The enzyme 3-hydroxykynurenine transaminase as potential target for 1,2,4-oxadiazoles with larvicide activity against the dengue vector Aedes aegypti

Article abstract of DOI:10.1016/j.bmc.2013.09.020

The mosquito Aedes aegypti is the vector agent responsible for the transmission of yellow fever and dengue fever viruses to over 80 million people in tropical and subtropical regions of the world. Exhaustive efforts have lead to a vaccine candidate with only 30% effectiveness against the dengue virus and failure to protect patients against the serotype 2. Hence, vector control remains the most viable route to dengue fever control programs. We have synthesized a class of 1,2,4-oxadiazole derivatives whose most biologically active compounds exhibit potent activity against Aedes aegypti larvae (ca. of 15 ppm) and low toxicity in mammals. Exposure to these larvicides results in larvae pigmentation in a manner correlated with the LC₅₀ measurements. Structural comparisons of the 1,2,4-oxadiazole nucleus against known inhibitors of insect enzymes allowed the identification of 3-hydroxykynurenine transaminase as a potential target for these synthetic larvicides. Molecular docking calculations indicate that 1,2,4-oxadiazole compounds can bind to 3-hydroxykynurenine transaminase with similar conformation and binding energies as its crystallographic inhibitor 4-(2-aminophenyl)-4- oxobutanoic acid.

Full text of DOI:10.1016/j.bmc.2013.09.020

Accepted Manuscript
The enzyme 3-hydroxykynurenine transaminase as potential target for 1,2,4-
oxadiazoles with larvicide activity against the Dengue vector Aedes aegypti
Vanessa S. Oliveira, Cecília Pimenteira, Diana C.B. da Silva-Alves, Laylla L.L.
Leal, Ricardo A.W. Neves-Filho, Daniela M.A.F. Navarro, Geanne K.N. Santos,
Kamilla A. Dutra, Janaína V. dos Anjos, Thereza A. Soares
PII:
S0968-0896(13)00800-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.09.020
BMC 11104
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Please cite this article as: Oliveira, V.S., Pimenteira, C., da Silva-Alves, D.C.B., Leal, L.L.L., Neves-Filho, R.A.W.,
Navarro, D.M.A., Santos, G.K.N., Dutra, K.A., dos Anjos, J.V., Soares, T.A., The enzyme 3-hydroxykynurenine
transaminase as potential target for 1,2,4-oxadiazoles with larvicide activity against the Dengue vector Aedes
aegypti, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.09.020
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The enzyme 3-hydroxykynurenine
transaminase as potential target for 1,2,4-
oxadiazoles with larvicide activity against the
Dengue vector Aedes aegypti.
Leave this area blank for abstract info.
Vanessa S. Oliveira, Cecília Pimenteira, Diana C. B. da Silva-Alves, Laylla L. L. Leal, Ricardo A. W. Neves-
Filho, Daniela M. A. F. Navarro, Geanne K. N. Santos, Kamilla A. Dutra, Janaína V. dos Anjos, Thereza A.
Soares. Department of Fundamental Chemistry, Federal University of Pernambuco, 50740-560, Brazil

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
The enzyme 3-hydroxykynurenine transaminase as potential target for 1,2,4-
oxadiazoles with larvicide activity against the Dengue vector Aedes aegypti.
Vanessa S. Oliveira, Cecília Pimenteira, Diana C. B. da Silva-Alves, Laylla L. L. Leal, Ricardo A. W.
Neves-Filho, Daniela M. A. F. Navarro, Geanne K. N. Santos, Kamilla A. Dutra, Janaína V. dos Anjos*,
Thereza A. Soares
Department of Fundamental Chemistry, Federal University of Pernambuco, Recife 50740-560, Brazil
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The mosquito Aedes aegyptii is the vector agent responsible for the transmission of yellow fever
and dengue fever viruses to over eighty million people in tropical and subtropical regions of the
world. Exhaustive efforts have lead to a vaccine candidate with only 30% effectiveness against
the dengue virus and failure to protect patients against the serotype 2. Hence, vector control
remains the most viable route to dengue fever control programs. We have synthesized a class of
1,2,4-oxadiazole derivatives whose most biologically active compounds exhibit potent activity
against Aedes aegyptii larvae (ca. of 15 ppm) and low toxicity in mammals. Exposure to these
larvicides results in larvae pigmentation in a manner correlated with the LC50 measurements.
Structural comparisons of the 1,2,4-oxadiazole nucleus against known inhibitors of insect
enzymes allowed the identification of 3-hydroxykynurenine transaminase as a potential target
for these synthetic larvicides. Molecular docking calculations indicate that 1,2,4-oxadiazole
compounds can bind to 3-hydroxykynurenine transaminase with similar conformation and
binding energies as its crystallographic inhibitor 4-(2-aminophenyl)-4-oxobutanoic acid. 2009
Elsevier Ltd. All rights reserved.
