Isoxazoles: Synthesis, evaluation and bioinformatic design as acetylcholinesterase inhibitors

Article abstract of DOI:10.1111/jphp.12180

Objectives Inhibition of acetylcholinesterase (AChE) is a common treatment for early stages of Alzheimer's disease. In this study, nine isoxazoles derivatives were tested for their in-vitro AChE activity. The molecular docking showed the interaction of the compounds with the active site. Methods The isoxazoles were synthesized using 1,3-dipolar cycloaddition in the presence of sodium hypochlorite. They were also isolated and characterized by spectroscopic methods. The in-vitro activity was measured by an adapted version of Ellman's assay. Key findings The isoxazoles are described as inhibitors of AChE. The most potent compound in the series exhibited a moderate inhibitory activity (50% inhibitory concentration = 134.87 μm). The design of new compounds was created by using the RACHEL module of the SYBYL software. Conclusions Our research provided enough evidence of the efficacy of isoxazoles as AChE inhibitors. The isoxazoles were synthesized and evaluated as inhibitors of AChE. The docking study based on a novel series of complexes isoxazole with AChE from Electroporus electricus has demonstrated that the ligand bind is similar to the compounds used as reference. To find new candidates with the isoxazole core that act as inhibitors of AChE, part of the structure of the compound 9 was used for de-novo design. Molecular docking models of the ligand-AChE complexes suggest that the compound 10 is located on the periphery of the AChE active site.

Full text of DOI:10.1111/jphp.12180

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Research Paper
Journal of Pharmacy
And Pharmacology
Isoxazoles: synthesis, evaluation and bioinformatic design
as acetylcholinesterase inhibitors
Margarita Gutiérreza, María Francisca Matusa, Tomas Pobletea, Jessica Amigoa, Gabriel Vallejosb and
Luis Astudilloa
aLaboratorio Síntesis Orgánica, Instituto de Química de Recursos Naturales, Universidad de Talca, Talca and bLaboratorio de Bio-Orgánica, Instituto
de Ciencias Químicas, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile
Keywords
acetylcholinesterase inhibitors; Alzheimer’s
Abstract
Objectives Inhibition of acetylcholinesterase (AChE) is a common treatment for
early stages of Alzheimer’s disease. In this study, nine isoxazoles derivatives were
tested for their in-vitro AChE activity. The molecular docking showed the interac-
tion of the compounds with the active site.
Methods The isoxazoles were synthesized using 1,3-dipolar cycloaddition in the
presence of sodium hypochlorite. They were also isolated and characterized by
spectroscopic methods. The in-vitro activity was measured by an adapted version
of Ellman’s assay.
disease; isoxazoles; ligand-protein interactions
Correspondence
Margarita Gutiérrez, Laboratorio Síntesis
Orgánica, Instituto de Química de Recursos
Naturales, Universidad de Talca, Casilla 747,
Talca 3460000, Chile.
E-mail: mgutierrez@utalca.cl
Received February 25, 2013
Accepted October 10, 2013
Key findings The isoxazoles are described as inhibitors of AChE. The most potent
compound in the series exhibited a moderate inhibitory activity (50% inhibitory
concentration = 134.87 μm). The design of new compounds was created by using
the RACHEL module of the SYBYL software.
doi: 10.1111/jphp.12180
Conclusions Our research provided enough evidence of the efficacy of isoxazoles
as AChE inhibitors. The isoxazoles were synthesized and evaluated as inhibitors of
AChE. The docking study based on a novel series of complexes isoxazole with
AChE from Electroporus electricus has demonstrated that the ligand bind is similar
to the compounds used as reference. To find new candidates with the isoxazole
core that act as inhibitors of AChE, part of the structure of the compound 9 was
used for de-novo design. Molecular docking models of the ligand-AChE com-
plexes suggest that the compound 10 is located on the periphery of the AChE
active site.
Introduction
The heterocyclic compounds are widely distributed in
nature and are essential to life in several ways. These com-
pounds are important because of the wide variety of activ-
ity associated with this class of substances.[1] Heterocyclic
rings are present in several compounds, for example, most
of the members of vitamin B complex, antibiotics, chloro-
phyll, haemin, other plant pigments, amino acids and pro-
teins, drugs, dye stuffs, enzymes and DNA. The isoxazole
is an azole with an oxygen atom bonded to a nitrogen
atom; these compounds are an important category of five-
membered unsaturated heterocyclic compounds. They
show numerous applications in diverse areas such as phar-
maceuticals, agro-chemistry and industry among others.
