Synthesis of 2′-(1,2,3-triazoyl)-acetophenones: molecular docking and inhibition of: In vitro monoamine oxidase activity

Article abstract of DOI:10.1039/d0nj04735j

We describe our results for the synthesis of 2′-(1,2,3-triazoyl)-acetophenones by copper catalyzed azide-alkyne cycloaddition of 2′-azidoacetophenone with alkynes. The corresponding 2′-(1,2,3-triazoyl)-acetophenones were obtained in moderate to excellent yields and according to our experiments, it was observed that ultrasound accelerates the reaction compared to conventional heating. In addition, the obtained compounds were screened by molecular docking for their possible inhibitory effect on the activity of monoamine oxidase (MAO), an enzyme responsible for the degradation of monoamine neurotransmitters. Three compounds were selected and presented an inhibitory effect on both MAO-A and B isoform activities in vitro. These new MAO inhibitors may be of interest to treat monoamine deficit related-neuropsychiatric disorders, such as depression and Parkinson's disease.

Full text of DOI:10.1039/d0nj04735j

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Synthesis of 2’-(1,2,3-triazoyl)-acetophenones: molecular docking
and in vitro monoamine oxidase activity inhibition
Gabriel P. Costa,a Ítalo F. C. Dias,a Mariana G. Fronza,b Evelyn M. Besckow,c Jenifer Fetter,b José
Edmilson R. Nascimento,a Raquel G. Jacob,a Lucielli Savegnago,b Cristiani F. Bortolatto,c César A.
Brüningc* and Diego Alvesa*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
We described our results for the synthesis of 2’-(1,2,3-triazoyl)-acetophenones by copper catalyzed azide-alkyne
cycloaddition of 2’-azidoacetophenone with alkynes. The corresponding 2’-(1,2,3-triazoyl)-acetophenones were obtained in
moderated to excellent yields and according to our experiments, it was observed that ultrasound accelerates the reaction
compared to conventional heating. In addition, the obtained compounds were screened by molecular docking for possible
inhibitory effect on monoamine oxidase (MAO) activity, an enzyme responsible for degradation of monoamine
neurotransmitters. Three compounds were selected and presented inhibitory effect on both MAO-A and B isoforms activities
in vitro. These new MAO inhibitors may be of interest to treat monoamine deficit related-neuropsychiatric disorders, such
as depression and Parkinson’s disease.
The 1,4-disubstituted 1,2,3-triazoles can be easily selectively
accessed via dipolar cycloaddition using organic azides and alkynes
through the copper catalysis (CuAAC reactions).7 Beyond the above,
Introduction
Among the various N-heterocycles, triazoles are undeniably
important due your different applications in materials chemistry1
such as corrosion inhibitors,2 and organic dyes.3 Meantime, they
have occupied a relevant position in the field of medicinal and
pharmaceutical chemistry,4 having a wide variety of biological
activities, such as amnesia-ameliorative effect, for treatment of
Alzheimer's disease, anticonvulsant, antinociceptive, anti-
inflammatory, among others.5 In addition to countless activities
already reported, this nucleus is also found in the structure of some
drugs, such as Rufinamide which acts as anticonvulsant agent, as well
as, Tazobactam and Cefatrizine which are antibiotics (Figure 1).6
they can effectively mimic trans-amide bonds that makes triazole a
privileged support for the construction of medicinal compounds. This
one effect is due to their similar size, planarity, H-bonding
capabilities, dipole moment and performing secondary interactions
which are able to bind to biological receptors.4a,4c,8 Analyzing the
structure potential of the 1,2,3-triazole nucleus, the presence of N2
and N3 nitrogen with lone electron pairs which may have potential
display hydrogen bond acceptor properties. Besides, π-π stacking
interactions of the aromatic ring and a weak hydrogen bonding
donor (C-H).4a,4c,8
In the medicinal chemistry, the ketone group is easily found and
shows important structural feature when referring at biological
recognition processes. The presence of two lone pairs of electrons
and the planarity of ketone group can be interact with a bonding site
through hydrogen bonding, in which carbonyl oxygen acts as a
hydrogen bond acceptor.9 Besides, the ketone group may also
perform dipole-dipole interaction with the binding site due to its
significant dipole moment.9 Considering the presented potential of
the compounds containing ketones and 1,2,3-triazole groups, hybrid
compounds with both structures would be an advantageous strategy
to search for new bioactive molecules.
In this sense, monoamine oxidases (MAO, E.C. 1.4.3.4.) are
flavoenzymes linked to the external mitochondrial membrane of
mammals. Since their discovery almost a century ago,10 these
enzymes have been the target of extensive biochemical and
pharmacological studies, mainly due to its central role in the
metabolism of neurotransmitters.11 MAO exists in two different
isoforms, MAO-A and MAO-B, which exhibit different specificities
regarding their substrates, inhibitors, and tissue locations.12 MAO-A
preferentially oxidizes serotonin (5-HT), norepinephrine (NE) and
HO
O
N
N
HN
O
N
H
O
F
O
S
N
O
S
N
O
N
N
N
N
OH
O
S
NH2
N
H
N
F
OH
O
H2N
Rufinamide®
Anticonvulsant agent
Tazobactam®
Antibiotics
Cefatrizine®
Antibiotics
Figure 1 Drugs containing 1,2,3-triazole nucleus.
a. Laboratório de Síntese Orgânica Limpa – LASOL, CCQFA, Universidade Federal de
Pelotas UFPel, P. O. Box 354, 96010-900, Pelotas, RS, Brazil
–
*diego.alves@ufpel.edu.br (D. Alves)
b. Grupo de Pesquisa em Neurobiotecnologia - GPN, CDTec, Universidade Federal de
Pelotas - UFPel, Pelotas, RS, Brazil.
c. Laboratorio de Bioquímica e Neurofarmacologia Molecular - LABIONEM,
Universidade Federal de Pelotas - UFPel, Pelotas, RS, Brazil.