Available online
Keywords:
Keyword_1 Larvae pigmentation
Keyword_2 Insecticide
Keyword_3 Molecular docking calculations
Keyword_4 Malaria vector Anopheles gambiae
Keyword_5 4-(2-aminophenyl)-4-oxobutanoic acid
1. Introduction
there are several reports of vertical transmission of the dengue
vírus in Aedes spp collected in the field.5-8 This latter finding
Aedes aegypti is the vector agent that spreads the yellow fever
implies that adult mosquitoes infected through transovarial
transmission do not have to feed in a viremic vertebrate host to
infect a naïve host, thus explaining the persistence of the virus in
nature in the absence of viremic vertebrate hosts.9 Therefore,
vector control will remain a crucial issue for dengue fever control
programs.
and the dengue fever viruses, and are endemic in tropical and
subtropical regions around the world.1, Yellow fever affects
2
annually 0.2 million people whereas about 80 million people are
infected every year by one of the four dengue virus types. The
disease is a serious worldwide public health treat due to the
presence of the mosquito in more than 100 countries. Yellow
fever vaccination is the most important and effective means to
prevent the occurrence of this disease, and carries a low risk of
serious adverse events. The live-attenuated 17D vaccine provides
protective immunity within one to two weeks in 95% of those
vaccinated.3 However, it has been recently shown that the leading
vaccine candidate was only 30% effective against dengue fever in
a first large clinical trial2. In addition, the vaccine has failed to
protect individuals against serotype 2. Currently, the sole
effective strategy to control the disease is the vector eradication
due to two main factors.4 First, the sickness incidence is closely
One of the major routes of detoxification (removal of reactive
oxygen and nitrogen species) in Aedes aegypti mosquitoes is the
kynurenine pathway. This is the chief catabolic pathway in
mosquitoes and the main route of tryptophan metabolism in
11
living organisms.10,
In this biochemical pathway, the most
important reaction is the conversion of the harmful metabolite 3-
hydroxy-kynurenine (3-HK) into xanthurenic acid (XA), a
chemically stable, non-toxic substance by the enzyme 3-hydroxy-
kynurenine transaminase (HKT).12-14 Previous studies have
shown that the 3-HK accumulation in adult insects leads to the
formation of reactive oxygen species with serious neuronal
related to domiciliary infestation by Aedes mosquitoes. Second,
———
 Corresponding authors. Tel.: +55-81-2126-8440; fax: +55-81-2126-8442; e-mail: thereza.soares@ufpe.br, janaina.anjos@ufpe.br

damage and insect death.14, 15 For this reason, the high conversion
efficiency of 3-HK to XA by HKT in mosquito larvae is
considered to be a mechanism by which mosquitoes detoxify the
chemically reactive 3-HK.16 Earlier studies have also shown that
3-HK content was quite different in insects at different
developmental stages.12, 17 3-HK is also the initial precursor for
the production of ommochromes, major eye pigments in
mosquitoes. Compound eye development and eye pigmentation
occur mainly during the pupal and early adult stages.18-21 For this
reason, the 3-HK to XA pathway must be down regulated in
pupae to allow for the accumulation and transport of some 3-HK
to the compound eyes for pigmentation.13, 19
2. Results and Discussion
2.1. Synthesis and Larvicidal Activity of Heterocyclic
Compounds
We have previously synthesized and established the larvicidal
activity of 3-(3-aryl-1,2,4-oxadiazol-5-yl)propionic acids against
A. aegypti.25,26 These compounds promote alterations in the
larvae spiracular valves of the siphon and anal papillae, leading
to the death of the insect. In this study, we have used 3-(3-aryl-
1,2,4-oxadiazol-5-yl)propionic acids as prototypes for chemical
modifications aiming at improved larvicide candidates (Table 1).
These modifications are depicted in Fig. 1B.
