Synthesis and leishmanicidal activity of eugenol derivatives bearing 1,2,3-triazole functionalities

Article abstract of DOI:10.1016/j.ejmech.2018.01.046

In this paper, it is described the synthesis and the evaluation of the leishmanicidal activity of twenty-six eugenol derivatives bearing 1,2,3-triazole functionalities. The evaluation of the compounds on promastigotes of Leishmania amazonensis (WHOM/BR/75/Josefa) showed that eugenol derivatives present leishmanicidal activities with varying degrees of effectiveness. The most active compound, namely 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-methylbenzyl)-1H-1,2,3-triazole (7k) (IC₅₀ = 7.4 ± 0.8 μmol L^?1), also targeted Leishmania parasites inside peritoneal macrophages (IC₅₀ = 1.6 μmol L^?1) without interfering with cell viability. The cytotoxicity of 7k against macrophage cells presented IC₅₀ of 211.9 μmol L^?1 and the selective index was equal to 132.5. Under similar conditions, compound 7k was more effective than glucantime and pentamidine, two drugs currently in the clinic. In addition, theoretical calculations showed that this compound also presents most physicochemical and pharmacokinetic properties within the ranges expected for orally available drugs. It is believed that eugenol bearing 1,2,3-triazole functionalities may represent a scaffold to be explored toward the development of new agents to treat leishmaniasis.

Full text of DOI:10.1016/j.ejmech.2018.01.046

European Journal of Medicinal Chemistry 146 (2018) 274e286
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and leishmanicidal activity of eugenol derivatives bearing
1,2,3-triazole functionalities
a
,
Robson Ricardo Teixeira *, Poliana Aparecida Rodrigues Gazolla a,
ꢀ
Adalberto Manoel da Silva b, Maria Paula Gonçalves Borsodi c, Bartira Rossi Bergmann c,
d
e
e
ꢀ
Rafaela Salgado Ferreira , Boniek Gontijo Vaz , Gessica Adriana Vasconcelos ,
Wallace Pacienza Lima c
a Departamento de Química, Universidade Federal de Viçosa, Av. P.H. Rolfs, S/N, Viçosa, MG, 36570-900, Brazil
Instituto Federal de Educaçao, Ciencia e Tecnologia Catarinense, Campus Araquari Rodovia BR 280, Km 27, Cx. Postal 21, Araquari, SC, 89245-000, Brazil
Instituto de Biofísica Carlos Chagas Filho, Laboratorio de Imunofarmacologia, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, Bloco
b
~
^
c
ꢀ
~
I2-038, Ilha do Fundao, Rio de Janeiro, 21941-902, RJ, Brazil
d
^
Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, Belo Horizonte, 31270-901, MG, Brazil
e
ꢀ
^
Instituto de Química, Universidade Federal de Goias, Av. Esperança, S/N, Campus Samambaia, Goiania, 74690-970, GO, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
In this paper, it is described the synthesis and the evaluation of the leishmanicidal activity of twenty-six
eugenol derivatives bearing 1,2,3-triazole functionalities. The evaluation of the compounds on promas-
tigotes of Leishmania amazonensis (WHOM/BR/75/Josefa) showed that eugenol derivatives present
leishmanicidal activities with varying degrees of effectiveness. The most active compound, namely 4-(3-
Received 18 August 2017
Received in revised form
11 January 2018
Accepted 15 January 2018
(4-allyl-2-methoxyphenoxy)propyl)-1-(4-methylbenzyl)-1H-1,2,3-triazole
(IC50 ¼ 7.4 0.8
mol Lꢀ1), also targeted Leishmania parasites inside peritoneal macrophages
(IC50 ¼ 1.6
mol Lꢀ1) without interfering with cell viability. The cytotoxicity of 7k against macrophage
cells presented IC50 of 211.9
mol Lꢀ1 and the selective index was equal to 132.5. Under similar condi-
(7k)
m
m
Keywords:
Eugenol
m
tions, compound 7k was more effective than glucantime and pentamidine, two drugs currently in the
clinic. In addition, theoretical calculations showed that this compound also presents most physico-
chemical and pharmacokinetic properties within the ranges expected for orally available drugs. It is
believed that eugenol bearing 1,2,3-triazole functionalities may represent a scaffold to be explored to-
ward the development of new agents to treat leishmaniasis.
Leishmaniasis
Antileishmanial
Leishmanicidal
Triazole
© 2018 Elsevier Masson SAS. All rights reserved.
1. Introduction
countries, with a global incidence of 0.9e1.6 million cases per year
[1]. Leishmania parasites are transmitted via the bites of infected
Parasitic diseases affect millions of hundreds of people annually
and constitute an important global health problem. These diseases
affect mainly the poorest population around the world and repre-
sent a significant issue in terms of human health and well-being.
Malaria, River blindness (Onchocerciasis), Lymphatic Filasis,
Schistosomiasis, Trypanosomiasis, and Leishmaniasis are some
examples of parasitic diseases.
Leishmaniasis is a complex of diseases caused by the protozoan
Leishmania and classified as one of the most important neglected
tropical diseases (NTDs) that affect 350 million people in 98
female phlebotomine sandflies. Three main clinical manifestations
of leishmaniasis, depending on the parasite species and the host-
parasite relationship, are known: visceral (often known as Kalazar
and the most serious form of the disease), cutaneous (the most
common), and mucocutaneous [2]. Visceral leishmaniasis is
responsible for 20,000e40,000 deaths annually [1].
Currently, several investigations have been conducted to find
alternative treatments for leishmaniasis [3] due to the small
number of available drugs and the development of resistance or
decreased the sensitivity of parasite strains to existing treatments
that have been utilized for human therapy [3]. These studies have
sought new methods and targets for diagnosis, new vaccine can-
didates and new rationally designed drugs that can be applied not
* Corresponding author.
E-mail address: robsonr.teixeira@ufv.br (R.R. Teixeira).
https://doi.org/10.1016/j.ejmech.2018.01.046
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

R.R. Teixeira et al. / European Journal of Medicinal Chemistry 146 (2018) 274e286
275
only in humans but also in dogs because canines are considered the
major reservoirs of several species of Leishmania [4,5].
regard, compounds obtained from nature can be used directly as a
pharmaceutical or can be chemically modified to afford derivatives
with improved pharmacological activity.
Historically, chemotherapy for leishmaniasis has relied on the
use of pentavalent antimonial drugs, such as N-methylglucamine
antimoniate (Fig. 1), which is one of the most widely used drugs [6].
However, this drug has limited clinical potential due to the occur-
rence of serious side effects and high incidence of disease recur-
rence [7]. Pentamidine (Fig. 1) is another antileishmanial agent, but
it is inadequate as a first-line treatment because of its high toxicity
[8]. Amphotericin B has been used as a second-choice drug in the
treatment of leishmaniasis since the 1960s, but the rate of resis-
tance against this drug is high [8]. Two other examples of anti-
leishmanial compounds are miltefosine and paromomycin (Fig. 1).
The major limitation of miltefosine is teratogenicity, excluding its
use in women of child-bearing age [9,10]. The most common side
effect associated with the paromomycin is the ototoxicity, as well as
problems in liver function. In patients treated with the ointment
formulation, skin rashes, local pruritus and burs have been
encountered [11]. Additionally, the drugs used in therapy have
some limitations in common: (i) they are toxic and their long-time
administration causes various undesirable side effects; (ii) they are
costly; (iii) they show low efficacy in endemic areas due to the
observed resistance of various parasite species [12]. The afore-
mentioned problems illustrate the need to develop new anti-
leishmanial drugs.
Eugenol (1) (Fig. 2) is a natural compound which has been
identified in several aromatic plants such as Myristica fragrans
Houtt. (nutmeg), Cinnamomum verum J.Presl (true cinnamon),
C. loureirii Nees. (Saigon cinnamon), Ocimum gratissimum Forssk.
(basil) and Ocimum basilicum L. (sweet basil). However, Eugenia
caryophyllata (¼Syzygium aromaticum) can be considered the
principal natural source of this compound as it represents between
45% and 90% of the composition of the essential oil derived from
this species. Several biological activities have been reported for
eugenol (1) including antibacterial, antifungal, antiplasmodial,
In the search for new drugs for the treatment of diseases, the
exploitation of natural products is a viable approach [13e18]. In this
Fig. 2. The structure of eugenol (1).
Fig. 1. Structures of drugs used for the treatment of leishmaniasis.

276
R.R. Teixeira et al. / European Journal of Medicinal Chemistry 146 (2018) 274e286
antiviral, anti-inflammatory, analgesic, antioxidant, anticancer and
anti-diabetes [19,20].
NMR spectra. The carbon chemical shifts are compatible with the
structures of the compounds. In the IR spectra, expected bands for
functional groups were noticed. The molecular formulas of the
eugenol triazolic derivatives were confirmed by high-resolution
mass spectrometry analyses.
An investigation conducted with Ocimum gratissimum revealed
that its eugenol-rich essential oil presents leishmanicidal activity.
The IC50 values of the oil against promastigotes and amastigotes
were, respectively, 135
was also evaluated and the activity against promastigote forms was
even more pronounced (IC50 value of 80
g mLꢀ1) [21]. Considering
m m
g mLꢀ1 and 100 g mLꢀ1. Eugenol (1) alone
2.2. Evaluation of leishmanicidal activity of eugenol derivatives
m
the leishmanicidal effect of eugenol (1) [21] and its derivatives [22]
as well as reports on the antileishmanial activity of 1,2,3-triazole
containing compounds [23e25], it is described in this paper the
synthesis and leishmanicidal activity evaluation of a series of
compounds derived from eugenol and containing 1,2,3-triazolic
portions.
Taking into consideration previous reports concerning the
leishmanicidal activity of eugenol (1) and triazole containing
compounds, we turned our attention to evaluate the leishmanicidal
activities of eugenol derivatives 6ae6n and 7ae7m. The treatment
of the parasites with the compounds resulted in a dose-dependent
growth inhibition of L. amazonensis promastigotes. The 50% growth
inhibitory activity values (IC50), after 72 h of treatment of each
compound, were determined by the Alamar Blue assay [34], and are
shown in Table 1. Eugenol derivatives 6a, 6b, 6k, 7a, 7b, and 7k
were the most active ones, presenting IC50 values of 30.2, 59.4, 49.2,
2. Results and discussion
2.1. Synthesis of eugenol derivatives
37.9, 32.2 and 7.4
tericin B were used as positive controls in the assays and presented
IC50 of 4.2 and 0.4
mol Lꢀ1, respectively. It is worth to mention that
the IC50 values of compounds 6a, 6b, 6k, 7a, 7b, and 7k are superior
to the IC50 of eugenol (1) (80
g mLꢀ1; 487 mol Lꢀ1) evaluated
m
mol Lꢀ1, respectively. Pentamidine and ampho-
The first step towards the preparation of eugenol 1,2,3-triazolic
compounds corresponded to the extraction of eugenol (1). This
compound was obtained via hydrodistillation from cloves pur-
chased in the local market in Viçosa, Minas Gerais State, Brazil. After
the extraction procedure, the compound was purified by silica gel
column chromatography. Next, eugenol (1) was submitted to
alkylation reactions affording the derivatives 2 and 4 as outlined in
Scheme 1 [26].
m
m
m
against promastigote forms of L. amazonensis [21].
After demonstrating leishmanicidal activity against extracel-
lular promastigotes, the next step was to investigate the effect
against intracellular amastigotes, the parasite form responsible for
the mammalian infection. We determined that compound 7k, the
most active against promastigote form, was able to target the
Leishmania parasites inside of mouse peritoneal macrophages
For the synthesis of eugenol derivatives containing triazole
rings, it was required the preparation of several benzyl azides
(compounds of general structure 5 in Scheme 2) which were ob-
tained in two steps from the corresponding benzyl alcohols uti-
lizing methodology previously described in the literature [27].
The key step involved in the synthesis of eugenol derivatives
6ae6n and 7ae7m (Scheme 3) corresponded to the Copper(I)-
catalyzed Azide-Alkyne Cycloaddition (CuAAC), also known as
click reaction [28e33], between the alkynylated compounds 2 and
4 and the benzyl azides 5. Compounds were obtained with syn-
thetically useful yields between 77% and 91% (Scheme 3). In gen-
eral, reactions took less than 1 min for their completion. The
synthesis of the compounds was planned so that the influence on
the leishmanicidal activity of different benzylic groups attached to
triazole ring as well as the length of the chain connecting the tri-
azole ring to the eugenol portion could be assessed. All the triazolic
compounds were fully characterized by NMR (13C and 1H), IR and
high-resolution mass spectrometry. In the 1H NMR spectra, the
signal for the hydrogen present in the triazolic ring was observed
within the 7.20e7.67 ppm range. The allylic protons were observed
as doublets, while the hydrogen atoms of methylene groups
attached to nitrogen or oxygen were noticed as singlets in the 1H
(IC50 ¼ 1.6
(Table 2). At similar conditions, pentamidine and glucantime
exhibited IC50 values of 1.8
mol Lꢀ1 and 45.5 g mLꢀ1
(124.3
mol Lꢀ1) respectively. Compound 7k was assayed against
uninfected murine intraperitoneal macrophages to evaluate its
cytotoxicity; it was found LD50 of 211.9
mol Lꢀ1. At similar condi-
tions, pentamidine and glucantime exhibited LD50 values of
8.5
mol Lꢀ1 and 75.5 g mLꢀ1 (206.3 mol Lꢀ1), respectively. Thus,
m
mol Lꢀ1), without interfering with host cell viability
m
m
m
m
m
m
m
the selectivity index (SI ¼ LD50/IC50 intracellular amastigotes) value
for compound 7k was 132.5, whereas SI ¼ 4.7 for pentamidine and
SI ¼ 1.6 for glucantime. Therefore, the derivative 7k presents lower
toxicity when compared to drugs currently used in the treatment of
leishmaniasis. In order to determine whether anti-amastigote ac-
tivity resulted from activation of the macrophage antileishmanial
mechanism, compound 7k was also evaluated with respect to the
nitric oxide production. It was observed that 7k did not signifi-
cantly stimulate the production of nitric oxide, suggesting a direct
and selective action of this derivative on the intracellular amasti-
gote (data not shown).
Scheme 1. Alkylation procedures of eugenol (1).

