Regioselective microwave synthesis and derivatization of 1,5-diaryl-3-amino-1,2,4-triazoles and a study of their cholinesterase inhibition properties

Article abstract of DOI:10.1039/c9ra04105b

Herein we describe the development of an efficient one-pot regioselective synthesis protocol to obtain N-protected or N-deprotected 1,5-diaryl-3-amino-1,2,4-triazoles from N-acyl-N-Boc-carbamidothioates. This improved protocol using microwave irradiation and low reaction times (up to 1 h) furnished desired compounds in yields ranging from 50 to 84%. This chemistry is useful for a variety of aromatic groups with electronically diverse substituents. The design and correct derivation of the amino group led to compounds able to inhibit cholinesterases with good IC₅₀ of up to 1 μM. Also, the mode of action (mixed-type) and SAR analysis for this series of compounds was described by means of kinetic and molecular modelling evaluations, showing potential for this class of compounds as new scaffolds for this biological activity.

Full text of DOI:10.1039/c9ra04105b

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Regioselective microwave synthesis and
derivatization of 1,5-diaryl-3-amino-1,2,4-triazoles
and a study of their cholinesterase inhibition
properties†
Cite this: RSC Adv., 2019, 9, 20356
Sabrina Neves Santos,ab Gabriela Alves de Souza,ab Thiago Moreira Pereira,ab
Daiana Portella Franco,ab Catarina de Nigris Del Cistia,b Carlos Mauricio R. Sant'Anna,b
ab
Renata Barbosa Lacerdaab and Arthur Eugen Kummerle
*
¨
Herein we describe the development of an efficient one-pot regioselective synthesis protocol to obtain N-
protected or N-deprotected 1,5-diaryl-3-amino-1,2,4-triazoles from N-acyl-N-Boc-carbamidothioates.
This improved protocol using microwave irradiation and low reaction times (up to 1 h) furnished desired
compounds in yields ranging from 50 to 84%. This chemistry is useful for a variety of aromatic groups
with electronically diverse substituents. The design and correct derivation of the amino group led to
compounds able to inhibit cholinesterases with good IC50 of up to 1 mM. Also, the mode of action
(mixed-type) and SAR analysis for this series of compounds was described by means of kinetic and
molecular modelling evaluations, showing potential for this class of compounds as new scaffolds for this
biological activity.
Received 30th May 2019
Accepted 22nd June 2019
DOI: 10.1039/c9ra04105b
rsc.li/rsc-advances
moiety (N-acylguanidines derived from benzotriazole or N-acyl
carbamidothioate) and a nucleophilic one (alkyl or substituted
Introduction
aryl hydrazines) (Fig. 2). Unfortunately, these processes suffer
from long reaction times, few examples, and low yields when
both substitutions are aryl groups. Furthermore, in many cases
they were unable to produce 3-N,N-unsubstituted amino tri-
azole compounds.
The second methodology, described by Szilagyi,2 is based
on a two steps mechanism: the opening of 1,3,4-oxadiazo-
lium perchlorate nucleus by the nucleophile cyanamide fol-
lowed by its recyclization (Fig. 2). Despite better in scope and
reactional yields compared to the Katritzky method, the
explosive nature of perchloric acid and perchlorate salts
The ve-membered azaheterocyle aminotriazole is widely
described in the literature and has received great attention from
the scientic community due to its diversity of biological
activities. This increased attention to this 1,2,4-triazole nucleus
comes from its possible non-classical bioisosteric replacement
of a cis-amide bond from bioactive compounds.1 3-amino-1,2,4-
triazoles have been described as an anti-inammatory agent
(A),2 as a treatment for bronchial asthma (B),3 as an inhibitor of
methionine aminopeptidase (C)4 and a NPY receptor,5 and as
a selective positive allosteric modulator of the a7 nicotinic
acetylcholine receptor (D)x (Fig. 1).6
Several procedures have already been described for the
synthesis of general 3-amino-1H-1,2,4-triazoles, with great
attention to solid phase, microwaves and combinatorial
synthesis.7 However, few of them explored the synthesis of 1,5-
diaryl-3-amino-1,2,4-triazoles. The rst methodology, described
by Katritzky8 and also adapted to solid phase synthesis by
Houghten,9 employs a cyclization reaction from an electrophilic
a
´
´
Laboratorio de Diversidade Molecular e Quımica Medicinal (LaDMol-QM, Molecular
Diversity and Medicinal Chemistry Laboratory), Chemistry Institute, Universidade
´
Federal Rural do Rio de Janeiro, Seropedica, Rio de Janeiro, 239897-000, Brazil.
E-mail: akummerle@hotmail.com
b
´
˜
´
Programa de Pos-Graduçao em Quımica (PPGQ), Universidade Federal Rural do Rio
´
de Janeiro, Seropedica, Rio de Janeiro, 239897-000, Brazil
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra04105b
Fig. 1 Bioactive compounds with 3-amino-1,2,4-triazole nucleus.
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Scheme 1 Synthesis of methyl N-acylisothiourea (3).
planned a three-steps protocol (Schemes 1 and 2). The rst step
was the synthesis of S-methylisothiourea (1) as previously
described,14 by the alkylation of urea with methyl iodide fol-
lowed by precipitation and ltration (99% yield). The second
step was the synthesis of S-methyl-N-benzoyl-isothiourea (3) in
73% yield from the reaction of S-methylisothiourea with an
activated benzotriazole intermediate, previously obtained from
the in situ reaction of benzoic acid (2a) with N,N0-diisopro-
pylcarbodiimide (DIC) and 1-hydroxybenzo-triazole (HOBt)
(Scheme 1).
The cyclization reaction leading to 1,5-diphenyl-3-amino-
1,2,4-triazole (6a) was performed in the last step by reacting
N-benzoylisothiourea (3) with phenylhydrazine (4). This reac-
tion was attempted in two different solvents and heating sour-
ces (Table 1). The best reacꢀtional yield was observed for the
conventional heating at 100 C in acetonitrile.
However, long reaction time (48 h) was required to observe
the consumption of reactants (Table 1, entry 3) and, although
75% of 6a could be obtained, 13% of its regioisomer (5) was also
formed, complicating their chromatographic separation.
Bearing in mind the possible low reactivity and the lack of
selectivity demonstrated by 3, we decided to introduce an
electron withdrawing group at –C]NH(S–CH3) moiety by
means of a labile tert-butiloxycarbonyl group (–BOC).
The synthesis of N-protected N-acyl-S-methylisothiourea (7)
was evaluated reacting 3 with di-tert-butyl dicarbonate in
dichloromethane at 70 ꢀC in a sealed borosilicate tube. However
only 40% of product 7 was furnished aer 48 h (Scheme 2A).
Aiming at to optimize the reaction process to obtain 7, a new
synthetic route was evaluated (Scheme 2B).
Instead of protecting the low reactivity N-acyl-S-methyl-
isothiourea (3) in the least step, the new process started with the
protection of S-methylisothiourea (1) in a similar condition as
above described, furnishing the N-Boc-S-methylisothiourea (8)
Fig. 2 Described synthetic strategies for the synthesis of 1,5-disub-
stituted 3-amino-1H-1,2,4-triazoles and our strategy for this work.
makes this methodology dangerous and not indicated to
scale-up synthesis.10,11
The third procedure was recently described by Shen,12 pre-
senting an interesting strategy for the C-arylation of amino-
triazoles at position 1 by means of a Negish coupling (Fig. 2).
Despite of good yields and scope demonstrated, the high cost of
this reaction associated with its drastic conditions in the nal
ꢀ
deprotection (6 M H2SO4, 80 C, 16 h) can make this reaction
limited in some cases. Thus, new simpler and more effective
methodologies for the synthesis of 1,5-diaryl-3-amino-1,2,4-
triazoles are still required.
Our goals in this work were: (1) to develop a simple one-pot
regioselective synthesis protocol to obtain N-protected (E) or
N-deprotected (F) 1,5-diaryl-3-amino-1,2,4-triazoles (Fig. 2) in
low reaction times and suitable for diverse R1 and R2 aromatic
groups, through an improvement of Katritzky procedure using
N-acyl-N-Boc-carbamidothioates; (2) given the importance of
microwave irradiation to heterocyclic chemistry,13 we decided
to evaluate its inuence in our synthesis. Furthermore, this
technique proved to be suitable to furnish different 1,2,3 and
1,2,4 triazole derivatives in good yields and low reaction
times;7b (3) derivatization studies of N-protected compounds E
to furnish series F, when R3 is an alkyl group; and (4) use of N-
derivatizations to correct design and obtain new 1,5-diaryl-3-
amino-1,2,4-triazole
cholinesterases.
derivatives
as
inhibitors
of
Results and discussion
Synthesis of triazoles
Our initial objective was to obtain the 1,5-diaryl-3-amino-1,2,4-
triazoles (6) without any protection. For this purpose, we Scheme 2 Synthesis of N-Boc-S-methylisothiourea derivatives (7).
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Table 1 Synthesis of 1,5-diphenyl-3-amine-1,2,4 triazole (6a)
isolated in its both protected (with BOC group) (9a), aer
reaction of up to 40 min (Table 2, entries 1 and 3), and depro-
tected form (6a), in the case when DMF was used as solvent in
48 h of reaction (Table 2, entry 2). Curiously, longer reaction
times in acetonitrile led to a by-product that we were not able to
identify, without any trace of targeted 1,5-diphenyl-3-amino-
1,2,4-triazoles (6a and 9a) by the end of reaction (Table 2,
entry 4).
In fact, as previous related for others amino-triazoles,7b the
use of microwave irradiation proved to be efficacious in
increasing reactional yields, leading to the protected product 9a
in yields of up to 90% in 40 minutes (Table 2, entry 7), as well as
being very efficient in obtaining the nal deprotected 1,5-
diphenyl-3-amino-1,2,4-triazole (6a), in shorter reaction time (1
h) and higher yield (82%) (Table 2, entry 6) compared to
conventional heating. Once more, the reaction in acetonitrile at
150 ꢀC led to the formation of the unknown by-product as
previous described for the conventional heating (Table 2, entry
8). Therefore, DMF was chosen as the best solvent to synthesise
the desired products, allowing the selective formation of the
protected and deprotected 1,5-diphenyl-3-amino-1,2,4-triazoles,
depending only on the temperature and reaction time.
With this set of conditions, the scope of reaction was eval-
uated varying the aromatic rings coming from both benzoic acid
and phenylhydrazine reactants (Table 3). The cyclo-
condensations furnished the functionalized 1,5-diaryl-3-amino-
1,2,4-triazoles in theirs protected and deprotected forms (9a–h
and 6a–f) in reasonable to good yields ranging from 47 to 90%
in reactions of up to 1 hour. For all compounds, the
Entry
Solv.
