Synthesis and anticholinesterase activity of new substituted benzo[d]oxazole-based derivatives

Article abstract of DOI:10.1111/cbdd.12902

A series of novel benzo[d]oxazole derivatives (6a–n) have been synthesized and biologically evaluated as potential inhibitors of acetylcholinesterases (AChE) and butyrylcholinesterase (BChE). The chemical structures of all final compounds were confirmed by spectroscopic methods. In vitro studies showed that most of the synthesized compounds are potent acetylcholinesterase and butyrylcholinesterase inhibitors. Among them, compounds 6a and 6j strongly inhibited AChE and BChE activities with IC₅₀ values of 1.03–1.35 and 6.6–8.1?μm, respectively. Docking studies also provided the binding modes of action and identified hydrophobic pi forces as the main interaction.

Full text of DOI:10.1111/cbdd.12902

Received Date : 18-Jul-2016
Revised Date : 23-Oct-2016
Accepted Date : 25-Oct-2016
Article type : Research Article
Synthesis and anti-cholinesterase activity of new substituted benzo[d]oxazole-based
derivatives
Behjat Pouramiri1, Setareh Moghimi2, Mohammad Mahdavi2, Hamid Nadri3, Alireza Moradi3,
Esmat Tavakolinejad-Kermani1, Loghman Firoozpour2, Ali Asadipour4, Alireza Foroumadi4,5
1 Department of Chemistry, Faculty of Science, Shahid Bahonar University of Kerman, Kerman,
Iran
2
Drug Design and Development Research Center, Tehran University of Medical Sciences,
Tehran, Iran
3
Department of Medicinal Chemistry, Faculty of Pharmacy, Shahid Sadoughi University of
Medical Sciences, Yazd, Iran
4 Department of Medicinal Chemistry, Faculty of Pharmacy and Neuroscience Research Center,
Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran
5
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences
Research Center, Tehran University of Medical Sciences, Tehran, Iran
 Corresponding author. Tel.: +98-21-66419413; Fax: +98-21-66461178.
E-mail address: aforoumadi@yahoo.com (A Foroumadi).
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differences between this version and the Version of Record. Please cite this article as doi:
10.1111/cbdd.12902
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Keywords: Acetylcholinesterase, Alzheimer’s disease, Benzo[d]oxazol, Docking study
Running title: Synthesis and anticholinesterases of benzoxazoles
Abstract: A series of novel benzo[d]oxazole derivatives (6a–n) have been synthesized and
biologically evaluated as potential inhibitors of acetylcholinesterases (AChE) and
butyrylcholinesterase (BChE). The chemical structures of all final compounds were confirmed
by spectroscopic methods. In vitro studies showed that most of the synthesized compounds are
potent acetylcholinesterase and butyrylcholinesterase inhibitors. Among them, compounds 6a
and 6j strongly inhibited AChE and BChE activities with IC50 values of 1.03-1.35 µM and 6.6-
8.1 µM, respectively. Docking studies also provided the binding modes of action, identified
hydrophobic pi forces as the main interaction.
Introduction
Alzheimer's disease (AD) is one of the critical health problems in the elderly population of the
world. AD is an age-related and complex neurodegenerative disorder of the brain characterized
by dementia, cognitive impairment, and memory loss (1, 2). Based on World Alzheimer Report
in 2010 (3), the estimated number of people who are afflicted by this disease is more than 30
million.
The pathogenesis of Alzheimer's disease is not completely clear; the typical pathological marks
are amyloid-β (Aβ) deposits, neurofibrillary tangles (NFT), oxidative stress, and decreased levels
of acetylcholine (ACh) in the brain (4). Acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE) with 65% homology in their structure, are responsible enzymes for the metabolic
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hydrolysis of ACh at the cholinergic synapses. In same condition, meaning temperature and pH,
ACh is more effectively hydrolyzed by AChE compared to BChE (5). The increased levels of
ACh is observed as a result of an effective inhibitory activity against these enzymes which led to
attenuated levels of behavioral and cognitive symptoms in AD patients (6). Most of the FDA
approved drugs are based on the inhibition of AChE. Cholinesterase inhibitor drugs (ChEI)
involving donepezil, rivastigmine and galantamine have been proposed as the most important
therapeutic option for the treatment of Alzheimer's disease (7). The main focuses of these agents
are their simultaneous action as disease-modifying agents and symptomatic therapy (8).