R.R. Teixeira et al. / European Journal of Medicinal Chemistry 146 (2018) 274e286
277
Scheme 2. Synthesis and structures of benzyl azides.
Infected amastigote macrophages treated with 7k, glucantime
3. Conclusion
and without treatment (negative control) were observed by
microphotography and revealed that the parasites were killed but
that the host cells remained unaffected (Fig. 3).
In conclusion, a series of compounds derived from eugenol and
containing 1,2,3-triazolic portions were prepared in good yields
and described for the first time. Among all the evaluated derivatives
against
L.
amazonensis,
compound
4-(3-(4-allyl-2-
methoxyphenoxy)propyl)-1-(4-methylbenzyl)-1H-1,2,3-triazole
(7k) showed significant antileishmanial activity and selectivity
index superior to those of pentamidine and glucantime, while also
having most physicochemical and pharmacokinetic properties
within the ranges expected for orally available drugs. The results
suggest that these compounds can be used as promising novel
prototypes for drug development against leishmaniasis.
2.3. Analysis of estimated physicochemical and pharmacokinetic
properties
During drug development, it is important to consider several
relevant properties, in addition to leishmanicidal activity. There-
fore, to evaluate if the most potent compounds against
L. amazonensis (6a, 6b, 6k, 7a, 7b, 7k) are suitable for further steps
of lead optimization, a set of 44 properties were estimated
employing the software QikProp [35]. Among those, we highlight
the analysis of those referring to Lipinski's [36] and Veber's rules
[37], which are considered as reference parameters for orally
available drugs. The six highlighted compounds obey Veber's rules
and most of the Lipinski's rules, except for clogP (Table 3). These
results indicate that the compounds herein investigated obey most
physicochemical drug-like properties.
Other key properties for drug development, such as the esti-
mated affinity to HERG Kþ channels (QPlogHERG), permeability
through Caco-2 (QPPCaco) and MDCK cells (QPPMDCK) and skin
(QPlogKp) and the number of predicted metabolic sites (#metab),
were also calculated (Table 4). This analysis reveals properties
which would probably have to be improved when optimizing these
compounds. For example, a key concern would be to reduce the
affinity for HERG, a Kþ channel commonly associated with cardiac
toxicity [38]. Skin permeability should also be improved in case a
topical application is desired, as indicated by the QPlogKp values.
On the other hand, high predicted permeability through Caco-
2 cells, a model for the gut-blood barrier, and MDCK cells, which
model the blood brain barrier, indicate good overall permeability in
biological membranes, which is a desirable property for orally
available drugs. The predicted number of metabolites is also within
the desired range, observed for most drugs. Altogether, most of the
calculated properties seem favorable for drug development.
4. Material and methods
4.1. Extraction and purification of eugenol and synthesis of 1,2,3-
triazole derivatives
4.1.1. Generalities
Solvents were purchased from Vetec (Rio de Janeiro, Brazil).
Benzyl alcohols, pent-4-yn-1-ol, methanesulfonyl chloride, sodium
azide, triethylamine, propargyl bromide, geneticin and dimethyl
sulfoxide hybri-max (DMSO) were purchased from Sigma Aldrich
and used as received. M199 medium powder, fetal calf serum
(HIFCS), penicillin, and streptomycin were procured from Cultilab.
Eugenol (1) was extracted via hydrodistillation from cloves pur-
chased in the local market in Viçosa, Minas Gerais State, Brazil, and
subsequently purified by column chromatography (vide infra). 1H
and 13C NMR spectra were recorded on a Varian Mercury 300 in-
strument at 300 MHz and 75 MHz, respectively, using CDCl3 or C6D6
as solvents. NMR data are presented as follows: chemical shift (d) in
ppm, multiplicity, the number of protons, J values in Hertz (Hz), and
proton assignment. Multiplicities are shown as the following ab-
breviations: s (singlet), brs (broad singlet), d (doublet), dd (double
of a doublet), t (triplet), quartet, brd (broad doublet), ddtap
(apparent doublet of doublets of triplets), q (quartet), quint
(quintet), m (multiplet). IR spectra were obtained using Varian 660-
IR equipped with GladiATR scanning from 4000 to 500 cmꢀ1. High-

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R.R. Teixeira et al. / European Journal of Medicinal Chemistry 146 (2018) 274e286
Scheme 3. Click reactions involved in the preparation of eugenol triazolic derivatives.
resolution mass spectra were recorded on a Q-Exactive (Thermo
Scientific, Bremen, Germany). The spectra were acquired using the
following conditions. Ionization source: Electron spray (þ) and (ꢀ);
Spray voltage: 3.5 kV; Capillary temperature: 275 ꢁC; Sheath gas: 5
(arbitrary units); Auxiliary gas: 0 (arbitrary units). For the mass
spectrometry analyses, the samples were prepared as follows: a
mass of 1 mg of the compound to be analyzed was dissolved in 1 mL
of acetonitrile. Then, the solution was diluted with 1 mL of meth-
anol so that the final concentration corresponded to 1 ppm. The
resulting solution was directly injected in the Q-Exactive equip-
QP5050A instrument under electron impact (70 eV) conditions of
positive ion mode. Analytical thin layer chromatography analyses
were conducted on aluminum backed precoated silica gel plates
using different solvent systems. TLC plates were visualized using
potassium permanganate solution, phosphomolybdic acid solution
and/or UV light. Flash column chromatography was performed
using silica gel 60 (60e230 mesh). Solvents were dried using
standard procedures described in the literature [39].
4.1.2. Extraction and purification of eugenol (1)
Eugenol was extracted via hydrodistillation from dried flower
buds of Eugenia caryophyllata, known as cloves, purchased in the
ment at 5
Low-resolution mass spectra were recorded on Shimadzu GCMS-
m
mL minꢀ1. The spectra were recorded in full MS mode.

R.R. Teixeira et al. / European Journal of Medicinal Chemistry 146 (2018) 274e286
279
Table 1
4.1.3. Synthesis of 4-allyl-2-methoxy-1-(prop-2-yn-1-yloxy)
benzene (2)
Effect of eugenol derivatives 6ae6n and 7ae7m on promastigote form of
L. amazonensis.
A 50 mL round bottom flask was charged with eugenol (1)
(1.20 g, 7.32 mmol), sodium hydroxide (0.313 g, 7.83 mmol) and
25 mL of methanol. The resulting mixture was heated at 40 ꢁC and
magnetically stirred for 30 min. After this time, methanol was
removed under reduced pressure and 10.0 mL of anhydrous ethanol
was added to the removal of the residual water. The ethanol was
removed under reduced pressure. Then, the round bottom flask,
under a nitrogen atmosphere, was charged with anhydrous aceto-
Compound
Promastigote IC50 (m )
mol Lꢀ1
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
30.2 1.5
59.4 1.7
1780
993
3
3
2013
3
130.2 2.1
424.9 2.6
322.1 2.5
nitrile (25.0 mL) and propargyl bromide (800 mL, 8.79 mmol) was
2013
969
3
added slowly. The mixture was magnetically stirred at room tem-
perature for 18 h. TLC analysis revealed the completion of the re-
action after this time. The reaction mixture was concentrated under
reduced pressure and the residue was partitioned between 25.0 mL
of sodium hydroxide solution (0.1 mol Lꢀ1) and 25 mL of diethyl
ether. The layers were separated and the aqueous phase was
extracted with diethyl ether (2 ꢂ 25.0 mL). The organic extracts
were combined and the resulting organic layer was washed with
brine (25.0 mL), dried over anhydrous sodium sulfate, filtrated and
concentrated under reduced pressure. The residue was purified by
column chromatography eluted with hexane-ethyl acetate (6:1 v/
v). The described procedure afforded 1.29 g (6.37 mmol, 87% yield)
of compound 2. The structure of 2 is supported by the following
data.
3
49.2 1.7
106.2 2.1
159.6 2.2
155.8 2.1
37.9 1.5
32.2 1.5
817
3
286.7 2.5
278.9 2.4
173.3 2.3
119.5 2.0
145.9 2.1
295.2 2.5
177.5 2.3
7.4 0.8
7k
7l
7m
913.3 2.9
980.2 2.9
4.2 0.3
Yellow oil, TLC: Rf ¼ 0.45 (hexane-ethyl acetate 6:1 v/v). 1H NMR
(300 MHz, CDCl3)
d
: 2.49 (t, 1H, J ¼ 2.4 Hz), 3.35 (d, 2H, J ¼ 6.7 Hz),
pentamidine
amphotericin B
3.86 (s, 3H), 4.73 (d, 2H, J ¼ 2.4 Hz), 5.03e5.14 (m, 2H), 5.96 (ddtap
,
0.4 0.1
1H, J1 ¼16.8 Hz, J2 ¼ 10.2 Hz, J3 ¼ 6.7 Hz), 6.70e6.76 (m, 2H), 6.97 (d,
1H, J ¼ 8.6 Hz). 1H NMR (300 MHz, C6D6)
: 1.99 (t, 1H, J ¼ 2.4 Hz),
d
3.14 (d, 2H, J ¼ 6.6 Hz), 3.31 (s, 3H), 4.36 (d, 2H, J ¼ 2.4 Hz),
4.93e5.02 (m, 2H), 5.85 (ddtap, 1H, J1 ¼17.0 Hz, J2 ¼ 10.4 Hz and
J3 ¼ 6.6 Hz), 6.52 (d, 1H, J ¼ 1.7 Hz), 6.57 (dd, 1H, J1 ¼8.1 Hz and
local market in Viçosa, Minas Gerais State, Brazil. Thus, 60.0 g of
cloves were mixed with 500 mL of distilled water in a round bottom
flask which was connected to the hydrodistillation apparatus. The
mixture was heated for three hours. The obtained hydrolate was
transferred to a separatory funnel and the aqueous layer was
extracted with dichloromethane (3 ꢂ 30 mL). The organic extracts
were combined and the resulting organic layer was dried over
anhydrous sodium sulfate, filtrated, and concentrated under
reduced pressure. The resulting oil was submitted to column
chromatography eluted with hexane-ethyl acetate (6:1 v/v). The
described procedure afforded 7.12 g of eugenol (1) which corre-
sponded to approximately 12% yield in relation to the initial mass of
cloves used in the extraction process. The structure of eugenol (1) is
in agreement with the following data.
J2 ¼ 1.7 Hz), 6.83 (d,1H, J ¼ 8.1 Hz). 13C NMR (75 MHz, CDCl3)
d: 39.8,
55.8, 56.9, 75.5, 78.7, 112.3, 114.6, 115.7, 120.3, 134.2, 137.4, 145.0,
149.6. MS, m/z (%) 202 (
, C13H14O2, 40), 163 (100), 135 (9), 103
(42), 91 (33), 77 (19), 65 (13), 55 (9), 41 (23).
4.1.4. Synthesis of pent-4-yn-1-yl methanesulfonate (3)
Pent-4-yn-1-ol (1.68 g, 20.0 mmol) and dichloromethane
(20 mL) were added to a 100 mL round-bottom flask under nitrogen
atmosphere. The mixture was cooled to ꢀ50 ꢁC and triethylamine
(5.60 mL, 40.0 mmol) was added. After that, methanesulfonyl
chloride was added slowly (2.32 mL, 30.0 mmol) to the reaction
mixture under continuous stirring. The progress of the reaction was
monitored by TLC. After completion of it, 10 mL of distilled water
were added. The organic phase was washed with 1% HCl solution
(3 ꢂ 15 mL) followed by saturated aqueous NaHCO3 (3 ꢂ 5 mL),
dried over anhydrous sodium sulfate, and concentrated under
reduced pressure. The residue was purified by column chroma-
tography eluted with hexane-ethyl acetate-dicloromethane (3:1:3
Yellow oil, TLC: Rf ¼ 0.44 (hexane-ethyl acetate 6:1 v/v). 1H NMR
(300 MHz, CDCl3)
d
: 3.33 (d, 2H, J ¼ 6.7 Hz), 3.88 (s, 3H), 5.02e5.13
(m, 2H), 5.54 (s, 1H, OH), 5.97 (ddtap, 1H, J1 ¼16.8 Hz, J2 ¼ 10.2 Hz
and J3 ¼ 6.7 Hz), 6.67e6.72 (m, 2H), 6.86 (d, 1H, J ¼ 8.5 Hz). 13C NMR
(75 MHz, CDCl3) d: 39.8, 55.8, 111.1, 114.2, 115.5, 121.1, 131.9, 137.8,
143.8,146. MS, m/z (%) 164 (
103 (32); 91 (28); 77 (34); 65 (13); 55 (24); 41 (7).
, C10H12O2, 100); 149 (35); 131 (29);
Table 2
In vitro antileishmanial activity of 7k against promastigotes, intracellular amastigotes of L. amazonensis after 72 h of treatment, and its cytotoxicity against macrophages.
Compound
Promastigotes IC50
(
m
mol Lꢀ1
)
Amastigotesa IC50
(
m
mol Lꢀ1
)
Macrophagesb LD50
(
m
mol Lꢀ1
)
Selectivity index (SI)c
7k
7.4 0.8
ndd
4.2 0.4
1.6 0.2
45.5 1.6
1.8 0.2
211.9 2.3
75.5 2.6
8.5 0.6
132.5
1.6
4.7
glucantimee
pentamidine
Arithmetic means SD (n ¼ 3).
a
Intracellular amastigotes.
Murine peritoneal macrophages.
b
c
SI ¼ LD50/IC50 intracellular amastigotes.
d
nd: not determined.
e
Glucantime [mg/ml].

