6.92 (t, J = 7.5 Hz, 1H, Ph), 6.86 (s,
1H, H4), 4.12 (t, J = 5.0 Hz, 2H, piperazine), 3.99 (t, J = 5.0 Hz, 2H, piperazine), 3.30 (t, J = 5.0
13
Hz, 2H, piperazine), 3.26 (t, J = 5.0 Hz, 2H, piperazine). C NMR (125 MHz, CDCl3): 168.1,
159.4, 158.6, 150.5, 130.7, 129.3, 129.1, 126.8, 125.9, 120.7, 116.8, 100.9, 50.1, 49.5, 46.9, 42.7.
MS (m/z, %): 333 (M+, 14), 228 (18), 200 (16), 172 (18), 159 (38), 105 (95), 91 (36), 77 (100),
56 (61), 42 (19). Anal. Calcd for C20H19N3O2: C, 72.05; H, 5.74; N, 12.60. Found: C, 72.22; H,
5.40; N, 12.41.
AChE and BChE inhibition assay
All chemicals required for the ChE assay were obtained from Sigma-Aldrich. The assay
was performed according to our previous reports[19,20] based on the Ellman’s method.[25]
Kinetic studies of AChE and BChE inhibition
For estimates of the inhibition model and inhibition constant Ki, reciprocal plots
of 1/V versus 1/[S] were obtained using different concentrations of the substrate. For this
purpose, all experiments were performed similar to enzyme inhibition assay.[18] The rate of
enzymatic reaction was obtained with different concentrations of inhibitor and in the absence of
inhibitor. For each experiment, reaction was initiated by adding substrate and progress curves
were recorded at 405 nm within 2 min. Next, double reciprocal plots (1/v vs. 1/[s]) were made
using the slopes of progress curves to obtain the type of inhibition. Slopes of these reciprocal
14
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10.1002/cbdv.201800433
Chemistry & Biodiversity
plots were then plotted against the concentration of compound, and Ki was determined as the
intercept on the negative x-axis.
BACE1 enzymatic assay
The BACE1 enzyme inhibition assay was achieved using a FRET (Forster resonance energy
transfer) kit, from Invitrogen (former Pan Vera corporation, Madison, WI) comparing with
OM99-2 as the reference inhibitor based on the literature [31]
.
Neuroprotection effect against Aβ-induced damage
The ability of compound 5c in protecting neuronal PC12 cells against damage induced by
Aβ25-35 was examined by the MTT assay as previously described [31]
.
Molecular modeling and docking internal validation
The 3D X-ray structure was taken from the protein data bank encoded 1EVE. For the
preparation of receptor, the hydrogen atoms were added, all water molecules were removed and
pH of the protein was adjusted to almost neutral (7.4). Molecular docking was performed using
smina which is a fork of AutoDock Vina in Linux platform [32].The autobox ligand option is used
for defining bounding box for docking. It generates a box with an 8 Å around the reference
ligand in the active site. In order to confirm the validity of the docking protocol, RMSD value
was measured between the crystal ligand pose and the generated nine poses of redocked crystal
ligand pose. The minimized affinity values for generated poses of redocked crystal ligand pose
were −11.62 to −9.81 and -8.55 to -7.71 kcal/mol for AChE (1EVE) and BACE1 (2qp8)
inhibitory, respectively.
15
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10.1002/cbdv.201800433
Chemistry & Biodiversity
Acknowledgement
This work was supported by grants from the Research Council of Tehran University of
Medical Sciences with project No. 95-04-33-32993. The authors also wish to thank the support
of the Vice-Chancellor for Research, Shiraz University of Medical Sciences.
Author Contribution Statement
Mina Saeedi contributed to the design of compounds and preparation of manuscript. Dorrin
Mohtadi-Haghighi and Mohammad Mahdavi contributed to the synthesis and characterization of
compounds. Seyedeh Sara Mirfazli performed the docking study. Roshanak Hariri, Hania lotfian,
and Aida Iraji, performed biological tests. Najmeh Edraki and Omidreza Firuzi supervised
biological tests and contributed to the preparation of the manuscript. Tahmineh Akbarzadeh
supervised all phases of the study.
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Chemistry & Biodiversity
Table 1. Anti-cholinesterase activity of arylisoxazole-phenylpiperazines 5a-k.a
Entry
Product 5
X
2-F
AChEI (IC50, µM)
>100
BChEI (IC50, µM)
1
2
5a
51.66±0.15
>100
5b
4-F
69.12 ± 0.39
21.85 ± 0.15
70.06 ± 0.76
34.08 ±0.50
84.55±1.60
>100
3
5c
2-Cl
>100
4
5d
4-Cl
>100
5
5e
2,4-diCl
4-Br
>100
6
5f
>100
7
5g
4-Me
3-MeO
3-NO2
4-NO2
H
>100
8
5h
>100
>100
9
5i
5j
>100
>100
10
11
12
13
44.14±0.75
>100
>100
5k
72.27±2.79
5.19±0.38
7.72±0.02
Donepezil
Rivastigmine
0.079 ±0.002
11.07±0.01
a Inhibitor concentration (mean ± SD of three experiments) required for 50% inactivation of AChE and BChE.
22
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10.1002/cbdv.201800433
Chemistry & Biodiversity
Fig. 1. Marketed cholinesterase inhibitor drugs used for management of Alzheimer’s disease.
23
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10.1002/cbdv.201800433
Chemistry & Biodiversity
Fig. 2. Design of new isoxazole derivatives based on the anti-AChE activity of compounds A
and B.
24
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10.1002/cbdv.201800433
Chemistry & Biodiversity
Scheme 1. Synthesis of arylisoxazole-phenylpiperazine derivatives 5a-k.
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10.1002/cbdv.201800433
Chemistry & Biodiversity
120
100
80
0
60
27.24 µM
108.99 µM
217.98 µM
40
20
0
‐0.008
‐0.006
‐0.004
‐0.002
0
0.002
0.004
0.006
0.008
0.01
‐20
‐40
‐60
‐80
1/[S], mM-1
(A)
14
12
10
8
y = 0.0434x + 1.655
6
4
2
0
‐100
‐50
0
50
100
150
200
250
[I], µM
(B)
Fig. 3. (A): Lineweavere-Burk plot for the inhibition of AChE by compound 5c at different
concentrations of acetylthiocholine (ATCh). (B): Steady-state inhibition constant (Ki) of
compound 5c.
26
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10.1002/cbdv.201800433
Chemistry & Biodiversity
120
100
80
0
28.49 µM
113.96 µM
60
40
20
227.92 µM
0
‐0.008
‐0.006
‐0.004
‐0.002
0
0.002
0.004
0.006
0.008
1/[S], mM-1
0.01
‐20
‐40
‐60
‐80
(A)
14
12
10
8
y = 0.0481x + 0.8748
6
4
2
0
‐50
0
50
100
150
200
250
‐2
[I], µM
(B)
Fig. 4. (A): Lineweavere-Burk plot for the inhibition of BChE by compound 5a at different
concentrations of acetylthiocholine (ATCh). (B): Steady-state inhibition constant (Ki) of
compound 5a.
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10.1002/cbdv.201800433
Chemistry & Biodiversity
Fig. 5. a) The RMSD value between crystal ligand pose and the best pose of redocked crystal
ligand pose is 0.901 b) 3D analysis of the interactions between AChE (1EVE) and compound 5c
b) 2D presentation of different interactions of compound 5c and 1EVE
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10.1002/cbdv.201800433
Chemistry & Biodiversity
Fig. 6. The proposed orientation and binding mode of compound 5c in the active site of BACE-1
(2qp8).
29
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