Temp.
Time
(5)c %
(6a)c %
1
2
3
4
DMF
DMF
ACN
ACN
100a
100b
100a
100b
48 h
2 h
4 8h
2 h
—
—
13
Trace
46
41
75
35
a
Conventional heating. b Microwave heating. c Isolated yields.
as a white solid in 86% yield aer 15 h. Subsequently, 8 was
reacted with correspondent acid chlorides in dichloromethane
at room temperature for 24 hours, leading to the desired
intermediates (7a–f) in yields ranging from 58 to 80%. This new
synthetic route was more suitable for obtaining the N-acyl-S-
methylisothiourea derivatives (7a–f) with higher yields and
lower reaction times. In order to evaluate the reactivity and
selectivity of the N-protected N-acyl-S-methylisothiourea (7a), we
started the cyclocondensations maintaining the same temper-
ature and solvents described before (i.e. DMF or acetonitrile as
solvent and 100 ꢀC) and the Table 2 shows the main results
obtained.
Initially the reactions were done without microwave assis-
tance (Table 2, entries 1–4). Depending on the reaction time it
was possible to observe that the cyclocondensation has
occurred and the 1,5-diphenyl-3-amino-1,2,4-triazole could be
Table 3 Scope of microwave-assisted synthesis of 1,5-diphenyl-3-
amino-1,2,4-triazoles (6 and 9)
Table
2 Optimization of 1,5-diphenyl-3-amine-1,2,4 triazole (6a)
synthesis
Prod.
Ar
Y
Timea
Temp.b
Yieldc
9a
9b
9c
9d
9e
9f
9g
9h
6a
6b
6c
6d
6e
6f
Ph
H
H
H
H
40
40
40
40
40
40
40
40
60
60
60
60
60
60
100
100
100
100
100
100
100
100
150
150
150
150
150
150
87
62
71
80
61
84
90
79
82
47
68
52
73
69
4-OCH3–Ph
4-Cl–Ph
4-NO2–Ph
Ph
Entry
Solv.
Temp.
Time
6a %c
9a %c
NO2
SO2CH3
H
H
H
H
H
H
Ph
1
2
3
4
5
6
7
8
DMF
DMF
ACN
ACN
DMF
DMF
ACN
ACN
100a
100a
100a
100a
100b
150b
100b
150b
40 min
48 h
40 min
48 h
40 min
60 min
40 min
60 min
—
76
—
—
—
82
—
—
71
—
74
—
79
—
87
—
4,5-DiBr-2-pyrrol
2-Pyrrol
Ph
4-OCH3–Ph
4-Cl–Ph
4-NO2–Ph
Ph
NO2
SO2CH3
Ph
a
a
Conventional heating. b Microwave heating. c Isolated yields.
Time in minutes. b Temperature in oC. c Isolated yields in %.
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deprotection has been well controlled, based on the tempera-
ture as standardized for 6a and 9a (Table 3). Also, analysing the
reactional yields we could not identify a clear inuence of
electronic effects of substituents on the cyclocondensations,
except for the 4-metoxy group that furnished the lowest yields
for both 6b and 9b (Table 3).
Finally, as a goal of our medicinal chemistry development
program, we decided to explore N-alkylations on the 3-amino
group. First, we tried the methylation of the deprotected 1,5-
diphenyl-3-amine-1,2,4 triazole (6a) in some conditions, varying
the base, temperature, and molar proportion. Curiously, none
of these conditions led to the desired alkylated product (results
not shown). As we could selective synthetize the protected 1,5-
diphenyl-3-amine-1,2,4 triazole (9a), this compound was reac-
ted with methyl iodide, NaH and DMF in a borosilicate sealed
tube during around 1 h at 45 ꢀC. The consumption of start
material was observed and aer chromatographic isolation we
could obtained the protected N-metil-1,5-diphenyl-3-amine-
1,2,4 triazole (10a) in 62% (Table 4).
Design and synthesis of triazole cholinesterase inhibitors
The knowledge of neurotransmitter disorders in Alzheimer
Disease (AD) has led to the approval of drugs with symptomatic
effects.15
The cholinergic hypothesis of AD states that the degenera-
tion of cholinergic neurons in basal forebrain nuclei causes
disorders in the presynaptic cholinergic terminals in the
hippocampus and neocortex, regions of extreme importance for
memory disorders and other cognitive symptoms.16 Because of
neurodegeneration, the activity of cholinergic neurons and
levels of neurotransmitter ACh are reduced and the main
approach to improve cholinergic neurotransmission is to
increase the availability of ACh by inhibition of
cholinesterases.17
Bear in mind the total control in the synthesis and deriva-
tization of our N-alkyl-1,5-diphenyl-3-amine-1,2,4 triazoles and
the description of cholinesterase inhibition and neuro-
protection of triazine derivative (12),18 we proposed the
synthesis of triazoles (13). The design was carried out based on
the description that multipotent activity of diaryltriazines (12)
comes from the diaryltriazine moiety and not from the
piperazine-ethyl side chain.18 Thus, we imagined that our 1,5-
diphenyl-3-amine-1,2,4 triazoles could be envisaged from a non-
classic bioisosterism19 of ring contraction from diaryltriazines
(12). And nally, a molecular hybridization20 with the classical
drug donepezil (14) could furnished the desired triazoles as
cholinesterase inhibitors (Fig. 3).
The desired 1,5-diphenyl-3-amine-1,2,4 triazoles (13a–d)
were obtained in a two-step synthesis (Table 5): (1) a reaction of
compounds 10f–i with 3 equivalents of benzylpiperazine in
acetonitrile for 12 h at 60 ꢀC, furnishing products 15a–d in
yields ranging from 66 to 93% aer chromatographic purica-
tion; and (2) a deprotection of Boc group with TFA in
dichloromethane, as above described, leading to desired prod-
ucts 13a–d in yields ranging from 88 to 94%. In these cases, the
thermal deprotection at 150 ꢀC, despite of being faster (around
30–60 min) did not work very well originating some impurities.
This methodology was applied to other alkylating agents,
furnishing the N-alkylated products (10b–h) in reasonable to
good yields (41–82%) (Table 4). Bulky alkylating groups as
dibromoethane did not work in this synthesis. For bioactivity
analysis, it is not so interesting to use compounds with pro-
tecting groups. Thus, we rst tried a deprotection of Boc group
with TFA in dichloromethane at room temperature, leading to
11a in 92% yield. Aer, we proceeded with a one-pot process of
methylation and deprotection from the starting material (9a).
Aer the total methylation of 9a, observed by TLC, we heated the
ꢀ
reaction to 150 C for 30 min and we could observe the depro-
tection of 10a, furnishing 11 in 59% global yield (2 steps one pot
procedure) aer chromatographic isolation (Table 4).
Table 4 Synthesis of N-alkyl-1,5-diphenyl-3-amine-1,2,4 triazoles (10
and 11)
Biological prole
The cholinesterase (AChE and BuChE) inhibition activities of
the designed triazole derivatives (13a–d) and the non-
Entry
Comp.
R
Yielda
1
2
3
4
5
6
7
8
9
10a
10b
10c
10d
10e
10f
10g
10h
11
–CH3
62
65
65
41
79
82
57
65
92b
59c
–CH2CH3
CH2CH2CH3
–CH2CHCH2
–CH2Ph
–C4H8Br
–C5H10Br
–C6H12Br
–CH3
10
11
–CH3
a
b
c
Fig. 3 Structural design of N-alkyl-1,5-diphenyl-3-amine-1,2,4 tri-
azoles (13a–d) as cholinesterase inhibitors.
Isolated yields in %. Deprotection from 10a. Aer one-pot 2 steps
procedure from 9a.
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Table 5 Synthesis of N-alkyl-1,5-diphenyl-3-amine-1,2,4 triazoles (15
and 13)
activity were also observed in the literature for donepezil
analogues.22
Additionally, all the tested triazoles (13a–d) were also
capable of inhibiting BuChE (Table 6) with equipotent IC50
values, ranging from 1.63 to 2.21 mM (compounds 13b and 13d
respectively). These potency values were comparable to the
reference compound donepezil (IC50 ¼ 2.39 mM). Additional
results obtained by kinetic study with derivative 13a, revealed
a mixed-type inhibition with a very similar non-competitive
constant ðKi0Þ for AChE ðKi0 ¼ 1:29 mMÞ and BuChE
ðKi0 ¼ 1:29 mMÞ. Additionally, the competitive inhibition
constant (Ki) for this compound was only 1.7 times higher for
BuChE (Ki ¼ 0.62 mM) compared to acetylcholinesterase (Ki ¼
0.36 mM) (Fig. 4). The obtained results suggest that the assayed
triazoles (13a–d) presented good mixed-type inhibition potency,
as the prototypal triazine (12), but without showing selectivity
between AChE and BuChE enzymes. Nevertheless, with the
molecular understanding of how triazoles interact with
cholinesterases, further modications could lead to a better
selective prole.
Comp.
R0
Yielda
15a
15b
15c
15d
13a
13b
13c
13d
–C3H6–PipBn
–C4H8–PipBn
–C5H10–PipBn
–C6H12–PipBn
–C3H6–PipBn
–C4H8–PipBn
–C5H10–PipBn
–C6H12–PipBn
66
86
93
81
94
95
88
90
a
Isolated yields in %.
Furthermore, the planned derivatizations were correct and
important for the activity since 6a had no signicant cholines-
terase inhibition at 30 mM.
substituted triazole 6a were determined by the Ellman's
method21 using donepezil as reference compound.
As depicted in Table 6, compounds (13a–d) presented good
inhibitory activities against AChE with IC50 values varying from
1.14 to 11.00 mM for compounds 13a and 13c respectively.
The inhibition prole of triazoles (13a–d) against AChE was
quite similar of that related in the literature with a regular
decreasing of activity with the increasing of the number of
methylene spacers. Compound 13a, which has the smallest
methylene chain, was the most potent compound. Curiously,
compound 13c (IC50 ¼ 11.00 mM), which has ve methylenes in
the spacer chain, was an exception, being considerably less
potent than its upper homolog (13d, IC50 ¼ 5.22 mM). This result
may represent a tendency of an alternating, or serrated, curve as
the carbon spacer chain increases. Sometimes this curve can
occur in a homologous series when variations in activity are
observed according to whether the number of carbons is even or
odd, because of the different 3D disposition of attached func-
tional groups. This difference in activity when plotted versus the
methylene spacer number originate this “serrated” prole.