The isoxazoles are also found in natural sources such as
insecticides, plant growth regulators and pigment
functions.[2]
The isoxazole rings are found in natural products like the
ibotonic acid – an active constituent of the psychotropic fly
agaric mushroom Amanita muscaria – acting as ionotropic
and metabotropic (G-protein linked) glutamate receptor
subtypes.[3] (+)-Neopeltolide was isolated from a taxonomi-
cally uncharacterized sponge of the Neopeltidae family,
order ‘Lithistida’ collected in deep water of the Jamaica’s
coast.[4] The compound was assigned structurally as
macrolide with an isoxazole ring on a side chain and
showed some similarities in the structure to the compound
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Margarita Gutiérrez et al.
Isoxazoles as acetylcholinesterase inhibitors
previously known, leucascandrolide A.[5] The isoxazoles also
form the basis for a number of specific drugs, including the
cyclooxigenase-2 inhibitor valdecoxib (Bextra, Omaha, NE,
USA).[6] Thus, the isoxazoles have been widely used and
studied in the modern drug discoveries.[7]
The isoxazoles can be prepared in many ways, but the
most widely reported and researched synthesis of isoxazoles
is via [3 + 2] cycloaddition of a nitrile oxide and an alkyne.
The conventional generation of a nitrile oxide requires the
dehydration of a nitroalkane or a similar starting material.
Other methods for generating nitrile oxides are by reac-
tion of aldoximes with oxidizing agents or halogenating
species.[8]
A series of 1,2-benzisoxazoles was synthesized, and found
potent and selective inhibitors of acetylcholinesterase
(AChE) in vitro against targets from different species.[9] New
D-glyceraldehyde heterocyclic derivatives were synthesized
and evaluated as potential inhibitors of AChE, the most
active compounds possess the isoxazole ring.[10] Pyrrolo-
isoxazole benzoic acid derivatives were synthesized and
evaluated as potential AChE inhibitors for the management
of Alzheimer’s disease (AD).[11]
AD is a progressive neurodegenerative disorder associ-
ated with cognitive, functional and behavioural impair-
ments as well as the most common form of dementia in
the elderly.[12] It affects brain regions that control thoughts,
memory, thinking, behaviour, emotion and language, and
evolves to a devastating status for patients and caregivers.
Nearly 50 million people worldwide have AD. Social and
economic burdens of this disease are massive because of
the estimated direct and indirect annual costs of $100
billion in patient care per year only in the USA.[13] These
facts suggest that potent drugs are needed to slow down
the progression of AD or even to prevent it.
The current therapies for AD, either symptomatic or pal-
liative, rely mainly on the restoration of acetylcholine
levels[14] and on the partial antagonism of the N-methyl-D-
aspartate receptor.[15] AChE is the enzyme that terminates
the transmission of nerve impulses in cholinergic synapses
by hydrolysing the neurotransmitter acetylcholine to acetic
acid and choline. Because cholinergic transmission is
involved in a variety of physiological systems, some inhibi-
tors are used to treat various disorders, such as myasthenia
gravis, and as a symptomatic approach to the management
of AD.[16]
This paper describes the work undertaken to study
the effectiveness of synthesis, biological activity and
the mechanism of interactions by computational analysis.
The results can be highly useful and may provide a
convenient platform for the development of new inhi-
bitors of AChE towards the treatment of AD, which
can generate a massive reductions development time of
drugs.
Materials and Methods
Chemistry
All solvents used were of analytical grade. Melting points
were determined by open capillary methods on a Buchi
apparatus and are uncorrected. Infrared (IR) spectra, KBr
pellets, 500–4000 cm−1 were recorded on a Thermo Nicolet
NEXUS 670 FT-IR spectrophotometer (Madison, WI,
USA), with a 0.125 cm−1 spectral resolution. The 1H-
nuclear magnetic resonance (NMR) and 13C-NMR spectra
were recorded on
a Bruker AM 400 instruments
(Rheinstetten, Germany). Chemical changes are expressed
as values relative to tetramethylsilane as internal standard.
