Larvicidal isoxazoles: Synthesis and their effective susceptibility towards Aedes aegypti larvae

Article abstract of DOI:10.1016/j.bmc.2012.12.006

Twenty 3,5-disubstituted isoxazoles have been synthesized and tested against fourth instar Aedes aegypti larvae. In the synthesis of title compounds, modifications have been made in the C-5 side-chain with a view to test their larvicidal activity. These i

Full text of DOI:10.1016/j.bmc.2012.12.006

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Larvicidal isoxazoles: Synthesis and their effective susceptibility
towards Aedes aegypti larvae
⇑
Diana C. B. da Silva-Alves, Janaína V. dos Anjos , Nery N. M. Cavalcante, Geanne K. N. Santos,
⇑
Daniela M. do A. F. Navarro, Rajendra M. Srivastava
Departamento de Química Fundamental, Universidade Federal de Pernambuco, Cidade Universitária, 50740-560 Recife, PE, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty 3,5-disubstituted isoxazoles have been synthesized and tested against fourth instar Aedes aegypti
larvae. In the synthesis of title compounds, modifications have been made in the C-5 side-chain with a
view to test their larvicidal activity. These isoxazoles have been obtained by 1,3-dipolar cycloaddition
of arylnitrile oxides to terminal alkynes which furnished the desired products in 20% to 79% yields. A
comparative study of the larvicidal activity between 3-(3-aryl-isoxazol-5-yl)-propan-1-ols and 3-(3-
aryl-isoxazol-5-yl)-propionic acids clearly demonstrated that the latter compounds possess much better
larvicidal activity than the former. We also tested two esters, viz., methyl 3-[3-(phenyl)-isoxazole-5-yl]
propionate and methyl 3-[3-(4-chlorophenyl)-isoxazole-5-yl] propionate, where the latter presented
an excellent larvicidal profile.
Received 18 October 2012
Revised 30 November 2012
Accepted 5 December 2012
Available online xxxx
Keywords:
Isoxazole
Click chemistry
Aedes aegypti
Larvicidal activity
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
tance. About twelve years ago, this kind of resistance has been
found in mosquitoes in Rio de Janeiro, RJ, Brazil.8 Temephos-in-
Dengue is an endemic disease that occurs in tropical and sub-
tropical regions, transmitted by the female Aedes aegypti mos-
quito.1 About eighty million people are infected per year by one
of the four dengue virus types, which is becoming one of the
world’s major public health problems, since this mosquito is pres-
ent in more than 100 countries.2 A huge dengue epidemic was ex-
pected for 2012, once the major peaks of dengue activity are
observed in a four-year interval (the last big epidemic outbreak
in Brazil took place in 2008, with almost one million notified
cases).3 Although vaccines are being developed to combat the
infection caused by dengue viruses,4 the sole effective strategy to
control the disease is the vector eradication, since the increase in
the incidence of this illness is closely linked to Aedes aegypti mos-
quito dissemination.5
Aedes aegypti mosquito is quite adapted to domestic environ-
ment and makes use of clean water held in containers for reproduc-
tion, which is the fundamental cause of proliferation.4,6 Mosquito
control campaigns encourage people to reduce water storages and
to use water larvicides, aiming to control the adult mosquito popu-
lation. These integrated approaches help to reduce mosquitoes and
larvae population, which might decrease virus transmission.6
One of the most employed larvicides used for combating A. ae-
gypti mosquitoes is TemephosÒ.7 Although quite effective, this
organophosphate insecticide is related to mosquito strains resis-
duced resistance has also been reported by Indian researchers for
different stages of A. aegypti mosquitoes.9 This way, the develop-
ment of new, non-toxic and cheap substances capable of disrupting
A. aegypti life cycle is an urgent need.10,11
Our research group has been involved in the synthesis of larvi-
cidal compounds after we discovered that 3-(3-aryl-1,2,4-oxadiaz-
ole-5-yl) propionic acids possess such activity.6,12 Due to the great
effectiveness of the above-mentioned compounds against mos-
quito larvae (ca. 15 ppm) and their low genotoxicity in mammals,
these products have been patented and are the first prototypes of
synthetic larvicides containing the 1,2,4-oxadiazole nucleus.12
The mechanism of action is not yet fully elucidated, but some
experimental evidences show that this class of substances may
act in the kynurenine pathway,13–18 the major catabolic pathway
in insects, causing the death by apoptosis of neuronal cells.
Isoxazoles are five-membered heterocycles quite similar in
structure to 1,2,4-oxadiazoles. Indeed, the difference lies in the
replacement of N-4 atom of oxadiazole with a CH group to furnish
an isoxazole.19,20 This latter heterocycle is present in a large
number of pharmacologically active drugs, such as cefoxazole
and oxacillin, two beta-lactam antibiotics; isoxicam, an oxycam
anti-inflammatory agent and the antidepressant drug isocarbox-
azid, a monoaminooxidase (MAO) inhibitor.21 In 2011, an antiviral
compound (NITD-982) containing an isoxazole–pyrazole core has
been found to inhibit dengue virus by supressing dihydroorotate-
dehydrogenase (DHODH), an enzyme required for pyrimidine bio-
synthesis in the host cell.22 Besides being highly active compounds
⇑
Corresponding author. Tel.: + 55 81 2126 5015; fax: + 55 81 2126 8480.
E-mail address: janaversiani@hotmail.com, janaversiani@hotmail.com (J.V. dos
Anjos), rms@ufpe.br (R.M. Srivastava).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.006
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2
D. C. B. da Silva-Alves et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
in biological systems and very similar to 1,2,4-oxadiazoles in struc-
ture, isoxazole-containg molecules have not been used to control A.
aegypti mosquito dissemination.
As indicated above, isoxazoles are highly effective in biological
systems and are widely used as drugs.23 In fact, this heterocycle is
found even in some natural products such as ibotenic acid.24 In
addition, a number of drugs including the COX-2 inhibitor valdec-
oxib (BextraÒ) belong to this class of compounds.
We are inspired by our past research work on 1,2,4-oxadiazoles
and decided to investigate isoxazoles because of their similarities
with the former except that isoxazoles have only one nitrogen
atom in the five-membered ring instead of two atoms found in
oxadiazoles.