The use of competitive antagonists for HKT can prevent the
conversion of the toxic 3-HK to xanthurenic acid in mosquito
larvae and adults, causing the accumulation of neurotoxic 3-HK
(Fig. 1A).22-24 As part of a drug discovery effort aiming at the
identification of new larvicide candidates, we have found a class
Modifications on the aryl group located at the C-3 position of
1,2,4-oxadiazole resulted in nine bioactive compounds, with the
halogen-containing compounds 2f (chlorine) and 2g (bromine)
exhibiting good larvicidal activities (below 30.0 ppm). On the
other hand, the fluoro-compound 2e had only weak activity (81.2
ppm). These findings indicate that electronic effects are as
important as steric effects, since there is a significant difference
in the larvicidal profile of the three compounds (Table 1).25 We
have also constructed a homologous series of the prototype 3-(3-
phenyl-1,2,4oxadiazol-5-yl)-propionic acid 2a. Hence we have
reduced and elongated, respectively, the aliphatic alkyl chain
located at C-5 of 1,2,4-oxadiazole nucleus for compounds 1a-d
and 5a-g. The 3-(3-aryl-1,2,4-oxadiazol-5-yl)-etanoic acids (1a-
d) were synthesized according to known literature procedures.27
The larvicidal activity for this series follows an analogous pattern
to 2a-i with the best results obtained for halogen-containing
compounds 1c-d. The reduction of the aliphatic alkyl chain
length led to less potent larvicides (LC50 above 45 ppm). In
contrast to these findings, the elongation of the alkyl chain,
which yielded the 3-(3-aryl-1,2,4-oxadiazol-5-yl)-butanoic acids
(compounds 5a-g) (Scheme 1) provides the best larvicidal
candidates (Table 1). Once again, the halogen-containing 1,2,4-
oxadiazoles presented the best biological activities, with
compound 5d (bromo-compound) having a LC50 value of 10.6
ppm.
of heterocycle derivatives with larvicidal activity against A.
26
aegypti (Fig. 1B).25,
Due to the potent activity against the
mosquito larvae (ca. of 15 ppm) and low toxicity in mammals,
these compounds are the first prototypes of synthetic larvicides
containing the 1,2,4-oxadiazole nucleus (Fig. 1B). Despite the
potential as anti-dengue larvicide, the biological mechanism of
action of this class of compounds has not been elucidated. The
identification of a molecular target in A. aegypti for 1,2,4-
oxadiazole is a pivotal step towards the design and synthesis of
novel or modified compounds with improved potency or
selectivity. In this work, we assess the prospect of having HKT
as a potential inhibitory target for a total of 27 chemical variants
of the original scaffold (Table 1). We perform molecular docking
calculations to ascertain whether these compounds can bind to
the HKT active site in the same conformation observed for the
inhibitor 4-(2-aminophenyl)-4-oxobutanoic acid (4-OB) by the
means of high-resolution X-ray crystalography.23 In addition, we
have also synthesized six novel oxadiazole compounds and
evaluated their larvicide activity against the A. aegypti as
function of systematic substitution/replacement of chemical
functions in specific regions of the 1,2,4-oxadiazole scaffold. Our
findings are presented thereafter.
A
Scheme 1. Synthesis of 3-(3-aryl-1,2,4-oxadiazol-5-yl)-butanoic acids (5a-g).
We have also changed the central nucleus to an isoxazole
moiety. As mentioned earlier, the difference between these two
five-membered heterocyclic compounds lies in the replacement
of N-4 atom of oxadiazole with a CH group in isoxazole.
B
Compounds 7-9 were synthesized in previous work.28
A
comparison can be made when analysing the LC50 values of
compounds 2a and 8 and 9 and 10 (Table 1), where the sole
difference is the heterocyclic ring. In both cases, the isoxazole-
containing compounds are more potent than the 1,2,4-oxadiazole
ones. These results show that the replacement of the 1,2,4-
oxadiazole for a isoxazole nucleus results in better larvicidal
compounds.
Figure 1. Top panel: antagonists for the HKT enzyme: (I) 4-(2-
aminophenyl)-4-oxobutanoic acid, (II) (+)-(1S,2S)-2-(3,4-dichlorobenzoyl)-
cyclopropyl-1-carboxilic acid, and (III) amino-oxyacetic acid, (IV) 1,2,4-
oxadiazole scaffold. Bottom panel: Chemical modifications performed on the
oxadiazole scaffold.
.

Table 1. 1,2,4-oxadiazole derivatives. Chemical structures, larvicidal activities against the mosquito Aedes aegypti, and binding
affinities against the enzyme 3-hydroxy-kynurenine transaminase (HKT).