Interestingly serrated curve variations for AChE inhibitory
Molecular modeling
All four scoring functions available in GOLD 5.6 were able to
produce docking poses in the redocking of N,N0-di-1,2,3,4-
tetrahydroacridin-9-yl-pentane-1,5-diamine with RMSD values
lower than 1.00 A. GoldScore23 was the only function for which
˚
˚
the pose with the lowest RMSD value (0.62 A) was also the pose
with the highest docking score, so it was chosen for the
subsequent docking procedure.
In the GOLD program, the docking functions yield the
“tness scores”, which are dimensionless values. The score
values are a guide of how good the docking pose is, with
a higher score indicating a better interaction between the ligand
and the binding site. All compounds were predicted to interact
effectively with EeAChE and EcBuChE using the GoldScore
tness score function.
Comparing compounds 13a and 13c, which presented the
greater difference in activity as EeAChE inhibitors, it was ob-
tained a better tness score for 13a using the 1C2O EeAChE
structure and the ligands protonated at the N atom attached to
Table 6 Cholinesterase inhibition of compounds (13a–d)
a,c
Compound
AChE IC50a,b (mM)
BuChE IC50 (mM)
13a
13b
13c
13d
1.14 ꢁ 0.031
3.79 ꢁ 0.061
11.00 ꢁ 0.251
5.22 ꢁ 0.188
NAd
1.79 ꢁ 0.060
1.63 ꢁ 0.111
1.65 ꢁ 0.059
2.21 ꢁ 0.068
NAd
6a
Donepezil
0.007 ꢁ 0.0002
2.39 ꢁ 0.105
a
Concentration required for 50% inhibition of ChEs, data were shown
b
Fig. 4 Lineweaver–Burk plots of EeAChE (A) and EcBuChE (B) inhi-
bition kinetics of compound 13a. Inset: concentrations used for 13a are
depicted with [I] graphic symbol.
in mean ꢁ SD of triplicate independent experiments. AChE from
c
d
electric eel. BuChE from horse serum. NA – not active, inhibition <
20% at 30 mM.
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the benzyl group (Table 7). The tness score was higher for
compound 13c but closer to that of compound 13a (both with the
same protonation mode used for the EaAChE docking) when they
were docked in the EcBuChE model. All these results are in
qualitative accordance with the experimental activity data.
Similarly to what was observed in the complex between N,N0-
di-1,2,3,4-tetrahydroacridin-9-yl-pentane-1,5-diamine in T. cal-
ifornica AChE by Rydberg and collaborators24 and also in
accordance with our previous results,25 it could be observed that
the present compounds were able to spread from the peripheral
site to the active site inside the EeAChE structure, completely
occupying the gorge connecting them. This is in accordance
with the experimental results that showed a mixed-type inhi-
bition for these compounds.
In Fig. 5, it is presented a superposition of the docking poses
of the strongest (13a) and the weakest (13c) EeAChE inhibitors
of the series. It can be observed that in the active site, the phenyl
substituents of the triazole ring interact by means of T-stacking
interactions with Trp86 for both inhibitors, in a similar way as
described for the prototype 12.18 Most of interactions involve the
side chains of aromatic rings, including some located at the
gorge, which is lined with aromatic residues. However,
conversely from 12, one of these residues (Tyr124) is donating
a hydrogen bond to one of the N atoms of the triazole ring in our
compounds.
Fig. 5 Superposition of the interaction poses of compounds 13a (A,
carbon atoms in yellow) and 13b (B, carbon atoms cyan) with EeAChE
obtained by molecular docking (Goldscore function). Color code:
oxygen atoms, red; nitrogen atoms, blue; hydrogen atoms, white;
carbon atoms, green. Dashed yellow line: hydrogen bond distance (A˚).
Figure generated with PyMol 0.99 (DeLano Scientific LLC).
geometries favors the interaction with the ligand with a shorter
spacer, 13a.
The main difference between the interaction modes of these
compounds (13a and 13c) occur at the peripheral binding site
(PAS). Because of its smaller spacer chain, the protonated
piperazine ring of compound 13a is located exactly over the
Trp286 side chain, which enables the formation of a strong
cation-p interaction. However, because compound 13c side
chain is two methylenes longer, a strong interaction between its
protonated piperazine ring and Trp286 is impaired, which
explains its weaker interaction with the enzyme. Worth of note,
this interaction differs from others cholinesterase inhibitors
bearing cationic groups like piperazine and piperidine, once
these groups normally interact with the catalytic site (CAS)
instead of PAS like ours.
Predicted ADMET prole analysis
To accede the drugability of tested triazoles, we proceeded with
in silico evaluations that showed a good ADMET prole for these
compounds. Parameters as topological polar surface area
(TPSA), consensus log P, log S, human intestinal absorption
(HIA), blood brain barrier permeation (BBB), and P-glycoprotein
(P-gP) substrate and drug-likeness prole were analyzed
(ESI†).26 TPSA value was calculated as 49.22 A and consensus
2
˚
log P ranged from 4.27 to 5.18. The moderate polarity and
relative lipophilicic characteristics put our compounds in the
yellow compartment of BOILED-Egg model (Fig. 6), having
a high probability to gastrointestinal absorption and access to
the CNS,27 which is fundamental for the distribution of
To better quantify the effect of the difference in the inter-
action pattern with the PAS, we implemented PM6 calculations
for 13a and 13c at the docking geometries of the inhibitor/
Trp286 pairs.
From the resulting enthalpies of formation (DHf), the DHf of
the corresponding inhibitor and of the Trp286 residue were
subtracted, resulting in the interaction enthalpy (DHint
)
between each inhibitor and the Trp286 residue. For the 13c/
Trp286 pair, the calculated DHint was ꢂ41.5 kJ molꢂ1, whereas
for the 13a/Trp286 pair, DHint was ꢂ59.5 kJ molꢂ1. With these
values, it is clear how the differences in the interaction
Table 7 Fitness scores (Goldscore)* obtained by molecular docking in
EeAChE and EcBuChE
Ligand
EeAChE
EcBuChE
Fig. 6 BOILED-Egg ADMET model27 for triazole compounds 13a–d.
(HIA) gastrointestinal absorption; (BBB) brain penetration.
13a
13c
83.7
79.7
69.7
71.5
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central-acting molecules. Additionally, the most potent a rotary evaporator to give 216 mg of a beige solid in 71% yield
compound 13a presented a good drug-likeness prole accord- that was used without any further purication.
ing to Lipinski,28 Veber29 and Egan30 rules.
Synthesis of S-methyl-N-benzoyl-isothiourea (3)
To a solution of methylisothiourea (0.06 g, 0.28 mmol) in
saturated sodium bicarbonate (2 mL) was slowly added under
Conclusions
We could develop an efficient and regioselective one-pot
synthesis protocol for obtaining N-protected or N-deprotected
functionalized 1,5-diaryl-3-amino-1,2,4-triazoles starting from
N-acyl-N-Boc-carbamidothioates. This protocol demonstrated
that microwave irradiation was very useful reducing reaction
times to up to 1 h and presenting yields ranging from 50 to 84%.
This chemistry summed to bioisosteric and molecular hybrid-
ization techniques allowed us to discover a new series of
compounds able to inhibit cholinesterases (without selectivity)
with good IC50 of up to 1 mM acting by a mixed-type way.
Molecular modelling evaluations demonstrated the importance
of phenyl substituents as well as the triazole ring to the inter-
action with CAS site of AChE, showing potential for this class of
compounds as new scaffolds for this biological activity.
stirring a solution of 2,5-dioxopyrrolidine-1-benzoate (0.06 g,
0.28 mmol) in dichloromethane (10 mL). The reaction mixture
was stirred at room temperature for 18 hours. Aer its
completion, the reaction was diluted in dichloromethane (20
mL) and washed with water (3 ꢃ 10 mL). The organic phase was
dried over anhydrous sodium sulphate and concentrated on
a rotary evaporator to give a white amorphous solid in 72%
yield.1H NMR (400 MHz, CDCl3) (d ppm): 8.28–8.26 (d, 2H),
7.53–7.51 (m, 1H), 7.45–7.41 (m, 2H), 2.63 (sl, 3H); DEPT-Q (100
MHz, CDCl3) (d ppm): 137.27, 132.03 (2x), 129.68 (2x), 128.04,
13.84.
General procedure for 3-amine-1,2,4 triazoles preparation (5
and 6a) from 3
In a sealed borosilicate tube, methyl N0-benzoylcarbamido-
thioate (3) (1 eq.) and phenylhydrazine (4) (1.1 eq.) were solu-
bilized in the correspondent solvent (2 mL). The reaction
mixture was stirred at temperature and time determined in
Table 1. Aer completion of reaction the slurry was diluted with
ethyl acetate (20 mL) and partitioned with water (10 mL). The
organic phase was dried over anhydrous sodium sulphate and
concentrated on a rotary evaporator. The products were puried
by silica gel column chromatography (FLASH Chromatography
System – Biotage), using an ultra-10 g snap column (n-hexane:
ethyl acetate, 5–75% gradient elution).
Experimental
Materials and instruments
All chemicals were purchased from commercial suppliers and
used without further purication. The1 H and 13C NMR spectra
were recorded using a 400 MHz or 500 MHz Brucker Avance
spectrometer (AC-400 or AC-500), using DMSO-d6 or CDCl3 and
TMS as an internal standard. The melting points recorded are
uncorrected. Elemental analyzes were carried out on a Thermo
Scientic Flash EA 1112 Series CHN-Analyzer. A Biotage Initi-
ator 2 Reactor was used for microwave reactions using an
internal IR temperature probe.
1,3-diphenyl-1,2,4-triazol-5-amine (5)
All commercial reagents were used without prior purica-
tion. Reactions were monitored by thin layer chromatography
(TLC) using silica gel plates and visualized in UV light (254 nm
and 365 nm). The multiplicities were called sl (singlet),
d (doublet), t (triplet), q (quartet), dd (double doublet) and m
(multiplet).
1
ꢀ
Brown solid; 13% yield; mp ¼ 159–160 C; H NMR (500 MHz,
CDCl3) (d ppm): 8.06–8.06 (d, 2H), 7.64–7.62 (d, 2H), 7.56–7.54
(t, 2H), 7.44–7.40 (m, 5H), 4.80 (s, 2H); DEPT-Q (125 MHz,
CDCl3) (d ppm): 159.28, 153.92, 136.81, 133.35, 130.76, 129.88,
129.21, 128.50, 128.23, 127.39, 126.16 (2X), 123.45 (2X).
Elemental analysis calcd (%) for C14H12N4: C, 71.17; H, 5.12; N,
23.71; found: C 71.05, H 5.22, N 23.63.