Considering the above mentioned advantages of isoxazole-con-
taining compounds and the increasing drug resistance of usual
compounds towards A. aegypti larvae, we decided to synthesize
evaluated: the type of heterocycle in the structure and the pres-
ence or absence of a carbonyl group in the molecules.
Prior to the synthesis of the isoxazoles, imidoyl chlorides have
been prepared from corresponding aryloximes and N-chlorosuc-
cinimide (NCS). The oximes were prepared from aromatic alde-
hydes and hydroxylamine.20 Reaction of the imidoyl chlorides
with an appropriate terminal alkyne (4-pentyn-1-ol, 4, or sodium
4-pentynoate, 6), using Cu(OAc)2 and sodium ascorbate as catalyst
(click conditions),25 in the presence of base (KHCO3), in 1:1 tert-
butanol/water yielded the desired products in moderate to good
yields (Schemes 1 and 2). Our efforts to synthesize the 3-[3-(3,4-
dichlorophenyl)-isoxazol-5-yl]-propionic acid failed. Even after
making several attempts to obtain this product, we had to abandon
its preparation because of extensive decomposition and/or side
products.
For all synthesized isoxazoles, only one regioisomer could be
3-(3-aryl-isoxazol-5-yl)-propan-1-ols,
3-(3-aryl-isoxazol-5-yl)-
detected by 1H NMR, because only one singlet was observed in
propionic acids and two of their esters capable of inhibiting L4
stage larvae growth in aqueous media. To the best of our knowl-
edge, no study has been reported employing such compounds
against A. aegypti larvae.
the 1H NMR spectra for C-4 ring proton between
d 6.16–
6.85 ppm. According to earlier literature for the copper catalyzed
isoxazole synthesis, it is believed that the obtained products are
3,5-disubstituted regioisomers.25 We employed HMBC (Heteronu-
clear Multiple Bond Correlation) experiments to determine the
regioisomers formed during the reaction. In the HMBC spectrum,
C–H interactions up to three bonds can be detected. To illustrate
how this magnetic resonance tool can be useful to differentiate
the regioisomers (3,5 and 3,4-disubstituted isoxazoles), a HMBC
experiment was performed for compound 7a (Fig. 1). Thus, if an
imaginary line is set in the carbon spectrum axis at 99.3 ppm, par-
allel to the hydrogen spectrum axis, it intersects three peaks. The
intersection with the peak in 3.2 ppm in the hydrogen axis corre-
sponds to a coupling between the methylene group adjacent to
the isoxazole ring, showing a 3JCH coupling. The other two interac-
tions represent a doublet which shows a direct correlation be-
tween this carbon and the hydrogen attached to it (1JCH
coupling). Another line parallel to hydrogen axis at 161.8 ppm
2. Results and discussion
2.1. Chemistry
Earlier results of our research group showed that 3-(3-aryl-
1,2,4-oxadiazole-5-yl) propionic acids had good yield in the
synthesis, a very good larvicidal activity and no genotoxicity.6
However, no further studies were carried out to discover the phar-
macophore, i.e. the minimal structure responsible for the biological
activity. Then, with the goal of finding out the pharmacophore
structure and, as a consequence, developing more potent larvicidal
prototypes, we have synthesized n-propanols and propionic acids
containing an isoxazole-5-yl ring attached at their C-3 atoms. This
way, two parameters in the prototype structure could be
2
intercepts two signals: one at 6.82 ppm representing a JCH cou-
pling and the other signal, at 8.00 ppm, for aromatic hydrogen,
which confirms that the carbon signal at 161.8 ppm is due to C-3
of the isoxazole ring (3JCH coupling). This piece of evidence pro-
vides strong support that compound 7a is the 3,5-disubstituted
regioisomer and not the 3,4-disubstituted one.
Previous results of our research group indicated that esters of
propionic acids containing an oxadiazole nucleus possess more po-
tent larvicidal activity than the parent acids.18 In order to verify the
effectiveness of propionic acid esters containing an isoxazol-5-yl as
substituent, we decided to prepare two such products. The acids
7a–b were subjected to the classical Fisher esterification condi-
tions, which furnished methyl esters in good yields (Scheme 3).
2.2. Bioassays
As mentioned earlier, we decided to synthesize these 3,5-disub-
stituted isoxazoles, which are similar to 1,2,4-oxadiazoles in struc-
ture, except for having CH function instead of N-4. This
modification will show which core (1,2,4-oxadiazole or isoxazole)
Scheme 1. Synthesis of 3-(3-aryl-isoxazol-5-yl)-propan-1-ols (5a–j).
Scheme 2. Synthesis of 3-(3-aryl-isoxazol-5-yl)-propionic acids (7a–h).
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D. C. B. da Silva-Alves et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
Figure 1. HMBC spectrum for compound 7a.
was 35.7 ppm. Just for comparison, the corresponding alcohol (5e),
presented a LC50 value above 100 ppm.
Table 3 shows the observed LC50 values for 1,2,4-oxadiazole
propionic acids (8a–i).6 These results clearly indicate that the
replacement of the 1,2,4-oxadiazole nucleus with a isoxazole ring
results in better larvicidal activity. It is interesting to note that
the substitution of N-4 in 1,2,4-oxadiazole with a CH moiety
caused a dramatic increase in larvicidal activity.
Scheme 3. Synthesis of methyl 3-[3-(aryl)-isoxazol-5-yl]-propionates (9a–b).
is more important for the maintenance or for the improvement of
larvicidal activity. Larvicidal profile of compounds 5a–j is de-
scribed in Table 1. It can be observed that only compounds 5b,
5d, and 5j showed significant larvicidal activity (LC50 bellow
100 ppm). Isoxazole 5j having 3,4-dichlorophenyl substituent
proved to be the most active in this series of compounds
(LC50 = 17.2 ppm), indicating the significant contribution of two
chlorine atoms as substituents in the phenyl ring.
It was observed that the addition of electronegative atoms in
the phenyl ring has some influence in improving the larvicidal
activity (see LC50 values for 7b (p-ClPh), 7c (p-FPh) and 7d
(p-BrPh)). It can be noticed that, for these isoxazoles, the electronic
effects are as important as the steric effects, since there is a huge
difference in the larvicidal profile when 7a (Ph) and 7c (p-FPh)
are compared. This way, the isoxazole containing the bromine
atom, bulky and electronegative as a substituent in the aryl
group, was the most active in this series (7d, LC50 = 13.9 ppm).