However, toxicity (9) and severe side effects in clinical studies diminished their effectiveness
(10). So, BChE should be alternatively pursued as a remarkable target due to its hydrolyzing role
in advanced stages of the disease and fewer side effects of selective BChEIs (11). The design of
new ligands targeting different molecular abnormalities, has directed many research projects to
discover effective and novel ChEIs agents (12-14) with improved pharmacokinetics properties.
Oxazoles are important scaffold in heterocyclic compounds, which are extensively found in
diverse pharmacologically active substances and in naturally occurring compounds. Furthermore,
benzo[d]oxazoles play an important role as a key building block in β-adrenergic receptor
antagonists (15), anti-tumor (16), antihyperglycemic (17), antibacterial (18), anti-HIV (19) and
anti-inflammatory agents (20). In this case, oxazole derivatives have raised considerable
attention of medicinal chemists, hence, a large number of investigations on their synthesis and
biological activities have been reported during recent years (21–25).
The presence of heterocyclic rings in AD drugs promoted medicinal chemists to examine
different skeletons in novel hybrid molecules. In addition to being in newly discovered anti-
cholinesterase agents, benzofuran presented a venerable pharmacophore in molecules with
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inhibitory activity against the formation of Aβ fibril. Furthermore, hydrophobic and π-π
interactions from aromatic residue on the entrance and inside of AChE gorge, add much interests
to find novel benzofuran-phenyl analogues (26-29). In this regard, we rationally combined
benzoxazole and phenyl ring system, bearing cyclic and acyclic nitrogen-containing moieties
which are logically essential for effective enzyme-compound interactions (Figure 1). Following
our interests in designing novel cholinesterase inhibitors (30), we synthesized a novel series of
N-((4-(benzo[d]oxazol-2-yl)phenyl)methyl)-amine derivatives as potential acetylcholinesterase
and butyrylcholinesterase inhibitors. Further, molecular docking studies interpreted the
inhibitory activity of the enzyme.
Methods and Materials
General chemistry
Melting points are uncorrected and were determined with a Kofler hot-stage apparatus (Reichert,
1
Vienna, Austria). H and 13C NMR spectra were recorded using Bruker 500 spectrometers.
Chemical shifts (δ) are reported in ppm relative to TMS as internal standard. Coupling constant
(J) values are presented in Hz and spin multiplicities are given as s (singlet), d (doublet), t
(triplet) and m (multiplet). The IR spectra were obtained on a Nicolet Magna FT-IR 550
spectrophotometer (potassium bromide disks). Mass spectra were determined on an Agilent
Technology (HP) mass spectrometer operating at an ionization potential of 70 eV. The elemental
analysis for C, H, N was carried out with an Elementar Analysen system GmbH. All reagents
and solvents used in this study were commercially available (Merck) and were used without
further purification.
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General procedure for the synthesis 2-p-tolylbenzo[d]oxazole (3):
A mixture of 2-aminophenol (1, 5 mmol) and 4-methylbenzoyl chloride (2, 5 mmol) in a mixed
o
solvent system (toluene 20 mL and triethyl amine 0.5 mL) was irradiated at 110 C for two
cycles of 30 min. The precipitate was removed by filtration. The filtrate was concentrated, and
1
the residue was recrystallized from ethanol to give compound 3 (90%). H NMR (500 MHz,
DMSO-d6): δ = 2.41 (s, 3H, CH3), 7.40-7.41 (m, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.75-7.79 (m,
2H), 8.09 (d, J = 8.0 Hz, 2H).
General procedure for the synthesis 2-(4-(bromomethyl)phenyl)benzo[d]oxazole (4):
A solution of 3 (10 mmol) and NBS (10 mmol) in toluene (30 mL) was refluxed under nitrogen
for 24 h. The solvent was evaporated and solid was recrystallized from ethanol to give compound
4 as a cream solid. (85%). 1H NMR (500 MHz, DMSO-d6): δ = 4.80 (s, 2H), 7.40-7.46 (m, 2H),
7.69 (d, J = 8.0 Hz, 2H), 7.78-7.86 (m, 2H), 8.19 (d, J = 8.0 Hz, 2H).