Synthesis of S-methyl-urea (1)
1,5-Diphenyl-1,2,4-triazol-3-amine (6a)
Methyl iodide (0.82 mL, 13.13 mmol) was added in a sealed tube
containing thiourea (0.50 g, 6.56 mmol) in methanol (5 mL).
The mixture was stirred at 65 C for 2 hours. At the end of the
1
ꢀ
Brown solid; 75% yield, mp ¼ 150–152 C; H NMR (400 MHz,
CDCl3) (d ppm): 7.47–7.45 (d, 2H), 7.39–7.32 (m, 8H), 4.27 (sl,
2H); DEPT-Q (100 MHz, CDCl3) (d ppm): 162.67, 153.09, 138.28,
129.85, 129.22 (2x), 128.74 (2x), 128.46 (2x), 128.28, 128.04,
125.18 (2x). Elemental analysis calcd (%) for C14H12N4: C, 71.17;
H, 5.12; N, 23.71; found: C 71.21, H 5.35, N 23.48.
ꢀ
reaction the mixture was concentrated on a rotary evaporator to
1
give 1.42 g of a brown solid in 99% yield. H NMR (500 MHz,
DMSO) (d ppm): 2.58 (s, 3H); DEPT-Q (125 MHz, DMSO) (d ppm):
171.09, 13.55.
Synthesis of 2,5-dioxopyrrolidine-1-benzoate
General procedure for synthesis of 3-amine-1,2,4-triazoles
(6b–6f) from 7a–d
Benzoic acid (0.10 g, 0.82 mmol), N,N0-dicyclohexylcarbodi-
imide (DCC) (0.22 g, 0.82 mmol) and N-hydroxysuccinimide In
a
sealed borosilicate tube containing Boc-S-methyl-N-
(0.12 g, 0.82 mmol) were solubilized in THF (10 mL). The benzoyl-isothiourea intermediates 7a–d (1 eq.) in DMF (2 mL) it
resulted solution was kept under stirring at room temperature was added the corresponding hydrazine (1.1 eq.). The reaction
for 20 hours. Reaction mixture was ltered and evaporated on was carried out in microwave irradiation at 150 ꢀC for 60
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minutes. Aer the end of the reaction, the slurry was partitioned
between ethyl acetate (20 mL) and water (10 mL). The organic
phase was dried over anhydrous sodium sulphate and concen-
trated on a rotary evaporator. Products were separated by FLASH
Chromatography System (Biotage), using an ultra-10 g snap
column (n-hexane: ethyl acetate, 5–75% gradient elution).
General synthesis of compounds (7a–7d)
In a ask containing benzoic acid (1 eq.) and dichloromethane
(5 mL) it was added oxalyl chloride (4 eq.) and catalytic amount
of dimethylformamide (DMF). Aer total consumption of the
correspondent carboxylic acid, the reaction was concentrated
on a rotary evaporator and diluted with dichloromethane (5
mL). This solution was added dropwise to another solution
containing the protected S-metil-isothiurea (8) (1 eq.) and trie-
thylamine (2 eq.) in dichloromethane (10 mL) and kept under
stirring overnight. Then, mixture was diluted with dichloro-
methane (20 mL) and washed with aqueous sodium bicar-
bonate 10% (3 ꢃ 10 mL). Organic phase was dried over sodium
sulphate and evaporated on a rotary evaporator.
1,5-Diphenyl-1,2,4-triazol-3-amine (6a)
Brown solid; 82% yield; spectral data described before.
5-(4-methoxyphenyl)-1-phenyl-1,2,4-triazol-3-amine (6b)
1
ꢀ
Brown solid; 47% yield, mp ¼ 198–200 C; H NMR (500 MHz,
CDCl3) (d ppm): 7.44–7.37 (m, 7H), 6.94–6.93 (d, 2H), 6.86–6.84
(d, 2H), 3.82 (sl, 3H); DEPT-Q (125 MHz, CDCl3) (d ppm): 161. 23,
153.04, 130.42, 129.48, 129.20, 129.06, 125.50, 114.06, 113.35,
55.34. Elemental analysis calcd (%) for C15H16N4O: C, 67.65; H,
5.30; N, 21.04; found: C 67.62, H 5.21, N 21.28.
tert-Butyl(benzamido(methylthio)methylene)carbamate (7a)
White solid; 70% yield; 1H NMR (500 MHz, CDCl3) (d ppm):
8.29–8.28 (d, 2H), 7.56–7.46 (dd, 3H), 2.59 (sl, 3H), 1.54 (sl, 9H);
DEPT-Q (125 MHz, CDCl3) (d ppm): 172.03, 132.81, 130.12 (2X),
128.25 (2X), 27.98 (3X), 14.92. Elemental analysis calcd (%) for
5-(4-chlorophenyl)-1-phenyl-1,2,4-triazol-3-amine (6c)
C
14H18N2O3S: C, 57.12; H, 6.16; N, 9.52; found: C 56.89, H
1
ꢀ
6.38, N 9.33.
Beige solid; 68% yield, mp ¼ 151–153 C; H NMR (400 MHz,
CDCl3) (d ppm): 7.43–7.42 (m, 5H), 7.33–7.31 (m, 4H); DEPT-Q
(100 MHz, CDCl3) (d ppm): 161.76, 151.26, 137.66, 136.53,
130.01 (2X), 128.98 (2X), 128.90, 125.43, 125.20 (2X), 101.30.
Elemental analysis calcd (%) for C14H11N4Cl: C, 62.11; H,
4.10; N, 20.70; found: C 62.00, H 4.31, N 20.63.
tert-Butyl((4-methoxybenzamido)(methylthio)methylene)-
carbamate (7b)
White solid; 68% yield; 1H NMR (400 MHz, CDCl3) (d ppm):
8.11–8.09 (d, 2H), 7.06–7.04 (d, 2H), 3.85 (sl, 3H), 2.49 (sl, 3H),
1.46 (sl, 9H). DEPT-Q (100 MHz, CDCl3) (d ppm): 171.00, 163.44,
151.12, 132.32 (2X), 129.31, 113.49 (2X), 83.26, 55.43, 27.98 (3X),
14.86. Elemental analysis calcd (%) for C15H20N2O4S: C, 55.54;
H, 6.21; N, 8.64; found: C 55.72, H 6.29, N 8.77.
5-(4-nitrophenyl)-1-phenyl-1,2,4-triazol-3-amine (6d)
1
ꢀ
Yellow solid; 52% yield, mp ¼ 202–204 C; H NMR (500 MHz,
CDCl3) (d ppm): 8.19–8.17 (d, 2H), 7.65–7.64 (d, 2H), 7.45 (sl,
3H), 7.32 (m, 2H), 4.33 (sl, 2H); DEPT-Q (125 MHz, CDCl3) (d
ppm): 162.97, 150.72, 148.25, 137.64, 133.77, 129.68 (2X), 129.55
(2X), 129.17, 125.32 (2X), 123.73 (2X). Elemental analysis calcd
(%) for C14H11N5O2: C, 59.78; H, 3.94; N, 24.90; found: C 59.55,
H 4.02, N 24.95.
tert-Butyl(4-chlorobenzamido(methylthio)methylene)-
carbamate (7c)
White solid; 63% yield; 1H NMR (500 MHz, CDCl3) (d ppm):
12.54 (sl, 1H), 8.22–8.20 (d, 2H), 7.43–7.41 (d, 2H), 2.58 (sl, 3H),
1.53 (sl, 9H); DEPT-Q (125 MHz, CDCl3) (d ppm): 174.93, 172.63,
150.95, 139.17, 135.14, 131.48 (2X), 128.55 (2X), 83.64, 27.98
(3X), 14.97. Elemental analysis calcd (%) for C14H17ClN2O3S: C,
51.14; H, 5.21; N, 8.52; found: C 51.00, H 5.39, N 8.41.
1-(4-nitrophenyl)-5-phenyl-1,2,4-triazol-3-amine (6e)
1
ꢀ
Brown solid; 73% yield, mp ¼ 173–176 C; H NMR (500 MHz,
CDCl3) (d ppm): 8.27–8.26 (d, 2H), 7.54–7.49 (m, 5H), 7.45–7.43
(m, 2H), 4.99 (sl, 2H); DEPT-Q (125 MHz, CDCl3) (d ppm):
160.70, 146.97, 142.07, 131.72, 129.35 (2X), 128.95 (2X), 125.25,
125.00 (2X), 124.93 (2X). Elemental analysis calcd (%) for
tert-Butyl((4-nitrobenzamido)(methylthio)methylene)
carbamate (7d)
White solid; 58% yield; 1H NMR (500 MHz, CDCl3) (d ppm): 7.04
(s, 1H); 2.51 (s, 3H); 1.52 ppm (s, 9H); DEPT-Q (125 MHz, CDCl3)
(d ppm): 174.28, 173.68, 150.76, 150.21, 142.03, 130.95 (2X),
123.27 (2X), 84.06, 27.95 (3X), 15.12. Elemental analysis calcd
(%) for C14H17N3O5S: C, 49.55; H, 5.05; N, 12.38; found: C 49.21,
H 5.24, N 12.02.
C
14H11N5O2: C, 59.78; H, 3.94; N, 24.90; found: C 59.43, H
4.14, N 24.78.
1-(4-(methylsulfonyl)phenyl)-5-phenyl-1,2,4-triazol-3-amine
(6f)
1
ꢀ
Brown solid; 69% yield, mp ¼ 213–215 C; H NMR (500 MHz)
CDCl3 (d ppm): 7.94–7.92 (d, 2H), 7.53 (d, 2H), 7.45 (m, 3H),
7.42–7.39 (m, 2H), 4.38 (sl, 2H), 3.08 (sl, 3H); DEPT-Q (125 MHz,
CDCl3) (d ppm): 163.04, 153.84, 142.25, 139.25, 130.60 (2X),
tert-Butyl(1H-pyrrole-2-carboxamido(methylthio)-methylene)
carbamate (7e)
128.94, 128.80 (2X), 128.60 (2X), 127.56, 124.78 (2X), 44.46. White solid; 80% yield; 1H NMR (500 MHz, CDCl3) (d ppm):
Elemental analysis calcd (%) for C15H14N4O2S: C, 57.31; H, 12.49 (s, 1H), 9.55 (s, 1H), 7.10 (s, 1H), 7.02 (s, 1H), 6.30 ppm (s,
4.49; N, 17.82; found: C 57.32, H 4.54, N 17.66.