Multiplicities are designated as singlet (s), doublet (d),
triplet (t), quadruplet (q) and multiplet (m). High-
resolution mass spectrometry electrospray ionization mass
spectrometry (ESI-MS) and ESI-tandem mass spectrometry
(ESI-MS/MS) analysis were conducted in a high-resolution
hybrid quadrupole (Q) and an orthogonal time-of-flight
(TOF) mass spectrometer (Waters/Micromass Q-TOF
micro, Manchester, UK), with a constant nebulizer tempera-
ture of 100°C. The experiments were carried out in positive
ion mode, and the cone and extractor potentials were set at
10 and 3.0 V, respectively, with a scan range of m/z 100–600.
MS/MS experiments were carried out by mass selection of a
specific ion in Q1, which was then submitted to collision-
induced dissociation with helium in the collision chamber.
The product ion MS analysis was accomplished with the
high-resolution orthogonal TOF analyser. The samples were
directly infused into the ESI source via a syringe pump at
flow rates of 5 μl/min through the instrument’s injection
valve. The elemental analysis has been obtained using a
LECO CHNS-900 and Thermo Finnigan FlashEA1112
CHNS-O (STIUJA, Hemel, Hempstead, UK) elemental ana-
lysers. TLC was done on precoated silica gel 60 F254 plates
(Merck, Darmstadt, Germany).
Synthetic methodologies
The isoxazole derivatives were efficiently synthesized
according to the protocol outlined in Figure 1. This proto-
col can be divided in two steps:
Step 1: preparation of oxime derivatives
An aqueous solution of hydroxylamine (NH2OH 50%)
24 ml (12.0 mmol) and sodium acetate 1.0 g (12.2 mmol)
were dissolved. The aldehyde (10 mmol) was dissolved in
ethyl alcohol (10 ml); both solutions were mixed and kept
from 30 to 40 min at room temperature. Then the oxime
was extracted with dichloromethane (3 × 15 ml) and sepa-
rated from the aqueous layer, and the volume was reduced
half under decreased pressure. The formation of oxime was
analysed for TLC.
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1796–1804
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Isoxazoles as acetylcholinesterase inhibitors
Margarita Gutiérrez et al.
OH
H
N
O
NaClO, 5%
Et3N
R
O
NH2OH(ac)
EtOH
N
R1
R
H
R
R1
Figure 1 Protocol for synthesis of isoxazole derivatives.
Step 2: preparation of 3,5-disubstituted isoxazoles
USA). IC50 of isoxazole derivatives against AChE was calcu-
lated with a regression analysis. Statistical analyses were
performed by one-way analysis of variance (ANOVA). Sig-
nificant differences between groups were determined at
P < 0.05.
Oxime (10 mmol) in dichloromethane (20 ml) at 0°C
was dropwise added to a mixture of alkyne (10 mmol),
triethylamine 1.5 ml (11 mmol) and 5% of aqueous sodium
hypochlorite (20 ml) after stirring for 60 min in iced water.
Then, the mixture was extracted with dichloromethane
(3 × 15 ml) and separated from the aqueous layer. Organic
extracts were combined, dried over Na2SO4 and the solvent
was removed under vacuum. The solid obtained was sus-
pended in hot ethyl alcohol and crystallized, obtaining the
isoxazole. TLC checked the reaction completion.
Docking studies
Molecular docking studies on acetylcholinesterase
The computational process of searching for a ligand that is
able to fit both geometrically and energetically the binding
site of a protein is called molecular docking.[17] Docking
studies were performed using Glide[18] version 5.5. Glide
docking uses a series of hierarchical filters to find the best
possible ligand binding locations in a receptor grid space
previously built. The filters include a systematic searching
approach, which samples the positional, conformational
and orientational space of the ligand before evaluating the
energy interactions of the ligand with the protein. The
Glide program is contained in Maestro 9.0 suite (New York,
NY, USA). The protein coordinates were extracted from the
X-ray crystal structure of AChE from the E. electricus organ-
ism that was acquired from the Protein Data Bank (PDB ID:
1EEA). Extra-precision module of Glide was used. The
docking hierarchy begins with the systematic conforma-
tional expansion of the ligand, followed by the placement in
the receptor site. Then, minimization of the ligand in the
field of the receptor is carried out using the OPLS-AA[19]
force field, with a distance-dependent dielectric 2.0. Subse-
quently, the lowest energy poses are subjected to a Monte
Carlo procedure that samples nearby torsional minima. The
best pose for a given ligand is determined by the Emodel
score while different compounds are ranked using Glide
Score (a modified version of the Chem Score function of
Eldridge et al.[20]) that includes terms for buried polar
groups and steric clashes.
