Full text of DOI:10.1039/d0md00305k

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RESEARCH ARTICLE
A second generation of 1,2,4-oxadiazole
derivatives with enhanced solubility for inhibition
of 3-hydroxykynurenine transaminase (HKT) from
Aedes aegypti†
Cite this: DOI: 10.1039/
d0md00305k
Larissa G. Maciel,‡a Andrey da S. Barbosa, ‡a Edilson B. de Alencar-Filho,b
a
a
*
*
Thereza A. Soares
and Janaína V. dos Anjos
The most widely used method for the control of the Aedes aegypti mosquito population is the chemical
control method. It represents a time- and cost-effective way to curb several diseases (e.g. dengue, Zika,
chikungunya, yellow fever) through vector control. For this reason, the discovery of new compounds with
a distinct mode of action from the available ones is essential in order to minimize the rise of insecticide
resistance. Detoxification enzymes are an attractive target for the discovery of new insecticides. The
kynurenine pathway is an important metabolic pathway, and it leads to the chemically stable xanthurenic
acid, biosynthesized from 3-hydroxykynurenine, a precursor of reactive oxygen and nitrogen species, by
the enzyme 3-hydroxykynurenine transaminase (HKT). Previously, we have reported the effectiveness of
1,2,4-oxadiazole derivatives acting as larvicides for A. aegypti and AeHKT inhibitors from in vitro and in silico
studies. Here, we report the synthesis of new sodium 4-[3-(aryl)-1,2,4-oxadiazol-5-yl] propanoates and the
cognate HKT-inhibitory activity. These new derivatives act as competitive inhibitors with IC50 values in the
range of 42 to 339 μM. We further performed molecular docking simulations and QSAR analysis for the
previously synthesized sodium 4-[3-(aryl)-1,2,4-oxadiazol-5-yl] butanoates reported earlier by our group
and the data produced herein. Most of the 1,2,4-oxadiazole derivatives, including the canonical
compounds for both series, showed a similar binding mode with HKT. The binding occurs similarly to the
co-crystallized inhibitor via anchoring to Arg356 and positioning of the aromatic ring and its substituents
outwards at the entry of the active site. QSAR analysis was performed in search of more than 770
molecular descriptors to establish a relationship between the lowest energy conformations and the IC50
values. The five best descriptors were selected to create and validate the model, which exhibited
parameters that attested to its robustness and predictability. In summary, we observed that compounds
with a para substitution and heavier groups (i.e. CF3 and NO2 substituents) had an enhanced HKT-inhibition
profile. These compounds comprise a series described as AeHKT inhibitors via enzymatic inhibition
experiments, opening the way to further the development of new substances with higher potency against
HKT from Aedes aegypti.
Received 3rd September 2020,
Accepted 17th November 2020
DOI: 10.1039/d0md00305k
rsc.li/medchem
or to the Togaviridae family, such as the alphavirus, which
causes chikungunya fever.2 These viruses cause asymptomatic
Introduction
Arboviruses are a heterogeneous group of viruses transmitted
by hematophagous arthropods. The most disseminated
arboviruses in Brazil belong to the Flaviviridae family,
comprising viruses that cause yellow fever, dengue and Zika;1
infections or pyretic diseases in humans both in the wild and
in urban cycles and are quite ubiquitous in tropical regions
due to geographical distribution of the vector insect.3
A
consequential discovery for public health was the discovery
that Zika virus causes a congenital syndrome in newborns
whose mothers were infected during pregnancy.4,5 This
syndrome is responsible for infantile microcephaly and cases
of malformation and can be sexually transmitted by infected
human males.6–9 The inexistence of effective vaccines for
immunization against all the viruses and their serotypes
makes vector species population control the most operative
a Department of Fundamental Chemistry, Federal University of Pernambuco (UFPE),
Recife – PE, Brazil. E-mail: thereza.soares@ufpe.br, janaina.anjos@ufpe.br
b College of Pharmacy, Federal University of San Francisco Valley (UNIVASF),
Petrolina – PE, Brazil
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0md00305k
‡ Both authors contributed equally to this work.
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and affordable alternative to disease control. Therefore,
coordinated efforts have been focused on containing the
main transmitting vector of those arboviruses, the Aedes
aegypti mosquito. Since A. aegypti is extremely adapted to the
domestic environment,10–12 the development of products
capable of eliminating larvae or the adult insect in household
water reservoirs offers one viable alternative to slow down the
transmission of arboviruses.
advancements in our endeavor to develop specific inhibitors
for AeHKT led us to further explore the synthesis of new
water-soluble oxadiazole salts with inhibitory activity against
recombinant AeHKT in vitro and to characterize the structural
features of the resulting complex through computational
methods.
Results and discussion
Numerous expedients can be used to control A. aegypti
dissemination, such as biological, chemical, mechanical and
biotechnological control, and these can be used alone or in
an integrated manner.13 Among these, chemical control can
be highlighted as the cheapest of the effective approaches to
limit mosquito propagation.14–17 Notably, because A. aegypti
larvae are mostly found at the surface of small domestic
water reservoirs, the potential environmental toxicity
associated to chemical control is greatly minimized.
Chemical control involves the use of organic or inorganic
insecticides, the latter not being environmentally viable,
since most inorganic pesticides contain heavy metals in their
formulae, thus showing high toxicity to animals.18 The most
commonly used organic insecticides are organochlorines,
organophosphates, carbamates and pyrethroids, all acting
directly on the insect nervous system.19–21 Hence, there is an
unceasing need for the development of new insecticides once
some of these larvicides lose potency through the
development of insect resistance. The search for
environment-friendly products with less harmful toxicological
profiles is essential to achieve new candidates for A. aegypti
control.
Mechanisms that lead to accumulation of reactive oxygen
and nitrogen species in the insect organism are a powerful
therapeutic weapon against A. aegypti. One of the most
important metabolic pathways in detoxifying reactive oxygen
species is the so-called kynurenine pathway, the major
tryptophan catabolism pathway in living organisms. In this
biochemical pathway, tryptophan is oxidized to quinolinic or
kynurenic acid or even xanthurenic acid, depending on the
organism.22–24 These endogenous acids can be totally oxidized
to CO2 and water or even used in the biosynthesis of other
substances, such as nicotinamide.25 In mosquitoes, this
pathway leads to a chemically stable species, xanthurenic acid
(XA). This acid is biosynthesized from 3-hydroxykynurenine
Chemistry
This second generation of water-soluble oxadiazole salts
belong to a unique class of insecticides possessing a 1,2,4-
oxadiazole nucleus. 1,2,4-Oxadiazoles are five-membered
heterocycles with two nitrogen atoms and one oxygen, and
they have been reported as amides and ester bioisosteres
with numerous biological activities attributed to molecules
containing this heterocycle as part of its scaffold.34
The interest in this recently discovered class of AeHKT
inhibitors began with the recognition of
a structural
similarity between a known HKT crystallographic inhibitor,
4-(2-aminophenyl)-4-oxobutyric acid (4-OB), and some of the
1,2,4-oxadiazole derivatives with larvicide activity developed
by our research group. Previous in silico studies showed that
these heterocyclic compounds could have similar binding
modes and affinity for HKT as the crystallographic
inhibitor.32 Further studies demonstrated that the oxadiazole
derivatives de facto worked as inhibitors of HKT from Aedes
aegypti, thus confirming our previously formulated
hypothesis (Fig. 1).33
In order to obtain more potent water-soluble AeHKT
inhibitors, we evaluated the effect of two types of
modifications in the oxadiazole scaffold (Fig. 2):
modifications in the aromatic ring bound to the C-3 position
of the heterocycle and variations on the length of the
carboxylate side chain. These structural changes provided a
useful way to investigate potential structure–activity
relationships to enhance the AeHKT antagonist activity of
1,2,4-oxadiazole derivatives.
The inhibitors were synthesized from arylamidoximes and
succinic anhydride to form 3-[3-(aryl)-1,2,4-oxadiazol-5-yl]
propanoic acids. Arylamidoximes were prepared from the
corresponding arylnitriles and hydroxylamine.30 After
purification, the acids reacted with sodium hydroxide in
methanol, yielding the corresponding sodium salts, which were
tested for inhibitory activity against AeHKT (Scheme 1).
Alternatively, 4-[3-(aryl)-1,2,4-oxadiazol-5-yl] butanoic acids can
be synthesized by a reaction of glutaric anhydride with its
(3-HK) in
a process catalyzed by 3-hydroxykynurenine
transaminase (HKT),26–29 the sole enzyme responsible for
3-HK regulation in mosquitoes.29 Therefore, inhibition of the
enzymatic conversion of 3-HK to xanthurenic acid in the
kynurenine pathway consists in a powerful approach to
control A. aegypti development via the accumulation of 3-HK.