General procedure for the synthesis of N-((4-(benzo[d]oxazol-2-yl)phenyl)methyl)-amine
(6a-n):
Treatment of 2-(4-(bromomethyl)phenyl)benzo[d]oxazole 4 (1 mmol) with potassium carbonate
(1.5 mmol) and amines 5a-n (1 mmol) in DMF (10 ml) afforded corresponding compounds 6a-n
in good yields.
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2-(4-((Piperidin-1-yl)methyl)phenyl)benzo[d]oxazole (6a)
Pale yellow solid; yield: 0.17 g (60 %); mp. 100-102 oC. IR (νmax, cm−1): 3041, 2930, 1450, 851,
1
742. H NMR (500 MHz, DMSO-d6) δ = 1.40 (s, 2H), 1.51 (s, 4H), 2.36 (s, 4H), 3.52 (s, 2H,
CH2), 7.38 (t, J = 7.0 Hz, 2H), 7.52 (d, J = 7.5 Hz, 2H), 7.77 (t, J = 8.0 Hz, 2H), 8.15 (d, J = 7.5
Hz, 2H). 13C NMR (125 MHz, DMSO-d6): δ = 23.8, 25.5, 53.9, 62.3, 110.7, 119.6, 124.7, 125.3,
127.1, 129.5, 140.6, 143.2, 144.2, 156.7, 161.9. Anal. Calcd. For C19H20N2O: C, 78.05; H, 6.89;
N, 9.58 %. Found: C, 77.98; H, 6.78; N, 9.65 %.
2-(4-((4-Benzylpiperidin-1-yl)methyl)phenyl)benzo[d]oxazole (6b)
Pale yellow solid; yield: 0.25 g (65 %); mp. 120-121 oC. IR (νmax, cm−1): 3025, 2912, 1450, 835,
745. 1H NMR (500 MHz, DMSO-d6) δ = 1.22-1.26 (m, 1H), 1.50-1.55 (m, 4H), 1.92 (t, J = 11.0
Hz, 4H), 2.79 (d, J = 11.0 Hz, 2H), 3.52 (s, 2H, CH2), 7.15 (t, J = 7.5 Hz, 3H), 7.25 (t, J = 7.1
Hz, 2H), 7.38-7.43 (m, 2H), 7.51 (d, J = 8 Hz, 2H), 7.75-7.79 (m, 2H), 8.13 (d, J = 8.1 Hz, 2H).
13C NMR (125 MHz, DMSO-d6): δ = 31.6, 37.1, 42.2, 53.2, 61.8, 110.7, 119.6, 124.8, 125.2,
125.5, 127.0, 127.8, 127.9, 128.8, 129.2, 140.2, 141.4, 143.2, 150.3, 162.1. Anal. Calcd. For
C26H26N2O: C, 81.64; H, 6.85; N, 7.32 %. Found: C, 81.55; H, 6.76; N, 7.44 %.
2-(4-(Morpholinomethyl)phenyl)benzo[d]oxazole (6c)
Pale yellow solid; yield: 0.19 g (65 %); mp. 106-108 oC. IR (νmax, cm−1): 3033, 2953, 1451, 822,
750. 1H NMR (500 MHz, DMSO-d6) δ = 2.40 (s, 4H), 3.56 (s, 2H, CH2), 3.60 (s, 4H), 7.39-7.44
13
(m, 2H), 7.54 (d, J = 7.5 Hz, 2H), 7.76-7.81 (m, 2H), 8.15 (d, J = 7.5 Hz, 2H). C NMR (125
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MHz, DMSO-d6): δ = 53.1, 61.9, 66.1, 110.7, 119.6, 124.7, 126.1, 127.1, 129.5, 130.1, 141.4,
142.2, 150.1, 162.2. Anal. Calcd. For C18H18N2O2: C, 73.45; H, 6.16; N, 9.52 %. Found: C,
73.52; H, 6.25; N, 9.61 %.