Biological activity
In the 96-well plates, 50 ml of the sample dissolved in phos-
phate buffer (8 mmol/l K2HPO4, 2.3 mmol/l NaH2PO4,
150 mmol/l NaCl, and 0.05% Tween 20 at pH 7.6) as well
as 50 ml of the AChE solution (0.25 unit/ml), from
Electroporus electricus, in the same phosphate buffer were
added. The assay solutions except the substrate were
preincubated with the enzyme for 30 min at room tempera-
ture. After preincubation, the substrate was added. The
solution substrate consists of Na2HPO4 (40 mmol/l),
acetylthiocholine (0.24 mmol/l) and 5,5’-dithio-bis-(2-
nitrobenzoic acid) (0.2 mmol/l, Ellman’s reagent). The
absorbance of the yellow anion product due to the sponta-
neous hydrolysis of substrate was measured at 405 nm for
5 min on a Microtiter plate reader (Multiskan EX, Thermo,
Finland). The AChE inhibition was determined for each
compound. The enzyme activity was calculated as a per-
centage compared with a control using only the buffer and
enzyme solution. The compounds were assayed in the dilu-
tion interval of 500–15 μg/ml, and the galantamine alkaloid
was used as the reference compound. Each assay was run in
triplicate, and each reaction was repeated at least three inde-
pendent times. The 50% inhibitory concentration (IC50)
values were calculated by means of regression analysis.
De-novo design
Statistical analysis
This technique enables design inhibitors from the binding
site on the target or the pharmacophore, that is, the infor-
mation of spatial points relevant of the receptor–ligand
interaction. Once knowing both, the binding mode and the
structural relationship between the compound 9 and AChE
Three or more independent experiments were performed.
The data were analysed and expressed as mean standard
error of the mean (SEM) using Statistical Product and
Service Solutions version 17th (SPSS, Inc., Chicago, IL,
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Margarita Gutiérrez et al.
Isoxazoles as acetylcholinesterase inhibitors
and their experimental activity, we proceeded to the optimi-
zation of the compound 9 through the RACHEL module of
SYBYL (TRIPOS, St Louis, MO, USA). Starting from a
ligand/receptor complex, RACHEL performs automated
combinatorial optimization of lead compounds by systemi-
cally derivatizing the user-defined sites on the ligand. These
compounds are conformationally searched within the active
site and then evaluated; only those that bind tightly with the
receptor are retained. Afterwards, this new population of
compounds is processed to form the next generation of
derivatives. Over time, a lead compound is iteratively
refined into a set of high-affinity structures.
997. Anal.Calcd for C12H8N2OS: C, 63.14; H, 3.53. Found C,
63.16; H, 3.51.
3-(5-Thiophen-3-yl-isoxazol-3-yl)pyridine (5): Yield
1
81%. Mp198-199°C. H NMR δppm (CDCl3-400 MHz):
9.16 (1H, d, J = 1.6 Hz), 9.06 (1H, d, J = 1.6 Hz), 8.83 (1H,
dd, J = 5.0, 1.2 Hz), 8.71 (1H, dd, J = 4.8, 1.6 Hz), 8.20
(2H,m), 7.86 (1H, dd, J = 2.6, 1.2 Hz), 6.74 (1H, s). MS m/z
(EI): 228.93 (M++1). IR (KBr)/cm: 3438; 2072; 1648, 78.
Anal. Calcd for C12H8N2OS: C, 63.14; H, 3.53. Found C,
63.17; H, 3.54.
4-(5-Thiophen-3-yl-isoxazol-3-yl)pyridine (6): Yield
1
83%. Mp162-163°C. H NMR δppm (CDCl3-400 MHz):
8.76 (2H, d, J = 6.0 Hz), 7.88 (1H, s), 7.74 (2H, d,
J = 6.0 Hz), 7.48 (2H, m), 6.75(1H, s). MS m/z (EI): 228.93
(M++1). IR (KBr)/cm: 3438; 2075; 1640; 948. Anal. Calcd for
C12H8N2OS: C, 63.14; H, 3.53. Found C, 63.16; H, 3.51.