Our research group has been involved in the synthesis
and characterization of larvicidal heterocycles for
a
while.30–33 Recently, we have reported the discovery of new
1,2,4-oxadiazole derivatives as inhibitors of HKT from A.
aegypti (AeHKT), providing direct evidence that HKT can be
explored as a molecular target for the discovery of a novel
class of chemical insecticides against A. aegypti.32,33 The past
Fig. 1 Structures of HKT inhibitors 4-(2-aminophenyl)-4-oxobutyric
acid (4-OB) and sodium 4-[3-(4-iodo-phenyl)-[1,2,4] oxadiazol-5-yl]-
butanoate.
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Fig. 2 Modifications made on oxadiazole derivatives in this work.
respective arylamidoxime, and then the corresponding sodium salts
can be obtained as previously reported by Maciel and colleagues.33
AeHKT inhibition assays
The mechanism of enzymatic inhibition was investigated for
recombinant AeHKT with the saturation curve presented in
Fig. 3A. It can be verified that the enzymatic reaction both in
the presence and in the absence of the inhibitor 4a satisfies the
Michaelis–Menten model. From the double-reciprocal plot
(Lineweaver–Burk plot), the type of inhibition mechanism was
estimated (Fig. 3B) in accordance with the competitive
inhibition kinetics. In this type of inhibition, 4a occupies the
active site, preventing the substrate 3-HK from binding to the
AeHKT enzyme and therefore suppressing the transamination
activity of the recombinant AeHKT enzyme. The addition of
compound 4a caused changes in the regression slope and
y-intercept, indicating the competitive inhibition.
Fig.
3
Inhibition mechanism of the canonical compound 4a. (A)
Kinetic model of Michaelis–Menten inhibition in the presence and
absence of inhibitor 4a. (B) Lineweaver–Burk plot for the enzymatic
reaction in the presence and absence of inhibitor 4a.
Enzymatic assays were conducted using
a
recent
standardized methodology developed by Maciel and
colleagues.33,35 The assays allowed the quantification of
xanthurenic acid (XA) converted from 3-hydroxykynurenine
(3-HK) by AeHKT in the presence of the inhibitor. The
reaction was initiated by the combination of 2 μg of
recombinant AeHKT, 2 mM of amino donor substrate (3-D,
L-HK), 2 mM of sodium pyruvate as an amino acceptor and
40 μM of the cofactor pyridoxal-5′- phosphate (PLP) in a final
volume of 100 μL. Reactions were stopped by adding 100 μL
of a 10 mM FeCl3·6H2O solution in 0.1 M HCl. The assays
were performed in duplicate, and a calibration curve was
prepared to quantify the amount of XA generated by AeHKT
(R2 = 0.98). The complex absorbance values were read at 570
nm after 5 minutes of incubation at 50 °C. Two tests were
performed in the absence of XA, in order to verify if ferric ion
could also complex with PLP and/or sodium pyruvate. Those
assays showed no absorbance (<0.1), confirming the
specificity of the complexation of Fe3+ to XA. The inhibitory
activity of salts 4a–4s was assessed in duplicate by adding
500–50 μM of each diluted salt to 200 mM HEPES buffer pH
7/100 mM NaCl. In each assay, a negative control was taken
into account when calculating the final activity percentage.
The IC50 values for each of the salts are compiled in Table 1.
The two most active compounds displayed inhibition activity in
the micromolar range (4b, IC50 = 35 μM; 4h, IC50 = 42 μM). A
few trends can be identified concerning the relationship
between chemical structure and inhibitory activity.
The modification of the 6-membered aromatic group (4a,
phenyl) into 5-membered aromatic rings in 4n (2-furyl) and/
or 4o (2-thiophenyl) was positive for the AeHKT inhibition
Scheme 1 Synthesis of oxadiazole salts tested as AeHKT inhibitors. (i) NH2OH.HCl and NaHCO3 (4 eq.), ethanol/H2O, r.t.; (ii) succinic anhydride
(1.5 eq.), neat, 140 °C; (iii) NaOH (1 eq.) and methanol, r.t; (iv) glutaric anhydride (1.5 eq.), neat, 140 °C; (v) NaOH (1 eq.) and methanol, r.t.
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Table 1 The molecular structure of 1,2,4-oxadiazole sodium salts tested as AeHKT inhibitors with their biological activity (IC50) and estimated ΔKI values
Code
R
IC50 (μM)
ΔKIa
−5.2
Code
R
IC50 (μM)
ΔKIa
−5.1
4a
139
1
4b
35
1
4c
4e
86
1
−7.0
−6.1
4d
4f
119
1
−6.5
−6.4
73
1
110
1
4g
4i
124
76
1
1
1
−4.9
−6.4
−6.4
4h
4j
42
85
1
1
2
−5.1
−6.2
−6.2
4k
83
4l
157
4m
4o
339
68
2
1
1
−5.9
−3.8
−4.4
4n
4p
4r
84
85
61
1
1
1
−2.4
−7.3
−6.8
4q
116
4s
77
1
−6.4
6a
6c
72
64
1
1
−6.2
−7.3
6b
6d
101
110
1
1
−6.2
−7.0
6e
88
1
−6.8
6f
54
1
−6.9
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Table 1 (continued)
Code
R
IC50 (μM)
ΔKIa
−7.4
Code
R
IC50 (μM)
ΔKIa
−6.2
6g
63
1
6h
70
1
6i
42
87
1
1
−7.2
−7.1
6j
294
1
−6.8
6k
a
ΔKI = KI (4-OB) − KI (compound).
reaction, given that the IC50 value decreased in the sequence
4a (139 μM) > 4n (84 μM) > 4o (68 μM). In fact, the
thiophene ring is considered a phenyl group bioisostere since
the thiophene ring is also aromatic and has similar electronic
characteristics.36 This may be related to ring sizes, favoring a
better fit of the smaller rings on the AeHKT active site.
via soaking and cocrystallization procedures using several
1,2,4-oxadiazole compounds (manuscript in preparation). For
this reason, molecular docking simulations were performed
to investigate the binding mode and interactions between
AeHKT and the present set of 1,2,4-oxadiazole compounds.
We have validated our molecular docking protocol through
redocking tests for the co-crystallized ligand 4-OB with both
AgHKT and AeHKT X-ray structures. It should be noted that
the two enzymes share a sequence identity of 79%. Ligand
conformations resulting from these simulations were
analyzed and compared to the crystallographic structure
obtained for the 4-OB–AgHKT complex (Fig. 4).
In addition to the nineteen sodium 1,2,4-oxadiazolyl-
propanoates reported here, eleven sodium 1,2,4-oxadiazolyl-
butanoates described by Maciel and colleagues were added to
this set to evaluate their interactions with AeHKT at the
atomic level.33 The 30 oxadiazole derivatives were placed in
the active site, and after 200 generational runs with 2.5 × 106
evaluated interactions per generation, the lowest energy
conformations and inhibition constants were analyzed. The
canonical compounds for both series (4a and 6a) showed
similar binding modes to Arg356, which is consistent with the
experimental 4-OB binding mode (Fig. 5A). In these
compounds, the residues Arg356/C, Ser154/C and Asn44/D
interact through electrostatic and hydrogen bonding with the
oxadiazole ring and the carboxylate, stabilizing the
compounds in these lowest energy conformations. These are
key residues for substrate and inhibitor recognition as
described in the literature.37 In the binding conformation,
the aromatic ring and its substituents point to the entry of
the active site, allowing interactions with the residues located
at this entry. Compounds 4a–4s, most of them with para
substituents, exhibited similar conformation to 4a, with 4g
Compounds having electron donor substituents on the
aromatic ring exhibited higher IC50 values according to the
proximity of the 1,2,4-oxadiazolic ring and to the placement of
the substituent, with the para position IC50 < meta position
IC50 < ortho position IC50. This is the case for 4e (73 μM), 4 m
(110 μM) and 4f (339 μM) compounds with a methyl group as a
substituent on the aromatic ring. The same was observed for
compounds with a methoxy group, 4b and 4l, in which the para
compound had better performance. Even for nitro compounds,
well-known electron-withdrawing substituents, it was observed
that para-substituted oxadiazoles have lower IC50 values than
the meta-substituted ones, 4i (76 μM) and 4p (85 μM),
indicating that in these cases para-substitution leads to better
AeHKT inhibitors. On the other hand, double substitution on
the aromatic ring was not very effective in enhancing inhibitory
activity against AeHKT. For example, compound 4c (3,4-
dichlorophenyl) showed an IC50 value of 86 μM, which is
similar to that of 4j (para-chloro, 85 μM) and 4s (ortho-chloro,
77 μM), the latter two being slightly more effective inhibitors
with a single substitution on the aromatic ring.