aKi (µM)
Compound
Chemical structure
LC50 (ppm)
Lowest energy conformer
O
OH
4-OB
-
Reference
0
O
NH2
O
N O
N
OH
1a
1b
>100
92.3
Multiple
Multiple
36.21
10.14
18.99
O
N O
N
OH
O
O
N O
OH
1c
49.3
Multiple
N
Cl
N O
OH
OH
1d
2a
2b
67.9
98.6
70.9
Multiple
Multiple
Multiple
13.01
12.98
19.01
N
Br
N O
N
O
N O
N
OH
O
N O
N
OH
2c
2d
2e
2f
63.8
65.8
81.2
28.1
Multiple
Multiple
Multiple
Multiple
6.89
7.76
18.75
8.26
9.48
O
N O
N
OH
OH
O
N O
N
O
F
N O
OH
OH
N
O
Cl
N O
2g
2h
2i
15.2
71.5
50.5
Multiple
Multiple
Multiple
N
O
Br
N O
OH
30.83
6.94
N
O
O
N O
OH
N
O
O2N
O
N O
N
OH
5a
5b
5c
92.7
75.9
34.9
Inhibitor-like
Inhibitor-like
Inhibitor-like
-1.24
-2.5
O
N O
N
OH
O
N O
OH
-2.34
N
Cl

O
N O
N
OH
OH
OH
OH
5d
5e
5f
10.6
16.2
>100
72.9
Inhibitor-like
Inhibitor-like
Inhibitor-like*
Inhibitor-like*
-2.74
-3.17
-1.37
-2.53
-1.45
Br
O
N O
N
I
O
N O
N
O
O
N O
N
O
5g
O
O
N O
O
6a
59.9
Inhibitor-like
N
O
N O
Cl
Cl
O
6b
7
6.8
Inhibitor-like
-3.2
N
N O
OH
OH
>100
Inhibitor-like**
1.72
N O
N O
8
82.8
13.2
30.6
>100
Multiple
14.65
5.47
-2.9
O
O
9
Multiple
O
N O
N
O
10
11
Inhibitor-like**
Inhibitor-like**
O
N O
N
2.08
O
aKi = Ki(oxadiazoles) - Ki(4-OB) where Ki is estimated from the molecular docking calculations. The more negative Ki, the stronger the oxadiazole compound
binds to HKT compared to the crystallographic inhibitor 4-OB.
*The inhibitor-like conformation is among the lowest energy conformers, but it is not the lowest energy one.
**Compound binds to HKT in inhibitor-like conformation, but the carbonyl group or hydroxyl interacts preferentially with the side-chain of Gln204 instead of
Arg356.
Derivatizations on the carboxylate moiety were made. Esters,
an alcohol and a ketone were synthesized to evaluate the the
effect of these scaffold modifications on larvicide activity. The
transformation of carboxylate moiety into an ester moiety is the
most beneficial one. Esters 9 and 10 had better larvicidal
activities than their parent acids (2a and 8, respectively).
unavailable in the Protein Database. The HKT sequence of A.
aegypti (AeHKT) and A. gambiae (AgHKT) shares an identity of
73%.29 Previous studies have also shown that the amino acid
sequence in the active site of both enzymes is nearly identical
with the triad Ser43-Asn44-Phe45 involved in the substrate
recognition process in the two species. Given the high identity
between AeHKT and AgHKT primary sequences, we have built a
homology-based structural model for AeHKT using the X-ray
structure of AeHKT (PDB ID 2CH2) as template. Superposition
of the model and template structures showed that all active-site
residues within 7 Å cutoff from the atomic coordinates of the 4-
OB inhibitor present in the X-ray structure 2CH2 are preserved in
the sequence of AeHKT. These residues define the first and
second spheres of interaction between inhibitors and the enzyme,
and therefore AeHKT and AgHKT can be reliably represented by
the same active site conformation, i.e. by the AgHKT high-
resolution X-ray structure (PDB ID 2CH2). The identical active
2.2. Binding of Heterocyclic Derivatives to 3-Hydroxykynurenine
Transaminase
The HKT sequence has been isolated from several
invertebrate species including Aedes aegypti, Neobelleria bullata,
Locusta migratoria and Anopheles gambiae.12, 16 High-resolution
X-ray structures are available for HKT from A. gambiae at 2.7 Å
resolution (PDB IDs 2CH1 and 2CH2).23 Experimentally
determined structures of HKT from other species are currently

site residues in the two enzymes are Met21, Gly23, Pro24,
Gly25, Pro26, His79, Leu42, Ser43, Asn44, Phe45, His46, His79,
Ile103, Trp104, Ser154, Ser155, Tyr256, His257, His258,
His259, Glu342, Val343, Gln344, Gly345, Gly346, Leu347.
In order to assess the ability of our docking procedure to
correctly predict the binding modes of heterocyclic derivatives at
the AgHKT crystal structure, a validation procedure was devised
to demonstrate that the undertaken procedure is able to reproduce
the bound conformation of inhibitor 4-OB in the crystallographic
structure of the complex. This procedure is described in the
computational simulations section. The 4-OB conformations
resulting from these simulations were analyzed and compared to
its crystallographic conformation bound to the active site of
HKT. In all tests, the conformation of the co-crystallized
inhibitor in the active site of AgHKT X-ray structure was
successfully reproduced with a root-mean-square deviation
(RMSD) of less that 0.09 nm (Fig. 3).
Figure 4. Comparative binding of the co-crystallized inhibitor 4-OB (A) and
inhibitor-like conformers of heterocyclic compounds (B-D) to AgHKT.
Conformers are representative of the most populated, lowest-energy
conformational clusters. Canonical inhibitor-like conformers (B and D), and a
variation (C) where Arg356 interacts with the heterocycle atoms.