The corresponding isoxazole alcohol (5d) did not show the same
effect.
Larvicidal profile for compounds 7a–h is summarized in Table 2.
In this series, only one compound (7h) has not shown significant
larvicidal activity. As an example, for compound 7e, the LC50 value
Table 1
Larvicidal activity for compounds 5a–j
Compound
Ar
LC50 (ppm)
Standard error
Confidence interval (ppm)
5a
5b
5c
5d
5e
5f
5g
5h
5i
Phenyl
4-ClPh
4-FPh
4-BrPh
4-CH3OPh
4-NO2Ph
p-Tolyl
m-Tolyl
o-Tolyl
3,4-Cl2Ph
>100
28.9
>100
33.3
>100
81.9
>100
>100
>100
17.2
—
1.1
—
0.7
—
1.6
—
—
—
0.5
—
26.8–31.0
—
32.0–34.7
—
78.7–85.1
—
—
—
5j
16.1–18.2
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D. C. B. da Silva-Alves et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Table 2
Larvicidal activity for compounds 7a–h
Compound
Ar
LC50 (ppm)
Standard error
Confidence interval (ppm)
7a
7b
7c
7d
7e
7f
Phenyl
4-ClPh
4-FPh
4-BrPh
4-CH3OPh
4-NO2Ph
p-Tolyl
m-Tolyl
82.8
37.7
33.7
13.9
35.7
73.1
38.8
>100
1.5
1.2
0.7
1.0
0.5
1.4
0.7
—
79.9–85.7
35.2–40.1
32.4–35.1
12.0–15.7
34.7–36.6
70.4–75.8
37.4–40.2
—
7g
7h
Table 3
Larvicidal activity for 3-[3-(aryl)-1,2,4-oxadiazol-5-yl]-propionic acids (8a–h)
Compound
Ar
LC50 (ppm)
Standard error
Confidence interval (ppm)
8a
8b
8c
8d
8e
8f
Phenyl
4-ClPh
4-FPh
4-BrPh
4-CH3OPh
4-NO2Ph
p-Tolyl
m-Tolyl
98.6
28.1
81.2
15.2
71.5
50.5
65.8
73.8
2.9
1.9
1.3
0.8
1.5
2.4
3.2
2.9
95.7–101.5
26.2–30.0
79.9–82.5
14.4–16.0
70.0–73.0
48.1–52.9
62.6–69.0
70.9–76.7
8g
8h
Table 4
Larvicidal activity for compounds 9a–b
Compound
Ar
LC50 (ppm)
Standard error
Confidence interval (ppm)
9a
7a
9b
7b
Phenyl
Phenyl
4-ClPh
4-ClPh
13.2
82.8
3.4
0.5
1.5
0.1
1.2
12.5–14.3
79.9–85.7
3.2–3.6
37.7
35.2–40.1
Our earlier research has shown that 3-(3-aryl-isoxazol-
5-yl)-propionic acids are more effective than their corresponding
oxadiazole derivatives (8a–i), because LC50 values of the former
are lower than the latter.6 For example, the LC50 value of
3-[3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl] propionic acid (8e) is
81.2 ppm6 whereas 3-[3-(4-fluorophenyl)-isoxazol-5-yl] propionic
acid (7c) has LC50 = 33.7 ppm, clearly demonstrating the increase
of larvicidal activity (See Tables 2 and 3). We can conclude that
the substitution of nitrogen atom in the position 4 of the oxadiaz-
ole ring shows a positive effect in the larvicidal activity.
Finally, we evaluated the mortality of the larvae using two
methyl esters of isoxazoles (9a,b) and compared their behavior
with the parent acids 7a,b. We found that 9a,b are even more ac-
tive than the acids, once the LC50 values are 13.2 and 3.4 ppm,
and the acids 7a,b have the LC50 values of 82.8 and 37.7 ppm,
respectively (Table 4). We have chosen two compounds (7a,b) to
be transformed into their esters to evaluate their behavior towards
the mosquito larvae. Both acids have moderate activity, but when
transformed into their methyl esters, they become good larvicidal
candidates.
The larvicidal activity of the synthesized compounds is evalu-
ated against fourth instar A. aegypti larvae in a small amount of
water. Once A. aegypti larvae are immersed in water and since
the larvae skin is lipophilic in nature, the drug penetration in their
system is much easier compared to their predecessor acids, hence
the esters are more active. This fact can be correlated to larvae life-
time when exposed to isoxazole containing acids and esters in
their C-3 side chains. When exposed to acids, larvae die within
48 h, but when in contact with esters, larvae are almost all dead
24 h after the beginning of the test.
route in insects like Aedes aegypti. In this biochemical pathway,
tryptophan is oxidized to kynurenic or xanturenic acids, which
can then be oxidized to CO2 and water or even be used in the
biosynthesis of other substances.13 One of intermediates in this
biochemical pathway is 3-hydroxy-kynurenine, harmful metabo-
lite, which can disproportionate to nitrogen and oxygen reactive
species.26,27 One enzyme in this pathway, the 3-hydroxy-kynuren-
ine transaminase (3-HKT) mediates the conversion of 3-hydroxy-
kynurenine to xanturenic acid.17 Once this enzyme is blocked,
3-hydroxy-kynurenine cannot be converted to xanturenic acid
and this harmful substance can accumulate and cause neuronal cell
apoptosis by inducing oxidative stress.28–31
3-Hydroxy-kynurenine direct inhibition assays could not be
performed to prove this hypothesis. But the HPLC (high perfor-
mance liquid chromatography) assays,32 an indirect approach,
can show if there is a decrease in the levels of xanturenic acid
and an increase of 3-hydroxy-kynurenine in the insects when they
get in contact with the larvicidal compounds synthesized in this
work. In addition, molecular docking studies have shown that
3-(3-aryl-1,2,4-oxadiazole-5-yl) propionic acids do interact with
3-HKT. Such studies are being currently performed and the results
will be published in the future.