Oxadiazole derivative binding modes and estimated binding
energies
We have successfully determined the X-ray structure of
AeHKT (PDB ID 6MFB) but we have struggled with twinned
data sets from macromolecular crystallography experiments
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canonical 6a and the most active compound 6i (Fig. 5C),
except for 6g. Considering that 6g is the bulkiest compound
in this series and there is no difference in conformation for
smaller substituents in para or meta positions (e.g. 6e and
6f), steric effect is
a plausive reason for this lowest
conformation outside of the active site (Fig. 5E). Even so, the
carboxylate portion is still interacting with Arg356 and Gln344
(residues located at the active site). The same happens to 4i,
but here the nitro substituent is interacting with Arg356 and
Gln344 instead of the carboxylate anion. The latter group is
closer to another Arg residue (Fig. 5E). The competition
between the nitro substituent and the carboxylate anion for
interaction with Arg356 is not favorable to the stabilization of
the compounds in the active site. Besides the previously
described interactions, van der Waals forces play an
important role at close range for stabilization and binding of
these compounds in the AeHKT active site (ESI†).
The calculated KI and measured IC50 values were
compared to determine if these two measurements behave
similarly, since for both, the lower the values, the better the
inhibitor (Fig. 6). Although there is no clear-cut relationship
between the calculated KI and experimentally measured IC50,
most compounds with a low KI also exhibit a low IC50 and
vice-versa (Fig. 6). Yet, a few outliers were observed, mostly
within the oxadiazolyl-propanoic derivative set (i.e. 4b, 4h,
4m, 4n, 4o). As previously reported,32 molecular docking
calculations suggested that the oxadiazolyl-propanoic
derivatives (4a–4s) do not bind to the active site of AeHKT in
one single conformation as seen for oxadiazolyl-butanoic
derivatives (6a–6k), but rather through multiple conformers.
We have interpreted this pattern as indicative of less efficient
inhibitory activity since some of the bound conformations
will be unproductive and will require unbinding–rebinding to
the active site in order to effect inhibitory action on the
enzyme. However, due to the conformational diversity of the
oxadiazolyl-propanoic derivatives, the calculated KI value,
Fig. 4 The 4-OB ligand binding modes in AgHKT and AeHKT active
sites after the redocking procedure. (A) AgHKT in light yellow, showing
the PLP cofactor and Arg356 residue (important for substrate and ligand
binding and recognition). Co-crystallized 4-OB in orange and the
lowest energy conformation from molecular docking calculations in
purple. (B) AeHKT in grey, showing the PLP cofactor and Arg356
residue. Co-crystallized 4-OB in green and the lowest energy
conformations (dark blue and cyan) from calculations started with the
ligand placed outside the active site. Distances in angstroms.
chosen for the lowest energy conformer of
a given
and 4i being the exception. The electronegative/negatively
charged substituents may be responsible for differences in
their binding mode due to favorable electrostatic interactions
with the positively charged residues Arg or Lys. Compounds
4n and 4o also showed different conformations compared to
4a. The exchange of the six-membered to five-membered ring
compound, will not be as representative of the diversity of
conformations as it is for the conformationally more
homogenous compounds. It should be noted, however, that a
fairly proportional relation was observed for 24 of the 30
compounds, which indicates that chemical modifications of
the 1,2,4-oxadiazole scaffold appear to enhance the inhibitory
activity of AeHKT.
resulted in
a
conformation with
a
180-degree turn,
suppressing the interactions with Ser154/C and Asn44/D
(Fig. 5B). This behavior was also observed in all
ortho-substituted compounds (Fig. 5D). The lowest energy
conformation for these compounds is slightly different from
that of 4a but still well placed in the active site.
This search for compounds with short carboxylate linkers
is consistent with our previous work, since the 1,2,4-
oxadiazolyl-propanoic derivatives (4a–4s) can display multiple
conformations in the AgHKT active site, most of which are
expected to be unproductive for inhibition.32
This demonstrates that better oxadiazole-based candidates
for in vitro and in vivo HKT inhibition can be further
developed and discovered for this new class of potential A.
aegypti larvicides. Furthermore, we calculated the difference
ΔKI between the calculated values of KI for the oxadiazole
derivatives and for the lowest-energy docked conformation of
4-OB (Table 1). It can be seen that all 30 compounds exhibited
a negative ΔKI, indicating that these oxadiazole derivatives
can bind similarly or better than the co-crystallized inhibitor
4-OB in terms of estimated binding energy. The combined
analysis of ΔKI values, binding modes in the AeHKT active site
and experimental inhibitory activities places this set of 30
Nevertheless, most of the 1,2,4-oxadiazolyl-butanoic
derivatives (6a–6k) show similar conformation to the
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Fig. 5 Calculated binding modes for oxadiazole derivatives in AeHKT active sites via molecular docking simulations. (A) The lowest energy
conformation for compounds 4a (red) and 4-OB (dark blue), showing similar interactions with residues Arg356, Ser154 and Asn44. (B) The lowest
energy conformation of compound 4n (pink), resulting in a conformation with a 180-degree turn in comparison to the canonical 4a. (C) The lowest
energy conformation of compound 6i (blue), which is representative for most compounds of this series except for 6g. (D) All ortho-substituted
compounds 4m, 4q and 4s (yellow) exhibited the lowest energy conformations similar to 4n, with the aromatic ring pointing to the inner side of
the active site. (E) Compounds 6g and 4i displayed the lowest energy conformation outside of the active site, but still interacting with Arg356
.
compounds as a prototype to design new compounds as
AeHKT inhibitors with higher potency and specificity.
not discriminate one single binding site in KAT I and KAT II
for a canonical variant of 1,2,4-oxadiazoles.33 However, it is
important to highlight that this series of 1,2,4-oxadiazoles are
prototypes that will require further structural modifications
for enhanced efficiency as well as selectivity for HKT. In
addition, the anthropophilic behavior and great adaptability
of A. aegypti to urban environments makes possible the use
of oxadiazole-based larvicides in water-filled containers
outside buildings which are used by the female mosquitoes
to oviposition. For this purpose, high selectivity for HKT
versus its human orthologs is desirable but not essential
since ingestion by humans or higher animals would be
accidental and in very small amounts (millimolar range).
After overcoming first-pass metabolism and biological
It should be noted that HKT is a functional ortholog of
human kynurenine aminotransferases (KATs) which have
been shown to catalyze the irreversible transamination of
3-HK to XA.37 Therefore, there is a concern that 1,2,4-
oxadiazoles can undesirably inhibit these orthologs. However,
it was previously shown that the 4-(2-aminophenyl)-4-
oxobutyric acid (4-OB) inhibitor co-crystallized in the active
site of AgHKT does not inhibit the human KAT I isozyme in
the millimolar range.37 Given the extraordinary structural
similarity of 1,2,4-oxadiazoles with 4-OB, it is reasonable to
assume that the former will not bind efficiently to KATs.
Furthermore, previous molecular docking calculations could
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0.73; Q2
= 0.61; Q2
= 0.77. Results of leave-N-out cross-
EXT
LOO
validation analysis (Q2LNO) can be visualized in Fig. 7,
showing that the values fluctuate around 0.1 from the Q2
LOO
value, which is a good profile.
The Y-scrambling results are displayed in Fig. 8 and show
validation parameters for the scrambled models (blue dots)
and for the original valid model (red triangle). In this
analysis, 50 shuffles were performed only on the Y column
and models were built from these new matrices, which must
have poor validation parameters if there is no random
correlation between Y and X. Once again, we can observe the
success of this analysis, where most false models show values
smaller than 0.4.
The physical sense of the molecular descriptors and a
structure–activity relationship, from a qualitative standpoint,
is essential to understand the QSAR analysis. Taking into
account the nature and interpretation of the selected
variables, a compilation of these parameters is presented
below. It is noteworthy to highlight that the analysis was
performed individually, but it is the descriptor's overall
contribution that is responsible for model predictability.
HATS7i: It is part of the GETAWAY descriptors (GEometry,
topology, and atom-weights assemblY), which are derived
from the molecular influence matrix (H).38 This matrix is
constructed starting from the geometric distance matrix (M)
between all the atoms in a structure, containing three
columns regarding the x, y and z coordinates and rows
corresponding to the total number of the atoms. Then, the
matrix is obtained by H = M·(MT·M)−1·MT.39 The generic
descriptor HATSkw implies that k is the lag of the 1–8 bonds
between two atoms i and j, and w corresponds to the
weighting scheme. Thus, the descriptor can be calculated as:
Fig. 6 Plot of inhibitory activity of the oxadiazole derivative sodium
salts (IC50) versus estimated KI values. Compounds 4a–4s are shown as
filled symbols, whereas compounds 6a–6k are shown as empty
symbols. The shape of the symbols is equivalent to the position of the
substitution (para, meta, ortho or other). The same substituents have
matching colors for both series.
barriers, such
a small amount is expected to exhibit
considerably lower bioavailability. In addition, structurally
related compounds have already shown toxicological safety in
preliminary assays.30 Therefore, for use as synthetic
larvicides, these compounds can be considered safe.