2-(4-(Thiomorpholinomethyl)phenyl)benzo[d]oxazole (6d)
Pale yellow solid; yield: 0.18 g (60 %); mp. 110-112 oC. IR (νmax, cm−1): 3029, 2907, 1450, 803,
745. 1H NMR (500 MHz, DMSO-d6) δ = 2.64 (s, 4H), 2.66 (s, 4H), 3.60 (s, 2H, CH2), 7.39-7.44
13
(m, 2H), 7.53 (d, J = 8.0 Hz, 2H), 7.76-7.80 (m, 2H), 8.15 (d, J = 8.0 Hz, 2H). C NMR (125
MHz, DMSO-d6): δ = 27.1, 54.4, 62.0, 110.7, 119.6, 124.7, 125.0, 127.1, 129.4, 130.1, 141.4,
142.5, 150.1, 162.2. Anal. Calcd. For C18H18N2OS: C, 69.65; H, 5.84; N, 9.02 %. Found: C,
69.70; H, 5.77; N, 9.12 %.
2-(4-((Piperazin-1-yl)methyl)phenyl)benzo[d]oxazole (6e)
Pale yellow solid; yield: 0.20 g (70 %); mp. 273-275 oC. IR (νmax, cm−1): 3401, 2930, 1450, 812,
742. 1H NMR (500 MHz, DMSO-d6) δ = 2.47 (s, 4H), 3.15 (s, 4H), 3.67 (s, 2H, CH2), 7.33 (t, J
= 7.2 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.55-7.81 (m, 2H), 8.41 (d, J = 8.2 Hz, 2H), 10.13 (s,
1H, NH). 13C NMR (125 MHz, DMSO-d6): δ = 23.1, 53.4, 59.1, 110.7, 119.6, 124.7, 125.2,
127.1, 127.8, 129.0, 130.1, 143.8, 150.1, 161.5. Anal. Calcd. For C18H19N3O: C, 73.69; H, 6.53;
N, 14.32 %. Found: C, 73.78; H, 6.46; N, 14.42 %.
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2-(4-((4-Methylpiperazin-1-yl)methyl)phenyl)benzo[d]oxazole (6f)
Pale yellow solid; yield: 0.21 g (67 %); mp. 140-142 oC. IR (νmax, cm−1): 3025, 2912, 1450, 821,
1
745. H NMR (500 MHz, DMSO-d6) δ = 2.16 (s, 3H, CH3), 2.34 (s, 4H), 2.41 (s, 4H), 3.55 (s,
2H, CH2), 7.40 (t, J = 7.5 Hz, 2H), 7.41 (d, J = 7.3 Hz, 2H), 7.76-7.79 (m, 2H), 8.13 (d, J = 7.3
Hz, 2H). 13C NMR (125 MHz, DMSO-d6): δ = 45.6, 52.5, 54.6, 61.5, 110.7, 120.1, 124.7, 125.3,
126.1, 127.8, 129.4, 130.1, 142.8, 150.1, 162.2. Anal. Calcd. For C19H21N3O: C, 74.24; H, 6.89;
N, 13.67 %. Found: C, 74.38; H, 7.00; N, 13.55 %.
2-(4-((4-Ethylpiperazin-1-yl)methyl)phenyl)benzo[d]oxazole (6g)
Pale yellow solid; yield: 0.23 g (71 %); mp. 168-170 oC. IR (νmax, cm−1): 3061, 2904, 1452, 810,
1
739. H NMR (500 MHz, DMSO-d6) δ = 0.97 (t, J = 11.0 Hz, 3H), 2.30 (q, J = 12.1 Hz, 2H),
2.36-2.40 (m, 8H), 3.49 (s, 2H, CH2), 7.25 (t, J = 7.5 Hz, 1H), 7.28 (t, J = 8.1 Hz, 1H), 7.32 (d, J
13
= 8.0 Hz, 2H), 7.63 (d, J = 7.7 Hz, 1H), 7.67 (d, J = 7.7 Hz, 1H), 7.86 (d, J = 8.0 Hz, 2H). C
NMR (125 MHz, DMSO-d6): δ = 12.4, 51.5, 52.3, 52.6, 61.6, 111.0, 119.6, 120.1, 124.7, 125.0,
126.1, 127.8, 129.4, 142.8, 152.1, 160.9. Anal. Calcd. For C20H23N3O: C, 74.74; H, 7.21; N,
13.07 %. Found: C, 74.65; H, 7.19; N, 12.92 %.