3. Conclusions
In brief, we have accomplished the synthesis of twenty isoxaz-
oles via copper catalyzed cycloaddition reaction between aryloxi-
mes and terminal alkynes, where sixteen are new (5b, e–j; 7b–h;
9a–b). All compounds presented as single 3,5-disubstituted regioi-
somers, as proven by HMBC experiments. Larvicidal data show that
the replacement of the 1,2,4-oxadiazole moiety by a ring of
isoxazole has a positive effect in larvae mortality. The propyl chain
We suggest that the compounds described in this paper could
act to inhibit the kynurenine pathway,13–17 the major catabolic
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D. C. B. da Silva-Alves et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
present in the compounds does enhance the larvicidal activity,
which can be correlated with an increase of lipophilicity of the ter-
minal groups in the order: ester > acid > alcohol. The compounds
with electronegative substituents in the phenyl ring exhibited
the best activity. Methyl 3-[3-(4-chlorophenyl)isoxazole-5-yl] pro-
pionate (9b) presented an excellent larvicidal profile among the
substances examined in this work.
4.1.2. 3-[3-(4-Chlorophenyl)-isoxazol-5-yl)]-propan-1-ol (5b)
Colorless crystals; yield: 20%; Rf: 0.74 (hexanes/AcOEt, 1:1 v/v);
mp: 71–72 °C. 1H NMR (CDCl3, 300 MHz) d: 1.99 (q, 2H, J 7.5 Hz,
6.3 Hz, CH2), 2.08 (s, 1H, OH); 2.91 (t, 2H, J 7.5 Hz, CH2), 3.73 (t,
2H, J 6.3 Hz, CH2OH), 6.29 (s, 1H, Hisox); 7.40 (d, 2H, J 6.9 Hz, Harom),
7.70 (d, 2H, J 6.9 Hz, Harom). 13C NMR (CDCl3, 75 MHz) d: 23.1; 30.2;
61.3; 99.0; 127.6; 127.9; 128.8; 129.1; 135.8; 161.4;173.8. I.R. (KBr
pellet)
m
max/cmÀ1: 3260 (O–H); 2939, 2873(C–H); 1658 (C@N);
4. Experimental section
4.1. General methods
1430 (N–O). ESI-HRMS m/z: 305.9989 (Calcd for C11H12BrN3ONa
[M+Na]+: 304.0061).
4.2.3. 3-[3-(4-Fluorophenyl)-isoxazol-5-yl)]-propan-1-ol (5c)
Colorless crystals; yield: 33%; Rf: 0.40 (hexanes/AcOEt, 1:1 v/v);
mp: 74–75 °C. 1H NMR (DMSO, 400 MHz) d (ppm): 1.81 (q, 2H, J
7.6 Hz, 6.0 Hz, CH2); 2.81 (t, 2H, J 7.6 Hz, CH2); 3.35 (s, 1H, OH);
3.47 (t, 2H, J 6.0 Hz, CH2OH); 6.77 (s, 1H, Hisox), 7.30 (dd, 2H, J
8.8 Hz, 6.8 Hz, Harom); 7,87 (dd, 2H, J 8.8 Hz, 6.8 Hz, Harom). 13C
NMR (DMSO, 100 MHz) d: 22.8; 30.3; 59.6; 99.2; 115.9; 116.1;
125.4; 125.5; 128.7; 128.8; 160.9; 161.8; 164.3; 174.2. I.R. (KBr
All commercially available reagents were used without any fur-
ther purification and the reactions were monitored by TLC analysis
with TLC plates containing GF254 (E. Merck). Melting points were
determined on a Büchi apparatus and are uncorrected. Column
chromatography was performed on Silica Gel 60 (70–230 mesh,
E. Merck). NMR spectra were recorded with a Varian Unity Plus
300 MHz or a Varian UNMRS 400 MHz spectrometer. Infrared spec-
tra were obtained on a Brucker IFS66 FT-IR, using KBr pellets. Ele-
mental analysis was performed with a Carlo Erba instrument
model E-1110. Exact mass measurements of the molecular ions
pellet)
m
max/cmÀ1: 3252 (O–H); 2932, 2870 (C–H); 1613, 1594
(C@N); 1434 (N–O); 1243 (C–F). Anal. Calcd for C12H12FNO2
(C,H,N): C, 65.15%; H, 5.47%; N, 6.33%. Found: C, 65.35%; H,
5.82%; N, 6.39%.
were obtained on
a
Shimadzu LC/MS-IT-TOF Eletrospray.
Aryloximes 1a–j were prepared according to known literature
procedures.25 Compounds 5a, 5c33 and 7a34 had their physical–
chemical data compared to earlier literature for these substances.
4.2.4. 3-[3-(4-Bromophenyl)-isoxazol-5-yl)]-propan-1-ol (5d)
Colorless crystals; yield: 20%; Rf: 0.74 (hexanes/AcOEt, 1:1 v/v);
mp: 71–72 °C. 1H NMR (CDCl3, 300 MHz) d (ppm): 1.68 (s, 1H, OH);
2.00 (q, 2H, J 6.3 Hz, CH2); 2.93 (t, 2H, J 7.5 Hz, CH2); 3.74 (t, 2H, J
6.3 Hz, CH2); 6.30 (s, 1H, Hisox); 7.57 (dd, 2H, J 8.7 Hz, 2.1 Hz, Har-
om); 7.65 (dd, 2H, J 8.7 Hz, 2.1 Hz, Harom). 13C NMR (CDCl3,
75 MHz) d: 23.2; 30.2; 61.5; 99.0; 124.1; 128.2; 132.1; 153.5;
4.2. Synthesis of 3-(3-aryl-isoxazol-5-yl)-propan-1-ols (5a–j)
To a solution of an appropriate aryloxime 1a–j (4.0 mmol) in
20 mL of dimethylformamide (DMF), 4.8 mmol (0.64 g) of N-chlo-
rosuccinimide (NCS) was added dropwise in 30 min. The resulting
solution was stirred at room temperature until TLC analysis con-
firmed the disappearance of the starting oxime. The mixture was
diluted with water (20 mL) and ethyl acetate (20 mL). The organic
layer was separated, and the aqueous phase was extracted again
with AcOEt (20 mL). Drying of the combined organic layers over
anhydrous Na2SO4 and solvent removal in vacuo furnished the
crude imidoyl chloride which was used in the next step without
further purification (3a–j). 4-Pentyn-1-ol (4, 0.4 g, 4.8 mmol) and
the imidoyl chloride (3a–j, 4.0 mmol) were suspended in 1:1 mix-
ture of tert-butanol and water (10 mL). To this solution, a mixture
of Cu(OAc)2 (40 mg, 0.2 mmol) and sodium ascorbate (120 mg,
0.6 mmol) was added followed by the dropwise addition of an
aqueous solution of KHCO3 (18.0 mmol) to it. The contents were al-
lowed to stir at room temperature until TLC analysis indicated the
complete consumption of imidoyl chloride. The mixture was then
diluted with water (10 mL) and AcOEt (10 mL). The organic layer
was separated, and the water phase was extracted with AcOEt
(2 Â 10 mL). The combined organic layers were dried over anhy-
drous Na2SO4, filtered and the solvent evaporated under reduced
pressure. The residue was chromatographed over silica gel using
cyclohexane/EtOAc as eluant (1:1, v/v), crystallized from chloro-
form and hexane, which after work-up furnished the desired
3-(3-aryl-isoxazol-5-yl)-propan-1-ol.