280
R.R. Teixeira et al. / European Journal of Medicinal Chemistry 146 (2018) 274e286
Fig. 3. Microphotographs of the L. amazonensis-infected macrophages treated with compound 7k, glucantime and those without treatment. After 48 h of treatment, the slides were
stained and parasites quantified by optical microscopy. (A) Negative control; (B) uninfected macrophages; (C) 1
mol Lꢀ1, (D) 10 mol Lꢀ1 and (E) 100 mol Lꢀ1 of 7k; (F) 1 g mLꢀ1
(2.73
mol Lꢀ1), (G) 10 g mLꢀ1 (27.3 mol Lꢀ1) and (H) 100 g mLꢀ1 (273 mol Lꢀ1) of glucantime.
m
m
m
m
m
m
m
m
m
Table 3
Calculated molecular properties for compounds most active against Leishmania. Estimated properties are considered relevant for drug-likeness according to the Lipinski's and
Veber's rules.
Compound
Molecular weight (g/mol)
clogP
# H-bond donors
# H-bond acceptors
# Lipinski's rule violations
# of rotatable bonds
PSA (Å3)
Ideal values
ꢃ500
335.4
353.4
404.3
363.5
381.4
377.5
ꢃ5
ꢃ5
0
0
0
0
ꢃ10
4
4
4
4
ꢃ1
1
1
1
1
ꢃ10
8
8
ꢃ75
45.6
45.6
45.2
46.0
45.9
46.1
6a
6b
6k
7a
7b
7k
5.15
5.39
6.05
5.95
6.19
6.28
8
10
10
10
0
0
4
4
1
1
Table 4
Additional predicted pharmacokinetic properties for compounds most active against Leishmania.
QPlogHERGa
QPPCaco
(nm/s)b
QPPMDCK
(nm/s)c
QPlogKp
#metabe
d
Compound
Rangef or reference values
>ꢀ5
ꢀ6.8
ꢀ6.7
ꢀ6.6
ꢀ7.1
ꢀ7.0
ꢀ7.0
>500
3136
3147
3176
3474
3497
3490
>500
1701
3091
8274
1901
3464
1910
ꢀ8.0 to ꢀ1.0
1 to 8
6a
6b
6k
7a
7b
7k
ꢀ0.42
ꢀ0.55
ꢀ0.69
ꢀ0.16
ꢀ0.29
ꢀ0.35
5
5
5
5
5
6
a
Predicted logIC50 for HERG Kþ blockage.
Predicted apparent Caco-2 cell permeability for non-active transport.
Predicted apparent MDCK cell permeability for non-active transport.
Predicted skin permeability, log Kp.
Number of likely metabolic sites.
Range observed for 95% of known drugs.
b
c
d
e
f