The 1,2,4-oxadiazole compounds containing a butanoic acid
moiety in series 5a-g bind to AgHKT via a single, predominant
conformation much alike the inhibitor 4-OB (Fig. 4B). The Ki
values obtained for the lowest energy conformer via molecular
docking calculations are in the low micromolar range, similar to
the calculated Ki for 4-OB (Table 1). Indeed, all the compounds
of this class exhibited calculated Ki values smaller (Ki negative)
than that of 4-OB. The replacement of the carboxylate group by
an ester in compounds 6a-b has not altered the interaction mode
of the inhibitor with AgHKT. A decrease in the number of carbon
atoms in the carboxylate linker of compounds in the series 1a-d
and 2a-i leads to an increase of the respective Ki values in about
one order of magnitude (Table 1). This effect is associated with
the occurrence of multiple conformations instead of a single one
(Fig. 4C). The chemical substitutions in the aromatic ring do not
appear to affect significantly the calculated affinity of the 1,2,4-
oxadiazole derivatives for AgHKT. Upon binding to AgHKT, the
aromatic ring is loosely positioned in the entry of active site (Fig.
4).
Figure 3. Cartoon representation of the X-ray structure of AgHKT with the
co-crystallized inhibitor 4-OB in cpk. The experimental and calculated
conformations are show in white and green, respectively. Residues involved
in the substrate recognition are also shown in white.
A critical comparison of atomic interactions in different
docked complexes against those observed in the 4-OB-AgHKT
crystallographic structure was undertaken. A twofold criteria has
been used to select potentially effective inhibitors out of the pool
of the synthesized compounds. First, potential inhibitors should
interact with AgHKT active site analagously to 4-OB. Second,
the estimated Ki for these inhibitors should be smaller or
equivalent to the value estimated for 4-OB using the same
molecular docking procedure (Table 1). The competitive
inhibitor 4-OB binds to AgHKT with Ki = 300 M, and cannot
be processed by the enzyme due to the absence of a reactive
amino group at the C2 position equivalent to the Cꢀatom of the
natural amino acid substrates.23 Likewise, reactive amino groups
are absent in the heterocyclic compounds.
2.3. Larvae Pigmentation upon Exposure to Oxadiazole-based
Larvicides
Upon exposition of fourth-instar A. aegypti larvae to a
solution of compound 2g at 100 ppm significant alterations in the
pigmentation pattern were observed (Fig. 5A and 5B). Likewise,
a similar pigmentation pattern is seen when larvae were treated
with solutions of isoxazoles 8 and 9 at 100 ppm (Fig. 5D and
5E). Such pigmentation alterations in A. aegypti pattern correlate
with the respective LC50 measurements, i.e. larvae treated with
the most potent larvicides (LC50 below 20 ppm, 2g and 9)
exhibited the most intense pigmentation.

which exhibits a decrease from parent to derived compounds.
However, compounds 5a-g exhibit average LC50 values which
are higher than their cognate esters while binding to HKT with
comparable Ki constants (Table 1). This effect may be due to the
enhanced lipophilicity of esters, which facilitates the penetration
of this class of compounds into the larvae system.30 Likewise, the
addition of halogens (Fluorine, Chlorine, Bromine and Iodine) to
the aromatic ring increases the larvicidal activity of the 1,2,4-
oxadiazole scaffold without having much effect on the
corresponding binding affinities (Table 1). In fact, chemical
modifications of the aromatic ring are not expected to impact
significantly the binding of 1,2,4-oxadiazole derivatives to HKT
because of its location near the entry of the active site (Fig. 4).
Our report suggests that the heterocyclic compounds presented
herein have a potent larvicide activity against the mosquito A.
aegypti. Computational calculations suggest that this heterocyclic
scaffold can bind to the enzyme HKT of A. gambiae and A.
aegypti in an analogous conformation to the co-crystallized
inhibitor 4-OB, and with comparable binding affinities (Fig. 4,
Table 1). HKT has been previously identified as a new target for
malaria treatment due to its role in the conversion of the toxic 3-
HK into XA.22-24 The high sequence identity between HKT from
A. gambiae and A. aegypti implies that the enzyme is also a
potential new target for dengue fever. Our findings suggest that
HKT is a plausible molecular target for inhibition by this type of
heterocyclic ring scaffold. Kinetics assays for recombinant HKT
and 1,2,4-oxadiazole derivatives are currently under way in order
to substantiate these results.
Figure 5. Pigmentation pattern. Optical microscopy images of four-instar
larvae of A. aegypti. Images made with Olympus SZ 51 stereo microscopes.
Top panel: (A) Control and upon 24 h exposure to 100 ppm solution of
compound 2g (B). Bottom panel: (C) Control and upon 24 h exposure to 100
ppm solution of compounds 8 (D) and 9 (E).