QSAR modeling
n
n
X X
ꢀ
ꢁ ꢀ
ꢁ
QSAR modeling was performed to investigate molecular
descriptors related to the IC50 inhibitory activity, allowing
better comprehension of biological activity, design, and
prediction of new compounds to be synthesized. After OPS
and systematic search algorithms, the selected variables of
the best model are presented on Table 2, excluding the
outlier compounds, 4m and 6j. These compounds were
evaluated by the statistical method of “interquartile
distances” in the range of the activities, revealing that they
are true outliers.
Thus, these samples were disregarded from the QSAR
modeling. In the same table, a correlation matrix is displayed
based on the training set, showing a weak correlation
between the selected descriptors (<0.6). The regression vector
is shown below with the respective regression coefficients:
HATSkw ¼
ðwihiiÞ· wjhjj ·δ k; dij
i¼1 j¼1
where δ(k; dij) is the Kronecker delta, i.e. δ(k; dij) = 1, if the
ijth entry in the topological level matrix is = k, and δ(k; dij) =
0.39 For the HATS7i, the w parameter is the ionization
potential (IP) of the atoms, in a lag of seven (k) bonds. The
regression coefficient in the QSAR equation is negative,
indicating that higher values for this descriptor decrease
pIC50 and, subsequently, the activity. This means that atoms
with higher ionization potentials at a distance of seven bonds
tend to decrease biological activity. We can take as example
compounds 4b, 6b ( p-methoxy substituted) and 4l
(m-methoxy substituted). The former two compounds have
the oxygen from the methoxy group at a distance of seven
bonds of the oxadiazolyl oxygen. On the other hand,
compound 4l has the oxygen from the methoxy group at a lag
of seven bonds of oxygen or nitrogen of the oxadiazole ring,
even in different combinations. Then, the descriptor value
for 4l is higher and the inhibitory activity is worse. It is
indicative that a substitution at the para position of the
aromatic ring is preferable, including specific relationships
pIC50 = −1.8894 ( 1.0232) HATS7i + 0.0449 ( 0.0229) RDF050m
− 0.0554 ( 0.0347) RDF070u + 2.4498 ( 1.4924) G2u
+ 2.9769 ( 2.9260) H8m + 4.3958 ( 0.5623)
The values of some validation parameters attested to a good
degree of robustness and predictability for the model: R2
=
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Table 2 Molecular descriptors selected after OPS/systematic search algorithms
Entry
Compound
pIC50
HATS7i
RDF050m
RDF070u
G2u
H8m
1
2
3
4
5
6
7
8
4a
4e
4da
4b
4j
3.857
4.137
3.924
4.456
4.071
3.804
3.907
4.081
4.119
4.071
4.377
4.066
4.076
4.167
3.959
3.936
4.215
4.114
4.143
3.996
4.194
3.959
4.056
4.268
4.201
4.155
4.377
4.060
0.4429
0.3881
0.4405
0.3092
0.4431
0.4622
0.3793
0.3907
0.4050
0.3368
0.4145
0.3871
0.3978
0.3847
0.4013
0.3954
0.3927
0.4030
0.4312
0.3169
0.3751
0.4185
0.3953
0.3704
0.2983
0.4151
0.4129
0.3974
3.6412
3.6746
4.5166
4.1684
3.3415
3.7805
3.7471
3.9885
4.1037
7.9366
7.6887
3.6384
1.7998
3.3366
3.1145
1.9689
3.6949
4.3525
5.0707
4.5514
6.6453
7.6206
3.8126
6.9577
8.1453
8.8637
9.3951
3.9394
4.1757
5.3230
4.5978
3.0959
3.9394
3.9770
5.0978
6.1007
6.7380
6.6552
3.8661
5.3099
2.8134
2.6781
6.5438
5.2898
4.8954
4.3739
4.2567
6.9781
5.4440
7.6289
6.9982
6.4733
7.5686
5.2402
5.4779
5.4625
0.1772
0.2119
0.1772
0.2086
0.1772
0.1707
0.1772
0.1926
0.1951
0.1738
0.2021
0.2008
0.1832
0.2110
0.1722
0.2008
0.1772
0.1772
0.1830
0.1667
0.2119
0.1722
0.2397
0.2027
0.1643
0.1679
0.2678
0.2397
0.0213
0.0402
0.0393
0.0338
0.0267
0.0163
0.0030
0.0355
0.0546
0.0005
0.0311
0.0179
0.0042
0.0029
0.0245
0.0203
0.0118
0.0161
0.0015
0.0041
0.0043
0.0016
0.0018
0.0004
0.0082
0.0019
0.0014
0.0017
4l
4g
4k
4i
4p
4ha
4c
4n
4oa
4f
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
4q
4r
4sa
6a
6b
6c
6d
6e
6f
6g
6h
6i
6k
Correlation matrix
HATS7i
RDF050m
RDF070u
G2u
H8m
HATS7i
RDF050m
RDF070u
G2u
1
0.223
1
—
—
—
0.339
0.445
1
—
—
0.043
0.075
0.015
1
0.080
0.456
0.132
0.036
1
—
—
—
—
H8m
—
a
Test set.
in this position for other atoms with considerable IP
(halogens).
RDF070u has a negative one. Thus, the former indicates that
atoms with higher mass in the molecular scaffold tend to
contribute positively to activity, in contrast to the latter,
which indicates that a greater number of atoms in the
structure's periphery tends to decrease activity. In other
words, heavier atoms as substituents contribute positively to
biological activity. Compounds 4h, 6h and 6i are good
examples that explain this archetype, presenting a high value
RDF050m and RDF070u: These descriptors are related to
the radial distribution function in the molecular scaffold.38,39
The first one means the probability of finding an atom in a
spherical volume of radius 50.0 Å, weighted by the atomic
mass, the second one, in a radius of 50.0 Å, unweighted.38
RDF050m has
a positive regression coefficient, while
Fig. 8 Y-scrambling results (blue dots: results for scrambled models;
Fig. 7 Leave-N-out cross-validation results.
red triangle: results from the original model).
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for the first descriptor and a low value for the second one.
These three compounds display heavy substituents at the
para position of the aromatic moiety.
external matrix consisting of alpha-cyano-4-hydroxycinnamic
acid (10 mg mL−1) in 50% acetonitrile and 0.3%
trifluoroacetic acid was used. Arylamidoximes were
synthesized as described in the literature.40
G2u: The second component symmetry directional WHIM
index/unweighted. WHIM descriptors capture relevant
molecular 3D information.38,39
A
principal component
Synthesis of 3-(3-aryl-1,2,4-oxadiazol-5-yl) propanoic acids
analysis is performed centered on Cartesian coordinates of a
compound by using a weighted covariance matrix.39 In this
Succinic anhydride (30 mmol) and the corresponding
arylamidoxime (20 mmol) were put in a round-bottom flask
in an oil-bath at 473 K. After one hour, the end of the
reaction was verified by TLC analysis in a 7 : 3 hexane/ethyl
acetate mobile phase. Then, saturated NaHCO3 solution (10
mL) and 10 mL of dichloromethane were added to the cold
flask and the reaction mixture was stirred overnight. After
the phase separation, the aqueous phase was acidified until
precipitation of the title compound occurred and then
dissolved in dichloromethane, dried over Na2SO4 and
recrystallized from a mixture of chloroform and hexane. The
substances 3a–3e, 3g, 3h, 3j, 3p, 3r (ref. 27) and 3f, 3i, 3m, 3o
(ref. 28) were synthesized previously and their data were
compared to previous literature.
specific case, the descriptor has
a positive regression
coefficient, indicating that higher values tend to increase the
activity. Compounds 4e, 6e, 6i and 6k, all presenting high
values for this descriptor, are para-substituted compounds.
H8m: H autocorrelation of lag 8/weighted by atomic
masses.38 It is also a GETAWAY descriptor, as the HATS7i,
obtained by the equation:
n
n
X X
ꢀ
ꢁ ꢀ
ꢁ
Hkw ¼
hij·wi·wj ·δ k; dij; hij
i¼1 j¼1
where δ(k; dij; hij) = 1 if dij = k (in the topological level
matrix) and hij > 0 (considering the H matrix), and zero
otherwise.39 This way, heavier atoms at a distance of eight
bonds increase the value of this descriptor, which has a
positive regression coefficient. Compound 4i has the highest
value due to the nitro substitution in the para position,
allowing the combination of oxygen from this group with
the heavy atoms of the oxadiazole ring, thus increasing the
value. The para-CF3 substituted compound, 4h, follows the
same trend, but with a higher value on this descriptor. On
the other hand, compound 4p has a very low value, and it
presents the nitro substituent on the meta position of the
aromatic ring.