R.R. Teixeira et al. / European Journal of Medicinal Chemistry 146 (2018) 274e286
281
v/v) to give compound 3 in 92% yield (3.00 g, 18.0 mmol).
4.1.6.1. 4-((4-Allyl-2-methoxyphenoxy)methyl)-1-(4-fluorobenzyl)-
1H-1,2,3-triazole (6b). White solid, obtained in 87% yield (0.218 g,
0.620 mmol) from the reaction of alkyne 2 (0.143 g, 0.710 mmol), 4-
fluorobenzyl azide (0.107 g, 0.710 mmol), sodium ascorbate
(0.0560 g, 0.280 mmol) and CuSO4$5H2O (0.0350 g, 0.140 mmol).
Purified by column chromatography eluted with hexane-ethyl ac-
etate-dichloromethane (3:1:3 v/v), m.p. 92.1e92.7 ꢁC, TLC:
Rf ¼ 0.39 (hexane-ethyl acetate-dichloromethane 3:1:3 v/v). IR
(ATR) nmax/cmꢀ1: 3069, 3005, 2935, 2914, 1588, 1511, 1259, 1223,
Yellow oil, TLC: Rf ¼ 0.76 (hexane-ethyl acetate-dicloromethane
3:1:3 v/v). 1H NMR (300 MHz, CDCl3)
d
:1.93 (quint, 2H, J ¼ 6.5 Hz),
1.99 (t, 1H, J ¼ 2.7 Hz), 2.33 (td, 2H, J1 ¼6.8 Hz and J2 ¼ 2.7 Hz), 3.00
(s, 3H, CH3), 4.32 (t, 2H, J ¼ 6.1 Hz), 13C NMR (75 MHz, CDCl3)
d:14.5,
27.6, 37.1 (CH3), 68.2, 69.7, 82.0.
4.1.5. Data for 4-allyl-2-methoxy-1-(pent-4-yn-1-yloxy)benzene
(4)
1143, 1017, 905, 771. 1H NMR (300 MHz, CDCl3)
d: 3.32 (d, 2H,
This compound was prepared using a similar procedure to that
described for the preparation of compound 2. In this case, the
alkylating agent corresponded to compound 3. Substance 4 was
obtained in 78% yield (1.64 g, 7.13 mmol) after purification by col-
umn chromatography eluted with hexane-ethyl acetate (6:1 v/v).
The structure of 4 is supported by the following data.
Yellow oil, TLC: Rf ¼ 0.55 (hexane-ethyl acetate 6:1 v/v). 1H NMR
J ¼ 6.7 Hz), 3.81 (s, 3H), 5.02e5.12 (m, 2H), 5.23 (s, 2H), 5.47 (s, 2H),
5.94 (ddtap, 1H, J1 ¼17.0 Hz, J2 ¼ 10.5 Hz and J3 ¼ 6.7 Hz), 6.67e6.70
(m, 2H), 6.93 (d, 1H, J ¼ 7.9 Hz), 7.04 (tap, 2H, J ¼ 8.6 Hz), 7.20e7.30
(m, 2H), 7.54 (s, 1H). 13C NMR (75 MHz, CDCl3)
d: 39.7, 53.4, 55.7,
63.4, 112.2, 114.5, 115.7, 116.1 (d, J ¼ 21.8 Hz), 120.4, 122.6, 129.9 (d,
J ¼ 8.3 Hz), 130.3 (d, J ¼ 3.0 Hz), 133.8, 137.4, 145.0, 145.8, 149.5,
162.8 (d,
C
J
¼
246.0 Hz). HRMS (M
þ
Hþ): Calculated for
(300 MHz, CDCl3)
d:1.97 (t, 1H, J ¼ 2.6 Hz), 2.04 (quint, 2H,
20H21FN3O2, 354.16178; found: 354.16077. HRMS (M þ Naþ):
J ¼ 6.6 Hz), 2.42 (td, 2H, J1 ¼7.0 Hz and J2 ¼ 2.6 Hz), 3.34 (d, 2H,
J ¼ 6.6 Hz), 3.85 (s, 3H), 4.10 (t, 2H, J ¼ 6.3 Hz), 5.03e5.13 (m, 2H),
5.97 (ddtap, 1H, J1 ¼16.8 Hz, J2 ¼ 10.2 Hz and J3 ¼ 6.7 Hz), 6.68e6.75
Calculated for C20H20FN3NaO2. 376.14372; found: 376.14240.
4.1.6.2. 4-((4-Allyl-2-methoxyphenoxy)methyl)-1-(4-chlorobenzyl)-
1H-1,2,3-triazole (6c). White solid, obtained in 84% yield (0.210 g,
0.570 mmol) from the reaction of alkyne 2 (0.137 g, 0.680 mmol), 4-
chlorobenzyl azide (0.1114 g, 0.680 mmol), sodium ascorbate
(0.0540 g, 0.270 mmol) and CuSO4$5H2O (0.0340 g, 0.140 mmol).
Purified by column chromatography eluted with hexane-ethyl ac-
etate-dichloromethane (3:1:3 v/v), m.p. 112.3e112.6 ꢁC, TLC:
Rf ¼ 0.37 (hexane-ethyl acetate-dichloromethane 3:1:3 v/v). IR
(ATR) nmax/cmꢀ1: 3065, 3001, 2936, 2915, 1588, 1513, 1943, 1258,
1228, 1143, 1016, 907, 803, 722, 693, 592. 1H NMR (300 MHz, CDCl3)
(m, 2H), 6.85 (d, 1H, J ¼ 8.7 Hz). 13C NMR (75 MHz, CDCl3)
d: 15.2,
28.2, 39.8, 55.9, 67.5, 68.8, 83.6, 112.4, 113.6, 115.6, 120.5, 133.0,
137.6, 146.7, 149.5. MS, m/z (%) 230 (
, C15H18O2, 100), 164 (68),
149 (44), 131 (29), 121 (16), 103 (34), 91 (31), 77 (23), 65 (16), 55
(15), 41 (34).
4.1.6. General procedure for the preparation of compounds 6aꢀ6n
and 7aꢀ7m exemplified by the synthesis of compound 4-((4-allyl-
2-methoxyphenoxy)methyl)-1-benzyl-1H-1,2,3-triazole (6a)
A 10 mL round bottom flask was charged with 4-allyl-2-
methoxy-1-(prop-2-yn-1-yloxy)benzene (2) (0.150 g, 0.740 mmol),
benzyl azide (0.0990 g; 0.740 mmol), sodium ascorbate (0.0590 g,
0.300 mmol), 1.00 mL of ethanol and 1.00 mL of distilled water.
Then, CuSO4$5H2O (0.0370 g, 0.150 mmol) was added. The reaction
mixture was vigorously stirred at room temperature. After
completion of the reaction as noticed by TLC analysis, the mixture
was extracted with dichloromethane (3 ꢂ 10.0 mL). The organic
extracts were combined and the resulting organic layer was
washed with aqueous sodium carbonate saturated solution, dried
over anhydrous sodium sulfate, filtrated and concentrated under
reduced pressure. The residue was purified by column chroma-
tography eluted with hexane-ethyl acetate-dichloromethane (3:1:3
v/v). Compound 6a was obtained in 91% yield (0.228 g,
0.680 mmol). The structure of 6a is supported by the following
data.
d
: 3.32 (d, 2H, J ¼ 6.7 Hz), 3.81 (s, 3H), 5.02e5.12 (m, 2H), 5.23 (s,
2H), 5.47 (s, 2H), 5.94 (ddtap, 1H, J1 ¼17.0 Hz, J2 ¼ 10.2 Hz and
J3 ¼ 6.7 Hz), 6.65e6.73 (m, 2H), 6.93 (d, 1H, J ¼ 7.9 Hz), 7.18 (d, 2H,
J ¼ 8.5 Hz), 7.33 (d, 2H, J ¼ 8.5 Hz), 7.54 (s, 1H). 13C NMR (75 MHz,
CDCl3) d: 39.8, 53.4, 55.7, 63.4, 112.2, 114.6, 115.7, 120.4, 122.7, 129.2,
129.3, 132.9, 133.9, 134.7, 137.4, 145.0, 145.8, 149.5. HRMS (M þ Hþ):
Calculated for C20H21ClN3O2, 370.13223; found: 370.13132. HRMS
(M þ Naþ): Calculated for C20H20ClN3NaO2, 392.11417; found:
392.1l285.
4.1.6.3. 4-((4-Allyl-2-methoxyphenoxy)methyl)-1-(4-bromobenzyl)-
1H-1,2,3-triazole (6d). White solid, obtained in 80% yield (0.216 g,
0.520 mmol) from the reaction of alkyne 2 (0.132 g, 0.650 mmol), 4-
bromobenzyl azide (0.138 g, 0.650 mmol), sodium ascorbate
(0.0510 g, 0.260 mmol) and CuSO4$5H2O (0.0330 g, 0.130 mmol).
Purified by column chromatography eluted with hexane-ethyl ac-
etate-dichloromethane (3:1:3 v/v), m.p. 119.1e120.2 ꢁC, TLC:
Rf ¼ 0.59 (hexane-ethyl acetate-dichloromethane 3:1:3 v/v). IR
(ATR) nmax/cmꢀ1: 3065, 3001, 2935, 2915, 1588, 1513, 1385, 1258,
White solid, m.p. 90.5e91.3 ꢁC, TLC: Rf ¼ 0.49 (hexane-ethyl
acetate-dichloromethane 3:1:3 v/v). IR (ATR) nmax/cmꢀ1: 3086,
3034, 2999, 2929, 2912, 1589, 1511, 1446, 1259, 1229, 1143, 1016,
1229, 1144, 1015, 803, 738, 592, 488. 1H NMR (300 MHz, CDCl3)
d:
921, 831, 713, 696. 1H NMR (300 MHz, CDCl3)
d
: 3.32 (d, 2H,
3.32 (d, 2H, J ¼ 6.7 Hz), 3.82 (s, 3H), 5.02e5.12 (m, 2H), 5.23 (s, 2H),
5.45 (s, 2H), 5.94 (ddtap,1H, J1 ¼17.1 Hz, J2 ¼ 10.2 Hz and J3 ¼ 6.7 Hz),
6.65e6.73 (m, 2H), 6.92 (d, 1H, J ¼ 7.9 Hz), 7.11 (d, 2H, J ¼ 8.4 Hz),
J ¼ 6.7 Hz), 3.81 (s, 3H), 5.02e5.12 (m, 2H), 5.23 (s, 2H), 5.50 (s, 2H),
5.94 (ddtap, 1H, J1 ¼16.9 Hz, J2 ¼ 10.2 Hz and J3 ¼ 6.7 Hz), 6.65e6.71
(m, 2H), 6.94 (d, 1H, J ¼ 8.0 Hz), 7.21e7.40 (m, 5H), 7.54 (s, 1H). 13
C
7.48 (d, 2H, J ¼ 8.4 Hz), 7.54 (s,1H). 13C NMR (75 MHz, CDCl3)
d: 39.8,
NMR (75 MHz, CDCl3)
d
: 39.8, 54.1, 55.7, 63.5, 112.2, 114.6, 115.7,
53.4, 55.7, 63.4, 112.2, 114.6, 115.7, 120.4, 122.7, 122.8, 129.6, 132.2,
133.4, 133.9, 137.4, 145.1, 145.7, 149.5. HRMS (M þ Hþ): Calculated
for C20H21BrN3O2, 414.08171; found: 414.08072. HRMS (M þ Naþ):
Calculated for C20H20BrN3NaO2, 436.06366; found: 436.06058.
120.4, 122.7, 128.1, 128.7, 129.0, 133.8, 134.4, 137.4, 144.8, 145.8,
149.5. HRMS (M þ Hþ): Calculated for C20H22N3O2, 336.17120;
found: 336.17020. HRMS (M þ Naþ): Calculated for C20H21N3NaO2.
358.15315; found: 358.15164.
Compounds 6be6n and 7ae7m were prepared using a pro-
cedure similar to that described for the synthesis of 6a. The struc-
tures of these compounds were confirmed by NMR (1H and 13C), IR,
and HRMS analyses. Information regarding the reactions involved
in the preparation of the aforementioned compounds and data that
support their structures are described below.
4.1.6.4. 4-((4-Allyl-2-methoxyphenoxy)methyl)-1-(4-iodobenzyl)-
1H-1,2,3-triazole (6e). White solid, obtained in 87% yield (0.200 g,
0.430 mmol) from the reaction of alkyne 2 (0.101 g, 0.500 mmol), 4-
iodobenzyl azide (0.130 g, 0.500 mmol), sodium ascorbate
(0.0400 g, 0.200 mmol) and CuSO4$5H2O (0.0250 g, 0.100 mmol).
Purified by column chromatography eluted with hexane-ethyl