1H), 2.52 (s, 3H), 1.51 (s, 9H). DEPT-Q (125 MHz, CDCl3) (d
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ppm): 170.5, 151.1, 130.5, 116.8, 110.9, 83.5, 27.9, 14.6. ppm): 161.01, 155.94, 151.28, 138.11, 130.43 (2X), 129.25 (2X),
Elemental analysis calcd (%) for C12H17N3O3S: C, 50.87; H, 128.77, 125.82 (2X), 113.94 (2X), 81.34, 55.31, 28.18 (3X).
6.05; N, 14.83; found: C 50.71, H 5.98, N 14.96.
Elemental analysis calcd (%) for C20H22N4O3: C, 65.56; H,
6.05; N, 15.29; found: C 65.90, H 6.07, N 14.99.
tert-Butyl(4,5-dibromo-1H-pyrrole-2-
carboxamido(methylthio)-methylene)carbamate (7f)
tert-Butyl(5-(4-chlorophenyl)-1-phenyl-1,2,4-triazol-3-yl)
White solid; 69% yield; 1H NMR (500 MHz, CDCl3) (d ppm):
12.53 (sl, 1H), 8.43–8.42 (d, 2H), 8.30–8.28 (d, 2H), 2.61 (sl, 3H),
1.55 (sl, 9H); DEPT-Q (125 MHz, CDCl3) (d ppm): 119.3; 84.0;
28.0; 15.1. Elemental analysis calcd (%) for C12H15Br2N3O3S: C,
32.67; H, 3.43; N, 9.53; found: C 32.38, H 3.64, N 9.48.
carbamate (9c)
1
ꢀ
White solid; 71% yield, mp ¼ 140–142 C; H NMR (400 MHz,
CDCl3) (d ppm): 7.94 (sl, 1H), 7.43–7.41 (m, 4H), 7.38 (d, 2H),
7.31–7.25 (m, 3H), 1.49 (s, 9H); DEPT-Q (100 MHz, CDCl3) (d
ppm): 156.47, 151.93, 151.33, 137.75, 136.37, 130.18 (2X), 129.38
(2X), 129.05 (2X), 128.82 (2X), 125.74, 125.78, 81.37, 28.14 (3X).
Elemental analysis calcd (%) for C19H19ClN4O2: C, 61.54; H,
5.16; N, 15.11; found: C 61.23, H 5.34, N 14.82.
Synthesis of tert-butyl [amine(methylthio)methylene]
carbamate (8)
In a ask containing methyl isothiourea (1 g, 4.58 mmol),
dichloromethane (10 mL) and triethylamine (639 mL, 4.58
mmol), in an ice-bath, it was slowly added a solution of di-tert-
butyl dicarbonate (Boc–O–Boc) (0.5 g, 2.29 mmol) in dichloro-
methane (5 mL). Then, ice bath was removed and reaction was
le under stirring overnight at room temperature. Aer
consumption of reactants, the reaction mixture was diluted
with dichloromethane (15 mL), washed with water (2 ꢃ 10 mL),
dried over sodium sulphate and concentrated on a rotary
evaporator. A white solid product was obtained in 86% yield and
used without any further purication.
tert-Butyl(5-(4-nitrophenyl)-1-phenyl-1,2,4-triazol-3-yl)
carbamate (9d)
1
ꢀ
White solid; 80% yield, mp ¼ 166–168 C; H NMR (400 MHz,
CDCl3) (d ppm): 8.19–8.17 (d, 2H), 7.69–7.66 (d, 2H), 7.47–7.45
(m, 3H), 7.39–7.36 (m, 3H), 1.54 (s, 9H); DEPT-Q (100 MHz,
CDCl3) (d-ppm): 156.71, 151.10, 148.41, 137.40, 133.22, 129.78
(2X), 129.67 (2X), 129.62, 125.79 (2X), 123.72 (2X), 81.76, 28.18
(3X). Elemental analysis calcd (%) for C19H19N5O4: C, 59.84; H,
5.02; N, 18.36; found: C 59.78, H 5.12, N 18.21.
1H NMR (500 MHz, DMSO) (d ppm): 8.58 (sl, 2H), 2.32 (sl,
3H), 1.41 (sl, 9H); DEPT-Q (125 MHz, DMSO) (d ppm): 171.87,
160.98, 78.11, 26.17 (3X), 13.13.
tert-Butyl (1-(4-nitrophenyl)-5-phenyl-1,2,4-triazol-3-yl)
carbamate (9e)
1
ꢀ
Brown solid; 61% yield, mp ¼ 147–150 C; H NMR (400 MHz,
DMSO) (d ppm): 10.12 (s, 1H), 8.35–8.33 (d, 2H), 7.65 (d, 2H),
7.49–7.47 (m, 5H), 1.49 (s, 9H); DEPT-Q (100 MHz, DMSO) (d
ppm): 157.53, 153.88, 152.38, 147.19, 143.08, 131.21, 129.52
(2X), 127.85, 126.48 (2X), 125.52 (2X), 80.19, 28.61 (3X).
Elemental analysis calcd (%) for C19H19N5O4: C, 59.84; H,
5.02; N, 18.36; found: C 59.95, H 5.01, N 18.14.
Synthesis of compounds 9a–9e
In a sealed borosilicate tube containing triazole (7a–7f) (1 eq.) in
2 mL of acetonitrile, it was added the corresponding hydrazine
(1.1 eq.). Reaction was stirred at 100 ꢀC for 40 minutes as shown
in Table 3. Aer the end of the reaction, the reactional slurry
was partitioned between ethyl acetate (20 mL) and water (10
mL). The organic phase was dried over anhydrous sodium
sulphate and concentrated on a rotary evaporator. The products
were separated with a FLASH Chromatography System (Biot-
age), using an ultra-10 g snap column (n-hexane: ethyl acetate,
5–75% gradient elution).
tert-Butyl (1-(4-(methylsulfonyl)phenyl)-5-phenyl-1,2,4-triazol-
3-yl)carbamate (9f)
1
ꢀ
Brown solid; 84% yield, mp ¼ 179–181 C; H NMR (400 MHz,
CDCl3) (d ppm): 7.96–7.94 (d, 2H), 7.90 (s, 1H), 7.61–7.60 (d, 2H),
7.46 (m, 3H), 7.41–7.38 (m, 2H), 3.07 (s, 3H), 1.50 (s, 9H); DEPT-
Q (100 MHz, CDCl3) (d ppm): 156.94, 153.56, 151.12, 142.09,
140.00, 130.85, 128.96 (2X), 128.64 (2X), 126.82, 125.73 (2X),
81.66, 44.46, 28.11 (3X). Elemental analysis calcd (%) for
tert-Butyl(1,5-diphenyl-1,2,4-triazol-3-yl)carbamate (9a)
1
ꢀ
Brown solid; 87% yield, mp ¼ 147–149 C; H NMR (400 MHz,
CDCl3) (d ppm): 7.49 (m, 3H), 7.39 (sl, 6H), 7.34–7.32 (d, 2H),
1.52 (s, 9H); DEPT-Q (100 MHz, CDCl3) (d ppm): 156.33, 153.05,
151.32, 138.08, 130.11, 129.23 (2X), 128.93 (2X), 128.50 (2X),
125.75 (2X), 81.34, 28.21 (3X). Elemental analysis calcd (%) for
C
20H22N4O4S: C, 57.96; H, 5.35; N, 13.52; found: C 57.59, H
5.55, N 13.28.
C
19H20N4O2: C, 67.84; H, 5.99; N, 16.66; found: C 68.02, H
tert-Butyl (1-phenyl-5-(1H-pyrrol-2-yl)-1-phenyl-1H-1,2,4-
6.07, N 16.62.
triazol-3-yl)carbamate (9g)
1
ꢀ
Brown solid; 90% yield, mp ¼ 170–172 C; H NMR (500 MHz,
CDCl3) (d ppm): 12.49 (s, 1H), 9.55 (s, 1H), 7.10 (s, 1H), 7.02 (s,
1H), 6.30 ppm (s, 1H), 2.52 (s, 3H), 1.51 (s, 9H). DEPT-Q (125
tert-Butyl(5-(4-methoxyphenyl)-1-phenyl-1,2,4-triazol-3-yl)
carbamate (9b)
1
ꢀ
Brown solid; 62% yield, mp ¼ 164–167 C; H NMR (400 MHz, MHz, CDCl3) (d ppm): 170.5, 151.1, 130.5, 116.8, 110.9, 83.5,
CDCl3) (d ppm): 7.59 (sl, 1H), 7.43–7.39 (m, 7H), 6.84–6.82 (d, 27.9, 14.6. Elemental analysis calcd (%) for C17H19N5O2: C,
2H), 3.81 (s, 3H), 1.51 (s, 9H); DEPT-Q (100 MHz, CDCl3) (d 62.75; H, 5.89; N, 21.52; found: C 62.79, H 5.95, N 21.38.
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tert-Butyl (5-(4,5-dibromo-1H-pyrrol-2-yl)-1-phenyl-1H-1,2,4-
triazol-3-yl)carbamate (9h)
tert-Butyl allyl (1,5-diphenyl-1,2,4-triazol-3-yl) carbamate (10d)
White solid; 41% yield, mp ¼ 82–84 ꢀC; 1H NMR (500 MHz,
Brown solid; 79% yield; 1H NMR (500 MHz, CDCl3) (d ppm): CDCl3) (d ppm): 7.49–7.47 (d, 2H), 7.40–7.7.31 (m, 8H), 6.06–
12.98 (sl, 1H), 9.77 (sl, 1H), 7.59–7.48 (m, 5H), 5.65 (s, 1H), 1.45 5.98 (m, 1H), 5.32–5.15 (dd, 2H), 4.47–4.46 (d, 2H), 1.53 (sl, 9H);
(s, 9H). DEPT-Q (125 MHz, CDCl3) (d ppm): 155.9, 145.9, 137.5, DEPT-Q (125 MHz, CDCl3) (d ppm): 159.40, 153. 20, 153, 12,
130.3, 129.8, 126.7, 120.2, 114.0, 105.5, 100.0, 28.5. Elemental 138.00, 133.85, 129.97, 129.24 (2X), 128.94 (2X), 128.44 (2X),
analysis calcd (%) for C17H17Br2N5O2: C, 42.26; H, 3.55; N, 14.49; 127.69, 125.50 (2X), 116.61, 81.36, 50.79, 28.24 (3X). Elemental
found: C 42.13, H 3.58, N 14.25.
analysis calcd (%) for C22H24N4O2: C, 70.19; H, 6.43; N, 14.88;
found: C 70.22, H 6.52, N 14.97.