3-[3-(4-Trifluormethylphenyl)-1,2,4-oxadiazol-5-yl]-
propanoic acid (3h) (3.53 g, 62%); mp 110–112 °C. 1H NMR
(CDCl3, 400 MHz) δ (ppm): 3.02 (t, 2H, J = 7.2 Hz, CH2); 3.27
(t, 2H, J = 7.2 Hz, CH2); 7.73 (d, 2H, J = 8.0 Hz Harom); 8.18
(d, 2H, J = 8.0 Hz, Harom); 13C NMR (CDCl3, 400 MHz) δ
(ppm): 21.7; 30.1; 122.3; 125.0; 125.8; 125.8; 127.7; 130.0;
133.1; 167.3; 176.8; 178.6. MALDI-TOF HRMS m/z: 285.1118
(calcd for C12H9F3N2O3 [M − H]+: 285.0487).
3-[3-(2-Furyl)-1,2,4-oxadiazol-5-yl]- propanoic acid (3n)
(2.58 g, 63%), mp 118–120 °C; 1H NMR (CDCl3, 400 MHz) δ
(ppm): 2.99 (t, 2H, J = 7.6 Hz, CH2); 3.23 (t, 2H, J = 7.6 Hz,
CH2); 7.11 (dd, 1H, J = 4.4; 4.4 Hz, Harom); 7.67 (s, 2H,
Harom).13C (CDCl3, 400 MHz) δ (ppm): 21.6; 30.0; 111.8;
113.8; 141.9; 145.2; 161.2; 176.5; 178.2. MALDI-TOF HRMS m/
z: 209.6012 (calcd for C9H8N2O4 [M + H]+: 209.0562).
Considering the analysis exposed above for the oxadiazole
series described herein, we can summarize that compounds
presenting heavy atoms as substituents at the para position
of the aromatic ring have an inclination to be more active as
4-[3-(3-Nitrophenyl)-1,2,4-oxadiazol-5-yl)- propanoic acid
(3p) (4.2 g, 80%); mp 121–122 °C (from CHCl3). 1H NMR
(CDCl3, 400 MHz) δ (ppm): 3.04 (t, 2H, J = 7.2 Hz, CH2); 3.29
(t, 2H, J = 7.2 Hz, CH2); 7.68 (d, 1H, J = 8.4 Hz, Harom); 8.36
(d, 2H, J = 8.40 Hz, Harom); 8.91 (s, 1H, Harom). 13C NMR
(CDCl3, 400 MHz) δ (ppm): 21.7; 29.9; 122.6; 125.7; 128.5;
130.0; 133.0; 166.7; 175.7; 178.9. MALDI-TOF HRMS m/z:
262.1154 (calcd for C11H8N3O5 [M − H]+: 262.0464).
HKT antagonists. These findings were validated by
a
mathematical model depicted in this work, which allows for
a better understanding of structure–activity relationships and
can be used in the future for synthetic approaches.
Experimental
Synthesis and characterization of oxadiazole salts – general
methods
3-[3-(2-Fluorphenyl)-1,2,4-oxadiazol-5-yl]- propanoic acid
1
The reactions were monitored by TLC analysis with TLC
plates containing GF254 from E. Merck©. Compounds were
characterized using an Electrothermal Mel-Temp© apparatus
to determine the melting points. NMR spectra were recorded
using a Varian Unity Plus 300 MHz or a Varian UNMRS 400
MHz spectrometer. Infrared spectra were obtained on a
Shimadzu model IR Prestige-21 FT-IR instrument using KBr
pellets. The exact mass of the synthesized compounds was
obtained on a MALDI-TOF Autoflex III Mass Spectrometer
(3q) (2.5 g, 53%); mp 83–85 °C. H NMR (CDCl3, 400 MHz) δ
(ppm): 3.02 (t, 2H, J = 6.8 Hz, CH2); 3.29 (t, 2H, J = 6.8 Hz,
CH2); 7.29–7.20 (m, 2H, Harom); 7.25–7.46 (m, 1H, Harom);
8.02–8.06 (m, 1H, Harom). 13C NMR (CDCl3, 400 MHz) δ
(ppm): 21.6; 30.2; 116.8; 124.4; 130.7; 132.8; 159.4; 161.9;
165.1; 176.8; 177.7. MALDI-TOF HRMS m/z: 237.7582 (calcd
for C11H9FN2O3 [M + H]+: 237.0669).
3-[3-(2-Chlorophenyl)-1,2,4-oxadiazol-5-yl]- propanoic acid
1
(3s) (3.68 g, 73%); mp 78–80 °C. H NMR (CDCl3, 400 MHz)
(Bruker Daltonics, Billerica, MA, USA) with
wavelength Nd:YAG laser at a frequency of 100 Hz. An
a
355 nm
δ (ppm): 3.02 (t, 2H, J = 7.2 Hz, CH2); 3.30 (t, 2H, J = 7.2
Hz, CH2); 7.35–7.45 (m, 2H, Harom); 7.53 (dd, 2H, J = 7.6,
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1.2 Hz, Harom); 7.90 (dd, 1H, J = 7.2; 2.0 Hz, Harom). 13C
NMR (CDCl3, 400 MHz) δ (ppm): 21.6; 30.2; 125.8; 126.4;
130.8; 131.6; 131.7; 133.4; 167.0; 176.9; 177.6. MALDI-TOF
HRMS m/z: 252.7416 (calcd for C11H9ClN2O3 [M + H]+:
253.0374).
2H, J = 7.5 Hz, CH2); 7.11 (d, 2H, J = 8.1 Hz, Harom); 7.50 (d,
2H, J = 8.1 Hz, Harom). 13C NMR (D2O, 75 MHz) δ (ppm):
20.5; 22.9; 32.9; 122.2; 126.8; 129.5; 142.5; 167.3; 179.6; 180.4.
FT-IR (KBr pellet) νmáx/cm−1: 2939 (C–Harom); 1650 (CO);
1561 (CN); 1223 (N–O). MALDI-TOF HRMS m/z: 255.0982
(calcd for C12H11N2NaO3 [M + H]+: 255.0740).
Synthesis of sodium 3-(3-phenyl-1,2,4-oxadiazol-5-yl)
propionic salts
Sodium 3-[3-(3-tolyl)-1,2,4-oxadiazol-5-yl]propanoate (4f)
(0.24 g, 93%); mp 95–97 °C. 1H NMR (D2O, 300 MHz) δ
(ppm): 2.70 (t, 2H, J = 7.8 Hz, CH2); 3.12 (t, 2H, J = 7.8 Hz,
CH2); 7.33–7.37 (m, 3H, Harom); 7.56 (s, 1H, Harom). 13C
NMR (D2O, 75 MHz) δ (ppm): 20.3; 23.0; 33.0; 123.9; 125.1;
127.3; 128.8; 132.3; 139.1; 167.4; 179.6; 180.8. FT-IR (KBr
pellet) νmáx/cm−1: 2946 (C–Harom); 1691 (CO); 1589 (CN);
1216 (N–O). MALDI-TOF HRMS m/z: 254.7474 (calcd for C12-
H11N2NaO3 [M]+: 254.0661).
In a round-bottom flask, the respective 4-(3-phenyl-1,2,4-
oxadiazol-5-yl)propionic acid (1 mmol) and 4.8 mL of 1%
NaOH methanol solution (freshly prepared) were mixed and
the reaction was stirred for one hour at room temperature.
After that, the methanol was evaporated and the product was
recrystallized from chloroform.
Sodium 3-(3-phenyl-1,2,4-oxadiazol-5-yl)propanoate (4a)
(0.22 g, 93%); mp 171–172 °C. 1H NMR (D2O, 300 MHz) δ
(ppm): 2.56 (t, 2H, J = 7.35 Hz, CH2); 2.98 (t, 2H, J = `7.35 Hz,
CH2); 7.32–7.44 (m, 3H, Harom); 7.66 (d, 2H, J = 7.80 Hz,
Harom). 13C NMR (D2O, 75 MHz) δ (ppm): 23.0; 33.0; 125.2;
126.9; 129.0; 131.6; 167.4; 179.6; 180.6. FT-IR (KBr pellet)
νmáx/cm−1: 2925 (C–Harom); 1691 (CO); 1582 (CN); 1205
(N–O). MALDI-TOF HRMS m/z: 241.0707 (calcd for C11H9N2-
NaO3 [M + H]+: 241.0583.
Sodium 3-[3-(4-fluorphenyl)-1,2,4-oxadiazol-5-yl]propanoate
(4g) (0.25 g, 96%); mp 190–192 °C. H NMR (D2O, 300 MHz) δ
1
(ppm): 2.58 (t, 2H, J = 7.3 Hz, CH2); 3.02 (t, 2H, J = 7.3 Hz,
CH2); 7.02–7.07 (m, 2H, Harom); 7.61–7.66 (m, 2H, Harom).
13C NMR (D2O, 75 MHz) δ (ppm): 22.9; 33.0; 115.8; 116.1;
121.56; 121.6; 129.3; 129.4; 162.7; 166.0; 166.7; 179.6, 180.8.
FT-IR (KBr pellet) νmáx/cm−1: 2939 (C–Harom); 1608 (CO);
1554 (CN); 1241 (N–O). MALDI-TOF HRMS m/z: 259.0794
(calcd for C11H8FN2NaO3 [M + H]+: 259.0489).