282
R.R. Teixeira et al. / European Journal of Medicinal Chemistry 146 (2018) 274e286
acetate-dichloromethane (3:1:3 v/v), m.p. 129.2e131.1 ꢁC, (3:1:3 v/
v), TLC: Rf ¼ 0.37 (hexane-ethyl acetate-dichloromethane 3:1:3 v/
v). IR (ATR) nmax/cmꢀ1: 3124, 3063, 2932, 1587, 1512, 1486, 1385,
1258, 1228, 1143, 1017, 1008, 911, 801, 769, 592, 485. 1H NMR
J1 ¼17.0 Hz, J2 ¼ 10.2 Hz and J3 ¼ 6.7 Hz), 6.65e6.73 (m, 2H), 6.93 (d,
1H, J ¼ 7.9 Hz), 7.20 (d, 2H, J ¼ 8.5 Hz), 7.28 (d, 2H, J ¼ 8.5 Hz), 7.57 (s,
1H). 13C NMR (75 MHz, CDCl3)
d: 39.7, 53.2, 55.8, 63.4, 112.2, 114.5,
115.8, 120.3 (q, J ¼ 256.3 Hz, OCF3), 120.4, 121.5, 122.7, 129.5, 133.2,
133.9, 137.4, 145.1, 145.8, 149.3e149.2 (m), 149.5. HRMS (M þ Hþ):
Calculated for C21H21F3N3O3, 420.15350; found: 420.15250. HRMS
(M þ Naþ): Calculated for C21H20F3N3NaO3, 442.13545; found:
442.13422.
(300 MHz, CDCl3)
d
: 3.32 (d, 2H, J ¼ 6.6 Hz), 3.82 (s, 3H), 5.02e5.12
(m, 2H), 5.20 (s, 2H), 5.44 (s, 2H), 5.94 (ddtap, 1H, J1 ¼17.0 Hz,
J2 ¼ 10.5 Hz and J3 ¼ 6.6 Hz), 6.65e6.73 (m, 2H), 6.92 (d, 1H,
J ¼ 7.9 Hz), 6.98 (d, 2H, J ¼ 8.3 Hz), 7.54 (s, 1H), 7.69 (d, 2H,
J ¼ 8.3 Hz). 13C NMR (75 MHz, CDCl3)
d: 39.8, 53.5, 55.7, 63.4, 94.5,
112.2, 114.6, 115.7, 120.4, 122.7, 129.8, 133.9, 134.1, 137.4, 138.2, 145.1,
4.1.6.8. 4-((4-Allyl-2-methoxyphenoxy)methyl)-1-(4-(tri-
fluoromethyl)benzyl)-1H-1,2,3-triazole (6i). White solid, obtained in
84% yield (0.210 g, 0.520 mmol) from the reaction of alkyne 2
(0.125 g, 0.620 mmol), 4-trifluoromethylbenzyl azide (0.125 g,
0.620 mmol), sodium ascorbate (0.0490 g, 0.250 mmol) and
CuSO4$5H2O (0.0310 g, 0.120 mmol). Purified by column chroma-
tography eluted with hexane-ethyl acetate-dichloromethane (3:1:3
v/v), m.p. 142.7e143.0 ꢁC, TLC: Rf ¼ 0.43 (hexane-ethyl acetate-
dichloromethane 3:1:3 v/v). IR (ATR) nmax/cmꢀ1: 3060, 3003,
2977, 2944, 1589, 1515, 1422, 1328, 1229, 1142, 1117, 1017, 913, 799,
145.8, 149.5. HRMS (M
þ
Hþ): Calculated for C20H21IN3O2,
462.06784; found: 462.06711. HRMS (M þ Naþ): Calculated for
C
20H20IN3NaO2, 484.04979; found: 484.04898.
4.1.6.5. 4-((4-Allyl-2-methoxyphenoxy)methyl)-1-(4-nitrobenzyl)-
1H-1,2,3-triazole (6f). White solid, obtained in 89% yield (0.223 g,
0.590 mmol) from the reaction of alkyne 2 (0.133 g, 0.660 mmol), 4-
nitrobenzyl azide (0.118 g, 0.660 mmol), sodium ascorbate
(0.0520 g, 0.260 mmol) and CuSO4$5H2O (0.0330 g, 0.130 mmol).
Purified by column chromatography eluted with hexane-ethyl ac-
etate-dichloromethane (3:1:3 v/v),m.p. 120.9e121.9 ꢁC, TLC:
Rf ¼ 0.14 (hexane-ethyl acetate-dichloromethane 3:1:3 v/v). IR
(ATR) nmax/cmꢀ1: 3063, 3005, 2950, 2917, 1591, 1516, 1350, 1258,
624. 1H NMR (300 MHz, CDCl3)
d
: 3.32 (d, 2H, J ¼ 6.7 Hz), 3.81 (s,
3H), 5.02e5.12 (m, 2H), 5.25 (s, 2H), 5.57 (s, 2H), 5.94 (ddtap, 1H,
J1 ¼17.0 Hz, J2 ¼ 10.2 Hz and J3 ¼ 6.7 Hz), 6.65e6.73 (m, 2H), 6.93 (d,
1H, J ¼ 7.8 Hz), 7.34 (d, 2H, J ¼ 8.1 Hz), 7.58 (brs, 1H), 7.61 (d, 2H,
1230, 1140, 1019, 835, 720. 1H NMR (300 MHz, CDCl3)
d: 3.32 (d, 2H,
J ¼ 8.1 Hz). 13C NMR (75 MHz, CDCl3)
d: 39.7, 53.4, 55.8, 63.4, 112.2,
J ¼ 6.7 Hz), 3.81 (s, 3H), 5.01e5.12 (m, 2H), 5.25 (s, 2H), 5.62 (s, 2H),
5.93 (ddtap, 1H, J1 ¼17.0 Hz, J2 ¼ 10.3 Hz and J3 ¼ 6.7 Hz), 6.65e6.73
(m, 2H), 6.92 (d, 1H, J ¼ 7.9 Hz), 7.37 (d, 2H, J ¼ 8.6 Hz), 7.63 (s, 1H),
114.6, 115.7, 120.4, 122.8, 123.7 (q, J ¼ 270.8 Hz, CF3), 126.0 (q,
J ¼ 3.8 Hz), 128.1, 131.0 (q, J ¼ 32.5 Hz), 133.9, 137.4, 138.4, 145.2,
145.7, 149.5. Calculated for
C21H21F3N3O2, 404.15859; found:
8.20 (d, 2H, J ¼ 8.6 Hz). 13C NMR (75 MHz, CDCl3)
d
: 39.7 53.0, 55.7,
404.15771 HRMS (M þ Naþ): Calculated for C21H20F3N3NaO2,
63.3, 112.3, 114.5, 115.7, 120.4, 123.0, 124.2, 128.5, 134.0, 137.4, 141.5,
426.14053; found: 426.13928.
145.4, 145.7, 148.0, 149.5. HRMS (M
C
þ
Hþ): Calculated for
20H21N4O4, 381.15628; found: 381.15533. HRMS (M þ Naþ):
4.1.6.9. 4-((4-Allyl-2-methoxyphenoxy)methyl)-1-(3,4-
difluorobenzyl)-1H-1,2,3-triazole (6j). White solid, obtained in 78%
yield (0.195 g, 0.530 mmol) from the reaction of alkyne 2 (0.136 g,
0.670 mmol), 3,4-difluorobenzyl azide (0.113 g, 0.670 mmol), so-
dium ascorbate (0.0530 g, 0.270 mmol) and CuSO4$5H2O (0.0340 g,
0.130 mmol). Purified by column chromatography eluted with
Calculated for C20H20N4NaO4, 403.13822; found: 403.13715.
4.1. 6. 6. 4-((4-Allyl-2-methoxyphenoxy)methyl)-1-(4-
methoxybenzyl)-1H-1,2,3-triazole (6g). White solid, obtained in 80%
yield (0.200 g, 0.550 mmol) from the reaction of alkyne 2 (0.138 g,
0.680 mmol), 4-methoxybenzyl azide (0.110 g, 0.680 mmol), so-
dium ascorbate (0.0540 g, 0.270 mmol) and CuSO4$5H2O (0.0340 g,
0.140 mmol). Purified by column chromatography eluted with
hexane-ethyl
acetate-dichloromethane
(3:1:3
v/v),
m.p.
104.9e105.2 ꢁC,
TLC:
Rf ¼ 0.30
(hexane-ethyl
acetate-
dichloromethane 3:1:3 v/v). IR (ATR) nmax/cmꢀ1: 3065, 3003,
hexane-ethyl
acetate-dichloromethane
(3:1:3
v/v),
m.p.
2939, 2915, 1589, 1516, 1447, 1287, 1259, 1213, 1142, 1018, 835, 756,
92.4e93.1 ꢁC,
TLC:
Rf ¼ 0.36
(hexane-ethyl
acetate-
575. 1H NMR (300 MHz, CDCl3)
d
: 3.32 (d, 2H, J ¼ 6.7 Hz), 3.82 (s,
dichloromethane 3:1:3 v/v). IR (ATR) nmax/cmꢀ1: 3068, 3005,
3H), 5.01e5.12 (m, 2H), 5.24 (s, 2H), 5.45 (s, 2H), 5.94 (ddtap, 1H,
J1 ¼17.0 Hz, J2 ¼ 10.3 Hz and J3 ¼ 6.7 Hz), 6.65e6.73 (m, 2H), 6.92 (d,
1H, J ¼ 7.9 Hz), 6.97e7.19 (m, 3H), 7.57 (s, 1H). 13C NMR (75 MHz,
2936, 2832, 1613, 1587, 1512, 1463, 1251, 1227, 1142, 1016, 903, 805,
774, 702, 548. 1H NMR (300 MHz, CDCl3)
d
: 3.31 (d, 2H, J ¼ 6.7 Hz),
3.80 (s, 3H), 3.81 (s, 3H), 5.01e5.12 (m, 2H), 5.21 (s, 2H), 5.43 (s, 2H),
5.94 (ddtap, 1H, J1 ¼16.9 Hz, J2 ¼ 10.2 Hz and J3 ¼ 6.7 Hz), 6.65e6.73
(m, 2H), 6.88 (d, 2H, J ¼ 8.6 Hz), 6.94 (d, 1H, J ¼ 7.8 Hz), 7.21 (d, 2H,
CDCl3)
d
: 39.7, 53.0 (brd, J ¼ 1.0 Hz), 55.7, 63.4, 112.2, 114.6, 115.7,
117.2 (d, J ¼ 18.0 Hz), 117.9 (d, J ¼ 18.0 Hz), 120.4, 122.7, 124.2 (dd,
J1 ¼6.8 Hz and J2 ¼ 3.8 Hz), 131.4e131.5 (m), 133.9, 137.4, 145.2,
145.6, 149.5, 148.7e148.9 (m), 152.0e152.2 (m). Calculated for
J ¼ 8.6 Hz), 7.51 (s, 1H). 13C NMR (75 MHz, CDCl3)
d: 39.8, 53.7, 55.3,
55.7, 63.5, 112.2, 114.4, 114.5, 115.6, 120.4, 122.5, 126.4, 129.7, 133.7,
C
20H20F2N3O2, 372.15236; found: 372.15137. HRMS (M þ Naþ):
137.4, 144.7, 145.9, 149.5, 159.9. HRMS (M þ Hþ): Calculated for
Calculated for C20H19F2N3NaO2, 394.13430; found: 394.13312.
C
21H24N3O3, 366.18177; found: 366.18069. HRMS (M þ Naþ):
Calculated for C21H23N3NaO3, 388.16371; found: 388.16229.
4.1.6.10. 4-((4-Allyl-2-methoxyphenoxy)methyl)-1-(2,5-
dichlorobenzyl)-1H-1,2,3-triazole (6k). White solid, obtained in 81%
yield (0.203 g, 0.500 mmol) from the reaction of alkyne 2 (0.125 g,
0.620 mmol), 2,5-dichlorobenzyl azide (0.125 g, 0.620 mmol), so-
dium ascorbate (0.0490 g, 0.250 mmol) and CuSO4$5H2O (0.0310 g,
0.120 mmol). Purified by column chromatography eluted with
4.1.6.7. 4-((4-Allyl-2-methoxyphenoxy)methyl)-1-(4-(tri-
fluoromethoxy)benzyl)-1H-1,2,3-triazole (6h). White solid, obtained
in 85% yield (0.213 g, 0.510 mmol) from the reaction of alkyne 2
(0.121 g, 0.600 mmol), 4-trifluoromethoxybenzyl azide (0.130 g,
0.600 mmol), sodium ascorbate (0.0480 g, 0.240 mmol) and
CuSO4$5H2O (0.0300 g, 0.120 mmol). Purified by column chroma-
tography eluted with hexane-ethyl acetate-dichloromethane (3:1:3
v/v), m.p. 126.4e127.3 ꢁC, TLC: Rf ¼ 0.49 (hexane-ethyl acetate-
dichloromethane 3:1:3 v/v). IR (ATR) nmax/cmꢀ1: 3065, 3004,
2978, 2941, 1589, 1510, 1467, 1387, 1256, 1218, 1142, 1017, 915, 804.
hexane-ethyl
acetate-dichloromethane
(3:1:3
v/v),
m.p.
87.9e88.4 ꢁC,
TLC:
Rf ¼ 0.59
(hexane-ethyl
acetate-
dichloromethane 3:1:3 v/v). IR (ATR) nmax/cmꢀ1: 3143, 3008,
2962, 2871, 1587, 1508, 1460, 1391, 1256, 1213, 1137, 1030, 1003, 905,
821, 797, 645, 560. 1H NMR (300 MHz, CDCl3)
d: 3.32 (d, 2H,
J ¼ 6.7 Hz), 3.83 (s, 3H), 5.01e5.12 (m, 2H), 5.23 (s, 2H), 5.60 (s, 2H),
5.94 (ddtap, 1H, J1 ¼16.9 Hz, J2 ¼ 9.9 Hz and J3 ¼ 6.7 Hz), 6.68e6.71
(m, 2H), 6.93 (d, 1H, J ¼ 7.8 Hz), 7.12 (d, 1H, J ¼ 2.3 Hz), 7.27 (dd, 1H,
1H NMR (300 MHz, CDCl3)
5.02e5.12 (m, 2H), 5.24 (s, 2H), 5.51 (s, 2H), 5.94 (ddtap, 1H,
d: 3.32 (d, 2H, J ¼ 6.7 Hz), 3.81 (s, 3H),