2-(4-((4-Phenylpiperazin-1-yl)methyl)phenyl)benzo[d]oxazole (6h)
Pale yellow solid; yield: 0.27 g (72 %); mp. 100-103 oC. IR (νmax, cm−1): 3064, 2930, 1450, 855,
742. 1H NMR (500 MHz, DMSO-d6) δ = 2.56 (s, 4H), 3.15 (s, 4H), 3.63 (s, 2H, CH2), 6.76 (t, J
= 7.1 Hz, 1H), 6.91 (d, J = 8.1 Hz, 2H), 7.19 (t, J =7.5 Hz, 2H), 7.42 (t, J = 6 Hz, 2H), 7.58 (d, J
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= 8.1 Hz, 2H), 7.71-7.81 (m, 2H), 8.18 (d, J = 8.0 Hz, 2H). 13C NMR (125 MHz, DMSO-d6): δ
= 48.1, 52.5, 61.5, 110.7, 115.3, 118.6, 119.6, 124.7, 125.1, 125.3, 127.1, 128.7, 129.5, 141.5,
142.5, 150.1, 150.9, 162.2. Anal. Calcd. For C24H23N3O: C, 78.02; H, 6.27; N, 11.37 %. Found:
C, 78.08; H, 6.18; N, 11.45 %.
2-(4-((4-Benzylpiperazin-1-yl)methyl)phenyl)benzo[d]oxazole (6i)
Pale yellow solid; yield: 0.27 g (70 %); mp. 160-163 oC. IR (νmax, cm−1): 3438, 2931, 1448, 801.
1H NMR (500 MHz, DMSO-d6) δ = 2.41 (s, 8H), 3.46 (s, 2H, CH2), 3.56 (s, 2H, CH2), 6.76 (t, J
= 7.5 Hz, 1H), 6.91 (t, J = 8.1 Hz, 2H), 7.19 (t, J = 7.5 Hz, 2H), 7.36-7.44 (m, 2H), 7.57 (d, J =
13
8.1 Hz, 2H), 7.77-7.79 (m, 2H), 8.17 (d, J = 8.0 Hz, 2H). C NMR (125 MHz, DMSO-d6): δ =
52.5, 61.5, 110.7, 115.3, 118.6, 119.6, 124.7, 125.1, 125.3, 127.1, 128.7, 129.5, 141.5, 142.5,
150.1, 150.9, 162.2. Anal. Calcd. For C25H25N3O: C, 78.30; H, 6.57; N, 10.96 %. Found: C,
78.19; H, 6.42; N, 11.10 %.
2-(4-((Pyrrolidin-1-yl)methyl)phenyl)benzo[d]oxazole (6j)
Pale yellow solid; yield: 0.18 g (65 %); mp. 110-112 oC. IR (νmax, cm−1): 3031, 2950, 1450, 829,
1
749. H NMR (500 MHz, DMSO-d6) δ = 1.70-1.72 (m, 4H), 2.43-2.45 (m, 4H), 3.62 (s, 2H,
CH2), 7.25 (t, J = 7.5 Hz, 1H), 7.32 (t, J = 7.5 Hz, 1H), 7.43 (d, J = 8.0 Hz, 2H), 7.63 (d, J = 7.2
Hz, 1H), 7.65 (d, J = 7.2 Hz, 1H), 7.86 (d, J = 8.0 Hz, 2H). 13C NMR (125 MHz, DMSO-d6): δ
= 23.1, 53.4, 59.1, 110.7, 119.6, 124.8, 125.2, 127.1, 127.8, 129.0, 130.1, 143.8, 150.1, 162.3.
Anal. Calcd. For C18H18N2O: C, 77.67; H, 6.52; N, 10.06 %. Found: C, 77.79; H, 6.45; N, 10.16
%.