161.5; 173.8. I.R. (KBr pellet)
m
max/cmÀ1: 3252 (O–H); 2931 (C–
12H12BrNO2
H); 1613 (C@N); 1434 (N–O). Anal. Calcd for
C
(C,H,N): C, 51.09%; H, 4.29%; N, 4.96%. Found: C, 51.42%; H,
4.37%; N, 4.97%.
4.2.5. 3-[3-(4-Methoxyphenyl)-isoxazol-5-yl)]-propan-1-ol (5e)
Colorless crystals; yield: 20%; Rf: 0.60 (hexanes/AcOEt, 1:1 v/v);
mp: 60–62 °C. 1H NMR (CDCl3, 300 MHz) d: 1.94–2.03 (m, 2H, CH2),
2.89 (t, 2H, J 7.5 Hz, CH2), 3.72 (t, 2H, J 6.3 Hz, CH2), 3.83 (s, 3H,
OCH3), 3.93 (s, 1H, OH), 6.26 (s, 1H, Hisox); 6.94 (dd, 2H, J 9.0 Hz,
2.1 Hz, Harom); 7.70 (dd, 2H, J 9.0 Hz, 2.1 Hz, Harom). 13C NMR
(CDCl3, 75 MHz) d: 23.1; 30.3; 55.3; 61.4; 98.9; 112.0; 114.2;
121.6; 126.2; 128.0; 160.8; 173.2. I.R. (KBr pellet)
m :
max/cmÀ1
3257 (O–H); 3123 (C–H); 1610 (C@N); 1431 (N–O). ESI-HRMS m/
z: 256.1184 (Calcd for C13H15NO3Na [M+Na]+: 256,0950).
4.2.6. 3-[3-(4-Nitrophenyl)-isoxazol-5-yl)]-propan-1-ol (5f)
Yellow crystals; yield: 44%; Rf: 0.63 (hexanes/AcOEt, 1:1 v/v);
mp: 80–82 °C. 1H NMR (CDCl3, 400 MHz) d (ppm): 1.67 (s, 1H,
OH); 2.03 (q, 2H, J 7.6 Hz, 6.4 Hz, CH2); 2.97 (t, 2H, J 7.6 Hz, CH2);
3.76 (t, 2H, J 6.4 Hz, CH2OH); 6.41 (s, 1H, Hisox), 7.96 (dd, 2H, J
8.8 Hz, 2.0 Hz, Harom); 8.30 (dd, 2H, J 8.8 Hz, 2.0 Hz, Harom). 13C
NMR (CDCl3, 100 MHz) d: 23.2; 30.2; 61.4; 99.3; 124.1; 127.6;
135.4; 148.6; 160.6; 174.6. I.R. (KBr pellet)
H); 3358 (C–H); 1662 (C@N); 1516 (N–O). Anal. Calcd for
12H12N2O4 (C,H,N): C, 58.06%; H, 4.87%; N, 11.29%. Found: C,
m
max/cmÀ1: 3462 (O–
4.2.1. 3-(3-Phenyl-isoxazol-5-yl)-propan-1-ol (5a)
C
Colorless crystals; yield: 54%; Rf: 0.40 (hexanes/AcOEt, 1:1 v/v);
mp: 49–51 °C. 1H NMR (CDCl3, 300 MHz) d (ppm): 1.80 (s, 1H, OH);
2.01 (q, 2H, J 7.5 Hz, 6.3 Hz, CH2); 2.93 (t, 2H, J 7.5 Hz, CH2); 3.75 (t,
2H, J 6.3 Hz, CH2OH); 6.33 (s, 1H, Hisox), 7.42-7.46 (m, 3H, Harom);
7.78 (dd, 2H, J 9.0 Hz, 2.1 Hz, Harom). 13C NMR (CDCl3, 75 MHz) d:
23.2; 30.3; 61.5; 99.2; 126.7; 128.8; 129.1; 129.9; 162.4; 173.4.
58.17%; H, 5.23%; N, 11.30%.
4.2.7. 3-(3-p-Tolyl-isoxazol-5-yl)-propan-1-ol (5g)
Colorless crystals; yield: 52%; Rf: 0.78 (hexanes/AcOEt, 1:1 v/v);
mp: 48–50 °C. 1H NMR (CDCl3, 300 MHz) d (ppm): 1.59 (s, 1H, OH);
2.01 (q, 2H, J 7.5 Hz, 6.0 Hz; CH2); 2.39 (s, 3H, CH3Ph); 2.92 (t, 2H, J
7.5 Hz, CH2); 3.75 (t, 2H, J 6.0 Hz, CH2); 6.30 (s, 1H, Hisox), 7.25 (dd,
I.R. (KBr pellet)
(C@N); 1471 (N–O). Anal. Calcd for C12H13NO2 (C,H,N): C, 70.92%;
m : 3259 (O–H); 3124 (C–H); 1610
max/cmÀ1
2H, J 8.1 Hz, 0.6 Hz, Harom); 7.68 (dd, 2H, J 8.1 Hz, 0.6 Hz, Harom). 13
C
H, 6.45%; N, 6.89%. Found: C, 71.19%; H, 6.69%; N, 6.99%.