R.R. Teixeira et al. / European Journal of Medicinal Chemistry 146 (2018) 274e286
283
J1 ¼8.6 Hz and J2 ¼ 2.3 Hz), 7.35 (d, 1H, J ¼ 8.6 Hz), 7.67 (s, 1H). 13
C
4.1.6.14. 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-benzyl-1H-
1,2,3-triazole (7a). White solid, obtained in 79% yield (0.198 g,
0.540 mmol) from the reaction of alkyne 3 (0.158 g, 0.690 mmol),
benzyl azide (0.0920 g, 0.690 mmol), sodium ascorbate (0.0550 g,
0.280 mmol) and CuSO4$5H2O (0.0350 g, 0.140 mmol). Purified by
column chromatography eluted with hexane-ethyl acetate-
dichloromethane (3:1:3 v/v), m.p. 71.4e72.6 ꢁC, TLC: Rf ¼ 0.16
NMR (75 MHz, CDCl3) d: 39.8, 50.9, 55.8, 63.5, 112.3, 114.7, 115.7,
120.5, 123.1, 130.0, 130.2, 130.9, 131.5, 133.5, 133.97, 133.99, 137.4,
145.1, 145.7, 149.5. Calculated for C20H20Cl2N3O2, 404.09326; found:
404.09232. HRMS (M þ Naþ): Calculated for C20H19Cl2N3NaO2,
426.07520; found: 426.07388.
4.1.6.11. 4-((4-Allyl-2-methoxyphenoxy)methyl)-1-(4-methylbenzyl)-
1H-1,2,3-triazole (6l). White solid, obtained in 82% yield (0.205 g,
0.590 mmol) from the reaction of alkyne 2 (0.145 g, 0.720 mmol), 4-
methylbenzyl azide (0.106 g, 0.720 mmol), sodium ascorbate
(0.0570 g, 0.290 mmol) and CuSO4$5H2O (0.0360 g, 0.140 mmol).
Purified by column chromatography eluted with hexane-ethyl ac-
etate-dichloromethane (3:1:3 v/v), m.p. 81.4e82,3 ꢁC, TLC:
Rf ¼ 0.66 (hexane-ethyl acetate-dichloromethane 3:1:3 v/v). IR
(ATR) nmax/cmꢀ1: 3064, 3003, 2917, 1588, 1513, 1446, 1257, 1229,
(hexane-ethyl acetate-dichloromethane 3:1:3 v/v). IR (ATR) nmax
cmꢀ1: 3059, 3007, 2958, 2830, 2876, 1588, 1513, 1461, 1332, 1260,
1230, 1140, 1026, 915, 799, 709, 663. 1H NMR (300 MHz, CDCl3)
/
d:
2.17 (quint, 2H, J ¼ 7.0 Hz), 2.90 (t, 2H, J ¼ 7.5 Hz), 3.32 (d, 2H,
J ¼ 6.7 Hz), 3.81 (s, 3H), 4.01 (t, 2H, J ¼ 6.4 Hz), 5.01e5.11 (m, 2H),
5.48 (s, 2H), 5.95 (ddtap
,
1H, J1 ¼16.8 Hz, J2 ¼ 10.2 Hz and
J3 ¼ 6.7 Hz), 6.65e6.73 (m, 2H), 6.77 (d, 1H, J ¼ 7.9 Hz), 7.22e7.37
(m, 6H). 13C NMR (75 MHz, CDCl3)
d: 22.1, 28.7, 39.7, 53.9, 55.8, 68.1,
112.3, 113.4, 115.5, 120.4, 120.9, 127.9, 128.5, 129.0, 132.9, 134.8,
137.6, 146.6, 147.7, 149.3. Calculated for C22H25N3O2, 364.20250;
found: 364.20148. HRMS (M þ Naþ): Calculated for C22H25N3NaO2,
386.18445; found: 386.18341.
1142, 1017, 907, 803, 756. 1H NMR (300 MHz, CDCl3)
d: 3.34 (s, 3H,
CH3), 3.32 (d, 2H, J ¼ 6.6 Hz), 3.81 (s, 3H), 5.01e5.12 (m, 2H), 5.22 (s,
2H), 5.45 (s, 2H), 5.94 (ddtap, 1H, J1 ¼16.9 Hz, J2 ¼ 10.2 Hz and
J3 ¼ 6.6 Hz), 6.65e6.73 (m, 2H), 6.93 (d, 1H, J ¼ 7.9 Hz), 7.15 (s, 4H),
7.52 (s, 1H). 13C NMR (75 MHz, CDCl3)
d
: 21.1 (CH3), 39.8, 53.9, 55.7,
4.1.6.15. 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-
fluorobenzyl)-1H-1,2,3-triazole (7b). White solid, obtained in 86%
yield (0.215 g, 0.560 mmol) from the reaction of alkyne 3 (0.151 g,
0.660 mmol), 4-fluorobenzyl azide (0.100 g, 0.660 mmol), sodium
ascorbate (0.0520 g, 0.260 mmol) and CuSO4$5H2O (0.0330 g,
0.130 mmol). Purified by column chromatography eluted with
63.5, 112.2, 114.6, 115.6, 120.4, 122.6, 128.1, 129.7, 131.4, 133.8, 137.5,
138.6, 144.7, 145.9, 149.5. Calculated for C21H24N3O2, 350.18685;
found: 350.18582. HRMS (M þ Naþ): Calculated for C21H23N3NaO2,
372.16880; found: 372.16776.
4.1.6.12. 4-((4-Allyl-2-methoxyphenoxy)methyl)-1-(2-bromobenzyl)-
1H-1,2,3-triazole (6m). White solid, obtained in 77% yield (0.208 g,
0.500 mmol) from the reaction of alkyne 2 (0.132 g, 0.650 mmol), 2-
bromobenzyl azide (0.138 g, 0.650 mmol), sodium ascorbate
(0.0510 g, 0.260 mmol) and CuSO4$5H2O (0.0330 g, 0.130 mmol).
Purified by column chromatography eluted with hexane-ethyl ac-
etate-dichloromethane (3:1:3 v/v), m.p. 67.5e68.6 ꢁC, TLC:
Rf ¼ 0.70 (hexane-ethyl acetate-dichloromethane 3:1:3 v/v). IR
(ATR) nmax/cmꢀ1: 3141, 3079, 3003, 2944, 2873, 1587, 1509, 1421,
1389,1254,1213,1134,1050,1026,1000, 920, 759, 649, 596. 1H NMR
hexane-ethyl
acetate-dichloromethane
(3:1:3
v/v),
m.p.
96.4e96,8 ꢁC,
TLC:
Rf ¼ 0.21
(hexane-ethyl
acetate-
dichloromethane 3:1:3 v/v). IR (ATR) nmax/cmꢀ1: 3111, 3061, 3008,
2951, 1587, 1511, 1455, 1260, 1222, 1140, 1015, 905, 798, 772, 654. 1H
NMR (300 MHz, CDCl3)
d
: 2.17 (quint, 2H, J ¼ 6.9 Hz), 2.90 (t, 2H,
J ¼ 7.5 Hz), 3.32 (d, 2H, J ¼ 6.7 Hz), 3.81 (s, 3H), 4.01 (t, 2H,
J ¼ 6.4 Hz), 5.00e5.13 (m, 2H), 5.44 (s, 2H), 5.95 (ddtap, 1H,
J1 ¼16.8 Hz, J2 ¼ 10.8 Hz and J3 ¼ 6.7 Hz), 6.65e6.73 (m, 2H), 6.77 (d,
1H, J ¼ 7.9 Hz), 7.03 (t, 2H, J ¼ 8.6 Hz), 7.16e7.30 (m, 3H). 13C NMR
(75 MHz, CDCl3) d: 22.1, 28.7, 39.7, 53.2, 55.8, 68.0, 112.3, 113.4,
(300 MHz, CDCl3)
d
: 3.32 (d, 2H, J ¼ 6.6 Hz), 3.82 (s, 3H), 5.00e5.11
115.6, 116.0 (d, J ¼ 21.8 Hz), 120.4, 120.8, 129.7 (d, J ¼ 8.3 Hz), 130.7
(d, J ¼ 3.0 Hz),132.9,137.6,146.6,147.8,149.3,162.7 (d, J ¼ 246.8 Hz).
Calculated for C22H25FrN3O2, 382.19308; found: 382.19211. HRMS
(M þ Naþ): Calculated for C22H24FN3NaO2, 404.17502; found:
404.17404.
(m, 2H), 5.25 (s, 2H), 5.64 (s, 2H), 5.94 (ddtap, 1H, J1 ¼16.9 Hz,
J2 ¼ 10.2 Hz and J3 ¼ 6.6 Hz), 6.65e6.73 (m, 2H), 6.95 (d, 1H,
J ¼ 7.8 Hz), 7.12 (dd, 1H, J1 ¼7.4 Hz and J2 ¼ 1.7 Hz), 7.17e7.33 (m,
2H), 7.60 (dd, 1H, J1 ¼7.8 Hz and J2 ¼ 1.2 Hz), 7.66 (s, 1H). 13C NMR
(75 MHz, CDCl3) d: 39.7, 53.7, 55.7, 63.5, 112.2, 114.7, 115.6, 120.4,
123.1, 123.4, 128.1, 130.30, 130.33, 133.1, 133.8, 134.0, 137.4, 144.7,
4.1.6.16. 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-
chlorobenzyl)-1H-1,2,3-triazole (7c). White solid, obtained in 87%
yield (0.218 g, 0.550 mmol) from the reaction of alkyne 3 (0.145 g,
0.630 mmol), 4-chlorobenzyl azide (0.106 g, 0.630 mmol), sodium
ascorbate (0.0480 g, 0.250 mmol) and CuSO4$5H2O (0.0320 g,
0.130 mmol). Purified by column chromatography eluted with
145.7, 149.5. Calculated for
C20H21BrN3O2, 414.08171; found:
414.08096. HRMS (M þ Naþ): Calculated for C20H20BrN3NaO2,
436.06366; found: 436.06290.
4.1.6.13. 4-((4-Allyl-2-methoxyphenoxy)methyl)-1-(3-bromobenzyl)-
1H-1,2,3-triazole (6n). White solid, obtained in 79% yield (0.213 g,
0.520 mmol) from the reaction of alkyne 2 (0.132 g, 0.650 mmol), 3-
bromobenzyl azide (0.138 g, 0.650 mmol), sodium ascorbate
(0.0510 g, 0.260 mmol) and CuSO4$5H2O (0.0330 g, 0.130 mmol).
Purified by column chromatography eluted with hexane-ethyl ac-
etate-dichloromethane (3:1:3 v/v), m.p. 97.5e97.9 ꢁC, TLC: Rf ¼ 0.70
hexane-ethyl
acetate-dichloromethane
(3:1:3
v/v),
m.p.
81.8e82.2 ꢁC,
TLC:
Rf ¼ 0.46
(hexane-ethyl
acetate-
dichloromethane 3:1:3 v/v). IR (ATR) nmax/cmꢀ1: 3111, 3063,
3000, 2937, 2856, 1589, 1514, 1446, 1259, 1227, 1140, 1015, 909, 799,
776, 739, 641. 1H NMR (300 MHz, CDCl3)
d: 2.17 (quint, 2H,
J ¼ 6.9 Hz), 2.90 (t, 2H, J ¼ 7.5 Hz), 3.32 (d, 2H, J ¼ 6.7 Hz), 3.82 (s,
3H), 4.01 (t, 2H, J ¼ 6.4 Hz), 5.00e5.11 (m, 2H), 5.44 (s, 2H), 5.95
(ddtap, 1H, J1 ¼17.0 Hz, J2 ¼ 10.2 Hz and J3 ¼ 6.7 Hz), 6.65e6.73 (m,
2H), 6.77 (d, 1H, J ¼ 7.9 Hz), 7.16 (d, 2H, J ¼ 8.5 Hz), 7.24 (s, 1H), 7.32
(hexane-ethyl acetate-dichloromethane 3:1:3 v/v). IR (ATR) nmax
cmꢀ1: 3065, 3011, 2936, 1588, 1512, 1463, 1387, 1226, 1142, 1017,
835, 735, 692. 665. 1H NMR (300 MHz, CDCl3)
: 3.32 (d, 2H,
/
d
J ¼ 6.6 Hz), 3.82 (s, 3H), 5.00e5.13 (m, 2H), 5.24 (s, 2H), 5.47 (s, 2H),
(d, 2H, J ¼ 8.5 Hz). 13C NMR (75 MHz, CDCl3)
d: 22.1, 28.7, 39.7, 53.2,
55.8, 68.0, 112.3, 113.4, 115.6, 120.4, 120.9, 129.2, 132.9, 133.3, 134.6,
137.6, 146.6, 147.9, 149.3. Calculated for C21H25ClN3O2, 398.16353;
5.94 (ddtap, 1H, J1 ¼16.9 Hz, J2 ¼ 10.8 Hz and J3 ¼ 6.6 Hz), 6.65e6.73
(m, 2H), 6.93 (d, 1H, J ¼ 7.8 Hz), [7.13e7.26 (m), 7.40 (brs), 7.48 (dap
,
J ¼ 7.7 Hz), 4H], 7.57 (s, 1H). 13C NMR (75 MHz, CDCl3)
d
: 39.8, 53.3,
found: 398.16272. HRMS (M
þ
Naþ): Calculated for
55.7, 63.4, 112.2, 114.6, 115.7, 120.5, 122.7, 123.0, 126.6, 130.6, 131.0,
131.9, 133.9, 136.6, 137.4, 145.1, 145.8, 149.5. Calculated for
C22H24ClN3NaO2, 420.14547; found: 420.14474.
C
20H21BrN3O2, 414.08171; found: 414.08069. HRMS (M þ Naþ):
4.1.6.17. 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-
bromobenzyl)-1H-1,2,3-triazole (7d). White solid, obtained in 84%
Calculated for C20H20BrN3NaO2, 436.06366; found: 436.06265.