*cabruning@yahoo.com.br (C. A. Brüning)
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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epinephrine (EN) and is irreversibly inhibited by low doses of
In order to explore the generality, the reaction scope was
View Article Online
DOI: 10.1039/D0NJ04735J
clorgyline, while MAO-B uses phenylethylamine as a substrate and is extended using both conventional heating (method A, Table S1,
irreversibly blocked by low doses of selegiline .12 Besides, dopamine entry 5) and the ultrasound irradiation (method B, Table S2,
(DA) and tyramine are common substrates for both isoforms.13
These biogenic amines, such as 5-HT, NE and DA, play a crucial terminal alkynes. The reaction tolerated arylalkynes containing
role in modulating mood and emotion, as well as in motor, electron-donating groups, such as p-methyl, p-ethyl, p-tbutyl
perceptual and cognitive function control, so abnormal levels of and p-methoxy, giving the desired compounds in a good to
these monoamines have been associated with several excellent yields in both methods (Figure 2, method A: 85-98%,
neuropsychiatric disorders,14 such as Parkinson's disease,15 method B: 86-97%, compounds 3a-e).
depression16 and Alzheimer's disease.17 In this context, MAO
entry 2). Initially, the reactivity was evaluated using different
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O
method A
O
CuI (10 mol%),
DMSO, N2, 60 ºC, 24 h
conventional heating
inhibitors have long been developed and are widely used in clinics for
thiourea (20 mol%)
Et3N (2 equiv)
+
N
R
R
the treatment of many neuropsychiatric and neurodegenerative
disorders. For example, MAO-A inhibitors have been shown to be
effective as antidepressant drugs18 and MAO-B inhibitors have been
used for the treatment of Parkinson’s disease.19 2’-(1,2,3-Triazoyl)-
acetophenones are not structurally related to classical MAO
inhibitors, that usually have structure similar to phenylethylamine
and most of them with reactive groups, like hydrazine,
propargylamine and cyclopropylamine. However, triazol-containing
molecules have already demonstrated inhibitory effect on both
MAO-A and MAO-B activities.20
In view of what was explained above and according to our studies
on the development of new functionalized 1,2,3-triazoles, we report
herein the results of the synthesis of 2’-(1,2,3-triazoyl)-
acetophenones by a CuAAC of 2’-azidoacetophenone with alkynes
using thiourea as a ligand. Molecular docking and MAO activity
analyses of synthesized compounds were performed to evaluate
whether the 2’-(1,2,3-triazoyl)-acetophenones inhibit MAO isoforms.
method B
N3
O
N
N
2a-s
1a
DMSO:H2O (3:1)
US 40%, 30 min
3a-s
40-98%
O
O
N
N
N
N
N
N
N
N
N
3a
3b
3c
method A = 98%
method B = 97%
method A = 98%
method B = 86%
method A = 89%
method B = 96 %
O
O
O
N
O
N
N
N
N
N
N
N
N
O
3d
3e
3f
method A = 94%
method B = 85%
method A = 85%
method B = 88%
method A = 71%
method B = 78%
O
O
O
CF3
Cl
N
N
N
N
N
N
N
N
N
3g
3h
3i
Results and discussion
Synthesis
method A = 48%
method B = 45%
method A = 50%
method B = 54%
method A = 89%
method B = 92%
O
O
O
Focused on proceed the studies using the thiourea as ligand to
form a new class of 1,2,3-triazoles by CuAAC reactions, the initial
conditions were based in a previous repot in literature by our
research group.21 Thus, we carried out a long study to obtain the
optimized conditions for the synthesis of triazole 3a through the
reaction between 2’-azidobenzophenone 1a and phenylacetylene 2a
using conventional heating, as well as the use of ultrasound
irradiation (US)22 as an alternative energy source (Scheme 1) (for
more details see support information, Tables S1 and S2).
N
N
N
N
N
N
N
N
N
3j
3k
3l
method A = 71%
method B = 40%
method A = 77%
method B = 61%
method A = 71%
method B = 65%
O
O
O
N
HO
N
N
N
N
NH2
N
N
N
N
N
O
O
3m
3n
3o
catalyst, ligand
method A = 63%
method B = 68%
method A = N.F.
method B = N.F.
method A = N.F.
method B = N.F.
temperature, base,
solvent, 24 h
+
N
O
N3
O
O
N
1a
2a
N
3a
S
Se
Reaction Parameters
N
COOR
N
N
N
N
N
N
N
N
Under conventional heating
3p
method A, , R = Et (N.F.)
3r
3s
Catalyst: CuI
, CuBr, CuBr2, Cu(OAc)2
3q
, R = H (N.F.)
method A = 75%
method B = 78%
method A = 93%
method B = 89%
3p
method B, , R = Et (N.F.)
Catalyst (mol%): 10
5, , 15, without
3q
, R = H (N.F.)
Temperature (°C):
60
25, 50, , 70, 80, 100, 120
Figure 2: Generality in the synthesis of 2’-(1,2,3-triazoyl)-acetophenone
3. Method A: the reactions were performed with 2’-azidoacetophenone
(1a) (0.25 mmol), alkyne (2a-t) (0.25 mmol), thiourea (20 mol%), TEA
(0.5 mmol), CuI (10 mol%) and DMSO (0.5 mL) at 60 ºC for 24 h under
N2 atmosphere. Method B: the reactions were performed with 2’-
azidoacetophenone (1a) (0.25 mmol), alkyne (2a-t) (0.25 mmol),
thiourea (20 mol%), TEA (0.5 mmol), CuI (10 mol%) and DMSO:H2O (3:1)
(0.5 mL) under US irradiation (40% of amplitude) for 30 min.
Solvent: DMSO
, 1,4-dioxane, DMF, Ethanol, H2O, DMSO:H2O (3:1)
Ligand: Thiourea
, Bipy
20
Ligand (mol%):
10,
Base: Et N
, DBU
3
Under US irradiation
Solvent:
DMSO:H O (3:1)
2
DMSO,
Amplitude (%):
40
20, 30, , 50, 60
Time (min):
30
20,
Scheme 1
2 | J. Name., 2012, 00, 1-3
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Indeed, a slight decreasing in the yield was obtained when compound 3b made hydrophobic interactions with Phe352, Ile335,
arylalkyne containing methoxy group in the orto position at Tyr407 and a hydrogen bond with Ala68 in MAO-A (Figure 3a and 3d).
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DOI: 10.1039/D0NJ04735J
aromatic ring was used, probably due to steric hindrance (Figure
2, method A: 71%, method B: 78%, compound 3f).
Unluckily, when arylalkynes having electron-withdrawing
groups (p-Cl, m-CF3) bonded in aromatic ring were used, a
Table 1 Molecular docking scores of 2’-(1,2,3-triazoyl)-acetophenones on the
isoforms of monoamine oxidase (MAO) -A (PDB ID: 2Z5X) and MAO-B (PDB
ID: 5MRL) given by Autodock Vina software.