NMR (DMSO, 100 MHz) d: 21.3; 23.2; 30.3; 61.5; 99.0; 126.3;
Please cite this article in press as: da Silva-Alves, D. C. B.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2012.12.006

6
D. C. B. da Silva-Alves et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
126.6; 129.5; 139.9; 162.3; 173.3. I.R. (KBr pellet)
m
max/cmÀ1: 3264
161.8; 172.9; 173.0. I.R. (KBr pellet)
3048–2934 (O–H); 1695 (C@O); 1601 (C@N). Anal. Calcd for
12H11NO3 (C,H,N): C, 66.35%; H, 5.10%; N, 6.45%. Found: C,
m
max/cmÀ1: 3116 (C–H);
(O–H); 2931, 2872 (C–H); 1608 (C@N); 1433 (N–O). ESI-HRMS m/z:
240.0951 (Calcd for C13H15NO2Na [M+Na]+: 240.1000).
C
66.12%; H, 5.60%; N, 6.63%.
4.2.8. 3-(3-m-Tolyl-isoxazol-5-yl)-propan-1-ol (5h)
Yellow oil; yield: 57%; Rf: 0.73 (hexanes/AcOEt, 1:1 v/v). 1H
NMR (CDCl3, 300 MHz) d: 1.95–2.04 (m, 3H, CH2, OH); 2.39 (s,
3H, CH3); 2.91 (t, 2H, J 7.5 Hz, CH2); 3.73 (t, 2H, J 6.3 Hz, CH2);
6.30 (s, 1H, Hisox); 7.22-7.35 (m, 2H, Harom); 7.54-7.61 (m, 2H, Har-
om). 13C NMR (CDCl3, 75 MHz) d: 21.3; 23.1; 30.2; 61.4; 99.2; 123.8;
127.3; 128.7; 129.0; 130.6; 138.5; 162.5; 173.4. I.R. (KBr pellet)
4.3.2. 3-[3-(4-Chlorophenyl)-isoxazol-5-yl]-propionic acid (7b)
Colorless crystals; yield: 50%; Rf: 0.30 (chloroform/methanol,
9:1 v/v); mp: 189–190 °C. 1H NMR (CDCl3, 300 MHz) d (ppm):
2.70 (t, 2H, J 7.2 Hz, CH2); 3.02 (t, 2H, J 7.2 Hz, CH2); 6.85 (s, 1H, Hi-
sox); 7.57 (dd, 2H, J 8.7 Hz, 1.8 Hz, Harom); 7.87 (dd, 2H, J 8.7 Hz,
1.8 Hz, Harom). 13C NMR (CDCl3, 75 MHz) d (ppm): 22.8; 32.1;
100.5; 128.6; 129.3; 130.2; 135.8; 161.9; 174.0; 174.4. I.R. (KBr
m
max/cmÀ1: 3285 (O–H); 2940, 2872 (C–H); 1604 (C@N); 1420
(N–O). ESI-HRMS m/z: 256.0904 (Calcd for C13H15NO2K [M+K]+:
pellet) m
max/cmÀ1: 3120 (C–H); 2935 (O–H); 1706 (C@O), 1602
256.0740).
(C@N). Anal. Calcd for C12H10ClNO3 (C,H,N): C, 57.27%; H, 4.01%;
N, 5.57%. Found: C, 57.08%; H, 4.51%; N, 5.46%.
4.2.9. 3-(3-o-Tolyl-isoxazol-5-yl)-propan-1-ol (5i)
Yellow oil; yield: 22%; Rf: 0.48 (hexanes/AcOEt, 1:1 v/v). 1H
NMR (CDCl3, 300 MHz) d: 1.98 (q, 2H, J 7.5 Hz, 6.3 Hz; CH2); 2.22
(s, 1H, OH); 2.43 (s, 3H, CH3Ph); 2.89 (t, 2H, J 7.5 Hz, CH2); 3.71
(t, 2H, J 6.3 Hz, CH2); 6.16 (s, 1H, Hisox), 7.20–7.30 (m, 3H, Harom);
7.2 (bd, 1H, J 7.2 Hz, Harom). 13C NMR (CDCl3, 75 MHz) d: 21.0;
23.1; 30.2; 61.4; 101.8; 125.9; 128.9; 129.3; 130.9; 136.7; 163.0;
4.3.3. 3-[3-(4-Fluorophenyl)-isoxazol-5-yl]-propionic acid (7c)
Colorless crystals; yield: 77%; Rf: 0.37 (chloroform/methanol,
9:1 v/v); mp: 165–168 °C. 1H NMR (CDCl3, 300 MHz) d (ppm):
2.70 (t, 2H, J 7.2 Hz, CH2); 3.01 (t, 2H, J 7.2 Hz, CH2); 3.34 (s, 1H,
OH); 6.84 (s, 1H, Hisox); 7.35 (dd, 2H, J 8.4 Hz, 2.4 Hz, Harom); 7.89
(dd, 2H, J 8.4 Hz, 2.4 Hz, Harom). 13C NMR (CDCl3, 75 MHz) d
(ppm): 22.5; 31.8; 100.1; 116.7; 117.0; 126.0; 129.4; 129.6;
172.5. I.R. (KBr pellet)
1597 (C@N); 1403 (N–O). ESI–HRMS m/z: 240.0927 (Calcd for
13H15NO2Na [M+Na]+: 240.1000).
m
max/cmÀ1: 3383 (O–H); 2951, 2876 (C–H);
161.6; 165.4; 173.8. I.R. (KBr pellet)
m
max/cmÀ1: 3115 (C–H);
C
2936 (O–H); 1696 (C@O); 1607 (C@N). ESI-HRMS m/z: 234.0501
(Calcd for C12H9FNO3 [MÀH]+: 234.0567).