General synthesis of compounds 10a–10h
tert-Butyl benzyl (1,5-diphenyl-1,2,4-triazol-3-yl) carbamate
The corresponding alkyl halide (2 eq.) was added in a sealed
borosilicate tube containing triazole 9a (1 eq.) and sodium
hydride (4 eq.) in anhydrous DMF (2 mL). The reaction was
stirred at 45 ꢀC for 60–90 minutes. When the end of reaction was
determined by TLC (hexane: ethyl acetate 30%), mixture was
diluted in 20 mL of ethyl acetate and washed with water (3 ꢃ 10
mL). The organic phase was dried over sodium sulphate,
concentrated and puried by an isolation with FLASH Chro-
matography System (Biotage), using an ultra-10 g snap column
(n-hexane: ethyl acetate, 10–50% gradient elution).
(10e)
1
ꢀ
White solid; 79% yield, mp ¼ 110–113 C; H NMR (500 MHz,
CDCl3) (d ppm): 7.48–7.47 (m, 4H), 7.39 (m, 5H), 7.32–7.31 (m,
6H), 7.25–7.23 (m, 1H), 5.10 (sl, 2H), 1.47 (sl, 9H); DEPT-Q (125
MHz, CDCl3) (d ppm): 159.68, 153.27, 153.07, 138.52, 137.96,
129.95, 129.21 (2X), 128.91 (2X), 128.42 (2X), 128.18 (2X), 127.66,
126.91, 125.49 (2X), 125.14, 118.96, 112.82, 81.54, 56.52, 51.84,
47.84, 28.16 (3X). Elemental analysis calcd (%) for C26H26N4O2:
C, 73.22; H, 6.14; N, 13.14; found: C 72.97, H 6.42, N 13.23.
tert-Butyl(4-bromobutyl)(1,5-diphenyl-1,2,4-triazol-3-yl)
tert-Butyl (1,5-diphenyl-1,2,4-triazol-3-yl)(methyl) carbamate
carbamate (10f)
(10a)
Brown oil; 82% yield; 1H NMR (500 MHz, CDCl3) (d ppm): 7.50–
Yellow oil; 62% Yield; 1H NMR (400 MHz, CDCl3) (d ppm): 7.50– 7.48 (d, 2H), 7.41–7.32 (m, 8H), 3.89–3.87 (t, 2H), 3.48–3.46 (t,
7.48 (d, 2H), 7.40–7.33 (m, 8H), 3.43 (sl, 3H), 1.55 (sl, 9H); DEPT- 2H), 2.01–1.97 (m, 2H), 1.90–1.87 (m, 2H), 1.54 (sl, 9H); DEPT-Q
Q (100 MHz, CDCl3) (d ppm): 160.11, 153.56, 153.11, 137.97, (125 MHz, CDCl3) (d ppm): 159.40, 153.50, 153.30, 137.99,
130.01, 129.28 (2X), 128.91 (2X), 128.47 (2X), 127.64, 125.46 (2X), 130.08, 129.33 (2X), 128.96 (2X), 128.53 (2X), 127.66, 125.51 (2X),
81.29, 35.54, 28.26 (3X). Elemental analysis calcd (%) for 81.39, 47.41, 33.51, 29.94, 28.31 (3X), 27.47. Elemental analysis
C
20H22N4O2: C, 68.55; H, 6.33; N, 15.99; found: C 68.32, H calcd (%) for C23H27BrN4O2: C, 58.60; H, 5.77; N, 11.89; found: C
6.51, N 15.88.
58.55, H 5.84, N 11.68.
tert-Butyl(5-bromopentyl)(1,5-diphenyl-1,2,4-triazol-3-yl)
carbamate (10g)
tert-Butyl (1,5-diphenyl-1,2,4-triazol-3-yl)(ethyl) carbamate
(10b)
Brown oil; 52% yield; 1H NMR (400 MHz, CDCl3) (d ppm): 7.50–
7.48 (d, 2H), 7.41–7.23 (m, 7H), 3.86–3.82 (t, 2H), 3.44–3.40 (t,
2H), 1.94–1.90 (m, 2H), 1.79–1.77 (m, 2H), 1.53 (sl, 9H); DEPT-Q
(100 MHz, CDCl3) (d ppm): 159.48, 153.49, 153.22, 137.96,
130.03, 129.29 (2X), 128.48 (2X), 127.64, 125.46 (2X), 81.21,
48.13, 33.77, 32.38, 28.29 (3X), 27.88, 25.30. Elemental analysis
calcd (%) for C24H29BrN4O2: C, 59.38; H, 6.02; N, 11.54; found: C
59.01, H 5.89, N 11.34.
Beige solid; 65% yield, mp ¼ 98–99 ꢀC; 1H NMR (500 MHz,
CDCl3) (d ppm): 7.50–7.48 (d, 2H), 7.40–7.31 (m, 8H), 3.91–3.86
(m, 2H), 1.53 (sl 9H), 1.33–1.30 (t, 3H); DEPT-Q (125 MHz,
CDCl3) (d ppm): 159.40, 153.34, 137.99, 129.96, 129.24 (2X),
128.91 (2X), 128.44 (2X), 127.70, 125.45 (2X), 80.99, 43.58, 28.28
(2X), 14.07. Elemental analysis calcd (%) for C21H24N4O2: C,
69.21; H, 6.64; N, 15.37; found: C 69.00, H 6.68, N 15.49.
tert-Butyl (1,5-diphenyl-1,2,4-triazol-3-yl)(propyl) carbamate
tert-Butyl(6-bromohexyl)(1,5-diphenyl-1,2,4-triazol-3-yl)
(10c)
carbamate (10h)
Beige solid; 65% Yield, mp ¼ 97–99 ꢀC; 1H NMR (500 MHz, Brown oil; 65% yield; 1H NMR (400 MHz, CDCl3) (d ppm): 7.50–
CDCl3) (d-ppm): 7.50–7.48 (d, 2H), 7.41–7.32 (m, 8H), 3.80–3.77 7.48 (d, 2H), 7.41–7.32 (m, 8H), 3.84–3.81 (t, 2H), 3.42–3.39 (t,
(t, 2H), 1.79–1.72 (m, 2H), 1.53 (sl, 9H), 0.98–0.95 (t, 3H); DEPT- 2H), 1.89–1.85 (m, 2H), 1.76–1.75 (m, 2H), 1.53 (sl, 9H); DEPT-Q
Q (125 MHz, CDCl3) (d ppm): 159.60, 153.62, 153.18, 130.01, (100 MHz, CDCl3) (d ppm): 159.51, 153.55, 153.21, 137.97,
129.98, 129.26 (2X), 128.94 (2x), 128.46 (2X), 127.73, 125.48 (2X), 130.01, 129.29 (2X), 128.93 (2X), 128.48 (2X), 127.66, 125.46 (2X),
80.98, 50.19, 28.29 (3X), 22.09, 11.23. Elemental analysis calcd 101.13, 81.11, 48.28, 33.91, 32.70, 28.51, 28.29 (3X), 27.82, 25.80.
(%) for C22H26N4O2: C, 69.82; H, 6.92; N, 14.80; found: C 69.99, Elemental analysis calcd (%) for C25H31BrN4O2: C, 60.12; H,
H 6.95, N 14.58.
6.26; N, 11.22; found: C 59.86, H 6.43, N 11.17.
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General synthesis of compounds 15a–15d
General synthesis of compounds (13a–13d)
Benzylpiperazine (3 eq.) was added in a sealed borosilicate tube In a sealed borosilicate tube containing triazole 12a–d (1 eq.)
containing triazole 10f–h (1 eq.) and acetonitrile (2 mL). Reac- solubilized dichloromethane (4 mL) in an ice-bath, it was added
tion was stirred at 60 ꢀC until the end of the reaction, deter- triuoracetic acid (35 eq.). The ice bath was removed and
mined by TLC (DCM : methanol 10%). Then, mixture was reaction stirred at room temperature overnight. The end of
concentrated and puried by an isolation with FLASH Chro- reaction was determined by TLC and saturated sodium bicar-
matography System (Biotage), using an ultra-10 g snap column bonate solution was added until pH 7 and then, extracted with
(DCM : methanol, 1–50% gradient elution).
dichloromethane, dried over sodium sulphate and concen-
trated on a rotary evaporator.
tert-Butyl (3-(4-benzylpiperazin-1-yl)propyl)(1,5-diphenyl-1H-
1,2,4-triazol-3-yl)carbamate (15a)
N-(3-(4-benzylpiperazin-1-yl)propyl)-1,5-diphenyl-1H-1,2,4-
triazol-3-amine (13a)
Brown oil; 66% yield; 1H NMR (500 MHz, CDCl3) (d ppm): 7.47–
7.45 (d, 2H), 7.40–7.30 (m, 13H), 3.92–3.89 (t, 2H), 3.61 (s, 2H),
2.81–2.76 (m, 8H), 2.12 (m, 2H), 1.51 (sl, 9H), 1.25 (sl, 2H);
DEPT-Q (125 MHz, CDCl3) (d ppm): 159.03, 153.43, 153.33,
137.78, 130.09, 129.41 (2X), 129.28 (2X), 128.84 (3X), 128.47 (2X),
128.42 (2X), 127.66, 127.40, 125.39 (2X), 81.59, 62.11, 55.17,
52.07, 50.88, 46.12, 28.21, 24.56. Elemental analysis calcd (%)
for C27H35N5O2: C, 70.25; H, 7.64; N, 15.17; found: C 69.99, H
7.86, N 15.01.
Brown oil; 94% yield; 1H NMR (400 MHz, CDCl3) (d ppm): 7.45–
7.43 (d, 2H), 7.37–7.30 (m, 13H), 3.54 (sl, 2H), 3.44–3.42 (t, 2H),
2.66–2.43 (m, 10H), 1.89–1.84 (m, 2H); DEPT-Q (100 MHz,
CDCl3) (d-ppm): 163.98, 152.82, 138.83, 129.79, 129.23, 128.46,
128.16, 128.11, 127.64, 125.24, 62.21, 57.34, 43.06, 27.53.