2-(5-Cyclohex-1-enyl-isoxazol-3-yl)-pyridine (7): Yield
Results
Nine isoxazoles disubstituted, with yield around 80%, were
synthesized using cycloadittion 1,3-dipolar of aldehyde and
alkyne with hydroxylamine, triethylamine and sodium
hypochlorite. Structures of all compounds with alkynes and
aldehydes used for synthesis are showed in Table 1.
1
85%. Oil. H NMR δppm (CDCl3-400 MHz): 8.66 (1H, d,
J = 8.0 Hz), 8.06 (1H, d, J = 8.0 Hz) 7.77 (1H, t, J = 7.6 Hz),
7.32 (1H, t, J = 7.6 Hz), 6.73 (1H, s); 6.67 (1H, brs), 2. 31
(4H, m) 1.72 (4H, m). 13Cδppm (CDCl3-100 MHz): 171.78,
163.12, 149.56, 148.79, 136.77, 130.17, 125.32, 124.26,
121.52, 96.94, 25.36, 25.07, 22.01, 21.63. MS m/z (EI):
226.94 (M++1). IR (KBr) cm-13435; 2102; 1670; 864. Anal.
Calcd for C14H14N2O: C, 74.31; H, 6.24. Found C, 74.33; H,
6.20.
Structural identification
2-(5-Phenyl-isoxazol-3-yl)pyridine (1): Yield 87%. Mp80-
1
82°C. H NMR δppm (CDCl3-400 MHz): 8.73 (1H, ddd,
J = 8.0; 4.8; 1.2 Hz); 8.15(1H, d, J = 8.0); 7.87 (2H, dd,
J = 8.0; 1.6 Hz), 7.84 (1H, dt, J = 7.6, 1.6), 7.49 (3H, m), 7.38
(1H, ddd, J = 7.5, 5.2, 1.2 Hz), 7.20 (1H, s). 13C δppm
(CDCl3-100 MHz): 170.64; 163.76; 149.71; 148.55; 136.91;
130.24; 129.02; 127.24; 125.84; 124.52; 121.66; 98.31. MS
m/z (EI): 222.98 (M++1). IR (KBr)/cm: 3413; 1649;1456.
Anal. Calcd for C14H10N2O:C, 75.66; H, 4.50. Found: C,
75.67; H, 4,51.
3-(5-Cyclohex-1-enyl-isoxazol-3-yl)-pyridine (8): Yield
1
83%. Mp186-187°C. H NMR δppm (CDCl3-400 MHz):
9.36 (1H, s), 8.91 (2H, m), 8.03 (1H, t, J = 8.0), 6.74 (2H,
brs), 2.34 (4H, m), 1.75 (4H,m). MSm/z (EI): 226.95
(M++1). IR(KBr)/cm: 3397; 2097; 1644; 949. Anal. Calcd for
C14H14N2O: C, 74.31; H, 6.24. Found C, 74.32; H, 6.23.
4-(5-Cyclohex-1-enyl-isoxazol-3-yl)-pyridine (9): Yield
1
3-(5-Phenyl-isoxazol-3-yl)pyridine (2): Yield 88%.
Mp134-135°C. 1H NMR δppm (CDCl3-400 MHz): 9.09
(1H, d, J = 1.2 Hz), 8.72 (1H, dd, J = 4.8, 1.6 Hz), 8.22 (1H,
dt, J = 8.0, 2.0 Hz), 7.86 (2H, dd, J = 8.0, 1.2 Hz), 7.51 (3H,
m), 7.44 (1H, dd, J = 8.0, 4.8 Hz), 6.89 (1H, s). MS m/z (EI):
222.98 (M++1). IR (KBr)/cm: 3417; 2106; 1642; Anal. Calcd
for C14H10N2O: C, 75.66; H, 4.50. Found: C, 75.67; H, 4,51.
4-(5-Phenyl-isoxazol-3-yl)pyridine (3): Yield 82%.
78%. Oil. H NMR δppm (CDCl3-400 MHz):8.41 (2H, d,
J = 8.0 Hz), 7.92 (2H, d, J = 8.0 Hz), 6.31 (1H, brs), 6.25
(1H, s), 2.02 (4H, m), 1.39 (4H, m). MSm/z (EI): 227.054
(M++1). IR(KBr)/cm 3418; 2130; 1649; 832. Anal. Calcd for
C14H14N2O: C, 74.31; H, 6.24. Found C, 74.34; H, 6.25.