4.2.10. 3-[3-(3,4-Dichlorophenyl)-isoxazol-5-yl)]-propan-1-ol
(5j)
4.3.4. 3-[3-(4-Bromophenyl)-isoxazol-5-yl]-propionic acid (7d)
Colorless crystals; yield: 45%; Rf: 0.17 (chloroform/methanol,
9:1 v/v); mp: 152–153 °C. 1H NMR (DMSO-d6, 400 MHz) d (ppm):
2.70 (t, 2H, J 7.2 Hz, CH2); 3.02 (t, 2H, J 7.2 Hz, CH2); 6.84 (s, 1H, Hi-
sox); 7.69 (dd, 2H, J 8.8 Hz, 2.4 Hz, Harom); 7.81 (dd, 2H, J 8.8 Hz,
2.4 Hz, Harom). 13C NMR (CDCl3, 100 MHz) d (ppm): 21.7; 31.1;
99.3; 123.4; 127.9; 128.5; 132.1; 160.9; 166.9; 173.3. I.R. (KBr pel-
Colorless oil; yield: 27%; Rf: 0.70 (hexanes/AcOEt, 1:1 v/v). 1H
NMR (CDCl3, 300 MHz) d (ppm): 1.86 (s, 1H, OH); 2.01 (q, 2H, J
7.5 Hz, 6.3 Hz, CH2); 2.93 (t, 2H, J 7.5 Hz, CH2); 3.74 (t, 2H, J
6.3 Hz, CH2); 6.30 (s,1H, Hisox); 7.51 (d, 1H, J 8.4 Hz, Harom); 7,61
(d, 1H, J 8.4 Hz, Harom); 7.86 (s, 1H, Harom). 13C NMR (CDCl3,
75 MHz) d (ppm): 23.2; 30.2; 61.4; 99.0; 125.8; 128.5; 129.1;
130.9; 133.1; 133.9; 160.5; 174.2. I.R. (KBr pellet)
m
max/cmÀ1
:
let) m
max/cmÀ1: 3119 (C–H); 2934 (O–H); 1706 (C@O); 1600 (C@N).
3382 (O–H); 2938 (C–H); 1604 (C@N); 1421 (N–O). Anal. Calcd
for C12H11Cl2NO2 (C,H,N): C, 52.96%; H, 4.07%; N, 5.15%. Found: C,
52.65%; H, 4.06%; N, 5.07%.
ESI-HRMS m/z: 293.9715 (Calcd for
293.9766).
C
12H9BrNO3 [MÀH]+:
4.3.5. 3-[3-(4-Methoxyphenyl)-isoxazol-5-yl]-propionic acid
(7e)
4.3. Synthesis of 3-(3-aryl-isoxazol-5-yl)-propionic acids (7a–h)
Colorless crystals; yield: 43%; Rf: 0.14 (chloroform/methanol,
9:1 v/v); mp: 159–161 °C. 1H NMR (DMSO, 400 MHz) d (ppm):
2.69 (t, 2H, J 7.2 Hz, CH2); 3.00 (t, 2H, J 6.8 Hz, CH2); 3.81 (s, 3H,
OCH3); 6.73 (s, 1H, Hisox); 7.04 (d, 2H, J 8.4 Hz, Harom); 7.76 (d,
2H, J 8.4 Hz, Harom). 13C NMR (DMSO, 100 MHz) d (ppm): 21.8;
31.1; 55.2; 56.3; 99.1; 113.2; 114.4; 121.1; 126.6; 127.7; 127.9;
The imidoyl chlorides were prepared as described for the syn-
thesis of 5a–j. Sodium 4-pentynoate (6, 0.58 g, 4.8 mmol) and the
appropriated imidoyl chloride (3a–h, 4.0 mmol) were suspended
in 1:1 mixture of tert-butanol and water (10 mL). To this solution
was added a mixture of Cu(OAc)2 (40 mg, 0.2 mmol) and sodium
ascorbate (120 mg, 0.6 mmol). Finally, an aqueous solution of
potassium hydrogen carbonate (1.8 g, 18.0 mmol) was added drop-
wise to the reaction mixture. The contents were allowed to stir at
room temperature until TLC analysis indicated complete consump-
tion of the imidoyl chloride. To the reaction contents were added
10 mL of an aqueous potassium bicarbonate solution and 10 mL
of AcOEt and this resulting mixture was allowed to stir overnight.
The phases were separated and the aqueous phase was neutralized
with saturated citric acid solution. This solution was then extracted
with AcOEt (2 Â 10 mL). Drying the combined organic layers over
anhydrous Na2SO4, filtration and solvent evaporation under
reduced pressure left the product as a solid which could be crystal-
lized from cold ethanol in all cases.
160.6; 161.4; 172.6; 173.0. I.R. (KBr pellet) m
max/cmÀ1: 2839 (C–
H); 1696 (C@O); 1612 (C@N); 1435 (N–O). ESI–HRMS m/z:
248.0860 (Calcd for C13H14NO4 [M+H]+: 248.0923).
4.3.6. 3-[3-(4-Nitrophenyl)-isoxazol-5-yl]-propionic acid (7f)
Yellow oil; yield: 46%; Rf: 0.38 (chloroform/methanol, 9:1 v/v);
1H NMR (CDCl3, 300 MHz) d (ppm): 2.97 (t, 2H, J 7.2 Hz, CH2); 3.76
(t, 2H, J 6.3 Hz, CH2); 6.41 (s, 1H, Hisox); 7.96 (dd, 2H, J 8.7 Hz,
1.8 Hz, Harom); 8.30 (dd, H, J 8.7 Hz, 1.8 Hz, Harom). 13C NMR (CDCl3,
75 MHz) d (ppm): 23.2; 30.2; 61.4; 99.3; 124.1; 127.5; 135.3;
148.5; 160.6; 174.6. I.R. (KBr pellet)
m
max/cmÀ1: 3133 (C–H);
2874 (O–H); 1706 (C@O), 1602 (C@N). ESI-HRMS m/z: 263.0668
(Calcd for C12H11N2O5 [M+H]+: 263.0690.