Elemental analysis calcd (%) for C28H32N6O6: C, 74.30; H,
7.13; N, 18.57; found: C 74.15, H 7.39, N 18.28.
tert-Butyl (4-(4-benzylpiperazin-1-yl)butyl)(1,5-diphenyl-1H-
1,2,4-triazol-3-yl)carbamate (15b)
N-(4-(4-benzylpiperazin-1-yl)butyl)-1,5-diphenyl-1H-1,2,4-
triazol-3-amine (13b)
Brown oil; 86% yield; 1H NMR (500 MHz, CDCl3) (d ppm): 7.48–
7.47 (d, 2H), 7.40–7.31 (m, 12H), 3.83 (m, 2H), 3.56 (s, 2H), 2.69
(m, 7H), 1.78 (m, 4H), 1.51 (sl, 9H), 1.26 (m, 1H), 0.88–0.83 (m,
1H); DEPT-Q (125 MHz, CDCl3) (d ppm): 163.24, 137.86, 130.10
(2X), 129.26 (3X), 128.89 (2X), 128.51 (3X), 128.37 (2X), 127.44
(2X), 125.43, 81.35, 62.35, 57.47, 52.29, 51.02, 47.71, 28.26, 26.30
(2X). Elemental analysis calcd (%) for C28H37N5O2: C, 70.71; H,
7.84; N, 14.72; found: C 70.56, H 8.01, N 14.58.
Brown oil; 95% yield; 1H NMR (400 MHz, CDCl3) (d ppm): 7.44–
7.43 (d, 2H), 7.38–7.30 (m, 13H), 3.60 (sl, 2H), 3.39–3.37 (t, 2H),
2.75 (m, 6H), 1.76–1.70 (m, 10H); DEPT-Q (100 MHz, CDCl3) (d
ppm): 164.12, 152.79, 138.90, 137.90, 129.70, 129.25, 129.19,
128.76, 128.42, 128.06, 125.25, 62.92, 56.61, 53.14, 52.86, 42.62,
29.67, 26.34. Elemental analysis calcd (%) for C29H34N6: C,
74.65; H, 7.34; N, 18.01; found: C 74.48, H 7.51, N 17.82.
tert-Butyl (5-(4-benzylpiperazin-1-yl)pentyl)(1,5-diphenyl-1H-
1,2,4-triazol-3-yl)carbamate (15c)
N-(5-(4-benzylpiperazin-1-yl)pentyl)-1,5-diphenyl-1H-1,2,4-
triazol-3-amine (13c)
Brown oil; 93% yield; 1H NMR (500 MHz, CDCl3) (d ppm): 7.48–
7.46 (d, 2H), 7.40–7.30 (m, 13H), 3.83–3.80 (t, 2H), 3.61 (s, 2H),
2.86 (m, 6H), 1.89 (m, 2H), 1.76–1.72 (m, 2H), 1.51 (sl, 9H), 1.45–
1.42 (m, 2H); DEPT-Q (125 MHz, CDCl3) (d ppm): 159.33, 153.58,
153.28, 137.86, 130.06, 129.30, 128.49 (2X), 127.54 (2X), 125.43,
81.25, 61.88, 57.28, 49.58, 47.77, 28.26, 27.94, 23.81. Elemental
analysis calcd (%) for C29H39N5O2: C, 71.13; H, 8.03; N, 14.30;
found: C 70.89, H 8.22, N 14.29.
Brown oil; 88% yield; 1H NMR (400 MHz, CDCl3) (d ppm): 7.42–
7.41 (d, 2H), 7.35–7.29 (m, 13H), 4.82 (m, 1H), 4.69 (m, 1H),
4.32–4.31 (sl, 1H), 3.60 (sl, 2H), 3.37–3.33 (m, 2H), 2.82 (m, 10H),
1.84 (m, 2H), 1.72–1.66 (m, 2H), 1.48–1.42 (m, 2H); DEPT-Q (100
MHz, CDCl3) (d ppm): 163.95, 152.75, 138.28, 129.74, 129.20,
128.68, 128.41, 128.11, 128.05, 127.71, 125.22, 61.93, 57.30,
49.95, 43.05, 29.63, 29.21, 24.05. Elemental analysis calcd (%)
for C30H36N6: C, 74.97; H, 7.55; N, 17.48; found: C 74.66, H
7.68, N 17.27.
tert-Butyl (6-(4-benzylpiperazin-1-yl)hexyl)(1,5-diphenyl-1H-
1,2,4-triazol-3-yl)carbamate (15d)
N-(6-(4-benzylpiperazin-1-yl)hexyl)-1,5-diphenyl-1H-1,2,4-
triazol-3-amine (13d)
Brown oil; 81% yield; 1H NMR (500 MHz, CDCl3) (d ppm): 7.48–
7.47 (d, 2H), 7.40–7.31 (m, 12H), 3.81–3.78 (t, 2H), 3.65–3.57 (m,
2H), 3.88 (m, 2H), 2.95–2.80 (m, 7H), 1.88 (m, 2H), 1.73–1.70 (m, Brown oil; 90% yield 1H NMR (400 MHz, CDCl3) (d ppm): 7.45–
2H), 1.51 (sl, 9H), 1.41 (m, 4H), 0.94–0.89 (m, 1H); DEPT-Q (125 7.43 (d, 2H), 7.37–7.31 (m, 12H), 4.27 (sl, 1H), 3.54 (sl, 2H), 3.37–
MHz, CDCl3) (d ppm): 159.40, 153.59, 153.26, 137.90, 130.05, 3.33 (m, 2H), 2.59–2.50 (m, 10H), 1.67–1.57 (m, 4H), 1.45–1.35
129.30 (2X), 128.90 (2X), 128.80 (2X), 128.56 (2X), 127.59, 125.44 (m, 4H); DEPT-Q (100 MHz, CDCl3) (d ppm): 164.11, 152.79,
(2X), 81.15, 61.80, 57.21, 48.11, 46.80, 31.50, 29.56, 28.30, 28.27, 138.42, 129.76, 129.29, 128.77, 128.46, 128.31, 128.20, 128.12,
26.38, 25.87, 25.27, 22.61, 13.93, 11.80. Elemental analysis calcd 127.27, 125.31, 62.73, 58.20, 52.73, 52.07, 43.56, 29.77, 27.11,
(%) for C30H41N5O2: C, 71.54; H, 8.20; N, 13.90; found: C 71.30, 26.68, 25.91. Elemental analysis calcd (%) for C31H38N6: C,
H 8.45, N 13.71.
75.27; H, 7.74; N, 16.99; found: C 74.99, H 7.96, N 16.75.
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the type of inhibition observed. Ki, Ki0 (competitive and non-
competitive inhibition constants respectively), Km (Michaelis–
Menten constant) and Vmax values were calculated with Graph-
pad Prism 7.0 using the non-linear regression models for
enzyme kinetics – inhibition and enzyme Kinect – substrate vs.
velocity.
Molecular modeling. A molecular docking study was imple-
mented with the acetylcholinesterase of the electric eel (Elec-
trophorus electricus) (EeAChE) and with the horse (Equus
caballus) butyrylcholinesterase (EcBChE) in order to get insight
of possible reasons for the observed enzyme inhibition data at
the molecular level. Cholinesterases have two known binding
sites: the active site, where the enzymes hydrolyse their
substrates; and the peripheral site, both located at the extrem-
ities of a gorge in the enzyme structure.31 Both sites were
explored in the docking study, to determine which one is the
most suitable for the binding of these compounds.
We implemented redocking experiments as a previous test
for the ability of the docking program GOLD 5.6 (CCDC So-
ware Ltd., Cambridge, UK) to nd reliable solutions for the
docking to cholinesterases. Since all EeAChE structures acces-
sible in the Protein Data Bank (PDB) do not have ligands in their
crystallographic structures, the scoring functions available in
the program were tested through redocking procedures with the
PDB structure 2CMF (Torpedo californica AChE co-crystallized
with alkylene-linked bis-tacrine dimer).24 Hydrogen atoms
were added to proteins structures based on ionization and
tautomeric states dened by GOLD. The bonds of groups
capable to donate hydrogen bonds present in serine, threonine,
tyrosine and lysine side chains were set free to rotate during the
docking procedure to allow the best orientation of the hydrogen
bonds. In the course of the searching procedure, 100 000
genetic operations (crossover, migration, mutation) were used
for each docking run. Radius of binding sites for the enzymes
Biological assessment
Anticholinesterase activity assays. The anticholinesterase
activity was determined according to adapted Ellman's
method.21,25 All of the solutions were prepared in 0.02 M Tris–
HCl buffer (pH ¼ 7.5), stock solutions of test compounds
prepared in DMSO (50 mM), and the experiment conducted in
triplicate. To a at-bottom 96-well transparent plate were added
150 mL of treatment solutions with inhibitors and donepezil at
eight different concentrations serially diluted. Negative
(without treatment) and vehicle (DMSO, nal concentration
0.2% v/v for AChE and 0.8% v/v for BuChE) controls were kept
for reference. Following, there were added 60 mL of 5,50-
dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent) at
1.1 mM and 30 mL of electric eel acetylcholinesterase (EeAChE)
or equine serum butyrylcholinesterase (EqBuChE) at 0.20 U
mLꢂ1 in the presence of 1 mg mLꢂ1 bovine serum albumin
(BSA). Absorbance was then recorded using an iMark plate
reader (Bio-Rad) equipped with a l ¼ 415 nm light lter and this
measure used as a blank reference. Aer a 10 minute incubation
at 30 ꢀC, 24 mL of 2.75 mM acetylthiocholine iodide (ACTI) or S-
butyrylthiocholine iodide (BCTI) were added and the absor-
bance recorded aer a 10 minute incubation at 30 ꢀC at l ¼
415 nm for 3 times within 30 seconds. Enzyme activity was
calculated as percentage of untreated control discounting the
blank reference. The nal inhibitor concentration ranged from
100–0.00001 mM for AChE (dilution factor ¼ 10) and from 400–
0.1 mM for BuChE (dilution factor ¼ 4). IC50 values were calcu-
lated on Graphpad Prism 7.0 using the non-linear regression
model for dose–response inhibition.
Enzymatic kinetic study. The enzymatic kinetic of cholines-
terase inhibition was determined according to adapted Ell-
man's method.21,25 All of the solutions were prepared in 0.02 M
Tris–HCl buffer (pH ¼ 7.5), stock solutions of test compounds
prepared in DMSO (50 mM), and the experiment conducted in
triplicate. To a at-bottom 96-well transparent plate were added
150 mL of treatment solutions with inhibitors at two different
concentrations distributed in eight sets of triplicates each.
Eight sets of untreated triplicates were used as negative control.