Docking Scores (Kcal/mol)
significant decrease in the yield of 1,2,3-triazoles 3g-h were
Compounds
MAO-A
-7.4
-9.7
-9.7
-7.9
-7.6
-7.2
-7.1
-9.2
-11.3
-6.5
-7.2
-8.9
-8.2
-7.0
-9.1
MAO-B
-7.2
-8.6
-8.5
-7.1
-6.2
-7.0
-6.7
-7.7
-8.4
-7.0
-5.7
-7.3
-6.4
-6.6
-7.4
observed (Figure 2, method A: 48-50%, method B: 45-54%,
compounds 3g-h, respectively). Interestingly, the 1,2,3-triazole
3i was obtained in good yield when 2-ethynylnaphthalene 2i
was used as staring material (Figure 2, method A: 89%, method
B: 92%, compound 3i). We extended the reaction scope using
alkyl alkynes 2j-l such as 3,3-dimethylbut-1-yne, hept-1-yne, 1-
ethynylcyclohex-1-ene, which provided the desired products 3j-
l from moderated to good yields (Figure 2, method A: 71-77%,
method B: 40-65%, compounds 3j-l). Also, this methodology
tolerated the propargyl alcohol derivative 2m giving the 1,2,3-
triazole 3m in moderated yield (Figure 2, method A: 63%,
method B: 68%, compound 3m).
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3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3r
3s
Unfortunately, when we used as starting materials the
corresponding alkynes 2n-q, such as N,N-dimethylprop-2-yn-1-
amine, 2-methylbut-3-yn-2-amine, ethyl propiolate and
propionic acid, the desired products 3n-q were not formed in
both methods. Finally, aiming to obtain products containing
chalcogen atom in their structures, we used propargyl sulfides
and selenides as starting materials and the compounds 3r-s
were obtained in good to excellent yield (Figure 2, method A:
75-93%, method B: 78-89%, compounds 3r-s, respectively).
In the Figures 3b and 3e are shown the interactions made by
compound 3c, such as hydrophobic interactions with Phe352,
Ile180, Phe280, Ile335 and Tyr407. Similarly, compound 3i made
multiple hydrophobic interactions with the residues Tyr407,
Tyr444, Ile335, Arg51, Cys406 and a hydrogen bond with Ala68
(Figure 3c and 3f).
Noteworthy, it has been described that Tyr407 and Tyr444
are essential residues in MAO-A catalytic activity and may be
involved in the non-covalent binding to FAD, forming an
aromatic sandwich that stabilizes the substrate binding.23
In this sense, these tyrosines have been proposed to orient
the substrate for oxidation or to activate the amine by
enhancing its nucleophilicity, while the rotation of Phe208 acts
like a gatekeeper and allows the two cavities to be fused into
one larger site.24 The Ile335 has also been proposed to have a
crucial role in substrate/inhibitor selectivity, accommodating its
chemical structure into the catalytic cavity.25 These reported
molecular interactions might suggest 3b, 3c and 3i as MAO-A
inhibitors.
Molecular Docking
With the purpose of select the molecules most probable to
have an inhibitory effect in MAO activity, a virtual screening
using the molecular docking tool was performed. Our results
demonstrated that the 2’-(1,2,3-triazoyl)-acetophenones 3b, 3c
and 3i showed the strongest affinity in both isoforms of MAO
(Table 1).
Compounds 3b and 3c showed an affinity of -9.7 kcal/mol, while
the compound 3i, -11.3 kcal/mol in MAO-A and stayed all in similar
position in the catalytic site of the enzyme. More specifically,
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Figure 3. Molecular docking results expressed by protein ligand interactions in (a) compound 3b, (b) compound 3c, and (c) compound 3i and the tridimensional
binding position in (d) compound 3b, (e) compound 3c and (f) compound 3i in MAO-A isoform (PDB ID: 2Z5X).
The compounds 3b, 3c and 3i also showed strong affinity Arg36, Pro277, Glu391, Tyr393 and Pro234 (Figure 4b and 4e).
with the isoform MAO-B, with docking scores of -8.6, -8.5 and - As is shown in Figure 4c and 4f, the compound 3i stay in a similar
8.4 kcal/mol, respectively (Table 1). In protein-ligand position of 3c, making predominantly hydrophobic interactions,
interactions, 3b made hydrogen bonds with Tyr112 and Asn116 such as Pro277, Tyr393, Pro234 and Leu250. This data can be
surrounded by hydrophobic interactions with Phe103, Arg120 visualized in Figure 5b, where all the ligands are superimposed
and Glu483 (Figure 4a and 4d). Despite the similar docking to show the differences in the the binding mode between
scores, compound 3c stay in different position in MAO-B, compound 3b in comparison with the others.
stabilized through hydrophobic interactions with residues
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Figure 4. Molecular docking results expressed by protein ligand interactions in (a) compound 3b, (b) compound 3c and (c) compound 3i and
the tridimensional binding position in (d) compound 3b, (e) compound 3c and (f) compound 3i in MAO-B isoform (PDB ID: 5MRL).
New proposed MAO-B inhibitors stay in similar position of results demonstrated that compounds 3b, 3c and 3i inhibited
compound 3b, as Diels-Alder type adducts26 through both isoforms of MAO (Figure 6 and 7), confirming the
interactions with Tyr112, Phe103, Arg120 and Glu483, which molecular docking prediction. Table 2 shows the calculated IC50
might suggest a different inhibitory mechanism of action, not values of the compounds 3b, 3c, 3i and the positive controls
necessarily related to active site. Besides, it is known that clorgyline and pargyline on MAO-A and MAO-B activities. In
Phe103 is located in the loop guarding the active site cavity and relation to MAO-A activity, the lowest IC50 value was obtained
is shown to be one of the targets of MAO-B inhibitors such as with the compound 3c (2.64 µM), which has the ethyl radical.
pioglitazone and safinamide.27 Regarding to the interactions Regarding the MAO-B isoform, the compound 3i, which has an
performed by 3c and 3i, they presented similar binding mode to aromatic ring as substituent, showed the lowest IC50 value
coumarin derivates with inhibitory MAO-B effect.28 Interesting, (41.47 µM). The IC50 values obtained for 2’-(1,2,3-triazoyl)-
Tyr393 is shown to be crucial for MAO-B inhibition, since acetophenones were higher than those obtained for the
participate in covalent binding of FAD directly affecting the positive controls clorgyline (0.0015 µM) and pargyline (0.0668
catalytic activity of the enzyme.23
µM), inhibitors of MAO-A and MAO-B, respectively. However,
pargyline and clorgyline and the available MAO inhibitors
presented many side effects, resulting in poor compliance and
hence therapeutic failures.29 In this way, the discover of new
In vitro results
Monoamine oxidase activity in vitro
Considering the molecular docking results, the compounds MAO inhibitors is noteworthy.