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(4-(Benzo[d]oxazol-2-yl)phenyl)-N-benzylmethanamine (6k)
o
Pale yellow solid; yield: 0.24 g (76 %); mp. 120-123 C. IR (νmax, cm−1): 3405, 3030, 2968,
1
1450, 833, 743. H NMR (500 MHz, DMSO-d6) δ = 3.72 (s, 2H, CH2), 3.79 (s, 2H, CH2), 7.22
(t, J = 7.2 Hz, 1H), 7.31 (t, J = 7.5 Hz, 2H), 7.36 (d, J = 7.5 Hz, 2H), 7.41 (t, J = 7.1 Hz, 2H),
7.58 (d, J = 7.5 Hz, 2H), 7.76-7.80 (m, 2H), 8.15 (d, J = 7.5 Hz, 2H), 8.60 (s, 1H, NH). 13C NMR
(125 MHz, DMSO-d6): δ = 51.8, 52.2, 110.7, 119.6, 124.7, 125.2, 126.4, 127.0, 127.5, 127.8,
128.0, 128.5, 140.6, 141.5, 145.3, 150.1, 162.3. Anal. Calcd. For C21H18N2O: C, 80.23; H, 5.77;
N, 8.91 %. Found: C, 80.12; H, 5.63; N, 8.98 %.
(4-(Benzo[d]oxazol-2-yl)phenyl)-N-benzyl-N-methylmethanamine (6l)
Pale yellow solid; yield: 0.22 g (68 %); mp. 106-107 oC. IR (νmax, cm−1): 3022, 2983, 1465, 819,
744. 1H NMR (500 MHz, DMSO-d6) δ = 2.12 (s, 3H, CH3), 3.54 (s, 2H, CH2), 3.60 (s, 2H, CH2),
7.26 (t, J = 6.5 Hz, 2H), 7.33-7.36 (m, 4H), 7.42 (t, J = 7.3 Hz, 1H), 7.58 (d, J = 8.1 Hz, 2H),
7.77 (d, J = 7.4 Hz, 1H), 7.79 (d, J = 7.4 Hz, 1H), 8.17 (d, J = 8.1 Hz, 2H). 13C NMR (125 MHz,
DMSO-d6): δ = 41.6, 60.5, 61.1, 110.7, 119.6, 124.7, 125.3, 126.8, 127.1, 128.1, 128.5, 129.2,
138.8, 141.5, 143.5, 150.1, 154.2, 162.2. Anal. Calcd. For C22H20N2O: C, 80.46; H, 6.14; N, 8.53
%. Found: C, 80.56; H, 6.25; N, 8.52 %.
N-((4-(benzo[d]oxazol-2-yl)phenyl)methyl)-N-benzylethanamine (6m)
Pale yellow solid; yield: 0.26 g (76 %); mp. 120-122 oC. IR (νmax, cm−1): 3028, 2976, 1449, 879,
744. 1H NMR (500 MHz, DMSO-d6) δ = 1.04 (t, J = 7.0 Hz, 3H, CH3), 2.48 (q, J = 6.3 Hz, 2H,
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CH2), 3.58 (s, 2H, CH2), 3.64 (s, 2H, CH2), 7.23 (t, J = 7.1 Hz, 1H), 7.33 (t, J = 7.5 Hz, 2H), 7.36
(d, J = 7.5 Hz, 2H), 7.41 (t, J = 7.1 Hz, 2H), 7.59 (d, J = 7.5 Hz, 2H), 7.76 (d, J = 7.2 Hz, 1H),
13
7.78 (d, J = 7.3 Hz, 1H), 8.15 (d, J = 7.5 Hz, 2H). C NMR (125 MHz, DMSO-d6): δ = 11.5,
46.5, 56.7, 57.1, 110.7, 119.6, 124.7, 124.9, 125.2, 126.7, 127.1, 128.1, 128.4, 129.1, 139.3,
141.5, 144.2, 150.1, 162.2. Anal. Calcd. For C23H22N2O: C, 80.67; H, 6.48; N, 8.18 %. Found: C,
80.78; H, 6.46; N, 8.07 %.