Sodium
3-[3-(4-methoxyphenyl)-1,2,4-oxadiazol-5-yl]
Sodium 3-[3-(4-trifluoromethylphenyl)-1,2,4-oxadiazol-5-yl]
propanoate (4h) (0.3 g, 98%); mp 194–195 °C. H NMR (D2O,
1
propanoate (4b) (0.25 g, 92%); mp 214–215 °C. 1H NMR (D2O,
300 MHz) δ (ppm): 2.63 (t, 2H, J = 6.9 Hz, CH2); 3.07 (t, 2H, J
= 6.9 Hz, CH2); 3.77 (s, 3H, CH3); 6.98 (d, 2H, J = 8.7 Hz,
Harom); 7.76 (dd, J = 8.7 Hz,1.8 Hz, Harom). 13C NMR (D2O,
75 MHz) δ (ppm): 20.8; 30.8; 53.2; 112.1; 115.7; 126.5; 159.2;
164.9; 177.5; 178.2. FT-IR (KBr pellet) νmáx/cm−1: 2966 (C–
Harom); 1609 (CO); 1423 (CN); 1237 (N–O). MALDI-TOF
HRMS m/z: 271.1074 (calcd for C12H11N2NaO4 [M + H]+:
271.0689.
300 MHz) δ (ppm): 2.66 (t, 2H, J = 7.3 Hz, CH2); 3.13 (t, 2H, J
= 7.3 Hz, CH2); 7.73 (d, 2H, J = 8.1 Hz Harom); 7.96 (d, 2H, J
= 8.1 Hz, Harom). 13C NMR (D2O, 75 MHz) δ (ppm): 23.1;
33.1; 125.4; 127.4; 128.8; 132.4; 166.5; 179.6; 181.2. FT-IR
(KBr pellet) νmáx/cm−1: 2939 (C–Harom); 1568 (CO); 1437
(CN); 1320 (N–O). MALDI-TOF HRMS m/z: 309.0762 (calcd
for C12H8F3N2NaO3 [M + H]+: 309.0457).
Sodium 3-[3-(4-nitrophenyl)-1,2,4-oxadiazol-5-yl]propanoate
1
Sodium
4-[3-(3,4-dichlorophenyl)-1,2,4-oxadiazol-5-yl]
(4i) (0.28 g, 97%); mp 225–226 °C. H NMR (D2O, 300 MHz) δ
1
propanoate (4c) (0.3 g, 98%); mp 270–272 °C. H NMR (D2O,
300 MHz) δ (ppm): 2.77 (t, 2H, J = 7.3 Hz, CH2); 3.23 (t, 2H, J
= 7.3 Hz, CH2); 7.40 (d, 1H, J = 8.1 Hz, Harom); 7.51 (d, 1H, J
= 8.1 Hz, Harom); 7.64 (s, 1H, Harom). 13C NMR (D2O, 75
MHz) δ (ppm): 23.1; 33.1; 125.1; 126.2; 128.3; 130.8; 132.4;
134.9; 165.6; 179.5; 181.0. FT-IR (KBr pellet) νmáx/cm−1: 2925
(C–Harom); 1692 (CO); 1582 (CN); 1272 (N–O). MALDI-
TOF HRMS m/z: 308.7603 (calcd for C11H7Cl2N2NaO3 [M +
H]+: 308.9804).
(ppm): 2.67 (t, 2H, J = 7.2 Hz, CH2); 3.15 (t, 2H, J = 7.2 Hz,
CH2); 8.05 (d, 2H, J = 8.7 Hz, Harom); 8.25 (d, 2H, J = 8.7 Hz,
Harom). 13C NMR (D2O, 75 MHz) δ (ppm): 23.1; 33.1; 124.1;
128.2; 131.6; 148.9; 166.2; 179.7; 181.6. FT-IR (KBr pellet)
νmáx/cm−1: 2925 (C–Harom); 1589 (CO); 1506 (CN); 1306
(N–O). MALDI-TOF HRMS m/z: 284.0463 (calcd for C11H8N3-
NaO5 [M − H]+: 284.0277).
Sodium
3-[3-(4-chlorophenyl)-1,2,4-oxadiazol-5-yl]
1
propanoate (4j) (0,26 g, 97%); mp 217–218 °C. H NMR (D2O,
300 MHz) δ (ppm): 2.64 (t, 2H, J = 7.2 Hz, CH2); 3.10 (t, 2H, J
= 7.2 Hz, CH2); 7.40 (d, 2H, J = 8.7 Hz, Harom); 7.73 (d, 2H, J
= 8.7 Hz, Harom). 13C NMR (D2O, 75 MHz) δ (ppm): 20.9;
30.9; 121.6; 126.2; 126.8; 134.8; 164.5; 177.4; 178.7. FT-IR
(KBr pellet) νmáx/cm−1: 2962 (C–Harom); 1589 (CO); 1423
(CN); 1195 (N–O). MALDI-TOF HRMS m/z: 275.0217 (calcd
for C11H8ClN2NaO3 [M + H]+: 275.0193).
Sodium
3-[3-(4-bromophenyl)-1,2,4-oxadiazol-5-yl]
propanoate (4d) (0.29 g, 90%); mp 238–240 °C. 1H NMR
(D2O, 300 MHz) δ (ppm): 2.62 (t, 2H, J = 7.5 Hz, CH2); 3.06 (t,
2H, J = 7.5 Hz, CH2); 7.36 (d, 2H, J = 8.7 Hz Harom); 7.43 (d,
2H, J = 8.7 Hz, Harom). 13C NMR (D2O, 75 MHz) δ (ppm):
23.1; 33.0; 124.1; 125.4; 128.4; 131.9; 166.7; 179.6; 180.8. FT-
IR (KBr pellet) νmáx/cm−1: 2943 (C–Harom) 1589 (CO); 1473
(CN); 1195 (N–O). MALDI-TOF HRMS m/z: 318.8952 (calcd
for C11H8BrN2NaO3 [M + H]+: 319.0866).
Sodium
3-[3-(benzo[d][1,3]dioxol-5-yl)-1,2,4-oxadiazol-5-yl]
propanoate (4k) (0.27 g, 94%); mp 185–186 °C. 1H NMR (D2O,
300 MHz) δ (ppm): 2.62 (t, 2H, J = 7.5 Hz, CH2); 3.07 (t, 2H, J =
7.5 Hz, CH2); 5.94 (s, 2H, OCH2O); 6.86 (d, 1H, J = 8.4 Hz,
Harom); 7.23 (d, 1H, J = 1.2 Hz, Harom); 7.36 (dd,1H, J = 8.7
Sodium 3-[3-(4-tolyl)-1,2,4-oxadiazol-5-yl]propanoate (4e)
(0.25 g, 98%); mp 179–180 °C. 1H NMR (D2O, 300 MHz) δ
(ppm): 2.19 (s, 3H, CH3); 2.54 (t, 2H, J = 7.5 Hz, CH2); 2.95 (t,
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Hz,1.2 Hz, Harom). 13C NMR (D2O, 75 MHz) δ (ppm): 20.8;
30.9; 99.7; 104.6; 106.5; 116.8; 120.0; 145.3; 147.7; 164.9; 177.5;
178.3. FT-IR (KBr pellet) νmáx/cm−1: 2902 (C-Harom); 1582
(CO); 1458 (CN); 1230 (N–O); 1023 (C–O). MALDI-TOF
HRMS m/z: 284.7558 (calcd for C12H9N2NaO5 [M]+: 284.0403.
1H, J = 7.2; 7.5 Hz, Harom). 13C NMR (D2O, 75 MHz) δ (ppm):
22.9; 33.1; 34.2; 113.3; 116.6; 124.7; 130.0; 133.6; 158.3; 161.6;
164.2; 179.6; 180.2. FT-IR (KBr pellet) νmáx/cm−1: 3049 (C–Harom);
1685 (CO); 1596 (CN); 1285 (N–O). MALDI-TOF HRMS m/z:
259.1279 (calcd for C11H8FN2NaO3 [M + H]+: 259.0489.