284
R.R. Teixeira et al. / European Journal of Medicinal Chemistry 146 (2018) 274e286
yield (0.210 g, 0.480 mmol) from the reaction of alkyne 3 (0.130 g,
0.570 mmol), 4-bromobenzyl azide (0.120 g, 0.570 mmol), sodium
ascorbate (0.0450 g, 0.230 mmol) and CuSO4$5H2O (0.0290 g,
0.110 mmol). Purified by column chromatography eluted with
0.140 mmol). Purified by column chromatography eluted with
hexane-ethyl
acetate-dichloromethane
(3:1:3
v/v),
m.p.
88.9e90.4 ꢁC,
TLC:
Rf ¼ 0.22
(hexane-ethyl
acetate-
dichloromethane 3:1:3 v/v). IR (ATR) nmax/cmꢀ1: 3114, 3062,
hexane-ethyl
acetate-dichloromethane
(3:1:3
v/v),
m.p.
3005, 2951, 2868, 1587, 1512, 1455, 1295, 1251, 1227, 1131, 1032,
89.4e90.3 ꢁC,
TLC:
Rf ¼ 0.46
(hexane-ethyl
acetate-
1015, 917, 801, 749, 652. 1H NMR (300 MHz, CDCl3)
d: 2.16 (quint,
dichloromethane 3:1:3 v/v). IR (ATR) nmax/cmꢀ1: 3110, 3063,
2H, J ¼ 7.0 Hz), 2.88 (t, 2H, J ¼ 7.5 Hz), 3.32 (d, 2H, J ¼ 6.6 Hz), 3.80 (s,
3H), 3.82 (s, 3H), 4.01 (t, 2H, J ¼ 6.4 Hz), 5.00e5.11 (m, 2H), 5.40 (s,
2H), 5.95 (ddtap, 1H, J1 ¼16.8 Hz, J2 ¼ 9.9 Hz and J3 ¼ 6.6 Hz),
6.65e6.73 (m, 2H), 6.77 (d, 1H, J ¼ 7.9 Hz), 6.87 (d, 2H, J ¼ 8.6 Hz),
2999, 2950, 1588, 1514, 1459, 1420, 1260, 1227, 1141, 1013, 908, 797,
733, 489. 1H NMR (300 MHz, CDCl3)
d: 2.17 (quint, 2H, J ¼ 6.9 Hz),
2.90 (t, 2H, J ¼ 7.5 Hz), 3.32 (d, 2H, J ¼ 6.7 Hz), 3.82 (s, 3H), 4.01 (t,
2H, J ¼ 6.3 Hz), 5.03e5.13 (m, 2H), 5.43 (s, 2H), 5.95 (ddtap, 1H,
J1 ¼16.8 Hz, J2 ¼ 10.2 Hz and J3 ¼ 6.7 Hz), 6.65e6.73 (m, 2H), 6.77 (d,
1H, J ¼ 7.9 Hz), 7.10 (d, 2H, J ¼ 8.4 Hz), 7.24 (s, 1H), 7.47 (d, 2H,
7.15e7.23 (m, 3H), 13C NMR (75 MHz, CDCl3)
d: 22.1, 28.7, 39.7, 53.5,
55.3, 55.8, 68.1, 112.3, 113.4, 114.4, 115.5, 120.4, 120.6, 126.8, 129.5,
132.9, 137.6, 146.6, 147.6, 149.3, 159.8. Calculated for C23H28N3O3,
394.21307; found: 394.21225. HRMS (M þ Naþ): Calculated for
J ¼ 8.4 Hz). 13C NMR (75 MHz, CDCl3)
d: 22.1, 28.7, 39.7, 53.2, 55.8,
68.0, 112.3, 113.4, 115.6, 120.4, 120.9, 122.7, 129.5, 132.2, 133.0, 133.9,
137.6, 146.6, 147.9, 149.4. Calculated for C22H25BrN3O2, 442.11301;
C23H27N3NaO3, 416.19501; found: 416.19415.
found: 442.11227 HRMS (M
þ
Naþ): Calculated for
4.1.6.21. 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-(tri-
fluoromethoxy)benzyl)-1H-1,2,3-triazole (7h). White solid, obtained
in 84% yield (0.210 g, 0.470 mmol) from the reaction of alkyne 3
(0.129 g, 0.560 mmol), trifluoromethoxybenzyl azide (0.129 g,
0.560 mmol), sodium ascorbate (0.0440 g, 0.220 mmol) and
CuSO4$5H2O (0.0280 g, 0.110 mmol). Purified by column chroma-
tography eluted with hexane-ethyl acetate-dichloromethane (3:1:3
v/v), m.p. 93.4e94.9 ꢁC, TLC: Rf ¼ 0.49 (hexane-ethyl acetate-
dichloromethane 3:1:3 v/v). IR (ATR) nmax/cmꢀ1: 3118, 3063,
2949, 2878, 1590, 1511, 1454, 1259, 1225, 1138, 1034, 1014, 911, 800,
C
22H24BrN3NaO2, 464.09496; found: 464.09402.
4.1.6.18. 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-iodobenzyl)-
1H-1,2,3-triazole (7e). White solid, obtained in 91% yield (0.246 g,
0.500 mmol) from the reaction of alkyne 3 (0.127 g, 0.550 mmol), 4-
iodobenzyl azide (0.143 g, 0.550 mmol), sodium ascorbate
(0.0440 g, 0.202 mmol) and CuSO4$5H2O (0.0280 g, 0.110 mmol).
Purified by column chromatography eluted with hexane-ethyl ac-
etate-dichloromethane (3:1:3 v/v), m.p. 98.3e99.2 ꢁC, TLC:
Rf ¼ 0.45 (hexane-ethyl acetate-dichloromethane 3:1:3 v/v). IR
(ATR) nmax/cmꢀ1: 3110, 3062, 2999, 2951, 1588, 1513, 1461, 1419,
1260, 1227, 1140, 1009, 910, 797, 731, 655, 484. 1H NMR (300 MHz,
743, 652. 1H NMR (300 MHz, CDCl3)
d
: 2.18 (quint, 2H, J ¼ 6.9 Hz),
2.91 (t, 2H, J ¼ 7.5 Hz), 3.32 (d, 2H, J ¼ 6.7 Hz), 3.81 (s, 3H), 4.01 (t,
2H, J ¼ 6.3 Hz), 5.01e5.13 (m, 2H), 5.48 (s, 2H), 5.95 (ddtap, 1H,
J1 ¼16.8 Hz, J2 ¼ 10.2 Hz and J3 ¼ 6.7 Hz), 6.65e6.73 (m, 2H), 6.77 (d,
1H, J ¼ 7.9 Hz), 7.19 (d, 2H, J ¼ 8.6 Hz), 7.23e7.31 (m, 3H). 13C NMR
CDCl3)
d
: 2.17 (quint, 2H, J ¼ 6.9 Hz), 2.90 (t, 2H, J ¼ 7.5 Hz), 3.33 (d,
2H, J ¼ 6.6 Hz), 3.82 (s, 3H), 4.01 (t, 2H, J ¼ 6.3 Hz), 5.00e5.12 (m,
2H), 5.41 (s, 2H), 5.95 (ddtap, 1H, J1 ¼16.8 Hz, J2 ¼ 10.2 Hz and
J3 ¼ 6.6 Hz), 6.65e6.73 (m, 2H), 6.77 (d, 1H, J ¼ 7.9 Hz), 6.96 (d, 2H,
J ¼ 8.3 Hz), 7.24 (s, 1H), 7.68 (d, 2H, J ¼ 8.3 Hz). 13C NMR (75 MHz,
(75 MHz, CDCl3) d: 22.1, 28.7, 39.7, 53.0, 55.8, 68.0, 112.3, 113.4,
115.6, 120.3 (q, J ¼ 257.0 Hz, OCF3), 120.4, 121.0, 121.4, 129.3, 133.0,
133.6, 137.6, 146.6, 148.0, 149.2e149.3 (m), 149.4. Calculated for
CDCl3)
d
: 22.1, 28.7, 39.7, 53.3, 55.8, 68.0, 94.3, 112.3, 113.4, 115.6,
C
23H25F3N3O3, 448.18480; found: 448.18399 HRMS (M þ Naþ):
120.4, 120.9, 129.6, 132.9, 134.5, 137.6, 138.1, 146.6, 147.9, 149.3.
Calculated for C22H25OIN3O2, 490.09915; found: 490.09836 HRMS
(M þ Naþ): Calculated for C22H24IN3NaO2, 512.08109; found:
512.08038.
Calculated for C23H24F3N3NaO3, 470.16675; found: 470.16602.
4.1.6.22. 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-(tri-
fluoromethyl)benzyl)-1H-1,2,3-triazole (7i). White solid, obtained in
92% yield (0.230 g, 0.530 mmol) from the reaction of alkyne 3
(0.133 g, 0.580 mmol), trifluoromethylbenzyl azide (0.117 g,
0.580 mmol), sodium ascorbate (0.0460 g, 0.230 mmol) and
CuSO4$5H2O (0.0290 g, 0.120 mmol). Purified by column chroma-
tography eluted with hexane-ethyl acetate-dichloromethane (3:1:3
v/v), m.p. 111.3e112.5 ꢁC, TLC: Rf ¼ 0.50 (hexane-ethyl acetate-
dichloromethane 3:1:3 v/v). IR (ATR) nmax/cmꢀ1: 3110, 3062, 2947,
2910, 1589, 1516, 1466, 1421, 1328, 1263, 1224, 1142, 1116, 1014, 908,
4.1.6.19. 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-nitrobenzyl)-
1H-1,2,3-triazole (7f). White solid, obtained in 82% yield (0.205 g,
0.500 mmol) from the reaction of alkyne 3 (0.141 g, 0.610 mmol), 4-
nitrobenzyl azide (0.109 g, 0.610 mmol), sodium ascorbate
(0.0480 g, 0.240 mmol) and CuSO4$5H2O (0.0310 g, 0.120 mmol).
Purified by column chromatography eluted with hexane-ethyl ac-
etate-dichloromethane (3:1:3 v/v), m.p. 79.7e80.4 ꢁC, TLC: Rf 0.29
(hexane-ethyl acetate-dichloromethane 3:1:3 v/v). IR (ATR) nmax
cmꢀ1: 3111, 3062, 2949, 2911, 1590, 1518, 1465, 1348, 1227, 1139,
1014, 907, 849, 798, 713, 592. 1H NMR (300 MHz, CDCl3)
: 2.19
/
798, 740, 669. 1H NMR (300 MHz, CDCl3)
d: 2.19 (quint, 2H,
J ¼ 6.9 Hz), 2.92 (t, 2H, J ¼ 7.5 Hz), 3.32 (d, 2H, J ¼ 6.7 Hz), 3.81 (s,
3H), 4.02 (t, 2H, J ¼ 6.3 Hz), 5.00e5.13 (m, 2H), 5.54 (s, 2H), 5.94
(ddtap, 1H, J1 ¼16.8 Hz, J2 ¼ 10.2 Hz and J3 ¼ 6.74 Hz), 6.65e6.73 (m,
2H), 6.77 (d, 1H, J ¼ 7.9 Hz), 7.28 (s, 1H), 7.32 (d, 2H, J ¼ 8.1 Hz), 7.61
d
(quint, 2H, J ¼ 6.9 Hz), 2.94 (t, 2H, J ¼ 7.5 Hz), 3.31 (d, 2H, J ¼ 6.7 Hz),
3.81 (s, 3H), 4.01 (t, 2H, J ¼ 6.3 Hz), 5.00e5.12 (m, 2H), 5.59 (s, 2H),
5.94 (ddtap, 1H, J1 ¼16.8 Hz, J2 ¼ 10.2 Hz and J3 ¼ 6.7 Hz), 6.65e6.73
(m, 2H), 6.77 (d, 1H, J ¼ 7.8 Hz), 7.31e7.38 (m, 3H), 8.19 (d, 2H,
(d, 2H, J ¼ 8.1 Hz). 13C NMR (75 MHz, CDCl3)
d: 22.1, 28.8, 39.7, 53.2,
55.9, 68.0,112.3, 113.4,115.6, 120.4,121.1, 123.8 (q, J ¼ 270.8 Hz, CF3),
126.0 (q, J ¼ 3.8 Hz), 128.0, 130.9 (q, J ¼ 32.5 Hz), 133.0, 137.6, 138.8,
146.7, 148.1, 149.4. Calculated for C23H25F3N3O2, 432.18989; found:
443.18903. HRMS (M þ Naþ): Calculated for C23H24F3N3NaO2,
454.17183; found: 454.17093.
J ¼ 8.7 Hz). 13C NMR (75 MHz, CDCl3)
d: 22.1, 28.6, 39.7, 52.9, 55.8,
68.0, 112.4, 113.4, 115.6, 120.4, 121.3, 124.2, 128.4, 133.0, 137.5, 141.9,
146.6, 147.9, 148.3, 149.3. Calculated for C22H25N4O4, 409.18758;
found: 409.18677. HRMS (M þ Naþ): Calculated for C22H24N4NaO4,
431.16952; found: 431.16867.
4.1.6.23. 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(3,4-
difluorobenzyl)-1H-1,2,3-triazole (7j). White solid, obtained in 90%
yield (0.225 g, 0.560 mmol) from the reaction of alkyne 3 (0.144 g,
0.630 mmol), 3,4-difluorobenzyl azide (0.107 g, 0.630 mmol), so-
dium ascorbate (0.0500 g, 0.250 mmol) and CuSO4$5H2O (0.0320 g,
0.130 mmol). Purified by column chromatography eluted with
4.1.6.20. 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-
methoxybenzyl)-1H-1,2,3-triazole (7g). White solid, obtained in 83%
yield (0.224 g, 0.570 mmol) from the reaction of alkyne 3 (0.158 g,
0.690 mmol), 4-methoxybenzyl azide (0.113 g, 0.690 mmol), so-
dium ascorbate (0.0550 g, 0.280 mmol) and CuSO4$5H2O (0.0350 g,