3b, 3c and 3i were selected to test their effect on MAO-A and
These results combined with molecular docking analyses
MAO-B activities in vitro, using mice brain homogenates. The show that indeed compound 3b, 3c and 3i have inhibitory
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activity of MAO-A and B. However, compound 3b and 3c molecular docking approach is used a crystalographic isoform of
View Article Online
demonstrated same affnity with MAO-A, compound 3b the enzyme to predict the affinity of thDeOcI:o1m0.1p0o3u9n/Dd0NthJ0ro47u3g5hJ
presented a higher potency in in vitro evaluation. The same computational calculations, excluding several physicochemical
occurs in MAO-B activity, since compound 3i demonstrated parameters that are involved in in vitro evaluation utilizing
similar docking scores when compared to compound 3b and 3c, tissue as substrate, such as solubility and lipophilicity. In this
but showed a lower efficiency. Although, is worth to be sense, although molecular docking is a great aproach to predict
highlighted that all the tested compounds demonstrated similar the affinity of compounds with the target protein and to better
efficacy. This could be explained for several reasons, since there undestand the interactions and ligand binding mode, the in
is a high discrepancy between the two adopted methods for vitro evaluation might generate data with closer aproximation
evaluation, not making them strictly comparable themselves. In of reality.
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Figure 5. Superimposed binding modes of compounds 3b (yellow), 3c (orange) and 3i (pink) in (a) MAO-A (PDB ID: 2Z5X) and (b) MAO-B (PDB ID:
5MRL).
Figure 6. Effect of compounds (a) 3b, (b) 3c, (c) 3i and (d) clorgyline on cerebral MAO-A activity in vitro. Data were expressed as means ± S.E.M.
of four to six individual experiments. MAO-A activity was expressed as nanomole of 4-hydroxyquinoline per milligram of protein per minute. The
values found were considered statistically significant when p<0.05. *p < 0.05, **p < 0.01 and ***p < 0.001 when compared with control group
(One-way ANOVA followed of the Newman–Keul’s test). C: control; V: vehicle.
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Figure 7. Effect of compounds (a) 3b, (b) 3c, (c) 3i and (d) pargyline on cerebral MAO-B activity in vitro. Data were expressed as means ± S.E.M.
of four to six individual experiments. MAO-B activity was expressed as nanomole of 4-hydroxyquinoline per milligram of protein per minute. The
values found were considered statistically significant when p<0.05. **p < 0.01 and ***p < 0.001 when compared with control group (one-way
ANOVA followed of the Newman–Keul’s test). C: control; V: vehicle.
Table 2 Half-maximal inhibitory concentration (IC50) values of 2’-(1,2,3-
triazoyl)-acetophenones on MAO-A and MAO-B activities
azidoacetophenone with alkynes. Overall, reactions were
performed under mild conditions and the reactions worked well
in the presence of catalytic amount of copper iodide as catalyst
IC50 (µM)
Compounds
MAO-A
47.29
02.64
ND
0.0015
-
MAO-B
173.5
77.18
41.47
-
and thiourea as ligand. When we employed the ultrasound
irradiation as alternative source of energy a significant decrease
in the time reactional was observed, noting that there were no
large differences in yields when compared to those obtained
when using conventional heating. In addition, three compounds
presented inhibitory effect on MAO-A and MAO-B activities and
may be of interest to develop new therapeutic strategies for
monoamine deficit related-neuropsychiatric disorders, such as
depression and Parkinson’s disease.
3b
3c
3i
Clorgyline
Pargyline
0.0668
ND: not determined
MAO inhibitors have contributed to the improvement of
several pathologies such as depression, Parkinson's disease,
Alzheimer's disease, prostate cancer, and heart disease,30
pointed the therapeutic relevance of these compounds.
Despite, their clinical use also showed some limitation due to
adverse effects, as well as interactions with food and
medicines.31 In this way, the search for more suitable
compounds is particularly important. The 2’-(1,2,3-triazoyl)-
acetophenones through molecular docking and in vitro studies
demonstrated to inhibit both isoforms of MAO, MAO-A and B,
and could be explored as new MAO inhibitors.
Experimental
General Information: The reactions were monitored by TLC carried
out on Merck silica gel (60 F254) by using UV light as visualizant agent
and 5% vanillin in 10% H2SO4 and heat as developing agents. Baker
silica gel (particle size 0.040-0.063 mm) was used for flash
chromatography. The ultrasound-promoted reactions were
performed using a Cole Parmer-ultrasonic processor Model CPX 130,
with a maxim power of 130 W, operating at amplitude of 40% and a
frequency of 20 kHz. Hydrogen nuclear magnetic resonance (1H
NMR) spectra were obtained on a Bruker Avance III HD 400
spectrometer at 400 MHz. The spectra were recorded in CDCl3
solutions. The chemical shifts are reported in ppm, referenced to
Conclusions
In summary we developed two general methods for
accessing a series of new 2’-(1,2,3-triazoyl)-acetophenones by a
copper catalyzed azide-alkyne cycloaddition of 2’-
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tetramethylsilane as the internal reference. Coupling constants (J) 1-(2-(4-(p-tolyl)-1H-1,2,3-triazoyl)phenyl)ethan-1-one 3b: method
View Article Online
DOI: 10.1039/D0NJ04735J
are reported in hertz. Abbreviations to denote the multiplicity of a A: yield: 0.068 g (98%), method B: yield: 0.059 g (86%); white solid;
1
particular signal are s (singlet), d (doublet), t (triplet), quint (quintet), mp 104-106 ºC; H NMR (400 MHz, CDCl3) δ= 8.06 (s, 1H), 7.79 (d, J
sext (sextet), and m (multiplet). 13C NMR spectra were obtained on a = 8.1 Hz, 2H), 7.71 (dd, J = 7.6, 1.4 Hz, 1H), 7.66 – 7.59 (m, 2H), 7.51
Bruker Avance III HD 400 spectrometer at 100 MHz. The chemical (dd, J = 7.6, 1.4 Hz, 1H), 7.26 (d, J = 8.1 Hz, 2H), 2.40 (s, 3H), 2.20 (s,
shifts are reported in ppm, referenced to the solvent peak of CDCl3 3H). 13C {1H} NMR (100 MHz, CDCl3) δ= 199.9, 148.6, 138.6, 136.6,
Low-resolution mass spectra were obtained with a Shimadzu GC-MS- 134.6, 132.0, 130.0, 129.7, 129.1, 127.2, 125.9, 125.6, 120.7, 29.4,
QP2010 mass spectrometer. High resolution mass spectra (HRMS) 21.4. MS (relative intensity) m/z: 277(3.8), 249(86.5), 234(87.0),
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were recorded on a Bruker Micro TOF-QII spectrometer 10416.