Sodium
3-[3-(3-methoxyphenyl)-1,2,4-oxadiazol-5-yl]
Sodium
3-[3-(3-bromophenyl)-1,2,4-oxadiazol-5-yl]
1
1
propanoate (4l) (0,21 g, 76%); mp 148–150 °C. H NMR (D2O,
300 MHz) δ (ppm): 2.64 (t, 2H, J=7.35 Hz, CH2); 3.09 (t, 2H, J
= 7.35 Hz, CH2); 3.76 (s, 3H, OCH3); 7.05 (dd, 2H, J = 8.1 Hz;
1.2 Hz, Harom); 7,33–7,44 (m, 3H, Harom). 13C NMR (D2O,
75 MHz) δ (ppm): 14.6; 20.9; 30.9; 37.0; 53.2; 109.6; 115.3;
117.5; 124.4; 128.2; 156.8; 165.1; 177.5; 178.5; 180.2. FT-IR
(KBr pellet) νmáx/cm−1: 2953 (C–Harom); 1582 (CO); 1452
(CN); 1230 (N–O). MALDI-TOF HRMS m/z: 271.0772 (calcd
for C12H11N2NaO4 [M + H]+: 271.0689).
propanoate (4r) (0.20 g, 62%); mp 118–120 °C. H NMR (D2O,
300 MHz) δ (ppm): 2.75 (t, 2H, J = 7.3 Hz, CH2); 3.19 (t, 2H, J =
7.35 Hz, CH2); 7.26 (dd, 1H, J = 8.1 Hz; 7.5 Hz, Harom); 7.53 (d,
1H, J = 8.1 Hz, Harom); 7.64 (d, 1H, J = 8.1 Hz, Harom); 7.74 (s,
1H, Harom). 13C NMR (D2O, 75 MHz) δ (ppm): 23.1; 33.1;
122.2; 125.6; 127.0; 129.5; 130.6; 134.3; 166.2; 179.6; 180.8. FT-
IR (KBr pellet) νmáx/cm−1: 2939 (C–Harom); 1671 (CO); 1561
(CN); 1202 (N–O). MALDI-TOF HRMS m/z: 318.7591 (calcd for
C11H8BrN2NaO3 [M + H]+: 318.9688).
Sodium 3-[3-(2-tolyl)-1,2,4-oxadiazol-5-yl]propanoate (4m)
(0.23 g, 92%) mp 170 °C (dec.) 1H NMR (D2O, 300 MHz) δ
(ppm): 2.31 (s, 3H, CH3); 2.63 (t, 2H, J = 6.9 Hz, CH2); 3.08 (t,
2H, J = 6.9 Hz, CH2); 7.22–7.37 (m, 3H, Harom); 7.56 (d, 1H, J
= 7.5 Hz, Harom). 13C NMR (D2O, 75 MHz) δ (ppm): 18.0;
20.8; 30.9; 32.0; 122.7; 123.9; 127.4; 128.9; 130.0; 135.7; 165.8;
177.5; 177.8. FT-IR (KBr pellet) νmáx/cm−1: 2973 (C–Harom);
1575 (CO); 1444 (CN); 1195 (N–O). MALDI-TOF HRMS m/
z: 254.7268 (calcd for C12H11N2NaO3 [M]+: 254.0661).
Sodium
3-[3-(2-chlorophenyl)-1,2,4-oxadiazol-5-yl]
1
propanoate (4s) (0.27 g, 98%); mp 118–120 °C. H NMR (D2O,
400 MHz) δ (ppm): 2.61 (t, 2H, J = 6.8 Hz, CH2); 3.06 (t, 2H, J
= 6.8 Hz, CH2); 7.23–7.36 (m, 3H, Harom); 7.53 (d, 1H, J = 7.6
Hz, Harom). 13C NMR (D2O, 100 MHz) δ (ppm): 22.9; 33.0;
34.2; 124.4; 127.3; 130.6; 131.3; 132.4; 166.2; 179.6; 180.3. FT-
IR (KBr pellet) νmáx/cm−1: 2944 (C–Harom); 1721 (CO); 1558
(CN); 1292 (N–O). MALDI-TOF HRMS m/z: 274.6786 (calcd
for C11H8ClN2NaO3 [M + H]+: 274.6355).
Sodium 3-[3-(2-furyl)-1,2,4-oxadiazol-5-yl]propanoate (4n)
(0.12 g, 51%) mp 239–240 °C. 1H NMR (D2O, 300 MHz) δ
(ppm): 2.77 (t, 2H, J = 7.1 Hz, CH2); 3.22 (t, 2H, J = 7.1 Hz,
CH2); 6.70 (s, 1H, Harom); 7.18 (s, 1H, Harom); 7.76 (s, 1H,
Harom). 13C NMR (D2O) δ (ppm): 22.9; 32.9; 34.2; 112.0;
114.5; 140.8; 146.1; 160.3; 179.7; 180.9. FT-IR (KBr pellet)
νmáx/cm−1: 2910 (C–Harom); 1635 (CO); 1549 (CN); 1222
(N–O); 1024 (C–O–C). MALDI-TOF HRMS m/z: 230.7160 (calcd
for C9H7N2NaO4 [M]+: 230.0298).
Biochemical assay
The methodology was developed by our research group in a
previous work.33,35 Stock solutions were freshly prepared,
except for 3-hydroxy-D,L-kynurenine (3-D,L-HK) which was kept
at −20 °C. 3-D,L-HK and sodium pyruvate were directly diluted
in 200 mM HEPES buffer pH 7.5/100 mM NaCl, while 20 mM
pyridoxal 5′-phosphate (PLP) was first dissolved in 0.1 M
NaOH and then diluted in HEPES/NaCl buffer until a
concentration of 0.5 mM is obtained. A solution of 20 mM
xanthurenic acid was prepared in 0.1 M NaOH to perform
calibration curves. The reactions were carried out in a 96-well
round-bottom microplate containing 8 μL of 25 mM 3-D,L-HK,
8 μL of 0.5 mM PLP and 4 μL of 50 mM sodium pyruvate, 2
μg of recombinant AeHKT and HEPES/NaCl buffer to a 100
μL final volume. The plates were incubated at 50 °C for 5
minutes and then 100 μL of 10 mM FeCl3·6H2O in 0.1 M HCl
was added to interrupt the reaction and generate the XA–Fe3+
complex. The temperature (30–70 °C) and pH (6–10) of the
AeHKT enzyme were evaluated to establish optimum
conditions, and kinetic parameters were estimated in our
previous work.33 Mixes were prepared to avoid differences
between replicates. Samples were performed in duplicate and
absorbances were read at 570 nm. All the data were analyzed
using the GraphPad Prism7.0 software package using the
Michaelis–Menten equation. Kinetics and inhibition
parameters were determined by non-linear regression.
Sodium 3-[3-(2-thiophenyl)-1,2,4-oxadiazol-5-yl]propanoate
1
(4o) (0.24 g, 96%); mp 188–190 °C. H NMR (D2O, 300 MHz) δ
(ppm): 2.73 (t, 2H, J = 7.2 Hz, CH2); 3.16 (t, 2H, J = 7.2 Hz,
CH2); 7.20 (dd, 1H, J = 4.2 Hz, 4.2 Hz, Harom); 7.67 (s, 2H,
Harom). 13C NMR (D2O, 75 MHz) δ (ppm): 23.0; 33.0; 126.4;
128.2; 130.1; 130.4; 163.4; 179.6; 180.7. FT-IR (KBr pellet)
νmáx/cm−1: 2925 (C–Harom); 1572 (CO); 1417 (CN); 1313
(N–O). MALDI-TOF HRMS m/z: 247.0458 (calcd for C9H7N2-
NaO3S [M + H]+: 247.0147).
Sodium 3-[3-(3-nitrophenyl)-1,2,4-oxadiazol-5-yl]propanoate
1
(4p) (0.27 g, 96%); mp 202–203 °C. H NMR (D2O, 300 MHz)
δ (ppm): 2.88 (t, 2H, J = 7.5 Hz, CH2); 3.33 (t, 2H, J = 7.5 Hz,
CH2); 7.73 (dd, 1H, J = 8.1 Hz, 8.4 Hz, Harom); 8.20 (d, 1H, J
= 8.4 Hz, Harom), 8.34 (d, 1H, J = 8.1 Hz, Harom), 8,53 (s,
1H, Harom). 13C NMR (D2O, 75 MHz) δ (ppm): 23.1; 33.0;
121.7; 126.0; 126.9; 130.5; 133.1; 147.7; 166.0; 179.6; 181.5.
FT-IR (KBr pellet) νmáx/cm−1: 2953 (C–Harom); 1592 (CO);
1506 (CN); 1327 (N–O). MALDI-TOF HRMS m/z: 284.0625
(calcd for C11H8N3NaO5 [M − H]+: 284.0277).