3-HK is the initial precursor for the production of the
ommochromes responsible for the development of pigmented
wings and eyes.13, 19 During the pupal and early adult stages, the
3-HK to XA pathway must be down-regulated in pupae to allow
for the accumulation and transport of some 3-HK to the
compound eyes for pigmentation.13, 19 We hypothesize that larvae
pigmentation upon exposure to oxadiazole derivatives may be
due to the accumulation and conversion of 3-HK into pigments.
In this context, the transaminase HKT is a plausible molecular
target for the presented heterocyclic larvicides based on two
considerations. First, there is a noticeable chemical similarity
between the heterocyclic nucleus and the 4-(2-aminophenyl)-4-
oxobutanoic acid (4-OB) antagonist of HKT (Fig. 1A and 1B).
Second, our biological activity assays show that upon contact
with the synthesized larvicides, the dead larvae exhibit dark
pigmentation (Fig. 5). These findings coupled to our molecular
docking calculations suggest that 1,2,4-oxadiazole derivatives
can rationally designed as inhibitors of HKT, an essential enzyme
of kynurenine pathway, the chief catabolic pathway in
mosquitoes and the main route of tryptophan metabolism in
4. Experimental
4.1. Synthesis and Characterization
All commercially available reagents were used without any
further purification and the reactions were monitored by TLC
analysis with TLC plates containing GF254 (E. Merck). Melting
points were determined on Electro-thermal (Mel-temp) apparatus
and are uncorrected. Column chromatography was performed on
Silica Gel 60 (70-230 mesh, E. Merck). NMR spectra were
recorded with a Varian UNMRS 400 MHz spectrometer and
1
referenced as follows: H (400 MHz), internal SiMe4
13
ppm,
Elemental analysis was performed with a Carlo Erba instrument
model E-1110. Compounds 1a-d were synthesized and their
physical-chemical data were compared to earlier literature.27
Compounds 2a-i were synthesized by our group and published
elsewhere.25 Arylamidoximes 3a-g were prepared according to
known literature procedures.31 Substances 5a,32 5c,28 5d,33 7-9,30
10,34 and 1135 were synthesized and had their physical-chemical
data compared to previous literature.
living organisms.10, 11
.
4.1.1. Synthesis of 3-(3-aryl-1,2,4-oxadiazol-5-yl)-
butanoic acids (5a-g)
3. Conclusions
A suitable arylamidoxime 3a-g (1.0 mmol) was allowed to
react with glutaric anhydride 4 (1.2 mmol) in a round bottom
flask, with no solvent, at 130°C (oil bath) for 3-4 hours. When
TLC analysis indicated the complete consumption of the starting
amidoxime, the reaction was cooled to room temperature, The
reaction mixture was then diluted with a concentrated solution of
NaHCO3 (10 mL) and AcOEt (10 mL) and this resulting
suspension was allowed to stir overnight. The aqueous layer was
separated and then a concentrated solution of citric acid was
added until total precipitation of the desired compounds. The
obtained product was dissolved in chloroform, dried over
A direct comparison between measured lethal concentration
(LC50) and calculated Ki is not possible. LC50 values express
pharmacodynamical properties, and are not an accurate indicator
of binding affinity as is the case for Ki. However, analysis of
converging trends between the two quantities can be useful in the
deconvolution of various quantities contributing to the LC50.
Accordingly, measured LC50 values indicate that the
transformation of the carboxylate moiety into an ester moiety is
advantageous. Esters 9 and 10 display improved larvicidal
activities compared to their parent acids (2a and 8, respectively)
(Table 1). This pattern is also observed for the calculated Ki

anhydrous Na2SO4 and re-crystallized from chloroform-hexane to
afford the pure products (5a-g).
Methyl
4-[3-(3,4-dichlorophenyl)-1,2,4-oxadiazol-5-
yl]butanoate (6b): Yield: 62%, m.p. (oC): - (oil). 1H NMR (400
MHz, CDCl3) δ (ppm): 2.22 (q, J = 7.6 Hz, 6.8 Hz, 2H); 2.50 (t, J
= 6.8 Hz, 2H); 3.03 (t, J = 7.6 Hz, 2H); 3.69 (s, 3H); 7.55 (d, J =
8.8 Hz, 1H); 7.90 (dd, J = 8.8 Hz, 1.6 Hz, 1H); 8.17 (d, J = 1.6
Hz, 1H).13C NMR (100 MHz, CDCl3) δ(ppm): 21.6, 25.7, 32.7,
51.8, 126.4, 126.7, 129.3, 130.9, 133.3, 135.4, 166.7, 172.8;
179.5. Elemental Analysis for C13H12Cl2N2O3: Calc. C, 49.54; H,
3.84; N, 8.89. Found C, 49.92; H, 4.04 ; N, 8.64.