220(30.6), 207(46.9), 167(20.3), 149(61.9), 130(68.2), 119(100.0).
HRMS (APCI-QTOF) calculated mass for C17H16N3O [M+H]+: 278.1288,
found: 278.1297.
General procedure for the synthesis of 2’-(1H-1,2,3-triazol-1-yl)
acetophenone 3a-s (Method A)
1-(2-(4-(4-ethylphenyl)-1H-1,2,3-triazoyl)phenyl)ethan-1-one 3c:
In
a glass vial was added 0.25 mmol (0.040 g) of 2’-
method A: yield: 0.064 g (89%), method B: yield: 0.069 g (96%);
azidoacetophenone 1, 0.25 mmol of alkyne 2a-s, 20 mol% thiourea,
DMSO (0.5 mL), TEA (2 equiv.) and CuI (10 mol%). The glass vial was
placed under heating in an oil bath at 60 ºC on a magnetic stirrer and
under a nitrogen atmosphere. The reaction medium was maintained
under stirring for 24 hours. After this time, the mixture was washed
with saturated aqueous NaCl solution (3x50 mL) and
dichloromethane (DCM) (50 mL). The organic phase was separated
and dried by the addition of anhydrous MgSO4. Subsequently, the
solvent was evaporated under low pressure. The products were
purified through a silica gel chromatographic column with a Hex /
AcOEt mixture as the eluent.
1
white solid; mp 101-103 ºC; H NMR (400 MHz, CDCl3) δ= 8.08 (s,
1H), 7.82 (d, J = 8.2 Hz, 2H), 7.70 (dd, J = 7.5, 1.6 Hz, 1H), 7.64 – 7.55
(m, 2H), 7.50 (dd, J = 7.7, 1.2 Hz, 1H), 7.28 (d, J = 8.2 Hz, 2H), 2.69 (q,
J = 7.6 Hz, 2H), 2.18 (s, 3H), 1.26 (t, J = 7.6 Hz, 3H).13C {1H} NMR (100
MHz, CDCl3) δ= 199.8, 148.6, 144.9, 136.5, 134.5, 132.0, 129.9, 129.0,
128.5, 127.4, 125.8, 125.5, 120.7, 29.3, 28.8, 15.6. MS (relative
intensity) m/z: 291(2.0), 263(42.3), 248(100.0), 234(28.7),
206(28.8), 133(60.5). HRMS (APCI-QTOF) calculated mass for
C18H18N3O [M+H]+: 292.1444, found: 292.1443.
1-(2-(4-(4-(tbutyl)phenyl)-1H-1,2,3-triazoyl)phenyl)ethan-1-one 3d:
method A: yield: 0.075 g (94%), method B: yield: 0.068 g (85%);
General procedure for the synthesis of 2’-(1H-1,2,3-triazol-1-yl)
acetophenone 3a-s (Method B)
1
yellowish solid; mp 135-137 ºC; H NMR (400 MHz, CDCl3) δ= 8.30
(s, 1H), 7.85 (d, J = 8.3 Hz, 2H), 7.72 – 7.54 (m, 4H), 7.47 (d, J = 8.4 Hz,
2H), 2.17 (s, 3H), 1.35 (s, 9H). 13C {1H} NMR (100 MHz, CDCl3) δ=
199.0, 151.1, 147.8, 135.7, 133.8, 131.5, 129.3, 128.5, 126.7, 125.3,
125.1, 124.7, 120.3, 34.2, 30.8, 28.7. MS (relative intensity) m/z:
319(1.4), 291(33.9), 276(100.0), 261(9.0), 248(10.2), 234(44.3),
161(54.7), 131(34.2). HRMS (APCI-QTOF) calculated mass for
C20H22N3O [M+H]+: 320.1757, found: 320.1757.
In glass vial was added 0.25 mmol (0.040 g) of 2’-
azidoacetophenone 1, 0.25 mmol of alkyne 2a-s, 20 mol% thiourea,
DMSO:H2O (3:1) (0.5 mL), TEA (2 equiv.) and CuI (10 mol%). An
ultrasound probe was placed in the glass vial containing the reaction
mixture. The amplitude of the ultrasound waves was fixed at 40%.
Then, the reaction mixture was sonicated for min. After this time, the
mixture was washed with saturated aqueous NaCl solution (3x50 mL)
and dichloromethane (DCM) (50 mL). The organic phase was
separated and dried by the addition of anhydrous MgSO4.
Subsequently, the solvent was evaporated under low pressure. The
products were purified through a silica gel chromatographic column
with a Hex / AcOEt mixture as the eluent.
1-(2-(4-(4-methoxyphenyl)-1H-1,2,3-triazoyl)phenyl)ethan-1-one
3e: method A: yield: 0.062 g (85%), method B: yield: 0.064 g (88%);
1
white solid; mp 122-124 ºC; H NMR (400 MHz, CDCl3) δ= 8.02 (s,
1H), 7.83 (d, J = 8.8 Hz, 2H), 7.71 (dd, J = 7.5, 1.6 Hz, 1H), 7.66-7.62
(m, 1H), 7.61-7.57 (m, 1H), 7.51 (dd, J = 7.7, 1.2 Hz, 1H), 6.99 (d, J =
8.8 Hz, 2H), 3.86 (s, 3H), 2.21 (s, 3H). 13C {1H} NMR (100 MHz, CDCl3)
δ= 199.9, 160.0, 148.4, 136.5, 134.6, 132.0, 129.9, 129.1, 127.3,
125.5, 122.7, 120.2, 114.5, 55.5, 29.4. MS (relative intensity) m/z:
293(5.9), 265(33.2), 250(83.6), 208(24.8), 180(5.8), 135(100.0),
91(70.7). HRMS (APCI-QTOF) calculated mass for C17H16N3O2 [M+H]+:
294.1243, found: 294.1237.