Following, there were added 60 mL of 5,50-dithiobis(2-
nitrobenzoic acid) (DTNB, Ellman's reagent) at 1.1 mM and 30
mL of electric eel acetylcholinesterase (EeAChE) or equine serum
butyrylcholinesterase (EqBuChE) at 0.20 U mLꢂ1 in presence of
1 mg mLꢂ1 bovine serum albumin (BSA). Absorbance was then
recorded using an iMark plate reader (Bio-Rad) equipped with
a l ¼ 415 nm light lter and this measure used as a blank
reference. Aer 10 minute incubation at room temperature, 24
mL of acetylthiocholine iodide (ACTI) or S-butyrylthiocholine
iodide (BCTI) at eight concentrations serially diluted (factor ¼
1.3) from 2.75–0.44 mM (nal concentration: 0.25–0.04 mM)
were added to all wells and the absorbance recorded aer
incubation for 0, 5, 10, 15 and 20 minutes at room temperature
at l ¼ 415 nm. The Lineweaver–Burk reciprocal plots were ob-
tained by plotting a 1/velocity versus 1/[substrate] graph for two
different inhibitor concentrations and untreated control. The
linear regression of each data-set shows a convergent behav-
iour, in ways the region to where the curves converge determine
˚
˚
was tested at 15 A and 20 A around atoms from adequate amino
acids selected based on literature information for each binding
site. The same score function, corresponding amino acids and
radius were adopted for the docking studies with the EcBChE
model.
Three EeAChE structures are available in PDB, with codes
1EEA,32 1C2B33 and 1C2O34 and all of them were evaluated in our
docking studies. There is no available crystallographic structure
for EcBChE, so it was necessary the construction of a 3D model
from a sequence available in the UniProtKB/Swiss-Prot protein
sequence database (entry Q9N1N9) with the protein structure
homology-modelling server, Swiss-Model,31 as previously
described,24 using as a template the human BChE with PDB
code 4TPK.35 The resolution of the template is 2.52 A, the
˚
identity between sequences is 90.40%, with a coverage 0.95. The
global and per-residue model quality has been assessed using
the QMEAN scoring function.
In the GOLD program, the docking functions yield the
“tness scores”, which are dimensionless values. The score of
each pose identied is calculated as the negative of the sum of
a series of energy terms involved in the protein–ligand inter-
action process, so that the more positive the score, the better is
the interaction. The score values are a guide of how good the
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docking pose is, with a higher score indicating a better inter-
action between the ligand and the binding site.
9 Y. Yu, J. M. Ostresh and R. A. Houghten, Tetrahedron Lett.,
2003, 44, 7841.
Spartan'14 program [Wavefunction, Inc.] was utilized to 10 D. G. Churchill, J. Chem. Educ., 2006, 83, 1798.
construct and optimize the molecules in study, with the PM6 11 A. A. Schiltand, L. C. McBride, Perchloric Acid and
method. Since these compounds have two plausible proton-
ation sites in the piperazine ring, structures protonated at each
Perchlorates, ed. G. F. Smith, Chemical Co, Columbus
(USA), 2003.
of them and also with both of them protonated were considered 12 J. Shen, B. Wong, C. Gu and H. Zhang, Org. Lett., 2015, 17,
in our studies. Several poses were obtained for each compound 4678.
in all proteins, and the best-ranked pose for each one was 13 (a) D. Garella, E. Borretto, A. Di Stilo, K. Martina, G. Cravotto
chosen for analysis of the interactions with the amino acid
residues.
and P. Cintas, Med. Chem. Commun., 2013, 4, 1323; (b)
´
¨
N. M. Nascimento-Junior, A. E. Kummerle, E. J. Barreiro
and C. A. M. Fraga, Molecules, 2011, 16, 9274.
14 S. M. Hickey, T. D. Ashton, S. K. Khosa, R. N. Robson,
J. M. White, J. Li, R. L. Nation, H. Y. Yu, A. G. Elliott,
M. S. Butler, J. X. Huang, M. A. Cooperd and F. M. Pfeffer,
Org. Biomol. Chem., 2015, 13, 6225.
Conflicts of interest
There is no conict to declare.
15 R. Anand, K. D. Gill and A. A. Mahdi, Neuropharmacology,
2014, 76, 27.
Acknowledgements
Fellowship and nancial support for this study was provided by 16 A. V. Terry and J. J. Buccafusco, J. Pharmacol. Exp. Ther.,
´
Conselho Nacional de Desenvolvimento Cientıco e Tec-
2003, 306, 821.
´
˜
`
nologico - Brasil (CNPq), Fundaçao de Amparo a Pesquisa do 17 C. P. Ferri, M. Prince, C. Brayne, M. Ganguli, K. Hall,
˜
Estado do Rio de Janeiro – Brasil (FAPERJ) and Coordenaçao de
K. Hasegawa, H. Hendrie, Y. Huang, A. Jorm, C. Mathers,
P. R. Menezes, E. Rimmer and M. Scazufca, Lancet, 2005,
366, 2112.
´
Aperfeiçoamento de Pessoal de Nıvel Superior – Brasil (CAPES) –
Finance Code 001.
18 A. Sinha, R. S. Tamboli, B. Seth, A. M. Kanhed, S. Kant
Tiwari, S. Agarwal, S. Nair, R. Giridhar, R. K. Chaturvedi
and M. R. Yadav, Mol. Neurobiol., 2015, 52, 638.
Notes and references
¨
1 S. Borg, G. Estenne-Bouhtou, K. Luthman, I. Csoregh, 19 L. M. Lima and E. J. Barreiro, Curr. Med. Chem., 2005, 12, 23.
W. Hesselink and U. Hacksell, J. Org. Chem., 1995, 60, 3112. 20 C. Viegas-Junior, A. Danuello, V. d. S. Bolzani, E. J. Barreiro
2 E. Bozo, G. Szilagyi and J. Janaky, Arch. Pharm., 1989, 322,
583.
3 Y. Naito, F. Akahoshi, S. Takeda, T. Okada, M. Kajii,
and C. A. Fraga, Curr. Med. Chem., 2007, 14, 1829.
21 G. L. Ellman, K. D. Courtney, V. Andres and
R. M. Featherstone, Biochem. Pharmacol., 1961, 7, 88.
H. Nishimura, M. Sugiura, C. Fukaya and Y. J. Kagitani, J. 22 H. Sugimoto, Y. Iimura, Y. Yamanishi and K. Yamatsu, J.
Med. Chem., 1996, 39, 3019.
Med. Chem., 1995, 24, 4821.
4 J. P. Marino, P. W. Fisher, G. A. Hofmann, R. B. Kirkpatrick, 23 G. Jones, P. Willett, R. C. Glen, A. R. Leach and R. Taylor, J.
C. A. Janson, R. K. Johnson, C. Ma, M. Mattern, T. D. Meek,
Mol. Biol., 1997, 267, 727.
M. D. Ryan, C. Schulz, W. W. Smith, D. G. Tew, 24 E. H. Rydberg, B. Brumshtein, H. M. Greenblatt, D. M. Wong,
T. A. Tomazek, D. F. Veber, W. C. Xiong, Y. Yamamoto,
K. Yamashita, G. Yang and S. K. Thompson, J. Med. Chem.,
2007, 50, 3777.
5 J.-L. Fauchere, J.-C. Ortuno, J. Duhault, J. A. Boutin and
N. Levens, European Patent EP 1044970, 2000.
6 G. Winterer, J. Gallinat, J. Brinkmeyer, F. Musso,
D. Shaya, L. D. Williams, P. R. Carlier, Y. P. Pang, I. Silman
and J. L. Sussman, J. Med. Chem., 2006, 49, 5491.
25 G. A. de Souza, S. J. da Silva, C. N. Del Cistia, P. Pitasse-
Santos, L. O. Pires, Y. M. Passos, Y. Cordeiro,
C. M. Cardoso, R. N. Castro, C. M. R. Sant'Anna and
¨
A. E. Kummerle, J. Enzyme Inhib. Med. Chem., 2019, 34, 631.
J. Kornhuber, N. Thuerauf, D. Rujescu, R. Favis, Y. Sun, 26 D. Antoine, M. Olivier and Z. Vincent, Sci. Rep., 2017, 7,
M. A. Franc, S. Ouwerkerk-Mahadevan, L. Janssens, 42717.
M. Timmers and J. R. Streffer, Neuropharmacology, 2013, 27 D. Antoine and Z. Vincent, ChemMedChem, 2016, 11, 1117.
64, 197.
28 C. A. Lipinski, F. Lombardo, B. W. Dominy and P. J. Feeney,
7 (a) S. Maddila, R. Pagadala and S. B. Jonnalagadda, Lett. Org.
Adv. Drug Delivery Rev., 2001, 46, 3.
Chem., 2013, 10, 693; (b) J. Meng and P. P. Kung, Tetrahedron 29 D. F. Veber, R. J. Stephen, C. Hung-Yuan, B. R. Smith,
Lett., 2009, 50, 1667; (c) G. M. Makara, Y. Ma and
K. W. Ward and K. D. Kopple, J. Med. Chem., 2002, 45, 2615.
L. Margarida, Org. Lett., 2002, 4, 1751; (d) 30 W. J. Egan, K. M. Merz and J. J. Baldwin, J. Med. Chem., 2000,
A. V. Bogolyubsky, O. Savych, A. V. Zhemera, S. E. Pipko,
43, 3867.
A. V. Grishchenko, A. I. Konovets, R. O. Doroshchuk, 31 L. Pezzementi, F. Nachon and A. Chatonnet, PLoS One, 2011,
D. N. Khomenko, V. S. Brovarets, Y. S. Moroz and
M. Vybornyi, ACS Comb. Sci., 2018, 20, 461.
8 A. R. Katritzky, B. V. Rogovoy, V. Y. Vvedensky, K. Kovalenko,
P. J. Steel, V. I. Markov and B. Forood, Synthesis, 2001, 6, 897.
6, e17396.
32 M. L. Raves, K. Giles, J. D. Schrag, M. F. Schmid,
G. N. Phillips Jr, W. Chiu, A. J. Howard, I. Silman and
J. L. Sussman, Quaternary Structure of Tetrameric
20368 | RSC Adv., 2019, 9, 20356–20369
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Paper
RSC Advances
Acetylcholinesterase, in Structure and Function of 34 M. Biasini, S. Bienert, A. Waterhouse, K. Arnold, G. Studer,
Cholinesterases and Related Proteins, ed. B. P. Doctor, P.
Taylor, D. M. Quinn, R. L. Rotundo and M. K. Gentry,
Springer, Boston, MA, 1998.
T. Schmidt, F. Kiefer, T. G. Cassarino, M. Bertoni,
L. Bordoli and T. Schwede, Nucleic Acids Res., 2014, 42, W252.
35 B. Brus, U. Kosak, S. Turk, A. Pislar, N. Coquelle, J. Kos,
J. Stojan, J. P. Colletier and S. Gobec, J. Med. Chem., 2014,
57, 8167.
33 Y. Bourne, J. Grassi, P. E. Bougis and P. Marchot, J. Biol.
Chem., 1999, 274, 30370.
This journal is © The Royal Society of Chemistry 2019
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