4.3.1. 3-(3-Phenyl-isoxazol-5-yl)-propionic acid (7a)
4.3.7. 3-(3-p-Tolyl-isoxazol-5-yl)-propionic acid (7g)
Colorless crystals; yield: 63%; Rf: 0.32 (chloroform/methanol,
9:1 v/v); mp: 148–150 °C. 1H NMR (CDCl3, 300 MHz) d (ppm):
2.85 (t, 2H, J 7.5 Hz, CH2); 3.16 (t, 2H, J 7.5 Hz, CH2); 6.37 (s, 1H, Hi-
sox); 7.45 (m, 3H, Harom); 7.78 (m, 2H, Harom). 13C NMR (CDCl3,
75 MHz) d (ppm): 21.8; 31,1; 99.3; 126.5; 128.7; 129.0; 130.0;
Colorless crystals; yield: 48%; Rf: 0.36 (chloroform/methanol,
9:1 v/v); mp: 168–169 °C. 1H NMR (CDCl3, 300 MHz) d (ppm):
2.39 (s, 3H, CH3); 2.84 (t, 2H, J 7.3 Hz, CH2); 3.14 (t, 2H, J 7.3 Hz,
CH2); 6.33 (s, 1H, Hisox); 7.25 (d, 2H, J 8.4 Hz, Harom); 7.67 (d, 2H,
J 8.4 Hz, Harom). 13C NMR (CDCl3, 75 MHz) d (ppm): 21.4; 21.9;
Please cite this article in press as: da Silva-Alves, D. C. B.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2012.12.006

D. C. B. da Silva-Alves et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
31.3; 45.7; 99.5; 126.1; 126.6; 129.6; 162.4; 176.0. I.R. (KBr pellet)
out for every sample concentration. A negative control for each as-
say, using distilled water containing the same amount of co-sol-
vent as the test sample has been used. Mortality of the larvae
was determined after 48 h of incubation at 28 2 °C. Larvae were
considered dead when no response to stimuli or not rising to the
solution surface was observed. Lethal concentration (LC50) values
were calculated through probit analysis using the Status Plus
2006 software program.36
m
max/cmÀ1: 3122 (C–H); 2944 (O–H); 1696 (C@O); 1602 (C@N).
ESI-HRMS m/z: 230.0768 (Calcd for C13H12NO3 [MÀH]+: 230.0817.
4.3.8. 3-(3-m-Tolyl-isoxazol-5-yl)-propionic acid (7h)
Colorless crystals; yield: 52%; Rf: 0.19 (chloroform: methanol,
9:1 v/v); mp: 148–150 °C. 1H NMR (DMSO, 400 MHz) d (ppm):
2.71 (t, 2H, J 7.2 Hz, CH2); 3.02 (t, 2H, J 7.2 Hz, CH2); 3.33 (s, 3H,
CH3); 6.81 (s, 1H, Hisox); 7.50 (d, H, J 5.2 Hz, Harom); 7.83 (dd, H, J
5.2 Hz, 2.4 Hz, Harom). 13C NMR (DMSO, 100 MHz) d (ppm): 21.8;
31.1; 99.3; 126.4; 128.7; 129.0; 130.0; 161.8; 172.9; 173.0. I.R.
Acknowledgments
(KBr pellet)
m
max/cmÀ1: 3125 (C–H); 2907 (O–H); 1703 (C@O),
Our thanks are due to the Brazilian National Research Council
(CNPq) and Pernambuco State’s Scientific and Research Foundation
(FACEPE, PRONEX-0788-1.06/08 CNPq, PRONEX/FACEPE/2008-
0981-1.06/08, PPP/FACEPE/APQ-1225-1.06/10, PPSUS/FACEPE,
INCT Dengue) for providing financial assistance. D.C.B.S.-A. is
grateful to FACEPE for a research fellowship. N.N.M.C. is thankful
to CNPq for an undergraduate fellowship. One of us (R.M.S.) is in-
debted to J.V.d.A., the co-advisor of D.C.B.S.-A. for helping and
supervising her during his absence.
1607 (C@N). ESI–HRMS m/z: 230.0768 (Calcd for C13H12NO3
[MÀH]+: 230.0801.
4.4. Synthesis of methyl esters of 3-[3-(aryl)-isoxazol-5-yl]-
propionic acids (9a–b)
To an appropriate solution of the 3-[(3-(aryl)-isoxazol-5-yl]-
propionic acid (5 mmol) in 40 mL of methanol, five drops of
concentrated sulfuric acid were added followed by reflux under
stirring until TLC analysis indicated the total consumption of the
starting acid. Dilution of the contents with water (20 mL) followed
by solvent removal under reduced pressure gave the crude prod-
uct. Extraction of the residue with AcOEt (2 Â 20 mL) and drying
solvent over anhydrous Na2SO4, pursued by solvent removal in va-
cuo furnished the crude product. Crystallization from methanol/
water provided the desired methyl 3-[3-(aryl)-isoxazol-5-yl]-
propionate.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.12.006.
References and notes
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4.4.2. Methyl 3-[3-(4-chlorophenyl)isoxazol-5-yl] propionate
(9b)
Colorless crystals; yield: 80%; Rf: 0.94 (chloroform/ethyl ace-
tate, 7:3 v/v); mp: 103–104 °C. 1H NMR (CD3OD, 400 MHz) d
(ppm): 2.77 (t, 2H, J 7.2 Hz, CH2); 3.12 (t, 2H, J 7.2 Hz, CH2); 3.66
(s, 3H, CH3); 6.58 (s, 1H, Hisox), 7.45 (dd, 3H, J 8.4 Hz, 2.0 Hz, Harom);
7.76 (dd, 2H, J 8.4 Hz, 2.0 Hz, Harom). 13C NMR (CD3OD, 100 MHz) d:
23.0; 32.3; 52.3; 100.4; 129.1; 129.3; 130.2; 137.1; 162.9; 173.9;
174.4. I.R. (KBr pellet) m
max/cmÀ1: 3117 (C–H); 1732 (C@O); 1605
(C@N); 1439 (N–O). Anal. Calcd for C13H12ClNO3 (C,H,N): C,
58.77%; H, 4.55%; N, 5.27%. Found: C, 59.09%; H, 4.65%; N, 5.16%.
4.5. Larvicidal bioassay
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dimethylsulfoxide or acetone (analytical grade) or 2 drops of
Tween 80, and completing to a volume of 50 mL with distilled
water. In order to test the effect of the compounds on the larvae
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vae per beaker) containing the synthesized compounds in a range
of concentrations obtained by appropriate dilution of the stock
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Please cite this article in press as: da Silva-Alves, D. C. B.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2012.12.006