1-(2-(4-phenyl-1H-1,2,3-triazoyl)phenyl)ethan-1-one 3a: method A:
yield: 0.064 g (98%), method B: yield: 0.063 g (97%); white solid;
1
mp 106-108 ºC; H NMR (400 MHz, CDCl3) δ= 8.11 (s, 1H), 7.90 (d, J
= 7.1 Hz, 2H), 7.71 (dd, J = 7.4, 1.7 Hz, 1H), 7.66 – 7.57 (m, 2H), 7.51
(dd, J = 7.7, 1.3 Hz, 1H), 7.47 – 7.43 (m, 2H), 7.39 – 7.34 (m, 1H), 2.22
(s, 3H). 13C {1H} NMR (100 MHz, CDCl3) δ= 199.8, 148.5, 136.5, 134.5,
132.0, 130.0, 130.0, 129.1, 129.0 (2C), 128.6, 126.0 (2C), 125.6,
121.1, 29.4. MS (relative intensity) m/z: 263(2.9) 235(99.2),
220(100.0), 206(62.2), 193(56.2), 165(47.1), 130(19.1), 116(56.8),
105(99.5) HRMS (APCI-QTOF) calculated mass for C16H14N3O [M+H]+:
264.1131, found: 264.1142.
1-(2-(4-(2-methoxyphenyl)-1H-1,2,3-triazoyl)phenyl)ethan-1-one
3f: method A: yield: 0.052 g (71%), method B: yield: 0.057 g (78%);
1
yellowish solid; mp 109-111 ºC; H NMR (400 MHz, CDCl3) δ= 8.43
(dd, J = 7.7, 1.7 Hz, 1H), 8.38 (s, 1H), 7.72 (dd, J = 7.5, 1.5 Hz, 1H),
7.67-7.63 (m, 1H), 7.60 – 7.54 (m, 2H), 7.38 – 7.33 (m, 1H), 7.13-7.09
(m, 1H), 7.01 (d, J = 8.4, 1H), 3.95 (s, 3H), 2.15 (s, 3H). 13C {1H} NMR
(100 MHz, CDCl3) δ= 200.0, 155.9, 144.0, 136.6, 134.8, 132.0, 129.8,
129.5, 129.1, 127.9, 125.5, 124.3, 121.2, 118.8, 110.9, 55.5, 29.3. MS
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(relative intensity) m/z: 293(2.8), 264(12.1), 250(65.4), 222(43.8), (m, 3H), 7.46 (dd, J = 7.7, 1.2 Hz, 1H), 2.80 (t, J = 7.8 HVzi,ew2HAr)t,ic2le.1O2nli(nse,
DOI: 10.1039/D0NJ04735J
208(51.7), 180(37.2), 135(100), 91(88.0). HRMS (APCI-QTOF) 3H), 1.75 (q, J = 7.4 Hz, 2H), 1.40 – 1.37 (m, 4H), 0.93 – 0.90 (m, 3H).
calculated mass for C17H16N3O2 [M+H]+: 294.1243, found: 294.1239.
13C NMR {1H} (100 MHz, CDCl3) δ= 199.9, 149.3, 136.5, 134.8, 131.9,
129.7, 128.9, 125.4, 122.2, 31.5, 29.2, 29.1, 25.6, 22.5, 14.1. MS
(relative intensity) m/z: 229(1.1), 214(5.6), 186(20.5), 172(100.0),
146(12.0), 130(13.8), 91(58.8). HRMS (APCI-QTOF) calculated mass
for C15H20N3O [M+H]+: 258.1601, found: 258.1608.
1-(2-(4-(4-chlorophenyl)-1H-1,2,3-triazoyl)phenyl)ethan-1-one 3g:
method A: yield: 0.035 g (48%), method B: yield: 0.033 g (45%);
1
yellowish solid; mp 115-117 ºC; H NMR (400 MHz, CDCl3) δ= 8.09
(s, 1H), 7.84 (d, J = 8.5 Hz, 2H), 7.74 (dd, J = 7.4, 1.6 Hz, 1H), 7.68 –
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7.60 (m, 2H), 7.52 (d, J = 7.6 Hz, 1H), 7.43 (d, J = 8.5 Hz, 2H), 2.27 (s, 1-(2-(4-(cyclohex-1-en-1-yl)-1H-1,2,3-triazoyl)phenyl)ethan-1-one
3H). 13C {1H} NMR (100 MHz, CDCl3) δ= 199.7, 147.4, 136.5, 134.5, 3l: method A: yield: 0.047 g (71%), method B: yield: 0.043 g (65%);
134.4, 132.1, 130.2, 129.3, 129.1, 128.6, 127.3, 125.8, 121.3, 29.5. yellow solid; mp 64-66 ºC; 1H NMR (400 MHz, CDCl3) δ= 7.74 (s, 1H),
MS (relative intensity) m/z: 297(4.9), 269(99.3), 254(29.7), 7.68 (dd, J = 7.5, 1.3 Hz, 1H), 7.63 – 7.54 (m, 2H), 7.45 (d, J = 7.8 Hz,
240(33.0), 234(72.0), 227(50.1), 219(57.4), 150(52.9), 139(100.0). 1H), 6.64 (s, 1H), 2.43-2.41 (m, 2H), 2.24 – 2.22 (m, 2H), 2.15 (s, 3H),
HRMS (APCI-QTOF) calculated mass for C16H13ClN3O [M+H]+: 1.83-1.77 (m, 2H), 1.72-1.67 (m, 2H). 13C {1H} NMR (100 MHz, CDCl3)
298.0742, found: 298.0746.
δ= 199.9, 150.2, 136.4, 134.6, 131.9, 129.7, 129.0, 126.8, 126.2,
125.3, 119.6, 29.3, 26.4, 25.4, 22.5, 22.2. MS (relative intensity)
m/z: 267(1.5), 238(38.1), 224(33.4), 210(61.0),196(61.8),
168(64.4), 146(29.3), 120(33.9), 109(62.0), 91(100.0). HRMS
(APCI-QTOF) calculated mass for C16H18N3O [M+H]+:268.1444, found:
268.1447.