Biological results
1
Mp123. H NMR δppm (CDCl3-400 MHz): 8.68 (2H, d,
The concentration of the compound required for 50%
enzyme inhibition (IC50) was calculated by means of
regression analysis. All tabulated results in Table 2 are
expressed in micrograms per millilitre and micromoles per
litre as means SEM, and was compared by using ANOVA
analysis. A P-value of less than 0.05 was considered signifi-
cant. Details for pharmacological experiments are described
in Materials and Methods as well as in previous works.[21]
The series of compounds with 1-ethynylcyclohexene used as
alkyne (7–9) showed the highest activity with IC50 values
upper to 200 μm. Although the activity of compounds is
deficient comparable with the reference employed, it is
J = 8.0 Hz), 8.63 (2H, d, J = 8.0 Hz), 8.58 (2H, d,
J = 8.0 Hz), 7.71 (1H, t, J = 8.0 Hz), 7.66 (2H, d, J = 8.0 Hz),
6.76 (1H, s). MS m/z (EI): 222.98 (M++1). IR (KBr)/
cm:3442; 2102; 1632; 998. Anal. Calcd for C14H10N2O: C,
75.66; H, 4.50. Found: C, 75.67; H, 4,51.
2-(5-Thiophen-3-yl-isoxazol-3-yl)pyridine (4): Yield
78%. Mp119°C. H NMR δppm (CDCl3-400 MHz): 8.72
(1H, ddd, J = 8.0, 4.8, 1.2 Hz); 8.13 (1H, d, J = 8.0),7.83
(2H, m), 7.47 (1H, t, J = 6.0 Hz), 7.45 (1H, dd, J = 5.0,
3.2 Hz), 7.37 (1H, ddd, J = 7.5, 4.8, 1.2 Hz),7.06 (1H, s).MS
m/z (EI): 228.93 (M++1). IR (KBr)/cm: 3433; 2078; 1641;
1
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1796–1804
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Isoxazoles as acetylcholinesterase inhibitors
Margarita Gutiérrez et al.
Table 1 Aldehydes and alkynes required for the synthesis of compounds 1–10 and structure to obtain
Compound
Aldehyde (R)
Alkyne (R1)
Structure
1
Pyridine-2-carbaldehyde
Phenylacetylene
N
N
O
2
3
4
Pyridine-3-carbaldehyde
Pyridine-4-carbaldehyde
Pyridine-2-carbaldehyde
Phenylacetylene
Phenylacetylene
3-ethynylthiophene
N
N
O
N
N
O
N
N
S
O
5
Pyridine-3-carbaldehyde
3-ethynylthiophene
N
N
N
S
O
6
7
8
9
Pyridine-4-carbaldehyde
Pyridine-2-carbaldehyde
Pyridine-3-carbaldehyde
Pyridine-4-carbaldehyde
3-ethynylthiophene
1-ethynylcyclohexene
1-ethynylcyclohexene
1-ethynylcyclohexene
N
S
O
N
N
O
N
N
N
O
N
O
10
Pyridine-4-carbaldehyde
5-hexyn-1-ol
N
OH
N
O
1800
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Margarita Gutiérrez et al.
Isoxazoles as acetylcholinesterase inhibitors
Table 2 IC50 of isoxales derivatives and galantamine on
acetylcholinesterase evaluated by a microplate assay
E199, the carbonylic oxygen is the HB acceptor, and the
amino group of the aldehyde derivative portion in com-
pound 10 is the HB donor group. In residue F330, the
carbonylic oxygen is the HB acceptor, and the hydroxyl
group in the compound 10 is the HB donor group. The resi-
dues H440 and W84 (cyan representation) established π-π
stacking interactions with the aldehyde derivative portion
and with the isoxazolic ring. The residues S200 and F331
(green representation) are key residues in the active site of
AChE. These residues do not establish any bond, but they
were found to be less than 3 Å, similarly to the compound
9-AChE complex.
Compound
IC50 (μg/ml)
IC50 (μM)
1
2
3
4
69.19
79.65
205.06
>250
311.67 2.54
358.78 2.85
923.69 3.07
5
81.46
357.28 0.17
6
>250
7
8
9
44.53*
41.53*
30.48*
1.1
197.04 0.28*
183.76 0.44*
134.87 0.29*
0.54 0.10
Galantamine
Values are average SEM from three experiments. Bold values show
the most active compound. *P < 0.05.