Sodium
3-[3-(2-fluorphenyl)-1,2,4-oxadiazol-5-yl]propanoate
1
Inhibition assays
(4q) (0.22 g, 86%); mp 170–172 °C. H NMR (D2O, 300 MHz) δ
(ppm): 2.75 (t, 2H, J = 7.3 Hz, CH2); 3.20 (t, 2H, J = 7.3 Hz, CH2);
7.21–7.31 (m, 2H, Harom); 7.52–7.58 (m, 1H, Harom); 7.80 (dd,
The inhibition assays were performed with pre-incubation of
each compound in five concentrations ranging from 500 μM
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to 50 μM with PLP and AeHKT. To achieve this, solution
mixes were prepared to ensure that the same amount of
enzyme and co-factor was present in the different inhibitor
concentrations. Blank samples and negative controls were
incubated at the same time as the samples. After 30 minutes
of incubation at 50 °C, the samples were mixed with the
other reaction components and the assays were performed as
described in the Biochemical assay subsection. All
compounds tested were diluted in 200 mM HEPES buffer pH
7.5/100 mM NaCl and the samples were evaluated in
duplicate. In each test, the negative control was added to
calculate the activity percentage.
probability of search for one individual. Ligand
conformations presenting the most favorable binding energy
were selected in each step of the simulation in such a way
that at the end of the calculation, atomic coordinates of the
200 conformers that better fitted the binding site were
selected. The final lowest energy structures were clustered
based on a RMS positional deviation of 2.0 Å. These
structures were examined, and the figures were generated by
PyMOL.47 Final atomic coordinates for docked complexes are
available in the ESI.†
QSAR methods
Molecular docking procedure
The 3D geometry of the compounds was obtained from the
lowest energy conformations of the docking results.
Molecular descriptors of diverse nature (1D, 2D and 3D) were
generated by the Dragon® software,38 providing an initial
matrix of 5.000 descriptors. This amount was initially
reduced by a cutoff of min = 0.1 in the standard deviation
and a correlation of <0.9 between the variables. After this
process, 781 variables remained.
An initial analysis was performed in the IC50 values, where
two samples with very different values were detected:
compounds 4m and 6j. Statistical analysis of interquartile
distances reveals that they behave as outliers. Considering
the 30 IC50 values; taking the median, the first (Q1) and third
(Q3) interquartile values (84.5, 68 and 110, respectively) as
well as calculating the interquartile range (IQR = 42), the
calculation of the limits for the possible lower and upper
outliers were, respectively: Q1 − 1.5 × (IQR) and Q2 + 1.5 ×
(IQR). These calculations resulted in values of 5 and 173 for
the lower and the upper limits, respectively. In this way, we
verified that none of the samples showed a value smaller
than 5 and the two evidenced compounds showed values
greater than 173 (4m and 6j). Thus, these samples were not
considered for the QSAR analysis.
The original IC50 values were then converted to −log(IC50),
or pIC50, reducing the standard deviation so the highest
values correspond to the best activities. The descriptor matrix
of the 28 compounds was submitted to a variable selection
procedure using the ordered predictor selector (OPS)
algorithm48 implemented in the QSAR modeling free
software.49 This algorithm performs the construction of
incremental partial least squares (PLS) models, starting from
a reordering of the original descriptor matrix, using the X
variables most correlated with the Y variable in the first
columns.48,50 Considering the specific parameters used for
this work, the incremental models started from the first 10
reordered columns, increasing by a factor of 5, with a
maximum of 30% of the total variables. At the end, the best
models were presented chosen by the standard deviation of
the prediction error sum of squares (SPRESS)48,51 value
calculated for each incremental set of variables. After OPS,
the original matrix was reduced to 30 descriptors. In this
step, training and test sets were randomly chosen,
Molecular docking calculations were performed as described
in our previous studies.32,33 The programs AutoGrid v.4.0 and
AutoDock v.4.0 were used during these calculations.41,42 The
protein structure was treated as rigid, while the ligands were
treated flexibly. Atomic coordinates of the AeHKT crystal
structure in its holo form were retrieved from PDB (ID
6MFB). Polar hydrogens were added to the protein
considering amino acid protonation states at pH 7. Partial
charges for protein atoms were assigned according to
AMBER86 force field parameters,43 while PLP and ligand
charges were assigned using the Gasteiger method,44
ensuring that all residues have presented integer charges.
The AMBER86 atom types were assigned to all atoms.43
Overall, 30 oxadiazole derivatives were evaluated in terms of
binding affinity to AeHKT in a complex. In addition to the
sodium 1,2,4-oxadiazolyl-propionates reported here, the
eleven sodium 1,2,4-oxadiazolyl-butanoates described by
Maciel and colleagues were docked in the AeHKT active
site.33 Aiming at the reproducibility of the present docking
protocol, redocking experiments were conducted in three
steps: (i) docking of co-crystallized 4-OB in the AgHKT active
site (PDB ID 2CH2);37 (ii) docking of co-crystallized 4-OB in
the AeHKT active site; and (iii) docking of co-crystallized
4-OB in AeHKT partially outside the active site, all sampled
through 200 generation cycles. The
C monomer was
arbitrarily chosen to perform the docking calculations in
both enzymes. A grid with dimensions of 28 × 28 × 28 Å and
spacing of 0.22 Å was centered at the co-crystallized ligand
4-OB, located at the active site. The binding modes for all the
ligands were predicted based on affinity maps generated by
AutoGrid v4.0. The Lamarckian genetic algorithm45,46 was
employed in all molecular dockings with the following
parameters:45,46 an initial population of 150 individuals per
generation, a maximum number of 27 000 generations, and a
total of 2.5 × 106 energy evaluations per generation. Thus, a
total of 4.05 × 106 conformations for each of the ligands were
generated in each of the 200 steps of the simulation. An
elitism rate of 1 was applied to ensure that the top individual
always survives into the next generation in conjunction with
mutation and crossover rates of 0.02 and 0.08, respectively.
In the local search, 300 steps were applied with a 0.06
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considering 4d, 4h, 4o, and 4s for the test compounds and
the remaining 24 compounds for the training set.
potential as prototypes for the design of new HKT inhibitors
with greater potency and specificity.
The training set was submitted to a new variable selection
using the systematic search algorithm implemented in the
Build QSAR program.52 At the end, five descriptors were
The QSAR modeling revealed five molecular descriptors
that could explain the variability in oxadiazole biological
responses. From
a
qualitative viewpoint, compounds
selected in
a
multiple linear regression (MLR) model,
presenting heavy atoms as substituents on the para position
of the aromatic ring tend to be more active. The equation
can be used to predict compounds not yet tested, considering
the general adjustment of the numerical information
provided by the molecular descriptors.
considering the recommended 1 : 5 ratio between descriptors
and compounds.53
The validation parameters53 for the model were defined
as R2 values (the coefficient of determination for
calibration); Q2
(the coefficient of determination for
LOO
leave-one-out cross validation); Q2
(the coefficient of Conflicts of interest
LNO
determination for leave-N-out cross validation); Q2
(the
EXT
There are no conflicts to declare.
coefficient of determination for external validation) and the
results of Y-scrambling. The first two and the Q2
EXT
Acknowledgements
parameters were obtained based on the standard equations.
The Q2
results were expressed in a plot as the means of
LNO
We thank Dr. Andrew Oliveira and Dr. Rafael Guido for the
collaboration in cloning, expression and purification
experiments of recombinant HKT and also Dr. Tatiany Romão
and Dr. Maria Helena Neves for the use of the
spectrophotometer and providing the DNA samples to achieve
the cloning step. Financial support for this research project
was provided by the Brazilian funding agencies FACEPE (APQ-
0732-1.06/14), BioMol/CAPES (BioComp 23038.004630/2014-
35), INCTFx/CNPq (465259/2014-6) and FAPESP (2013/07600-
3). TAS acknowledges CNPq for a productivity fellowship.
10 random runs in each N chosen – which was from one to
eight samples taken – considering the training set. For the
Y-scrambling procedure, the dependent variable column was
mixed 50 times to build alternative models and determine
R2 and Q2
for them.53 If the descriptors used in the
LOO
modeling do not correspond to a random correlation with
the activity, it is expected for most values of the models
with scrambled Y to be calculated with a value smaller than
0.4.
Conclusions
Notes and references
Nineteen sodium 3-[3-(aryl)-1,2,4-oxadiazol-5-yl]-propanoates
were synthesized and obtained in yields ranging from 51% to
98% and comprise a second generation of 1,2,4-oxadiazoles
planned to act as Aedes aegypti larvicides. These salts were
tested for A. aegypti HKT inhibition and from this reaction, it
was possible to conclude that these compounds are part of a
new class of HKT inhibitors. Compounds containing p-anisoyl
and p-trifluoromethylphenyl groups exhibited the best IC50
values, 35.0 and 42.3 mM, respectively. These results showed
that inhibitors with high electron density substituents have
lower IC50 values and therefore are more promising candidates
for inhibitors of the HKT from A. aegypti. Inhibition assays
showed that the synthesized salts are competitive inhibitors of
the enzyme, promoting accumulation of the toxic metabolite
3-HK in the reaction medium.
Molecular docking calculations provided a 3D map of
essential interactions for the molecular recognition of the 30
oxadiazole derivatives by HKT with a diverse repertoire of
side chains and aromatic ring substituents. Although some
derivatives showed lowest energy conformations slightly
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4-OB, the most promising compounds with para substituents
showed similar conformation and interaction with the Arg356
residue. Remarkably, all 1,2,4-oxadiazole derivatives docked
exhibited an estimated KI smaller than the corresponding
value for 4-OB under the same simulation conditions. This
finding indicates that this class of compounds has great
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