1-(2-(4-(3-(trifluoromethyl)phenyl)-1H-1,2,3-
triazoyl)phenyl)ethan-1-one 3h: method A: yield: 0.041 g (50%),
method B: yield: 0.045 g (54%); brown solid; mp 67-69 ºC; 1H NMR
(400 MHz, CDCl3) δ= 8.12 (s, 1H), 8.08 (s, 1H), 8.00 (d, J = 7.5 Hz, 1H),
7.65 (dd, J = 7.5, 1.7 Hz, 1H), 7.59 – 7.46 (m, 4H), 7.43 (dd, J = 7.6, 1.3
Hz, 1H), 2.20 (s, 3H). 13C NMR {1H} (100 MHz, CDCl3) δ= 199.5, 147.0, 1-(2-(4-(1-hydroxycyclopentyl)-1H-1,2,3-triazoyl)phenyl)ethan-1-
136.3, 134.3, 132.1, 131.4 (q, J = 32.5 Hz) , 130.9, 130.2, 129.6, 129.1, one 3m: method A: yield: 0.043 g (63%), method B: yield: 0.046 g
1
128.1, 125.8, 125.4, 125.2, 125.2, 125.12, 125.14 (q, J = 3.7 Hz), 124.1 (68%); white solid; mp 59-61 ºC; H NMR (400 MHz, CDCl3) δ= 7.77
(q, J = 273.7 Hz), 122.8 (q, J = 3.8 Hz), 121.8, 120.0, 29.4. MS (relative (s, 1H), 7.61 (dd, J = 7.3, 1.9 Hz, 1H), 7.56 – 7.47 (m, 2H), 7.39 (dd, J =
intensity) m/z: 331(1.1), 303(100.0), 288(28.6), 274(48.3), 7.6, 1.6 Hz, 1H), 2.93 (s, 1H), 2.16 – 1.75 (m, 11H). 13C {1H} NMR (100
261(36.9), 234(25.5), 173(55.5), 120(24.7), 91(74.7). HRMS (APCI- MHz, CDCl3) δ= 199.8, 155.1, 136.3, 134.55, 132.0, 129.9, 129.0,
QTOF) calculated mass for C17H13F3N3O [M+H]+: 332.1005, found: 125.6, 121.7, 79.0, 41.4, 29.2, 23.7. MS (relative intensity) m/z:
332.1015.
242(12.0), 224(24.9), 214(37.7), 200(32.4), 196(18.6), 186(40.2),
172(50.8), 146(100.0), 120(53.7). HRMS (APCI-QTOF) calculated
mass for C15H18N3O2 [M+H]+: 272.1394, found: 272.1382.
1-(2-(4-(naphthalen-2-yl)-1H-1,2,3-triazoyl)phenyl)ethan-1-one 3i:
method A: yield: 0.069 g (89%), method B: yield: 0.072 g (92%);
white solid; mp 158-160 ºC; H NMR (400 MHz, CDCl3) δ= 8.33 (s, 1-(2-(4-((phenylthio)methyl)-1H-1,2,3-triazoyl)phenyl)ethan-1-one
1H), 8.12 (s, 1H), 7.89 (dd, J = 8.5, 1.6 Hz, 1H), 7.83 – 7.74 (m, 3H), 3r: method A: yield: 0.058 g (75%), method B: yield: 0.060 g (78%);
7.63 (dd, J = 7.5, 1.6 Hz, 1H), 7.56 – 7.38 (m, 5H), 2.16 (s, 3H). 13C {1H} redish solid; mp 54-56 ºC; H NMR (400 MHz, CDCl3) δ= 7.60 – 7.57
1
1
NMR (100 MHz, CDCl3) δ= 199.8, 148.5, 136.4, 134.5, 133.6, 133.4, (m, 2H), 7.53 – 7.45 (m, 2H), 7.29-7.27 (m, 3H), 7.21-7.17 (m, 2H),
132.0, 130.0, 129.1, 128.8, 128.4, 127.9, 127.3, 126.7, 126.5, 125.6, 7.13 – 7.08 (m, 1H), 4.23 (s, 2H), 1.93 (s, 3H). 13C {1H} NMR (100 MHz,
124.9, 123.9, 121.4, 29.4. MS (relative intensity) m/z: 313(12.5), CDCl3) δ= 199.5, 145.9, 136.3, 135.1, 134.4, 132.0, 130.0, 129.9,
285(100.0), 270(36.5),243(55.7), 215(18.1), 166(70.3), 155(82.1), 129.11, 129.08, 126.8, 125.6, 123.7, 29.0, 28.8. MS (relative
139(46.8), 127(23.6). HRMS (APCI-QTOF) calculated mass for intensity) m/z: 309(19.1), 238(55.6), 172(77.5), 154(16.9),
C20H16N3O [M+H]+: 314.1288, found: 314.1287.
146(16.0), 130(65.3), 109(21.1), 91(100.0). HRMS (APCI-QTOF)
calculated mass for C17H16N3OS [M+H]+: 310.1009, found: 310.1019.
1-(2-(4-(tbutyl)-1H-1,2,3-triazoyl)phenyl)ethan-1-one 3j: method A:
yield: 0.043 g (71%), method B: yield: 0.024 g (40%); white solid; 1-(2-(4-((phenylselanyl)methyl)-1H-1,2,3-triazoyl)phenyl)ethan-1-
1
mp 93-95 ºC; H NMR (400 MHz, CDCl3) δ= 7.67 (dd, J = 7.5, 1.6 Hz, one 3s: method A: yield: 0.083 g (93%), method B: yield: 0.079 g
1
1H), 7.63 – 7.53 (m, 3H), 7.47 (dd, J = 7.7, 1.1 Hz, 1H), 2.10 (s, 3H), (89%); yellowish solid; mp 62-64 ºC; H NMR (400 MHz, CDCl3) δ=
1.42 (s, 9H). 13C {1H} NMR (100 MHz, CDCl3) δ= 200.0, 158.6, 136.5, 7.60 (dd, J = 7.4, 1.8 Hz, 1H), 7.54 – 7.42 (m, 5H), 7.27 (dd, J = 7.3, 1.7
134.8, 131.8, 129.6, 128.9, 125.4, 120.2, 31.0, 30.4, 29.1. MS Hz, 1H), 7.20 – 7.17 (m, 3H), 4.17 (s, 2H), 1.97 (s, 3H). 13C {1H} NMR
(relative intensity) m/z: 214(2.6), 200(100.0), 182(15.9), 157(8.1), (100 MHz, CDCl3) δ= 199.5, 146.5, 136.4, 134.5, 133.6, 132.0, 130.0,
146(16.1), 105(12.0), 91(24.9). HRMS (APCI-QTOF) calculated mass 129.5, 129.3, 129.1, 127.7, 125.6, 123.4, 29.1, 20.4. 77Se {1H} NMR
for C14H18N3O [M+H]+: 244.1444, found: 244.1447.
(76 MHz, CDCl3) δ= 368.1. MS (relative intensity) m/z: 357(8.1),
286(4.9), 172(47.4), 154(26.5), 149(35.1), 130(100.0), 105(13.9),
91(78.8). HRMS (APCI-QTOF) calculated mass for C17H16N3OSe
[M+H]+: 358.0454, found: 358.0463.