Discussion
These coupling reactions were performed under mild con-
ditions (room temperature, 24 h) in the presence of
aqueous sodium hypochlorite (5%). The isoxazoles
products were obtained in good yield with almost no
by-products. All isoxazoles were purified by recrystallization
process.
show as interesting pharmacophores to be used for the
design of new biologically active compounds against
enzymes related with neurodegenerative diseases. Com-
pound 9 showed the major activity against AChE, with IC50
of 134.87 μM. With the aim of knowing the location and
interactions generated with the active site of the enzyme,
compound 9 was subjected to molecular docking, and then,
these compounds were used from de-novo design.
Isoxazoles 1–9 were characterized by 1H-NMR, 13C-NMR
and mass spectra. 1H-NMR spectra of all isoxazoles synthe-
sized were quite similar and characterized by the presence
of three groups of signals (aromatics protons, protons near
heteroatoms and aliphatic protons), which resonated in dif-
Docking studies on acetylcholinesterase
1
ferent zones. All H-NMR spectra showed a characteristic
Figure 2 depicts the compound 9 (gray representation)
docked at the active site of AChE using Glide software. The
binding energy of the best pose obtained from docking was
−9.32 kcal/mol. Compound 9 to establish a hydrogen bond
(HB) interaction with the residue E199 (yellow representa-
tion), the amine group of the aldehyde derivative portion in
the compound 9 is the HB donor group, and the carbonylic
oxygen in this residue is the HB acceptor group. Further-
more, the residues H440 and W84 (cyan representation)
established π-π stacking interactions with this portion, and
the residue F330 established π-π stacking interactions with
the isoxazolic ring. The residues S200 and F331 (green rep-
resentation) are key residues in the AChE active site. These
residues do not establish any bond, but they were found to
be less than 3 Å.
proton singlet of the isoxazole ring with shift near seven.
The mass spectra showed similar fragmentation patterns
between compounds, all showing the molecular ion
[M + H+]+ and the gain or loss of small fragments of low
molecular mass.
All compounds were evaluated as AchE inhibitors showed
values.
All compounds were evaluated as inhibitors of AChE,
showing the differences statistically significant of com-
pounds 7–9 with the rest of the analysed groups. Com-
pound 9 was the one with the most activity; with IC50 of
134.87 μm, the results suggest that rational modifications of
the substituent in the scaffold isoxazoles would provide a
rational basis for the development of novel AChE inhibi-
tors. Compound 10 showed to be an interesting compound
to block this protein. This molecule (as compound 9) was
able to establish interactions and bonds with key residues
that belong to the active site of AChE. The interactions with
these residues have been reported before[22] in AChE – the
galantamine complex. Although isoxazol9 showed better
binding energy than 10, this difference is not significant.
Compound 10 was able to establish more numbers of
H-bond, which explains that this molecule would be closer
related to AChE than the compound 9. It is important to
analyse the differences of the alkyne derivative portion in
both compounds. Compound 9 has a ring, which estab-
De novo design
Compound 10 was the most potent compound (score:
11.75) found. This novel compound is a derivative of
isoxazol 9, with modifications on the cyclohexen ring.
Molecular docking studies showed better affinity with active
site of AChE. The binding energy of the best pose obtained
from docking was −8.45 kcal/mol. Figure 3 depicts the most
important interactions between this inhibitor and AChE.
Compound 10 was able to established HB interactions with
residues E199 and F330 (yellow representation). In residue
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1796–1804
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Isoxazoles as acetylcholinesterase inhibitors
Margarita Gutiérrez et al.
W 84
E 199
F 330
S 200
F 331
H 440
Figure 2 Molecular docking models for compounds 9 within the AChE binding site highlighting the protein residues that form the main interac-
tions with the inhibitors.
W 84
E 199
F 330
S 200
F 331
H 440
Figure 3 Molecular docking models for compounds 10 within the AChE binding site highlighting the protein residues that form the main interac-
tions with the inhibitors.
lishes π-π stacking interactions with some residues.
However, this ring structure leads to the ligand to be
anchored to the active site in a stiffer way. In contrast, com-
pound 10 has a more flexible tail in this part, which estab-
lishes H-bonds with some key residues. Therefore, the
whole structure has more mobility, which suggests that if
the protein suffers a conformational change, this tail could
be adapted and fit better than the compound 9 into the
active site because of its greater numbers of freedom of
degrees, maintaining this interaction over the time.