N-((4-(benzo[d]oxazol-2-yl)phenyl)methyl)-1-benzylpiperidin-4-amine (6n)
o
Pale yellow solid; yield: 0.29 g (74 %); mp. 114-115 C. IR (νmax, cm−1): 3421, 3028, 2937,
1451, 834, 744. 1H NMR (500 MHz, DMSO-d6) δ = 1.31-1.33 (m, 4H), 1.94 (t, J = 10.2 Hz, 4H),
2.74 (t, J = 10.2 Hz, 1H), 3.42 (s, 2H, CH2), 3.84 (s, 2H, CH2), 7.22 (t, J = 7.3 Hz, 1H), 7.26-
7.28 (m, 3H), 7.31 (t, J = 7.4 Hz , 1H), 7.38-7.43 (m, 2H), 7.58 (d, J = 8.5 Hz, 2H), 7.75-7.78
(m, 2H), 8.13 (d, J = 8.5 Hz, 2H), 8.50 (s, 1H, NH). 13C NMR (125 MHz, DMSO-d6): δ = 32.0,
49.4, 51.3, 53.7, 62.1, 110.7, 119.6, 124.7, 125.2, 126.6 (2C), 127.0, 127.4, 127.9, 128.5, 128.6
(2C), 138.6, 145.9, 162.2. Anal. Calcd. C26H27N3O: C, 78.56; H, 6.85; N, 10.57 %. Found: C,
78.58; H, 6.89; N, 10.48 %.
Cholinesterases inhibition assay
Acetylcholinesterase (AChE, E.C. 3.1.1.7, Type VeS, lyophilized powder, from electric eel,
1000 unit), butyrylcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum) and
butyrylthiocholine iodide (BTCh) were provided from Sigma-Aldrich. 5,50-Dithiobis-(2-
nitrobenzoic acid) (DTNB), potassium dihydrogen phosphate, dipotassium hydrogen phosphate,
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potassium hydroxide, sodium hydrogen carbonate, and acetylthiocholine iodide (ATCh) were
purchased from Fluka. The stock solutions of the target compounds were prepared in a mixture
of DMSO (1 mL) and ethanol (9 mL) and diluted with 0.1 MKH2PO4/K2HPO4 buffer (pH ¼ 8.0)
to obtain final concentrations. 20 mL of substrate (acetylthiocholine iodide 0.075 M) was added
to the test solution to obtain final concentration of 466 mM. All experiments were performed
based on the previously described method (31). Spectrophotometric measurements were
performed on a UV Unico Double Beam Spectrophotometer. The same method was also taken
for BuChE inhibition assay.
Results and Discussion
Chemistry
The synthesis of target compounds 6a-n was accomplished using the pathway, illustrated in
Scheme 1. First, the condensation reaction between 2-aminophenol 1 and 4-methyl benzoyl
chloride 2 in toluene under microwave irradiation at 110 oC under microwave irradiation resulted
in 2-p-tolylbenzo[d]oxazole 3, which was brominated in CCl4 by using NBS to afford the
corresponding 2-(4-(bromomethyl)phenyl)benzo[d]oxazole 4 in high yield. Further reaction with
proper amines 5a-n (Table 1) in the presence of K2CO3 produced N-((4-(benzo[d]oxazol-2-
yl)phenyl)methyl)-amine derivatives (6a-n). The structures of all the newly synthesized
compounds were characterized by IR, 1H NMR and 13C NMR spectroscopy. The spectra of target
compounds are provided in Appendix S1 file.
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Pharmacology
Cholinesterase inhibitory activity
The inhibitory potency of compounds 6a-n toward AChE and BChE were determined in terms of
IC50 values, using modified Ellman’s colorimetric method (32), demonstrated in Table 1. All the
synthesized compounds were active against AChE except 6b, 6h and 6i. Less number of
compounds showed remarkable anti-BChE activities, but, more promising results were obtained
in comparison to donepezil. Five- and six-membered heterocycles containing nitrogen were more
favorable than cycles with more than one heteroatom. In this regard, piperidine and pyrolidine
substituted benzoxazoles, meaning 6a and 6j exhibited excellent activities against the AChE and
BChE with IC50 values of 1.03, 1.35 and 6.6, 8.1 µM, respectively. Utilizing acyclic amines led
to totally inactive compounds against BChE.