4-(3-m-Tolyl-1,2,4-oxadiazol-5-yl)butanoic
acid
(5b):
Yield:70%, m.p. (oC):107-108. H NMR (400 MHz, CDCl3) δ
(ppm): 2.21 (q, J = 7.5 Hz, 2H); 2.41 (s, 3H); 2.55 (t, J = 7.5 Hz,
2H); 3.04 (t, J = 7.5 Hz, 2H); 7.28 (d, J = 7.5 Hz, 2H); 7.95 (d, J
= 7.5 Hz, 2H) 9.96 (bs, 1H). 13C NMR (100 MHz, CDCl3) δ
(ppm): 21.28; 21.47, 25.55, 32.67, 123.70, 127.27, 129.50,
141.48, 178.46, 178.75. Elemental Analysis for C13H14N2O3:
Calc. C, 63.40; H, 5.73; N, 11.38. Found C, 63.45; H, 5.48; N,
11.55.
1
4.2. Larvicide Activity Measurements
4-[3-(4-Iodophenyl)-1,2,4-oxadiazol-5-yl]butanoic acid (5e):
The larvicidal activity of the synthesized drugs was evaluated
using an adaptation of the method recommended by the World
Health Organization (WHO).36 A stock solution (100 ppm) was
prepared by diluting 0.005 g of test-compounds in 0,7 mL of
ethanol, 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 survival, fourth instar A. aegypti was added to the beakers
(20 larvae per beaker) containing the synthesized compounds in a
range of concentrations obtained by appropriate dilution of the
stock solution with distilled water. Four replicate assays were
carried out for every sample concentration. A negative control for
each assay, using distilled water containing the same amount of
co-solvent 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.
1
Yield: 55%, m.p. (oC): 117-118. H NMR (400 MHz, CDCl3) δ
(ppm): 2.01 (q, J = 7.5 Hz, 2H); 2.56 (t, J = 7.5 Hz, 2H); 3.05 (t,
J = 7.5 Hz, 2H); 7.77-7.84 (m, 4H) 13C NMR (100 MHz, CDCl3)
δ (ppm): 21.27; 25.99, 32.67, 97.90, 126.15, 128.83, 138.07,
167.70, 167.07, 167.7, 178.32, 179.12. Elemental Analysis for
C12H11IN2O3 : Calc. C, 40.24; H, 3.10; N, 7.82. Found C, 40.21;
H, 3.32; N, 7.44.
4-[3-(4-Methoxyphenyl)-1,2,4-oxadiazol-5-yl]butanoic acid
1
(5f): Yield: 69%, m.p. (oC): 108-109. H NMR (400 MHz,
CDCl3) δ (ppm): 2.20 (q, J = 7.5 Hz, 2H); 2.55 (t, J = 7.5 Hz,
2H); 3.02 (t, J = 7.5 Hz, 2H); 3.85 (s, 3H); 6.99 (d, J = 8.7 Hz,
2H); 7.99 (d, J = 7.5 Hz, 2H); 9.80 (bs, 1H). 13C NMR (100
MHz, CDCl3) δ(ppm): 21.31, 25.56, 32.68, 55.32, 114.19,
119,02, 128.96, 161.84, 167.86, 178.40, 178.63. Elemental
Analysis for C13H14N2O4: Calc. C, 59.54; H, 5.38; N, 10.68.
Found C, 60.06; H, 5.12; N, 10.93.
4-[3-(Benzo[d][1,3]dioxol-5-yl)-1,2,4-oxadiazol-5-yl]butanoic
acid (5g): Yield: 60%, m.p. (oC): 112-113. 1H NMR (400 MHz,
CDCl3) δ (ppm): 2.21 (q, J = 7.6 Hz, 6.8 Hz, 2H); 2.56 (t, J = 7.6
Hz, 2H); 3.03 (t, J = 6.8 Hz, 2H); 6.03 (s, 2H); 6.89 (dd; J = 8.4
Hz, 1.6Hz, 1H); 7.51 (d, J = 1.6 Hz, 1H); 7.63 (dd, J = 8.4 Hz,
1H).13C NMR (100 MHz, CDCl3) δ (ppm): 21.4, 25.6, 32.6,
101.6, 107.5, 108.6, 120.6, 122.3, 148.1, 150.1, 167.9, 177.8,
178.7. Elemental Analysis for C13H12N2O5: Calc. C, 56.52; H,
4.38; N, 10.14. Found C, 56.70; H, 4.08 ; N, 9.82.