R.R. Teixeira et al. / European Journal of Medicinal Chemistry 146 (2018) 274e286
285
hexane-ethyl
acetate-dichloromethane
(3:1:3
v/v),
m.p.
hexane-ethyl
acetate-dichloromethane
(3:1:3
v/v),
m.p.
97.1e97.8 ꢁC, TLC: Rf ¼ 0.41 (hexane-ethyl acetate-dichloromethane
65.9e67.1 ꢁC,
TLC:
Rf ¼ 0.32
(hexane-ethyl
acetate-
3:1:3 v/v). IR (ATR) nmax/cmꢀ1: 3111, 3061, 2949, 1588, 1515, 1454,
dichloromethane 3:1:3 v/v). IR (ATR) nmax/cmꢀ1: 3110, 3062,
1261, 1288, 1228, 1141, 1017, 915, 754. 1H NMR (300 MHz, CDCl3)
d
:
2949, 2881, 1589, 1514, 1462, 1260, 1227, 1140, 1013, 798, 726, 689,
2.18 (quint, 2H, J ¼ 6.9 Hz), 2.92 (t, 2H, J ¼ 7.5 Hz), 3.32 (d, 2H,
586. 1H NMR (300 MHz, CDCl3)
d: 2.18 (quint, 2H, J ¼ 6.9 Hz), 2.91 (t,
J ¼ 6.7 Hz), 3.82 (s, 3H), 4.01 (t, 2H, J ¼ 6.3 Hz), 5.00e5.12 (m, 2H),
2H, J ¼ 7.5 Hz), 3.32 (d, 2H, J ¼ 6.7 Hz), 3.82 (s, 3H), 4.02 (t, 2H,
J ¼ 6.3 Hz), 5.00e5.13 (m, 2H), 5.44 (s, 2H), 5.94 (ddtap, 1H,
J1 ¼16.8 Hz, J2 ¼ 10.2 Hz and J3 ¼ 6.7 Hz), 6.65e6.73 (m, 2H), 6.78 (d,
1H, J ¼ 7.9 Hz), [7.10e7.30 (m), 7.38 (brs), 7.46 (dap), J ¼ 7.8 Hz, 5H].
5.43 (s, 2H), 5.95 (ddtap
,
1H, J1 ¼16.8 Hz, J2 ¼ 10.2 Hz and
J3 ¼ 6.7 Hz), 6.65e6.71 (m, 2H), 6.77 (d, 1H, J ¼ 7.9 Hz), 6.92e7.20
(m, 3H), 7.27 (s, 1H). 13C NMR (75 MHz, CDCl3)
d: 22.1, 28.7, 39.7,
52.5, 55.9 (brd, J ¼ 8.3 Hz), 68.0, 112.5, 113.4, 115.6, 117.0 (d,
J ¼ 17.3 Hz), 117.9 (d, J ¼ 17.3 Hz), 120.4, 120.9, 124.0 (dd, J1 ¼6.8 Hz
and J2 ¼ 3.8 Hz), 131.8e131.9 (m), 133.0, 137.5, 146.5, 148.1, 149.3,
[148.6e148.9 (m), 151.9e152.2 (m)]. Calculated for C22H24F2N3O2,
400.18366; found: 400.18274. HRMS (M þ Naþ): Calculated for
13C NMR (75 MHz, CDCl3)
d: 22.1, 28.7, 39.7, 53.1, 55.8, 68.0, 112.3,
113.4, 115.6, 120.4, 121.0, 123.0, 126.4, 130.6, 130.8, 131.7, 132.9, 137.1,
137.6, 146.6, 148.0, 149.3. Calculated for C22H25BrN3O2, 442.11301;
found: 442.11218. HRMS (M
þ
Naþ): Calculated for
C22H24BrN3NaO2, 464.09496; found: 464.09412.
C
22H23F2N3NaO2, 422.16560; found: 422.16473.
4.2. Leishmanicidal assays
4.2.1. Parasites
4.1.6.24. 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-
methylbenzyl)-1H-1,2,3-triazole (7k). White solid, obtained in 80%
yield (0.216 g, 0.570 mmol) from the reaction of alkyne 3 (0.165 g,
0.720 mmol), 4-methylbenzyl azide (0.106 g, 0.720 mmol), sodium
ascorbate (0.0570 g, 0.290 mmol) and CuSO4$5H2O (0.0360 g,
0.140 mmol). Purified by column chromatography eluted with
Promastigotes of L. amazonensis (WHOM/BR/75/Josefa) trans-
fected with the gene of green fluorescent protein (GFP) were
cultured in M199 medium, supplemented with 50 UI/mL of peni-
cillin, 50
fetal calf serum and 2% of human urine at 26 ꢁC. Promastigotes were
periodically cultured in 1000
g mLꢀ1 of geneticin (G418) for bright
m
g mLꢀ1 of streptomycin and 10% (v/v) of heat-inactivated
hexane-ethyl
acetate-dichloromethane
(3:1:3
v/v),
m.p.
59.6e60.9 ꢁC,
TLC:
Rf ¼ 0.42
(hexane-ethyl
acetate-
m
dichloromethane 3:1:3 v/v). IR (ATR) nmax/cmꢀ1: 3111, 3061, 2953,
green fluorescence selection [40].
2916, 2880, 1588, 1514, 1449, 1420, 1260, 1228, 1140, 1014, 904, 845,
799, 75, 720, 650. 1H NMR (300 MHz, CDCl3)
d: 2.16 (quint, 2H,
4.2.2. Antipromastigote assay
J ¼ 7.0 Hz), 2.34 (s, 3H, CH3), 2.88 (t, 2H, J ¼ 7.5 Hz), 3.32 (d, 2H,
For evaluation of anti promastigote activity, fluorescent pro-
mastigotes were plated in triplicate at 5 ꢂ 105 parasites/well with
varying concentrations of the tested compounds (0, 0.1, 1, 10, and
J ¼ 6.7 Hz), 3.81 (s, 3H), 4.01 (t, 2H, J ¼ 6.4 Hz), 5.00e5.13 (m, 2H),
5.43 (s, 2H), 5.95 (ddtap
,
1H, J1 ¼16.8 Hz, J2 ¼ 10.2 Hz and
J3 ¼ 6.7 Hz), 6.65e6.73 (m, 2H), 6.77 (d, 1H, J ¼ 7.9 Hz), 7.10e7.18 (m,
100 m mL of medium M199 con-
mol Lꢀ1) in a final volume of 200
4H), 7.21 (s, 1H). 13C NMR (75 MHz, CDCl3)
d: 21.0 (CH3), 22.1, 28.7,
taining 5% HIFCS. It is important to mention that the compounds
were dissolved in dimethyl sulfoxide (DMSO) and the final con-
centration of DMSO was ꢃ1% in all treated variables. After 72 h at
27 ꢁC, the fluorescence intensity of the cultures was measured us-
ing a plate-reader fluorometer (Fluoroskan) set at 435 nm excita-
tion/538 nm emissions. All treatments were performed in triplicate,
and the results were expressed as percent inhibition in relation to
control treatment in medium alone.
39.7, 53.7, 55.8, 68.0, 112.3, 113.4, 115.5, 120.4, 120.7, 127.9, 129.6,
131.7, 132.8, 137.5, 138.4, 146.6, 147.6, 149.3. Calculated for
C
23H28N3O2, 378.21815; found: 378.21719. HRMS (M þ Naþ):
Calculated for C23H27N3NaO2, 400.20010; found: 400.19922.
4.1.6.25. 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(2-
bromobenzyl)-1H-1,2,3-triazole (7l). White solid, obtained in 79%
yield (0.198 g, 0.450 mmol) from the reaction of alkyne 3 (0.130 g,
0.570 mmol), 2-bromobenzyl azide (0.120 g, 0.570 mmol), sodium
ascorbate (0.0450 g, 0.230 mmol) and CuSO4$5H2O (0.0290 g,
0.110 mmol). Purified by column chromatography eluted with
4.2.3. Antiamastigote assay
For antiamastigote activity, mouse peritoneal macrophages
were collected from the peritoneal cavities of BALB/c mice in ice-
cold DMEM medium (Sigma). The cells were plated at 2 ꢂ 106/mL
in Lab-Tek 8-chamber slides (Nunc, Naperville, Ill.) and incubated at
37 ꢁC and 4% CO2 for 1 h. No adherent cells were removed by
washing with pre warmed phosphate-buffered saline (PBS).
Adherent macrophages were infected with L. amazonensis pro-
mastigotes (stationary growth phase) at a 5:1 parasite/macrophage
ratio and incubated for 1 h at 35 ꢁC and 5% CO2. After 4 h, the
chambers were washed three times with pre warmed PBS to
remove free parasites. The monolayers were incubated with com-
hexane-ethyl
acetate-dichloromethane
(3:1:3
v/v),
m.p.
47.8e49.1 ꢁC,
TLC:
Rf ¼ 0.61
(hexane-ethyl
acetate-
dichloromethane 3:1:3 v/v). IR (ATR) nmax/cmꢀ1: 3104, 3054,
2931, 1587, 1511, 1467, 1419, 1230, 1135, 1027, 948, 804, 744, 653. 1H
NMR (300 MHz, CDCl3)
d
: 2.19 (quint, 2H, J ¼ 7.0 Hz), 2.92 (t, 2H,
J ¼ 7.5 Hz), 3.32 (d, 2H, J ¼ 6.7 Hz), 3.82 (s, 3H), 4.02 (t, 2H,
J ¼ 6.4 Hz), 5.00e5.13 (m, 2H), 5.61 (s, 2H), 5.95 (ddtap, 1H,
J1 ¼16.8 Hz, J2 ¼ 10.2 Hz and J3 ¼ 6.7 Hz), 6.65e6.73 (m, 2H), 6.78 (d,
1H, J ¼ 7.8 Hz), 7.08 (dd, 1H, J1 ¼7.5 Hz and J2 ¼ 1.6 Hz), 7.15e7.31
(m, 2H), 7.35 (s, 1H), 7.59 (dd, 1H, J1 ¼7.8 Hz and J2 ¼ 1.1 Hz). 13C
NMR (75 MHz, CDCl3)
d: 22.1, 28.7, 39.7, 53.6, 55.8, 68.1, 112.3, 113.4,
pound 7k up to 100 m
mol Lꢀ1 for 72 h at 37 ꢁC and 5% CO2. The
115.5, 120.3, 121.3, 123.2, 128.1, 130.1, 130.2, 132.9, 133.1, 134.4, 137.6,
146.6, 147.7, 149.3. Calculated for C22H25BrN3O2, 442.11301; found:
442.11197. HRMS (M þ Naþ): Calculated for C22H24BrN3NaO2,
464.09496; found: 464.09390.
reference drugs pentamidine (Sigma-Aldrich) and glucantime
(Sanofi-aventis) were used at concentrations varying from 0.1 to
100 m m
mol Lꢀ1 and 0.1 to 100 g mLꢀ1, respectively. The anti amas-
tigote activity was evaluated microscopically after staining the
chambers with the Instant Prov hematological dye system (New-
prov, Curitiba, Brazil); at least 200 macrophages were counted per
sample. The results are expressed as the infection index (IF) using
the following equation: IF ¼ % infected cells X number of amasti-
gotes/total macrophages. The calculation of the IC50 was performed
by logarithmic regression, and the statistical analysis was per-
formed using Student's t-test in GraphPad Prism 5.0 software.
4.1.6.26. 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(3-
bromobenzyl)-1H-1,2,3-triazole (7m). White solid, obtained in 81%
yield (0.203 g, 0.460 mmol) from the reaction of alkyne 3 (0.130 g,
0.570 mmol), 3-bromobenzyl azide (0.120 g, 0.570 mmol), sodium
ascorbate (0.0450 g, 0.230 mmol) and CuSO4$5H2O (0.0290 g,
0.110 mmol). Purified by column chromatography eluted with

286
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Acknowledgements
This work was supported by the National Program for Academic
Cooperation (PROCAD) of the Coordination for the Improvement of
Higher Education Personnel-CAPES/Brazil, and by the agencies
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~
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