Docking study
The ligand-protein docking was done by AutoDock Vina (1.1.2) (33) to predict the binding mode
of the ligands in the active site of AChE. The 3D coordinate of the AChE (PDB ID: 1eve) in
complex with donepezil was retrieved from Protein Data Bank (PDB) at
http://www.rcsb.org/pdb/home/home.do. For protein preparation, all of the non-protein atoms
were removed and then minimized using OPLS3 force field (RMSD= 0.3 Å). The grid box with
the size of 25×25×25 was determined and the center of box was fixed on co-crystal ligand. After
docking the best pose was selected for further analysis.
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The binding model of titled compound against AChE was represented in Figure 2. All
compounds were docked in the active site and then overlaid. Since the orientation of the
compounds were similar therefore compound 6a, the most active compound, was selected for
further analysis. The hydrophobic pi forces are the main feature of interactions. The ligand was
anchored in the active site through a π-cation interaction between positively charged nitrogen of
piperidine ring and Trp83 and Phe329. Moreover π-alkyl forces may occur in contact with
Trp83. The phenyl ring in the middle of the molecule made a T-shaped π-π interaction with
Tyr333. According to the docking data the benzoxazole moiety was oriented to the rim of the
active site and made a hydrogen bond with Phe287.
Conclusion
In this study, a range of new benzoxazole derivatives have been synthesized and evaluated for
their anti-cholinesterase activities. Our preliminary data revealed that most of the synthesized
compounds had inhibitory activity against AChE and BChE. Particularly, piperidine and
pyrolidine derivatives (6a, 6j) was the most potent compound against AChE and BChE.
Keeping in mind that dual AChE/BChE inhibitory activity is necessary to improve the signs of
AD, our promising results would be considered as a new lead in this research field.
Supporting Information
1
Full experimental details and H and 13C NMR spectra can be found in the Appendix S1
section of this article’s web page.
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Acknowledgment
This study was supported and funded by grants from the Research Council of Tehran University
of Medical Sciences (Grant no. 95-02-45-32579) and Iran National Science Foundation (INSF).
Conflict of interest
The authors declare no conflict of interest
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Legends:
Figure 1: Phenyl-benzoheterocyclic moieties as anti-cholinesterase agents.
Scheme 1: Synthesis of compounds 6a-n. Reagents and conditions: (a) Toluene, NEt3, MW, 110
oC, 1 h; (b) NBS, CCl4, reflux, 24 h; (c) NHR1R2 (5a-n), K2CO3, DMF, r.t.
Figure 2: The interaction of compound 6a with AChE active site.
COCl
OH
Br
O
N
O
N
a
b
+
NH2
4
2
3
1
c
NR1R2
O
N
6
Scheme 1.
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Table 1: In vitro inhibitory activity of compounds 6a-n against AChE and BuChE.
NR1R2
O
N
6a-n
Compound
NHR1R2
AChE
BuChE
IC50 (µM)
6.6±0.9
IC50 (µM) a
1.03±0.2
6a
NH
>100
>100
6b
Ph
NH
20.10±2.7
2.55±0.5
3.45±0.7
62.23±3.5
38.17±3.1
>100
>100
16.9±2.6
21.5±2.1
>100
6c
6d
6e
6f
O
S
NH
NH
HN
N
NH
NH
NH
NH
>100
6g
6h
6i
N
>100
Ph N
Ph
>100
>100
N
NH
1.35±0.2
8.1±0.6
6j
NH
25.44±1.8
30.76±2.1
>100
>100
6k
6l
Ph
NH2
NH
Ph
Ph
56.36±3.4
7.76±0.7
>100
6m
6n
NH
50.1±4.2
12.0±1.4
N
NH2
Ph
Donepezil
-
0.018±0.002
a
Data are expressed as Mean ± SD (three independent experiments).
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O
O
-
N+
Br
O
N
O
O
n
O
X
O
OMe
Y
X = N, O, S
N
NR1R2
O
NH
X
n
O
N
Y
New designed
compounds
X = N, O, S
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