Values of IC50 obtained for the compound 9 of this
series may be comparable with other reported in literature
for active ingredients of commercial pharmaco as
galantamine, donepezil, rivastigmine and tacrine.[23] In
addition, it will be expected to obtain IC50 values for the
compound 10 lower than those reported in this work for
the compound 9. Moreover, the goal of the search for new
inhibitors aims has a decline in side effects showed by the
current drugs used in the treatment of this disease;[24,25] as
well as based in previous reports for compounds with
isoxazolic ring as effective drugs in other diseases treat-
ments with low side effects.[26,27] It is expected a decreased of
these to analyse in vivo.
Values of IC50 show some grade of relation between the
substituents that possess the molecule and its biological
activity; for instance, the presence of structures with a lower
degree of saturation promotes interaction with the active
site, whereas the position of the nitrogen in the pyridine
ring seems not to influence the activity of the compounds.
The presence of rings with heteroatoms or aromatics rings
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Margarita Gutiérrez et al.
Isoxazoles as acetylcholinesterase inhibitors
in the portion derived from alkyne used is unfavourable for
the interactions with active site of the enzyme. This is cor-
roborated by de-novo design because of compound 10
obtaining a greater flexibility and thus facilitating the entry
into the active site as well as the rapprochement between the
compound and the amino acidic residues.
isoxazol-3-yl)-pyridine would provide a rational basis for
the development of novel AChE inhibitors. Further efforts
aimed to develop potent AChE inhibitors based on the
modification of compound 9 will be continued in our
laboratory. This group of compounds could be an interest-
ing lead for AChE inhibitors.
Our analysis also shows that synthetic isoxazoles could be
the potential target molecules for the inhibition of AChE.
Hence, the more active of a series could be used as the tem-
plate for designing therapeutic lead molecule. We strongly
hope that the ingenuity and success of the computational
efforts discussed above bode well for the future prospects of
finding new inhibitors, which could result into massive
reductions in therapeutics development time.
Currently, our working group is synthesizing new
isoxazoles with the substitution pattern of compound 10
obtained by de-novo design to assess whether improve-
ments are experimentally observed in the activity. Com-
pound 10 can be synthesized from pyridine-4-carbaldehyde
and 5-hexyn-1-ol. This compound could be suggested as a
new possible inhibitor of AChE depending on the experi-
mental results. In addition, this compound could be sug-
gested as a member of a promising class of next-generation
derivatives, improving even more its features against AChE.
To conclude, in this report, an innovative method for
the synthesis of isoxazoles, a biologically important hetero-
cyclic scaffold, was described. This synthesis is based on
cycloaddition 1–3 dipolar in presence of sodium
hypochlorite, which offers a faster, cheaper and safer way to
construct isoxazoles with biological activity. This study
shows the effectiveness that may have the use of
bioinformatics tools for the rational design of drugs, which
implies a reduction in the time devoted to the synthesis and
the available resources.
Conclusions
In summary, a series of 1,3-disubstituted isoxazoles deriva-
tives were designed, synthesized and evaluated as AChE
inhibitors. Structure–activity studies showed that the AChE
inhibitory activity of compounds was influenced by the
substituent pattern at position 3 of isoxazol. The most
potent compound (9) contains a ciclohexil group in this
position. The molecular modelling study of compound 9
indicated that this is accommodated inside the active-site
gorge of AChE, adopting the same interactions that were
described for the AChE–galantamine complex.
Evidence was acquired demonstrating that isoxazoles 1,3-
disubstituted is a potential scaffold for the design of novel
AChE inhibitors. A detailed analysis of the experimental
results obtained so far allows us to conclude that the affinity
or selectivity of the isoxazoles towards AChE may be greatly
influenced by the nature of their substituents. Compound 9
exhibits the best performance and can be considered as the
starting point for the design and synthesis of new AChE
inhibitors. The results suggest that rational modifications of
the substituent in the scaffold 4-(5-cyclohex-1-enyl-
Declarations
Conflict of interest
The Author(s) declare(s) that they have no conflicts of
interest to disclose.
Funding
We are grateful to the Fondecyt 1 100 481 project for finan-
cial support. The authors thank the grant from Universidad
de Talca-VAC 600-569.
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7. Bylund J et al. Novel bioactivation
mechanism of reactive metabolite for-
mation from phenyl methylisoxazoles.
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