1-(2-(4-pentyl-1H-1,2,3-triazoyl)phenyl)ethan-1-one 3k: method A:
yield: 0.049 g (77%), method B: yield: 0.039 g (61%); yellow oil; 1H
NMR (400 MHz, CDCl3) δ= 7.68 (dd, J = 7.5, 1.7 Hz, 1H), 7.64 – 7.54
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Molecular docking
M/KH2PO4 0.0106 M isotonized with KCl 0.0036 M, pH 7.4), the
specific inhibitors pargyline (a MAO-B inhibitor, 250 nM) or clorgyline
View Article Online
DOI: 10.1039/D0NJ04735J
The 2D structures of chemical compounds were draw with
Chemdraw, converted to 3D using the software Avogadro 0.9.4 and
their geometry was optimized following GAFF method.32 Auto Dock
Tools 1.5.4 program was used to set all rotatable bonds of ligands
rotate freely and the protein receptors were considered rigid.33
The 3D X-ray crystal structures of MAO isoforms were retrieved
from Protein Data Bank (MAO-A PDB ID: 2Z5X; MAO-B PDB ID: 5MRL)
and prepared using the software Auto Dock Tools 1.5.4. The protein
preparation consisted of fixing structures, deleting molecules, ions,
and water, fixing hetero groups and finally optimize the structure
using Gasteiger charges with 500 steps of minimization. CHIMERA
1.5.3 software was used previously to remove ligands in 3D
structure.34 The molecular docking using the software autodock vina
(version 1.1.1) was conducted with a grid box centered in all atom
structure, allowing the program to search for additional places of
probable interactions.35 The protein−ligand interactions were
analyzed by Discovery studio visualizer 2016.
(a MAO-A inhibitor, 250 nM) to differentiate MAO isoforms, and 2’-
(1,2,3-triazoyl)-acetophenones, dissolved in DMSO (final
concentrations of 0.01 to 500 µM). The inhibitory effect of the
positive controls clorgyline (final concentrations of 0.00001 to 0.1
µM) and pargyline (final concentrations of 0.001 to 0.25 µM) on
MAO-A and MAO-B activities, respectively, was evaluated
simultaneously. Then submaximal concentrations of kynuramine
dihydrobromide (90 μM to MAO-A and 60 μM to MAO-B assay) were
added to the reaction mixture as a nonselective substrate. Samples
were then incubated at 37 °C for 30 min. After incubation, the
reaction was terminated by adding trichloroacetic acid (TCA) 10%.
After cooling and centrifugation at 16,000×g for 5 min, an aliquot
of supernatant was added to NaOH 1 M. The fluorescence intensity
was measured spectrofluorimetrically with excitation at 315 nm and
emission at 380 nm. The concentration of 4-hydroxyquinoline was
estimated from its corresponding standard fluorescence curve. MAO
activity was expressed as nanomole of 4-hydroxyquinoline per
milligram of protein per minute. All the chemical reagents were of
analytical grade and obtained from standard commercial suppliers.
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Monoamine oxidase (MAO) activity
MAO was obtained from brain mice homogenate. Male adult
Swiss mice (25–30 g), from the Central Biotery of the Federal
University of Pelotas, were kept in a separate animal room at 22 ± 2
°C under a 12-h light/ dark cycle, lights turning on at 7:00 a.m.
Commercial diet and tap water were supplied ad libitum. All animal
procedures were performed in accordance with the Guidelines for
Care and Use of Laboratory Animals of Federal University of Pelotas
and experiments were approved by the Animal Ethics Committee of
Federal University of Pelotas (CEEA – number of approval 50890-
2019), affiliated to the Council for Control of Animal Experiments
(CONCEA) and the animal care was according to the National
Institutes of Health Guide for the care and use of laboratory animals
(NIH Publications No. 8023, revised 1978). All efforts were made to
minimize animals suffering and to reduce the number of animals
used in the experiments.
Mice were euthanized and the whole brains were quickly
removed and washed in ice cold isolation medium (Na2HPO4 0.0168
M/KH2PO4 0.0106 M isotonized with sucrose 0.32 M, pH 7.4).
Preparations of cerebral mitochondria were used for MAO assay as
described by Sampaio et al. and Soto-Otero et al..36 Briefly, after
removing blood vessels and pial membranes, brains were manually
homogenized with four volumes (w/v) of the isolation medium. Then,
the homogenate was centrifuged at 900×g at 4 °C for 5 min. The
supernatant was centrifuged at 12,500×g at 4 °C for 15 min. The
mitochondria pellet was then washed once with isolation medium
and recentrifuged under the same conditions. Finally, the
mitochondrial pellet was reconstituted in a buffer solution (Na2HPO4
0.0168 M/KH2PO4 0.0106 M isotonized with KCl 0.0036 M, pH 7.4)
and stored in aliquots at −20 °C for up to 48 h before enzyme assay.
The protein concentration of brain homogenates was measured
by the method of Bradford,37 using bovine serum albumin as the
standard, and fixed at 1 mg/mL. MAO activity was determined as
described by Sampaio et al. and Soto-Otero et al. with some
modifications.36 Aliquots of samples were incubated at 37 °C for 5
min in a medium containing buffer solution (Na2HPO4 0.0168
Statistical analysis
Statistical analysis of data was performed using a one-way
analysis of variance (ANOVA), followed by the Newman-Keul’s test.
Half-maximal inhibitory concentration (IC50) was determined by
nonlinear regression from individual experiments using ‘‘GraphPad
Software’’ (GraphPad software, San Diego, CA, USA). Data of MAO
assay in vitro were expressed as means ± S.E.M. Values of p<0.05
were considered statistically significant.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The authors are grateful for the financial support and
scholarships from the Brazilian agencies CNPq and FAPERGS
(PRONEM 16/2551-0000240-1). CNPq is also acknowledged for the
fellowship for R.G.J., L.S. and D.A. This study was partially financed
by the Coordenação de Aperfeiçoamento de Pessoal de Nivel
Superior-Brasil (CAPES)-Finance Code 001. We are grateful to Prof.
Thiago Barcellos from University of Caxias do Sul (UCS) for providing
the HRMS analysis.
Notes and references
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The synthesis of 2’-(1,2,3-triazoyl)-acetophenones by a CuAAC using thiourea as a ligand,
molecular docking and MAO activity analyses were performed.