4.2.1. Computational Calculations
Molecular docking calculations were performed for 22
oxadiazole derivatives against the X-ray structure of AgHKT and
the comparative model of AeHKT (Figure 3). The programs
AutoGrid v.4.037 and AutoDock v.4.038 were used during the
calculations. Atomic coordinates of AgHKT crystal structure
were retrieved from PDB (ID: 2CH2).23 A dimer containing the
cofactor N-pyridoxyl-lysine-5-monophosphate (PLP) linked to
Lys205 side chain at the protein binding site was employed after
removal of the co-crystallized inhibitor 4-(2-aminophenyl)-4-
oxobutanoic acid (4-OB). Polar hydrogens were added to the
protein considering amino acid protonation states at pH 7. C-
terminal carbonyl oxygens were added when absent. Partial
charges for protein atoms were assigned according to AMBER86
force field parameters,39 while PLP and ligand charges were
calculated with the Gasteiger method,40 ensuring that all residues
have presented integer charges. The AMBER86 atom types were
assigned to all atoms.39 In order to assess the predictive quality
of our docking procedure, a validation methodology was devised
aiming to show that the undertaken procedure is able to
reproduce in detail the crystallographic structure of 4-OB in the
active site of AgHKT, and generate affinity maps for heterocycle
derivatives based on the ones achieved for the crystallographic
inhibitor 4-OB. This procedure was performed in three steps: (1)
First simulation, with all ligand dihedrals fixed, and 10
simulation steps; (2) Second simulation, with all ligand dihedrals
free, except for the ones formed by ring-amine group and ring-
carbonyl, and 10 simulation steps; (3) Third simulation, with all
ligand dihedrals free (except for the ones formed by ring-amine
group and ring-carbonyl), and the ligand being positioned out of
the binding site, with 200 simulation steps. Then, ligand
conformations resulting from these simulations were analyzed
and compared to the crystallographic conformation. The
4.1.2. Synthesis of 3-(3-aryl-1,2,4-oxadiazol-5-yl)-
butanoic acids methyl esters (6a-b)
To an appropriate solution of 3-(3-aryl-1,2,4-oxadiazol-5-yl)-
butanoic acid (5 mmol) in 10 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 product. Extraction of the residue with AcOEt (2 x 20 mL)
and drying solvent over anhydrous Na2SO4, pursued by solvent
removal in vacuo furnished the crude product. The residue was
chromatographed over silica gel using hexane:EtOAc as eluant
(4:1, v/v), crystallized from chloroform and hexane, which after
work-up furnished the desired methyl 3-(3-aryl-1,2,4-oxadiazol-
5-yl)-butanoates (6a-b).
Methyl 4-(3-phenyl-1,2,4-oxadiazol-5-yl)butanoate (6a):
1
Yield: 50%, m.p. (oC): 42-43. H NMR (400 MHz, CDCl3) δ
(ppm): 2.21 (q, J = 7.6 Hz, 7.2 Hz, 2H); 2.50 (t, J = 7.2 Hz, 2H);
3.02 (t, J = 7.6 Hz, 2H); 3.68 (s, 3H); 7.46 (m, 3H); 8.06 (m,
2H).13C NMR (100 MHz, CDCl3) δ(ppm): 21.4, 25.7, 32.8, 51.7,
126.8, 128.8, 131.1, 168.3, 172.9; 178.9. Elemental Analysis for
C13H14N2O3: Calc. C, 63.40; H, 5.73; N, 11.38. Found C, 63.31;
H, 6.06 ; N, 11.33.

aforementioned computational procedure was able to
successfully reproduce the crystallographic conformation (Fig.
3). A grid with dimensions of 28 Å x 28 Å x 28 Å and spacing of
0.22 Å was centered at the binding site of the enzyme. The
binding modes for the oxadiazole derivatives in the active site of
AgHKT were predicted based on the same affinity maps
generated for compound 4-OB. For atom types in the heterocycle
derivatives that were not present in 4-OB, additional affinity
maps were calculated using the same settings as for previous
affinity maps. During molecular docking calculations, binding
energies between the protein and a given ligand configuration
were determined based on potential energies stored as affinity
maps. Lamarckian genetic algorithm was employed in all
molecular dockings with the following parameters:41, 42 an initial
population of 150 individuals/ generation, a maximum number of
energy were selected in each step of the simulation, in such a way
that, at the end of calculation, atomic coordinates of the 100
conformers that better fitted the binding site were selected. These
conformers were structurally compared through their RMSDs,
and clustered into groups of similar conformations. A tolerance
of 2 Å for RMSD was employed to assign conformers to the
same cluster.
Acknowledgments
This work was supported by the Brazilian funding agencies
FACEPE, CNPq, INCT-INAMI and nBioNet. V.S. Oliveira
acknowledges an undergraduate PET fellowship from the
Brazilian Ministry of Education.
27,000 generations, and
a total of 2.5 ×
107 energy
evaluation/generation. Thus, a total of 4.05 × 106 conformations
for each of the ligands were generated in each of the 100 steps of
the simulation. Mutation and crossover rates corresponded to
0.02 and 0.08, respectively. In the local search, 300 steps were
applied with a 0.3 probability of search for one individual.
Ligand conformations presenting the most favorable binding
Supplementary Material
Supplementary data associated with this article can be
found at http...

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